Bp2023 - Volume IV

British
Pharmacopoeia
2023
Volume IV
General Notices
Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal Products
Materials for use in the Manufacture of Homeopathic Preparations
Blood-related Products
Immunological Products
Radiopharmaceutical Preparations
Surgical Materials
Incorporating the requeriments of the 10th edition of the

European Pharmacopoeia as amended by Supplements 10.1 to 10.8
British Pharmacopoeia 2023
Volume IV
British Pharmacopoeia 2023
Volume IV
The British Pharmacopoeia Commission has caused this British Pharmacopoeia 2023 to be prepared
under regulation 317(1) of the Human Medicines Regulations 2012 and, in accordance with
regulation 31 7 (4), the Ministers have arranged for it to be published.
The monographs of the Tenth Edition of the European Pharmacopoeia (2019), as amended by
Supplements 10.1 to 10.8, 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 2023
see Notices
London: The Stationery Office

In respect of Great Britain:
THE DEPARTMENT OF HEALTH AND SOCIAL CARE

In respect of Northern Ireland:
THE DEPARTMENT OF HEALTH (NI)
© Crown Copyright 2022

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: bpcom@mhra.gov.uk.
First Published 2022

ISBN 978 011 3230 914
British Phannacopoeia Commission Office:
Medicines and Healthcare products Regulatory Agency
10 South Colonnade,
Canary Wharf,
London E 14 4PU
E-mail: bpcom@mhra.gov.uk
Web site: http://www.pharmacopoeia.com
Laboratory:
British Pharmacopoeia Commission Laboratory
Queen's Road
Teddington
Middlesex TWll 0LY
Telephone: +44 (0)20 8943 8960
E-mail: bpcrs@mhra.gov.uk
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 Advisory Groups, Panels of Experts, Working Parties, Ad-hoe Group
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 CT - Z)
Contents of Volume III
NOTICES
GENERAL NOTICES
MONOGRAPHS
Formulated Preparations: General Monographs
Formulated Preparations: Specific Monographs
Contents of Volume IV
NOTICES
IV-v
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 Tenth Edition of the
European Pharmacopoeia comprising, unless otherwise stated, the main volume, published in 2019, 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 2023. 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 the Human
Medicines Regulations 2012, as amended, and the Veterinary Medicines Regulations 2013, as
amended.
IV-vii
2023 General Notices IV-1
General Notices
IV-2 General Notices 2023
CONTENTS OF THE GENERAL NOTICES

Part I Implementation of Pharmacopoei::11 Methods
Italic introduction Conventional Terms
European Pharmacopoeia Interchangeable Methods
References to Regulatory Documents
Part II 1.2 Other Provisions Applying to General Chapters
Italic introduction and Monographs
Official Standards Quantities
Definition of Terms Apparatus and Procedures
Expression of Standards Water-bath
Temperature Drying and Ignition to Constant Mass
Weights and Measures Reagents
Atomic Weights Solvents
Constant Weight Expression of Content
Expression of Concentrations Temperature
Water Bath 1. 3 General Chapters
Reagents Containers
Indicators 1.4 Monographs
Caution Statements Titles
Titles Relative Atomic and Molecular Masses
Chemical Formulae Chemical Abstracts Service (CAS) Registry
Definition Number
Production Definition
Manufacture of Formulated Preparations Limits of Content
Freshly and Recently Prepared Herbal Drugs
Methods of Sterilisation Production
Water Choice of Vaccine Strain, Choice of Vaccine
Excipients Composition
Colouring Agents Potential Adulteration
Antimicrobial Preservatives Characters
Characteristics Solubility
Solubility Identification
Identification Scope
Reference Spectra First and Second Identifications
Assays and Tests Powdered Herbal Drugs
Biological Assays and Tests . Tests and Assays
Reference Substances and Reference Preparat10ns Scope
Chemical Reference Substances Calculation
Biological Reference Preparations Limits
Storage Indication of Permitted Limit of Impurities
Labelling Herbal Drugs
Action and Use Equivalents
Crude Drugs; Traditional Herbal and Culture Media
Complementary Medicines Storage
Monograph Title Labelling
Definition Warnings
Characteristics Impurities
Control Methods Functionality-related Characteristics of
Homoeopathic Medicines Excipients
Unlicensed Medicines Reference Standards
Part III 1.5 Abbreviations and Symbols
Italic introduction . Abbreviations used in the Monographs on
t1 1• _ T _ _ 1 T Y~ .,, • • - -,
Generai Notices of the European Pharmacopoeia Immw1og10ounns, 1mmunos~ra anu v accmes

1.1 General Statements rn11Prtinrn: of Micro-organisms
Quality Systems 1.6 Units of the International System (SI) used in
Alternative Methods the Pharmacopoeia and Equivalence with
Demonstration of Compliance with the other Units
Pharmacopoeia International System of Units (SI)
Grade of Materials Notes
General Monographs
Validation of Pharmacopoeia! Methods
2023 General Notices IV- 3
Part I
The British Pharmacopoeia comprises the entire text within this publication. The word 'officiaf is used in the
Phannacopoeia to signify 'of the Phannacopoeia '. It applies to any title, substance, preparation, method or
statenzent included in the general notices, monographs and appendices of the Pharmacopoeia. The abbreviation
for British Pharmacopoeia is BP.
European Pharmacopoeia
Monographs of t_he European Pharmacopoeia are reproduced in this edition 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 (I 974) CMND 5763) as amended by the Protocol to the
Convention (Treaty Series No. MISC16 (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 Bur' 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 III of these notices.
IV-·4 General Notices 2023
Part II
The following general notices apply to ihe staternents made in the monographs of the Brilish Pharmacopoeia
other than those reproduced from the European Phannacopoeia and to the staiements 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 Phannacopoeia.
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 pharmacopoeial 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 pharmacopoeia! 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 pharmacopoeia! 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.
IL urny, fo1. cxa111pk., be a liLcu:si11M, auillv1.ity u1 au vffi.Lial Lv11uvl labvnuury. Fur a furmulau:d
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,
thP rhnlrP of snrh str::iinj test, method, suhstance, etc.j is m::ide in ::iccord::ince 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).
\X'here 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 Standards
Where the standard for the content of a substance described in a monograph is expressed in terms of
the chemical 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 C 20 H 24N 20 2,HCI' 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 C 20H 24 N 2 O 2 ,HC1.
Where the result of an assay or test is required to be calculated with reference to the 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 Measures

The metric system of weights and measures is employed; SI Units have 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.
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).
IV-6 Genera] Notices 2023
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 O. 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 (I hour is usually suitable).
Expression of Concentrations
The term 'per cent' or more usually the symbol {%' is used with one of four 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 w/w (% w/w) (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 aione authoritative.
The y_muuiLy uf au iudi'-.:atur :suluLiuu avvrupriau:: for u:st: iu at:id-ba:s<:: Lit.ratiuu:s d<:::sctibi::d in a:s:say:s
or tests is O.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.
2023 General Notices IV- 7
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 itaiicised statement;
the absence of such a statement should nor however be taken ro mean rhar 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.
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 titles. A cumulative list of
such Approved Synonyms is provided in Appendix XXI B.
Where the names of pharmacopoeia} 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 are 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 the context that the formulae denote the chemically pure substances.
Where the absolute stereochemical configuration is specified, the International Union of Pure and
Applied Chemistry (IUPAC) RIS and E!Z 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 othenvise indicated. The three-letter
and one-letter symbols used for amino acids in peptide and protein sequences are those recommended
by the Joint Commission on Biochemical Nomenclature of the International Union of Pure and
Applied Chemistry and the International Union l)f Bincht:mistry and Molecular Biology,
In the graphic formulae the following abbreviations are used:
Me --CH 3 Bus -CH(CH 1 )CH 2 CH,,

Et -CH 2 CH1 Bu11 --CH 2 CH 2 CH 2 CH,
Pr1 -CH(CH 3) 2 Bu' -C(CH 3) 3
11
Pr -CH 2 CH 2 CH3 Ph -C6Hs
Bu' -CH2CH(CR3h Ac -COCH 3
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. 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 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. The 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 Formulated Preparations

Attention is drawn to the need to observe adequate hygienic precautions in the preparation and
dispensing of pharmaceutical formulations. The principles of good pharmaceutical manufacturing
pr5irtirP '-hnn lrl hP oh'-PruPrl.
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 stricdy 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 Exdpients 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 Recently Prepared

The direction, given under the heading Extemporaneous Preparation, that a 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 Sterilisation
The methods of sterilisation used in preparing the sterile materials 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 \Vithout 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 unsufrable for a particular preparation.
IV -10 General Notices 2023
Excipients
Where an excipient for which there is a pharmacopoeia! 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 Preservatives
When the term 'suitable antimicrobial preservative' is used it is implied that 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.
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 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 of which only some of the components
dissolve.
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''.
Rejerence spectra Wbere 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 the 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.
\X'hen 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 the 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 ingredient. This means that 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.
IV-12 GeneraJ Notices 2023
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) secondary spot or peak) 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 the monograph.
In all cases where an impurity limit is given in parentheses, the figures given are approximations for
information only; conformity with the 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 and Tests

Methods of assay described as Suggested methods are not obligatory, but 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 (JU) 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 the 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 the assay is such that the fiducial limits of error, expressed as a
percentage of the estimated potency, are within a range not wider than that 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).

Wnere the biological assay is being used to ascertain the purity of the material, the stated potency
means the potency stated on the 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 the 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 (HJ) 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 the 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 primary standard.
Unless otherwise directed, animals used in an assay or a test are healthy 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 stated, 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 Substances and Reference Preparations

Certain monographs require the use of a reference substance, a reference preparation or a reference
spectrum. These are chosen with regard to their intended use as prescribed in the monographs of the
Pharmacopoeia and are 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 III 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 \'Xl orld 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) ,vhere appropriate. Where applicable, the
of the Biningica] Reference Preparations is expressed in Imern::1ti0nal Units, For some
IV-14 General Notices 2U23
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
mactf' of a mMnial that ahsorhs ;1ct1nic light ~mfficie-nt1y to prote-rt the- contt-nts frnm rh::mge- inctnf'e-ci
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.
Labelling
The labelling requirements of the Pharmacopoeia are 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, as amended, in respect of their labelling and
packaging 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 the
most recent version of the MHRA "Best Practice Guidance on the Labelling and Packaging of
Medicines"). 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
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expressed in terms of the official medicinal substance used in maki..flg tl-ie 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; Traditional Herbal and Complementary Medicines

Herbal and complementary medicines are classed as medicines under the Human Medicines Regulations 2012,
as amended. It is emphasised that, although requirements for the quality of the material are provided in the
monograph to assist the 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 title 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 title.
Definition Under the 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 chemicaVchromatographic 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, Sulfated 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(s).
The macroscopical characteristics include those features that can be seen by the unaided eye or by
the use of a hand lens. When two 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 on
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% w/w. 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 Medicines
Honweoparhic medicines are classed as medicines under the Human Medidnes Regulations 2012) as amended.
It £s emphasised that, although requirements for the qualit)' of the niaterial are provided in the relevant
in order to assist the sinzplified registration scheme by the UK Licensing Authority, the British
Pharmacopoeia Co,nmission has not assessed the safe()) 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.
Wnere 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(s); 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 Medicines
The General Monograph for Unlicensed Medicines applies to those 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 pharmacopoeia! compliance when tested, it is recognised that it might
not be practicable to carry out the pharmacopoeia! 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 alone authoritative.
Part III
Monographs and other texts of the European Pharmacopoeia that are incorporated in this edition of the British
Phamzacopoeia are governed by the general notices of the European Pharmacopoeia; these are reproduced
below.
GENERAL NOTICES OF THE EUROPEAN PHARMACOPOEIA
1.1 GENERAL STATEMENTS
1.1.1 General principles
1.1.1.1 Quality systems
1.1.1.2 Conventional terms
1.1.1.3 References to regulatory documents
1.1. 2 Compliance with the Ph. Eur.
1.1.2.1 Scope
1.1.2.2 Demonstration of compliance with the Ph. Eur.
1.1.2.3 Demonstration of suitability of monographs
1.1.2.4 Validation and implementation of Ph. Eur. analytical procedures
1.1 .2. 5 Alternative analytical procedures
1.1.2.6 Pharmacopoeial harmonisation
1.2 OTHER PROVISIONS APPLYING TO MONOGRAPHS AND GENERAL CHAPTERS
1.2.1 Quantities
1.2.2 Glassware
1.2.3 Temperature
1.2.4 Water-bath
1.2.5 Drying and ignition to constant mass
1.2.6 Solutions
1.2. 7 Reagents and solvents
1.2.8 Expression of content
1.2. 9 Caution statements
1.3 GENERAL CHAPTERS
1.3.1 Materials for containers and containers
1.4 GENERAL MONOGRAPHS AND GENERAL MONOGRAPHS ON DOSAGE FORM.S
1.5 INDMDUAL MONOGRAPHS
1.5.1 GENERAL PRINCIPLES
1. 5 .1.1 Titles
1.5.1.2 Relative atomic and molecular masses, formulae
1.5.1.3 CAS registry number
1.5.1.4 Definition
1. 5 .1. 5 Production
1.5.1.6 Potential adulteration
1. 5.1. 7 Characters
1. 5 .1. 8 Identification
1.5.1.9 Tests and assays
1. 5 .1.10 Storage
1. 5 .1.11 La belling
1. 5.1.12 Impurities
1. 5. l .13 Functionality-related characteristics of excipients
1.5.2 MONOGRAPHS ON HERBAL DRUGS
1.5.3 MONOGRAPHS ON MEDICINAL PRODUCTS CONTAINING CHEMICALLY

DEFINED ACTIVE SUBSTANCES
1.5.3.1 Related substances
1.5.3.2 Dissolution / Disintegration
1.5.3.3 Impurities
1.5.3.4 Storage
1.6 REFERENCE STANDARDS
1.7 ABBREVIATIONS AND SYMBOLS
1.8 UNITS OF THE INTERNATIONAL SYSTEM (SI) USED IN THE PH. EUR. AND
EQUIVALENCE WITH OTHER UNITS
1.1 GENERAL STATEMENTS
L 1.1 General principles
The General Notices apply to all texts of the European Pharmacopoeia.
The 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 published by the EDQM are alone
authoritative.
The date on which texts of the European Pharmacopoeia are to be implemented is fixed by a
resolution of the European Committee on Pharmaceuticals and Pharmaceutical Care (Partial
Agreement) of the Council of Europe, following a recommendation by the Ph. Eur. Commission. This
date is usually 1 year after adoption and about 6 months after publication. Where a text needs to be
implemented at a date earlier than the next publication date of a new edition or supplement of the
European Pharmacopoeia, a resolution of the European Committee on Pharmaceuticals and
Pharmaceutical Care is issued, giving the full text to be implemented. The text is also published in
Pharmeuropa Online for information and posted on the EDQM website as part of the resolution.
In the texts of the European Pharmacopoeia, the word 'Pharmacopoeia, without qualification means
the European Pharmacopoeia. The official abbreviation 'Ph. Bur.' may also be used for this purpose.
1.1.1.1 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 Ph. Eur.
1.1.1.2 Conventional terms

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.
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 have 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 and antioxidants are examples of
excipients.
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.
Competent authority 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 (NPA), a licensing authority or an official medicines control
laboratory (OMCL).
'Unless otherwise justified and authorised' This expression means that the requirements
must be met, unless the competent authority authorises a modification (e.g. of an analytical procedure
nr llmit) nr ~n t>vi=>mptinnj if j11"rlfit><l hy the mim11factnrer in a particular c::t~e.
'Should' Statements containing the word 'should' are informative or advisory.
'Suitable', 'appropriate' In certain texts, the terms 'suitable' and 'appropriate' are used to
describe a reagent, test, micro-organism, etc.; in such cases, if criteria for suitability are not described
in the text, suitability is demonstrated to the satisfaction of the competent authority.
1.1.1.3 References to regulatory documents

Monographs and general chapters may contain references to documents issued by regulatory
authorities for medicines, for example directives and notes for guidance of the European Union. These
references are provided to users of the Ph. Eur. for information. Inclusion of such a reference does not
modify the status of the documents referred to, unless explicitly stated in the text.
1.1.2 Compliance with the Pb. Eur.

1.1.2.1 Scope
The use of the title or the Latin subtitle of a monograph implies that the article complies with the
requirements of that monograph. Such references to monographs in the texts of the Ph. Bur. are
shown using the monograph title and reference number in italics.
The scope of a monograph is stated in its definition.
Medicinal products whose labels state the modified international nonproprietary name (INNM) of
their active substance (e.g. raltegravir potassium) must comply with the relevant monograph on the
medicinal product, even if the monograph title only refers to the unmodified INN (e.g. Raltegravir
tablets (2938)).
Shelf life and re-test period A medicinal product must comply with the relevant monograph
throughout its shelf life; the shelf life and the time point from which that period is to be calculated are
proposed by the manufacturer in light of experimental results of stability studies and are approved by
the competent authority. A distinct shelf life 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 re-test period, except for some
substances known to be labile and for certain antibiotics where a shelf life is established rather than a
re-test period.
Monographs on medicinal products provide shelf-life specifications that may differ from release
specifications indicated in marketing authorisations. Monographs on other articles provide
specifications that must be fulfilled until the end of the re-test period.
Human and/or veterinary use The active substances, excipients, medicinal products and other
articles described in monographs are intended for human and veterinary use unless explicitly restricted
to one of these uses in the title or the definition.
Grades Certain articles that are the subject of a monograph may exist in different grades that are
suitable for different purposes. Unless otherwise indicated in the monograph, the requirements apply
to all grades of the article.
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.
Analytical procedures for determination of one or more of these characteristics may be given, also for
information.
1. 1.2.2 Demonstration of compliance with the Ph. Eur.

Unless othenvise indicated in the General Notices or in the monographs, statements in monographs
constitute mandatory requirements.
(I) An article is of Ph. Eur. quality if it complies \Vith all of the requirements stated in the monograph.
This does not imply that a manufacturer must perform all of the tests described in a monograph when
assessing compliance with the Ph. Eur. before release. The manufacturer may obtain assurance that an
article is of Ph. Eur. quality on the basis of its design, together with its control strategy and data
derived, for example, from validation studies of the manufacturing process.
In certain monographs, the sentence 'The following procedure is given as an example' means that the
analytical procedure described has been validated and may be implemented as is or may be replaced
by a suitable, validated procedure (without having to demonstrate its equivalence to the 'example'
procedure), subject to approval by the competent authority.
(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 Ph. Eur.
(3) Reduction of animal testing: the Ph. Eur. is committed 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 Ph. Eur. as indicated above (I), 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 Ph. Eur. when
animal tests are prescribed is established in such a way that animal usage is kept to a minimum.
1.1.2.3 Demonstration of suitability of monographs

The manufacturer must evaluate the suitability of the monograph for the quality control of their
substance or medicinal product, since the choice of analytical procedures may be influenced by the
manufacturing process and/or the composition of the medicinal product. In cases where the
specification described in a monograph is considered to be insufficient to ensure the quality of the
product or substance by a competent authority, the latter may request more-appropriate specifications
from the manufacturer in line with national or regional regulations. In such cases, the competent
authority informs the Ph. Eur. Commission through either the national pharmacopoeia authority or
the Secretariat of the Ph. Eur. Commission (EDQM). The manufacturer is requested to provide the
national pharmacopoeia authority or the EDQM with the details of the alleged insufficiency and the
additional specifications applied, so that the Ph. Eur. Commission can decide on the need to revise
the monograph in question.
1.1.2.4 Validation and implementation of Pb. Eur. analytical procedures

The analytical procedures given in an individual monograph have been validated in accordance with
accepted scientific practice and recommendations on analytical validation. Unless otherwise stated in
the individual monograph or in the corresponding general chapter, validation of these procedures by
the user is not required.
The analytical procedures provided in general chapters may be used for active substances,
Pvrin1Pnt,;:
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When implementing a Ph. Eur. analytical procedure, the user must assess whether and to what
extent its suitability under the actual conditions of use needs to be demonstrated according to relevant
monographs, general chapters and quality systems.
1.1.2.5 Alternative analytical procedures

The tests and assays described are the official analytical procedures upon which the standards of the
Ph. Eur. are based. With the agreement of the competent authority, alternative analytical procedures
may be used for control purposes, provided that they enable an unequivocal decision to be made as to
whether compliance with the standards of the monographs would be achieved if the official procedures
were used. In the event of doubt or dispute, the analytical procedures of the Ph. Eur. are alone
authoritative.
1. 1.2.6 Pharmacopoeia! harmonisation

The Ph. Eur. is engaged in a process of pharmacopoeia! harmonisation with the Japanese
Pharmacopoeia and the United States Pharmacopeia, within an informal structure referred to as the
Pharmacopoeia! Discussion Group (PDG). More information is available in general chapter 5.8.
Pharmacopoeia! harmonisation.
1.2 OTHER PROVISIONS APPLYING TO MONOGRAPHS AND GENERAL
CHAPTERS
1.2.1 Quantities
In tests with numerical limits and assays, the quantity stated to be taken for examination corresponds
to the quantity used during development of the analytical procedure. The amount actually used may
deviate by not more than 10 per cent from the stated quantity. In all cases, the amount used is
accurately measured and the result of the test is calculated from this exact quantity.
In tests where the limit is not numerical, which usually depend instead 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.
The number of significant figures of a quantity value implies specific requirements for the quantities
(masses and volumes) to be measured, as detailed below.
For masses, requirements for balances for analytical purposes are provided in general chapter 2.1. 7
and are applicable to all texts. In addition, during weighing, the indication of a balance must match
the target mass value given in the text when mathematically rounded to the same number of
significant figures. For example, if the target mass value in the text is 50.0 mg, the 'minimum weight'
(mmin) of the balance used must be smaller and the weighing is done within ± 5 subunits after the
last figure of the stated mass value (for example, 50.0 mg is to be interpreted as 49.95 mg to
50.04 mg or 49.950 mg to 50.049 mg, depending on the readability of the balance).
For 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 target voiume is measured using a volumetric 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 number of significant figures with which
quantities are stated does not correspond to the number of significant figures stated in a specified
numerical limit. The stated quantities are then measured with a sufficiently improved accuracy.
1.2.2 Glassware
Volumetric glassware complies with Class A requirements of the appropriate International Standard
issued by the International Organisation for Standardisation (ISO).
Unless otherwise prescribed, visual comparative tests are carried out using identical colourless,
transparent, neutral glass tubes 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 (see general chapter 2.1. 5. Tubes for comparative tests). 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.
1.2.3 Temperature
Unless otherwise prescribed, analytical procedures are carried out at a temperature between 15 :c and
25 ~c.
Where a text describes a temperature without giving 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.
1.2.4 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.
1.2.5 Drying and ignition to constant mass

The terms 'dried to constant mass' and 'ignited to constant mass' mean that 2 consecutive weighings do
not differ by more than 0.5 mg, the second weighing carried out after an additional period of drying
or ignition appropriate to the nature and quantity of the residue.
Where drying is prescribed using one of the expressions 'in a desiccator' or 'in vacua', it is carried
out using the conditions described in general chapter 2. 2. 32. Loss on drying.
1.2.6 Solutions
'Freshly prepared solution' means that the solution is prepared each time the test/assay is to be carried
out and is used within 24 h.
'Immediately before use' indicates that the stability of the corresponding solution(s) has been found to
be critical during the elaboration of the text. The time between preparation and use must be
minimised.
1.2. 7 Reagents and solvents

The proper conduct of the analytical procedures described in the Ph. Eur. and the reliability of the
results depend, in part, upon the quality of the reagents used. The reagents are described in the
Ph. Eur. in 4. Reagents and subsections. It is assumed that reagents of analytical grade are used; for
some reagents, tests to determine suitability are included in the description.
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 determination is prescribed.
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 Ph. Eur.
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 (C 2 H 6 0)
required.
1.2.8 Expression of content

In defining content, the expression 'per cent' is used according to circumstances with one of two
meanings:
'per cent mlm' (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 expressions 'parts per million' (ppm) and 'parts per billion' (ppb) refer to mass in mass, unless
otherwise specified.
1.2.9 Caution statements

Articles described in monographs and reagents specified for use in the Ph. Eur. may be injurious to
health unless adequate precautions are taken. The principles of good quality control laboratory
practice and the provisions of any appropriite regulations are to be observed at all times. Attention is
drawn to particular hazards in certain monographs or general chapters by means of a caution
statement; absence of such a statement is not to be taken to mean that no hazard exists.
1.3 GENERAL CHAPTERS
General chapters (see sections 2, 3 and 5 of the Ph. Eur.) become mandatory when referred to in a
monograph, unless the wording clearly indicates that it is not the intention to make the text referred to
mandatory but rather to cite it for information.
When a general chapter is not referred to in any monograph or general chapter, it is given for
information; this is usually indicated in the preamble to the general chapter.
General chapters also become mandatory when referred to in another general chapter that is itself
referred to in a monograph, unless otherwise stated.
Requirements included in general chapters are not repeated in the individual monographs, unless
specific to the article in question (e.g. symmetry factor, signal-to...noise ratio of general chapter 2.2.46.
Chromatographic separation techniques).
1.3.1 Materials for containers and containers

Materials used for containers are described in 3.1. Materials used for the manufacture of containers and
subsections. Each subsection covers a defined plastic material with a positive list of accepted additives.
The specifications for each of these 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 corresponding tests and limits applied to them, are
subject to approval by the competent authority.
The specifications for containers in 3. 2. Containers and subsections 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 approval by the
competent authority.
Containers for human blood and blood components, transfusion sets and syringes that are not
designed to serve as primary packaging for medicinal products are described in 3. 3. Containers for
human blood and blood components> and materials used in their manufacture>· transfusion sets and materials
used in their manufacture; syringes and subsections. Most of these texts are published for information
only.
Reference may be made within the monographs of the Ph. Eur. to the definitions and specifications
for containers. The general monographs on dosage forms may, in the Definition/Production section,
require the use of certain types of container, as described in 3. 2. Containers and subsections; certain
other llhJrh.:,graphs may, in the Storage section, indicate the type of container that is recommended for
use.
1.4 GENERAL MONOGRAPHS AND GENERAL MONOGRAPHS ON DOSAGE

FORMS
General monographs and individual monographs are complementary.
\Xlhenever an individual monograph is used, it is essential to ascertain whether there are one or
more general monographs applicable to the article in question.
Substances and medicinal products that are the subject of an individual monograph are also
required to comply with relevant, applicable general monographs. Cross-references to applicable
general monographs are not given in individual monographs. However, exceptions are possible; for
example, individual monographs on medicinal products containing chemically defined active
substances include a reference to the relevant general monograph on the dosage form.
General monographs give requirements that are applicable to all articles in the given class or, in
some cases, to any article in the given class for which there is an individual monograph in the Ph. Eur.
Where no restriction on scope of a general monograph is given in a preamble, it is applicable to all
articles in the class defined, irrespective of whether there is an individual monograph for the article in
the Ph. Eur.
If the provisions of a general monograph do not apply to a particular article, this is expressly stated
in the individual monograph.
General monographs on dosage forms apply to all medicinal products of the type defined. The
requirements are not necessarily comprehensive for a specific medicinal product and requirements
additional to those prescribed in the general monograph may be imposed by the competent authority.
1.5 INDIVIDUAL MONOGRAPHS

1.5.1 GENERAL PRINCIPLES
1.5.1.1 Titles
Monograph titles are in English or French in the respective versions and there is a Latin subtitle.
Where available, the international nonproprietary name (INN) is used, unless there are justifiable
reasons for not doing so. If needed, it is supplemented by the name of the anion or cation and by the
degree of hydration. Additional qualifiers may limit the applicability of the monograph to certain
classes or forms (e.g. veterinary use, dosage form, route of administration).
1.5.1.2 Relative atomic and molecular masses, formulae

The relative atomic mass (Ar) or the relative molecular mass (Mr) is shown, 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.
1.5.1.3 CAS registry number

Chemical Abstract Service (CAS) registry numbers are included for information in monographs, where
applicable, to provide convenient access to useful information for users. CAS Registry NumberR' is a
registered trademark of the American Chemical Society.
1.5.1.4 Definition
This section provides the official definition of the article that is the subject of the monograph.
Limits of content If prescribed, limits of content are those determined using the analytical
procedure described under Assay.
1. 5.1. 5 Production
Statements in the Production section draw attention to particular aspects of the manufacturing process
but are not necessarily exhaustive. 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 process-related heterogeneity of the article, to in-process testing, or
to tests that are to be carried out by the manufacturer on the final article, either on selected batches or
on each batch prior to release. These requirements 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 examining data received from the manufacturer, through
inspection or by testing 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 vaccine
monograph may define the characteristics of a vaccine strain or vaccine composition. Unless otherwise
stated, analytical procedures given for verification of these characteristics are provided for information
as examples of suitable procedures. Subject to approval by the competent authority, other procedures
may be used without validation against the procedure shown in the monograph.
1.5.1.6 Potential adulteration

Due to the rise in fraudulent activities and cases of adulteration, information may be made available to
users of the Ph. Eur. to help them detect adulterated articles (i.e. active substances, excipients,
intermediate products, bulk products and medicinal products).
To this end, an analytical procedure for the detection of potential adulterants and relevant limits
may be included in this section of monographs on substances for which an incident has occurred or
which are at risk of deliberate contamination. In such cases, a reminder that a suitable quality system
is applied at all stages of production and sourcing is also provided. The frequency of testing by
manufacturers or by users (e.g. manufacturers of intermediate products, bulk products and medicinal
products) depends on a risk assessment, taking into account the level of knowledge of the whole
supply chain and national requirements.
The requirements listed in this section apply to the whole supply chain, from manufacturers to
users. The absence of this section does not imply that attention to features such as those referred to
above is not required.
1. 5.1. 7 Characters
The statements in the Characters section do not constitute Ph. Eur. requirements and are given for
information only.
Hygroscopicity, crystallinity, solubility See general chapter 5.11. Characters section in
monographs.
Polymorphism Where a substance shows polymorphism, this is usually stated. Except in rare
cases, no particular crystalline form is required in monographs. However, depending on the function
of a given substance in a medicinal product, it may be necessary for a manufacturer to ensure that a
particular crystalline form is used. The information given in the Characters section is intended to alert
users to the need to evaluate this aspect during the development of a medicinal product. See also
general chapter 5. 9. Polynwrphism.
1.5.1.8 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 article; they are intended to give confirmation, with an
acceptable degree of assurance, that the article conforms to the description on the label.
An identification test may refer to a test in the Tests section of the monograph.
If the monograph lists, for example, identification tests A, B and C, all three tests must be carried
out and must satisfy the requirements.
Certain monographs give two or more sets of identification tests that are equivalent and may be
used independently. They are preceded by a sentence of the type 'Carrt out either tests A, B or tests C,
1
D'. For example, one test determines enantiomeric purity by chromatography, while the other is a test
for specific optical rotation; the intended purpose of the two is the same, i.e. verification that the
correct enantiomer is present.
In some monographs the Identification section is subdivided as follmvs.
First ident~fication. The test(s) that constitute the first identification may be used in all
circumstances.
Second identification. The test(s) that constitute the second identification may be used in
pharmacies only, provided it can be demonstrated that the article is fully traceable to a batch
certified to comply with all the other requirements of the monograph. The implementation of the
tests under the second identification is subject to national regulation.
1.5.1.9 Tests and assays

Scope The requirements are not designed to take all possible impurities into account. 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 under
1.5.1.12. Impurities.
Calculation Where the result is to be calculated with reference to the dried or anhydrous
substance or on another specified basis, the determination of loss on drying, water content or another
property is carried out by the procedure prescribed 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 residual solvent content is taken into account when calculating the assay content
of the substance, the specific optical rotation and the specific absorbance. No further indication is
given in the individual monograph.
Limlts The prescribed limits are based on data obtained in routine analytical practice and are
intended to demonstrate that the article being examined complies with the requirements of the
monograph. They take account of normal analytical errors, of acceptable variations in manufacture/
preparation and of deterioration to an extent considered acceptable. No further tolerances are to be
applied to the prescribed limits.
In determining compliance with a numerical limit, the calculated result of an analytical procedure 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, 0 .15 indicates 2 significant figures and 140 indicates 3
significant figures). When rounding, only the digit immediately to the right of the last place in the
limit is to be considered. If this digit is smaller than 5, it is eliminated and the preceding digit is not
changed. If this digit is equal to or greater than 5, it is eliminated and the preceding digit is increased
by 1.
Indication of permitted limlts for impurities The limits for related substances are expressed
either in terms of comparison of peak areas (comparative method) or as numerical values (quantitative
method). For tests using the comparative method, the approximate tolerated content of the named
impurity, or of 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 limits.
If the use of a reference standard for the named impurity is not prescribed, the impurity content
may be expressed as a nominal concentration of the substance used to prepare the reference solution
specified in the monograph, unless otherwise described.
Chiral sitbstances Monographs describi..rig a particular enantiomer include a test to confirm
enantiomeric purity, either using specific optical rotation or a chromatograpliJc procedure.
A test for racemic character using optical rotation is included only if there is information on the
specific optical rotation of the enantiomers that indicates that such a test would be discriminating in
terms of enantiomeric purity.
Equivalents Where an equivalent is given, only the figures shown are to be used in applying the
requirements of the monograph. For example, for titrations: 1 mL of 1 M hydrochloric acid is
equivalent to 50.05 mg of CaCO 3 .
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 for optimal performance it may be necessary 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 seiective properties;
antibiotics, depending on their activity.
1.5.1.10 Storage
The information and recommendations given in the Storage section do not constitute a
pharmacopoeial requirement.
The articles described in the Ph. Eur. are stored in such a way as to prevent contamination and, as
far as possible, deterioration. \Xlhere special storage conditions are recommended, including the type
of container (see 1.3.1. Materials for containers and containers) and limits of temperature, they are
stated in the monograph.
The following expressions are used in monographs under Storage with the meaning shown below.
'In an airtight container' means that the article is stored in an airtight container (3.2. Containers).
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 article is avoided.
'Protected from light' means that the article is stored either in a container made of a material that
absorbs actinic light sufficiently to protect the contents from changes induced by such light, or in a
container enclosed in an outer cover that provides similar protection, or that the article is stored in a
place from which all such light is excluded.
1.5.1.11 Labelling
In general, labelling of medicinal products is subject to supranational and national regulation and to
international agreements.
The statements in the Labelling section are not therefore comprehensive. In addition, for the
purposes of the Ph. Eur., 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.
\Xlhen the term 'label' is used in the Ph. Eur., 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.
1. 5. 1. 12 Impurities
Monographs may include a list of all known and potential impurities that have been shown to be
detected by the tests. See also general 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.
1.5.1.13 Functionality-related characteristics of excipients

This section is included in some monographs on excipients. Its contents do not constitute mandatory
requirements, but the characteristics may be relevant for a particular use of an excipient and are given
for guidance. The decision to control a functionality-related characteristic of an excipient remains with
the manufacturer of the medicinal product and is taken with knowledge of the formulation of the
medicinal product in which it is to be used~ the analytical procedures, limits and tolerances are
determined on a contractual basis by the user and the supplier of the excipient (see also Grades under
1.1.2.1. Scope).
1.5.2 MONOGRAPHS ON HERBAL DRUGS

This section complements section 1.5.1. General principles.
Definition In monographs on herbal drugs, the definition indicates whether the subject of the
monograph is, for example, the whole drug or the fragmented drug. Where a monograph applies to
the drug in several states, for example both to the whole drug and to the fragmented drug, the
definition clearly indicates this.
Identification Monographs on herbal drugs may contain schematic drawings of the most
diagnostic microscopic botanical structures. These drawings complement the description given in the
relevant identification test.
Tests and Assay The sulfated ash, total ash, water-soluble matter, alcohol-soluble matter, water
content, and content of constituents with known therapeutic activity or markers are calculated with
reference to the drug that has not been specially dried, unless otherwise prescribed in the monograph.
1.5.3 MONOGRAPHS ON MEDICINAL PRODUCTS CONTAINING CHEMICALLY

DEFINED ACTIVE SUBSTANCES
This section complements section 1. 5.1. General principles,
Monographs on medicinal products are intended for human use only, unless otherwise indicated in
the monograph.
Medicinal products comply with the general monograph Pharmaceutical preparations (2619), the
relevant dosage form monograph, any relevant individual monograph, and any other relevant text.
1.5.3.1 Related substances

Medicinal product monographs limit degradation products arising during the manufacture and the
shelf life of the medicinal product, including any impurities of synthesis that are also degradation
products.
In certain circumstances, it is necessary to identify impurities of synthesis in the medicinal product,
for example when they are detected in the test for related substances at a level greater than the
reporting threshold in the medicinal product. Consequently, the monograph describes how to identify
any such known impurities of synthesis, so that they are disregarded and are not taken into account.
Monographs on medicinal products are not designed to control impurities of synthesis that are not
degradation products. However, tests provided in the monograph could be used to control impurities
of synthesis known to be detected by the monograph, if further validated for that purpose by the user.
It is acknowledged that additional controls may be required to monitor degradation products other
than those controlled by the monograph (e.g. degradation products related to different excipients or
containers used, or from a different manufacturing process).
1.5.3.2 Dissolution/Disintegration
The following terms are used hereafter:
monograph dissolution test: analytical procedure and acceptance criteria described in the individual
monograph;
product-spec~-'ic dissolution test: analytical procedure and acceptance criteria proposed by the
applicant in a marketing aut.11.orisation application (fv1.l-tA.) for a medicinal prnduct;
in-house dissolution test: analytical procedure developed and acceptance criteria defined by the
applicant.
In line with the relevant guidelines applied nationally or regionally (such as the ICH Q6A guideline)
and with the relevant Ph. Eur. dosage form monograph, a suitable product-specific dissolution test has
to be proposed by the applicant for routine quality control to confirm batch-to-batch consistency. This
test must be described in the MAA for submission to the competent authority, unless there is data
justifying the replacement of the dissolution test by a disintegration test (see below). The
demonstration of the suitability of the dissolution test has to be made by the applicant to the
satisfaction of the competent authority.
Where appropriate, a dissolution test is described in an individual monograph on a medicinal
product. In such cases, the applicant may either select the monograph dissolution test or develop an
in-house dissolution test as the product-specific dissolution test. In all cases, the applicant has to
demonstrate the suitability of the selected test to the satisfaction of the competent authority.
If an in-house dissolution test is proposed, justification for not selecting the monograph dissolution
test and demonstration of compliance with the monograph dissolution test is normally not requested
as part of the MAA.
However, when tested, the medicinal product has to comply with the monograph dissolution test,
unless otherwise justified by the applicant.
Where a given medicinal product does not comply with the monograph dissolution test and this
product is approvable by a competent authority, then the competent authority shall bring this to the
attention of the Ph. Bur. Commission so that it can review the monograph and revise it where
appropriate.
As outlined in the ICH Q6A guideline, for rapidly dissolving medicinal products containing active
substances that are highly soluble throughout the physiological range, a disintegration test may be
substituted for a dissolution test. Such a substitution has to be justified by the applicant to the
satisfaction of the competent authority.
1.5.3.3 Impurities
Impurities already listed in the monograph on the active substance, designated by a capital letter (A,
B, C, D, etc.), keep their name. Impurities specific to the medicinal product are designated by 'FP-'
followed by a letter of the alphabet (FP-A, FP-B, etc.).
1.5.3.4 Storage
As for other monographs, the statements included under Storage in a medicinal product monograph
constitute recommendations only; other conditions may be applied depending on the medicinal
product, subject to approval by the competent authority.
1.6 REFERENCE STANDARDS
Certain monographs require the use of reference standards, which can be chemical reference
substances (CRSs), herbal reference standards (HRSs), biological reference preparations (BRPs) or
reference spectra. See also general chapter 5.12. Reference standards. Unless otherwise stated, the
reference standards referred to in texts are alone authoritative in case of arbitration.
1.7. ABBREVIATIONS AND SYMBOLS
A Absorbance mp Melting puilll

Ai,m Specific absorbance 11jf Refractive index
Ar Relative atomic mass Ph. Eur. U. European Pharmacopoeia Unit
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 4. Reagents
CRS Chemical reference substance RF Retardation factor (see general chapter 2.2.46.
Relative density Chromatographic separation rechniques)
). Wavelength R_,1 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 (see general chapter 4. 2.1.
Mr Relative molecular mass Primary standards for 'Dolumetn'.c solutions)
Abbreviations used in the monographs on immunoglobulins, immunosera
Coiony-forming units Lo/10 dose The largest quantity of a toxin that, in the
The statistically determined quantity of a conditions of the test, when mixed \Vith 0.1 IU of
substance that, when administered by the antitoxin and administered by the specified route,
specified route, may be expected to cause the does not cause symptoms of toxicity in the test
death of 50 per cent of the test animals within a animals within a given period
given period Lf dose The quantity of toxin or toxoid that flocculates in
MLD Minimum lethal dose the shortest time with I IU of antitoxin
L+/10 dose The smallest quantity of a toxin that, in the CCIDso The statistically determined quantity of virus that
conditions of the test, when mixed with 0.1 IU of may be expected to infect 50 per cent of the cell
antitoxin and administered by the specified route, cultures to \Vhich it is added
causes the death of the test animals within a The statistically determined dose of a vaccine
given period that, in the conditions of the test, may be
L+ dose The smallest quantity of a toxin that, in the expected to induce specific antibodies for the
conditions of the test, when mixed with 1 IU of relevant vaccine antigens in 50 per cent of the
antitoxin and administered by the specified route, animals into which it is inoculated
causes the death of the test animals within a EIDso The statistically determined quantity of virus that
given period may be expected to infect 50 per cent of the
lr/100 dose The smallest quantity of a toxin that, in the fertilised eggs into which it is inoculated
conditions of the test, when mixed with 0.01 IU IDso The statistically determined quantity of a virus
of antitoxin and injected intracutaneously causes that may be expected to infect 50 per cent of the
a characteristic reaction at the site of injection animals into which it is inoculated
within a given period The statistically determined dose of a vaccine
PDso
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 of expected to protect 50 per cent of the animals
antitoxin and administered by the specified route, into which it is inoculated against a challenge
causes paralysis in the test animals within a given dose of the micro-organisms or toxins against
period which it is active
PFU Pock-forming units or plaque-forming units
SPF Specified-pathogen-free
Collections of micro-organisms
ATCC American Type Culture Collection NCIMB National Collection of Industrial Food and
CIP Collection des bacteries de l'Institut Pasteur Marine Bacteria Ltd
IMI International Mycological Institute NCPF National Collection of Pathogenic Fungi
IP Institut Pasteur, Collection Nationale de NCTC National Collection of Type Cultures

Cultures de Microorganismes (CNCM) NCYC National Collection of Yeast Cultures
NBRC NITE Biological Resource Center SSI Statens Serum Institut
1.8. UNITS OF THE INTERNATIONAL SYSTEM (SI) USED IN THE PH. EUR. AND
EQUIVALENCE WITH OTHER UNITS
INTERNATIONAL SYSTEM OF UNITS (SI)
The International System of Units comprises 2 main classes of units, namely base units and
derived units 1 . The base units are the metre, the kilogram, the second, the ampere, the kelvin, the
mole and the candela.
The derived units are formed as products of powers of the base units according to the algebraic
relationships linking the corresponding quantities. Some of these derived units have special names and
symbols. The derived units used in the Ph. Eur. are shown in Table 1.8.-1.
Some important and widely used units outside the International System are shown in Table 1.8.-2.
The prefixes shown in Table 1.8.-3 are used to form the names and symbols of the decimal
multiples and submultiples of SI units.
Table 1.8.-1. - Derived units used in the Ph. Bur. and equivalence with other units
Quantity Unit
Conversion of other units into SI
Name Symbol Name Symbol Expression F.xprt>!i:!i:ion in
: - ~, 1..~~~
units
.l.ll lJ..A. VQ.~~ other SI ur.its
units
Wave number t' one per metre 1/m m-1
Wavelength 2 micrometre µm 10- 6 m

nanometre nm 10- 9 m
Area A,S square metre m2 m2
Volume V cubic metre m3 m3 1 mL = 1 cm3 = 10- 6 m'
Frequency V hertz Hz s-1
3
Density p kilogram per kglm 3 kg·m- 1 1 g/mL = 1 g/cm3 = 103 kg-m
cubic metre
Velocity, speed V metre per m/s m-s- 1

second
Force F ne""'ton N m-kg·s 2 1 dyne= 1 g-cm·s 2 ::: 10 5 N

1 kp = 9.806 65 N
Pressure, stress p pascal Pa m- 1-kg-s 2

N-m 2
1 dyne/cm 2 = 10 1
Pa= 10 1
N·m 2
1 atm = 101 325 Pa= 101.325 kPa

1 bar= 10 5 Pa = 0.1 MPa
1 mm Hg = 133.322 387 Pa
1 Torr = 133.322 368 Pa
1 psi = 6.894 757 kPa
Dynamic 1/ pascal second Pa-s m- 1 -kg-s 1

N-s·m- 2 1 P = 10- 1 Pa-s = 10- 1 N-s·m- 2
viscosity 1 cP = I mPa-s
Kinematic V square metre m2/s m2,s-1 Pa-s•m 3-kg- 1 1 St= 1 cm 2 •s- 1

= 10- 4 m 2 -s-- 1
viscosity per second N·m·s-kg- I
1
The definitions of the units used in the Intenuuional Systenz are gz'.zJen in rhe booklet 'Le Syste'me lmemarional d 'Unite'.1 (SJ) ', published by the
Bureau lmemational des Poids a A,iesures, Pavillon de Breteml, F-92310 Se'.vres.
Quantity Unit
Conversion of other units into SI
Name Symbol Name Symbol Expression Expression in
units
in SI base other SI units
units
Energy w joule J m 2 -kg·s- 2 N-m 1 erg = 1 cm2·g·s-z =1 dyne-cm =

10-7 J
1 cal = 4.1868 J
Power} radiant p watt w m 2 -kg·s-3 N-m·s- 1 1 erg/s = 1 dyne-cm-s- =1
flux J•S-1 10-7 W = 10- N·m·s- = 10-

7 1 7
J,s- 1
Absorbed dose D gray Gy m2,s-2 J-kg-1 I rad= 10-2 Gy

(of radiant
energy)
Electric u volt V m 2 · kg·s- 3-A- 1 W-A- 1

potential
difference,
voltage
Electric R ohm Q m2· kg-s-3,A-2 V,A- 1

resistance
Electric charge Q coulomb C A-s
Activity A becquerel Bq s-1 1 Ci = 37-10 9 Bq =37·10 9

s- 1
referred to a
radionuclide
Concentration C mole per mol/m3 mol·m-3 1 mol!l... = 1 M = 1 mol/dm3 = 103

(of amount of cubic metre mol•m- 3
substance),
molar
concentration
Mass p kilogram per kg/m3 kg•m- 3 1 g/L = 1 g/dm3 =1 kg·m-3

concentration cubic metre
Catalytic z katal kat mol•s- 1

activity
Table 1.8.-2. - Non-SI units accepted

-
for use zvith the SI units
-·-··--· ··--·---
-•---. ,--.
Quantity Unit Value in SI units
Name Symbol
-- -
Time minute min 1 min = 60 s
hour h 1 h = 60 min = 3600 s
day d 1 d = 24 h = 86 400 s
Plane angle degree C

1" = (n/180) rad
Volume litre L 1 L = 1 dm3 = 10- 3 m 3
Mass tonne t 1 t = 103 kg
dalton Da 1 Da = 1.660539040(20) X 10-27 kg
Rotational
revolution per minute r/min 1 r/min = ( 1/60) s- 1
frequency
Energy electronvoit eV 1 eV = 1.602176634 x 10- 10 J
Table 1.8.-3. - Decimal multiples and sub-multiples of SI units

Factor Prefix Symbol Factor Prefix Symbol
10'8 exa E 10-1 deci d
1015 peta p 10--2 centi C
1012 tera T 10- 3 milli m
109 giga G 10-6

micro µ
10 6 mega M 10-9 nano n
10 3 kilo k 1ff 12 pico p
10 2 hecto h 10-15 femto f
10 1 deca da 10-18 atto a

NOTES
1. In the Pharmacopoeia, the Celsius temperature is used (symbol t). This is defined by the following
equation:
t === T- To
where T0 = 273.15 K by definition. The Celsius or centigrade temperature is expressed in degrees

Celsius (symbol :iq. The unit 'degree Ceisius' is equal to the unit 'kelvin'.
2. 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.
3. In the Ph. Eur., conditions of centrifugation are defined by reference to the acceleration of gravity (g):
g = 9.80665m · s2
4. Certain quantities without dimensions are used in the Ph. Eur.: relative density (2.2.5), absorbance
(2.2.25), specific absorbance (2.2.25) and refractive index (2.2.6).
5. The microkatal is defined as the enzymatic activity that, under defined conditions, produces the
transformation (e.g. hydrolysis) of 1 micromole of the substrate per second.
Monographs
Formulated Preparations: General

Monographs
2023 General Monographs IV-39
pharmacists) have, within their area of responsibilities, a duty

Pharmaceutical Preparations of care to the patient receiving the pharmaceutical
preparation.
(Ph. Bur. monograph 2619)
In considering the preparation of an unlicensed
pharmaceutical preparation, a suitable level of risk assessment
is undertaken.
INfRODUCTION
This monograph is intended to be a reference source of The risk assessment identifies:
standards in the European Pharmacopoeia on active - the criticality of different parameters (e.g. quality of active
substances, excipients and dosage forms, which are to be substances, excipients and containers; design of the
applied in the manufacture/preparation of pharmaceuticals, preparation process; extent and significance of testing;
but not a guide on how to manufacture as there is specific stability of the preparation) to the quality of the
guidance available covering methods of manufacture and preparation; and
associated controls. - the risk that the preparation may present to a particular
patient group.
It does not cover investigational medicinal products, but
competent authorities may refer to pharmacopoeia! standards Based on the risk assessment, the person responsible for the
when authorising clinical trials using investigational medicinal preparation must ensure, with a suitable level of assurance
products. that the pharmaceutical preparation is, throughout its sheJ-
life, of an appropriate quality and suitable and fit for its
DEFINITION purpose. For stock preparations, storage conditions and shelf-
Pharmaceutical preparations are medicinal products generally life have to be justified on the basis of, for example,
consisting of active substances that may be combined with analytical data or professional judgement, which may be
excipients, formulated into a dosage form suitable for the based on literature references.
intended use, where necessary after reconstitution, presented
in a suitable and appropriately labelled container. PRODUCTION
Manufacture/preparation must take place within the
Pharmaceutical preparations may be licensed by the
framework of a suitable quality system and be compliant with
competent authority, or unlicensed and made to the specific
the standards relevant to the type of product being made.
needs of patients according to legislation. There are 2
Licensed products must comply with the requirements of
categories of unlicensed pharmaceutical preparations:
their licence. For unlicensed products a risk assessment as
- extemporaneous preparations, i.e. pharmaceutical
outlined in the section 'Ethical considerations and guidance
preparations individually prepared for a specific patient or
in the preparation of unlicensed pharmaceutical preparations'
patient group, supplied after preparation;
is of special importance, as these products are not previously
- stock preparations, i.e. pharmaceutical preparations
assessed by the competent authority.
prepared in advance and stored until a request for a
supply is received. Where pharmaceutical preparations are
manufactured/prepared using materials of human or animal
In addition to this monograph, pharmaceutical preparations
origin, the general requirements of general chapters 5.1. 7.
also comply with the General Notices and with the relevant
Viral safety and 5.2. 6. Evaluation of safety of vetennary vaccines
general chapters of the Pharmacopoeia. General chapters are
and immunosera and of the general monograph Products with
normally given for information and become mandatory when
risk of transmitting agents of animal spongiform
referred to in a general or specific monograph, unless such
encephalopathies (1483) apply, where appropriate.
reference is made in a way that indicates that it is not the
intention to make the text referred to mandatory but rather Formulation
to cite it for information. During pharmaceutical development or prior to
Where relevant, pharmaceutical preparations also comply manufacture/preparation, suitable ingredients, processes, tests
and specifications are identified and justified in order to
with the dosage form monographs (e.g. Capsules (0016),
Tablets (0478)) and general monographs relating to
ensure the suitability of the product for the intended
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),
Homoeopathic pillules, coated (2786), Homoeopathic pillules
properties or process steps are critical to the required quality
of the pharmaceutical preparation.
impregnated (2079), Immunoserafor human use, animal (0084),
lmmunosera for veterinary use (0030), Live biotherapeutic Active substances and excipients
products for human use (3053), Monoclonal antibodies for human Active substances and excipients used in the formulation of
use (2031), Radiopharmaceutical preparations (0125), Vaccines pharmaceutical preparations comply with the requirements of
for human use (0153), Vaccines for veterinary use (0062)). the relevant general monographs, e.g. Substances for
pharmaceutical use (2034), Essential oils (2098), Herbal drug
ETHICAL CONSIDERATIONS AND GUIDANCE IN
extracts (0765), Herbal drugs (1433), Herbal drug
THE PREPARATION OF UNLICENSED
preparations (1434), Herbal drugs for homoeopathic
PHARMACEUTICAL PREPARATIONS
preparations (2045), Mother tinctures for homoeopathic
The underlying principle of legislation for pharmaceutical preparations (2029), Methods of preparation of homoeopathic
preparations is that, subject to specific exemptions, no stocks and potentisation (2371), Products of fermentation (1468),
pharmaceutical preparation may be placed on the market Products of recombinant DNA technology (0784), Vegetable fatty
without an appropriate marketing authorisation. oils (1579).
The exemptions from the formal licensing requirement allow
the supply of unlicensed products to meet the special needs In addition, where specific monographs exist, the quality of
of individual patients. However, when deciding to use an the active substances and excipients used complies with the
unlicensed preparation all health professionals involved corresponding monographs.
(e.g. the prescribing practitioners and/or the preparing
IV-40 General Monographs 2023
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 are 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 preservatives;
supplied in single-dose containers, or in multidose containers
- purity tests (e.g. investigation of degradation products,
that prevent microbial contamination of the contents after
residual solvents (2.4.24) or other related impurities,
opening.
sterility (2.6.J));
In the manufacture/preparation of non-sterile pharmaceutical - safety tests (e.g. safety tests for biological products).
preparations, suitable measures are taken to ensure their
microbial quality; recommendations on this aspect are Elemental impurities
provided in general chapters 5.1. 4. Microbiological quality of General chapter 5.20. Elemental impurities applies to
non-sterile pharmaceutical preparations and substances for pharmaceutical preparations except products for veterinary
pharmaceutical use and 5.1. 8. Microbiological quality of herbal use, unlicensed preparations and other products that are
medicinal products for oral use and extracts used in their excluded from the scope of this chapter.
preparation. For pharmaceutical preparations outside the scope of general
Sterile preparations are manufactured/prepared using chapter 5. 20, manufacturers of these products remain
materials and methods designed to ensure sterility and to responsible for controlling the levels of elemental impurities
avoid the introduction of contaminants and the growth of using the principles of risk management.
micro-organisms; recommendations on this aspect are If appropriate, testing is performed using suitable analytical
provided in general chapter 5.1.1. Methods of preparation of procedures according to general chapter 2.4.20. Determination
sterile products. of elemental impurities.
Containers Uniformity (2.9.40 or 2.9.5/2.9.6)
A suitable container is selected. Consideration is given to the Pharmaceutical preparations presented in single-dose units
intended use of the preparation, the properties of the comply with the test(s) as prescribed in the relevant specific
container, the required shelf-life, and product/container dosage form monograph. If justified and authorised, general
incompatibilities. Where applicable, containers for chapter 2. 9.40 can be applicable only at the time of release.
pharmaceutical preparations comply with the requirements Special uniformity requirements apply in the following cases:
for containers (3.2 and subsections) and materials used for - for herbal drugs and herbal drug preparations, compliance
the manufacture of containers (3.1 and subsections). with general chapter 2. 9. 40 is not required;
Stability - for homoeopathic preparations, the provisions of general
Stability requirements of pharmaceutical preparations are chapters 2. 9. 6 and 2. 9. 40 are normally not appropriate,
dependent on their intended use and on the desired storage however in certain circumstances compliance with these
time. chapters may be required by the competent authority;
- for single- and multivitamin and trace-element
Where applicable, the probability and criticality of possible
preparations, compliance with general chapters 2.9.6 and
degradation products of the active substance(s) and/or
2.9.40 (content uniformity only) is not required;
reaction products of the active substance(s) with an excipient
- in justified and authorised circumstances, for other
and/or the immediate container must be assessed. Depending
preparations, compliance with general chapters 2. 9. 6 and
on the result of this assessment, limits of degradation and/or
2.9.40 may not be required by the competent authority.
reaction products are set and monitored in the
pharmaceutical preparation. Licensed products require a
stability exercise.
Reference standards
Reference standards may be needed at various stages for
quality control of pharmaceutical preparations. They are
established and monitored taking due account of general
chapter 5.12. Reference standards.
ASSAY
Unless otherwise justified and authorised, contents of active
substances and specific excipients such as preservatives are
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 Drugs, Herbal Drug Preparations and

Herbal Medicinal Products
TESTS
Herbal Drugs
Foreign matter (2.8.2)
(Ph. Bur. monograph 1433) Carry out a test for foreign matter, unless otherwise
prescribed or justified and authorised. The content of foreign
Herbal Drugs comply with the requirements of the European matter is not more than 2 per cent mlm, unless otherwise
Pharmacopoeia. These requirements are reproduced below. prescribed or justified and authorised. An appropriate specific
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ test may apply to dried herbal drugs liable to be adulterated.
It may not be possible to perform the test for foreign matter
DEFINITION
on a dried herbal drug that is cut, as described under
Herbal drugs are mainly whole, fragmented or broken plants
Definition, for either a specific purpose or for extraction.
or parts of plants in an unprocessed state, usually in dried
Under these circumstances the cut material is presumed to
form but sometimes fresh. In this general monograph, the
comply with the test for foreign matter providing that the
word 'plant' is used in the broader sense to also include
dried herbal drug prior to cutting was compliant with this
algae, fungi and lichens. Certain exudates that have not been
test.
subjected to a specific treatment are also considered to be
herbal drugs. Herbal drugs are precisely defined by the Loss on drying (2.2.32)
botanical scientific name according to the binominal system Carry out a test for loss on drying, unless otherwise
(genus, species, variety and author). prescribed or justified and authorised.
W'hole Describes a herbal drug that has not been reduced in Water (2.2.13)
size and is presented, dried or undried, as harvested; for A determination of water may be carried out instead of a test
example: dog rose, bitter fennel or sweet fennel, Roman for loss on drying for dried herbal drugs with a high
chamomile flower. essential-oil content.
Fragmented Describes a herbal drug that has been reduced Pesticides (2. 8.13)
in size after harvesting to permit ease of handling, drying Dried herbal drugs comply with the requirements for
and/or packaging; for example: cinchona bark, rhubarb, pesticide residues. The requirements take into account the
passion flower. nature of the plant, where necessary the preparation in which
Broken Describes a herbal drug in which the more-fragile the plant might be used, and where available the knowledge
parts of the plant have broken during drying, packaging or of the complete treatment record of the batch of the plant.
transportation; for example: belladonna leaf, matricaria Heavy metals (2.4.27)
flower, hop strobile. Unless otherwise stated in an individual monograph or unless
Cut Describes a herbal drug that has been reduced in size, otherwise justified and authorised:
other than by powdering, to the extent that the macroscopic - cadmium: maximum 1.0 ppm;
description in the monograph of the herbal drug can no - lead: maximum 5.0 ppm;
longer be applied. When a herbal drug is cut for a specific - mercury: maximum 0.1 ppm.
purpose that results in the cut herbal drug being Where necessary, limits for other heavy metals may be
homogeneous, for example when cut for herbal teas, it is a required.
herbal drug preparation. Certain cut herbal drugs processed W'here necessary, dried herbal drugs comply with other tests, such
in this way may be the subject of an individual monograph. as the following, for example.
A herbal drug that complies with its monograph and is Total ash (2.4.16)
subsequently cut for extraction shall comply in its cut form,
except for its macroscopic description, with the monograph Ash insoluble in hydrochloric acid (2.8.1)
for that herbal drug, unless otherwise justified. Extractable matter
The term herbal drug is synonymous with the term herbal Swelling index (2.8.4)
substance used in European Community legislation on herbal Bitterness value (2. 8.15)
medicinal products.
Aflatoxin B 1 (2. 8.18)
DRIED HERBAL DRUGS Where necessary, limits for aflatoxins may be required.
PRODUCTION Ochratoxin A (2.8.22)
Dried herbal drugs are obtained from cultivated or wild Where necessary, a limit for ochratoxin A may be required.
plants. Suitable collection, cultivation, harvesting, drying,
fragmentation and storage conditions are essential to Radioactive contamination
guarantee their quality. In some specific circumstances, the risk of radioactive
contamination is to be considered.
Dried herbal drugs are, as far as possible, free from
impurities such as soil, dust, dirt and other contaminants Microbial contamination
such as fungal, insect and other animal contaminations. They Where a dried herbal drug is used whole, cut or powdered as
are not rotten. an ingredient in a medicinal product, the microbial
contamination is controlled (5.1.8. Microbiological quality of
If a decontaminating treatment has been used, it is necessary
herbal medicinal products for oral use and extracts used in their
to demonstrate that the constituents of the herbal drug are
preparation or 5.1. 4. Microbiological quality of non-sterile
not affected and that no harmful residues remain. The use of
pharmaceutical preparations and substances for pharmaceutical use
ethylene oxide is prohibited for the decontamination of
(e.g. for cutaneous use)).
herbal drugs.
ASSAY
IDENTIFICATION
Unless otherwise prescribed or justified and authorised, dried
Dried herbal drugs are identified using their macroscopic and
herbal drugs are assayed by an appropriate method.
microscopic descriptions and any further tests that may be
required (for example, thin-layer chromatography).
STORAGE of which may alter the quality of the herbal drug preparation,
Protected from light. including possible mycotoxin production.
Where used for the production of essential oils, some of the tests _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
prescribed for dried herbal drugs may no longer be necessary. This
is considered on a case-by-case basis depending on the provenance
of the dried herbal drug and the production process. The need for
testing (e.g. for potential contaminants) and the stage of testing Processed Herbal Drugs
(herbal drug/essential oil) is to be considered.
DEFINITION
FRESH HERBAL DRUGS Processed Herbal Drugs are obtained by subjecting Herbal
A fresh herbal drug is one that is intended to be processed Drugs to traditional processing methods.
into a herbal drug preparation (e.g. essential oil, juice,
Processed Herbal Drugs are defined precisely by the
tincture) within a relatively short period of time after
botanical scientific name according to the binomial system
harvesting. Under these circumstances, the extensive analysis
(genus, species, subspecies, variety, and author) and plant
prescribed for dried herbal drugs is not appropriate and the
part. Monographs for Processed Herbal Drugs may refer to
following analytical requirements, based on the provenance of
the relevant monograph for the unprocessed material where
the fresh herbal drug, are considered suitable, provided that
the binomial name is given.
processing to the herbal drug preparation takes place within a
validated time period after harvesting. PRODUCTION
(1) For a fresh herbal drug that has been cultivated from Processed Herbal Drugs are obtained by subjecting Herbal
seeds, cuttings, etc., whose origin and traceability can be Drugs to specific types of processing according to traditional
demonstrated, and where the complete history of the herbal processing methods. These traditional processing methods
drug from planting to harvesting is documented: have the potential to alter the physical characteristics and/or
- macroscopic identification of the plant and plant parts chemical constituents of a Herbal Drug. Traditional
to be processed; processing methods may require the addition of processing
- compliance with a suitable limit test for foreign aids to the herbal drug, for example, honey, vinegar, wine,
matter. milk and salt. The additional processing aids used should be
(2) For a cultivated fresh herbal drug where the information of a suitable quality or of pharmacopoeia! quality where a
on life cycle from seed to harvesting, as described under (1), monograph exists. The method of traditional processing is
is incomplete, the same analytical requirements as described provided under the Production section in individual
under (1) apply, as well as any additional tests that may be monographs.
necessary depending on the information available on the IDENTIFICATION
herbal drug to be processed and any potential or known Processed Herbal Drugs are identified using their
quality issues. macroscopical and, where appropriate, microscopical
(3) For a fresh herbal drug that is wild-crafted, the analytical descriptions and any further tests that may be required.
requirements should be assessed on a case-by-case basis and TESTS
will depend on the ease of identification or characterisation of A test for foreign matter, Appendix XI D, is carried out,
the herbal drug/herbal drug preparation and potential unless otherwise prescribed in the individual monographs.
adulterants, and the method of processing or type of herbal
A specific appropriate test may be prescribed to detect
drug preparation to be manufactured. For example, in the
potential contaminants in processed herbal drugs.
case of essential oils, the majority of the oil composition is
determined when it is analysed, whereas for a juice or a If appropriate, the Processed Herbal Drugs comply with
tincture, such analytical capabilities are limited. For the other tests, for example, total ash, Appendix XI J, Method II,
processing of plant parts, a wild-crafted fresh flower, easily ash insoluble in hydrochloric acid, Appendix XI K, Method II,
identifiable by its distinctive appearance and colour, would extractable matter, swelling index, Appendix XI C and bitterness
be expected to require fewer analytical control parameters value, Appendix XI N.
than a wild-crafted fresh root with few, if any, visually The test for loss on drying, Appendix IX D, is carried out on
distinctive features. Analytical requirements as described Processed Herbal Drugs, unless otherwise prescribed in the
under (1) may be acceptable when fully justified. individual monographs. A determination of water by distillation,
For fresh herbal drugs, where the extensive analysis described Appendix IX C, Method II, is carried out for Processed
for dried herbal drugs is not feasible prior to processing into Herbal Drugs with a high essential oil content.
the herbal drug preparation, appropriate tests Processed Herbal Drugs comply with the requirements for
(e.g. for contaminants) are performed on a suitable retained pesticide residues, Appendix XI L. The requirements take into
sample of the fresh herbal drug or on the herbal drug account the nature of the Processed Herbal Drugs, where
preparation. necessary the preparation in which the plant might be used,
Fresh herbal drugs may be frozen for storage purposes. and where available, the knowledge of the complete record of
Defined processes for freezing and thawing are required to treatment of the batch of the Processed Herbal Drugs during
ensure the quality of the herbal drug. Appropriate testing of cultivation, harvesting and processing. The content of
the herbal drug, justified on the basis of the freezing and pesticide residues may be determined by the method
manufacturing processes, is put in place and may include described in the annex to the general method.
testing prior to freezing and at the time of use. The risk of contamination of Processed Herbal Drugs by
When handling and processing fresh herbal drugs, it is heavy metals must be considered. In an individual
necessary to ensure, by visual inspection or other suitable monograph either a general limit for heavy metals or specific
means, the absence of unwanted fermentation, the presence limits for individual heavy metal may be required.
Where necessary limits for specific toxins, for example
aflatoxins or ochratoxins, may be applied.
2023 General Monographs IV-4 7
Where processing is carried out to remove or limit specific An essential oil may be subjected to further processing steps
constituents from the herbal drug a suitable limit test should (combining, rectification, etc.), that may or may not
be carried out. significantly affect its composition.
In some specific circumstances, the risk of radioactive An essential oil whose composition has been significantly
contamination is to be considered. modified may be known as:
- Rectified essential oil: an essential oil from which part of the
ASSAY
constituents has been partially or totally removed by
Unless otherwise justified and authorised Processed Herbal
rectification;
Drugs are assayed by an appropriate method.
- Deterpenated essential oil: an essential oil from which
monoterpene hydrocarbons have been partially or totally
removed by rectification or any other suitable process;
- Deterpenated and desesquiterpenated essential oil: an essential
Herbal Drug Preparations oil from which monoterpene and sesquiterpene
hydrocarbons have been partially or totally removed by
rectification or any other suitable process;
Herbal Drug Preparations comply with the requirements of the - 'X'-free or partially 'x '-free essential oil: an essential oil from
European Pharmacopoeia. These requirements are reproduced which one or more particular constituents have been
below. totally or partially removed by rectification or any other
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ suitable process.
The type of modification must be indicated in the Definition
DEFINITION
section of the individual monograph.
Herbal drug preparations are homogeneous products
obtained by subjecting herbal drugs to treatments such as A European Pharmacopoeia monograph for an essential oil
extraction, distillation, expression, fractionation, purification, covers the essential oil used as a constituent in a medicinal
concentration or fermentation. product.
Herbal drug preparations include, for example, extracts, PRODUCTION
essential oils, expressed juices, processed exudates, and Herbal drugs used for the preparation of essential oils are of
herbal drugs that have been subjected to size reduction for suitable quality and, where applicable, comply with the
specific applications, for example herbal drugs cut for herbal requirements of any relevant monograph in the European
teas or powdered for encapsulation. Pharmacopoeia.
Herbal teas comply with the monograph Herbal teas (1435). Depending on the monograph, the herbal drug may be fresh,
NOTE The term comminuted used in European Community lightly wilted, wilted, partially dried, dried, whole,
legislation on herbal medicinal products describes a herbal fragmented, broken or cut.
drug that has been either cut or powdered. Different batches of the herbal drug may be combined prior
The term herbal drug preparation is synonymous with the term to processing, for example to achieve the quantity required
herbal preparation used in European Community legislation for the production process. The herbal drug may also
on herbal medicinal products. undergo a preliminary treatment.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur Unless otherwise justified and authorised, as a minimum,
water used in the production of essential oils complies with
local drinking water standards or, in their absence, with
World Health Organization drinking water standards.
Essential Oils Steam distillatwn The essential oil is produced from the
herbal drug using steam and suitable distillation equipment.
(Ph. Bur. monograph 2098) The steam may be introduced from an external source or
generated by boiling water below the plant material or by
Essential Oils comply with the requirements of the European boiling water in which the plant material is immersed.
Pharmacopoeia. These requirements are reproduced below. The steam and oil vapours are condensed. The water and
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ essential oil are separated by decantation or any other
The statements in this monograph are intended to be read in suitable physical process.
conjunction with general chapter 5. 30 Monographs on essential oils Dry distillation The essential oil is produced by heating the
(informatwn chapter) and the individual monographs on essential herbal drug at a high temperature in suitable equipment
oils in the European Pharmacopoeia. The competent authority without adding water or steam.
may decide to apply the monograph to other essential oils. Mechanical process The essential oil, usually known as 'cold-
DEFINITION pressed', is produced by a mechanical process without any
Odorous product, usually of complex composition, obtained heating. This method is used for citrus fruits and involves
from a botanically defined herbal drug by steam distillation, expressing the essential oil from the pericarp, followed by
dry distillation, or a suitable mechanical process without separation using suitable physical means.
heating. If an aqueous phase is present, the essential oils are Rectificatwn The essential oil is subjected to distillation,
separated from it by a physical process that does not usually under vacuum. This additional processing step can be
significantly affect their composition. applied to remove, partially or totally, water or any other
Essential oils obtained from the primary production steps unwanted matter, or to significantly modify the composition.
may be subjected to a suitable subsequent treatment in order In certain cases, a suitable antioxidant may be added to the
to remove unwanted matter (e.g. insoluble matter) or essential oil.
remaining water, without significantly affecting their
composition.
2 6
5
9
3
411 I
-,
I I I ii,
I ,---r--y ··-·r·-·-·r-r-·1--;--,......,......-r I
0 10 '
20 40 50 60 70 80 min
I. Ot-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
CHARACTERS Pesticide residues (2. 8.13)

The appearance and odour of the essential oil are Where justified and authorised, individual testing of every
determined. batch may not be necessary.
IDENTIFICATION Aflatoxin B 1 (2.8.18)
Essential oils are identified by their gas chromatographic Where justified and authorised, individual testing of every
profile or by any other suitable test (e.g., high-performance batch may not be necessary.
thin-layer chromatography). Microbiological quality (5.1.4 or 5.1.8)
TESTS Where justified and authorised, individual testing of every
batch may not be necessary.
GENERAL TESTS
The essential oil complies with the prescribed limits for the SUPPLEMENTARY TESTS
following tests. If applicable and necessary, the essential oil complies with
the prescribed limits for the following tests.
Fatty oils and resinified essential oils (2.8. 7)
The test applies to essential oils obtained by steam distillation Relative density (2.2.5)
or dry distillation. Refractive index (2.2.6)
Heavy metals (2.4.27) Optical rotation (2.2. 7)
Unless otherwise stated in an individual monograph or unless
Freezing point (2.2.18)
otherwise justified and authorised:
- cadmium: maximum 1.0 ppm; Acid value (2.5.1)
- lead: maximum 5.0 ppm; Peroxide value (2.5.5)
- mercury: maximum 0.1 ppm. Foreign esters (2.8.6)
Where necessary, limits for other heavy metals may be Residue on evaporation (2.8.9)
required. Where justified and authorised, individual testing of
every batch may not be necessary. Water in essential oils (2.8.5)
Solubility in alcohol (2. 8.10)
Adulteration
If appropriate, tests are carried out by high-performance thin-
Herbal Drug Extracts
layer chromatography (2.8.25), thin-layer chromatography Extracts
(2.2.27) or gas chromatography (2.2.28) using a chiral
(Ph. Eur. monograph 0765)
column if necessary, or by any other suitable procedure.
Herbal Drug Extracts comply with the requirements of the
Chromatographic profile
Gas chromatography (2.2.28): use the normalisation European Pharmacopoeia. These requirements are reproduced
procedure. below.
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
In addition to the system suitability test described in the
individual monograph, the suitability of the chromatographic DEFINITION
system must be checked periodically during performance Herbal drug extracts are liquid (liquid extraction
qualification, using the test described below. preparations), semi-solid (soft extracts and oleoresins) or
The chromatogram shown in Figure 2098.-1 is given as an solid (dry extracts) preparations obtained from Herbal drugs
example. (1433) using suitable solvents.
Reference solution essential oil CRS. If necessary, the reference An extract is essentially defined by the quality of the herbal
solution can be diluted with heptane R. drug, by its production process (extraction solvent(s), method
Column: of processing, etc.) and by its specifications.
- material: fused silica; European Pharmacopoeia monographs for extracts cover the
=
- size: l 60 m, 0 = 0.25 mm; genuine (native) extract and, where present, excipients.
- stationary phase: macrogol 20 000 R (film thickness
Different types of extract may be distinguished.
0.25 µm).
Carrier gas helium for chromatography R. Standardised extracts
Are adjusted to a defined content of one or more
Flow rate 1.5 mUmin.
constituents with known therapeutic activity. This is achieved
Split ratio 1:500. The split ratio/injection volume can be
by adjustment of the extract with inert excipients or by
adjusted in order to fit the specific equipment used, provided
blending batches of the extract.
that the column load stays the same.
Temperature: Quantified extracts
Are adjusted to one or more active markers, the content of
Time Temperature which is controlled within a limited, specified range.
(min) CC) Adjustments are made by blending batches of the extract.
Column 0 - 15 70 Other extracts
15 - 100 70 ➔ 240 Are not adjusted to a particular content of constituents.
100 - 105 240 For control purposes, one or more constituents are used as
Injection port 250 analytical markers. The minimum content for these analytical
Detector 270 markers is given in an individual monograph.
Detection Flame ionisation. PRODUCTION

Injection 1 µL. Herbal drugs, solvents and other materials used for the
preparation of extracts are of suitable quality and where
Identification of components Use the chromatogram supplied
applicable comply with the requirements of any relevant
with essential oil CRS.
monograph in the European Pharmacopoeia. Where justified,
System suitability Reference solution:
herbal drugs used for the production of extracts may exceed
- resolution: minimum 1.5 between the peaks due to linalol
the limits for heavy metals specified in the monograph Herbal
and linalyl acetate;
drugs (1433) provided that the resulting extract satisfies the
- signal-to-noise ratio: minimum 100 for the peak due to
requirements for heavy metals (see Tests).
decanal;
- limits: the percentage content of each of the 9 components Different batches of the herbal drug which are compliant
is within the limits stated on the leaflet provided with with the relevant monograph, or in the absence of an
essential oil CRS. individual monograph with other suitable specifications, may
be combined prior to extraction, for example for the purpose
STORAGE of achieving the quantity of herbal drug required for the
In a well-filled, airtight container, protected from light. production process or, in the case of standardised and
LABELLING quantified extracts, to achieve a certain range of content for
The label states: one or more constituents in the herbal drug to be extracted.
- where applicable, the name according to the title of the The herbal drug may also undergo a preliminary treatment,
individual monograph; for example, grinding, inactivation of enzymes or defatting.
- where there is no individual Ph. Eur. monograph, the In addition, unwanted constituents (e.g. toxic constituents)
name of the essential oil, the scientific name of the herbal or unwanted matter (e.g. insoluble matter) may be removed
drug used and where applicable, the type of modification; at a suitable stage in the production process.
- where applicable, the type and/or the chemotype of the Where solvents are recovered from the production process,
essential oil; such recovered or recycled solvents may be used, provided
- where applicable, the name and concentration of any that the recovery procedures are controlled and monitored to
added antioxidant; ensure that solvents meet appropriate standards before re-use
- where applicable, additional processing steps that are not or admixture with other approved materials. Water used for
specified in the Definition section of the monograph; the production of extracts complies with the requirements of
- the storage conditions. the monograph Water for preparation of extracts (2249).
- - - - - - - - - - - - - - - - - - - - - - PhEur
Where applicable, miscella (extraction liquors) are acceptable tolerance. Where there is an individual
concentrated to the intended consistency using suitable monograph in the pharmacopoeia for a standardised extract
methods, usually under reduced pressure and at a with a defined range of content, the acceptable tolerance will
temperature at which deterioration of the constituents is be stated in the individual monograph (for example, for
reduced to a minimum. Essential oils that have been Frangula bark dry extract, standardised (1214), the acceptable
separated during processing may be restored to the extracts tolerance is stated as ± 10 per cent relative to the declared
at an appropriate stage in the production process. Suitable content).
excipients may be added at various stages of the production Quantified extracts
process for technological reasons (for example, as part of the The content of assayed constituents must be within the
drying process or to improve the homogeneity or consistency values given in the Definition section of an individual
of an extract). For standardised extracts, suitable inert monograph.
excipients may also be added to adjust one or more
constituents to a defined content. For quantified extracts and Other extracts
'other' extracts, the addition of inert excipients to adjust the The content of assayed constituents must not be lower than
content of assayed constituents is not permitted. Excipients the minimum value given in the Definition section of an
are included for technological reasons only, and the individual monograph. Where justified and authorised, this
manufacturer must declare the content of such excipients as does not preclude the selection of alternative constituents as
a fixed percentage. In some applications, an excipient may be a basis for assay using a corresponding validated analytical
added in a narrow percentage range (e.g. silicon dioxide method, which may be more appropriate to the physical
between 0.1-0.5 per cent, to improve flowability of the and/or chemical properties of the medicinal product into
extract). The proposed range must be justified by the which the extract is to be incorporated. Where alternative
manufacturer. Suitable stabilisers, antioxidants and constituents are selected for assay, a suitable minimum value
antimicrobial preservatives may be added to extracts where for such constituents must be established.
justified and authorised. LABELLING
Extraction with a given solvent leads to a typical content of The label states:
selected constituents in the extracted dry matter; during - the herbal drug used;
production of standardised and quantified extracts, - where applicable, that fresh herbal drug has been used;
purification procedures may be applied that increase the - the form of the extract (for example, liquid, tincture, soft,
content of these selected constituents with respect to the oleoresin or dry);
expected values; such extracts are referred to as 'refined'. - where applicable, that the extract is standardised or
IDENTIFICATION quantified;
Extracts are identified using suitable methods. - for standardised extracts, the defined content of
constituents with known therapeutic activity;
TESTS - for quantified extracts, the specified range of content of
Where applicable, as a result of analysis of the herbal drug active markers;
used for production and in view of the production process, - where applicable, that the extract is 'refined';
tests for microbiological quality (5.1.4 or 5.1.8), heavy metals - the first solvent or solvents used for extraction (for
(2.4.27), aflatoxins (2.8.18), ochratoxin A (2.8.22) and example, ethanol 60 per cent VIV);
pesticide residues (2. 8.13) in the extracts may be necessary. - the name and amount of any excipients present in the
Where a test for heavy metals is carried out, the same limits extract (for example, diluents, stabilisers, antimicrobial
for heavy metals as those given in the monograph Herbal preservatives, antioxidants);
drugs (1433) are applicable to extracts unless otherwise stated - for quantified extracts and 'other' extracts, the ratio of the
in an individual extract monograph or unless otherwise quantity of herbal drug to the quantity of genuine (native)
justified and authorised. extract (DERgenuim) 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 LIQUID EXTRACTION PREPARATIONS -
defined range of content. PRAEPARATIONES FLUIDAE AB
Defined single content For example, in the monograph EXTRACTIONE
lpecacuanha liquid extract, standardised (1875), the content of
Liquid extraction preparations are liquid preparations
assayed constituents is stated as 1.80 per cent to
consisting of a diverse range of products which are described
2.20 per cent. In this case, the declaration is based on a
by their extraction solvents, methods of production and drug
defined single content of 2.0 per cent with a tolerance of
solvent ratios or drug extract ratios. Included in this range
± 10 per cent. The acceptable tolerance is usually within the are products obtained using ethanol, water, glycerol,
range ± 5 per cent to ± 10 per cent taking into account the
propylene glycol and fatty oils as extraction solvents. Liquid
nature of the extract and the method of assay
(fluid) extracts and tinctures belong to this category and are
Defined range of content For example, in the monograph described below.
Frangula bark dry extract, standardised (1214), the content of
assayed constituents is stated as 15.0 per cent to LIQUID (FLUID) EXTRACTS - EXTRACTA FLUIDA
30.0 per cent. In this case, it is intended that an extract will DEFINITION
consistently be produced to a defined single content selected Quantified liquid (fluid) extracts and 'other' liquid (fluid)
from within the defined range taking into account an extracts are liquid extraction preparations of which, in
general, 1 part by mass or volume is equivalent to 1 part by PRODUCTION

mass of the dried herbal drug. Tinctures are usually prepared by either maceration or
Standardised liquid (fluid) extracts are only defined by their percolation, using ethanol of a suitable concentration to
content of constituents with known therapeutic activity. extract the herbal drug, or by dissolving a soft or dry extract
PRODUCTION of the herbal drug (which has been produced using the same
Liquid extracts are prepared using ethanol of a suitable extraction solvent as would be used to prepare the tincture
concentration and/or water together with, where necessary, by direct extraction) in ethanol of the required concentration.
other substances (e.g. glycerol or ammonia solution) to The tincture is tested for 2-propanol (2.9.11), with a
extract the herbal drug, or by dissolving a soft or dry extract maximum of 0.05 per cent VIV, unless assurance of
of the herbal drug (which has been produced using the same compliance with this limit is provided by a detailed
extraction solvent as would be used to prepare the liquid knowledge of the ethanol supply chain and the tincture
extract by direct extraction) in either ethanol of the required manufacturing process.
concentration or water. Except for standardised tinctures, tinctures produced from
Where the liquid extract contains ethanol, it is tested for soft or dry extracts do not contain any excipients other than
2-propanol (2.9.11), with a maximum of 0.05 per cent V/V, those that would be present in the tincture prepared by direct
unless assurance of compliance with this limit is provided by extraction. However, exceptions may be justified in certain
a detailed knowledge of the ethanol supply chain and the cases such as when the soft extract used to produce the
extract manufacturing process. tincture contains stabilisers, antioxidants or antimicrobial
Except for standardised liquid extracts, liquid extracts preservatives that have been added to ensure its stability.
produced from soft or dry extracts do not contain any Tinctures are adjusted, if necessary so that they satisfy the
excipients other than those that would be present in the requirements for content of solvent. Tinctures may be filtered
liquid extract prepared by direct extraction. However, if necessary.
exceptions may be justified in certain cases such as when the Tinctures are usually clear. A slight sediment may form on
soft extract used to produce the liquid extract contains standing.
stabilisers, antioxidants or antimicrobial preservatives that
have been added to ensure its stability. TESTS
Liquid extracts are adjusted, if necessary, so that they satisfy Relative density (2.2.5)
the requirements for content of solvent. Liquid extracts may Where applicable, the tincture complies with the limits
be filtered, if necessary. prescribed.
A slight sediment may form on standing. Ethanol (2. 9.10)
TESTS The ethanol content complies with the limits prescribed.
Relative density (2.2.5) Methanol (2. 9.11)
Where applicable, the liquid extract complies with the limits Maximum 0.05 per cent VIV, unless otherwise prescribed or
prescribed. justified and authorised.
Ethanol (2. 9.10) Dry residue (2.8.16)
For ethanolic liquid extracts, carry out the determination of Where applicable, the tincture complies with the limits
ethanol content. The ethanol content complies with the prescribed.
limits prescribed. STORAGE
Methanol (2.9.11) Protected from light.
Maximum 0.05 per cent V/V for ethanolic liquid extracts, LABELLING
unless otherwise prescribed or justified and authorised. The label states, in addition to the requirements listed above,
Dry residue (2.8.16) the ethanol content in per cent V/V.
Where applicable, the liquid extract complies with the limits
prescribed. SOFT EXTRACTS - EXTRACTA SPISSA
STORAGE
DEFINITION
Protected from light.
Soft extracts are semi-solid preparations obtained by
LABELLING evaporation or partial evaporation of the solvent used for
The label states in addition to the requirements listed above, production.
the ethanol content in per cent V/V, where applicable.
TESTS
TINCTURES - TINCTURAE
Dry residue (2.8.16)
DEFINITION
The soft extract complies with the limits prescribed.
Quantified tinctures and 'other' tinctures are liquid extraction
preparations that are obtained using either 1 part by mass of Solvents
herbal drug and 10 parts by mass or volume of extraction Residual solvents are controlled as described in chapter 5.4,
solvent, or 1 part by mass of herbal drug and 5 parts by mass unless otherwise prescribed or justified and authorised.
or volume of extraction solvent. Alternatively, they may be STORAGE
obtained using either 1 part by mass of herbal drug and In an airtight container, protected from light.
sufficient extraction solvent to produce 10 parts by mass or
volume of tincture or 1 part by mass of herbal drug and
OLEORESINS - OLEORESINA
sufficient extraction solvent to produce 5 parts by mass or
volume of tincture. Other ratios of herbal drug to extraction DEFINITION
solvent may be used. Oleoresins are semi-solid extracts composed of a resin in
Standardised tinctures are only defined by their content of solution in an essential and/or fatty oil and are obtained by
constituents with known therapeutic activity. evaporation of the solvent(s) used for their production.
This monograph applies to oleoresins produced by extraction DERgenuine are usually identical. For soft extracts and liquid
and not to natural oleoresins. extraction preparations, where the genuine (native) extract
does not exist without excipients and/or processing aids
TESTS
(e.g. usually 20-30 per cent of water in soft extracts,
Water (2.2.13)
ethanolic extraction solvent in tinctures), the DER, ,a1 and the
0
The oleoresin complies with the limits prescribed.
DERgenuine are identical.
Solvents
Drug solvent ratio (DSR)
Residual solvents are controlled as described in chapter 5.4,
The ratio between the quantity of herbal drug, expressed in
unless otherwise prescribed or justified and authorised.
mass, used in the manufacture of an extract and the quantity
STORAGE of the first extraction solvent, expressed in mass or volume.
In an airtight container, protected from light. Extraction solvents
Solvents which are used for the extraction process.
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
Dry extracts usually have a loss on drying of not greater than the genuine extract may contain variable amounts of
5 per cent mlm. 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 (2.5.12) the marker has been quantitatively determined in the herbal
Where a test for loss on drying is not applicable, the dry drug or herbal drug preparation.
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;
STORAGE - analytical markers are constituents or groups of
In an airtight container, protected from light. 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
are generally accepted to contribute substantially to the Production of tinctures by maceration
A process whereby, unless otherwise prescribed, the herbal
therapeutic activity of a herbal drug, a herbal drug
preparation or a herbal medicinal product. d~g to be extracted is reduced to pieces of suitable size,
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;
the number written after the colon is the relative quantity of Production of tinctures by percolation
the extract obtained. Two DERs can be differentiated: A process whereby, unless otherwise prescribed, the herbal
drug to be extracted is reduced to pieces of suitable size and
- Genuine (native) drug extract ratio (DERgenuine)-
The ratio between the quantity of herbal drug used in the mixed thoroughly with a portion of the prescribed extraction
manufacture of an extract and the quantity of genuine solvent and allowed to stand for an appropriate time.
The mixture is transferred to a percolator and more
(native) extract obtained.
extraction solvent is added until the herbal drug is covered
- Total drug extract ratio (DERtotaO· The ratio between
the quantity of herbal drug used in the manufacture of an with a layer of extraction solvent. The percolate is allowed to
extract and the quantity of whole extract (including flow slowly from the base of the percolator while extraction
excipients) obtained. solvent is slowly added to the top of the percolator, ensuring
that the herbal drug to be extracted is constantly covered
For example, DERgenuine 2.5-4.5:1 means that between 2.5 with extraction solvent, until all the extraction solvent has
and 4.5 parts of herbal drug are required to produce I part been added. Percolation continues until the percolate is
of genuine (native) extract. Where processing aids are added recovered. If the residue is pressed, the 2 liquids are
to the genuine (native) extract to produce, for example, a dry combined.
extract, the DERio,at and the DERgenuine will have different
values; where a dry extract is produced without the need for - - - - - - - - - - - - - - - - - - - - - - PhEur
any processing aids, the DER,0 ,a1 and the DERgenuine will be
identical. Oleoresins are usually produced without the need
to include processing aids, therefore the DER,0 ta1 and the
2023 Herbal Teas IV-53
Tinctures of the British Pharmacopoeia Instant Herbal Teas

In addition to the requirements for Tinctures of the European
Pharmacopoeia (stated under Extracts), the following statements
apply to those tinctures that are the subject of an individual ~~----------------------
monograph in the British Pharmacopoeia. DEFINITION
DEFINITION Instant herbal teas consist of 1 or more herbal drug
Certain preparations of the British Pharmacopoeia entitled preparations (primarily extracts with or without added
Tinctures do not conform strictly to the definition of the essential oils), and are intended for the preparation of an oral
European Pharmacopoeia and consequently application of solution immediately before use.
some of the above requirements is inappropriate. Instant herbal teas may also contain, in addition to herbal
Any necessary exceptions are stated in the relevant individual drug preparations, suitable excipients such as maltodextrin
monographs. and added flavourings.
Instant herbal teas are presented as a powder or granules and
are usually supplied in bulk form or in sachets.
Herbal Teas The herbal drug preparations used comply with the
appropriate individual European Pharmacopoeia monographs
(Ph. Bur. monograph 1435) or, in the absence of such individual monographs, with the
Herbal Teas comply with the requirements of the European general monograph Herbal drug preparations (1434) and with
Pharmacopoeia. These requirements are reproduced below. other appropriate general monographs, for example Herbal
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ drug extracts (0765) or Essential oils (2098).
DEFINITION IDENTIFICATION
Herbal teas consist exclusively of one or more herbal drugs The identity of herbal drug preparations present in instant
intended for oral aqueous preparations by means of herbal teas is checked by suitable methods.
decoction, infusion or maceration. The preparation is TESTS
prepared immediately before use. General chapter 5.1. 8 contains recommendations on the
Herbal teas are usually supplied in bulk form or in bags for microbiological quality of extract-containing herbal medicinal
single use. products such as instant herbal teas.
The herbal drugs used comply with the appropriate The proportion of herbal drug preparations present in instant
individual European Pharmacopoeia monographs or in their herbal teas is checked by suitable methods.
absence with the general monograph Herbal drugs (1433). Instant herbal teas in sachets comply with the following test.
IDENTIFICATION Uniformity of mass
The identity of herbal drugs present in herbal teas is checked Determine the individual and the average mass of the
by suitable methods such as botanical examinations and/or contents of 20 randomly chosen units as follows: weigh a
chromatographic profiles. single full sachet of instant herbal tea, open it without losing
TESTS any fragments. Empty it completely using a brush. Weigh the
Recommendations on the microbiological quality of herbal empty sachet and calculate the mass of the contents by
teas (5.1. 8) take into account the prescribed preparation subtraction. Repeat the operation on the 19 remaining
method (use of boiling or non-boiling water). sachets and calculate the average mass of the contents of the
The proportion of herbal drugs present in herbal teas is 20 units. Unless otherwise justified, not more than 2 of the
checked by appropriate methods. individual masses deviate from the average mass by more
than the percentage deviation shown in the table below and
Herbal teas in bags comply with the following test:
none deviates by more than twice that percentage.
Uniformity of mass
Determine the individual and the average mass of the Average mass Percentage deviation
contents of 20 randomly chosen units as follows: weigh a
less than 1.5 g 15 per cent
single full bag of herbal tea, open it without losing any
1.5 g to 2.0 g included 10 per cent
fragments. Empty it completely using a brush. Weigh the
more than 2.0 g 7.5 per cent
empty bag and calculate the mass of the contents by
subtraction. Repeat the operation on the 19 remaining bags
and calculate the average mass of the contents of the STORAGE
20 units. Unless otherwise justified, not more than 2 of the Protected from light.
20 individual masses deviate from the average mass by more _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
than the percentage deviation shown in the table below and
none deviates by more than twice that percentage.
Average mass Percentage deviation

less than 1.5 g 15 per cent
1.5 g to 2.0 g included 10 per cent
more than 2.0 g 7.5 per cent
STORAGE
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
IV-54 Abelmoschus Flower 2023
Abelmoschus Flower
(Abelmoschi Corolla, Ph. Bur. monograph 2827)
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried corolla of Abelmoschus manihot (L.) Medik.
Content
Minimum 1.0 per cent of hibifolin (C 21 H 18 0 14; Mr 494.4)
(dried drug).
IDENTIFICATION
A. The corolla is often crumpled and fragmented, and when
whole, consists of 5 petals slightly fused at their base. Each
petal is triangular and broadly obovate, with slightly sinuous
margins, about 7-20 cm long and 7-12 cm wide. They are
yellow or pale yellow, with a dark violet or brown unguis at
the base. The outer surface shows numerous pale green
radial longitudinal striations, and stamens united by their
filaments into a tube about 1.5-2.5 cm long bearing
numerous sessile anthers. At the centre of the stamen tube is
the style, ending in a dark violet or black stigma, with 5 lobes
rolled outwards.
B. Microscopic examination (2.8.23). The powder is yellow
or yellowish-brown. Examine under a microscope using
chloral hydrate solurion R. The powder shows the following
diagnostic characters (Figure 2827.-1): fragments of the
epidermis of the distal part of the corolla (surface view [BJ,
transverse section [F]) with papillose cells [Ba, Fa] and long
glandular trichomes, up to 800 µm long, with a stalk Figure 2827 .-1. - Illustration for identification test B of powdered
composed of 1 [Be] to 4 cells [HJ and a multicellular head herbal drug of abelmoschi corolla
whose diameter does not exceed that of the stalk, composed
of cells with slightly granular contents [Bb]; epidermal cells Plate TLC silica gel F254 plate R (2-10 µm) [or TLC silica gel
surrounding each glandular trichome that are not papillose F2 54 plate R (5-40 µm)].
[Bd]; fragments of the epidermis of the base of the Mobile phase anhydrous formic acid R, acetic acid R, water R,
corolla [A], often pink or violet, with elongated epidermal ethyl acetate R (11:11:27:100 VIVIVIV).
cells [Aa], unicellular covering trichomes either isolated [Ab]
or paired [Ac], up to 200 µm long, and glandular trichomes Application 3 µL as bands of 8 mm.
with a dub-shaped secretory head [E]; fragments of the Development 70 mm from the lower edge of the plate (or
characteristic layer of the pollen sac of the anthers with 120 mm).
polyhedral cells, whose walls show thickened parallel Drying In air.
striations [G], accompanied by oil droplets [Ga]; spherical Detection Heat at 100-105 °C for 3-4 min; spray the warm
pollen grains [DJ with finely pitted walls, exceeding 100 µm plate with a 10 g/L solution of diphenylboric acid aminoethyl
in diameter, spiny and with numerous germinal pores [Da]; ester R in methanol R and then with a 50 g/L solution of
numerous annular or spiral vascular bundles [CJ; fragments macrogol 400 R in methanol R; allow to dry in air and examine
of parenchyma consisting of polyhedral cells, some of which in ultraviolet light at 366 nm.
contain cluster crystals of calcium oxalate about 20 µm in System suitability Reference solution (c):
diameter m. - the chromatogram shows 2 distinct zones in the
C. High-performance thin-layer chromatography (2.8.25). middle third; both the lower zone (hibifolin) and the
Prepare the solutions immediately before use. upper zone (hyperoside) show a yellow fluorescence.
Test solution To 0.25 g of the powdered herbal drug (1000) Results See below the sequence of fluorescent zones present
(2. 9.12) add 10 mL of a mixture of 4 volumes of water R and in the chromatograms obtained with reference solution (a)
6 volumes of acetonitrile R, and sonicate for 15 min. and the test solution. Furthermore, in the chromatogram
Centrifuge the solution for 15 min and filter the supernatant obtained with the test solution, other faint fluorescent zones
through a membrane filter (nominal pore size 0.45 µm). may be present.
Reference solurion (a) Dissolve 4.0 mg of rutoside trihydrate R
and 5.0 mg of hibifoh'n R in methanol Rand dilute to 5.0 mL
with the same solvent.
Reference solution (b) Dilute 2.5 mL of reference solution (a)
to 10.0 mL with methanol R.
Reference solution (c) Dissolve 2.5 mg of hibifolin Rand
2.5 mg of hyperoside R in methanol R and dilute to 5 mL with
the same solvent.
Intensity marker Hibifolin.
2023 Acanthopanax Bark IV-55
Top of the plate Column:

=
- size: l 0.25 m, 0 4.6 mm; =
- stationary phase: phenylsilyl silica gel for chromatography R
(5 µm);
- temperature: 40 °C.
-- --
Mobile phase:
A blue zone, equivalent - mobile phase A: acetonitrile R, 0.07 per cent V!V solution of
phosphoric acid R (5:95 V/V);
- mobile phase B: 0.07 per cent V/V solution of phosphoric
acid R, acetonitrile R (5:95 V/V);
A yellow zone, equivalent
Time Mobile phase A Mobile phase B
A yellow zone, equivalent (min) (per cent V/Ji) (percent V/Ji)
(hyperoside)
0 - 10 89 11
Hibifolin: a yellow zone A yellow zone, equivalent (hibifolin) 10 - 15 89-> 87 11 -> 13
15 - 25 87-> 75 13-> 25
25 - 30 75-> 60 25-> 40
Rutoside: a yellow zone A yellowish zone, faint (rutoside) 30 - 33 60 40
-- --
A yellowish zone, very faint

Fkno rate 1.0 mIJmin.
Detection Spectrophotometer at 360 nm.
Autosampler Set at 10 °C.
Jnjectwn 10 µL of the test solution and reference
A blue zone, equivalent solutions (c) and (d).
Retention time Hibifolin = about 22 min; quercitrin = about
23 min.
Reference solution (a) Test solution System suitability Reference solution (c):
- resolution: minimum 2.5 between the peaks due to
hibifolin and quercitrin.
TESTS
Loss on drying (2.2.32) Calculate the percentage content of hibifolin using the
Maximum 12.0 per cent, determined on 1.000 g of the following expression:
powdered herbal drug (1000) (2.9.12) by drying in an oven
A1 xmz xpx 1.75
at 105 °C for 4 h.
A2xm1
Total ash (2.4.16)
Maximum 8 per cent. A1 area of the peak due to hibifolin in the chromatogram obtained
with the test solution;
ASSAY A2 area of the peak due to hibifolin in the chromatogram obtained
Liquid chromatography (2.2.29). Prepare the solutwns with reference solution (d);
immediately before use. m1 mass of the herbal drug to be examined used to prepare the test
solution, in grams;
Solvent mixture A water R, acetonitrile R (40:60 VIV). m2 mass of hilnfolin CRS used to prepare reference solution (a), in
Solvent mixture B acetonitrile R, water R (10:90 VIV). grams;
p percentage content of hibifolin in hibifolin CRS.
Test solutwn Reduce the herbal drug to a powder (1000)
(2. 9.12) using a refrigerated blade grinder. To 0.500 g add _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
20 mL of solvent mixture A and homogenise using a shaker.
Sonicate for 30 min. Centrifuge for 15 min and transfer the
supernatant to a 50 mL volumetric flask. Change the water
in the ultrasonic bath every 30 min to prevent heating and
repeat the extraction twice, adding to the residue 15 mL of Acanthopanax Bark
solvent mixture A. Combine the supernatants, cool, and
dilute to 50.0 mL with solvent mixture A. Dilute 2.0 mL of
the solution to 5.0 mL with solvent mixture B. Filter through Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
a membrane filter (nominal pore size 0.45 µm). DEFINITION
Reference solutwn (a) Dissolve 6.0 mg of hibifolin CRS in Dried root bark of Eleutherococcus nodifiorus (Dunn) S.Y.Hu
solvent mixture A and dilute to 10.0 mL with solvent (syn. Acanthopanax gracilistylus W.W.Sm.) collected in
mixture A. summer and autumn.
Reference solutwn (b) Dissolve 2.5 mg of quercitrin R in
IDENTIFICATION
solvent mixture A and dilute to 5 mL with solvent
A. The bark occurs in irregular quills, 5-15 cm long,
mixture A.
0.4-1.4 cm in diameter, about 2 mm thick. The outer surface
Reference solutwn (c) Mix 0.4 mL of reference solution (b) is greyish-brown, with slightly twisted longitudinal wrinkles
and 0.5 mL of reference solution (a), and dilute to 5 mL and transverse lenticel-like scars. The inner surface is pale
with solvent mixture B. yellow or greyish-yellow, with fine longitudinal striations.
Reference solutwn (d) Dilute 0.70 mL of reference The texture is light, fragile, easily broken. The fracture is
solution (a) to 5.0 mL with solvent mixture B. irregular, greyish-white.
IV-56 Achyranthes Bidentata Root 2023
TESTS
Periploca sepium
Thin-layer chromatography (2.2.27).
Test solution To 0.3 g of the powdered herbal drug (355)
(2. 9.12) add 3 mL of methanol R, heat in a water-bath at
60 °C for 1 min and filter.
Reference solution Dissolve 5 mg of thymol R and 8 mg of
borneol R in 5 mL of methanol R.
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
plate R (2-10 µm)].
Mobile phase ethyl acetate R, methylene chloride R (2:98 V/V).
Application 20 µL [or 1 µL] as bands of 10 mm [or 8 mm].
Development Over a path of 10 cm [or 6 cm].
Drying In air.
Detection Treat with anisaldehyde solution R, heat at 105 °C
for 5 min and examine in daylight.
Results The chromatogram obtained with the test solution
shows no intense coloured zones above the zone due to borneol
in the chromatogram obtained with the reference solution.
Acanthopanax giraldii
The presence of scaly covering trichomes on the outer
surface of the root bark indicates adulteration by
Acanthopanax giraldii.
,·
..
. Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 1.000 g of the
K ·. . . . . powdered herbal drug (355) (2.9.12) by drying in an oven at
5 µm · , · .· • 105 °C for 2 h.
>----< t , . ' .
Total ash (2.4.16)
Figure 2432.-1. - Illustration for identification test B of powdered Maximum 12.0 per cent.
herbal drug of acanthopanax bark Ash insoluble in hydrochloric acid (2.8.1)
Maximum 2.0 per cent.
B. Microscopic examination (2.8.23). The powder is greyish- Extractable matter
white. Examine under a microscope using chloral hydrate Minimum 16.0 per cent.
solution R. The powder shows the following diagnostic To 2.00 g of the powdered herbal drug (250) (2.9.12) add a
characters (Figure 2432.-1): cluster crystals of calcium mixture of 8 g of water R and 12 g of ethanol (96 per cent) R
oxalate, 8-64 µm in diameter, free [F] or included in and allow to macerate for 2 h, shaking frequently. Filter,
parenchyma cells [A, Ea, Ha] sometimes forming crystal evaporate the filtrate to dryness on a water-bath in vacua and
sheaths [J]; cork cells, rectangular or polygonal, thin-walled dry in an oven at 100-105 °C for 2 h. The residue weighs a
(surface view [BJ, transverse section [El), sometimes walls of minimum of 320 mg.
cork cells of older barks unevenly thickened, slightly _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
pitted [K]; fragments of secretory canals [H] consisting of
thin-walled cells [Hb] containing colourless or pale yellow
secretions; phloem fibres, free [C] or in bundles of 2-4
fibres [D] present in older barks. Examine under a Achyranthes Bidentata Root
microscope using a 50 per cent V/V solution of glycerol R.
The powder shows numerous starch granules, simple, (Ph. Bur. monograph 2999)
polygonal or subspherical, 2-8 µm in diameter, or 2-10 PhEur - - - - - - - - - - - - - - - - - - - - ~
compound, free or included in cells [G].
DEFINITION
C. Examine the chromatogram obtained in the test for Whole or fragmented, dried principal root of Achyranthes
Periploca sepium. bidentata Blume with secondary roots removed.
Results See below the sequence of zones present in the
Content
chromatograms obtained with the reference solution and the
Minimum O.1 per cent of total sterones, expressed as
test solution. Furthermore, other faint zones may be present
~-ecdysterone (C 27 H 44 0 7; Mr 480.6) (dried drug).
in the chromatogram obtained with the test solution.
IDENTIFICATION
Top of the plate A. Whole drug. The whole root is slender, cylindrical, straight
or slightly curved, 13-90 cm long and 4-10 mm in diameter.
-- --
The outer surface is greyish-yellow to greyish-brown or pale
Thymol: an orange zone brown to pale yellowish-brown with fine longitudinal
wrinkles, slightly twisted; transverse lenticels and sparse
-- --
protruding rootlet scars are present. The root is easily broken
Borneo!: a brown zone and has a hard, fragile texture. The fracture is pale yellowish-
A broad pink zone
brown, slightly horny. Numerous small vascular bundles
appear as dots arranged in 2-4 whorls surrounding 2-3 larger
Reference solution Test solution central bundles; the xylem vessels are yellowish-white.
2023 Achyranthes Bidentata Root IV-57
Fragmented drug The fragmented drug occurs as short chromatography R with 5 mL of methanol R and then with
cylindrical pieces or as oblique slices, 3-10 mm in diameter. 3 mL of water R at a rate of 1 drop per second, ensuring that
The outer surface is greyish-yellow to greyish-brown or pale the column does not dry out. Transfer the solution to the
brown to pale yellowish-brown with fine longitudinal SPE column. Allow to drain, ensuring that the column does
wrinkles, slightly twisted; transverse lenticels and sparse not dry out, and discard the eluate. Wash the column with
protruding rootlet scars may be visible. The texture is hard. 2 mL of water R. Elute the column using 1 mL of
The cut surface is pale yellowish-brown, yellowish-brown or methanol R. Collect the eluate.
brown, slightly horny. Numerous small vascular bundles Reference solutwn (a) Dissolve 1.0 mg of /3-ecdysterone Rand
appear as dots arranged in 2-4 whorls surrounding 2-3 larger 1.0 mg of ginsenoside Ro R in methanol Rand dilute to
central bundles; the xylem vessels are yellowish-white. 2.0 mL with the same solvent.
B. Microscopic examination (2.8.23). The powder is pale Reference solutwn (b) Mix 1.0 mL of reference solution (a)
brown or whitish-grey. Examine under a microscope using and 3.0 mL of methanol R.
ch/,oral hydrate solutwn R. The powder shows the following
Reference solutwn (c) Dissolve 1 mg of /3-ecdysterone R and
diagnostic characters (Figure 2999.-1): numerous fragments
1 mg of cyasterone R in methanol R and dilute to 2 mL with
of parenchyma [B, E] consisting of thin-walled, rounded,
the same solvent.
ovoid or subrectangular cells, some of which contain
triangular, pointed, subsquare or irregularly shaped sandy Intensity marker ~-ecdysterone.
calcium oxalate microcrystals [Ba, Ea]; bundles of vessels [C, Plate TLC silica gel F 254 plate R (2-10 µm).
F], reticulate [Fa] or bordered pitted (8-93 µm in diameter) Mobile phase anhydrous formic acid R, water R, methanol R,
[Ca, Fb], sometimes accompanied by fibres [Cb], with methylene chloride R (1:1:5:14 VIVIVIV).
slightly thickened walls and sparse, oblique-slit-shaped, cross- Application 3 µL as bands of 8 mm.
shaped or V-shaped pits; numerous fragments of isolated
Development 70 mm from the lower edge of the plate.
vessels [HJ; cork fragments with more or less square,
rectangular, rounded or polygonal cells (surface view [Al); Drying In a current of cold air for 5 min.
the cork layers (transverse section [G]) are superimposed and Detection Treat with a 100 g/L solution of sulfunc acid R in
associated with the 1st layers of parenchyma [Ga); numerous ethanol (96 per cent) R and heat at 100 °C for 5 min; examine
scattered microcrystals of calcium oxalate [DJ, clearly visible in ultraviolet light at 366 nm.
in polarised light. System suitability Reference solution (c):
- the chromatogram shows in the upper third 2 distinct
zones, which may be touching; the lower zone
(~-ecdysterone) shows a blue fluorescence and the
upper zone (cyasterone) shows a bright blue
fluorescence.
chromatograms obtained with reference solution (a) and the
test solution. Furthermore, in the chromatogram obtained
with the test solution, other faint fluorescent zones may be
present.
Top of the plate
3 reddish zones, faint
~-Ecdysterone: a blue fluorescent A blue fluorescent zone

zone (~-ecdysterone)
-- --
A green zone, faint to intense
A green zone, faint to equivalent
A green zone
Ginsenoside Ro: a green zone A green zone, faint to intense

(ginsenoside Ro)
Figure 2999 .-1. - Illustration for identificatwn test B of powdered -- --

herbal drug of Achyranthes bulentata root A reddish-zone, faint to equivalent
(maybe overlapping with the zone
C. High-performance thin-layer chromatography (2.8.25). below)
Test solutwn To 1.0 g of the powdered herbal drug (355) A reddish-violet zone, intense
(2. 9.12) add 5 mL of a mixture of 2 volumes of water R and
A reddish-violet zone, intense
8 volumes of methanol R. Sonicate for 30 min and centrifuge.
Take up the residue with 8 mL of water R. Sonicate for
1 min (or shake for 1 min), centrifuge and combine the
Reference solution (a) Test solution
supematants. Condition a solid phase extraction (SPE)
column containing 0 .200 g of octadecylsilyl silica gel for
IV-58 Agnus Castus Fruit 2023
TESTS sum of the peak areas due to sterones 2 and 3 and

~-ecdysterone in the chromatogram obtained with the test
Loss on drying (2.2.32) solution;
Maximum 15.0 per cent, determined on 1.000 g of the = area of the peak due to P-ecdysterone in the chromatogram
powdered herbal drug (355) (2. 9. 12) by drying in an oven at obtained with reference solution (a);
105 °C for 2 h. mass of the herbal drug to be examined used to prepare the test
solution, in grams;
Total ash (2.4.16) mass of P-ecdysterone CRS used to prepare reference
Maximum 9.0 per cent. solution (a), in grams;
p percentage content of P-ecdysterone in P-ecdysterone CRS.
Ash insoluble in hydrochloric acid (2.8.1)
Maximum 1.5 per cent. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
ASSAY
Liquid chromatography (2.2.29).
Solvent mixture water R, methanol R (20:80 V/V).
Test solution To 1.0 g of the powdered herbal drug (355) Agnus Castus Fruit
(2. 9.12) add 45 mL of the solvent mixture and 5 mL of
butanol R saturated with water R. Sonicate for 30 min and (Ph. Bur. monograph 2147)
filter. Evaporate the filtrate to dryness under reduced Preparation
pressure. Dissolve the residue in the solvent mixture and Agnus Castus Fruit Dry Extract
dilute to 10.0 mL with the solvent mixture. Filter through a PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
membrane filter (nominal pore size 0.45 µm).
DEFINITION
Reference solution (a) Dissolve 6.0 mg of /3-ecdysterone CRS
in the solvent mixture and dilute to 25.0 mL with the solvent Whole, ripe, dried fruit of Vitex agnus-castus L.
mixture. Dilute 2.0 mL of the solution to 5.0 mL with the Content
solvent mixture. Minimum 0.08 per cent of casticin (C 19H 18 0 8 ; Mr 374.3)
Reference solution (b) To 5 mg of Achyranthes bidentata root (dried drug).
dry extract for system suitability HRS add 5 mL of the solvent IDENTIFICATION
mixture. Sonicate for 10 min and shake well. Filter through a A. Agnus castus fruit is oval or almost globular, with a
membrane filter (nominal pore size 0.45 µm). diameter of up to 5 mm. The persistent calyx is greenish-
Column: grey, finely pubescent, ends in 4-5 short teeth and envelops
- size: l =0.15 m, 0 =4.6 mm; 2/3 to 3/4 of the surface of the fruit. The blackish-brown
- stationary phase: solid core octadecylsilyl silu:a gel for fruit consists of a pericarp that becomes progressively
chromatography R (2.7 µm); sclerous up to the endocarp. The style scar is often visible.
- temperature: 35 °C. Some of the fruits may retain a stalk, about 1 mm long.
Mobile phase: A transverse section of the fruit shows 4 locules, each
- mobile phase A: water for chromatography R; containing an elongated seed.
- mobile phase B: acetonitrile for chromatography R; B. Microscopic examination (2.8.23). Examine under a
microscope using chloral hydrate solution R. The powder
Time Mobile phase A Mobile phase B shows the following diagnostic characters: fragments of the
(min) (per cent V/JI) (per cent V/JI) outer epidermis of the calyx composed of polygonal cells
0-3 85 15 densely covered with short, bent or undulate, uni-, bi- or tri-
3 - 18 85---> 81 15---> 19 cellular uniseriate covering trichomes; cells of the epicarp
with thick walls and well-marked, large pits; isolated
Flow rate 0.8 mUmin. glandular trichomes with a unicellular stalk and a uni- or
multi-cellular head; layers of parenchyma from the outer part
of the mesocarp, some containing brown pigment, others
Injection 10 µL. extending into septa; fragments from the inner part of the
Identification of peaks Use the chromatogram supplied with mesocarp composed of thin-walled, pitted, sclerenchymatous
Achyranthes bidentata root dry extract for system suitability HRS cells and of typical isodiametric sclerous cells with very thick,
and the chromatogram obtained with reference solution (b) deeply grooved walls and a narrow, stellate lumen; small
to identify the peaks due to sterones 2 and 3 and peak 4; use brown cells of the endocarp; fragments of the testa
the chromatogram obtained with reference solution (a) to containing areas of fairly large, thin-walled lignified cells with
identify the peak due to P-ecdysterone (sterone 1). reticulate bands of thickening; numerous fragments of the
Relative retention With reference to P-ecdysterone (retention endosperm composed of thin-walled parenchymatous cells
time= about 12.5 min): peak 4 = about 1.04; containing aleurone grains and oil droplets.
sterone 2 = about 1. 11; sterone 3 = about 1.19. C. Thin-layer chromatography (2.2.27).
System suitability Reference solution (b): Test solution To 1.0 g of the powdered herbal drug (355)
- resolution: minimum 1.5 between the peaks due to (2. 9. 12) add 10 mL of methanol R. Heat in a water-bath at
P-ecdysterone and peak 4. 60 °C for 10 min. Allow to cool and filter.
Calculate the percentage content of total sterones expressed Reference solution Dissolve 0.5 mg of aucubin Rand 1 mg of
as P-ecdysterone using the following expression: agnuside R in methanol R and dilute to 1.0 mL with the same
solvent.
A1 xm2 x4xp
Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silu:a gel
A2 xm1 x25 F254 plate R (2-10 µm)J.
2023 Agnus Castus Fruit Preparations IV-59
Mobile phase water R, methanol R, ethyl acetate R Time Mobile phase A Mobile phase B
(8: 15:77 V/V/V). (min) (per cent V/V) (per cent V/V)
0 - 30 70--+ 45 30--+ 55
Drying In air.
Detection Treat with anhydrous formic acid R and heat at
120 °C for 10 min; examine in daylight. Injection 10 µL.
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 agnus castus fruit 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 penduletin and casticin.
Top of the plate - resolution: minimum 1.5 between the peaks due to
penduletin and casticin.
- - -- Calculate the percentage content of casticin using the
Agnuside: a blue zone A blue zone (agnuside) following expression:
- - - -
Aucubin: a blue zone A blue zone (aucubin)
Reference solution Test solution area of the peak due to casticin in the chromatogram obtained
area of the peak due to casticin in the chromatogram obtained
with the reference solution;
TESTS
mass of the herbal drug used to prepare the test solution, in
Foreign matter (2.8.2) grams;
Maximum 3.0 per cent. mass of agnus castus fruit dry extract HRS used to prepare the
reference solution, in grams;
Other species of Vitex, in particular Vitex negundo L p percentage content of casticin in agr,us casrus fruit dry
No fruit of other species with a much greater diameter is extract HRS.
present.
Total ash (2.4.16) - - - - - - - - - - - - - - - - - - - - - - PhEur
powdered herbal drug (355) (2.9.12) by drying in an oven at Agnus Castus Fruit Dry Extract
105 °C for 2 h.
ASSAY
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Test solution Extract 1.000 g of the powdered herbal drug DEFINITION
(355) (2. 9.12) with 40 mL of methanol R for 2 min using a Dry extract produced from Agnus castus fruit (2147).
suitable-speed homogeniser. Collect the supernatant liquid Content
and filter into a 250 mL flask. Repeat the extraction with a Minimum 0.1 per cent ofcasticin (C 19H 18 0 8 ; Mr 374.3)
further 40 mL of methanol R, collecting the supernatant (dried extract).
liquid and filtering as before. Rinse the residue carefully with
a small quantity of methanol R. Combine the methanol
PRODUCTION
extracts and rinsings and evaporate to dryness in vacua in a The extract is produced from the herbal drug by a suitable
water-bath at not more than 30 °C. With the aid of procedure using ethanol (40-80 per cent V/V).
ultrasound, dissolve the residue obtained in methanol R and CHARACTERS
dilute to 20.0 mL with the same solvent. Filter the solution Appearance
through a membrane filter (nominal pore size 0.45 µm). Brown, amorphous powder.
Dilute 2.0 mL of the solution to 10.0 mL with methanol R.
IDENTIFICATION
Reference solution Suspend a quantity of agnus castus fruit dry Thin-layer chromatography (2.2.27).
extract HRS corresponding to 0.10 mg of casticin in 7.5 mL
of methanol R, sonicate for 5 min and dilute to 10.0 mL with
Test solution Suspend 0.5 g of the extract to be examined in
the same solvent. Filter through a membrane filter (nominal 5 mL of methanol R and sonicate for 15 min. Shake the
mixture vigorously 3 or 4 times during the procedure. Filter.
pore size 0.45 µm).
Reference solution Dissolve 1 mg of cajfeic acid R and 4 mg
Column:
of homoorientin R in 10 mL of methanol R.
=
- size: l 0.125 m, 0 4.0 mm; =
- stationary phase: end-capped octadecylsilyl silica gel for Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica
chromatography R (5 µm). gel F254plate R (2-10 µm)].
Mobile phase: Mobile phase water R, anhydrous formic acid R, toluene R,
- mobile phase A: 5.88 g/L solution of phosphoric acid R; tetrahydrofuran R (1:2:8:16 VIVIVIV).
- mobile phase B: acetonitrile R; Application 12 µL [or 3 µL] as bands of 10 mm [or 8 mm].
Drying In air.
IV-60 Agrimony 2023
Detection Treat with a 10 g/1. solution of diphenylboric acid System suitability Reference solution:
aminoethyl ester R in methanol R and dry in a current of cold - resolution: minimum 1.5 between the peaks due to
air, then treat with a 50 g/1. solution of macrogol 400 R in penduletin and casticin.
methanol R, allow to dry and examine in ultraviolet light at Calculate the percentage content of casticin using the
365 nm. following expression:
Results See below the sequence of fluorescent zones present
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
test solution. with the test solution;
Top of the plate with the reference solution;
m1 mass of the extract to be examined used to prepare the test
solution, in grams;
m2 mass of agnus castus fruit dry extract HRS used to prepare the
Caffeic acid: a light blue fluorescent An orange fluorescent zone and a reference solution, in grams;
zone white or pale yellow fluorescent p percentage content of casticin in agnus castus fruit dry
zone, partly superimposed extract HRS.
-- -- _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Several light blue fluorescent zones
I or 2 yellow fluorescent zones
-- -- Agrimony
A light blue fluorescent zone
Homoorientin: a yellow fluorescent A yellow fluorescent zone
zone (homoorientin) Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
A yellow fluorescent zone
Dried flowering tops of Agrimonia eupatoria L.
Content
Reference solution Test solution
Minimum 2.0 per cent of tannins, expressed as pyrogallol
(C6H603; Mr 126.1) (dried drug).
ASSAY IDENTIFICATION
Liquid chromatography (2.2.29). A. The stem is green or, more usually, reddish, cylindrical
Test solution Suspend 0.200 g of the extract to be examined and infrequently branched. It is covered with long, erect or
in 15 mL of methanol R, sonicate for 5 min and dilute to tangled hairs. The leaves are compound imparipennate with
20.0 mL with the same solvent. Filter through a membrane 3 or 6 opposite pairs of leaflets, with 2 or 3 smaller leaflets
filter (nominal pore size 0.45 µm). between. The leaflets are deeply dentate to serrate, dark
Reference solution Suspend a quantity of agnus castus frnit dry green on the upper surface, greyish and densely tomentose
extract HRS corresponding to 0.10 mg of casticin in 7.5 mL on the lower face. The flowers are small and form a terminal
of methanol R, sonicate for 5 min and dilute to 10.0 mL with spike. They are pentamerous and borne in the axils of hairy
the same solvent. Filter through a membrane filter (nominal bracts, the calyces closely surrounded by numerous terminal
pore size 0.45 µm). hooked spires, which occur on the rim of the hairy
receptacle. The petals are free, yellow and deciduous. Fruit-
Column:
bearing obconical receptacles, with deep furrows and hooked
- size: l = 0.125 m, 0 = 4.0 mm;
bristles, are usually present at the base of the inflorescence.
- stationary phase: end-capped octadecylsilyl silica gel for
chromatography R (5 µm). B. Microscopic examination (2.8.23). The powder is
yellowish-green or grey. Examine under a microscope using
Mobile phase:
- mobile phase A: 5.88 g/1. solution of phosphoric acid R; chloral hydrate solution R. The powder shows the following
diagnostic characters (Figure 1587.-1): numerous straight or
- mobile phase B: acetonitrile R;
bent, unicellular, long, thick-walled (about 500 µm) covering
trichomes [Ab, Ca, F], finely warty, and sometimes spirally
(min) (per cent V/V} (per cent V/V) marked, often fragmented [F]; fragments of the epidermis of
70 ➔ 45 30 ➔ 55
the stems [A] with stomata [Aa], covering trichomes [Ab]
0 - 30
and glandular trichomes [Ac]; fragments of upper leaf
epidermis (surface view [C]) with straight walls bearing
Flow rate 1.0 mUmin. covering trichomes [Ca), accompanied by palisade
Detection Spectrophotometer at 348 nm. parenchyma [Cb], with some of the cells containing calcium
Injection 10 µL. oxalate prisms [Cc]; fragments of lower leaf epidermis in
Identification of peaks Use the chromatogram supplied with surface view fJ] with sinuous walls and abundant
agnus castus frnit dry extract HRS and the chromatogram stomata Ua], mostly anomocytic (2.8.3) but occasionally
obtained with the reference solution to identify the peaks due anisocytic, and glandular trichomes fjb]; ovoid to
to penduletin and casticin. subspherical pollen grains, with 3 pores and a smooth
exine [D]; glandular trichomes with a multicellular, uniseriate
stalk and a unicellular to quadricellular head [B, Jb];
fragments of the stems [H] with groups of fibres [Ha] and
2023 Akebia Stem IV-61
parenchymatous cells, some of which contain cluster crystals Top of the plate
of calcium oxalate [Hb]; small spiral vessels from the
An orange fluorescent zone may be
leaflets [G]; fragments of large, spiral or bordered-pined present (quercitrin)
vessels from the stem [E].
Isoquercitroside: an orange An orange fluorescent zone
fluorescent zone (isoquercitroside)
An orange fluorescent zone

(hyperoside)
Rutoside: an orange fluorescent zone An orange fluorescent zone
(rutoside)
TESTS
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Tannins (2.8.14)
Use 1.000 g of the powdered herbal drug (180) (2.9.12).
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
Akebia Stem
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or fragmented, dried stem of Akebia quinata (Houtt.)
Figure 1587.-1. - Illustration for identification test B of powdered Decne. or Akebia trifoliata (Thunb.) Koidz., or a mixture of
herbal drug of agrimony the 2 species.
Content
C. Thin-layer chromatography (2.2.27). Minimum 0.15 per cent of oleanolic acid (C 30H 48 O3;
Test solution To 2.0 g of the powdered herbal drug (355) Mr 456.7) (dried drug).
(2. 9.12) add 20 mL of methanol R. Heat with shaking at IDENTIFICATION
40 °C for 10 min. Filter.
A. Toe whole drug is cylindrical, often slightly twisted,
Reference solution Dissolve 1.0 mg of isoquercitroside Rand 30-70 cm long and 0.5-2 cm in diameter. Externally light
1.0 mg of rutoside trihydrate R in 2 mL of methanol R. grey or greyish-brown, rough, with irregular longitudinal and
Plate TLC silica gel plate R. horizontal cracks and irregular longitudinal furrows.
Mobile phase anhydrous formic acid R, water R, ethyl acetate R The outer bark is sometimes easily removed; the exposed
(10:10:80 VIVIV). inner surface is yellowish-brown or reddish-brown. Lenticels
Application 10 µL as bands. are distinct. Nodes are sometimes swollen, with scars of
lateral branches. Toe texture is light and compact.
Development Over a path of 12 cm. Toe fracture is uneven; the bark is relatively thick and easily
Drying At 100-105 °C. distinguished from the wood, sometimes with arched hollows
Detection Spray the still-warm plate with a 10 g/L solution around the outside of the wood; it is yellowish-brown or
of diphenylboric acid aminoethyl ester R in methanol R and then reddish-brown, with pale yellow, granular dots visible; the
with a 50 g/L solution of macrogol 400 R in methanol R; allow wood is yellowish-white, with distinct radial striations, the
the plate to dry in air for 30 min and examine in ultraviolet pith is small, occasionally hollowed, yellowish-white or
light at 365 nm. yellowish-brown.
Results See below the sequence of zones present in the Toe fragmented drug occurs as rounded, elliptical or
chromatograms obtained with the reference solution and the irregular slices, 0.5-2 cm in diameter. Externally light grey or
test solution. greyish-brown, rough, with irregular longitudinal and
horizontal cracks and occasional lenticels; the outer bark is
easily removed, the inner exposed surface is yellowish-brown
or reddish-brown. Toe texture is light, compact and easily
broken. Toe cut surface has a relatively thick, yellowish-
brown, greyish-brown or reddish-brown bark that is easily
distinguished from the wood and appears granular and
sometimes lamellar in transverse section. Hollows appearing
IV-62 Akebia Stem 2023
as arches may be present around the outside of the wood. Reference solution Dissolve 1 mg of hederagenin R and 1 mg
The wood is yellowish-white, with distinct radial striations of oleanolic acid R in 8 mL of methanol R.
and distinct xylem vessels. The pith is small, occasionally Plate TLC silica gel plate R (2-10 µm).
hollowed, yellowish-white or yellowish-brown.
Mobile phase glacial acetic acid R, acetone R, toluene R
B. Microscopic examination (2.8.23). The powder is pale (5:20:80 V/V/V).
yellow to light brown. Examine under a microscope using Applicatwn 15 µL as bands of 8 mm.
chloral hydrate solutwn R. The powder shows the following
diagnostic characters (Figure 24 72.-1 ): groups of fibres, Development Over a path of 6 cm.
frequently broken [C], whose narrow lumens contain small Drying In air.
prisms of calcium oxalate [Ca]; these groups are usually Detection Treat with a 10 per cent V/V solution of suljuric
surrounded by crystal sheaths of calcium oxalate [Cb]; acid R in ethanol (96 per cent) R, heat at 105 °C until the
groups of parenchymatous cells with thick, pitted walls [F], spots appear and examine in daylight.
some containing compact masses of large polyhedral or Results See below the sequence of zones present in the
prismatic crystals of calcium oxalate [Fa]; some of these cells chromatograms obtained with the reference solution and the
are isolated [G]; cork fragments consisting of reddish-brown test solution. Furthermore, other coloured zones may be
polygonal cells with brown contents (surface view [A]); present in the chromatogram obtained with the test solution.
fragments of parenchyma consisting of cells with thin
colourless walls (transverse section [K], longitudinal section Top of the plate
[L]); isolated cubic or prismatic crystals of calcium oxalate
[D]; rare tetragonal, polygonal, elongated or rounded A faint violet-blue zone
sclereids with very thick walls and granular contents [B, M];
abundant single or grouped narrow tracheids with oblique -- --
pits and very fine oblique or criss-cross lines [E]; vessels A violet-blue zone
often fragmented, mostly bordered pitted, up to 120-160 µm Oleanolic acid: a pink zone A faint pink zone (oleanolic acid)
in diameter ill; ligneous, more or less rectangular
parenchyma cells of the medullary rays with very thick, pitted Sequence of narrow violet-blue zones
walls [H]. Hederagenin: a pinkish-blue zone
A prominent violet-blue zone
-- --
TESTS
Total ash (2.4.16)
105 °C for 3 h.
Aristolochic acids (2.8.21, Metlwd A)
It complies with the test.
ASSAY
Test solutwn To 2.00 g of the powdered herbal drug (1400)
(2. 9.12) add 50 mL of methanol R, sonicate for 30 min and
filter. Wash the residue with an appropriate quantity of
methanol R, combine the filtrate and washings and evaporate
to dryness. Dissolve the residue in 10 mL of water R, extract
with 3 quantities, each of 20 mL, of butanol R saturated with
water R, combine the extracts and evaporate to dryness. Take
up the residue with a mixture of 2 mL of hydrochwric acid R
and 20 mL of methanol R and heat under a reflux condenser
Figure 2472.-1. - Illustratwn for identification test B of powdered for 2 h. Add 10 mL of water R, extract with 2 quantities,
herbal drug of akebia stem each of 20 mL, of methylene chwride R, combine the extracts
and evaporate to dryness. Dissolve the residue in methanol R
C. Thin-layer chromatography (2.2.27).
and dilute to 10.0 mL with the same solvent.
Reference solutwn (a) Dissolve 20.0 mg of oleanolic acid CRS
(2. 9.12) add 5 mL of methanol R. Sonicate for 10 min and
in methanol R and dilute to 20.0 mL with the same solvent.
filter or centrifuge. Use the filtrate or the supernatant as the
test solution.
2023 Alchemilla IV-63
Reference solution (b) Dissolve 10 mg of ursolic acid R in IDENTIFICATION

reference solution (a) and dilute to 10.0 mL with the same A. The greyish-green, partly brownish-green, radical leaves,
solution. which are the main part of the drug, are reniform or slightly
Reference solutions (c), (d), (e), (j), (g), (h) Dilute reference semicircular with a diameter generally up to 8 cm, seldom up
solution (a) with methanol R to obtain 6 reference solutions to 11 cm and have 7-9 or 11 lobes and a long petiole.
of oleanolic acid, the concentrations of which span the The smaller, cauline leaves, which have a pair of large
expected value in the test solution. stipules at the base, have 5-9 lobes and a shorter petiole or
Column: they are sessile. The leaves are densely pubescent especially
=
- size: l 0.25 m, 0 = 4.6 mm; on the lower surface and have a coarsely serrated margin.
Young leaves are folded with a whitish-silvery pubescence;
chromatography R (5 µm). older leaves are slightly pubescent and have a finely meshed
venation, prominent on the lower surface. The greyish-green
Mobile phase triethylamine R, glacial acetic acid R, water for
or yellowish-green petiole is pubescent, about 1 mm in
chromatography R, methanol R (0.02:0.04:13:87 V/V/V/V).
diameter, with an adaxial groove. The apetalous flowers are
Flow rate 1.0 mUmin. yellowish-green or light green and about 3 mm in diameter.
Detection Evaporative light-scattering detector: the following The calyx is double with 4 small segments of the epicalyx
settings have been found to be suitable; if the detector has alternating with 4 larger sepals, subacute or triangular. They
different setting parameters, adjust the detector settings so as are 4 short stamens and a single carpel with a capitate
to comply with the system suitability criterion for signal-to- stigma. The greyish-green or yellowish-green stem is
noise ratio: pubescent, more or less longitudinally wrinkled and hollow.
- carrier gas: nitrogen R; B. Microscopic examination (2.8.23). The powder is yellow
- pressure: 350 kPa; to yellowish-brown. Examine under a microscope using
- evaporator temperature: 60 °C. chloral hydrate solution R. The powder shows the following
Injection 10 µL. diagnostic characters (Figure 1387.-1): narrow, unicellular
Run time 24 min. covering trichomes, isolated or included in an epidermis,
Retention time Oleanic acid = about 19 min; ursolic whole [Ba] or sheared off [Ee], up to 1 mm long, with thick,
acid = about 20 min. lignified, spirally striated walls, bluntly pointed at the apex
and pitted at the base; fragments of leaves (transverse section
System suitability:
[E]) consisting of a slightly cuticularised and stomatiferous
- resolution: minimum 1. 7 between the peaks due to
upper epidermis [Ea], 2 layers of palisade parenchyma with
oleanolic acid and ursolic acid in the chromatogram
the cells of the upper layer [Eb] being 2-3 times longer than
obtained with reference solution (b);
those of the lower layer [Ee] (which contain cluster crystals
of calcium oxalate, up to 25 µm in diameter), and spongy
oleanolic acid in the chromatogram obtained with
parenchyma [Ed]; fragments of the upper epidermis of the
reference solution (a).
leaf (surface view (C]) consisting of somewhat sinuous cells
Establish a calibration curve with the logarithm of the with beaded anticlinal walls [Ca], a few anomocytic stomata
concentration (in milligrams per 10 mL) of reference (2.8.3) [Cb], and accompanied by palisade parenchyma [Cc];
solutions (c), (d), (e), (f), (g) and (h) (corrected by the fragments of the lower epidermis of the leaf (surface view
declared percentage content of oleanolic acid CRS) as the [A]) composed of sinuous cells with unevenly thickened
abscissa and the logarithm of the corresponding peak area as anticlinal walls [Aa], anomocytic stomata [Ab] and covering
the ordinate. trichomes or their scars [Ac], rounded, thick-walled and
Calculate the percentage content of oleanolic acid using the channelled; fragments from the petioles and stems [K],
following expression: consisting of spiral or bordered-pitted vessels [Ka] and
lignified fibres [Kb]; a few straight, conical hairs [DJ from
l<Y' the corolla, about 300 µm long, with regularly thickened
mx 10 walls; parenchymatous cells of the mesophyll [HJ, with thin
walls, containing cluster crystals of calcium oxalate [Ha], and
A logarithm of the concentration of oleanolic acid in the test
spiral xylem vessels [Hb] accompanied by short fibres [He];
solution, determined from the calibration curve;
m mass of the herbal drug to be examined used to prepare the test fragments of the epidermis of the stems [BJ consisting of
solution, in grams. polygonal cells with straight, thin walls; spherical pollen
grains [F], about 15 µm in diameter, with 3 distinct germinal
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur pores and a granular exine; fragments of the corolla m with
rectangular epidermal cells with sinuous walls [fa] and
parenchyma with polygonal cells, each containing a small
cluster crystal of calcium oxalate [fb]; occasional fragments of
Alchemilla the ovary [G] with cells each containing a prism crystal of
calcium oxalate.
(Ph. Bur. monograph 1387) C. Thin-layer chromatography (2.2.27).
(2. 9.12) add 5 mL of methanol R and heat in a water-bath at
DEFINITION 70 °C under a reflux condenser for 5 min. Cool and filter.
Whole or cut, dried, flowering, aerial parts of Alchemilla
Reference solutwn Dissolve 1 mg of caffeic acid R and 1 mg
vulgaris L. s.l.
of chlorogenic acid R in 10 mL of methanol R.
Content
Plate TLC silica gel plate R.
(C6H6O3; Mr 126.1) (dried drug).
IV-64 Alisma Plantago-Aquatica 2023
TESTS
powdered herbal drug (355) (2. 9.12) by drying in an oven at
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Tannins (2.8.14)
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Alisma Plantago-Aquatica
DEFINITION
Alisma Plantago-Aquatica is the dried rhizome of Alisma
plantago-aquatica L.
PRODUCTION
The rhizomes are collected in winter after the stem has
withered, washed clean, dried and fibrous roots removed.
May be sliced and dried, rhizomes may be salted. May be
processed with vinegar.
IDENTIFICATION
A. The whole rhizome is sub spherical, elliptical or ovate,
2 to 7 cm long and 2 to 6 cm in diameter. Externally
yellowish-white or yellowish-brown, with irregular transverse-
Figure 138 7. -1. - Illustration for identification test B of powdered annular shallow furrows and numerous small raised fibrous
herbal drug of alchemilla root scars, occasionally tuberculate bud scars attached to the
base. Texture compact, fracture yellowish-white, starchy,
Mobile phase anhydrous Jonnie acid R, water R, ethyl acetate R
with numerous small pores. The rhizome is frequently cut to
(8:8:84 VIVIV).
give roughly circular or oval discs up to about 7 cm along the
Application 20 µL of the test solution and 10 µL of the longest axis and 3 to 7 mm thick. The outer surface of the
reference solution, as bands. discs is dull pale or mid-brown, (sometimes reddish brown,)
Development Over a path of 10 cm. with paler markings and shallow grooves and occasional
Drying At 100-105 °C for 5 min. rootlets. The cut surface is very finely granular and buff (or
Detection Spray with a 10 g/L solution of diphenylhoric acid reddish) coloured.
aminoethyl ester R in methanol R, then spray with a 50 g/L B. Reduce to a powder, Appendix XVII A. Examine under a
solution of macrogol 400 R in methanol R; allow to dry in air microscope using chloral hydrate solution. The powder shows
for about 30 min and examine in ultraviolet light at 365 nm. the following characteristics: The powder is pale buff to
Results See below the sequence of zones present in the reddish buff coloured. Abundant parenchymous cells, many
chromatograms obtained with the reference solution and the sub rounded with elliptical pits aggregated into pit areas;
test solution. Furthermore, other fluorescent zones may be slightly smaller ovoid cells, often in clusters; more angular
present in the chromatogram obtained with the test solution. parenchyma cells often with beaded or pitted walls;
occasional endodermis cells with sinuous, thickened and
lignified walls; xylem elements with scalariform or reticulate
Top of the plate
thickening; oil cavities mostly broken, whole ones sub
2 red fluorescent zones (chlorophyll) rounded, 50 to 100 µm in diameter. Mount in water, the
following characteristics are observed. Numerous small starch
Caffeic acid: a light blue florescent I or 2 intense light blue fluorescent
zone zones
granules, ovoid, sub spherical or ellipsoid, hilum a short slit,
V-shaped or Y-shaped, often in small clusters.
I or several intense green or
greenish-yellow fluorescent zones
C. Carry out the method for high perfonnance thin-layer
chromatography, Appendix XI W, using the following
-- -- solutions.
Chlorogenic acid: a light blue An intense yellow or orange (1) Mix 1 g of freshly powdered herbal drug with 5 mL of
fluorescent zone fluorescent zone
methanol and mix with aid of ultrasound for 15 minutes and
-- --
filter.
(2) 0.025% w/v of alisol B 23-acetate in methanol and
0.05% w/v of alisol B in methanol.
(3) Dilute 1 volume of solution (2) to 4 volumes with
methanol.
2023 Aloes IV-65
(4) 0.025% w/v of alisol B 23-acetate and 0.025% w/v of

/3-sitosterol in methanol.
Barbados Aloes
CHROMATO GRAPHIC CONDITIONS Cura,;:ao Aloes
(a) Use as the coating high perfomzance silica gel F254 (Merck (Ph. Eur. monograph 0257)
silica gel 60 F 254 plates are suitable). Preparation
(b) Use the mobile phase as described below. Standardised Aloes Dry Extract
(c) Apply 8 µL of each solution, as 8 mm zones. Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
(d) Develop the plate to 7 cm. DEFINITION
(e) After removal of the plate, dip in methanolu sulfuru acid Concentrated and dried juice of the leaves of Awe barbadensis
10%, heat at 120° for 3 minutes and examine under Mill.
ultraviolet light (366 nm).
Content
MOBILE PHASE Minimum 28.0 per cent of hydroxyanthracene derivatives,
1 volume of cycwhexane and 2 volumes of ethyl acetate. expressed as barbaloin (C 21 H 22 O 9 ; Mr 418.4) (dried drug).
SYSTEM SUITABILITY CHARACTERS
The test is not valid unless the chromatogram obtained with Appearance
solution (4) shows two clearly separated zones under Dark brown masses, slightly shiny or opaque with a
ultraviolet light (366 nm). conchoidal fracture, or brown powder.
CONFIRMATION Solubility
The chromatogram obtained with solution (1) when observed Partly soluble in boiling water, soluble in hot ethanol
under UV light at 366 nm shows two zones in the top third (96 per cent).
of the plate, a faint zone corresponding to the purple IDENTIFICATION
~-sitosterol zone in the chromatogram obtained with solution Examine the chromatograms obtained in the test for Cape
(4), two intense to faint orange zones corresponding to aloes.
alisol B 23 acetate and alisol B in the chromatograms Results A See below the sequence of zones present in the
obtained with solutions (2) and (3) and a blue-white zone in chromatograms obtained with the reference solution and the
the bottom third of the plate. test solution. Furthermore, other faint fluorescent zones may
be present in the chromatogram obtained with the test
Top of the plate solution.
An intense to faint zone
Top of the plate
An intense to faint blue
zone Aloe emodin: a yellow fluorescent
A purple zone A faint to very faint zone
(~-sitosterol) zone
-- --
- - -
An orange zone An orange zone An intense to faint

(Alisa! B 23 acetate) (Alisol B 23 acetate) orange zone
-
Barbaloin: an orange fluorescent An orange fluorescent zone
An orange zone (Alisa! An intense to faint
zone (barbaloin)
B) orange zone
A blue-white zone -- --
A bluish-white fluorescent zone
A bluish-green fluorescent zone

Solution (4) Solution (2) and (3) Solution (1)
SST Intensity markers Test Solution Reference solution Test solution
TESTS Results B See below the sequence of zones present in the

Foreign matter chromatograms obtained with the reference solution and the
Not more than 2.0%, Appendix XI D. test solution. Furthermore, other faint zones may be present
Loss on drying in the chromatogram obtained with the test solution.
When dried for 2 hours at 105°, loses not more than 9.0% of
its weight, Appendix IX D. Use 1 g.
Total Ash
Not more than 3.0%, Appendix XI J, Method II.
Acid insoluble ash
Not more than 0.5%, Appendix XI K.
IV-66 Aloes 2023
Top of the plate separating funnel. Shake the contents of the separating funnel
with 3 quantities, each of 20 mL, of ether R. Wash the
Aloe emodin: a violet zone
combined ether layers with 2 quantities, each of 10 mL, of
-- --
water R. Discard the washings and dilute the organic phase to
100.0 mL with ether R. Evaporate 20.0 mL of the 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
methanol R. Measure the absorbance (2.2.25) at 512 nm
using methanol R as the compensation liquid.
Barbaloin: a brown zone A brown zone (barbaloin) Calculate the percentage content of hydroxyanthracene
derivatives, expressed as barbaloin, using the following
A violet zone
expression:
-- --
Ax 19.6
m
i.e. taking the specific absorbance ofbarbaloin to be 255.
TESTS A absorbance at 512 nm;
Loss on drying (2.2.32) m mass of the substance to be examined, in grams.
powdered herbal drug by drying in an oven at 105 °C. STORAGE
Total ash (2.4.16) In an airtight container.
Maximum 2.0 per cent. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Cape aloes
Test solution To 0.25 g of the powdered herbal drug add
20 mL of methanol R and heat to boiling in a water-bath. Cape Aloes
Shake for a few minutes and decant the solution. Store at
about 4 °C and use within 24 h. (Ph. Bur. monograph 0258)
Reference solutwn Dissolve 2 mg of aloe emodin R and 2 mg Preparation
of barbaloin R in methanol R and dilute to 1 mL with the Standardised Aloes Dry Extract
same solvent. PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
plate R (2-10 µm)]. DEFINITION
Concentrated and dried juice of the leaves of Aloe ferox Mill.
Mobile phase water R, methanol R, ethyl acetate R
(13:17:100 VIVIV). Content
Minimum 18.0 per cent ofhydroxyanthracene derivatives,
Applicatwn 10 µL [or 2 µL] as bands of 20 mm [or 8 mm].
expressed as barbaloin (C 21 H 22O 9 ; Mr 418.4) (dried drug).
CHARACTERS
Drying In air.
Appearance
Detection A Examine in ultraviolet light at 365 nm.
Dark brown masses tinged with green and having a shiny
Results A The chromatogram obtained with the test solution conchoidal fracture, or greenish-brown powder.
shows no blue fluorescent zones above the orange fluorescent
Solubility
zone due to barbaloin.
Partly soluble in boiling water, soluble in hot ethanol
Detection B Treat with a 100 g/L solution of potassium (96 per cent).
hydroxide R in methanol R, heat at 110 °C for 5 min and
examine in daylight. IDENTIFICATION
Examine the chromatograms obtained in the test for
ASSAY Barbados aloes.
Carry out the assay protected from bright light.
Results A See below the sequence of zones present in the
Introduce 0.300 g of the powdered herbal drug (180) chromatograms obtained with the reference solution and the
(2. 9.12) into a 250 mL conical flask. Moisten with 2 mL of test solution. Furthermore, other faint fluorescent zones may
methanol R, add 5 mL of water R warmed to about 60 °C, be present in the chromatogram obtained with the test
mix, then add a further 75 mL of water Rat about 60 °C solution.
and shake for 30 min. Cool, filter into a volumetric flask,
rinse the conical flask and filter with 20 mL of water R, add
the rinsings to the volumetric flask and dilute to 1.0 L with
water R. Transfer 10.0 mL of this solution to a 100 mL
round-bottomed flask containing 1 mL of a 600 g/L solution
of ferric chloride R and 6 mL of hydrochloric acid R. Heat in a
water-bath under a reflux condenser for 4 h, with the water
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 J M sodium
hydroxide and 4 mL of water R and add the rinsings to the
2023 Aloes Preparations IV-6 7
Top of the plate Total ash (2.4.16)

Aloe emodin: a yellow fluorescent
zone ASSAY
-- --
Introduce 0.400 g of the powdered herbal drug (180)
A blue fluorescent zone
(2. 9.12) into a 250 mL conical flask. Moisten with 2 mL of
methanol R, add 5 mL of water R warmed to about 60 °C,
mix, then add a further 75 mL of water R at about 60 °C
and shake for 30 min. Cool, filter into a volumetric flask,
Barbaloin: an orange fluorescent An orange fluorescent zone rinse the conical flask and filter with 20 mL of water R, add
zone (barbaloin) the rinsings to the volumetric flask and dilute to 1000.0 mL
with water R. Transfer 10.0 mL of this solution to a 100 mL
-- --
round-bottomed flask containing 1 mL of a 600 g/L solution
A bluish-white fluorescent zone of ferric chloride R and 6 mL of hydrochloric acid R. Heat in a
water-bath under a reflux condenser for 4 h, with the water
level above that of the liquid in the flask. Allow to cool,
transfer the solution to a separating funnel, rinse the flask
Results B See below the sequence of zones present in the successively with 4 mL of water R, 4 mL of 1 M sodium
chromatograms obtained with the reference solution and the hydroxide and 4 mL of water R and add the rinsings to the
test solution. Furthermore, other faint zones may be present separating funnel. Shake the contents of the separating funnel
in the chromatogram obtained with the test solution. with 3 quantities, each of 20 mL, of ether R. Wash the
combined ether layers with 2 quantities, each of 10 mL, of
Top of the plate water R. Discard the washings and dilute the organic phase to
100.0 mL with ether R. Evaporate 20.0 mL of the solution
Aloe emodin: a violet zone
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
Barbaloin: a brown zone A brown zone (barbaloin)
using methanol R as the compensation liquid.
-- - - Calculate the percentage content of hydroxyanthracene
derivatives, expressed as barbaloin, using the following
expression:
Ax 19.6
m
TESTS
Barbados aloes i.e. taking the specific absorbance of barbaloin to be 255.
Test solution To 0.25 g of the powdered herbal drug add A absorbance at 512 nm;
20 mL of methanol R and heat to boiling in a water-bath. m mass of the substance to be examined in grams.
about 4 °C and use within 24 h. STORAGE
Reference solutwn Dissolve 2 mg of aloe emodin R and 2 mg In an airtight container.
of barbaloin R in methanol R and dilute to 1 mL with the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
same solvent.
Plate TLC silica gel F 254 plate R (5-40 µm) [or TLC silica gel
F 254 plate R (2-10 µm)].
Mobile phase water R, methanol R, ethyl acetate R Standardised Aloes Dry Extract
(13:17:100 VIVIV).
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Drying In air. DEFINITION
Detection A Examine in ultraviolet light at 365 nm. Standardised dry extract prepared from Barbados aloes (0257)
Results A The chromatogram obtained with the test solution or Cape aloes (0258), or a mixture of both.
shows no prominent bluish-green fluorescent zone below the Content
bluish-white fluorescent zone in the lower third. 19.0 per cent to 21.0 per cent of hydroxyanthracene
Detection B Treat with a 100 g/L solution of potassium derivatives, expressed as barbaloin (C 21 H 22 0 9; Mr 418.4),
hydroxide R in methanol R, heat at 110 °C for 5 min and adjusted if necessary (dried extract).
examine in daylight. PRODUCTION
Results B The chromatogram obtained with the test solution The extract is produced from the herbal drug by a suitable
shows no violet zone just below the zone due to barbaloin. procedure using boiling water.
Loss on drying (2.2.32) CHARACTERS
Maximum 10.0 per cent, determined on 1.000 g of the Appearance
powdered herbal drug by drying in an oven at 105 °C. Brown or yellowish-brown powder.
IV-68 Aloes Preparations 2023
Solubility Top of the plate

Sparingly soluble in boiling water.
Aloe emodin: a violet
IDENilFICATION zone
-- -- --
Test solution To 0.25 g of the extract to be examined add
20 mL of methanol R and heat to boiling in a water-bath.
about 4 °C and use within 24 h.
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 Barbaloin: a brown A brown zone A brown zone
same solvent. zone (barbaloin) (barbaloin)
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel A violet zone
-- -- --
(13:17:100 VIVIV).
Application 10 µL [or 2 µL] as bands of 20 mm [or 8 mm]. Reference solution Test Test solution ( Cape
Development Over a path of 10 cm [or 6 cm]. solution (Barbados aloes)
aloes)
Drying In air.
TESTS
chromatograms obtained with the reference solution and the Loss on drying (2. 8.17)
test solution. Furthermore, other faint fluorescent zones may Maximum 4.0 per cent.
be present in the chromatogram obtained with the test Total ash (2.4.16)
solution. Maximum 2.0 per cent.
ASSAY
Top of the plate Carry out the assay protected from bright light.
Aloe emodin: a yellow Introduce 0.400 g of the extract to be examined into a
fluorescent zone 250 mL conical flask. Moisten with 2 mL of methanol R, add
5 mL of water R warmed to about 60 °C, mix, add a further
-- -- --
75 mL of water R at about 60 °C and shake for 30 min.
A blue fluorescent zone Cool, filter into a volumetric flask, rinse the conical flask and
the filter with 20 mL of water R, add the rinsings to the
volumetric flask and dilute to 1.0 L with water R. Transfer
A blue fluorescent zone 10.0 mL of this solution to a 100 mL round-bottomed flask
Barbaloin: an orange An orange fluorescent An orange fluorescent
containing 1 mL of a 600 g/L solution offerric chloride R and
fluorescent zone zone (barbaloin) zone (barbaloin) 6 mL of hydrochloric acid R. Heat in a water-bath under a
reflux condenser for 4 h, with the water 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 J M sodium hydroxide and 4 mL of water R,
A bluish-white A bluish-white
:fluorescent zone fluorescent zone
and add the rinsings to the separating funnel. Shake the
contents of the separating funnel with 3 quantities, each of
A bluish-green 20 mL, of ether R. Wash the combined ether layers with
fluorescent zone
2 quantities, each of 10 mL, of water R. Discard the
Reference solution Test Test solution (Cape washings and dilute the organic layer to 100.0 mL with
solution (Barbados aloes) ether R. Evaporate 20.0 mL carefully to dryness on a water-
aloes) bath and dissolve the residue in 10.0 mL of a 5 g/L solution
of magnesium acetate R in methanol R. Measure the
Detection B Treat with a 100 g/L solution of potassium absorbance (2.2.25) at 512 nm using methanol Ras the
hydroxide R in methanol R, heat at 110 °C for 5 min and compensation liquid.
examine in daylight. Calculate the percentage content of hydroxyanthracene
Results B See below the sequence of zones present in the derivatives, expressed as barbaloin, using the following
chromatograms obtained with the reference solution and the expression:
Ax 19.6
m
i.e. taking the specific absorbance ofbarbaloin to be 255.
A absorbance at 512 nm;

m mass of the substance to be examined, in grams.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
2023 Amomum Fruit IV-69
of calciwn oxalate; a few aggregates of small starch granules

Amomum Fruit are also present.
(Ph. Bur. monograph 2554) A. longiligulare Microscopic examination (2.8.23).
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
The powder is greyish-brown. Examine under a microscope
using chloral hydrate solutwn R. The powder shows the
DEFINITION following diagnostic characters: fragments of epicarp
Dried, whole or fragmented, peeled or unpeeled ripe fruit of consisting of brownish-orange, polyhedral or elongated cells
Amomum villosum Lour. or Amomum longiligulare T.L.Wu. containing microcrystals and prisms of calciwn oxalate,
Content clearly visible in polarised light; fragments of mesocarp
- essential oil: minimwn 30 mUkg for A. villosum composed of thin-walled polygonal cells and occasional
(anhydrous drug) and minimwn 10 mUkg for round oil cells with orange to dark brown contents; vascular
A. longiligulare (anhydrous drug); bundles composed of spiral or reticulate vessels and fibres
- bornyl acetate (C 12H 200 2 ; Mr 196.3): minimwn with thick and distinctly pitted walls; fragments of the aril
30.0 per cent of the essential oil. consisting of elongated, very thin-walled cells, some of which
contain prisms and microcrystals that are clearly visible in
IDENTIFICATION polarised light; fragments of the outer testa consisting of a
A. A. villosum. The fruit is an indehiscent capsule, ovoid or layer of cells, fusiform in surface view, with slightly and
ellipsoidal, indistinctly 3-ridged, up to 2 cm long and 1.5 cm regularly thickened walls, usually accompanied by a layer of
in diameter. The outer surface is brown and covered with rectangular or polyhedral cells with brown contents,
soft spiny protuberances. The apex bears the remains of the perpendicular to the previous layers; brownish-red fragments
perianth and the base usually bears a stalk. The pericarp is of the inner testa, composed of very thick-walled and very
thin and soft. The seeds are agglomerated into 3 masses, regularly polyhedral cells, in surface view; brownish-red
each consisting of 5-25 seeds, separated by whitish septa. fragments of the inner testa, in transverse section, composed
The seeds are hard, irregularly polyhedral, 2-3 mm in of rectangular or palisade cells with heavily thickened inner
diameter, reddish-brown or blackish-brown on the surface, and lateral walls. Examine under a microscope using a
finely wrinkled and covered with a pale brown, transparent, 50 per cent VIV solution of glycerol R. The powder shows
membranous aril; the endosperm is whitish-grey. nwnerous fragments of the endosperm with sub-rectangular
A. longiligulare The fruit is an indehiscent capsule, long, or polyhedral cells, filled with small starch granules
ovoid or ellipsoidal, distinctly 3-ridged, up to 2 cm long and aggregated into masses and free aggregates of starch granules.
1.2 cm in diameter. The outer surface is brown, C. Thin-layer chromatography (2.2.27).
longitudinally channelled and scaly, and bears nwnerous soft Test solution To 1 g of the powdered herbal drug (355)
spiny protuberances, sometimes branched. The apex bears a (2. 9.12) add 5 mL of a mixture of equal volwnes of toluene R
prominent stylopodiwn and the base shows the scar of the and xylene R. Sonicate for 10 min. Centrifuge and use the
stalk. The pericarp is thick and hard. The seeds are supernatant.
agglomerated into 3 relatively small masses, each consisting
Reference solution Dissolve 10 µL of bornyl acetate R, 10 µL
of about 2-24 seeds, separated by incomplete septa.
of cineole R and 10 mg of borneol R in 1 mL of toluene R.
The seeds are small, 1.5-2 mm in diameter, polyhedral with
rounded edges, reddish-brown or blackish-brown on the Plate TLC silica gel plate R (2-10 µm).
surface, finely wrinkled and covered with a pale brown, Mobile phase ethyl acetate R, toluene R (7:93 V/V).
transparent, membranous aril; the endosperm is whitish. Application 5 µL as bands of 8 mm.
B. A. villosum. Microscopic examination (2.8.23). Development Over a path of 6 cm.
The powder is greyish-brown. Examine under a microscope Drying In air.
using chloral hydrate solution R. The powder shows the
following diagnostic characters: fragments of epicarp Detection A Examine in ultraviolet light at 366 nm.
consisting of brownish-orange polyhedral cells containing Results A See below the sequence of zones present in the
microcrystals and prisms of calciwn oxalate, clearly visible in chromatogram obtained with the test solution. The reference
polarised light and sometimes long covering trichomes, about solution shows no spots at 366 nm. Furthermore, other faint
250 µm long, usually unicellular, straight or bent, with fluorescent zones may be present in the chromatogram
slightly and regularly thickened walls; fragments of mesocarp obtained with the test solution.
composed of thin-walled polygonal cells and round oil cells,
with orange to dark brown contents; vascular bundles Top of the plate
consisting of vessels, mainly spiral, and fibres with thick and
A faint blue fluorescent zone
pitted walls; fragments of the outer testa consisting of a layer
of cells, fusiform in surface view, with slightly and regularly -- --
thickened walls, usually accompanied by a layer of
I or 2 red fluorescent zones
rectangular or polyhedral cells, perpendicular to the previous
layers, and sometimes by underlying oil cells; brownish-red -- --
fragments of the inner testa, in surface view, composed of A red fluorescent zone
very regularly polyhedral cells with heavily thickened walls
and a punctiform lwnen; brownish-red fragments of the inner I or 2 red fluorescent zones
testa, in transverse section, composed of palisade cells with Reference solution Test solution
strongly thickened inner and lateral walls. Examine under a
The powder shows nwnerous fragments composed of sub- Detection B Treat with anisaldehyde solution R, heat at
rectangular or irregular cells, filled with aggregates of small 100-105 °C for 3 min and examine in daylight.
starch granules; some of these cells also contain small prisms
IV-70 Andrographis Herb 2023
Results B See below the sequence of zones present in the - statwnary phase: macrogol 20 000 R (film thickness
chromatograms obtained with the reference solution and the 0.25 µm).
test solution. Furthermore, other faint zones may be present Carrier gas helium for chromatography R.
Split ratw 1:50.
Top of the plate
Temperature:
A reddish-brown zone
Time Temperature
-- -- (min) ('C)
A bluish-violet zone Column 0 - 60 60 ➔ 210
Injection port 230
Bornyl acetate: a greyish-brown zone A greyish-brown zone (bomyl
acetate) Detector 250
Detection Flame ionisation.

A pink zone
Injection 1 µL.
Identification of peaks Use the chromatogram obtained with
reference solution (a) to identify the peaks due to camphor
and bomyl acetate.
1,8-Cineole: a bluish-violet zone
Relative retention With reference to camphor (retention
A greyish-brown zone time = about 26 min): bomyl acetate = about 1.1.
Borneo!: a greyish-brown zone at the A greyish-brown zone (bomeol) at System suitability Reference solution (a):
border between the middle and the border between the middle and - resolution: minimum 5 between the peaks due to camphor
lower thirds lower thirds and bomyl acetate.
-- -- Calculate the percentage content of bomyl 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.
I or 2 bluish-violet zones
TESTS
Water (2.2.13) Andrographis Herb
Maximum 120 mUkg, determined by distillation on 20.0 g
of the powdered herbal drug (355) (2.9.12). (Ph. Bur. monograph 2712)
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Total ash (2.4.16)
Maximum 9.0 per cent. DEFINITION
ASSAY Dried, whole or fragmented flowering and/or fruit-bearing
Essential oil (2.8.12) aerial parts of Andrographis paniculata (Burm.f.) Nees.
Use 10.0 g of the herbal drug reduced to a powder (1400) Content
(2. 9.12) immediately before the assay, a 500 mL round- Minimum 0.80 per cent of the sum of andrographolide
bottomed flask, 200 mL of water R as the distillation liquid (C2aH3aOs; Mr 350.4) and 14-deoxy-11,12-
and 0.5 mL of trimethylpentane R in the graduated tube. didehydroandrographolide (C 20H 280 4 ; Mr 332.4) (dried
Distil at a rate of 3-3.5 mUmin for 5 h. drug).
Bornyl acetate IDENTIFICATION
Gas chromatography (2.2.28): use the normalisation A. The whole aerial parts consist of the following: the green,
procedure. brown, or yellowish-green stems are mostly square in cross-
Test solutwn Dilute a volume of the essential oil- section with a white, central pith, about 50-70 cm long,
trimethylpentane mixture obtained in the assay of essential oil 1.2-5 mm (sometimes up to 9 mm) in diameter, frequently
corresponding to 150 µL of the essential oil in heptane R and branched, slightly swollen at the nodes, with longitudinal
dilute to 10.0 mL with the same solvent. wrinkles on the outer surface; the leaves are opposite, simple,
Reference solutwn (a) Dissolve 25 mg of camphor R in glabrous, shortly petiolate or nearly sessile with a lamina that
heptane R, add 25 µL of bornyl acetate Rand dilute to 5.0 mL is crumpled and easily broken; when whole, the lamina is
with heptane R. lanceolate to ovate-lanceolate, 2-15 cm long and 1-5 cm
Reference solutwn (b) Dilute 5 µL of bornyl acetate R to wide; the apex is acuminate, the base cuneate and decurrent,
100.0 mL with heptane R. Dilute 1.5 mL of the solution to the margin is entire or undulate, the upper surface is green to
10.0 mL with heptane R. dark almost blackish-green and the lower surface is greyish-
green or light green. Cystoliths may be visible as short,
Column:
lighter-coloured markings on the upper surface of the leaf
- material: fused silica;
and stem. The texture is fragile. The fragmented aerial parts
=
- size: l 60 m, 0 0.25 mm; = occur as small pieces of flattened green, brown, or yellowish-
2023 Andrographis Herb IV-71
Figure 2712.-1. - Illustration for identification test B of powdered herbal drug of andrographis herb
green stems, mostly square in cross-section with a white, trichomes [Ad, Be] and very rare, unicellular or multicellular
central pith, 1.2-5 mm (sometimes up to 9 mm) in diameter (2-4 cells) conical covering trichomes with a pointed or
and 0.5-5.0 cm in length, with longitudinal wrinkles on the rounded apex, thick, sometimes lignified cell walls and a
outer surface, and fragments of crumpled lamina, green to striated cuticle, up to 190 µm long and 40 µm wide at the
dark almost blackish-green on the upper surface and greyish- base (transverse section [Da]), which may be present on both
green or light green on the lower surface. Cystoliths may be epidermises; fragments of leaf mesophyll [DJ consisting of 1
visible as short, lighter-coloured markings on the upper or 2 layers of palisade parenchyma, one of which, under the
surface of the leaf and stem. The texture is fragile. Partial upper epidermis [Db], is composed of long narrow cells (up
remains of flowers are sometimes present consisting of fine to 150 µm long and 8-17 µm wide), cells containing
and delicate pedicels and sepals with a pointed apex bearing cystoliths, (surface view [Ab, Bd], transverse section [Del)
glandular hairs with a long stalk and dark head. The light and loosely arranged spongy parenchyma [Dd]; yellowish-
yellowish-brown or greenish-yellow fruits are narrow linear- brown or colourless fragments of stem with polygonal
lanceolate or oblong capsules, with 2 locules, widely opening epidermal cells, cystolithic cells, diacytic stomata and short
from the apex to form a V-shaped fruit, with 2 valves that glandular hairs; fragments of pith from the stem with
mostly no longer contain seeds; when seeds are present, they polygonal cells with pitted walls; numerous fragments of
are dark brown or brownish-yellow and sub-polyhedral to vascular tissue [E] from the stems, with reticulate or
square; funicle remains are often present. pitted [Ea] vessels and long, thick-walled and pitted
B. Microscopic examination (2.8.23). The powder is dark fibres [Eb]. If flowers are present, the powder also shows
green or greyish-green. Examine under a microscope using glandular trichomes (which may exceed 500 µm in length)
chloral hydrate solution R. The powder shows the following with a multicellular, uniseriate stalk and a multicellular cup-
diagnostic characters (Figure 2712.-1): fragments of stem shaped head [C], short glandular trichomes and fragmented
and leaf tissue with cystolithic cells, diacytic stomata, and or whole unicellular or multicellular covering trichomes [F]
short glandular trichomes. Ovoid, rounded or elongated from the outer surface of the flower (sepals) and pedicel.
cystolithic cells up to 250 µm long [leaf: Ab, Bd, De] and up If ripe fruits are present, the powder shows fragments of
to 39 µm wide, with concentric striations. Short glandular epicarp [G] consisting of elongated cells [Ga], covered by a
trichomes with a unicellular stalk and a rounded, thin striated cuticle, and diacytic stomata [Gb] and also
multicellular head, 27-37 µm in diameter, consisting of 6-8 shows groups of short, very thick-walled fibres arranged in
(sometimes 5) cells [leaf: Ad, Be]; green fragments of leaf, perpendicular layers [H], from the mesocarp. Tricolpate
fragments of the upper epidermis of the leaves (surface pollen with a fine spiculate exine may be present.
view [BJ) with rigid-walled polygonal cells [Ba], cystolithic C. Thin-layer chromatography (2.2.27).
cells [Bb] and rare diacytic stomata (2.8.3); fragments of the Test solution To 0.5 g of the powdered herbal drug (355)
lower epidermis of the leaves (surface view [Al) with slightly (2. 9.12) add 5 mL of methanol R. Sonicate for 10 min.
sinuous to very sinuous cells [Aa], cystolithic cells [Ab] and Centrifuge or filter; use the supernatant or filtrate.
numerous diacytic stomata [Ac]; rare glandular
IV-72 Andrographis Herb 2023
Reference solutwn Dissolve 1 mg of 14-deoxy-11,12- methanol R. Dilute 25.0 mL of the solution to 50.0 mL with
didehydroandrographolide R and 2 mg of andrographolide R in acetonitrile for chromatography R.
1 mL of methanol R. Column:
Plate TLC silica gel F254 plate R (2-10 µm). - size: l = 0.25 m, 0 = 4.6 mm;
Mobile phase methanol R, ethyl acetate R, methylene chloride R - statwnary phase: end-capped octadecylsilyl silica gel for
(4:30:40 VIVIV). chromatography with embedded polar groups R (5 µm);
Applicatwn 5 µL as bands of 8 mm. - temperature: 30 °C.
Development Over a path of 6 cm. Mobile phase:
- mobile phase A: water for chromatography R, adjusted to
Drying In air. pH 3.2 with anhydrous formic acid R;
Detection Treat with a 10 per cent V/V solution of sulfuric - mobile phase B: acetonitrile for chromatography R;
acid R in methanol R, heat at 100 °C for 5 min; examine in
ultraviolet light at 366 nm. Time Mobile phase A Mobile phase B
Results See below the sequence of zones present in the (min) (per cent V/JJ) (per cent V/JJ)
chromatograms obtained with the reference solution and the 0-3 90 10
test solution. Furthermore, other faint fluorescent zones may 3 - 11 90---+ 62 10--> 38
be present in the chromatogram obtained with the test 11 - 25 62 38
solution. 25 - 30 62---+ 50 38 -, 50
Top of the plate Flow rate 1.0 mUmin.

A red fluorescent zone
Injection 10 µL.
2 violet-brown fluorescent zones
Identification of peaks Use the chromatogram supplied with
-- -- andrographis herb HRS and the chromatogram obtained with
l 4-deoxy-11, 12- A pale violet fluorescent zone (I 4-
reference solution (b) to identify the peaks due to
didehydroandrographolide: a pale deoxy-11,12- 14-deoxyandrographolide and l 4-deoxy-11, 12-
violet fluorescent zone didehydroandrographolide) didehydroandrographolide.
--
Relative retention With reference to andrographolide
--
(retention time= about 16 min):
Andrographolide: a pale violet A pale violet fluorescent zone 14-deoxyandrographolide = about 1.5; 14-deoxy-11,12-
fluorescent zone (andrographolide)
didehydroandrographolide = about 1.6.
A violet-brown fluorescent zone System suitability Reference solution (b):
Reference solution Test solution - resolution: minimum 1.5 between the peaks due to
14-deoxyandrographolide and l 4-deoxy-11,12-
didehydroandrographolide.
TESTS Calculate the percentage content of the sum of
Loss on drying (2.2.32) andrographolide and 14-deoxy-11,12-
Maximum 10.0 per cent, determined on 1.000 g of the didehydroandrographolide using the following expression:
105 °C for 2 h. (A1 + (A 3 x 1.45)) x m2 xp
Total ash (2.4.16) A2 xm1 x5
area of the peak due to andrographolide in the chromatogram
ASSAY obtained with the test solution;
Liquid chromatography (2.2.29). area of the peak due to andrographolide in the chromatogram
obtained with reference solution (a);
Test solutwn Disperse 2.000 g of the powdered herbal drug area of the peak due to I 4-deoxy-11, I 2-
(355) (2.9.12) in 50 mL of methanol R. Heat on a water-bath didehydroandrographolide in the chromatogram obtained with
under a reflux condenser for 15 min. Cool. Collect the the test solution;
mass of the herbal drug to be examined used to prepare the test
methanolic extract. Repeat the extraction with 3 additional
solution, in grams;
quantities, each of 50 mL, of methanol R. Combine the mass of andrographolide CRS used to prepare reference
extracts. Filter. Evaporate under vacuum. Dilute to 50.0 mL solution (a), in grams;
with methanol R. Dilute 25.0 mL of the solution to 50.0 mL p percentage content of andrographolide in andrographolide CRS;
1.45 peak correlation factor between andrographolide and 14-deoxy-
with acetonitrile for chromatography R.
l I, l 2-didehydroandrographolide.
Reference solutwn (a) Dissolve 5.0 mg of
andrographolide CRS in methanol Rand dilute to 10.0 mL - - - - - - - - - - - - - - - - - - - - - - PhEur
with the same solvent. Dilute 1.0 mL of the solution to
50.0 mL with acetonitrile for chromatography R.
Reference solutwn (b) Disperse 2 g of andrographis herb HRS
in 50 mL of methanol R. Heat on a water-bath under a reflux
condenser for 15 min. Cool. Collect the methanolic extract.
Repeat the extraction with 3 additional quantities, each of
50 mL, of methanol R. Combine the extracts. Filter.
Evaporate under vacuum. Dilute to 50.0 mL with
2023 Anemarrhena Asphodeloides Rhizome IV-73
Anemarrhena Asphodeloides
Rhizome
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried, whole or fragmented, peeled or unpeeled, rhizome of
Anemarrhena asphodeloicks Bunge, with roots removed,
collected in spring or autumn.
Content
Minimum 0.5 per cent ofmangiferin (C 19H 18 0 11 ; Mr 422.3)
(dried drug).
IDENTIFICATION
A. The rhizome with cork intact (called maozhimu) is
somewhat flattened, slightly curved, occasionally branched,
2-15 cm long and 0.8-2 cm in diameter. The outer surface is
brownish-yellow, reddish-brown or brown, with the upper
surface generally showing a concave groove. It consists of
many annular nodes with dense brownish-yellow fibrous
remains of the vasculature of the leaf bases growing upwards
on both sides of the groove; at the branched end of the
rhizome, whitish-yellow or light brown leaf blade bases may
also be present; the lower surface is coarsely longitudinally
wrinkled, showing depressed or protruding dotted root scars;
the surface of the root scars is whitish-yellow or light brown
and may show a circular ring of xylem tissue protruding from
its centre. The rhizome with outer cork removed (called
guangzhimu) is 5-15 cm in diameter. The outer surface is
yellowish-white, with twisted grooves, sometimes with leaf Figure 2661.-1. - Illustratwn for identification test B of powdered
scars and root scars. herbal drug of Anemarrhena asphockloides rhizome
The fragmented rhizome occurs in slices about 1.1-4 mm
thick and 0.8-3.4 cm in diameter, often with a furrowed
(2. 9.12) add 5 mL of a mixture of glacial acetic acid R and
outline. It can be cut either longitudinally or transversely or
methanol R (10:90 V/V). Sonicate for 10 min. Centrifuge or
obliquely. The outer surface is yellowish-white (guangzhimu)
filter; use the supernatant or filtrate.
or brownish-yellow, reddish-brown or brown (maozhimu);
it shows fibrous (vascular bundles) remains of leaves as well Reference solutwn Dissolve 1 mg of arbutin R and 1 mg of
as annular striations and round root scars or short remnants aescin R in 1 mL of methanol R.
of the root bases. The section is pale yellow or brownish- Plate TLC silica gel plate R (2-10 µm).
yellow, marked with numerous whitish-yellow pits or lines Mobile phase water R, methanol R, methylene chloride R
corresponding to vascular bundles. (4:30:70 V/V/V).
B. Microscopic examination (2.8.23). The powder is pale Application 5 µL as bands of 8 mm.
yellow. Examine under a microscope using chloral hydrate Development Over a path of 6 cm.
solution R. The powder shows the following diagnostic
Drying In air.
characters (Figure 2661.-1): very numerous, whole or
fragmented raphides of calcium oxalate up to 150 µm long, Detection Treat with anisaldehyck solutwn R and heat at
free [DJ or included in oval or rounded parenchyma cells of 100 °C for 3 min; examine in ultraviolet light at 365 nm.
various sizes [A, E]; fragments of parenchyma, with thin Results See below the sequence of zones present in the
walls [BJ or with slightly thickened and pitted walls [HJ; chromatograms obtained with the reference solution and the
fragments of vessels [F] sometimes spiral [Fa], more often test solution. Furthermore, other faint zones may be present
reticulate [Fb] or pitted [Fe], often accompanied by cells in the chromatogram obtained with the test solution.
containing large acicular crystals of calcium oxalate, in
bundles, whole or broken, up to 300 µm long and 7 µm wide
[Fd]; free bundles of large acicular crystals may also be
observed W; a few fragments of cork, orange-brown, present
only in the unpeeled herbal drug [K]; a few fragments of
parenchyma with elongated or polygonal cells with heavily
lignified and pitted walls from the remains of the aerial parts
[C]; a few fibres in bundles, with slightly thickened walls and
rounded ends, from the remains of the vascular bundles of
the leaves [G].
IV- 7 4 Anethum Graveolens Sowa Fruit 2023
Top of the plate chromatogram obtained with reference solution (b) to

identify the peak due to isomangiferin.
A violet zone
Retention time Mangiferin = about 4.0 min;
A brown zone isomangiferin = about 4.9 min.
A green zone System suitability Reference solution (b):
-- -- mangiferin and isomangiferin.
A brown zone Calculate the percentage content of mangiferin using the
Aescin: a brownish zone
following expression:
A pale green zone A 1 x m2 xpx0.25

A2 xm1
A pale green zone AI area of the peak due to mangiferin in the chromatogram
obtained with the test solution;
-- -- A2 area of the peak due to mangiferin in the chromatogram
Arbutin: a greenish zone A brown zone m1 mass of the herbal drug to be examined used to prepare the test
solution, in grams;
A brownish-green zone
m2 mass of mangiferin CRS used to prepare reference solution (a),
A brown zone in grams;
p percentage content of mangiferin in mangiferin CRS.
A very broad light blue zone
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
TESTS
Loss on drying (2.2.32) Anethum Graveolens Sowa Fruit
DEFINITION
Anethum Graveolens Sowa Fruit is the dried ripe fruit of
105 °C for 2 h.
Anethum graveolens L. Sowa Group.
Total ash (2.4.16)
Content
It contains not less than 3.0% v/w of essential oil calculated
Ash insoluble in hydrochloric acid (2.8.1) with reference to the anhydrous drug.
IDENTIFICATION
ASSAY A. The dried fruits usually occur as separate mericarps,
Liquid chromatography (2.2.29). pedicels normally absent; broadly oval, highly compressed
Test solution Disperse 0.100 g of the powdered herbal drug dorsally, about 4 mm long, 2 to 3 mm broad, with 5 dorsal
(355) (2.9.12) in 25.0 mL of a 50 per cent V/V solution of ridges, each mericarp exhibiting 3 pale brown dorsal ridges,
methanol R, weigh. Sonicate for 30 min. Allow to cool and the two lateral ridges elongated into characteristic
weigh again. Compensate for the loss of solvent with a membranous wings; surface glabrous; remnants of the
50 per cent VIV solution of methanol R, mix and filter stylopod at the apex; commissural surface flat, often with
through a membrane filter (nominal pore size 0.45 µm). attached, paler brown carpophore; vittae visible as two
Reference solution (a) Dissolve 5.0 mg of mangiferin CRS in darker, arc-shaped, longitudinal bands.
a 50 per cent V/V solution of methanol R and dilute to B. Reduce to a powder, Appendix XVII A. The powder is
100.0 mL with the same solution. pale brown. Examine under a microscope using chloral
Reference solution (b) Disperse 0.1 g of Anemarrhena rhizome hydrate solution. The powder contains numerous fragments of
for system suitability HRS in 25.0 mL of a 50 per cent V/V the epicarp, with cuticular striations and infrequent
solution of methanol R, weigh. Sonicate for 30 min. Allow to anomocytic stomata; parquetry layer of endocarp in surface
cool and weigh again. Compensate for the loss of solvent view; endosperm of oval to rectangular thick-walled cells
with a 50 per cent V/V solution of methanol R, mix and filter containing oil globules and aleurone grains with embedded
through a membrane filter (nominal pore size 0.45 µm). microrosette crystals of calcium oxalate; fragments of
Column: yellowish-brown septate vittae; parenchyma of mesocarp
- size: l =0.15 m, 0 = 4.6 mm; consisting of elongated, lignified, reticulately thickened cells;
- stationary phase: end-capped octadecylsilyl silica gel for sclereids of mesocarp thick-walled with few pits.
chromatography R (5 µm). C. Carry out the method for thin-layer chromatography,
Mobile phase acetonitrile R, 0.2 per cent V/V solution of Appendix III A, using the following solutions.
glacial acetic acid R (15:85 V/V). (1) Add 10 mL of methanol (70%) to 0.5 g of the powdered
Flow rate 1.0 mLJmin. drug (355), mix and place in an ultrasonic bath for
30 minutes. Filter (a 0.45-µm PTFE is suitable) into a
Detection Spectrophotometer at 258 nm. 10 mL volumetric flask and dilute to 10 mL with methanol
Injection 10 µL. (70%).
Run time 3 times the retention time of mangiferin. (2) 0.05% w/v each of carvone and dillapiole in methanol
Identification of peaks Use the chromatogram supplied with (70%).
Anemarrhena rhizome for system suitability HRS and the
2023 Anethum Graveolens Sowa Fruit IV-75
CHROMATOGRAPHIC CONDITIONS Time (Minutes) Temperature Comment

(a) Use high-performance silica gel 60 F254 plates (Merck 0-5 60" isothermal
silica gel 60 F 254 HPTLC plates are suitable). 5-68 60'-+250° linear gradient
68-75 250" isothermal
(b) Use the mobile phase as described below.
(c) Apply 10 µL of each solution as 6 mm bands.
(d) Develop the plate to 8 cm. SYSTEM SUITABILITY
(e) After removal of the plate, dry in air, spray with vanillin The test is not valid unless, in the chromatogram obtained
reagent, heat the plate at 110° until the coloured bands with solution (4), the resolution factor between the peaks due
appear and examine in daylight. to B-myrcene and limonene, is at least 4.5.
In the chromatogram obtained with solution (4), the
MOBILE PHASE
substances elute in the following order: B-myrcene and
2 volumes of acetic acid, 10 volumes of ethyl acetate and limonene.
88 volumes of toluene.
The signal-to-noise ratio of the peak due to apiole in solution
SYSTEM SUIT ABILITY (3) is at least 3.
The test is not valid unless the chromatogram obtained with LIMITS
solution (2) shows two clearly separated bands.
In the chromatogram obtained with solution (1) the area of
CONFIRMATION any peak due to apiole is not more than the area of the peak
The chromatogram obtained with solution (1) shows a due to apiole in the chromatogram obtained with
purple band corresponding in position and colour to the solution (3).
band due to carvone in the chromatogram obtained with Water
solution (2); a brown band corresponding in position and Not more than 10.0% v/w, Appendix IX C, method II using
colour to the band due to dillapiole and other bands as 30 g of powdered drug, Appendix XVII A,.
shown in the table. Other bands may be present.
Total Ash
Top of the plate
Carry out the method for gas chromatography,
A faint brown band Appendix III B, using the following solutions.
( 1) Use the oil retained in the Assay of Essential oil.
(2) 0.4% w/v each of limonene, dihydrocarvone, carvone and
A brown band (dillapiole) A brown band (dillapiole) dillapiole in toluene.
A purple band (carvone) A purple band (carvone) (3) 0.05% v/v of /3-myrcene and 0.8% v/v of limonene in
toluene.
CHROMATOGRAPHIC CONDITIONS
A grey band
The chromatographic procedure described under the test for
Apiole may be used.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained
with solution (3), the resolution between the peaks due to
B-myrcene and limonene is at least 4.5.
Solution (1) Solution (2) In the chromatogram obtained with solution (2), the
substances elute in the following order: limonene,
dihydrocarvone isomer 1, dihydrocarvone isomer 2, carvone
TESTS and dillapiole.
Apiole In the chromatogram obtained with solution (3), the
Carry out the method for gas chromatography, substances elute in the following order: B-myrcene and
Appendix III B, using the following solutions. limonene.
(1) Use the oil retained in the Assay of Essential oil.
LIMITS
(2) 1.0% w/v of apiole in toluene. Calculate the content of limonene, dihydrocarvone, carvone
(3) 0.05% w/v of apiole in toluene. and dillapiole by normalisation. Disregard the peak due to
(4) 0.05% v/v of /3-myrcene and 0.8% v/v of limonene in toluene.
toluene. Limits:
CHROMATOGRAPHIC CONDITIONS - limonene: 15.0 to 28.0%,
(a) Use a fused silica capillary column (30 m x 0.53 mm) - sum of dihydrocarvone isomers 1 and 2: 5.0 to 30.0%,
bonded with a 1 µm film thickness, polyethylene glycol 20,000 - carvone: 20.0 to 45.0%,
(DB-Wax is suitable). - dillapiole: 15.0 to 35.0%.
(b) Use helium as the carrier gas at 1.5 mL per minute. Disregard any peak with an area less than 0.025 times the
area of the peak due to carvone in the chromatogram
(c) Use the gradient conditions described in the table.
obtained with solution (2).
(d) Use an inlet temperature of 2500.
(e) Use a flame ionisation detector at a temperature of 260°.
(e) Inject 1 µL of each solution.
IV- 7 6 Angelica Archangelica Root 2023
ASSAY
Essential oil
Carry out the method for Essential Oils in Herbal Drngs,
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.
ANNEX
This section is non-mandatory.
DNA reference sequence
A DNA reference sequence for the identity of Anethum
Graveolens Sowa Fruit is published in Supplementary Chapter
VII D.
Angelica Archangelica Root

Ph Eur - - - - - - - - - - - - - - - - - - - - - -
DEFINITION
Whole or cut, carefully dried rhizome and root of Angelica
archangelica L. (syn. A. officinalis Hoffi:n.). >.·.~
~'. .·.·~
f_;,..... .:.<
Content -~~_:_,__·
F
Minimum 2.0 mL'kg of essential oil (dried drug).
CHARACTERS
Bitter taste. Figure 1857 .-1. - Illustration for identification test B of powdered
herbal drng of angelica archangelica root
IDENTIFICATION
A. The rhizome is greyish-brown or reddish-brown, with Results A See below the sequence of zones present in the
transversely annulated thickenings. The base bears greyish- chromatograms obtained with the reference solution and the
brown or reddish-brown, cylindrical, longitudinally furrowed, test solution. Furthermore, other faint fluorescent zones may
occasionally branched roots often with incompletely be present in the chromatogram obtained with the test
encircling, transverse ridges. The apex sometimes shows solution.
remnants of stem and leaf bases. The fracture is uneven.
The transversely cut surface shows a greyish-white, spongy,
Top of the plate
distinctly radiate bark, in which the secretary channels are
visible as brown spots, and a bright yellow or greyish-yellow (Z)-Ligustilide: a bluish-white
wood which, in the rhizome, surrounds the greyish or fluorescent zone
brownish-white pith. -- --
B. Microscopic examination (2.8.23). The powder is
Osthole: a blue fluorescent zone A blue fluorescent zone
brownish-white. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic lmperatorin: a whitish fluorescent A whitish fluorescent zone
characters (Figure 1857.-1): fragments of cork consisting of zone
several layers of thin-walled, greyish-brown or reddish-brown A blue fluorescent zone

cells (surface view [C], transverse section [El); large,
yellowish-brown secretory channels, whole or fragmented -- --
(transverse section [A], longitudinal section [F]); fragments 3 blue fluorescent zones
of medullary rays, 2 or 4 cells wide [GJ; fragments of xylem
[BJ consisting of lignified vessels with reticulate thickening Reference solution Test solution
[Ba] occurring singly or in small groups, and unlignified
parenchyma in which some of the cells associated with the Results B See below the sequence of zones present in the
vessels are collenchymatously thickened. Examine under a chromatograms obtained with the reference solution and the
microscope using a 50 per cent V/V solution of glycerol R. test solution. Furthermore, other faint quenching zones may
The powder shows numerous, simple starch granules 2-4 µm be present in the chromatogram obtained with the test
in diameter, free or included in parenchyma cells [DJ. solution.
C. Examine the chromatograms obtained in the test for other
species of Angelica, Levisticum and Ligusticum described in the
European Pharmacopoeia.
2023 Angelica Dahurica Root IV-77
Top of the plate

Angelica Dahurica Root
(Z)-Ligustilide: a blue fluorescent
zone (Ph. Bur. monograph 2556)
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
-- - -
Ostbole: a quenching zone A quenching zone DEFINITION

Dried, whole or fragmented root, with rootlets removed, of
lmperatorin: a quenching zone A quenching zone
Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. &
A quenching zone Sav. collected in summer or autumn.
Content
-- - -
Minimum 0.08 per cent ofimperatorin (C 16H 1404;

Several quenching zones
Mr 270.3) (dried drug).
Reference solution Test solution IDENTIFICATION
A. The non-fragmented drug consists of conical roots, about
TESTS 10-25 cm long and 1.2-3.1 cm in diameter. The root crown
is sub-quadrangular and obtuse and shows stem scars on
Other species of Angelica, Levisticum and Ligusticum
prominences. It tapers to the tip. The outer surface is
described in the European Pharmacopoeia
brownish-grey or yellowish-brown and clearly striated
longitudinally, showing scars of the rootlets and lenticel-like
Test solution To 1 g of the freshly powdered herbal drug transverse protuberances, some of them arranged in 4
(35 5) (2. 9.12) add 4 mL of heptane R, close and sonicate for longitudinal rows. The texture is compact, hard and heavy.
5 min. Centrifuge the mixture and use the supernatant.
The fracture is white or whitish-grey and mealy.
Reference solution Dissolve 1 mg of imperatorin R, l mg of The cambium occurs as a fine-lined, distinct brown ring; it is
(Z)-ligustilide Rand 1 mg of osthole R in 10 mL of subrounded, subsquare or subrectangular. Numerous brown
methanol R. to yellowish dots, corresponding to secretory canals, are
Plate TLC silica gel F 254 plate R (2-10 µm). visible in the cortex.
Mobile phase glacial acetic acid R, ethyl acetate R, toluene R The fragmented drug occurs as subrounded or subsquare
(1:10:90 VIVIV). slices with an irregular margin, 1.2-3.1 cm in diameter and
Application 4 µL as bands of 8 mm. 0.1-0.4 cm thick. The cut surface is white, yellowish-white or
greyish-white and mealy. The cambium occurs as a fine-
Development Over a path of 6 cm.
lined, distinct brown ring; it is subrounded, subsquare or
Drying In air. subrectangular. Numerous brown to yellowish dots,
Detection A Examine in ultraviolet light at 365 nm. corresponding to secretory canals, are visible in the cortex.
Results A The chromatogram obtained with the test solution
shows no zone at the position of (Z)-ligustilide in the
chromatogram obtained with the reference solution.
Detection B Examine in ultraviolet light at 254 nm.
Results B The chromatogram obtained with the test solution
shows no zone at or just below the position of (Z)-ligustilide
in the chromatogram obtained with the reference solution.
Maximum 5 per cent of leaf bases and stem bases, maximum
5 per cent of discoloured pieces and maximum 1 per cent of
other foreign matter.
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Essential oil (2. 8.12)
Reduce the herbal drug to a powder (500) (2. 9.12) and
immediately use 40.0 g for the determination. Use a 2 L
round-bottomed flask, 10 drops of liquid paraffin R, 500 mL
of water R as distillation liquid and 0.50 mL of xylene R in
the graduated tube. Distil at a rate of 2-3 mUmin for 4 h.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Figure 2556.-1. - Illustration for identification test B of powdered

herbal drug of Angelica dahurica root
IV-78 Angelica Dahurica Root 2023
B. Microscopic examination (2.8.23). The powder is test solution. Furthermore, other faint zones may be present
yellowish-white. Examine under a microscope using chloral in the chromatogram obtained with the test solution.
hydrate solution R. The powder shows the following diagnostic
characters (Figure 2556.-1): reticulate vessels [B, G], free or Top of the plate
in groups of 2 or 3 [G], and accompanied by xylem
parenchyma cells with thin cellulose walls [Ba]; numerous 2 prominent reddish zones
fragments of parenchyma with ovoid cells [D]; a few orange (Z)-Ligustilide: a grey zone
cork fragments, consisting of several layers of superimposed
cells (surface view [Al); secretory canals (longitudinal section --- ---
[C], transverse section [F]), usually broken, with yellow or A faint blue zone
pale brown contents and droplets of essential oil. Examine
Osthole: a violet zone
under a microscope using a 50 per cent VIV solution of
glycerol R. The powder shows very numerous starch granules lmperatorin: a reddish-grey zone A yellow and violet double-zone
varying in size from 5 µm to 25 µm, simple or 2-8
compound; most are polyhedral, either due to compound --- ---
granules breaking up or to compression in the cells; they are A prominent violet zone
either isolated [E] or included in parenchyma cells [Ea].
A yellow zone
officinal species of Angelica, Levisticum and Ligusticum. Reference solution Test solution

chromatograms obtained with the reference solution and the TESTS
test solution. Furthermore, other faint fluorescent zones may Other officinal species of Angelica, Levisticum and
be present in the chromatogram obtained with the test Ligusticum
solution. Thin-layer chromatography (2.2.27).
Test solution To 1 g of the powdered herbal drug (355)
Top of the plate
(2. 9.12) add 4 mL of heptane R, close and sonicate for 5 min.
(Z)-Ligustilide: a bluish-white Centrifuge the mixture and use the supernatant.
fluorescent zone Reference solution Dissolve 1 mg of imperatorin R, 1 mg of
--- ---
(Z)-ligustilide Rand 1 mg of osthole R in 10 mL of
methanol R.
A whitish fluorescent zone
Plate TLC silica gel F254 plate R (2-10 µm).
Osthole: a blue fluorescent zone A blue fluorescent zone Mobile phase glacial acetic acid R, ethyl acetate R, toluene R
lmperatorin: a whitish fluorescent A whitish fluorescent zone (1:10:90 VIVIV).
zone (imperatorin) Application 4 µL as bands of 8 mm.
--- --
A whitish fluorescent zone
Drying In air.
shows no bluish-white fluorescent zone corresponding to the
Results B See below the sequence of zones present in the zone due to (Z)-ligustilide in the chromatogram obtained
chromatograms obtained with the reference solution and the with the reference solution.
test solution. Furthermore, other faint quenching zones may Detection B Examine in ultraviolet light at 254 nm.
solution, in particular in the lower third of the
shows no blue fluorescent zone corresponding to the zone
chromatogram, below the zone due to imperatorin.
due to (Z)-ligustilide in the chromatogram obtained with the
reference solution; the chromatogram obtained with the test
Top of the plate solution shows no quenching zone corresponding to the zone
(Z)-Ligustilide: a blue fluorescent due to osthole in the chromatogram obtained with the
zone reference solution.
Detection C Treat with a 10 per cent V/V solution of sulfuric
-- ---
acid R in methanol R, heat at 100 °C for 5 ruin and examine
A quenching zone in daylight.
Osthole: a quenching zone Results C The chromatogram obtained with the test solution
shows no violet zone corresponding to the zone due to
Imperatorin: a quenching zone A quenching zone (imperatorin)
osthole in the chromatogram obtained with the reference
--- -- solution.
Reference solution Test solution powdered herbal drug (355) (2. 9.12) by drying in an oven at
105 °C for 2 h.
Results C See below the sequence of zones present in the Total ash (2.4.16)
chromatograms obtained with the reference solution and the Maximum 6.0 per cent.
2023 Angelica Pubescens Root IV- 79

Angelica Pubescens Root
ASSAY (Ph. Bur. monograph 2557)
Liquid chromatography (2. 2. 29). Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Test solution Disperse 0.400 g of the powdered herbal drug
DEFINITION
(355) (2. 9.12) in 45 mL of methanol R and sonicate for I h.
Cool and dilute to 50.0 mL with methanol R. Filter through a Whole or fragmented dried root, without rootlets, of Angelica
membrane filter (nominal pore size 0.45 µm). biserrata (RH.Shan & C.Q.Yuan) C.Q.Yuan & RH.Shan
(syn. Angelica pubescens Maxim. f. biserrata RH.Shan et C.Q.
Reference solution (a) Dissolve 5.0 mg of imperatorin CRS in Yuan), collected in early spring before sprouting, or in the
methanol R and dilute to 50.0 mL with the same solvent. end of autumn when stem and leaves wither.
Content
Reference solution (b) Disperse 80 mg of Angelica dahurica
Minimum 0.50 per cent of osthole (C 15 H 16 0 3; Mr 244.3)
root HRS in 9 mL of methanol R and sonicate for 1 h. Cool
(dried drug).
and dilute to 10 mL with methanol R. Filter through a
membrane filter (nominal pore size 0.45 µm). IDENTIFICATION
Pre column: A. The taproot is more or less cylindrical, branching rapidly
- size: l = 4 mm, 0 = 4.0 mm; into 2-3 or more principal roots at the lower part; the whole
- statwnary phase: octadecylsilyl silica gel for chromatography R is about 5-30 cm long. The root crown is enlarged, with
(5 µm). transverse, annulated wrinkles and measures about
0.5-4.5 cm in diameter; it shows the remains of stems, leaves
Column:
- size: l = 0.125 m, 0 = 4.0 mm; or buds. The greyish-brown or dark brown outer surface is
longitudinally wrinkled and shows slightly prominent rootlet
- stationary phase: octadecylsilyl silica gel for chromatography R
(5 µm).
scars and transverse lenticel-like protuberances. The fracture
shows greyish-yellow bark, with abundant distinct brown dots
Mobile phase: due to secretory canals that may be radially arranged; the
- mobile phase A: water for chromatography R; cambium ring is brown and the wood is greyish-yellow or
- mobi7e phase B: acetonitrile Rl; yellowish-brown; fine rod-like crystals may be present on the
fractured surface.
(min) (per cent V/J/) (per cent VIJ!) The fragmented drug occurs as subrounded or irregular
0 - 15 45 55
slices, 0.4-3.9 cm in diameter and 0.2-0.6 cm thick.
15 - 33 45 ➔ 5 55 ➔ 95
Externally it is greyish-brown or brown, with wrinkles. Fine
33 - 35 5 95
rod-like crystals may be present on the cut surface. The bark
is greyish-white or greyish-brown, with abundant, distinct
brown or yellowish-brown dots due to secretory canals that
Flow rate 1.0 mUmin. may be radially arranged. The cambium ring is brown.
Detection Spectrophotometer at 210 nm. B. Microscopic examination (2.8.23). The powder is
Injection 20 µL. yellowish-brown or brown. Examine under a microscope
Identification of peaks Use the chromatogram supplied with using chloral hydrate solution R. The powder shows the
Angelica dahurica root HRS and the chromatogram obtained following diagnostic characters (Figure 2557.-1): fragments of
with reference solution (b) to identify the peak due to vessels up to 90 µm in diameter with spiral or reticulate
phellopterin. thickenings, free or in groups of 2 or 3 [El; fragments of
Relative retention With reference to imperatorin (retention parenchyma with polyhedral cells [BJ; fragments of phloem
time= about 5 min): phellopterin = about 1.1. parenchyma [C] with fine, sinuous, fusiform cells, about
7-38 µm in diameter, with slightly thickened walls and fine,
System suitability Reference solution (b):
oblique criss-cross striations [Ca], sometimes accompanied
by medullary rays [Cb]; orange-brown cork fragments,
imperatorin and phellopterin.
consisting of several layers of superimposed, somewhat
Calculate the percentage content of imperatorin using the polyhedral cells (surface view [Al); secretory canals, usually
following expression: broken, with yellow or pale brown contents and droplets of
essential oil (transverse section [D], longitudinal section [F]).
Examine under a microscope using a 50 per cent VIV
solution of glycerol R. The powder shows numerous small,
AI area of the peak due to imperatorin in the chromatogram
rounded or ovoid, simple starch granules, up to 10 µm in
obtained with the test solution; diameter, with a punctiform hilum that is visible on the
A2 area of the peak due to imperatorin in the chromatogram largest granules; a few starch granules consisting of 2-10
obtained with reference solution (a); components are also present [G].
m1 mass of the herbal drug to be examined used to prepare the test
solution~ in grams; C. Examine the chromatograms obtained in the test for other
m2 mass of imperarorin CRS used to prepare reference solution (a), officinal species of Angelica, Levisticum and Ligusticum.
in grams;
p percentage content of imperatorin in imperaton·n CRS.
test solution. Furthermore, other faint fluorescent zones may
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
solution.
IV-80 Angelica Pubescens Root 2023
Top of the plate
(Z)-Ligustilide: a blue fluorescent A faint quenching zone

zone
--- ---
Ostbole: a quenching zone A quenching zone (ostbole)
Imperatorin: a quenching zone A quenching zone (may be missing)
--- ---
2 or 3 quenching zones
Results C See below the sequence of zones present in the

Top of the plate
A prominent reddish zone
(Z)-Ligustilide: a grey zone
--- ---
Ostbole: a violet zone A violet zone ( ostbole)
lmperatorin: a grey zone A violet zone (may be missing)
--- ---
Figure 2557 .-1. - Illustration for identification test B of powdered A prominent violet zone
herbal drug of Angelica pubescens root
A yellow zone

Top of the plate
(Z)-Ligustilide: a bluish-white A bluish-white fluorescent zone

fluorescent zone TESTS
Other officinal species of Angelica, Levisticum and
--- --- Ligusticum
A very faint whitish zone Thin-layer chromatography (2.2.27).
Ostbole: a blue fluorescent zone A prominent blue fluorescent zone
(ostbole) (2. 9.12) add 4 mL of heptane R, close and sonicate for 5 min.
A whitish fluorescent zone (may be
Centrifuge the mixture and use the supernatant.
Imperatorin: a whitish fluorescent
zone missing) Reference solution Dissolve 1 mg of imperatorin R, 1 mg of
(Z)-ligustilide Rand 1 mg of osthole R in 10 mL of
--- ---
methanol R.
A blue fluorescent zone Plate TLC silica gel F254 plate R (2-10 µm).
3 blue fluorescent zones Mobile phase glacial acetic acid R, ethyl acetate R, toluene R
(1:10:90 VIVIV).
Application 4 µL as bands of 8 mm.
Results B See below the sequence of zones present in the
chromatograms obtained with the reference solution and the Drying In air.
test solution. Furthermore, other faint quenching zones may Detection A Examine in ultraviolet light at 365 nm.
be present in the chromatogram obtained with the test Results A The chromatogram obtained with the test solution
solution. shows no intense whitish fluorescent zone directly above the
position of osthole and no blue fluorescent zone just below
the position of imperatorin in the chromatogram obtained
with the reference solution.
shows no blue fluorescent zone corresponding to the zone
due to (Z)-ligustilide in the chromatogram obtained with the
reference solution.
2023 Angelica Sinensis Root IV-81
Detection C Treat with a 10 per cent VIV solution of suljuric

acid R in metharwl R, heat at 100 °C for 5 min and examine
Angelica Sinensis Root
in daylight. DEFINITION
Results C The chromatogram obtained with the test solution Angelica Sinensis Root is the dried whole root of Angelica
shows no zone corresponding to the zone due to sinensis (Oliv.) Diels. (Angelica polymorpha Maxim. var.
(Z)-ligustilide in the chromatogram obtained with the sinensis Oliv.).The dried root consists of the top (uppermost
reference solution. part), main body and small lateral roots (tails).
Loss on drying (2.2.32) It is collected in late autumn, removed from rootlets and
Maximum 10.0 per cent, determined on 1.000 g of the dried.
powdered herbal drug (355) (2.9.12) by drying in an oven at It contains not less than 0.1 % of Z-ligustilide (C 12H 14 O 2),
105 °c for 2 h. calculated with reference to the dried material.
Total ash (2.4.16) IDENTIFICATION
Maximum 8.0 per cent. A. The whole root is yellowish-brown to brown, up to 25 cm
long, irregularly cylindrical with 3 to 5 or more branch roots
arising from the lower end. The upper part is 1.5 to 5 cm in
diameter, annulated on the surface and rounded at the apex
ASSAY which may show purple or yellowish-green remains of stems
Liquid chromatography (2.2.29). and leaves; the surface of the remainder of the main root is
Test solution Disperse 0.500 g of the powdered herbal drug strongly longitudinally wrinkled and has pale, transverse
(355) (2.9.12) in 18 mL of metharwl Rand sonicate for lenticels; the branch roots are 0.3 to 1 cm in diameter in the
30 min. Cool and dilute to 20.0 mL with methanol R. upper part, twisted and tapering towards the base, the outer
Mix and filter. Dilute 5.0 mL of the filtrate to 20.0 mL with surface is strongly striated and has few rootlet scars.
methanol R. B. Reduce to a powder (355). The powder is pale yellowish
Rejl,rence solution (a) Dissolve 5.0 mg of osthole CRS in to buff. Examine under a microscope using chloral hydrate
methanol Rand dilute to 100.0 mL with the same solvent. solution. The powder shows brown fragments of cork
Reference solution (b) Disperse 0.250 g of Angelica pubescens composed of thin-walled cells; abundant thin-walled
root HRS in 9 mL of methanol R and sonicate for 30 min. parenchyma from the secondary cortex, phloem and
Cool and dilute to 10.0 mL with metharwl R. Mix and filter. medullary rays, some of the phloem cells fusiform with
Dilute 5.0 mL of the filtrate to 20.0 mL with methanol R. slightly thickened walls; lignified vessels in groups of 2 or 3
associated with small celled and pined xylem parenchyma;
Column:
the vessels are up to 80 µm in diameter and have reticulate
- size: l = 0.125 m, 0 = 2.0 mm;
or scalariform thickening. Examine under a microscope using
50% v/v of glycerol. The powder shows small groups of single
(4 µm).
starch granules, spherical to ovoid, up to about 8 µm in
Mobile phase water for chromatography R, acetonitrile R diameter.
(40:60 V/V). C. Carry out the method for thin-layer chromatography,
Flow rate 0.23 mL'min. Appendix III A, using the following solutions.
Detection Spectrophotometer at 322 nm. (1) Add 4 mL of heptane to 1.0 g of the powdered drug, mix
Injection 10 µL. with the aid of ultrasound for 5 minutes and filter (use a
Retention time Osthole = about 8 min. 0.22 µm membrane filter).
System suitability Reference solution (b): (2) 0.1 % w/v of linoleic acid in metharwl.
- resolution: minimum 1.5 between the peak due to osthole (3) 0.1 % w/v of ferulic acid in metharwl.
and peak 2; use the chromatogram supplied with Angelica (4) 0.1 % w/v of Z-ligustilide CRS in methanol.
pubescens root HRS to identify peak 2. CHROMATOGRAPHIC CONDITIONS
Calculate the percentage content of osthole using the (a) Use a silica gel F254 precoated plate (Merck silica gel 60
following expression: F2s4 HPTLC plates are suitable).
A 1 xm2 xpx0.8 (b) Use the mobile phase as described below.
A2 xm1 (c) Apply 10 µL of solution (1) and 5 µL of solutions (2) to
(4), as bands.
area of the peak due to osthole in the chromatogram obtained (d) Develop the plate to 15 cm.
area of the peak due to osthole in the chromatogram obtained (e) Remove the plate, allow to dry in a stream of warm air
with reference solution (a); for 5 minutes or until the solvents are completely removed.
mass of the herbal drug to be examined used to prepare the test Examine under ultraviolet light (254 nm). Spray the plate with
solution) in grams;
mass of osthole CRS used to prepare reference solution (a), in
metharwlic sulfuric acid (5%), heat at 105° for 3 minutes and
grams; examine in daylight.
p percentage content of osthole in ostho/e CRS. MOBILE PHASE
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
1 volume of formic acid, 10 volumes of ethyl acetate and
SYSTEM SUITABILITY
When examined under ultraviolet light (254 nm) the violet
band with an Rf value of approximately 0.7 the
chromatogram obtained with solution (4) corresponds in
colour and position to that in the chromatogram obtained
IV-82 Angelica Sinensis Root 2023
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 ASSAY
obtained with solution (1). Other bands may be present in Carry out the method for liquid chromatography,
the chromatogram obtained with solution (1). Appendix III D, using the following solutions.
CONFIRMATION
( 1) Finely powder not less than 5.0 g of the drug being
When sprayed with methanolic sulfuric 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-µm filter.
Carry out the method for gas chromatography, (2) 0.025% w/v of Z-ligustilide CRS in acetonitrile.
Appendix III B, using the following solutions. CHROMATOGRAPHIC CONDITIONS
(1) Extract approximately 20 g of the coarsely powdered (a) Use a stainless steel column (15 cm x 4.6 mm) packed
drug in a 500 mL round-bottomed flask by hydrodistillation with octadecylsilyl silica gel for chromatography (5 µm) (Hypersil
for 2 to 3 hours in 200 mL of water, collecting the distillate ODS is suitable).
in suitable glassware. Extract the oily drops on top of the
(b) Use isocratic elution and the mobile phase described
distillate with 5 mL of toluene.
below.
(2) 0.2% w/v each of coumarin and eugenol in toluene.
(c) Use a flow rate of 1.0 mL per minute.
(3) 0.1 % w/v of Z-ligustilide CRS in acetonitrile.
(d) Use a detection wavelength of 350 nm.
(4) 0.1 % w/v of benzyl alcohol in toluene.
(e) Inject 10 µL of each solution.
(5) 0.1 % w/v of (-)-carvone in toluene.
MOBILE PHASE
(6) 0.1 % w/v of octanoic acid in toluene.
A mixture of 8 volumes of water and 12 volumes of
(7) 0.1 % w/v of 3-propylidenephthalide in toluene. acetonitrile.
CHROMATOGRAPHIC CONDITIONS SYSTEM SUITABILITY
(a) Use a fused silica capillary column (50 m x 0.32 mm) Inject solution (2) not less than five times. The test is not
bonded with a film (1.05 µm) of 5% phenyl/95% valid unless the relative standard deviation of the peak areas
dimethylpolysiloxane (HP 5 is suitable). of the Z-ligustilide peak is not more than 3.0%, the relative
(b) Use helium as the carrier gas at 1 mL per minute. standard deviation of the retention times of the Z-ligustilide
(c) Use an oven maintained at an initial temperature of 40° peak is not more than 3.0%. The column effidency,
increasing linearly to 220° at a rate of 5° per minute, then determined on the Z-ligustilide peak, is not less than 5000
maintained at 220°. theoretical plates. The symmetry factor, determined on the
(d) Use a split injection system having a split ratio of 1:20 Z-ligustilide peak, is not more than 1.3.
maintained at 250°. Inject solution ( 1). The test is not valid unless the resolution
(e) Use a flame ionisation detector maintained at a factor between the Z-ligustilide peak and the closest peak
temperature of 250°. (relative retention about 0.9 with respect to Z-ligustilide) is
not less than 1.5.
(f) Inject 1 µL of each solution.
DETERMINATION OF CONTENT
(g) Record the chromatograms for a sufficient length of time
to elute all the peaks in the chromatogram obtained with Using the retention time and the peak area from the
solution (1) (55 minutes may be suitable). chromatograms obtained with solution (2), locate and
integrate the peak due to Z-ligustilide in the chromatogram
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained Calculate the content of Z-ligustilide in the sample using the
with solution (2), the resolution between coumarin (eluting declared content of Z-ligustilide (C 12H14O2) in
at approximately 34 minutes) and eugenol (eluting at Z-ligustilide CRS and the following expression:
approximately 37 minutes) is at least 3.0.
CONFIRMATION A, m2 V, 100
In the chromatogram obtained with solution (1): -x-x-xpx---
- there are no peaks corresponding to the principal peaks in ~ V2 m, 100-d
the chromatograms obtained with solutions (4), (5), (6)
Area of the peak due to Z-ligustilide in the chromatogram
and (7); obtained with solution (1).
- there is a peak corresponding to the principal peak in the Area of the peak due to Z-ligustilide in the chromatogram
chromatogram obtained with solution (3). obtained with solution (2).
Weight of the drug being examined in mg.
Loss on drying
Weight of Z-ligustilide CRS in mg.
When dried at 100° to 105° for 2 hours, loses not more than Dilution volume of solution (1) in mL.
12.0% of its weight. Use 1 g. Dilution volume of solution (2) in mL.
Total ash Percentage content of C 12 H 14 O 2 in Z-ligustilide CRS.
Percentage loss on drying of the herbal drug being examined.
Acid-insoluble ash STORAGE
Angelica Sinensis Root should be protected from moisture.
2023 Angelica Sinensis Root IV-83
Processed Angelica Sinensis Root

(Angelica Sinensis Root, Ph. Bur. monograph 2558)
~~----------------------
DEFINITION
Smoke-dried, whole or fragmented root, with rootlets
removed, of Angelica sinensis (Oliv.) Diels collected in late
autumn.
Content
Minimum 0.050 per cent of trans-ferulic acid (C 10H 1004;
IDENTIFICATION
A. Taproot branching rapidly into 10 or more conical
principal roots; the whole is about 15-25 cm long.
The annulated root crown is about 1.5-4 cm in diameter; its
blunt, rounded tip shows the yellowish-green remains of
stems and petioles of leaves. The outer surface is light
brownish-yellow or dark brown, lumpy, irregularly striated
longitudinally and shows scars of secondary roots and
transversal lenticel-like markings. The branching roots have a
thick upper part (0.3-1 cm in diameter) and a thin lower
part. They are frequently twisted and show few scars of
secondary roots. The texture is friable. The fracture,
yellowish-white or yellowish-brown, shows a thick bark with
some clefts and numerous brown dots due to secretory
canals. The cambium occurs as a yellowish-brown ring.
The wood is light coloured.
The fragmented roots occur as long strips about 1.5-2 mm Figure 2558.-1. - Illustration for identification test B of powdered
thick, 1.5-4 cm wide at the root crown and 10-15 cm long. herbal drug of Angelica sinensis root
Top of the plate
yellowish-white. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic (Z)-Llgustilide: a bluish-white A prominent bluish-white fluorescent
characters (Figure 2558.-1): reticulate or scalariform fluorescent zone zone ((Z)-ligustilide)
vessels [DJ up to 80 µm in diameter, free or in groups of
-- --
2 or 3 and accompanied by xylem parenchyma cells [Da]
and medullary rays [Db]; numerous fragments of Osthoie: a blue fluorescent zone
parenchyma with ovoid cells [C]; orange cork fragments, Imperatorin: a whitish fluorescent
consisting of several layers of superimposed cells, more or zone
less rectangular (surface view [A], transverse section [BJ);
very small calcium oxalate prisms in the cork [Aa], visible in - - --
polarised light; secretory canals, often broken, up to 170 µm
in diameter (transverse section [G], longitudinal section [F])
with orange-yellow contents in droplets [Fa, Ga]. Examine
under a microscope using a 50 per cent VIV solution of
glycerol R. The powder shows small (less than 10 µm), Results B See below the sequence of zones present in the
simple, rounded or ovoid starch granules, usually included in chromatograms obtained with the reference solution and the
parenchyma cells [E]. test solution. Furthermore, other faint quenching zones may
solution.
officinal species of Angelica, Levisticum and Ligusticum.
Results A See below the sequence of zones present in the Top of the plate
(Z)-Llgustilide: a blue fluorescent A prominent blue fluorescent zone
test solution. Furthermore, other faint fluorescent zones may ( (Z)-ligustilide)
zone
solution. A faint quenching zone
-- --
Osthole: a quenching zone A faint quenching zone
lmperatorin: a quenching zone
-- --

IV-84 Aniseed 2023
TESTS Retention time trans-ferulic acid = about 13 min; cis-ferulic

Other officinal species of Angelica, Levisticum and acid= about 14 min.
Ligusticum System suitability Reference solution (b):
Thin-layer chromatography (2.2.27). - resolution: minimum 1.3 between the peaks due to trans-
Test solution To 1 g of the powdered herbal drug (355) ferulic acid and cis-ferulic acid.
(2. 9.12) add 4 mL of heptane R, close and sonicate for 5 min. Calculate the percentage content of trans-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.
Mobile phase glacial acetic acid R, ethyl acetate R, toluene R area of the peak due to trans-ferulic acid in the chromatogram
(1:10:90 VIVIV). area of the peak due to trans-ferulic acid in the chromatogram
Application 4 µL as bands of 8 mm. obtained with reference solution (a);
Development Over a path of 6 cm. solution, in grams;
Drying In air. mass of ferulic acid CRS used to prepare reference solution (a),
in grams;
Detection A Examine in ultraviolet light at 365 nm. p percentage content of trans-ferulic acid in ferulic acid CRS.
shows no intense blue fluorescent zone at or below the - - - - - - - - - - - - - - - - - - - - - - Ph Eur
position of osthole in the chromatogram obtained with the
reference solution.
Results B The chromatogram obtained with the test solution Aniseed
shows no quenching zone at or below the position of
imperatorin in the chromatogram obtained with the reference Anise
solution. (Ph. Bur. monograph 0262)
Loss on drying (2.2.32) When Powdered Aniseed is prescribed or demanded,
Maximum 12.0 per cent, determined on 1.000 g of the material complying with the requirements below, with the
powdered herbal drug (355) (2. 9.12) by drying in an oven at exception of Identification test A and the test for Foreign
105 °C for 2 h. matter, shall be dispensed or supplied.
Total ash (2.4.16) Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Ash insoluble in hydrochloric acid (2.8.1) Whole, dry cremocarp of Pimpinella anisum L.
Content
ASSAY Minimum 20 mUkg of essential oil (anhydrous drug).
Liquid chromatography (2.2.29). CHARACTERS
Test solution Disperse 0.200 g of the powdered herbal drug Reminiscent odour of anethole.
(355) (2.9.12) in 20.0 mL of a 70 per cent VIV solution of
The fruit is a cremocarp and generally entire; a small
methanol R in a conical flask, stopper tightly and weigh. Heat
fragment of the thin, rigid, slightly curved pedicel is
under a reflux condenser for 30 min, cool and weigh again.
frequently attached.
Compensate the loss of solvent with a 70 per cent V/V
solution of methanol R, mix well and allow to stand. Filter the IDENTIFICATION
supernatant through a membrane filter (nominal pore size A. The cremocarp is ovoid or pyriform and slightly
0.45 µm); use the filtrate. compressed laterally, yellowish-green or greenish-grey,
Reference solution (a) In a brown-glass volumetric flask, 3-5 mm long and up to 3 mm wide, surmounted by a
dissolve 10.0 mg offerulic acid CRS in a 70 per cent VIV stylopod with 2 short, reflexed stylar points. The mericarps
solution of methanol Rand dilute to 100.0 mL with the same are attached by their tops to the carpophore with a plane
solvent. commissural surface and a convex dorsal surface, the latter
being covered with short, warty trichomes visible using a
Reference solution (b) In order to prepare cis-ferulic acid in
lens; each mericarp shows 5 primary ridges, running
situ, introduce 2 mL of reference solution (a) into a
longitudinally, comprising 3 dorsal ridges and 2 lateral ridges,
transparent vial and expose to ultraviolet light at 254 nm for
non-prominent, and lighter in colour.
about 60 min.
Column:
greenish-yellow or brownish-green. Examine under a
- size: l = 0.150 m, 0 = 2.0 mm;
shows the following diagnostic characters (Figure 0262.-1):
(4 µm);
fragments of epicarp in surface view [D] with a striated
cuticle, occasional anomocytic stomata (2.8.3) [Da], bases of
Mobile phase acetonitrile R, 0.085 per cent VIV solution of covering trichomes [De] and whole covering trichomes [Db],
phosphoric acid R (17:83 V/V). mostly unicellular, sometimes curved, with a blunt apex and
Flow rate 0.23 mUmin. a warty cuticle; isolated fragments of covering trichomes [E];
Detection Spectrophotometer at 316 nm. fragments [H] of numerous narrow, branched vittae [Ha],
Injection l O µL. often accompanied by elongated cells of the commissural
surface [Hb]; fragments of testa [B] consisting of a layer of
2023 Star Anise IV-85
brown, polyhedral, thin-walled cells; fragments of endosperm of the test solution, the spot due to anethole is intermediate
[G] containing oil droplets [Ga], aleurone grains and small in size between the corresponding spots in the
cluster crystals of calcium oxalate [Gb]; oblong sclereids from chromatograms obtained with 1 µL and 3 µL of the reference
the mesocarp [CJ or the commissural surface of the fruit; solution. The chromatograms obtained with the test solution
bundles of short sclerenchymatous fibres [A] from the show in the lower third a blue spot (triglycerides) similar in
carpophore and the pedicel [Ab], accompanied by vessels position to the spot in the lower third of the chromatograms
with spiral or annular thickening [Aa, F]. obtained with the reference solution (triglycerides of olive
oil).
TESTS
Water (2.2.13)
Maximum 90 mIJkg, determined on 20.0 g of the herbal
drug reduced to a coarse powder immediately before use.
Total ash (2.4.16)
B
Db ~
.,d-:::;:;:::=:;::::;::;~------
E ASSAY
Essential oil (2.8.12)
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
· .. ,!}l. liquid. Place 0.50 mL of xylene R in the graduated tube.
Distil at a rate of 2.5-3.5 mIJmin for 2 h.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Star Anise
Preparation
Concentrated Anise Water
PhE~ - - - - - - - - - - - - - - - - - - - ~
DEFINITION
Dried composite fruit of lllicium vernm Hook.f.
Content
- minimum 70 mIJkg of essential oil (anhydrous drug),
herbal drng of aniseed - minimum 86.0 per cent of trans-anethole in the essential
C. Thin-layer chromatography (2.2.27). oil.
Test solution Shake O.10 g of the powdered herbal drug CHARACTERS
( 1400) (2. 9.12) with 2 mL of methylene chloride R for 15 min. The fruit carpels are brown.
Filter and carefully evaporate the filtrate to dryness on a Odour of anethole.
water-bath at 60 °C. Dissolve the residue in 0.5 mL of
IDENTIFICATION
toluene R.
A. The fruit generally consists of 8 developed, one-seeded
Reference solution Dissolve 3 µL of anethole R and 40 µL of
follicles, each 12-22 mm long and 6-12 mm high, radially
olive oil R in 1 mL of toluene R. arranged around a short, central, blunt-ending columella.
Plate TLC silica gel GF254 plate R. In some fruits 1 or 2 follicles may be missing, but their
Mobile phase toluene R. position is clearly visible. Each follicle is boat-shaped or boot-
Application 2 µL and 3 µL of the test solution, then 1 µL, shaped, with a greyish-brown dorsal surface showing rough
2 µL and 3 µL of the reference solution, at 2 cm intervals. markings and lateral surfaces bearing scars from the
neighbouring follicles. One or more follicles are split open
along the ventral suture, exposing a single, lenticular, shiny,
Drying In air. reddish-brown seed about 8 mm in diameter. The markings
Detection A Examine in ultraviolet light at 254 nm. on the dorsal surface are not visible from the ventral surface.
Results A The chromatograms show a quenching zone Some of the follicles (1-3) may be imperfectly developed.
(anethole) in the central part against a light background. Isolated follicles, pedicels and seeds may be present.
Detection B Spray with a freshly prepared 200 g/L solution B. Microscopic examination (2.8.23). The powder is reddish-
of phosphomolybdic aci.d R in ethanol (96 per cent) R, using brown. Examine under a microscope using chloral hydrate
10 mL for a 200 mm square plate, and heat at 120 °C for solution R. The powder shows the following diagnostic
5 min. characters: brown epicarpal cells, polygonal in surface view,
Results B The spots due to anethole appear blue against a with a strongly striated cuticle and occasional anomocytic
yellow background. In the chromatogram obtained with 2 µL stomata (2. 8.3); fragments of the endocarp with long
IV-86 Star Anise 2023
palisade-like cells; fragments of the mesocarp with large Drying In a current of warm air.
parenchymatous cells, vessels, oil-containing cells and groups Detection Spray with a 10 g/L solution of diphenylboric acid
of stone cells; fragments of the seed testa with palisade-like, aminoethyl ester R in methanol R and then with a 50 g/L
sclerified, strongly pitted, yellow cells up to 200 µm long; solution of macrogol 400 R in methanol R; after 30 min,
fragments of the columella and the fruit stalk with strongly examine in ultraviolet light at 365 nm.
and irregularly thickened, star-shaped stone cells about
400 µm long and 150 µm wide; rhomboidal or rectangular
shows no brownish-yellow fluorescent zone at or above the
crystals of calcium oxalate.
position of the zone due to quercitrin in the chromatogram
C. Examine the chromatograms obtained in test B for Illicium obtained with the reference solution. No yellow fluorescent
anisatum (= I. religiosum) and certain other Illicium spp. zone is seen at or above the position of the zone due to
Results See below the sequence of the zones present in the caffeic acid in the chromatogram obtained with the reference
chromatograms obtained with the reference solution and the solution. No brownish-yellow fluorescent zone is seen directly
test solution. Furthermore, other weaker zones may be above the zone due to hyperoside in the chromatogram
present in the chromatogram obtained with the test solution. obtained with the reference solution.
Water (2.2.13)
Top of the plate
-- -- of the powdered herbal drug (355) (2.9.12).
Caffeic acid: a light blue fluorescent Total ash (2.4.16)
zone Maximum 4.0 per cent.
Quercitrin: a brownish-yellow ASSAY
fluorescent zone Essential oil (2.8.12)
A brownish-yellow fluorescent zone Use a 250 mL round-bottomed flask and 100 mL of water R
as the distillation liquid. Immediately before the
-- -- determination, reduce 50.0 g of the drug to a powder (1400)
A greenish fluorescent zone (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
Hyperoside: a brownish-yellow A brownish-yellow fluorescent zone powder for the determination. Introduce 0.50 mL of xylene R
fluorescent zone
into the graduated tube. Distil at a rate of 2-3 mUmin
Chlorogenic acid: a light blue for 2 h.
fluorescent zone
trans-Anethole
A green fluorescent zone Gas chromatography (2.2.28): use the normalisation
procedure.
Rutoside: a brownish-yellow A brownish-yellow fluorescent zone
fluorescent zone Test solution Dilute the mixture of essential oil and xylene R
obtained in the assay of essential oil to 5.0 mL with xylene R
by rinsing the apparatus.
Reference solution To 1.0 mL of xylene R add 20 µL of
TESTS estragole R, 20 mg of rx-terpineol R and 60 µL of anethole R.
Illicium anisatum (= I. religiosum) and certain other Column:
Illicium spp - material: fused silica;
A. Adulteration with Illicium anisatum or certain other = =
- size: l 30 m, 0 0.25 mm;
Jllicium spp. is indicated by the presence of fruits mainly - stationary phase: macrogol 20 000 R.
consisting of more than 8 follicles; fruits either smaller than Carrier gas helium for chromatography R.
2.5 cm or greater than 3.5 cm; follicles with the suture edged
with a thickening extending to the neighbouring follicle, or
with dorsal markings visible from the ventral surface; follicles Split ratio 1: 100.
somewhat undulate and ending in a fine beak or a small, Temperature:
ventrally turned hook; follicles with a profile fitting into a
Time Temperature
rectangle; pedicels more than 5 cm long; seedless fruits; seeds (min) CC)
either very flat or almost spherical.
Column 0-5 60
B. Thin-layer chromatography (2.2.27). 5 - 80 60 ---> 210
Test solution To 2.0 g of the powdered herbal drug (355) 80 - 95 210
(2. 9.12) add 10 mL of methanol R and heat under a reflux Injection port 200
condenser in a water-bath at 60 °C for 5 min. Allow to cool Detector 220
and filter.
Reference solution Dissolve 1 mg of caffeic acid R, 1 mg of Detection Flame ionisation.
chlorogenic acid R, 2.5 mg of quercitrin R, 2.5 mg of rutoside Injection 1 µL.
trihydrate Rand 2.5 mg of hyperoside R in 10 mL of
Elution order Order indicated in the preparation of the
methanol R.
reference solution.
Plate TLC silica gel plate R (2-10 µm).
Mobile phase anhydrous formic acid R, glacial acetic acid R, - resolution: minimum 5 between the peaks due to estragole
water R, ethyl acetate R (11:11:26:100 VIV/VIV). and cHerpineol.
Application 5 µL as bands. Use the retention times from the chromatogram obtained
Development Over a path of 6 cm. with the reference solution locate the components of the
2023 Star Anise Oil IV-87
reference solution in the chromatogram obtained with the test solution. Furthermore, other zones may be present in the
test solution. chromatogram obtained with the test solution.
Calculate the percentage content of trans-anethole. Disregard
any peak due to the solvent or with an area less than Top of the plate
0.05 per cent of the area of the principal peak in the
A violet-brown zone, not fully
chromatogram obtained with the test solution. separated
- - - - - - - - - - - - - - - - - - - - - - Ph Eur Anethole: a brown zone A very strong brown zone (anethole)
-- --
Anisaldehyde: a yellow zone A yellow zone (anisaldehyde)
Star Anise Oil --
--
(Ph. Bur. monograph 2108) Linalol: a grey zone A grey zone (linalol)
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Essential oil obtained by steam distillation from the dry ripe B. Examine the chromatograms obtained in the test for
fruits of fllicium verum Hook.f. chromatographic profile.
CHARACTERS Results The characteristic peaks in the chromatogram
Appearance obtained with the test solution are similar in retention time to
Clear, colourless or pale yellow liquid. those in the chromatogram obtained with the reference
solution.
IDENTIFICATION
First identification: B. TESTS
Second identification: A. Relative density (2.2.5)
0.979 to 0.985.
A. Thin-layer chromatography (2.2.27).
Refractive index (2.2.6)
Test solution Dissolve 1 g of the substance to be examined
1.553 to 1.556.
in toluene R and dilute to 10 mL with the same solvent.
Reference solution Dissolve 10 µL of linawl R, 30 µL of Freezing point (2.2.18)
anisaldehyde R and 200 µL of anethole R and in toluene R and 15 °C to 19 °C.
dilute to 15 mL with the same solvent. Dilute 1 mL of this Fenchone
solution to 5 mL with toluene R. Gas chromatography (2.2.28) as described in the test for
Plate TLC silica gel F254 plate R. chromatographic profile with the following modifications.
Mobile phase ethyl acetate R, toluene R (7:93 VIV). Test solution Dissolve 400 µL of the substance to be
examined in 2.0 mL of hexane R.
Application 5 µLas bands of 10 mm (for normal
TLC plates) or 2 µL as bands of 10 mm (for fine particle Reference solution (a) Dilute 10 µL offenchone R to 1.2 g
TLC plates). with hexane R.
Development Over a path of 15 cm (for normal TLC plates) Reference solution (b) Dilute 100 µL ofreference solution (a)
or over a path of 6 cm (for fine particle size plates). to 100 mL with hexane R.
Drying In air. System suitability Reference solution (b):
- signal-to-noise ratio: minimum 10 for the principal peak.
Limit.
- fenchone: maximum 0.01 per cent.
test solution. Furthermore, other zones may be present in the Pseudoisoeugenyl 2-methylbutyrate
chromatogram obtained with the test solution. Gas chromatography (2.2.28) as described in the test for
chromatographic profile with the following modifications.
Top of the plate Test solution The substance to be examined.
Reference solution (a) Dilute 10 mg of the test solution to
A quenching zone, partly separated
1.000 g with hexane R. Dilute 0.5 mL of this solution to
Anethole: a quenching zone A very strong quenching zone 100 mL with hexane R.
(anethole)
Reference solution (b) Pseudoisoeugenyl 2-methylbutyrate for
-- -- peak identification CRS.
Anisaldehyde: a quenching zone A quenching zone (anisaldehyde) System suitability:
- the chromatogram obtained with reference solution (b) is
-- -- similar to the chromatogram provided with
Reference solution Test solution pseudoisoeugenyl 2-methylbutyrate for peak identification CRS.
- signal-to-noise ratio: minimum 10 for the principal peak in
the chromatogram obtained with reference solution (a).
Detection B Spray with methyl 4-acetylbenzoate reagent R and
heat at 100-105 °C for 10 min; examine the still hot plate in Limit Locate the peak due to pseudoisoeugenyl
daylight within 10 min. 2-methylbutyrate by comparison with the chromatogram
provided with pseudoisoeugenyl 2-methylbutyrate for peak
identification CRS.
- pseudoisoeugenyl 2-methylbut)rrate: maximum 0.01 per cent.
IV-88 Star Anise Oil 2023
2 5
4
6
3
.I l 111 I
I
I ~ I. 11 I L1 LJ l I
I ' I I I ' ' I ' I I ' ' I I I

0 10 20 30 40 50 60 70 80 min
I. linalol 3. 0<-terpineol 5. trans-anethole 7. foeniculin

2. estragole 4. cis-anethole 6. anisaldehyde
Figure 2108.-1. - Chromatogram for the test for chromatographic profile of star anise oil
Fatty oils and resinified essential oils (2.8.7) Detection Flame ionisation.
It complies with the test for fatty oils and resinified essential Injection 0.2 µL.
oils. Elution order Order indicated in the composition of the
Chromatographic profile reference solution; record the retention times of these
Gas chromatography (2.2.28): use the normalisation substances.
procedure. System suitability Reference solution:
Test solution Dissolve 200 µL of the substance to be - resolution: minimum 1.5 between the peaks due to
examined in 1.0 mL of hexane R. estragole and Cl-terpineol.
Reference solution To 1.0 mL of hexane R, add 20 µL of Using the retention times determined from the
linalol R, 20 µL of estragole R, 20 µL of a-terpineol R, 60 µL chromatogram obtained with the reference solution, locate
of anethole R and 30 µL of anisaldehyde R. the components of the reference solution in the
Column: chromatogram obtained with the test solution and locate cis-
- material: fused silica, anethole and foeniculin using the chromatogram shown in
=
- size: l 30 m, 0 0.25 mm, = Figure 2108.-1 (disregard any peak due to hexane).
- stationary phase: macrogol 20 000 R (film thickness Determine the percentage content of these components.
0.25 µm). The percentages are within the following ranges:
Carrier gas helium for chromatography R. - linalol: 0.2 per cent to 2.5 per cent,
Flow rate 1.0 mLJmin. - estragole: 0.5 per cent to 6.0 per cent,
- a-terpineol: maximum 0.3 per cent,
Split ratio 1: 100.
- cis-anethole: 0.1 per cent to 0.5 per cent,
Temperature: - trans-anethole: 86 per cent to 93 per cent,
- anisaldehyde: 0.1 per cent to 0.5 per cent,
Time Temperature - foeniculin: 0.1 per cent to 3.0 per cent.
(min) CC)
Column 0-5 60 STORAGE
5 - 80 60-+ 210 At a temperature not exceeding 25 °C.
80 - 95 210 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Injection port 200
Detector 220
2023 Anise Oil IV-89
Anise Oil Top of the plate
A violet-brown zone (monoterpene

Aniseed Oil hydrocarbons) (solvent front)
(Ph. Bur. monograph 0804) Anethole: a brown zone A very strong brown zone (anethole),
distinctly separated
Preparation
Concentrated Anise Water - - - -
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ A grey zone
DEFINITION Anisaldehyde: a yellow zone A yellow zone (anisaldehyde)

Essential oil obtained by steam distillation from the dry ripe
-- --
fruits of Pimpinella anisum L.
Linalol: a grey zone A grey zone (linalol)
CHARACTERS
Appearance A grey zone
Clear, colourless or pale yellow liquid. Reference solution Test solution
IDENTIFICATION
First identification: B. B. Examine the chromatograms obtained in the test for
Second identificatwn: A. chromatographic profile.
A. Thin-layer chromatography (2.2.27). Results The characteristic peaks in the chromatogram
Test solutwn Dissolve 1 g of the substance to be examined obtained with the test solution are similar in retention time to
in toluene R and dilute to 10 mL with the same solvent. those in the chromatogram obtained with the reference
Reference solutwn Dissolve 10 µL of linalol R, 30 µL of solution.
anisaldehyde R and 200 µL of anethole R in toluene R and TESTS
dilute to 15 mL with the same solvent. Dilute 1 mL of this Relative density (2.2.5)
solution to 5 mL with toluene R. 0.980 to 0.990.
Plate TLC silica gel F2s 4 plate R. Refractive index (2.2.6)
Mobile phase ethyl acetate R, toluene R (7:93 V/V). 1.552 to 1.561.
Applicatwn 5 µL as bands of 10 mm (for normal Freezing point (2.2.18)
TLC plates) or 2 µLas bands of 10 mm (for fine particle 15 °C to 19 °C.
size plates).
Fenchone
Development Over a path of 15 cm (for normal TLC plates) Gas chromatography (2.2.28) as described in the test for
or over a path of 6 cm (for fine particle size plates). chromatographic profile with the following modifications.
Drying In air. Test solutwn Dissolve 400 µL of the substance to be
Detection A Examine in ultraviolet light at 254 nm. examined in 2.0 mL of hexane R.
Results A See below the sequence of zones present in the Reference solutwn (a) Dilute 10 µL of fenchone R to 1.2 g
chromatograms obtained with the reference solution and the with hexane R.
test solution. Furthermore, other zones may be present in the Reference solutwn (b) Dilute 100 µL of reference solution (a)
chromatogram obtained with the test solution. to 100 mL with hexane R.
Top of the plate - signal-to-noise ratw: minimum 10 for the principal peak.
Anethole: a quenching zone A very strong quenching zone Limit:
(anethole) - fenchone: maximum 0.01 per cent.
-- -- Foeniculin
A quenching zone Gas chromatography (2.2.28) as described in the test for
chromatographic profile with the following modifications.
Anisaldehyde: a quenching zone A quenching zone (anisaldehyde)
Test solutwn The substance to be examined.
- - -- Reference solutwn ( a) Dilute 10 mg of the test solution to
Reference solution Test solution 1.000 g with hexane R. Dilute 0.5 mL of this solution to
100 mL with hexane R.
Reference solutwn (b) Foeniculin for peak identification CRS.
Detection B Spray with methyl 4-acetylbenzoate reagent R and
heat at 100-105 °C for 10 min; examine the still hot plate in System suitability:
daylight within 5 min. - the chromatogram obtained with reference solution (b) is
similar to the chromatogram provided with foeniculin for
peak identification CRS,
- signal-to-noise ratw: minimum 10 for the principal peak in
test solution. Furthermore, other zones may be present in the
the chromatogram obtained with reference solution (a).
chromatogram obtained with the test solution.
Limit Locate the peak due to foeniculin by comparison with
the chromatogram provided with foeniculin for peak
identification CRS.
- foeniculin: maximum 0.01 per cent.
IV-90 Anise Oil 2023
2
6
3 7
I I I I I I I I I I I I I I
0 10 20 30 40 50 60 70 80 min
I. linalol 3. c,-terpineol 5. trans-anethole 7. pseudoisoeugenyl 2-methylbutyrate

2. estragole 4. cis-anethole 6. anisaldehyde
Figure 0804.-1. - Chramatogramfor the test for chramatographic profile of anise oil
Fatty oils and resinified essential oils (2.8. 7) Detection Flame ionisation.
It complies with the test for fatty oils and resinified essential Injection 0.2 µL.
oils. Elution order Order indicated in the composition of the
Chromatographic profile reference solution. Record the retention times of these
Gas chromatography (2.2.28): use the normalisation substances.
procedure. System suitability Reference solution:
Test solution Dissolve 200 µL of the substance to be - resolution: minimum 1.5 between the peaks due to
examined in 1.0 mL of hexane R. estragole and C(-terpineol.
Reference solution To 1.0 mL of hexane R, add 20 µL of Using the retention times determined from the
linalol R, 20 µL of estragole R, 20 µL of rx-terpineol R, 60 µL chromatogram obtained with the reference solution, locate
of anethole R and 30 µL of anisaldehyde R. the components of the reference solution in the
Column: chromatogram obtained with the test solution and locate cis-
- material: fused silica, anethole and pseudoisoeugenyl 2-methylbutyrate using the
=
- size: l 30 m, 0 0.25 mm, = chromatogram shown in Figure 0804.-1 (disregard any peak
- stationary phase: macrogol 20 000 R (film thickness due to hexane).
0.25 µm). Determine the percentage content of these components.
Carrier gas helium for chromatography R. The percentages are within the following ranges:
Flow rate 1.0 mUmin. - linalol: maximum 1.5 per cent,
- estragole: 0.5 per cent to 5.0 per cent,
Split ratio 1:100.
- rx-terpineol: maximum 1.2 per cent,
Temperature: - cis-anethole: 0.1 per cent to 0.4 per cent,
- trans-anethole: 87 per cent to 94 per cent,
Time Temperature - anisaldehyde: 0.1 per cent to 1.4 per cent,
(rnin) CC)
- pseudoisoeugenyl 2-methylbutyrate: 0.3 per cent to
Column 0-5 60 2.0 per cent.
5 - 80 60--> 210
80 - 95 210 STORAGE
Injection port 200 At a temperature not exceeding 25 °C.
Detector 220 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
2023 Amica Flower IV-91
Concentrated Anise Water A ~Aa

~-,.•··•.•·· -
;gr-
DEFINITION
Ab
Anise Oil or Star Anise Oil 20 mL
B~g D,
Ethanol (90 per cent) 700mL
Water Sufficient to produce 1000 mL
~
Extemporaneous preparation
The following directions apply.
Dissolve the Anise Oil or Star Anise Oil in the Ethanol
(90 per cent) and add gradually, with vigorous shaking after
each addition, sufficient Water to produce 1000 mL.
H
Add 50 g of previously sterilised Purified Talc, or other
suitable filtering aid, allow to stand for a few hours, shaking
occasionally, and filter.
The water complies with the requirements stated under Aromatic
Waters and with the following requirements.
TESTS
Ethanol content
60 to 64% v/v, Appendix VIII F.
Weight per mL
0.898 to 0.908 g, Appendix VG.
Amica Flower
Preparation
Amica Tincture
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or partially broken, dried flower-heads of Amica
montana L.
Content
Minimum 0.40 per cent of total sesquiterpene lactones,
expressed as dihydrohelenalin tiglate (C 20H 26O 5; Mr 346.4)
(dried drug).
CHARACTERS
The capitulum, when spread out, is about 5-8 mm in
diameter and about 15 mm deep, and has a peduncle 2-3 cm
long. The involucre consists of 18-24 elongated lanceolate
bracts, with acute apices, arranged in 1-2 rows: the bracts,
about 8-10 mm long, are green with yellowish-green external
hairs visible under a lens. The receptacle, about 6 mm in
diameter, is convex, alveolate and covered with hairs.
Its periphery bears about 20 ligulate florets 20-30 mm long;
the disc bears a greater number of tubular florets about
15 mm long. The ovary, 4-8 mm long, is crowned by a
pappus of whitish bristles 4-8 mm long. Some brown
achenes, crowned or not by a pappus, may be present.
IDENTIFICATION
A. The involucre consists of elongated oval bracts with acute
apices; the margin is ciliated. The ligulate floret has a
reduced calyx crowned by fine, shiny, whitish bristles,
bearing small coarse trichomes. The orange-yellow corolla
bears 7-10 parallel veins and ends in 3 small lobes.
The stamens, with free anthers, are incompletely developed.
The narrow, brown ovary bears a stigma divided into Figure 1391. -1. - Illustration for identification test B of powdered
2 branches curving outwards. The tubular floret is herbal drug of amica flower
actinomorphic. The ovary and the calyx are similar to those
B. Microscopic examination (2.8.23). Separate the capitulum
of the ligulate floret. The short corolla has 5 reflexed
into its different parts. Examine under a microscope using
triangular lobes; the 5 fertile stamens are fused at the
chloral hydrate solution R. The powder shows the following
anthers.
IV-92 Amica Flower 2023
diagnostic characters (Figure 1391.-1): the epidermises of the TESTS

bracts of the involucre [L, M, 0, Q] have stomata [Lb, Oa, Foreign matter (2.8.2)
Qa] and trichomes, more abundant on the outer (abaxial) Maximum 5.0 per cent.
surface. There are several different types of trichomes: Amica chamissonis Less. - Calendula officinalis L.
uniseriate multicellular covering trichomes, varying in length - Heterotheca inuloides Cass
from 50-500 µm, particularly abundant on the margins of the High-performance thin-layer chromatography (2. 8. 25).
bract, whole [La] or fragmented [P]; secretary trichomes with
uni- or biseriate multicellular stalks and with multicellular,
(2.9.12) add 10.0 mL of methanol R. Sonicate for 15 min,
globular heads, about 300 µm long, abundant on the outer
surface of the bract [Qb]; secretary trichomes with filter or centrifuge and use the filtrate or supernatant.
multicellular stalks and with multicellular, globular heads, Reference solution (a) Dissolve 2.0 mg of cajfeic acid Rand
about 80 µm long, abundant on the inner surface of the bract 2.5 mg of rutoside trihydrate R in methanol Rand dilute to
(surface view [Ob], side view [Ma]). The epidermis of the 10.0 mL with the same solvent.
ligulate corolla [C, G, H, TI consists of lobed or elongated Reference solution (b) Dilute 2.5 mL of reference solution (a)
cells covered by a striated cuticle [Ga], a few stomata and to 10.0 mL with methanol R.
trichomes of different types: covering trichomes, with very Reference solution (c) Dissolve 1 mg of chlorogenic acid R and
sharp ends, whose length may exceed 500 µm, consisting of 2.5 mg of hyperoside R in methanol Rand dilute to 10 mL
1-3 proximal, thick-walled cells and 2-4 distal, thin-walled with the same solvent.
cells [C, Hb]; secretary trichomes with biseriate multicellular Intensity markers Caffeic acid for the blue or greenish-blue
heads (surface view [Gb], side view Gal); secretary trichomes fluorescent zones and rutoside for the yellow and orange
with multicellular stalks and multicellular globular heads [K]. fluorescent zones.
The ligule ends in rounded papillose cells [Ha]. Fragments of
the epidermis of the ovary [A, B, D] are covered with
trichomes of 2 types: secretary trichomes with short stalks Mobile phase formic acid R, water R, ethyl acetate R
and multicellular globular heads (surface view [Aa], side (6:9:90 V/V/V).
view [Da]); twinned covering trichomes usually consisting of Application 2 µL as bands of 8 mm.
2 longitudinally united cells, with common pitted walls Development 70 mm from the lower edge of the plate.
(surface view [Ab], side view [Bal); their ends are sharp and Drying In a current of air at room temperature for 5 min.
sometimes bifid. The epidermises of the calyx consist of
elongated cells bearing short, unicellular, covering trichomes Detection Heat at 100-105 °C for 5 min; spray the warm
pointing towards the upper end of the bristle [E] . The pollen plate with a 10 g/L solution of diphenylboric acid aminoethyl
grains have a diameter of about 30 µm, are rounded, with a ester R in methanol R, then with a 50 g/L solution of macrogol
spiny exine, and have 3 germinal pores [F, N]. 400 R in methanol R; allow to dry in air for 5 min and
examine in ultraviolet light at 366 nm.
C. Examine the chromatograms obtained in the test for
Amica chamissonis Less. - Calendula officinalis L. - Heterotheca System suitability Reference solution (c):
inuloides Cass. - the chromatogram shows in the lower third 2 distinct
zones which may be partially overlapping; the lower zone
Results See below the sequence of fluorescent zones present (chlorogenic acid) shows a light blue fluorescence and the
in the chromatograms obtained with reference solution (a) upper zone (hyperoside) shows a yellow or orange
and the test solution. Furthermore, in the chromatogram fluorescence.
obtained with the test solution, other fluorescent zones may
be present, especially in the upper third of the Results The chromatogram obtained with the test solution
chromatogram. does not show an orange-yellow fluorescent zone
corresponding to the blue fluorescent zone due to caffeic acid
in the chromatogram obtained with reference solution (a),
Top of the plate
nor does it show an orange-yellow fluorescent zone
Caffeic acid: a blue zone corresponding to the zone due to rutoside in the
chromatogram obtained with reference solution (a).
2-4 blue zones, faint to intense
-- -- Maximum 10.0 per cent, determined on 1.000 g of the
A blue zone, faint to equivalent powdered herbal drug (355) (2.9.12) by drying in an oven at
105 °C for 2 h.
A yellowish-green zone, faint
Total ash (2.4.16)
- - -- Maximum 10.0 per cent.
A yellow or orange zone, equivalent ASSAY
Internal standard solution Dissolve immediately before use
A blue zone, equivalent
10.0 mg of santonin CRS and 20 mg of butyl
A yellow or orange zone, equivalent parahydroxybenzoate R in ethanol (96 per cent) R and dilute to
10.0 mL with the same solvent.
Rutoside: a yellow or orange zone
Test solution Introduce 1.00 g of the powdered herbal drug
Reference solution (a) Test solution (355) (2.9.12) into a 100 mL conical flask, add 40 mL of
ethyl acetate Rand sonicate for 5 min. Filter into a 250 mL
round-bottomed flask. Take up the residue with 40 mL of
ethyl acetate R. Sonicate and filter. Repeat this operation
once. Combine the filtrates, add 2.00 mL of the internal
2023 Amica Preparations IV-93
standard solution and evaporate to dryness. Dissolve the mass of the herbal drug to be examined used to prepare the
test solution, in grams;
residue in 15 mL of ethanol (96 per cent) R and add 15 mL mass of santonin CRS used to prepare the internal standard
of water R. Add 7 .0 g of neutral aluminium oxide R, shake for solution, in grams;
120 s, and centrifuge at 2500 g for 10 min. Transfer the p percentage content of santonin in santonin CRS;
supernatant into a 100 mL round-bottomed flask and 1.187 peak correlation factor between dihydrohelenalin tiglate and
santonin.
evaporate to dryness under reduced pressure in a water-bath
at a temperature not exceeding 50 °C. Dissolve the residue in - - - - - - - - - - - - - - - - - - - - - - - Ph Eur
5.0 mL of ethanol (96 per cent) R and filter through a
Reference solution Dissolve 20 mg of ethyl
parahydroxybenzoate R and 20 mg of methyl Amica Tincture
parahydroxybenzoate R in methanol R and dilute to 10 mL
with the same solvent. (Ph. Bur. monograph 1809)
Column: PhEw _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
- size: l= 0.12 m, 0 = 4 mm;
- stationary phase: end-capped octadecylsilyl silica gel for DEFINITION
chromatography R (4 µm); Tincture produced from Arnicafiower (1391).
- temperature: 20 °C. Content
Mobile phase: Minimum 0.04 per cent of total sesquiterpene lactones,
- mobile phase A: water for chromatography R; expressed as dihydrohelenalin tiglate (C 20 H 26 0 5 ; Mr 346.4).
- mobile phase B: methanol Rl; PRODUCTION
The tincture is produced from the herbal drug by a suitable
(min) (per cent V/V) (per cent V/V)
procedure using 10 parts of ethanol (60-70 per cent V/V) for
1 part of drug.
0- 3 62 38
3 - 20 62----> 55 38----> 45 CHARACTERS
20 - 30 55 45 Appearance
30 - 55 55----> 45 45----> 55 Yellowish-brown liquid.
IDENTIFICATION
Examine the chromatograms obtained in the test for Amica
Flow rate 1.2 mUmin. chamissonis Less. - Calendula officinalis L. - Heterotheca
Detection Spectrophotometer at 225 nm and at 350 nm. inuloides Cass.
Injection 10 µL of the test solution and the reference Results See below the sequence of fluorescent zones present
solution. in the chromatograms obtained with reference solution (a)
Relative retention With reference to santonin (retention and the test solution. Furthermore, in the chromatogram
time= about 8.2 min): methyl parahydroxybenzoate = about obtained with the test solution, other fluorescent zones may
0.6; ethyl parahydroxybenzoate = about 1.2; butyl be present, especially in the upper third of the
parahydroxybenzoate = about 4.5. chromatogram.
The assay is not valid unless:
Top of the plate
~: S: 0.05 Caffeic acid: a blue zone
2-4 blue zones, very faint to intense

A1 total area of peaks eluting between the peaks due to santonin
and butyl parahydroxybenzoate in the chromatogram obtained -- ---
with the test solution at 350 nm; disregard any peaks with an
area less than 0.1 times the area of the peak due to santonin; A blue zone, faint to equivalent
A yellowish-green zone, faint
and butyl parahydroxybenzoate in the chromatogram obtained
with the test solution at 225 nm.
--- --
System suitability: A yellow or orange zone, faint to

equivalent
- resolution: minimum 5.0 between the peaks due to methyl
parahydroxybenzoate and ethyl parahydroxybenzoate in
the chromatogram obtained with the reference solution at A blue zone, equivalent to intense
225 nm.
A yellow or orange zone, faint
Calculate the percentage content of total sesquiterpene
lactones, expressed as dihydrohelenalin tiglate, using the Rutoside: a yellow or orange zone
Azxmzxpxl.187 Reference solution (a) Test solution

A3 x mix 5
total area of peaks eluting between the peaks due to santonin TESTS
and butyl parahydroxybenzoate in the chromatogram
obtained with the test solution at 225 nm;
Amica chamissonis Less. - Calendula offidnalis L.
area of the peak due to santonin in the chromatogram - Heterotheca inuloides Cass.
obtained with the test solution at 225 nm; High-performance thin-layer chromatography (2.8.25).
IV-94 Amica Preparations 2023
Test solution The tincture to be examined. Reference solution Dissolve 20 mg of ethyl

Reference solution (a) Dissolve 2.0 mg of caffeic acid Rand parahydroxybenzoate R and 20 mg of methyl
2.5 mg of rutoside trihydrate R in methanol Rand dilute to parahydroxybenzoate R in methanol R and dilute to 10 mL
10.0 mL with the same solvent. with the same solvent.
Reference solution (b) Dilute 2.5 mL of reference solution (a) Column:
to 10.0 mL with methanol R. - size: l= 0.12 m, 0 = 4 mm;
Reference solution (c) Dissolve 1 mg of chwrogenic acid R and - stationary phase: end-capped octadecylsilyl silica gel for
2.5 mg of hyperoside R in methanol Rand dilute to 10 mL chromatography R (4 µm);
Mobile phase:
IntensilJ! markers Caffeic acid for the blue or greenish-blue
fluorescent zones and rutoside for the yellow and orange - mobile phase A: water for chromatography R;
fluorescent zones. - mobile phase B: methanol Rl;
Plate TLC silica gel F254 plate R (2-10 µm). Time Mobile phase A Mobile phase B
Mobile phase formic acid R, water R, ethyl acetate R (min) (per cent V/JI) (per cent V/JI)
(6:9:90 VIVIV). 0-3 62 38
Application 2 µL, as bands of 8 mm. 3 - 20 62 ➔ 55 38 ➔ 45
Development 70 mm from the lower edge of the plate. 20 - 30 55 45
30 - 55 55 ➔ 45 45 ➔ 55
Drying In a current of air at room temperature for 5 min.
Detection Heat at 100-105 °C for 5 min; spray the warm
plate with a 10 g/L solution of diphenylboric acid aminoethyl Fww rate 1.2 mL/min.
ester R in methanol R, then with a 50 g/L solution of macrogol Detector Spectrophotometer at 225 nm and at 350 nm.
400 R in methanol R; allow to dry in air for 5 min and Injection 10 µL of the test solution and the reference
examine in ultraviolet light at 366 nm. solution.
System suitability Reference solution (c): Relative retention With reference to santonin (retention
- the chromatogram shows in the lower third 2 distinct time = about 8.2 min): methyl parahydroxybenzoate = about
zones which may be partially overlapping; the lower zone 0.6; ethyl parahydroxybenzoate = about 1.2; butyl
(chlorogenic acid) shows a light blue fluorescence and the parahydroxybenzoate = about 4.5.
upper zone (hyperoside) shows a yellow or orange The assay is not valid unless:
fluorescence.
~:::; 0.05
does not show an orange-yellow fluorescent zone
corresponding to the blue fluorescent zone due to caffeic acid
in the chromatogram obtained with reference solution (a),
nor does it show an orange-yellow fluorescent zone with the test solution at 350 nm; disregard any peaks with an
corresponding to the zone due to rutoside in the area less than 0 .1 times the area of the peak due to santonin;
chromatogram obtained with reference solution (a). A2 total area of peaks eluting between the peaks due to santonin
Ethanol (2. 9.10) with the test solution at 225 nm.
The final ethanol concentration is not less than 90 per cent
of that of the initial extraction solvent. System suitability:
Methanol and 2-propanol (2.9.11) - resolution: minimum 5.0 between the peaks due to methyl
Maximum 0.05 per cent V/V of methanol and maximum parahydroxybenzoate and ethyl parahydroxybenzoate in
0.05 per cent V/V of 2-propanol. the chromatogram obtained with the reference solution at
Dry residue (2.8.16) 225 nm.
Minimum 1. 7 per cent. Calculate the percentage content of total sesquiterpene
lactones, expressed as dihydrohelenalin tiglate, using the
ASSAY following expression:
Internal standard solution Dissolve immediately before use Az x mz xp x 1.187
10.0 mg of santonin CRS and 20 mg of bulJ!l A3 x m1 x 5
parahydroxybenzoate R in ethanol (96 per cent) R and dilute to
10.0 mL with the same solvent. total area of peaks eluting between the peaks due to santonin
Test solution To 5.00 g of the tincture to be examined add with the test solution at 225 nm;
5 mL of water R and 2.00 mL of the internal standard area of the peak due to santonin in the chromatogram obtained
solution. Transfer the solution obtained to a 50 mL with the test solution at 225 nm;
m1 mass of the tincmre to be examined used to prepare the test
centrifuge tube containing 3 g of neutral aluminium oxide R.
solution, in grams;
Rinse the flask with 2 mL of water and transfer the rinsings mass of santonin CRS used to prepare the internal standard
to the centrifuge tube. Shake for 120 s and centrifuge at solution, in grams;
2500 g for 10 min. Transfer the supernatant into a 50 mL p percentage content of santonin in santonin CRS;
round-bottomed flask and evaporate to dryness under
1.187 peak correlation factor between dihydrohelenalin tiglate and
reduced pressure in a water-bath at a temperature not santonin.
exceeding 50 °C. Dissolve the residue in 3.0 mL of ethanol
(96 per cent) R and filter through a membrane filter (nominal _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
pore size 0.45 µm).
2023 Artichoke Leaf IV-95
in surface view; the upper epidermis [F] is composed of cells

Artichoke Leaf with straight or slightly sinuous walls [Fa], accompanied by
(Ph. Bur. monograph 1866) palisade parenchyma [Fb]; the lower epidermis [C] is
composed of more sinuous-walled cells; abundant
Preparation anomocytic stomata (2.8.3) on both surfaces [D] and
Artichoke Leaf Dry Extract multicellular, uniseriate covering trichomes in felted masses,
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ the majority fragmented [Ca] with a short stalk composed of
several cells and a very long, narrow and frequently curled
DEFINITION
terminal cell, others consisting of 4-6 cylindrical cells; very
Whole or cut, dried leaf of Cynara cardunculus L.
occasional glandular trichomes with a short stalk and a
(syn. C. scolymus L.).
uniseriate or biseriate head (surface view [E], transverse
Content section [Ba]); abundant fragments of covering trichomes [G];
Minimum 0.7 per cent of chlorogenic acid (C 16H 18 0 9 ; fragments of the lamina (transverse section [B]); abundant
M, 354.3) (dried drug). fragments of vascular tissue from the petiole and veins [A].
IDENTIFICATION C. Thin-layer chromatography (2.2.27).
A. The entire leaf may be up to 70 cm long and 30 cm wide. Test solution To 2.0 g of the powdered herbal drug (1000)
The lamina is deeply lobed in the upper part to within (2. 9.12) add 20 mL of ethanol (60 per cent V/Jl) R. Allow to
1-2 cm of the petiole on either side, in the lower part the leaf stand for 2 h with occasional stirring. Filter.
becomes pinnate; all the segments have markedly dentate Reference solution Dissolve 5 mg of luteolin-7-glucoside Rand
margins and taper at the apex. Spines are absent. The upper 5 mg of chlorogenic acid CRS in methanol R and dilute to
surface of the lamina is green with a fine covering of whitish 10 mL with the same solvent.
hairs, the lower surface is pale green or white and densely
tomentose with long, tangled hairs. The petiole and main
veins are flat on the upper surface, prominently raised and
longitudinally ridged on the lower surface, with conspicuous Mobile phase anhydrous formic acid R, glacial acetic acid R,
hairs on both surfaces. water R, ethyl acetate R (11 :11:27:100 VIVIVIV).
Drying In air.
Detection Heat at 100 °C for 5 min; treat the warm plate
with a 10 g/L solution of diphenylboric acid aminoethyl ester R
in methanol R followed by a 50 g/L solution of macrogol
400 R in methanol R; examine in ultraviolet light at 365 nm.
and the test solution. Furthermore, other fluorescent zones
may be present in the chromatogram obtained with the test
solution.
Top of the plate
-- --
Luteolin-7-glucoside: a yellow or A yellow or orange fluorescent zone
orange fluorescent zone (luteolin-7-glucoside)
Chlorogenic acid: a light blue A light blue fluorescent zone

fluorescent zone (chlorogenic acid)
-- --
TESTS
Total ash (2.4.16)
40 µm
Figure 1866.-1. - Illustration for identification test B of powdered powdered herbal drug (710) (2. 9.12) by drying in an oven at
herbal drug of artichoke leaf 105 °C for 2 h.
B. Microscopic examination (2.8.23). Reduce to a powder

ASSAY
( 1000) (2. 9.12). The powder is greenish-grey. Examine Liquid chromatography (2.2.29).
under a microscope using chloral hydrate solution R. Solvent mixture methanol R, water R (30:70 V/V).
The powder shows the following diagnostic characters Test solution To 0.500 g of the powdered herbal
(Figure 1866.-1): fragments of the epidermises of the lamina, drug (1000) (2.9.12) add 50.0 mL of methanol Rand heat
IV-96 Artichoke Leaf Preparations 2023
under a reflux condenser on a water-bath at 70 °C for 1 h.

Centrifuge and transfer the supernatant to a 200 mL
Artichoke Leaf Dry Extract
volumetric flask. Repeat the procedure and dilute to (Ph. Bur. monograph 2389)
200.0 mL with water R.
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 DEFINITION
solution to a volumetric flask, add 5 mL of methanol R and Dry extract produced from Artichoke leaf (1866).
dilute to 20.0 mL with water R. Content
Reference solution (b) Dissolve 30 mg of artichoke leaf dry Minimum 0.6 per cent of chlorogenic acid (C 16H 18 0 9;
extract HRS in the solvent mixture and dilute to 25.0 mL M, 354.3) (dried extract).
with the solvent mixture.
PRODUCTION
Column: The extract is produced from the herbal drug by a suitable
=
- size: l 0.25 m, 0 4.6 mm; = procedure using water of minimum 80 °C.
chromatography R (5 µm); CHARACTERS
- temperature: 40 °C. Appearance
Mobile phase: Light brown or brown, amorphous powder.
- mobile phase A: phosphoric acid R, water for IDENTIFICATION
chromatography R (0.5:99.5 V/V); Thin-layer chromatography (2.2.27).
- mobile phase B: phosphoric acid R, acetonitrile R Test solution Dissolve 1.0 g of the extract to be examined in
(0.5:99.5 VIV); 10 mL of ethanol (60 per cent Vlv,) R. Sonicate for 5 min and
filter.
(min) (per cent VIP) (per cent VIP) Reference solution Dissolve 5 mg of luteolin-7-glucoside Rand
0- I 92 8
5 mg of chlorogenic acid R in 10 mL of methanol R.
I - 20 92--+ 75 8--+ 25 Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
20 - 33 75 25 plate R (2-10 µm)].
33 - 35 75--+ 0 25--+ 100 Mobile phase anhydrous formic acid R, glacial acetic acid R,
water R, ethyl acetate R (11:11:27:100 VIVIVIV).
Flow rate 1.2 mUmin. Application 10 µL [or 2 µL] as bands of 10 mm [or 8 mm].
Detection Spectrophotometer at 330 nm. Development Over a path of 13 cm [or 6 cm].
Injection 25 µL. Drying In air.
System suitability Reference solution (b): Detection Heat at 100 °C for 5 min; spray the warm plate
- the chromatogram obtained is similar to the with a 10 g/L solution of diphenylboric acid aminoethyl ester R
chromatogram supplied with the artichoke leaf dry in methanol R followed by a 50 g/L solution of macrogol
extract HRS; 400 R in methanol R; examine in ultraviolet light at 365 nm.
- peak-to-valley ratio: minimum 2.5, where Hp = height Results See below the sequence of fluorescent zones present
above the baseline of the peak due to cryptochlorogenic in the chromatograms obtained with the reference solution
acid and Hv = height above the baseline of the lowest and the test solution. Furthermore, other fluorescent zones
point of the curve separating this peak from the peak due may be present in the chromatogram obtained with the test
to chlorogenic acid. solution.
Calculate the percentage content of chlorogenic acid using
the following expression: Top of the plate
-- --
A1 area of the peak due to chlorogenic acid in the chromatogram Luteolin-7-glucoside: a yellow or A yellow or orange fluorescent zone
obtained with the test solution; orange fluorescent zone (luteolin-7-glucoside)
A2 area of the peak due to chlorogenic acid in the chromatogram
solution, in grams;
m2 mass of ch/orogenic acid CRS used to prepare reference
solution (a), in grams;
p percentage content of chlorogenic acid in ch/orogenic acid CRS. -- --

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
TESTS
Total ash (2.4.16)
ASSAY
2023 Ash Leaf IV-97
Solvent mixture methanol R, water R (30:70 VIV).

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. Bur. 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 DEFINITION
solution to a volumetric flask, add 5 mL of methanol R and Dried leaf of Fraxinus excelsior L. or Fraxinus angustifolia Vah!
dilute to 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 dry 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 (C 16H 18 0 9 ;
- size: l = 0.25 m, 0 = 4.6 mm; Mr 354.3) (dried drug).
- stationary phase: end-capped octadecylsilyl silica gel for IDENTIFICATION
chromatography R (5 µm); 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 R, water for oblong, lanceolate, somewhat unequal at the base, acuminate
chromatography R (0.5:99.5 V/V); at the apex, with fine, acute teeth on the margins; the upper
- mobile phase B: phosphoric acid R, acetonitrile R surface is dark green and the lower surface is greyish-green.
(0.5:99.5 VIV); The midrib and secondary veins are whitish and prominent
on the lower surface.
Time Mobile phase A Mobile phase B B. Microscopic examination (2.8.23). The powder is greyish-
(min) (per cent V/V) (per cent V/ll)
green. Examine under a microscope using chloral hydrate
0- 1 92 8 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 (surface view [BJ), 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
Flow rate 1.2 mUmin. in surface view [A] consisting of cells covered by fine
Detection Spectrophotometer at 330 nm. cuticular striations [Aa], numerous anomocytic stomata
Injection 25 µL. (2.8.3) [Ab] and rare peltate glandular trichomes with a
unicellular stalk and a glandular head composed of radiating
cells [Ac]; fragments of lamina in transverse section [F] with
- the chromatogram obtained is similar to the
2 layers of palisade parenchyma [Fa], spongy
chromatogram supplied with the artichoke leaf dry
parenchyma [Fb] and, occasionally, glandular trichomes
extract HRS; embedded in the epidermis [Fe]; occasional multicellular,
- peak-to-valley ratio: minimum 2.5, where Hp = height
uniseriate, conical covering trichomes composed of cells with
above the baseline of the peak due to cryptochlorogenic
thick striated walls, either on an epidermis [C] or
acid and Hv = height above the baseline of the lowest fragmented [D]; fragments of vascular tissue from the
point of the curve separating this peak from the peak due
leaflets [E] composed of spiral vessels [Ea], short fibres [Eb]
to chlorogenic acid. and sometimes palisade parenchyma [Ee]; fragments of
Calculate the percentage content of chlorogenic acid using vascular tissue from the veins [G] composed of fibres [Ga],
the following expression: sometimes accompanied by cells with thick, pitted walls from
the medullary rays [Gb].
A1xm2xpx0.125
A 2 xm 1
Fraxinus omus.
A, area of the peak due to chlorogenic acid in the chromatogram Results See below the sequence of zones present in the
obtained with the test solution; chromatograms obtained with the reference solution and the
area of the peak due to chlorogenic acid in the chromatogram test solution. The intensity of the zones present in the
mass of the extract to be examined used to prepare the test chromatogram obtained with the test solution may vary
solution, in milligrams; depending on the presence of F. excelsior, F. angustifolia, their
mass of chlorogenic acid CRS used to prepare reference hybrids or their concentration in a mixture. Furthermore,
solution (a), in milligrams; other fluorescent zones may be present in the chromatogram
p percentage content of chlorogenic acid in chlorogenic acid CRS.
obtained with the test solution.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
IV-98 Astragalus Mongholicus Root 2023

Drying In air.
Detection Heat at 100 °C for 3 min; treat the still-warm
plate with a 10 g/L solution of diphenylboric acid aminoethyl
ester R in methanol R; dry in air; treat with a 50 g/L solution
of macrogol 400 R in methanol R; dry in air; examine in
ultraviolet light at 365 nm.
does not show any intense light blue fluorescent zones in the
upper third of the chromatogram.
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
G a volumetric flask and dilute to 100.0 mL with ethanol
(50 per cent V/V) R.
25 µm Test solution (b) To 1.0 mL of test solution (a) in a test
1---1
Gb tube, add 2 mL of 0. 5 M hydrochloric acid, 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 R; mix.
herbal drug of ash leaf
Immediately measure the absorbance (2.2.25) of test
solution (b) at 525 nm, using as compensation liquid a
Top of the plate solution prepared as follows: mix 1.0 mL of test solution (a),
2 mL of 0.5 M hydrochloric acid, 2 mL of dilute sodium
- - - -
hydroxide solution Rand dilute to 10.0 mL with water R.
A light blue fluorescent zone Calculate the percentage content of total hydroxycinnamic
(acteoside)
acid derivatives, expressed as chlorogenic acid, using the
Chlorogenic acid: a light blue A light blue fluorescent zone may be following expression:
fluorescent zone present (chlorogenic acid)
Ax5.3
- - --
m
Rutoside: an orange fluorescent zone An orange fluorescent zone taking the specific absorbance of chlorogenic acid to be 188.
(rutoside)
Reference solution Test solution m mass of the herbal drug to be examined, in grams.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
TESTS
Maximum 3.0 per cent of stems and maximum 2.0 per cent
of other foreign matter.
Fraxinus ornus
Astragalus Mongholicus Root
Thin-layer chromatography (2.2.27). (Ph. Bur. monograph 2435)
Test solution To 1 g of the powdered herbal drug (355) Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
(2. 9.12) add 20 mL of methanol R. Stir with a magnetic
stirrer for 10 min. Filter. DEFINITION
Reference solution Dissolve 5 mg of rutoside trihydrate R and Whole or fragmented, dried root of Astragalus mongholicus
5 mg of chlorogenic acid R in 10 mL of methanol R. Bunge with secondary roots and root crown removed.
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel Content
plate R (2-10 µm)]. Minimum 0.040 per cent of astragaloside N (C 41 H 68 0 14;
Mobile phase anhydrous formic acid R, water R, ethyl acetate R Mr 785) (dried drug).
(10:10:80 VIVIV).
2023 Astragalus Mongholicus Root IV-99
IDENTIFICATION containing octadecylsilyl silica gel for chromatography R

A. Cylindrical, often with branches, upper part relatively previously conditioned with 3 mL of methanol R and then
thick, 30-90 cm long and 1-3.5 cm in diameter. Externally with 3 mL of water R. Wash the column with 15 mL of
pale brownish-yellow or pale brown, with irregular, water R followed by 15 mL of a 30 per cent V/V solution of
longitudinal wrinkles or furrows. Texture hard and tenacious; methanol R. Discard the washings. Elute with 20 mL of
uneasily broken, fracture highly fibrous and weakly methanol R and collect the eluate. Evaporate the eluate under
(cultivated origin) or strongly starchy (wild origin), bark reduced pressure to dryness and take up the residue with
yellowish-white, wood pale yellow, with radiate striations and 2 mL of methanol R.
fissures; the central region is dark brown and in older roots Reference solution Dissolve 10.0 mg of daidzin Rand 5.0 mg
may be broken down to form a hollow surrounded by of daidzein R in 5.0 mL of methanol R.
fragments of disintegrating tissue. Plate TLC silica gel F254 plate R (2-10 µm).
Abt?; (10:13.5:100 VIV!V).

I, I I\ I
Drying In air.
! A 11
i ' \ Results A See below the sequence of zones present in the
\\\ I
~
I Ca
Top of the plate
1 Aa Daidzein: a quenching zone

\ ·.,,
A quenching zone
\g
\ ..' F
-- --
; ~
id A quenching zone
Daidzin: a quenching zone A quenching zone

H
-- --

25µni'
Detection B Treat with anisaldehyde solution R. Heat at
100 °C for 3 min. Examine in ultraviolet light at 366 nm.
herbal drng of astragalus mongholicus root Results B See below the sequence of zones present in the
yellowish-white. Examine under a microscope using chloral in the chromatogram obtained with the test solution.
characters (Figure 2435.-1): fibres, isolated [A] or in groups Top of the plate
[C], 8-30 µm in diameter, with thick walls having
A violet zone
longitudinal striations on the surface, the primary walls [Aa]
sometimes separated from the secondary walls [AbJ with the Daidzein: a pale blue zone
ends of the fibres often broken and fibrous [Ac], rounded or
A violet zone
slightly truncated [Ca]; vessels [G] are annular [Ga],
reticulate [Gb] or bordered-pitted with closely spaced -- --
pits [DJ, colourless or orange; rare sclereids [F], rounded,
A violet zone
oblong or irregular, with slightly thickened walls; cork
fragments with polygonal cells (surface view [BJ); numerous Daidzin: a pale blue zone A brown zone
medullary rays with thin-walled cells [EJ. Examine under a
5 brown zones
The powder shows small (less than 10 µm), simple, rounded -- --
or ovoid starch granules, usually included in parenchyma
cells [HJ.
Test solution Heat 3 g of the powdered herbal drug (355) TESTS
(2. 9.12) with 50 mL of methanol R for 50 min under reflux Foreign matter (2.8.2)
and then filter. Evaporate the filtrate under reduced pressure Maximum 5 per cent.
to dryness and take up the residue in 1 mL of water R. Apply
the solution to a 6 mL solid phase extraction column
IV-100 Atractylodes Lance a Rhizome 2023
Loss on drying (2.2.32) Injection 20 µL of the test solution and reference

Maximum 10.0 per cent, determined on 1.000 g of the solutions (b), (c), (d) and (e).
powdered herbal drug (355) (2. 9. 12) by drying in an oven at Relative retention With reference to ginsenoside Rb 1
105 °c for 3 h. (retention time = about 33.6 min):
Total ash (2.4.16) astragaloside N = about 1.05.
Maximum 5.0 per cent. System suitability:
Ash insoluble in hydrochloric acid (2.8.1) - resolution: minimum 4.0 between the peaks due to
Maximum 1.0 per cent. astragaloside N and ginsenoside Rb 1 in the
chromatogram obtained with reference solution (e).
ASSAY
Establish a calibration curve with the logarithm of the
concentration (mgimL) of reference solutions (b), (c) and (d)
Test solution To 4.000 g of the powdered herbal drug (355) (corrected by the declared percentage content of
(2.9.12) add 25 mL of methanol Rand sonicate at 80 °C for astragaloside IV CRS) as the abscissa and the logarithm of the
30 min. Centrifuge for 7 min and transfer the supernatant to corresponding peak area as the ordinate. Calculate the
a round-bottomed flask. Add to the residue 25 mL of percentage content of astragaloside N using the following
methanol R and sonicate at 80 °C for 30 min. Centrifuge for expression:
7 min and add the supernatant to the same round-bottomed
flask. Repeat this operation once. Combine the 3 fractions of l<Y1 X 0.5
supernatant and evaporate to dryness. Dissolve the residue in
m
10 mL of water R using sonication if necessary. Transfer the
solution to a separating funnel and shake with 4 quantities, A logarithm of the concentration corresponding to the
each of 40 mL, of butanol R saturated with water R. Combine astragaloside IV peak in the chromatogtam obtained with the
test solution, determined from the calibration curve;
the butanol extracts and wash with 2 quantities, each of
m mass of the herbal drug to be examined used to prepare the test
40 mL, of ammonia R. Discard the ammonia layers and solution, in grams.
evaporate the butanol layers to dryness. Dissolve the residue
in 5.0 mL of methanol Rand filter through a membrane filter
(nominal pore size 0.45 µm).
Reference solution (a) Dissolve 10.0 mg of
astragaloside IV CRS in methanol R and dilute to 10.0 mL
with the same solvent. Atractylodes Lancea Rhizome
Reference solutions (b), (c), (d) Dilute reference solution (a)
to obtain 3 reference solutions of astragaloside N, the (Ph. Bur. monograph 2559)
concentrations of which span the expected value in the test
solution.
Reference solution (e) Dissolve 5.0 mg of ginsenoside Rbl R in DEFINITION
5 mL of methanol Rand dilute to 10.0 mL with reference Dried, whole or fragmented rhizome of Atractylodes
solution (a). lancea (Thunb.) DC. (syn. Atractylodes chinensis (Bunge)
Koidz.) with the roots removed, collected in spring and
Column:
autumn.
- size: l = 0.25 m, 0 = 3.2 mm;
- stationary phase: octadecylsilyl silica gel for chromatography R Content
(3 µm); Minimum 14 mL'kg of essential oil (anhydrous drug).
- temperature: 25 °C. IDENTIFICATION
Mobile phase: A. WhDle drug. The whole rhizome is somewhat curved,
- mobile phase A: water R; irregularly moniliform or nodular-cylindrical, occasionally
- mobile phase B: acetonitrile R; branched, 3-10 cm long and 1-3 cm in diameter.
The external surface is wrinkled and irregularly transversely
Time Mobile phase A Mobile phase B segmented, dark greyish-brown or yellowish-brown; it shows
(min) (per cent V/V) (per cent V/V) numerous rounded protuberances and large circular stem
0-5 90 10 scars and smaller root scars. The texture is hard, easily
5 - 10 90---+ 80 10---+ 20 broken; the fractured surface is yellowish-white or brownish-
10 - 20 80---+ 75 20---+ 25 yellow, uneven, with many glistening yellowish-orange or
20 - 30 75 ---+ 67 25---+ 33 reddish-brown oil cavities appearing as dots scattered
30 - 40 67---+ 65 33---+ 35 throughout the tissue.
40 - 50 65---+ 40 35---+ 60 Fragmented drug The fragmented rhizome occurs in
50 - 55 40 60 transverse or longitudinal slices with a highly variable
diameter (1-4 cm) and a thickness up to 0.5 cm.
Flow rate 0.5 mL'min. The external surface is wrinkled, dark greyish-brown or
Detection Evaporative light-scattering detector; the following yellowish-brown and shows numerous small rootlet scars.
settings have been found to be suitable; if the detector has The cut surface is whitish-yellow or brownish-yellow, with
different setting parameters, adjust the detector settings so as particularly abundant yellowish-orange or reddish-brown oil
to comply with the system suitability criterion: cavities appearing as dots scattered throughout the tissue.
- carrier gas: air; Occasionally, some cavities crystallise out as fine white
- flow rate: 1.5 mL'min; needle- or hair-like crystals.
- evaporator temperature: 50 °C. B. Microscopic examination (2.8.23). The powder is
brownish-yellow. Examine under a microscope using chloral
2023 Atractylodes Lancea Rhizome IV- I O1
hydrate solution R. The powder shows the following diagnostic Top of the plate
characters (Figure 2559.-1): fragments of orange cork with
P-Caryophyllene: a pink zone A pink or violet zone
polyhedral cells (surface view [Kl); fragments of dermal
tissue (transverse section [H]), consisting of several layers of An orange zone may be present
cork [Ha] often accompanied by subrectangular or ovoid
--
sclereids from the phelloderm [Hb] with very thick, - -
channelled walls and a narrow lumen; fragments of An intense greyish-green zone

phelloderm containing groups of sclereids (surface view fJ]);
A very faint violet zone
isolated sclereids, variable in shape (20-51 µm in
diameter) [B, G]; fragments ofparenchyma with polyhedral, -- --
subrectangular or subrounded cells containing small needle-
Bomyl acetate: a brown zone A violet zone
shaped crystals of calcium oxalate (5-30 µm in length) clearly
visible in polarised light [D]; fragments of fibres in bundles, Several violet zones
with heavily thickened and slightly pitted walls (up to 40 µm
in diameter) and a narrow lumen, very often associated with
xylem vessels [A]; fragments of short, reticulate or pitted
vessels, usually included in parenchyma with thin-walled TESTS
cells [F]; fragments of oil glands with thin-walled cells and Atractylodes macrocephala
orange-brown contents [C] accompanied by parenchyma Thin-layer chromatography (2.2.27).
cells containing small needle-shaped crystals of calcium Test solution Introduce 0.5 g of the powdered herbal drug
oxalate [Ca]. Examine under a microscope, without heating, (355) (2. 9. 12) into a centrifuge tube, add 2 mL of
using glycerol R. The powder shows pieces of inulin, free [E] methanol R and stopper the tube. Sonicate at 25 °C for
or included in parenchyma cells. 15 min and centrifuge.
Reference solution Dissolve 10 mg of f]-caryophyllene R and
10 mg of bornyl acetate R in 5 mL of methanol R.
Mobile phase ethyl acetate R, heptane R (5:95 V/V).
Development In an unsaturated tank, over a path of 10 cm
[or 6 cm].
Drying In air.
Detection Treat with anisaldehyde solution R and heat at
105-110 °C for 5-10 min; examine in daylight.
,,G Results The chromatogram obtained with the test solution

shows an intense greyish-green zone in the middle third.
In the case of a substitution by Atractylodes macrocephala, no
intense greyish-green zone is present in the middle third.
Water (2.2.13)
Maximum 100 mUkg, determined on 20.0 g of the
powdered herbal drug (355) (2. 9. 12).
'
~~I Total ash (2.4.16)
J-·
ASSAY
Use 15.0 g of freshly powdered herbal drug (710) (2.9.12), a
500 mL round-bottomed flask, 200 mL of water Ras the
distillation liquid and 0.50 mL of xylene R in the graduated
tube. Distil at a rate of 2-3 mUmin for 2 h.
Figure 2559.-1. - Illustration for identification test B of powdered _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
herbal drug of Atractylodes lancea rhizome

Atractylodes macrocephala.
IV- I 02 Atractylodes Rhizome 2023
Largehead Atractylodes Rhizome

DEFINITION
Dried, whole or fragmented rhizome of Atractylodes
macrocephala Koidz. with the roots removed, collected in
winter when the lower leaves of the plant tum yellow and the
upper leaves become fragile.
Content
Minimum 9 mIJkg of essential oil (anhydrous drug).
IDENTIFICATION
A. Whole drng. The whole rhizome is irregularly shaped,
3-13 cm long and 1.5-7 cm in diameter, externally yellowish
greyish-brown or dark brown. Small knob-like protrusions are
present, concentrated at the rhizome apex and interspersed
with straighter narrower regions, whose outer surface is
covered with longitudinal wrinkles and grooves. The texture
is hard, difficult to break, and the fracture is uneven with
wide spaces between the tissues, especially in the central
region. The fracture is whitish-yellow to brownish, with
yellowish-brown to orange oil cavities scattered throughout,
particularly abundant in the external tissues. A greyish-brown
cambium is occasionally visible.
Fragmented drng The fragmented rhizome mostly occurs as
longitudinal slices with a highly variable diameter (1-7 cm)
and a thickness of about 0.5 cm. The external surface is
wrinkled or grooved, more or less dark yellowish greyish- Figure 2560.-1. - Illustration for identification test B of powdered
brown with root scars and knob-like protrusions. The texture herbal drng of Atractylodes rhizome, largehead
is hard. The cut surface is whitish-yellow to brownish,
consisting of tissues with wide spaces between them and C. Examine the chromatograms obtained in the test for
scattered with many yellowish-brown to orange oil cavities A tractylodes lancea.
appearing as dots that are particularly abundant in the Results See below the sequence of zones present in the
external tissues. chromatograms obtained with the reference solution and the
brownish-yellow. Examine under a microscope using chloral in the chromatogram obtained with the test solution.
characters (Figure 2560.-1): fragments of orange cork Top of the plate
(surface view [A], transverse section [B]), with superimposed
cells [Aa, Ba] sometimes accompanied by parenchyma cells ~-Caryophyllene: a pink zone A pink or violet zone
containing fine needles of calcium oxalate [Ab, Bb]; An orange zone
fragments of parenchyma with polyhedral, subrectangular or
subrounded cells, many of which contain small needle- -- --
shaped crystals of calcium oxalate (10-32 µm in length) A very faint violet zone
clearly visible in polarised light [El; sclereids, isolated or in
small groups, with very thick, channelled walls, variable in -- --
shape (35-65 µm in diameter) [D, F]; larger sclereids often Bomyl acetate: a brown zone
with thinner walls and a large lumen; rare fragments of
A very faint violet zone
fibres, isolated or in bundles, with moderately thickened and
slightly pitted walls (up to 40 µm in diameter) [C]; fragments Several faint violet zones
of xylem m consisting of short, reticulate or pitted
vessels Ua], usually included in parenchyma with thin-walled
cells Ub]; oil glands [G] or fragments of oil glands with thin-
walled cells [Ga] and orange-brown contents, accompanied D. To 0.5 g of the powdered herbal drug (355) (2.9.12) add
by parenchyma cells containing small, needle-shaped crystals 5 mL of ethanol (96 per cent) R, heat in a water-bath at 60 °C
of calcium oxalate [Gb]. Examine under a microscope, for 2 min and filter. To 1 mL of the filtrate add 0.25 mL of
without heating, using glycerol R. The powder shows a solution freshly prepared as follows: dissolve 5 mg of
abundant pieces of inulin, free [H] or included in vanillin R in 0.5 mL of ethanol (96 per cent) R, to this
parenchyma cells. solution add 0.5 mL of water R and 3 mL of hydrochloric
acid R. Shake immediately; a red or reddish-purple colour
develops and persists.
TESTS
Atractylodes lancea
2023 Aucklandia Root IV-103
Test solution Introduce 0.5 g of the powdered herbal drug scattered throughout, radially striated xylem, and a brown or
(355) (2.9.12) into a centrifuge tube, add 2 mL of greyish-brown cambium. The texture is hard, dense and
methanol Rand stopper the tube. Sonicate at 25 °C for difficult to break.
15 min and centrifuge. Fragmented drug It occurs as subrounded or irregular
Reference solution Dissolve 10 mg of P-caryophyllene R and transverse slices, oblique or longitudinal slices or as small,
10 mg of bornyl acetate R in 5 mL of methanol R. irregular pieces. The slices are mostly about 5 mm thick,
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel 6-33 mm in diameter, externally greyish-brown or brownish-
plate R (2-10 µm)]. yellow, with longitudinal wrinkles and furrows and occasional
lateral root scars. The cut surface has narrow, radially
Mobile phase ethyl acetate R, heptane R (5:95 VIV).
striated xylem with fine clefts occasionally present, which do
Application 5 µL [or 3 µL] as bands of 10 mm [or 6 mm]. not extend into the centre; the brownish cambium is mostly
Development In an unsaturated tank, over a path of 10 cm distinct; dark brown or greyish-brown annulations are
[or 6 cm]. occasionally visible at intervals. There are scattered dots and
Drying In air. oil cavities, that are brownish-yellow, reddish-brown or dark
Detection Treat with anisaldehyde solution R and heat at brown in colour. The peripheral outer layer is mostly
105-110 °C for 5-10 min; examine in daylight. yellowish-brown or greyish-brown. The older roots have a
broad pith sometimes forming a hollow. The texture is hard
and dense.
shows no greyish-green zone in the middle third, above the
very faint violet zone. B. Microscopic examination (2.8.23). The powder is
yellowish-green or yellow-brown. Examine under a
Water (2.2.13)
powdered herbal drug (710) (2.9.12).
yellowish-brown cork fragments consisting of several layers of
Total ash (2.4.16) thin-walled cells (surface view [BJ, transverse section [E]);
Maximum 5.0 per cent. fragments of oil canals with orange-brown granular contents,
Ash insoluble in hydrochloric acid (2.8.1) some about 50 µm in diameter; numerous fragments of
Maximum 1.0 per cent. parenchyma with ovoid cells [F, DJ, some associated with oil
canal fragments (transverse section [Da]); fragments of xylem
ASSAY
[A, G] with dense, cellulosic xylem parenchyma [Aa, Gb],
and mostly reticulate vessels [Ac, Ga) though occasionally
pitted, lignified or slightly lignified, and sometimes associated
500 mL round-bottomed flask, 200 mL of water R as the
with oil canal fragments (longitudinal section (Ab));
fragments of medullary ray, composed of aligned rectangular
tube. Distil at a rate of 2-3 mUmin for 2 h.
cells (longitudinal section m); occasional bundles of long,
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
fusiform fibres, usually fragmented, about 20 µm in diameter,
with walls showing slit-, V- or Y-shaped pits (surface view
[CJ). Examine under a microscope using a solution of
glycerol R. The powder shows abundant inulin fragments,
Aucklandia Root irregular, mostly angular, some wing-like, either free [HJ or
included in parenchymatous cells [Kl.
(Ph. Bur. monograph 1797) C. Examine the chromatograms obtained in the test for
Vladimiria souliei (Franch.) Y. Ling and Inula helenium L.
DEFINITION Results See below the sequence of zones present in the
Whole or fragmented dried root of Saussurea costus (Falc.)
test solution. Furthermore, other faint pink zones may be
Llpsch. (syn. Saussurea Zappa C.B.Clarke, Aucklandia Zappa
present below the zone due to costunolide in the
Decne., Aucklandia costus Falc.). It is collected in winter and
spring, and the rootlets removed.
Content
Top of the plate
Minimum 0.6 per cent of costunolide (C 15H 200 2 ; M, 232.3)
and minimum 1.8 per cent for the sum of costunolide and A faint pink zone
dehydrocostus lactone (C 15 H 18 0 2 ; M, 230.3) (dried drug).
-- - -
IDENTIFICATION
-- --
A. Whole drug. It consists of long, sub-cylindrical or sub-
conical, slightly flattened, occasionally branched pieces, Dehydrocostus lactone; a violet zone A violet zone (dehydrocostus
4-12 cm in length, 0.5-5 cm in diameter, with some pieces lactone)
slightly bent, and sometimes longitudinally cut. The stem Costunolide: a greyish-violet zone A greyish-violet zone (costunolide)
scar forms a dent at the crown. The outer surface is
yellowish-brown or greyish-brown, with coarse, longitudinal
wrinkles and furrows, and also additional fine, reticulate
furrows. The remains and scars of lateral roots are also
present. The bark is tightly adhering; occasional roots are
without bark, and have an exposed surface without a distinct
network of vessels. The fractured surface has brownish-
yellow, reddish-brown or dark brown oil dots and cavities
IV-104 Aucklandia Root 2023
Test solution Disperse 0.300 g of the freshly powdered

herbal drug (355) (2.9.12) in 50.0 mL of methanol Rina
conical flask, stopper tightly and weigh. Sonicate for 30 min
and shake for 1 h. Allow to cool and weigh again.
Compensate the loss of solvent with methanol R and mix
thoroughly. Filter through a membrane filter (nominal pore
size 0.45 µm).
Reference solution (a) Dissolve 5.0 mg of costunolide CRS in
5 mL of methanol R, shake vigorously, dilute to 50.0 mL with
the same solvent and mix thoroughly.
Reference solution (b) Dissolve 2.5 mg of dehydrocostus
lactone R in 5 mL of reference solution (a), shake vigorously,
dilute to 25.0 mL with reference solution (a) and mix
thoroughly.
Column:
- size: l = 0.15 m, 0 = 4.6 mm;
- stationary phase: end-capped octadecylsil,yl silica gel for
chromatography R (5 µm).
Mobile phase water for chromatography R, methanol R
(35:65 V/V).
Flow rate 1 mUmin.
Injection 10 µL.
Run time 20 min.
Retention time Costunolide = about 8 min; dehydrocostus
lactone = about 10 min.
Figure 1797.-1. - Illustration for identification test B of powdered costunolide and dehydrocostus lactone.
herbal drug of aucklandia root Calculate the percentage content of costunolide using the
TESTS
Vladimiria souliei (Franch.) Y. Ling and Inula
helenium L.
A1 area of the peak due to costunolide in the chromatogram
Test solution To 0.5 g of the powdered herbal drug (355) obtained with the test solution;
(2. 9.12) add 10 mL of methylene chloride R and sonicate for A2 area of the peak due to costunolide in the chromatogram
30 min; filter. obtained with reference solution (a);
Reference solution Dissolve 5 mg of dehydrocostus lactone R solution, in grams;
and 5 mg of costunolide R in methylene chloride R and dilute to m2 mass of costunolide CRS used to prepare reference solution (a),
10 mL with the same solvent. in grams;
p percentage content of costunolide in costunolide CRS.
Mobile phase cyclohexane R, methylene chloride R (1:5 V/V). Calculate the percentage content of the sum of costunolide
Application 2 µL as bands of 8 mm. and dehydrocostus lactone using the following expression:
(A1 + (1.52 xA3)) x m2 xp
Drying In air.
A2 x m1
100 °C for 5 min; examine in daylight. area of the peak due to costunolide in the chromatogram
area of the peak due to costunolide in the chromatogram
shows no zone in the middle third, and no violet zone obtained with reference solution (a);
directly above the zone due to dehydrocostus lactone. area of the peak due to dehydrocostus lactone in the
chromatogram obtained with the test solution;
Aristolochic acids (2.8.21, Method A) mass of the herbal drug to be examined used to prepare the test
It complies with the test. solution, in grams;
Loss on drying (2.2.32) mz mass of costunolide CRS used to prepare reference solution (a),
in grams;
Maximum 12.0 per cent, determined on 1.000 g of the p percentage content of costunolide in costunolide CRS;
powdered herbal drug (355) (2. 9.12) by drying in an oven at 1.52 correlation factor between dehydrocosrus lactone and
105 °C for 2 h. costunolide.
Total ash (2.4.16)
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
ASSAY
2023 Azadirachta Indica Leaf IV-105
0.6 corresponding in position to a purple band in the

Azadirachta lndica Leaf chromatogram for solution (2) is obtained for /3-sitosterol.
Nimba Leaf
DEFINITION Top of the plate
Azadirachta Indica Leaf is the dried leaf of Azadirachta indica
A. Juss.
It contains not less than 1.0% of tetranortriterpinoids,
expressed as salannin, calculated with reference to the dried
Purple band 13-sitosterol: a purple
drug. band
IDENTIFICATION
A. Leaflets thin and fragile, ovate to lanceolate, 3 to 10 cm
long and 1 to 2.5 cm wide, curved with a serrate margin;
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 Black band Salannin: an indigo
the margins; both surfaces glabrous. Fragments of the rachis band
may be present; these are pale brown, slender, up to about
10 cm long, cylindrical with faint longitudinal striations and
Black band Azadirachtin: a brown
bearing alternating pairs of scars where the leaflets were band
attached.
B. Reduce to a powder (355). The powder is green. Examine
Solution (1) Solution (2)
under a microscope using chloral hydrate solution. The powder
shows fragments of the epidermis composed of thin-walled
tangentially elongated cells with abundant anomocytic
TESTS
stomata, Appendix XI H; abundant fragments of single
Foreign matter
layered palisade and thin-walled parenchymatous cells of the
Not more than 2%, Appendix XI D.
spongy mesophyll present, some with associated vessels; some
fragments display rosette crystals of calcium oxalate often in Loss on drying
rows. When dried for 2 hours at 105°, loses not more than 10.0%
C. Carry out the method for thin-layer chromatography, of its weight. Use 1 g.
Appendix III A, using the following solutions. Ash
(1) Add 30 mL of methanol to approximately 5 g of Not more than 10.0%, Appendix XI J, method II.
powdered herbal drug, mix thoroughly by hand and with the Water-soluble extractive
aid of ultrasound for 30 minutes. Centrifuge at 3000 rpm for Not less than 20.0%, Appendix XI B2.
5 minutes and collect the clear supernatant liquid. Repeat the ASSAY
extraction twice, combine the supernatant liquid and dilute
Carry out the method for liquid chromatography,
to 100 mL with methanol. Filter approximately 30 mL of the
Appendix III D, using the following solutions.
solution through a 0.45-µm filter and use the filtrate.
(1) Add 30 mL of methanol to approximately 5 g of
(2) 0.025% w/v each of azadirachtin, salannin CRS and
powdered herbal drug, mix thoroughly by hand and with the
[3-sitosterol in methanol.
aid of ultrasound for 30 minutes. Centrifuge at 3000 rpm for
CHROMATOGRAPHIC CONDITIONS 5 minutes and collect the clear supernatant liquid. Repeat the
(a) Use a silica gel 60 or high-performance silica gel 60 extraction twice, combine the supernatant liquid and dilute
precoated plate [Merck silica gel 60 HPTLC plates are to 100 mL with methanol. Filter approximately 30 mL of the
suitable]. solution through a 0.45-µm filter and use the filtrate.
(b) Use the mobile phase as described below. (2) 0.0025% w/v of salannin CRS and 0.001 % w/v of
(c) Apply as bands 5 µL of each solution. azadirachtin-A CRS in methanol.
(d) Develop the plate to 15 cm [or 7 cm]. CHROMATOGRAPHIC CONDITIONS
(e) After removal of the plate, spray with vanillin reagent, heat (a) Use a stainless steel column (15 cm x 2.1 mm) packed
the plate at 100° for 3 minutes and examine in daylight. with octadecylsilyl silica gel for chromatography (5 µm)
(Spherisorb ODS! is suitable).
MOBILE PHASE
(b) Use gradient elution and the mobile phase described
3 volumes of hexane and 7 volumes of ethyl acetate.
below.
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with (d) Use a column temperature of 30°.
solution (2) shows three clearly separated spots.
(e) Use a detection wavelength of 217 nm.
CONFIRMATION
(t) Inject 10 µL of each solution.
In the chromatogram obtained with solution (1), a black
When the chromatograms are recorded under the prescribed
band with an Rf value of approximately O.15 corresponding
conditions the retention time of the peak due to azadirachtin-
in position to a brown band in the chromatogram for
A is about 15 minutes and the retention time of the peak due
solution (2) is obtained. A black band with an Rf value of
to salannin is about 22 minutes.
approximately 0.3 corresponding in position to the indigo
band in the chromatogram for solution (2) is obtained for
salannin. An indigo band with an Rf value of approximately
IV-106 Bacopa Monnieri 2023
MOBILE PHASE 3 to 8 mm wide, reniform, spathulate or oblanceolate,

Mobile phase A 0.1 volume of trifiuroacetic acid and margin entire or, rarely, dentate. If present, flowers are
100 volumes of water. axillary and solitary, on peduncles usually longer than the
Mobile phase B 0.1 volume of trifiuroacetic acid and leaves; corolla up to 1 cm long, five lobed, oblong, obtuse;
100 volumes of acetonitrile. fruit capsule ovoid-acuminate or slightly beaked at the apex,
glabrous, up to 5 mm long.
Time Mobile phase A Mobile phase B Comment B. Reduce to a powder (355). The powder is greenish or
(Minutes) (% v/v) (%v/v) yellowish-brown. Examine under a microscope using chloral
0-10 90➔70 10➔ 30 linear gradient
hydrate solution. The powder shows fragments of the
epidermis, with a thin striated cuticle, multicellular glandular
10-25 70➔30 30➔ 70 linear gradient
trichomes, anomocytic stomata; numerous xylem vessels with
25-40 30 70 isocratic reticulate thickening. Examine under a microscope using
40-45 30 ➔90 70➔ 10 linear gradient 50% v/v of glycerol in water. Starch granules are present,
45-50 90 10 re-equilibration
usually simple, round or ovoid, 4 to 14 µm in diameter,
without a visible hilum.
C. Carry out the method for thin-layer chromatography,
SYSTEM SUITABILITY
Appendix III A, using the following solutions.
(1) Add 30 mL of methanol (70%) to 5.0 g of the powdered
with solution (2):
herbal drug, heat on a water-bath under reflux for
the symmetry factor of the peak due to azadirachtin-A is at 30 minutes, cool, centrifuge at 3000 rpm for 5 minutes and
most 1.2; decant the supernatant liquid. Repeat the extraction
the symmetry factor of the peak due to salannin is at most 1.4. procedure with a further two 30-mL quantities of methanol
DETERMINATION OF CONTENT (70%). Combine the supernatant liquid, dilute to 100 mL
with methanol (70%) and filter (0.45 µm PTFE is suitable).
Calculate the total content of tetranortriterpinoids, expressed
as salannin, from the sum of the areas of the peaks eluting (2) 0.05% w/v of bacopaside II CRS in methanol (70%).
from three minutes before to three minutes after the (3) 0.05% w/v each of bacopaside I CRS and bacopaside
retention time of salannin and from the declared content of II CRS in methanol (70%).
salannin in salannin CRS using the following expression: CHROMATOGRAPHIC CONDITIONS
(a) Use silica gel 60 precoated plates or high-performance
A1 X --
--
m
2 X __
IX
V 100
p X ---- silica gel 60 (Merck silica gel 60 plates are suitable).
A2 v2 ml 100 - d (b) Use the mobile phase as described below.
(c) Apply 20 µL [or 10 µL] of each solution, as bands.
(d) Develop the plate to 15 cm [or 8 cm] .
combined areas of the peaks in the chromatogram obtained with (e) After removal of the plate, dip in anisaldehyde solution RJ,
solution (I) with retention times from three minutes before to heat in an oven at 105° for 5 minutes and examine in
three minutes after the retention time of peak due to salannin in daylight.
the chromatogram obtained with solution (2),
area of the peak due to salannin in the chromatogram obtained MOBILE PHASE
with solution (2),
m1 weight of the drug being examined in mg,
10 volumes of 1% v/v of formic acid, 20 volumes of methanol
m2 weight of sa/annin CRS in mg, and 70 volumes of ethyl acetate.
V1 dilution volume of solution (1),
SYSTEM SUITABILITY
V2 dilution volume of solution (2),
p percentage content of salannin in salannin CRS, The test is not valid unless the chromatogram obtained with
d percentage loss on drying of the herbal drug being examined. solution (3) shows two clearly separated bands.
CONFIRMATION
STORAGE
The chromatogram obtained with solution (1) shows a dark
Azadirachta Indica Leaf should be protected from moisture.
band with an Rf value of approximately 0.4 corresponding to
the band obtained with bacopaside II in the chromatogram
obtained with solution (3), a lighter band with an Rf value of
approximately 0.3 corresponding to the band obtained with
Bacopa Monnieri bacopaside I in the chromatogram obtained with solution (3).
DEFINITION Bands with Rf values of approximately 0.2 and 0.8 are also
Bacopa Monnieri is the dried aerial parts of Bacopa monnieri present. Other bands may be present in solution (1).
(L.) Wettst.
It contains not less than 1.0% w/w of bacopa saponins,
expressed as bacopaside II (C 47 H 760 8 ), calculated with
reference to the dried drug.
IDENTIFICATION
A. Pieces of herb, consisting mainly of stem and leaf; buff or
greenish brown, angular stems, 1 to 2 mm in diameter and
10 to 30 cm long, nodes prominent, often showing sprouting
rootlets and with numerous ascending branches; greenish
leaves sessile or short petioled, fleshy, glabrous on the upper
surface, simple, opposite, decussate, 0.6 to 2.5 cm long and
2023 Barbary Woltberry Fruit IV-107
Top of the plate to bacoside A3 and bacopaside II 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 in bacopaside II CRS.
bacopaside II: dark band bacopaside II: dark band bacopaside II: dark band
bacopaside I: 1;ght band bacopaside I: nght band
unknown: light band

Barbary Wolfberry Fruit
Solution (1) Solution (2) Solution (3)
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 (Figure 2612. -1): fragments of the
further 30-mL quantities of methanol (70%). Combine the epicarp (surface view [Al) with polygonal or elongated cells,
three supernatant liquids, dilute to 100 mL with methanol about 60 µm in diameter, with straight or slightly wavy walls,
(70%) and filter (0.45 µm PTFE is suitable). covered with a thick cuticle, with distinct, more or less
(2) 0.05% w/v of bacopaside II CRS in methanol (70%). parallel striations; fragments of the epicarp (transverse
section [F]) covered by a crenate cuticle [Fa], consisting of 1
(3) 0.05% w/v of bacoside A CRS in methanol (70%).
or 2 layers of cells [Fb] associated with the mesocarp [Fe];
CHROMATOGRAPHIC CONDITIONS fragments of the mesocarp [C] with thin-walled subpolygonal
(a) Use a stainless steel column (25 cm x 4.6 mm) packed cells containing reddish-orange or brownish-red spherical
with end-capped octadecylsilyl silica gel for chromatography granules [Ca, Fd] or microsphenoidal crystals of calcium
(5 µm) (Phenomenex Luna ClS is suitable). oxalate [Cb]; fragments of the seeds (surface view [B]) with a
(b) Use isocratic elution and the mobile phase described testa consisting of greenish-yellow sclereids with heavily
below. thickened, striated and lobed walls and (transverse
section [El) with a testa having thin external walls [Ea] and
deeply lobed, irregularly thickened, striated, radial internal
(d) Use a column temperature of 30°. walls [Eb]; fragments of endosperm containing oil
(e) Use a detection wavelength of 205 nm. droplets [D]; fragments of vascular tissue with narrow, spiral
(f) Inject 20 µL of each solution. or annular vessels [G].
(g) Record the chromatograms for 7 5 minutes. C. Thin-layer chromatography (2.2.27).
MOBILE PHASE Test solution To 0.1 g of the powdered herbal drug (355)
315 volumes of acetonitrile and 685 volumes of 0.71 % w/v (2. 9.12) add 7 mL of water R. Sonicate for 10 min and
anhydrous sodium sulfate, previously adjusted to pH 2.3 with centrifuge. Prepare a ready-to-use sample preparation
suljuric acid. cartridge containing 0.50 g of octadecylsilyl silica gel (50 µm)
using 3 mL of methanol R, drying with a stream of air, then
When the chromatograms are recorded under the prescribed
using 3 mL of water R. The flow rate does not exceed
conditions the retention time of bacopaside II is about
6 mUmin. Apply 4 mL of the supernatant to the top of the
36 minutes. The retention times relative to bacopaside II are:
cartridge. Wash the cartridge twice with 1 mL of a mixture
bacoside A3 , about 0.9; bacopaside X, about 1.2;
of 1 volume of methanol R and 9 volumes of water R. Elute
bacopasaponin C, about 1.3; bacopaside I, about 1.4.
the cartridge with 1 mL of methanol R; collect the eluate and
SYSTEM SUITABILITY use it as the test solution.
with solution (3), the resolution factor between the peaks due
IV-108 Holy Basil Leaf 2023
- -
Top of the plate
) - . - -
,, . . . ,' \1>
Scopoletin: a bright blue fluorescent A blue fluorescent zone
. ,' ' -
zone (scopoletin)
_,·-~
-- --
2 blue fluorescent zones

(rutoside)
3-4 blue fluorescent zones
-- --
TESTS
E Loss on drying (2.2.32)
105 °C for 2 h.
Total ash (2.4.16)
Extractable matter
Minimum 55.0 per cent.
To 2.00 g of the powdered herbal drug (355) (2.9.12) add
50.0 g of water R, boil under reflux for 1 h, compensate the
loss of water and filter. Evaporate 25.0 g of the filtrate to
Figure 2612.-1. - Illustration for identification test B of powdered dryness on a water-bath and dry in an oven at 105 °C for
herbal drng of barbary wolfberry fruit
3 h. The residue weighs a minimum of 0.55 g.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Reference solution Dissolve 1 mg of scopoletin R in 10 mL of
methanol R. Dilute 1 mL of the solution to 10 mL with
methanol R to obtain solution (a). Dissolve 1 mg of rutoside
trihydrate R in 5 mL of solution (a).
Plate TLC silica gel F254 plate R (2-10 µm). Holy Basil Leaf
Mobile phase anhydrous formic acid R, glacial acetic acid R, DEFINTI10N
water R, ethyl acetate R (11:11:27:100 VIVIVIV). Holy Basil Leaf is the dried leaves of Ocimum tenuijlorum
Application 2 µL as bands of 8 mm. Linn. (syn. Ocimum sanctum). The leaf content is not less
Development Over a path of 6 cm. than 70% of the material harvested.
Drying In air. IDENTIFICATION
Detection Heat at 100 °C for 3 min; treat the still-warm A. Pieces of herb consisting mainly of whole or fragmented
plate with a 5 g/L solution of diphenylboric acid aminoethyl leaf, petiole and stem, with some flower parts, mainly calyx.
ester R in ethyl acetate R, then treat with a 50 g/L solution of Leaves simple, opposite, petiolate, ovate or elliptical with an
macrogol 400 R in methylene chloride R; examine in ultraviolet acute or obtuse apex, up to 5 cm long and 3.5 cm wide,
light at 365 nm after 5 min. green or purple, with an entire or serrated margin and
Results See below the sequence of zones present in the pubescent on both surfaces. Fragments of petiole and stem
chromatograms obtained with the reference solution and the twisted, hairy, purplish-brown or dark green-black,
test solution. Furthermore, other faint zones may be present subquadrangular, petiole thin up to 3 cm long, stem
in the chromatogram obtained with the test solution. herbaceous or woody and fibrous, thicker and highly
branched. Fragments of calyx, if present, membranous,
veined, 3 to 4 mm long, ovoid or campanulate bi-lipped with
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
consisting of 4 nutlets enclosed in a calyx, each nutlet sub-
globose, slightly compressed, nearly smooth; pale brown or
reddish with a small black hilum and each with one seed.
Seed rounded to ovoid brown, about 0.1 cm long.
B. Reduce to a powder, Appendix XVII A. The powder is
brownish-green to greenish-brown. Examine under a
microscope using chloral hydrate solution. The powder shows
the following characteristics: leaf tissue fragments with
2023 Holy Basil Leaf IV-109
polygonal, more or less straight walled epidermal cells and fluorescent bands will be present as shown in the table.
occasional diacytic stomata; covering trichomes elongated, Other fluorescent bands may be present.
uniseriate with three to seven cells; glandular trichomes of
two types: (a) unicellular base with small, rounded unicellular Top of the plate
or bicellular head; (b) unicellular base with enlarged oval
head composed of eight radiating cells. Fragments of leaf
midrib and petiole have thin walled ovoid cells and A red fluorescent band
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 A turquoise-blue fluorescent band A turquoise-blue fluorescent band
epidermal cells with deeply sinuous walls, elongated over the (rosmarinic acid)
veins and occasional covering and glandular trichomes of the
same type as the leaf tissues. Seed fragments consist of
Two blue fluorescent bands may be
closely packed cells with thick wavy walls and blackish
present
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 collenchyma.
Two turquoise fluorescent bands
Partially clear a second mount with chloral hydrate solution,
may be present
then carefully remove the chloral hydrate solution whilst
retaining the powder. Irrigate the powder thoroughly with a
10 % v/v alcoholic solution of phloroglucinol, allow the
solution to evaporate, and then add 1 to 2 drops of
hydrochloric acid. Mix, then mount in a 50% v/v solution of
glycerol. The large, eight celled glandular trichomes are An orange fluorescent band
stained red due to the presence of eugenol. Lignified fibres (hyperoside)
and vessels also stain red. A yellow-orange fluorescent band
An orange fluorescent band (rutin)
(1) Add 5 mL of methanol to approximately 0.5 g of the
powdered herbal drug in a centrifuge tube. Mix using a
Solution (1) Solurum (2)
vortex mixer briefly and then with the aid of ultrasound for
10 minutes. Centrifuge at 2500 rpm for 10 minutes and use
the supernatant liquid. D. Carry out the method for thin-layer chromatography,
(2) 0.016% w/v each of rutin, hyperoside and rosmarinic acid in Appendix III A, using the following solutions.
methanol. (1) Add 5 mL of methanol to approximately 0.5 g of the
powdered herbal drug in a centrifuge tube. Mix using a
vortex mixer briefly and then with the aid of ultrasound for
(a) Use as the coating silica gel (Merck silica gel 10 minutes. Centrifuge at 2500 rpm for 10 minutes and use
HPTLC plates are suitable). the supernatant liquid.
(b) Use the mobile phase as described below. (2) 0.04% w/v of methyleugenol in methanol.
(c) Apply 10 µL of solution (1) and 2 µL of solution (2) as (3) 0.02% w/v of ursolic acid and 0.04% w/v of eugenol in
8 mm bands. methanol.
(d) Develop the plate to 7 cm.
(e) After removal of the plate, dry in air for 5 minutes and
(a) Use as the coating silica gel (Merck silica gel
heat at 100° for 3 minutes. Whilst the plate is hot dip the
HPTLC plates are suitable).
plate into a 0.5% w/v solution of diphenylboric acid aminoethyl
ester in ethyl acetate, dry and dip in a 5% w/v solution of (b) Use the mobile phase as described below.
polyethylene glycol 400 in dichloromethane. Examine under (c) Apply 10 µL of solution ( 1) and 2 µL each of solutions
ultraviolet light (366 nm). (2) and (3) as 8 mm bands.
MOBILE PHASE (d) Develop the plate to 7 cm.
1 volume of formic acid, l volume of water and 15 volumes of (e) After removal of the plate, dry in air for 5 minutes.
ethyl acetate. Dip in anisaldehyde solution and heat at 100° for 3 minutes.
Examine under white light and ultraviolet light (366 nm).
SYSTEM SUITABILITY
MOBILE PHASE
The test is not valid unless, the chromatogram obtained with
solution (2) shows three clearly separated bands of which two 15 volumes of ethyl acetate and 85 volumes of toluene.
are orange bands with Rf values of approximately 0.1 (rutin) SYSTEM SUITABILITY
and 0.2 (hyperoside). The test is not valid unless the chromatogram obtained with
CONFIRMATION solution (3) shows two clearly separated bands and the band
The chromatogram obtained with solution (1) shows a strong due to methyleugenol in the chromatogram obtained with
yellow-orange fluorescent band that elutes between the 2 solution (2) has an Rf of approximately 0.6.
orange bands due to rutin and hyperoside in the
chromatogram obtained with solution (2). Several other
IV-110 Bearberry Leaf 2023
CONFIRMATION TESTS
When examined under white light the chromatogram Foreign matter
obtained with solution (1), as shown in the table, shows a Not more than 2%, Appendix XI D.
pink band that elutes above the band due to methyleugenol Loss on drying
in the chromatogram obtained with solution (2) and a grey When dried at 100° to 105° for 2 hours, loses not more than
band corresponding to the grey band due to urosolic acid 10% of its weight, Appendix XI D. Use 1 g.
obtained with solution (3). A grey-green band corresponding
Total ash
to the band due to eugenol in the chromatogram obtained
Ignite for 18 hours at 450°. Not more than 17.7%,
with solution (3) may be present. A grey-green band
Appendix XI J, Method I.
corresponding to the band due to methyl-eugenol in the
chromatogram obtained with solution (2) may be present. Acid insoluble ash
Other bands may be present. Not more than 6.0%, Appendix XI K, Method II. Use a
second ignition temperature set at 900° for 1 hour (to ensure
Top of the plate
full combustion of the filter paper).
ANNEX
This section is non-mandawry.
A DNA reference sequence for the identity of Holy Basil Leaf
is published in Supplementary Chapter VII D.
A pink band
A grey-green band
(methyl eugenol)
A grey-green band Bearberry Leaf

(eugenol)
Uva Ursi
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
A grey band A grey band (ursolic
acid) DEFINITION
Whole or fragmented, dried leaf of Arcwstaphylos uva-
ursi (L.) Spreng.
Solution (1) Solution (2) Solution (3)
Content
Minimum 7.0 per cent of anhydrous arbutin (C 12 H 16 O 7 ; M,
When examined under ultraviolet light (366 nm), the 272.3) (dried drug).
chromatogram obtained with solution (1) shows the bands as
IDENTIFICATION
shown in the table. Other bands may be present.
A. The leaf, shiny and dark green on the adaxial surface,
lighter on the abaxial surface, is generally 7-30 mm Jong and
Top of the plate
5-12 mm wide. The entire leaf is obovate with smooth
margins, somewhat reflexed downwards, narrowing at the
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,
A red fluorescent band
giving it a characteristic grainy appearance. Only the young
A greyish-brown leaf has ciliated margins. Old leaves are glabrous.
fluorescent band
B. Microscopic examination (2.8.23). The powder is green,
(methyl eugenol)
greenish-grey or yellowish-green. Examine under a
A brownish fluorescent microscope using chloral hydrate solution R. The powder
band (eugenol) shows the following diagnostic characters (Figure 1054.-1):
A red fluorescent band fragments of adaxial epidermis (surface view [A]) showing
thick and irregularly pitted polygonal cells [Aa] usually
accompanied by palisade parenchyma [Ab]; fragments of
adaxial epidermis (transverse section [G]), showing straight-
A yellow fluorescent A yellow fluorescent
walled cells [Ga] covered by a thick smooth cuticle [Gb], and
band band (ursolic acid) accompanied by palisade parenchyma [Ge] consisting of 3 or
4 layers of cells of unequal lengths, some of which contain
numerous prisms of calcium oxalate [Gd]; fragments of
Solution (1) Solution (2) Solution (3) abaxial epidermis (surface view [B, El), showing anomocytic
stomata (2.8.3) [Ba] surrounded by 5-11 subsidiary cells,
scars of hair bases [Ea], and accompanied by spongy
When examined under both white light and ultraviolet light parenchyma [Eb]; groups oflignified fibres from the pericycle
(366 nm) at least one band due to either eugenol or methyl [D]; fragments of the vascular system [F] consisting of pitted
eugenol should be present in the chromatogram obtained vessels [Fa] and fibres [Fb] accompanied by rows of cells
with solution ( 1). containing prisms of calcium oxalate [Fe]; oil droplets are
2023 Bearberry Leaf IV-111
present in the parenchymatous cells; occasional fragments of Top of the plate

conical, unicellular covering trichomes [CJ.
Gallic acid: a brownish zone A brownish zone
- - - -
A brown zone
-- --
Arbutin: a blue zone An intense blue zone (arbutin)
TESTS
Maximum 5 per cent of stems and maximum 3 per cent of
Leaves of different colour
Maximum 10 per cent, determined in the same manner as
foreign matter (2.8.2).
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Figure 1054.-1. - Illustration for identification test B of powdered Liquid chromatography (2.2.29).
herbal drug of bearberry leaf Test solution In a 100 mL flask with a ground-glass neck,
place 0.800 g of the powdered herbal drug (250) (2.9.12).
C. Thin-layer chromatography (2.2.27). Add 20 mL of water R and heat under a reflux condenser on
Test solution To 0.5 g of the powdered herbal drug (355) a water-bath for 30 min. Allow to cool and filter the liquid
(2. 9.12) add 5 mL of a mixture of equal volumes of through a plug of absorbent cotton. Add the absorbent
methanol R and water R, and heat under a reflux condenser cotton to the residue in the 100 mL flask and extract with
for 10 min. Filter whilst hot. Wash the flask and the filter 20 mL of water R under a reflux condenser on a water-bath
with a mixture of equal volumes of methanol R and water R for 30 min. Allow to cool and filter through a paper filter.
and dilute to 5 mL with the same mixture of solvents. Combine the filtrates and dilute to 50.0 mL with water R.
Reference solution Dissolve 50 mg of arbutin Rand 25 mg of Filter through a paper filter. Discard the first 10 mL of the
gallic acid R in methanol Rand dilute to 20.0 mL with the filtrate.
same solvent. Reference solutwn (a) Dissolve 50.0 mg of arbutin CRS in
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel the mobile phase and dilute to 50.0 mL with the mobile
plate R (2-10 µm)]. phase.
Mobile phase anhydrous formic acid R, water R, ethyl acetate R Reference solution (b) Dissolve 2.5 mg of hydroquinone R in
(6:6:88 VIVIV). the mobile phase and dilute to 10.0 mL with the mobile
phase. To 5.0 mL of the solution, add 2.5 mL of reference
solution (a) and dilute to 10.0 mL with the mobile phase.
Development Over a path of 15 cm [or 6 cm] .
Column:
Drying At 105-110 °C until the mobile phase has - size: l = 0.25 m, 0 = 4 mm;
evaporated. - stationary phase: base-deactivated octadecylsilyl silica gel for
Detection treat with a 10 g/L solution of chromatography R (5 µm).
dichloroquinonechlorimide R in methanol R, then treat with a Mobile phase methanol R, water R (10:90 V/V).
20 g/L solution of anhydrous sodium carbonate R.
test solution. Furthermore, other blue or brown zones may Injection 20 µL.
be present in the chromatogram obtained with the test System suitability Reference solution (b):
solution. - resolution: minimum 4.0 between the peaks due to arbutin
and hydroquinone.
Calculate the percentage content of arbutin using the
IV-112 Belamcanda Chinensis Rhizome 2023
area of the peak due to arbutin in the chromatogram obtained

area of the peak due to arbutin in the chromatogram obtained
with reference solution (a);
solution, in grams;
m2 mass of arbutin CRS used to prepare reference solution (a), in
grams;
p percentage content of arbutin in arbutin CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Belamcanda Chinensis Rhizome

DEFINITION
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.
Content
Minimum 0.10 per cent ofirisflorentin (C 20H 18 0 8 ;
IDENTIFICATION
A. The whole rhizome is nodular, rounded, about 3-10 cm Figure 2561.-1. - Illustration for identification test B of powdered
long and 1-2 cm in diameter, irregular, more or less herbal drug of belamcanda chinensis rhizome
branched, with numerous annular striations; there are
crateriforrn, annular stem scars on the upper surface and C. Examine the chromatograms obtained in the test for Iris
small roots about 2-3 mm in diameter on the lower surface. tecwrum Maxim.
The longitudinally fragmented rhizome occurs as pieces Results A See below the sequence of zones present in the
about 2-8 cm long, 2 cm wide and 1 cm thick; stem scars chromatograms obtained with the reference solution and the
and root fragments are present. The orange-brown or dark test solution. Furthermore, other faint zones may be present
brown outer surface is the same colour as the fracture. in the chromatogram obtained with the test solution.
The central parenchyma has a pitted appearance due to the
numerous primary vascular bundles. The texture is hard. Top of the plate
The fracture is granular.
B. Microscopic examination (2.8.23). The powder is orange-
brown or dark brown. Examine under a microscope using Coumarin: a quenching zone A quenching zone
chloral hydrate solution R. The powder shows the following -- --

diagnostic characters (Figure 2561.-1): rare fragments of
brown cork with superimposed polyhedral cells (surface view Irisflorentin: a quenching zone A quenching zone (irisflorentin)
[B]); numerous fragments of parenchyma [D, G] containing
somewhat rounded cells with irregularly thickened and pitted
walls [Da], some of which are thin-walled and contain a very -- --
large calcium oxalate prism up to 250 µm long and about
50 µm in diameter [Db]; very numerous free calcium oxalate
prisms, usually broken [A]; annular, reticulate or pitted Reference solution Test solution
lignified vessels [C], sometimes accompanied by long fibres
[F] . Examine under a microscope using a 50 per cent V/V Results B See below the sequence of zones present in the
solution of glycerol R. The powder shows very numerous chromatograms obtained with the reference solution and the
rounded or ovoid starch granules, 3-15 µm in diameter, test solution. Furthermore, other faint fluorescent zones may
simple or rarely 2- to 5-compound; the punctiforrn hilum is be present in the chromatogram obtained with the test
sometimes visible [E]. solution.
2023 Belamcanda Chinensis Rhizome IV-113
Top of the plate Reference solution (a) Dissolve 5.0 mg of irisfiorentin CRS in
ethanol (70 per cent Vlv,) 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 Vlv,) R.
fluorescent zone
Reference solution (b) Disperse 0.10 g of belamcanda chinensis
-- --
rhizome HRS in 10 mL of ethanol (70 per cent Vlv,) R in a
50 mL centrifuge tube. Sonicate for 30 min. Mix and
A black zone
centrifuge for 5 min. Transfer the supernatant to a 25 mL
A broad blue fluorescent zone volumetric flask. Add to the residue 10 mL of ethanol
(70 per cent Vlv,) R and sonicate for 30 min. Filter and add
Irisflorentin: a blue fluorescent zone A blue fluorescent zone (irisflorentin)
the filtrate to the same volumetric flask. Dilute to 25.0 mL
-- -- with ethanol (70 per cent Vlv,) R and mix. Filter through a
Column:
Reference solution Test solution =
- size: l 0.25 m, 0 4.6 mm;=
- stationary phase: base-deactivated end-capped octadecylsilyl
silica gel for chromatography R (5 µm).
TESTS
Iris tectorum Maxim Mobile phase:
Thin-layer chromatography (2.2.27). - mobile phase A: 0.05 per cent V/V solution of phosphoric
acid R;
(2. 9.12) add 5 mL of methanol R and sonicate for 10 min.
Centrifuge and filter.
Reference solution Dissolve 1 mg of coumarin R and 1 mg of (min) (per cent VIV) (per cent VIV)
irisfiorentin R in 4 mL of methanol R. 0- 5 82 18
Plate TLC silica gel F254 plate R (2-10 µm). 5 - 20 82 ➔ 80 18 ➔ 20
Mobile phase glacial acetic acid R, cyclohexane R, ethyl 20 - 30 80---+ 67 20 ➔ 33
30 - 50 67 ➔ 60 33 ➔ 40
acetate R (1:20:80 VIVIV).
50 - 65 60 ➔ 47 40 ➔ 53
Flow rate 1.0 mL/min.
Drying In air.
Injection 10 µL.
shows no quenching zone between the zones due to Identification of peaks Use the chromatogram obtained with
coumarin and irisflorentin in the chromatogram obtained reference solution (a) to identify the peak due to irisflorentin;
with the reference solution; the chromatogram obtained with use the chromatogram supplied with belamcanda chinensis
the test solution shows no quenching zones in the lower rhizome HRS and the chromatogram obtained with reference
solution (b) to identify the peaks due to tectoridin and
third.
peak 2 (unknown).
Relative retention With reference to irisflorentin (retention
time =about 54 min): tectoridin = about 0.39;
shows no pale blue fluorescent zone above the zone due to
peak 2 = about 0.42.
coumarin in the chromatogram obtained with the reference
solution. System suitability Reference solution (b):
- resolution: minimum 1.5 between the peak due to
Loss on drying (2.2.32) tectoridin and peak 2.
Calculate the percentage content of irisflorentin using the
105 °C for 2 h.
Total ash (2.4.16)
Maximum 7 .0 per cent.
area of the peak due to irisflorentin in the chromatogram
ASSAY area of the peak due to itisflorentin in the chromatogram
Test solution Disperse 0.100 g of the powdered herbal drug solution, in grams;
(35 5) (2. 9.12) in 10 mL of ethanol (70 per cent Vlv,) R in a mass of irisfiorentin CRS used to prepare reference solution (a),
in grams;
50 mL centrifuge tube. Sonicate for 30 min. Mix and
p percentage content of irisflorentin in inSfiarentin CRS.
centrifuge for 5 min. Transfer the supernatant to a 25 mL
volumetric flask. Add to the residue 10 mL of ethanol
- - - - - - - - - - - - - - - - - - - - - ~ PhEur
(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
with ethanol (70 per cent Vlv,) R and mix. Filter through a
IV-114 Belladonna Leaf 2023
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 Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
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 (C 17H 23 NO 3; 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
C. Shake 1 g of the powdered herbal drug (180) (2.9.12)
singly the flowers or occasionally fruits. The flowers have a
with 10 mL of dilute sulfuric acid Rl for 2 min. Filter and add
gamosepalous calyx and campanulate corolla. The drug may
to the filtrate 1 mL of concentrated ammonia R and 5 mL of
contain fruits, as globular berries, green or brownish-black
water R. Shake cautiously with 15 mL of ether R, avoiding
and surrounded by the persistent calyx with widely spread
formation of an emulsion. Separate the ether layer and dry
lobes.
over anhydrous sodium sulfate R. Filter and evaporate the ether
B. Microscopic examination (2.8.23). The powder is dark in a porcelain dish. Add 0.5 mL of fuming nitric acid Rand
green. Examine under a microscope using chloral, hydrate evaporate to dryness on a water-bath. Add 10 mL of
solution R. The powder shows the following diagnostic acetone R and, dropwise, a 30 g/L solution of potassium
characters (Figure 0221.-1): fragments of the lamina showing hydroxide R in ethanol (96 per cent) R. A deep violet colour
sinuous-walled epidermal cells with striated cuticle [A, C] develops.
and part of the underlying palisade parenchyma [Aa]
D. Examine the chromatograms obtained in the
associated with the upper epidermis [A]; numerous
chromatography test.
stomata [Ca] more frequent on the lower epidermis [C],
anisocytic and also some anomocytic (2.8.3); 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 [BJ; parenchyma cells including rounded TESTS
cells, some of which contain microsphenoidal crystals of Chromatography
calcium oxalate [E]; annularly and spirally thickened Thin-layer chromatography (2.2.27).
vessels [K]. The powdered herbal drug may also show: fibres Test solution To 0.6 g of the powdered herbal drug (180)
and reticulately thickened vessels from the stems; (2.9.12) add 15 mL of dilute sulfuric acid Rl, shake for
subspherical pollen grains, 40-50 µm in diameter, with 15 min and filter. Wash the filter with dilute sulfuric acid Rl
3 germinal pores, 3 furrows and an extensively pitted until 20 mL of filtrate is obtained. To the filtrate add 1 mL
exine [HJ; fragments of the corolla with a papillose of concentrated ammonia R and shake with 2 quantities, each
epidermis [] or bearing numerous covering or glandular of 10 mL, of peroxide-free ether R. If necessary, separate by
trichomes of the types previously described [L]; fragments of centrifugation. Dry the combined ether layers over anhydrous
the brownish-yellow testa consisting of irregularly sclerified sodium sulfate R, filter and evaporate to dryness on a water-
cells [G]. 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
2023 Belladonna Preparations IV-115
hyoscine hydro bromide 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
sodium sulfate with 3 quantities, each of 10 mL, of
Mobile phase concentrated ammonia R, water R, acetone R
chloroform R. Add the washings to the chloroform extract,
(3:7:90 V/V/V).
evaporate to dryness on a water-bath and heat in an oven at
Application 10 µLand 20 µL, 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 with potassium iocwbismuthate solution R2, methyl red mixed solution Ras 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.
57.88 X (20 - n)
Results A The zones in the chromatograms obtained with
the test solution are similar in position (hyoscyamine in the (100 - d) x m
lower third, hyoscine in the upper third of the
d loss on drying, as a percentage;
chromatograms) and colour to the bands in the n volume of 0. 02 M sodium hydroxide, in millilitres;
chromatograms obtained with the reference solution. m mass of the powdered herbal drug, in grams.
The zones in the chromatograms obtained with the test
solution are at least equal in size to the corresponding zones _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
in the chromatogram obtained with the same volume of the
reference solution. Faint secondary zones may appear,
particularly in the middle of the chromatogram obtained with
20 µL of the test solution or near the starting point in the Prepared Belladonna
chromatogram obtained with 10 µL of the test solution.
Detection B Spray with sodium nitrite solution R until the Prepared Belladonna Herb
coating is transparent; examine after 15 min. (Ph. Bur. monograph 0222)
Results B The zones due to hyoscyamine in the PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
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
5mm. Content
0.28 per cent to 0.32 per cent of total alkaloids, expressed as
Total ash (2.4.16) hyoscyamine (Mr 289.4) (dried drug).
CHARACTERS
Slightly nauseous odour.
IDENTIFICATION
ASSAY
A. Microscopic examination (2.8.23) of the powder (180)
a) Determine the loss on drying (2.2.32) on 2.000 g of the
(2.9.12). The powder is dark green. Examine under a
powdered herbal drug (180) (2.9.12), by drying in an oven at
microscope, using chloral hydrate solution R. The powder
105 °C.
shows the following diagnostic characters: fragments of leaf
b) Moisten 10.00 g of the powdered herbal drug (180) lamina showing sinuous-walled epidermal cells, a striated
(2. 9.12) with a mixture of 5 mL of ammonia R, 10 mL of cuticle and numerous stomata predominantly present on the
ethanol (96 per cent) R and 30 mL of peroxide-free ether R and lower epidermis (anisocytic and also some anomocytic)
mix thoroughly. Transfer the mixture to a suitable percolator, (2.8.3); multicellular uniseriate covering trichomes with
if necessary with the aid of the extracting mixture. Allow to smooth cuticle, glandular trichomes with unicellular heads
macerate for 4 h and percolate with a mixture of 1 volume of and multicellular, uniseriate stalks or with multicellular heads
chloroform R and 3 volumes of peroxide-free ether R until the and unicellular stalks; parenchyma cells including rounded
alkaloids are completely extracted. Evaporate to dryness a cells containing microsphenoidal crystals of calcium oxalate;
few millilitres of the liquid flowing from the percolator, annular and spirally thickened vessels. The powdered herbal
dissolve the residue in 0.25 M sulfuric acid and verify the drug may also show the following: fibres and reticulately
absence of alkaloids using potassium tetraiocwmercurate thickened vessels from the stems; subspherical pollen grains,
solution R. Concentrate the percolate to about 50 mL by 40-50 µm in diameter, with 3 germinal pores, 3 furrows and
distilling on a water-bath and transfer it to a separating an extensively pitted exine; fragments of the corolla, with a
funnel, rinsing with peroxide-free ether R. Add a quantity of papillose epidermis or bearing numerous covering or
peroxide-free ether R equal to at least 2.1 times the volume of glandular trichomes of the types previously described;
the percolate to produce a liquid of a density well below that brownish-yellow seed fragments containing irregularly
of water. Shake the solution with no fewer than 3 quantities, sclerified and pitted cells of the testa. Examined in glycerol
each of 20 mL, of 0.25 M sulfuric acid, separate the 2 layers (85 per cent) R, the powder may be seen to contain lactose
by centrifugation if necessary and transfer the acid layers to a crystals.
2nd separating funnel. Make the acid layer alkaline with
B. Shake 1 g with 10 mL of dilute sulfuric acid Rl for 2 min.
ammonia R and shake with 3 quantities, each of 30 mL, of
Filter and add to the filtrate 1 mL of concentrated ammonia R
IV-116 Belladonna Preparations 2023
and 5 mL of water R. Shake cautiously with 15 mL of Total ash (2.4.16)

ether R, avoiding formation of an emulsion. Separate the Maximum 16.0 per cent.
ether layer and dry over anhydrous sodium sulfate R. Filter and Ash insoluble in hydrochloric acid (2.8.1)
evaporate the ether in a porcelain dish. Add 0.5 mL of Maximum 4.0 per cent.
fuming nitric acid R and evaporate to dryness on a water-bath.
Add 10 mL of acetone R and, dropwise, a 30 g/L solution of ASSAY
potassium hydroxide R in ethanol (96 per cent) R. A deep violet a) Determine the loss on drying (2.2.32) on 2.000 g by
colour develops. drying in an oven at 105 °C.
C. Examine the chromatograms obtained in the test b) Moisten 10.00 g with a mixture of 5 mL of ammonia R,
Chromatography. 10 mL of ethanol (96 per cent) R and 30 mL of peroxide-free
Results The principal zones in the chromatograms obtained ether R and mix thoroughly. Transfer the mixture to a
with the test solution are similar in position, colour and size suitable percolator, if necessary with the aid of the extracting
to the principal zones in the chromatogram obtained with the mixture. Allow to macerate for 4 h and percolate with a
mixture of 1 volume of chloroform R and 3 volumes of
same volume of the reference solution.
peroxide-free ether R until the alkaloids are completely
TESTS extracted. Evaporate to dryness a few millilitres of the liquid
Chromatography flowing from the percolator, dissolve the residue in 0.25 M
Thin-layer chromatography (2.2.27). suljuric acid and verify the absence of alkaloids using
Test solution To 0.6 g of the drug to be examined add potassium tetraiodomercurate solution R. Concentrate the
15 mL of dilute suljuric acid Rl, shake for 15 min and filter. percolate to about 50 mL by distilling on a water-bath and
Wash the filter with dilute suljuric acid Rl until 20 mL of transfer it to a separating funnel, rinsing with peroxide-free
filtrate is obtained. To the filtrate add 1 mL of concentrated ether R. Add a quantity of peroxide-free ether R equal to at
ammonia R and shake with 2 quantities, each of 10 mL, of least 2.1 times the volume of the percolate to produce a
peroxide-free ether R. If necessary, separate by centrifugation. liquid of a density well below that of water. Shake the
Dry the combined ether layers over anhydrous sodium solution with no fewer than 3 quantities, each of 20 mL, of
sulfate R, filter, and evaporate to dryness on a water-bath. 0.25 M suljuric acid, separate the 2 layers by centrifugation if
Dissolve the residue in 0.5 mL of methanol R. necessary and transfer the acid layers to a 2nd separating
Reference solution Dissolve 50 mg of hyoscyamine sulfate R in funnel. Make the acid layer alkaline with ammonia R and
9 mL of methanol R. Dissolve 15 mg of hyoscine shake with 3 quantities, each of 30 mL, of chloroform R.
hydrobromide R in 10 mL of methanol R. Mix 1.8 mL of the Combine the chloroform layers, add 4 g of anhydrous sodium
hyoscine hydrobromide solution and 8 mL of the sulfate R and allow to stand for 30 min with occasional
hyoscyamine sulfate solution. shaking. Decant the chloroform and wash the sodium sulfate
with 3 quantities, each of 10 mL, of chloroform R. Add the
Plate TLC silica gel G plate R.
washings to the chloroform extract, evaporate to dryness on a
Mobile phase concentrated ammonia R, water R, acetone R water-bath and heat in an oven at 100-105 °C for 15 min.
(3:7:90 VIV/V). Dissolve the residue in a few millilitres of chloroform R, add
Application 10 µL and 20 µL of each solution, as bands of 20.0 mL of 0.01 M suljuric acid and remove the chloroform
20 mm by 3 mm, leaving 1 cm between each band. by evaporation on a water-bath. Titrate the excess of acid
Development Over a path of 10 cm. with 0. 02 M sodium hydroxide using methyl red mixed
Drying At 100-105 °C for 15 min; allow to cool. solution R as indicator.
Detection A Spray with potassium iodobismuthate solution R2, Calculate the percentage content of total alkaloids, expressed
using about 10 mL for a plate 200 mm square, until orange as hyoscyamine, using the following expression:
or brown zones become visible against a yellow background. 57.88 X (20 - n)
Results A The zones in the chromatograms obtained with (100-d) x m
the test solution are similar in position (hyoscyamine in the
lower third, hyoscine in the upper third) and colour to those d loss on drying as a percentage;
n volume of 0. 02 M sodium hydroxide used, in millilitres;
in the chromatograms obtained with the reference solution; m = mass of the herbal drug used, in grams.
the zones in the chromatograms obtained with the test
solution are at least equal in size to the corresponding zones
STORAGE
in the chromatogram obtained with the same volume of the
In an airtight container.
reference solution; faint secondary zones may appear,
particularly in the middle of the chromatogram obtained with - - - - - - - - - - - - - - - - - - - - ~ PhEur
20 µL of the test solution or near the point of application in
the chromatogram obtained with 10 µL of the test solution.
Detection B Spray with sodium nitrite solution R until the
coating is transparent and examine after 15 min.
Results B The zones due to hyoscyamine in the
chromatograms obtained with the test solution and the
reference solution change from brown to reddish-brown but
not to greyish-blue (atropine), and any secondary zones
disappear.
Maximum 5.0 per cent, determined on 1.000 g by drying in
an oven at 105 °C.
2023 Belladonna Preparations IV-11 7
and evaporate to dryness on a water-bath. Dissolve the

Standardised Belladonna Leaf Dry residue in 0.5 mL of methanol R.
Extract Reference solution Dissolve 50 mg of hyoscyamine sulfate R in
(Ph. Bur. monograph 1294) 9 mL of methanol R. Dissolve 15 mg of hyoscine
hyoscine hydrobromide solution and 8 mL of the
DEFINITION hyoscyamine sulfate solution.
Standardised dry extract obtained from Belladonna Plate TLC silica gel plate R.
leaf (0221). Mobile phase concentrated ammonia R, water R, acetone R
Content (3:7:90 VIVIV).
0.95 per cent to 1.05 per cent of total alkaloids, expressed as Application 20 µL as bands.
hyoscyamine (C 17 H 23NO 3 ; Mr 289.4) (dried extract). Development Over a path of 10 cm.
PRODUCTION Drying At 100-105 °C for 15 min; allow to cool.
The extract is produced from the herbal drug by a suitable Detection A Treat with potassium iodobismuthate solution R2,
procedure using ethanol (70 per cent V/V). until orange or brown zones become visible against a yellow
CHARACTERS background.
Appearance Results A The zones in the chromatogram obtained with the
Brown or greenish, hygroscopic powder. test solution are similar in position (hyoscyamine in the lower
third, hyoscine in the upper third) and colour to those in the
IDENTIFICATION
chromatogram obtained with the reference solution. Other
faint zones may be present in the chromatogram obtained
Test solution To 1 g of the extract to be examined add with the test solution.
5.0 mL of methanol R. Shake for 2 min and filter.
Detection B Treat with sodium nitrite solution R until the
Reference solution Dissolve 1.0 mg of chlorogenic acid R and coating is transparent and examine after 15 min.
2.5 mg of rutoside trihydrate R in 10 mL of methanol R.
Results B The zones due to hyoscyamine in the
Plate TLC silica gel plate R. chromatograms obtained with the test solution and the
Mobile phase anhydrous formic acid R, water R, methyl ethyl reference solution change from orange or brown to reddish-
ketone R, ethyl acetate R (10:10:30:50 VIVIVIV). brown but not to greyish-blue (atropine).
Application 20 µL as bands. Loss on drying (2.8.17)
Development Over a path of 15 cm. Maximum 5.0 per cent.
Drying At 100-105 °C. ASSAY
Detection Treat the warm plate with a 10 g/L solution of At each extraction stage it is necessary to check that the
diphenylboric acid aminoethyl ester R in methanol R, then treat alkaloids have been completely extracted. If the extraction is
with a 50 g/L solution of macrogol 400 R in methanol R; allow into the organic phase this is done by evaporating to dryness
to dry in air for 30 min and examine in ultraviolet light at a few millilitres of the last organic layer, dissolving the
365 nm. residue in 0.25 M sulfuric acid and verifying the absence of
Results The chromatograms obtained with the reference alkaloids using potassium tetraiodomercurate solution R. If the
solution and the test solution show in the central part a light extraction is into the acid aqueous phase, this is done by
blue fluorescent zone (chlorogenic acid) and in the lower part taking a few millilitres of the last acid aqueous phase and
a yellowish-brown fluorescent zone (rutoside); furthermore, verifying the absence of alkaloids using potassium
the chromatogram obtained with the test solution shows a tetrawdomercurate solution R.
little above the start a yellowish-brown fluorescent zone and Disperse 3.00 gin a mixture of 5 mL of ammonia Rand
directly above that a yellow fluorescent zone, and a yellow or 15 mL of water R. Shake with no fewer than 3 quantities,
yellowish-brown fluorescent zone between the zone due to each of 40 mL, of a mixture of 1 volume of methylene
rutoside and the zone due to chlorogenic acid. Further zones chloride R and 3 volumes of peroxide-free ether R until the
may be present. alkaloids are completely extracted. Concentrate the combined
B. Examine the chromatograms obtained in the test for organic layers to about 50 mL by distilling on a water-bath
atropine. and transfer the resulting liquid to a separating funnel,
Results The principal zones in the chromatogram obtained rinsing with peroxide-free ether R. Add a quantity of peroxide-
with the test solution are similar in position and colour to the free ether R equal to at least 2.1 times the volume of the
principal zones in the chromatogram obtained with the liquid to produce a layer having a density well below that of
reference solution. water. Shake the resulting solution with no fewer than
3 quantities, each of 20 mL, of 0.25 M suljuric acid until the
TESTS alkaloids are completely extracted. Separate the layers by
Atropine centrifugation, if necessary, and transfer the acid layers to a
Thin-layer chromatography (2.2.27). 2 nd separating funnel. Make the combined acid layers
Test solution To 0.20 g of the extract to be examined add alkaline with ammonia R and shake with no fewer than
10.0 mL of dilute sulfuric acid Rl, shake for 2 min and filter. 3 quantities, each of 30 mL, of methylene chloride R until the
Add 1.0 mL of concentrated ammonia R and shake with alkaloids are completely extracted. Combine the organic
2 quantities, each of 10 mL, of peroxide-free ether R. layers, add 4 g of anhydrous sodium sulfate R and allow to
If necessary, separate by centrifugation. Dry the combined stand for 30 min with occasional shaking. Decant the
ether layers over about 2 g of anhydrous sodium sulfate R, filter methylene chloride and wash the sodium sulfate with
3 quantities, each of 10 mL, of methylene chloride R. Combine
IV-118 Belladonna Preparations 2023
the organic extracts and evaporate to dryness on a water- Top of the plate
bath. Heat the residue in an oven at 100-105 °C for 15 min.
Dissolve the residue in a few millilitres of methylene chloride R, -- --
evaporate to dryness on a water-bath and again heat the Chlorogenic acid: a light blue A light blue fluorescent zone
residue in an oven at 100-105 °C for 15 min. Dissolve the fluorescent zone (chlorogenic acid)
residue in a few millilitres of methylene chloride R, add A yellow or yellowish-brown
20.0 mL of 0.01 M suljuric acid and remove the methylene fluorescent zone
chloride by evaporation on a water-bath. Titrate the excess of
-- --
acid with 0. 02 M sodium hydroxide using methyl red mixed
solution R as indicator. Rutoside: a yellowish-brown A bluish-grey fluorescent zone
fluorescent zone
Calculate the percentage content of total alkaloids, expressed
as hyoscyamine, using the following expression: A yellow fluorescent zone
A yellowish-brown fluorescent zone

57.88 X (20 - n)
lOOxm Reference solution Test solution
n volume of 0.02 M sodium hydroxide used, in millilitres;

m mass of the extract to be examined, in grams. B. Examine the chromatograms obtained in the test for
atropine, detection A.
- - - - - - - - - - - - - - - - - - - - - - Ph Eur Results A See below the sequence of zones present in the
test solution. Faint secondary zones may appear, particularly
in the middle of the chromatogram obtained with 40 µL of
Belladonna Tincture the test solution or near the point of application in the
chromatogram obtained with 20 µL of the test solution.
(Standardised Belladonna Leaf Tincture, Ph. Bur.
monograph 1812) Top of the plate
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Hyoscine: a brownish-orange zone A brownish-orange zone (hyoscine)
DEFINITION
-- --
Tincture produced from Belladonna leaf (0221).
Faint secondary zones
Content
0.027 per cent to 0.033 per cent of total alkaloids, calculated -- --
as hyoscyamine (C 17H 23N0 3; Mr 289.4). The alkaloids
Hyoscyamine: a brownish-orange A brownish-orange zone
consist mainly of hyoscyamine together with small quantities zone (hyoscyamine)
of hyoscine.
Faint secondary zones
PRODUCTION
The tincture is produced from 1 part of the powdered herbal Reference solution Test solution
drug (355) (2.9.12) and 10 parts of
ethanol (70 per cent V/V) by a suitable procedure.
TESTS
IDENfIFICATION Atropine
A. Thin-layer chromatography (2.2.27). Thin-layer chromatography (2.2.27).
Test solution Evaporate to dryness 10.0 mL of the tincture Test solutwn To 15.0 mL of the tincture to be examined
to be examined in a water-bath at 40 °C under reduced add 15 mL of dilute sulfuric acid Rl. Filter. Add 1 mL of
pressure. Dissolve the residue in 1.0 mL of methanol R. concentrated ammonia R to the filtrate and shake with
Reference solutwn Dissolve 1.0 mg of chlorogenic acid R and 2 quantities, each of 10 mL, of peroxide-free ether R. Separate
2.5 mg of rutoside trihydrate R in 10 mL of methanol R. by centrifugation if necessary. Dry the combined ether layers
Plate TLC silica gel plate R. over anhydrous sodium sulfate R. Filter and evaporate to
dryness on a water-bath. Dissolve the residue in 0.5 mL of
Mobile phase anhydrous Jonnie acid R, water R, methyl ethyl
methanol R.
ketone R, ethyl acetate R (10:10:30:50 VIVIVIV).
Application 40 µL as bands.
Development Over a path of 15 cm. hydro bromide R in 10 mL of methanol R. Mix 1. 8 mL of the
Drying At 100-105 °C. hyoscine hydro bromide solution and 8 mL of the
Detection Spray the warm plate with a 10 g/L solution of hyoscyamine sulfate solution.
diphenylboric acid aminoethyl ester R in methanol R; Plate TLC silica gel plate R.
subsequently spray the plate with a 50 g/L solution of Mobile phase concentrated ammonia R, water R, acetone R
macrogol 400 R in methanol R; allow the plate to dry in air for (3:7:90 VIVIV).
30 min and examine in ultraviolet light at 365 nm.
Application20 µL and 40 µL of each solution, as bands.
test solution. Furthermore, other fluorescent zones may be Drying At 100-105 °C for 15 min.
present in the chromatogram obtained with the test solution. Detection A Spray with potassium iodobismuthate solutwn R2.
plate is transparent. Examine after 15 min.
2023 Siam Benzoin IV-119
Results B The zones due to hyoscyamine in the Content

chromatograms obtained with the test solution and the 35.0 per cent to 55.0 per cent of total acids, expressed as
reference solution change from brownish-orange to reddish- benzoic acid (C 7 H 6 O 2 ; M, 122.1) (dried drug).
brown but not to greyish-blue (atropine) and any secondary
CHARACTERS
zones disappear.
Reminiscent odour of vanillin.
Ethanol (2. 9.10)
64 per cent V/V to 69 per cent V/V. IDENTIFICATION
A. Siam benzoin occurs as opaque, often shiny, rounded,
ASSAY ovoid or angular masses (tears or almonds), usually broken
Evaporate 50.0 g of the tincture to be examined to a volume and varying in size from a few millimetres up to about 3 cm.
of about 10 mL. Transfer quantitatively to a separating Individual tears are yellowish-white or reddish externally,
funnel, with the minimum volume of alcohol often with brown or grey patches of discolouration, and have
(70 per cent VIV) R. Add 5 mL of ammonia R and 15 mL of a waxy, whitish fracture which becomes reddish on prolonged
water R. Shake with not fewer than 3 quantities each of exposure to air.
40 mL of a mixture of 1 volume of methylene chloride R and B. Examine the chromatograms obtained in test B for
3 volumes of peroxuie-free ether R, carefully to avoid emulsion,
Sumatra benzoin.
until the alkaloids are completely extracted. Combine the
organic layers and concentrate the solution to a volume of Results See below the sequence of zones present in the
about 50 mL by distilling on a water-bath. Transfer the chromatograms obtained with the reference solution and the
resulting solution quantitatively to a separating funnel, rinsing test solution. Furthermore, other faint quenching zones may
with peroxuie-free ether R. Add a quantity of peroxide-free be present in the chromatogram obtained with the test
ether R equal to at least 2.1 times the volume of the solution solution.
to produce a layer having a density well below that of water.
Shake the resulting solution with not fewer than 3 quantities Top of the plate
each of 20 mL of 0.25 M sulfuric acid until the alkaloids are
- - - -
completely extracted. Separate the layers by centrifugation if
necessary and transfer the layers to a separating funnel. Make Methyl cinnamate: a quenching zone
the combined layers alkaline with ammonia R and shake with
not fewer than 3 quantities each of 30 mL of methylene
chloride R until the alkaloids are completely extracted. Cinnamic acid: a quenching zone
Combine the organic layers, add 4 g of anhydrous sodium
sulfate 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 zone
chloride R. Combine the organic extracts, evaporate to
dryness on a water-bath. Heat the residue in an oven at -- --
100-105 °C for 15 min. Dissolve the residue in a few Vanillin: a quenching zone
millilitres of methylene chloride R, evaporate to dryness on a
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 3 quenching zones
methylene chloride R. Add 20.0 mL of 0.01 M sulfuric acui and
remove the methylene chloride by evaporation on a water-
bath. Titrate the excess of acid with 0. 02 M sodium hydroxide
using methyl red mixed solution R as indicator. TESTS
Calculate the percentage content of total alkaloids, expressed Sumatra benzoin
as hyoscyamine, using the following expression: A. To 0.2 g of the finely powdered herbal drug add 10 mL
of ethanol (96 per cent) R. Shake vigorously until almost
57.88 X (20 - n) completely dissolved and filter. Place 5 mL of the filtrate in a
I00xm test-tube and add 0.5 mL of a 50 g/L solution of ferric
chloride R in ethanol (96 per cent) R. A green colour is
n volume of 0. 02 M sodium hydroxide used, in millilitres,
m mass of the herbal drug used, in grams. produced. No yellow colour is produced.
B. Thin-layer chromatography (2.2.27).
Test solutwn Sonicate 0.2 g of the finely powdered herbal
drug in 5 mL of ethanol (96 per cent) R and centrifuge.
Use the supernatant.
Reference solution Dissolve 4 mg of vanillin R, 10 mg of
Siam Benzoin trans-cinnamic acid R and 10 mg of methyl cinnamate R in
10 mL of ethanol (96 per cent) R.
(Ph. Bur. monograph 2158) Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel
Preparation F254 plate R (2-10 µm)].
Siam Benzoin Tincture Mobile phase glacial acetic acid R, di-isopropyl ether R,
cycwhexane R (10:40:60 VIVIV).
DEFINITION Application 10 µL [or 2 µL] as bands of 15 mm [or 8 mm].
Resin obtained by incising the trunk of Styrax tonkinensis Development Over a path of 12 cm [or 6 cm] .
(Pierre) Craib ex Hartwich. Drying In air.
IV-120 Siam Benzoin Preparations 2023
Detection Examine in ultraviolet light at 254 nm. be present in the chromatogram obtained with the test
Results The chromatogram obtained with the test solution solution.
shows no zone in the same position as the zone due to
cinnamic acid in the chromatogram obtained with the Top of the plate
reference solution.
-- --
Matter insoluble in ethanol
Maximum 5 per cent. Methyl cinnamate: a very prominent
quenching zone
To 2.0 g of the powdered herbal drug add 25 mL of ethanol
(90 per cent VIV,) R. Boil until almost completely dissolved. Benzoic acid: a quenching zone A quenching zone (benzoic acid)
Filter through a previously tared sintered-glass filter (16) Cinnamic acid: a prominent
(2.1. 2) and wash with 3 quantities, each of 5 mL, of boiling quenching zone
ethanol (90 per cent VIV,) R. Heat the glass filter and its
contents in an oven at 100-105 °C for 2 h. Weigh after -- --
cooling. A quenching zone

Loss on drying (2.2.32) A very prominent quenching zone
coarsely powdered herbal drug by drying in vacua for 4 h. Vanillin: a quenching zone A quenching zone (vanillin)
Total ash (2.4.16) Series of unresolved zones including

a quenching zone
ASSAY Reference solution Test solution
Place 0. 7 50 g of the finely powdered herbal drug in a
250 mL borosilicate glass flask and add 15.0 mL of 0.5 M TESTS
alcoholic potassium hydroxide. Boil under a reflux condenser
Sumatra benzoin tincture
on a water-bath for 30 min. Allow to cool and rinse the
condenser with 20 mL of ethanol (96 per cent) R. Titrate the
excess of potassium hydroxide with 0. 5 M hydrochloric acid. Test solution The tincture to be examined.
Determine the end-point potentiometrically (2.2.20). Carry Reference solution Dissolve 20 mg of benzoic acid R, 10 mg of
out a blank titration. trans-cinnamic acid R, 4 mg of vanillin R and 20 mg of methyl
1 mL of 0.5 M alcoholic potassium hydroxide is equivalent cinnamate R in 20 mL of ethanol of the same concentration
to 61.05 mg ofbenzoic acid (C 7H 60 2). as that used for the production of the tincture.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel
F254 plate R (2-10 µm)).
Mobile phase glacial acetic acid R, di-isopropyl ether R,
hexane R (10:40:60 V!V!V).
Siam Benzoin Tincture Application 20 µL [or 8 µL] as bands.
(Ph. Bur. monograph 2157 Drying In air.
PhEw _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Detection Examine in ultraviolet light at 254 nm.
DEFINITION Results The chromatogram obtained with the test solution
Tincture produced from Siam benzoin (2158). does not show any zone in the same position as the zones
due to cinnamic acid and methyl cinnamate in the
Content
Minimum 5.0 per cent mlm of total acids, calculated as
benzoic acid (C1H6O2; Mr 122.1). Ethanol (2.9.10)
95 per cent to 105 per cent of the content stated on the
PRODUCTION label.
The tincture is produced from 1 part of the drug and 5 parts
of ethanol (75 per cent V!V to 96 per cent V/V) by a suitable ASSAY
procedure. Place 3.50 gin a 250 mL borosilicate glass flask and add
15.0 mL of 0.5 M alcoholic potassium hydroxide. Boil under
CHARACTERS a reflux condenser on a water-bath for 30 min. Allow to cool
Appearance and rinse the condenser with 20 mL of ethanol
Orange-yellow liquid. (96 per cent) R. Titrate the excess of potassium hydroxide
It has a characteristic odour of vanillin. with 1 M hydrochloric acid, determining the end-point
IDENTIFICATION potentiometrically (2.2.20). Carry out a blank titration.
A. Place 10 mL in a test tube; add 0.5 mL of a 50 g/L 1 mL of 0.5 M alcoholic potassium hydroxide is equivalent
solution offerric chloride R in ethanol (96 per cent) R. A green to 61.05 mg ofbenzoic acid (C 7H 6O 2).
colour is produced. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
B. Examine the chromatograms obtained in the test for

Sumatra benzoin tincture.
Results See below the sequence of the zones present in the
2023 Sumatra Benzoin IV-121
Detection Spray with anisaldehyde solution R and heat at

Sumatra Benzoin 100-105 °C for 5 min.
Benzoin Results The chromatogram obtained does not show any
(Ph. Bur. monograph 1814) prominent spot with an Rp between 0.4 and 1.0.
Preparations Styrax tonkinensis

Benzoin Inhalation A. To 0.2 g of the finely powdered herbal drug add 10 mL
of ethanol (96 per cent) R. Shake vigorously until almost
Compound Benzoin Tincture
completely dissolved and filter. Place 5 mL of the filtrate in a
Sumatra Benzoin Tincture test-tube and add 0.5 mL of a 50 g/L solution of ferric
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ chloride R in ethanol (96 per cent) R. A yellowish, slightly
green colour is produced.
DEFINITION
Resin obtained by incising the trunk of Styrax benzoin B. Thin-layer chromatography (2.2.27).
Dryander. Test solution Sonicate 0.2 g of the finely powdered herbal
drug in 5 mL of ethanol (96 per cent) R and filter. Collect the
Content
25.0 per cent to 50.0 per cent of total acids, calculated as filtrate.
benzoic acid (C 7 H 6 0 2 ; Mr 122.1) (dried drug). 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
IDENTIFICATION cinnamate R in 10 mL of ethanol (96 per cent) R.
A. Sumatra benzoin occurs as creamy white, rounded to
ovoid tears, which may be embedded in a dull greyish-brown
F2 54 plate R (2-10 µm)].
or reddish-brown matrix. It is hard and brittle and the
fractured surface is dull and uneven. Mobile phase glacial acetic acid R, di-isopropyl ether R,
hexane R (10:40:60 V/V/V).
B. Examine the chromatograms obtained in test B for Styrax
tonkinensis. Application 10 µL [or 2 µL] as bands.
Results See below the sequence of quenching zones present Development Over a path of 12 cm [or 5 cm].
in the chromatograms obtained with the reference solution Drying In air.
and the test solution. Furthermore, other faint quenching Detection Examine in ultraviolet light at 254 nm.
zones may be present in the chromatogram obtained with the Results The chromatogram obtained with the test solution
test solution. shows 2 faint zones in the same positions as the dark zones
due to benzoic acid and vanillin in the chromatogram
Top of the plate obtained with the reference solution.
A very intense dark zone Matter insoluble in ethanol
-- --
To 2.0 g of the powdered herbal drug add 25 mL of ethanol
Methyl cinnarnate: a very intense A dark zone (90 per cent V/V) R. Boil until almost completely dissolved.
dark zone
Filter through a tared sintered-glass filter (16) (2.1.2) and
Benzoic acid: a dark zone A very weak dark zone (benzoic wash with 3 quantities, each of 5 mL, of boiling ethanol
acid) (90 per cent V/V) R. Heat the glass filter and its contents in
Cinnamic acid: an intense dark zone A very intense dark zone ( cinnamic an oven at 100-105 °C for 2 h. Allow to cool and weigh.
acid)
-- -- Maximum 5.0 per cent, determined on 2.000 g of the
A dark zone
coarsely powdered herbal drug by drying in vacuo for 4 h.
Total ash (2.4.16)
A very intense dark zone
A dark zone
ASSAY
Vanillin: a dark zone A very weak dark zone (vanillin) Place O. 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 including
2 dark zones alcoholic potassium hydroxide. Boil under a reflux condenser
on a water-bath for 30 min. Allow to cool and rinse the
Reference solution 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.
Dammargum
Thin-layer chromatography (2.2.27). 1 mL of 0.5 M alcoholic potassium hydroxide is equivalent
to 61.05 mg ofbenzoic acid (C 7 H 6 0 2 ).
Test solution Dissolve 0.2 g of the drug to be examined with
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
gentle heating in 10 mL of ethanol (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 µL.
Drying In air.
IV-122 Benzoin Preparations 2023
Drying In air.
Sumatra Benzoin Tincture
(Ph. Bur. monograph 1813) Results The chromatogram obtained with the test solution
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ does not show zones due to benzoic acid and vanillin that are
more intense than the corresponding zones in the
DEFINITION chromatogram obtained with the reference solution.
Tincture produced from Sumatra benzoin (1814). Ethanol (2.9.10)
Content 95 per cent to 105 per cent of the content stated on the
Minimum 4.0 per cent mlm of total acids, calculated as label.
benzoic acid (C 7 H 6 0 2 ; 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 parts 15.0 mL of 0.5 M alcoholic potassium hydroxide. Boil under
of ethanol (75 per cent V/V to 96 per cent V/V) by a suitable a 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.
1 mL of 0.5 M alcoholic potassium hydroxide is equivalent
IDENTIFICATION to 61.05 mg ofbenzoic acid (C 7H 6 0 2 ).
Examine the chromatograms obtained in the test for Siam _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
benzoin tincture.
Results See below the sequence of quenching zones present
and the test solution. Furthermore, other faint quenching
zones may be present in the chromatogram obtained with the Compound Benzoin Tincture
test solution. Friars' Balsam
DEFINITION
Top of the plate
Barbados Aloes or Cape Aloes 20 g
A very intense dark zone 100 g
Prepared storax of commerce
Sumatra Benzoin crushed 100 g
-- -- Ethanol (90 per cent) Sufficient to produce 1000 mL
Methyl cinnarnate: a very intense A dark zone
dark zone Extemporaneous preparation
Benzoic acid: a dark zone A very weak dark zone (benzoic The following directions apply.
acid) Macerate the Barbados Aloes or Cape Aloes, the prepared
Cinnamic acid: an intense dark zone A very intense dark zone (cinnamic storax and the Sumatra Benzoin with 800 mL of Ethanol
acid) (90 per cent) in a closed vessel for not less than 2 days,
-- -- shaking occasionally, filter and pass sufficient Ethanol
(90 per cent) through the filter to produce 1000 mL.
A dark zone
The tincture complies with the requirements for Tinctures stated
A very intense dark zone under Extracts and with the following requirements.
A dark zone Content of total balsamic acids
Not less than 4.5% w/v, calculated as cinnamic acid,
Vanillin: a dark zone A very weak dark zone (vanillin)
C 9 Hs0z.
Series of unresolved dark zones
TESTS
Reference solution Test solution Ethanol content
70 to 76% v/v, Appendix VIII F, Method III.
TESTS Dry residue

15 to 19% w/v.
Siam benzoin tincture
Thin-layer chromatography (2.2.27). Relative density
Test solutwn The tincture to be examined. 0.880 to 0.910, Appendix VG.
Reference solutwn Dissolve 20 mg of benzoic acid R, 10 mg of ASSAY
trans-cinnamic acid R, 4 mg of vanillin R and 20 mg of methyl Carry out the Assay described under Benzoin Inhalation
cinnamate R in 20 mL of ethanol of the same concentration using 10 mL of the tincture. Each mL of 0.lM sodium
as that used for the production of the tincture. hydroxide VS is equivalent to 14.82 mg of total balsamic
Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel acids, calculated as cinnamic acid, C 9 H 8 0 2 •
F2s4plate R (2-10 µm)].
hexane R (10:40:60 V/V/V).
Application 20 µL [or 8 µL] as bands.
2023 Berberis Aristata IV-123
diameter and varying in length. The bark is soft, about

Benzoin Inhalation 4-8 mm thick, with a yellowish brown outer surface, finely
Benzoin Inhalation Vapour wrinkled longitudinally or deeply furrowed, peeling off in
DEFINITION places and exposing the inner dark yellow wood. Fracture
Benzoin Inhalation is an inhalatwn vapour, solution. short in the region of the bark, hard and fibrous in the wood.
B. Reduce to a powder. The powder is yellowish brown.
Sumatra Benzoin crushed 100 g
Prepared storax of commerce 50 g
Examine under a microscope using chloral hydrate solution.
Ethanol (96 per cent) Sufficient to produce I 000 mL The powder contains numerous fragments of xylem, the
vessels reticulately and spirally thickened, some tracheids;
In making Benzoin Inhalation, Ethanol (96 per cent) may be thick-walled, short, spindle-shaped, lignified, yellowish fibres
replaced by Industrial Methylated Spirit 1 • of the phloem and xylem with a wide lumen; stone cells
Extemporaneous preparation elongated with thick, pitted walls, some containing a single
The following directions apply. calcium oxalate crystal, normally present in groups;
parenchyma cells of the medullary rays, some with yellow-
Macerate the crushed Sumatra Benzoin and the prepared
brown contents, single prism crystals of calcium oxalate, or
storax with 750 mL of Ethanol (96 per cent) for 24 hours.
Filter and pass sufficient Ethanol (96 per cent) through the simple starch granules; cork cells yellowish-brown, thin-
walled; numerous scattered starch grains and calcium oxalate
filter to produce I 000 mL.
crystals.
The inhalatwn complies with the requirements stated under
Preparations for Inhalatwn and with the following requirements. C. Carry out the method for thin-layer chromatography,
Content of total balsamic acids
Not less than 3.0% w/v, calculated as cinnamic acid, (1) Add 4 mL of methanol (80%) to 250 mg of the powdered
C 9 HsOz. herbal drug (180) in a centrifuge tube. Mix with the aid of
ultrasound for I O minutes. Centrifuge at 3000 rpm for
Total solids
5 minutes and collect the clear supernatant. Repeat the
9.0 to 12.0% w/v when determined by drying at 105° for
extraction twice with a further two 2-mL portions of methanol
4 hours, Appendix XI A. Use 2 mL.
(80%). Combine the supematants and dilute to 20 mL with
ASSAY methanol (80%).
Boil 10 mL with 25 mL of 0.5M ethanolic potassium hydroxide (2) 0.04% w/v each of berben·ne chloride BPCRS and palmatine
under a reflux condenser for 1 hour. Evaporate the ethanol, chloride BPCRS in methanol (80%).
disperse the residue in 50 mL of hot water, cool, add 80 mL
of water and 1. 5 g of magnesium sulfate dissolved in 50 mL of CHROMATOGRAPHIC CONDITIONS
water. Mix thoroughly and allow to stand for 10 minutes. (a) Use as the coating silica gel.
Filter, wash the residue on the filter with 20 mL of water, (b) Use the mobile phase as described below.
acidify the combined filtrate and washings with hydrochloric (c) Apply 20 µL of each solution as 6 mm bands.
acid and extract with four 40-mL quantities of ether. Discard
the aqueous solution, combine the ether extracts and extract
with successive quantities of 20, 20, I 0, I O and I O mL of (e) After removal of the plate, dry in air and examine under
sodium hydrogen carbonate solution, washing each aqueous ultraviolet light (254 nm).
extract with the same 20 mL of ether. Discard the ether MOBILE PHASE
layers, carefully acidify the combined aqueous extracts with I O volumes of anhydrous formic acid, 10 volumes of water and
hydrochloric acid and extract with successive quantities of 30, 80 volumes of ethyl acetate.
20, 20 and 10 mL of chloroform, filtering each extract through
SYSTEM SUITABILITY
anhydrous sodium sulfate supported on absorbent cotton. Distil
the chloroform from the combined filtrates until 10 mL The test is not valid unless the chromatogram obtained with
remains and remove the remainder in a current of air. solution (2) shows two clearly separated bands.
Dissolve the residue, with the aid of gentle heat, in 10 mL of CONFIRMATION
ethanol (96%), previously neutralised to phenol red solutwn, The chromatogram obtained with solution (1) shows a
cool and titrate with O. IM sodium hydroxide VS using phenol principal yellow band corresponding in colour and position to
red solutwn as indicator. Each mL of O.IM sodium hydroxide the band obtained for berberine chloride in solution (2), a
VS is equivalent to 14.82 mg of total balsamic acids, yellow band corresponding in colour and position to the
calculated as cinnamic acid, C 9 H 8 O 2 . band obtained for palmatine in solution (2) and several other
bands as shown in the table. Other bands may be present.
Barberis Aristata Top of the plate

DEFINITION
Berberis Aristata is the dried, cut stem of Berberis aristata
DC.
It contains not less than 1.4% ofberberine (C 20 H 19NO 5),
Yellow band (berberine chloride) Yellow band (berberine chloride)
calculated with reference to the dried material.
Faint yellow band
IDENTIFICATION Yellow band (palmatine) Yellow band (palmatine)
A. The cut pieces of stem are subcylindrical, often branched
and somewhat swollen at the nodes, from about 15-20 mm
Purple band
1Staruwry regulations governing rhe use of Industrial Methylated Spirit
musl be observed.
IV-124 Bilberry 2023
TESTS SYSTEM SUITABILITY

o-Tetrahydropalmatine The test is not valid unless, in the chromatogram obtained
Carry out the method for liquid chromawgraphy, with solution (2), the resolution facwr between the peaks due
Appendix III D, using the following solutions. to palmatine and berberine chloride is at least 2.0.
(1) To 0.5 g of powdered sample, add 400 mL of a mixture DETERMINATION OF CONTENT
of equal volumes of acetonitrile and 0.1 % v/v orthophosphoric
Using the retention time and the peak area from the
acid. Mix with the aid of ultrasound for 40 minutes and chromatogram obtained with solution (2), locate and
allow to cool. Dilute to 500 mL with the mobile phase and integrate the peak due to berberine chloride in the
filter through a 0.45-µm filter. chromatogram obtained with solution (1). Calculate the
(2) 0.01 % w/v each of palmatine chloride BPCRS and berberine content of berberine in the sample using the declared content
chloride BPCRS in the mobile phase. of berberine in berberine chloride BPCRS and the following
(3) 0.01 % w/v of D-tetrahydropalmatine hydrochloride in the expression:
mobile phase.
CHROMATOGRAPHIC CONDITIONS A, m2 V, 100
-x-x-xpx---
(a) Use a stainless steel column (15 cm x 4.6 mm) packed Ai V2 m, 100-d
with end-capped octadecylsilyl silica gel for chromatography
(5 µm) (Phenomenex Luna Cl8 is suitable).
(b) Use isocratic elution and the mobile phase described A1 area of the peak due to berberine in the chromatogram
below. obtained with solution (!),
A2 area of the peak due to berberine in the chromatogram
(c) Use a flow rate of 1.2 mL per minute. obtained with solution (2),
(d) Use an ambient column temperature. m1 weight of the drug being examined in mg,
m2 weight of berberine chloride BPCRS in mg,
(e) Use a detection wavelength of 235 nm. V1 dilution volume of solution (I) in mL,
(f) Inject 10 µL of each solution. V2 dilution volume of solution (2) in mL,
p percentage content of berberine in berberine chloride BPCRS,
When the chromatograms are recorded under the prescribed d percentage loss on drying of the herbal drug being examined.
conditions the retention times relative to palmatine (retention
time= about 8 minutes) are berberine chloride = about 1.1;
STORAGE
D-tetrahydropalmitine = about 1.6.
Berberis 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 Bilbeny Fruit, Ph. Bur. monograph 1588)
with solution (2), the resolution factor between the peaks due
to palmatine and berberine chloride is at least 2.0. Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
CONFIRMATION DEFINIDON
In the chromatogram obtained with solution (1), there are no Dried ripe fruit of Vaccinium myrtillus L.
peaks corresponding to the peak due to Content
D-tetrahydropalmitine in the chromatogram obtained with Minimum 0.8 per cent of tannins, expressed as pyrogallol
solution (3). (C6H6O3; Mr 126.1) (dried drug).
Loss on drying IDENTIFICATION
When dried for 2 hours at 105°, loses not more than 10.0% A. Dried bilberry is a blackish-blue, subglobular, shrunken
of its weight, Appendix IX D. Use 1 g. berry about 5 mm in diameter covered in a whitish bloom.
Total Ash Its lower end shows a scar or, rarely, a fragment of the
Not more than 3.0%, Appendix XI J, Method II. pedicel. The upper end is slightly depressed and is
ASSAY surmounted by the remains of the style and of the persistent
Carry out the method for liquid chromatography, calyx, which appears as a circular fold. The deep violet,
Appendix III D, using the following solutions. fleshy mesocarp contains numerous (more than 10) small,
brown, ovoid seeds.
( 1) To 0 .5 g of powdered sample, add 400 mL of a mixture
of equal volumes of acewnitrile and 0.1 % v/v orthophosphoric B. Microscopic examination (2.8.23). The powder is violet-
acid. Mix with the aid of ultrasound for 40 minutes and red. Examine under a microscope using chloral hydrate
allow to cool. Dilute to 500 mL with the mobile phase and solution R. The powder shows the following diagnostic
filter through a 0.45-µm filter. characters (Figure 1588.-1): fragments of dark red
epicarp [A] consisting of small groups of 2-4 polygonal,
(2) 0.01 % w/v each of palmatine chloride BPCRS and berberine
rectangular or quadrangular cells; in each group, the inner
chloride BPCRS in the mobile phase. walls are thin [Aa] whereas the outer walls are regularly
CHROMATOGRAPHIC CONDITIONS thickened [Ab ]; fragments of mesocarp [BJ consisting of
The chromatographic conditions described under the test for large, rounded, thin-walled cells associated with fine spiral
D-tetrahydropalmatine may be used. vessels [Ba]; numerous sclereids, isolated or in small groups,
MOBILE PHASE
from the mesocarp [C]; groups of sclereids from the
endocarp [DJ; yellowish-brown fragments from the testa,
27 volumes of acetonitrile and 73 volumes of a 1.36% w/v with large sclereids having walls that are extensively
solution of potassium dihydrogen orthophosphate. channelled and pitted (surface view [E]) and with U-shaped
2023 Bilberry IV-125
thickenings (transverse section [F]); irregular cluster crystals Top of the plate
of calcium oxalate [G) and isolated prisms of calcium oxalate
-- --
[HJ from the mesocarp.
A violet-red zone A violet-red zone
A violet zone A violet zone
A broad unresolved bluish-violet A broad unresolved bluish-violet zone

zone
-- --
TESTS
Maximum 2 per cent, with no fruit of Sambucus nigra
present.
Adulteration with S. nigra L. is indicated by the presence of
glossy, violet-black, ovoid berries without the circular fold
due to the calyx, and containing not more than 4 seeds.
powdered herbal drug by drying in an oven at 105 °C
for 2 h.
Total ash (2.4.16)
ASSAY
Tannins (2.8.14)
Use 1.500 g of the powdered herbal drug (355) (2. 9.12).
Figure 1588.-1. ~ Illustration for identification test B of powdered
herbal drug of dried bilberry frnit - - - - - - - - - - - - - - - - - - - - - PhEur

(2. 9.12) add 20 mL of methanol R. Shake for 15 min and Fresh Bilberry
filter.
Reference solution Dissolve 0.10 g of bilberry dry extract HRS (Fresh Bilberry Fruit, Ph. Bur. monograph 1602)
in 25 mL of methanol R. Stir for 15 min and filter. Preparation
Plate TLC sz7ica gel F254 plate R (5-40 µm) [or TLC silica gel Refined and Standardised Fresh Bilberry Fruit Dry Extract
F2s4 plate R (2-10 µm)]. PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Mobile phase anhydrous formic acid R, water R, butanol R DEFINITION

( 16: 19:65 VIVIV). Fresh or frozen, ripe fruit of Vaccinium myrtillus L.
Content
Development Over a path of 10 cm [or 6 cm]. Minimum 0.30 per cent of anthocyanins, expressed as
Drying In air. cyanidin 3-0-glucoside chloride (chrysanthemin,
Detection Examine in daylight. C 21 H 21 ClO 11 ; Mr 484.8) (dried drug).
Results See below the sequence of zones present in the IDENTIFICATION
chromatograms obtained with the reference solution and the A. The fresh fruit is a blackish-blue globular berry about
test solution. Furthermore, other faint zones may be present 5 mm in diameter, covered in a whitish bloom. Its lower end
in the chromatogram obtained with the test solution. shows a scar or, rarely, a fragment of the pedicel. The upper
end is slightly depressed and is surmounted by the remains of
the style and of the persistent calyx, which appears as a
circular fold. The violet, fleshy mesocarp includes 4 to 5
locules containing numerous (more than 10) small, brown,
ovoid seeds.
B. The crushed fresh fruit is violet-red. Examine small
fragments under a microscope using chloral hydrate solutwn R.
They show the following diagnostic characters (Figure
1602.-1): fragments of dark red epicarp [A] consisting of
small groups of 2-4 polygonal, rectangular or quadrangular
IV-126 Bilberry 2023
cells; in each group, the inner walls are thin [Aa] whereas the Top of the plate
outer walls are regularly thickened [Ab]; fragments of
mesocarp [BJ consisting of large, rounded, thin-walled cells -- --
associated with fine spiral vessels [Ba]; numerous sclereids, A violet-red zone A violet-red zone
isolated or in small groups, from the mesocarp [C]; groups of
sclereids from the endocarp [D]; yellowish-brown fragments
from the testa, with large sclereids having walls that are A violet zone A violet zone
extensively channelled and pitted (surface view [El) and with
A broad unresolved bluish-violet A broad unresolved bluish-violet zone
U-shaped thickenings (transverse section [F]); irregular zone
cluster crystals of calcium oxalate [G] and isolated prisms of
calcium oxalate [HJ from the mesocarp.
-- --
TESTS
Maximum 2 per cent, with no fruit of Sambucus nigra
present.
Adulteration with S. nigra L. is indicated by the presence of
glossy, violet-black, ovoid berries without the circular fold
due to the calyx, and containing not more than 4 seeds.
80.0 per cent to 90.0 per cent, determined on 5.000 g of the
freshly crushed drug by drying in an oven at 105 °C.
Total ash (2.4.16)
ASSAY
Crush 50 g immediately before use. To about 5.00 g of the
crushed, accurately weighed drug, add 95 mL of methanol R.
Stir mechanically for 30 min. Filter into a 100.0 mL
volumetric flask. Rinse the filter and dilute to 100.0 mL with
methanol R. Prepare a SO-fold dilution of this solution in a
0.1 per cent V/V solution of hydrochloric acid R in methanol R.
Measure the absorbance (2.2.25) of the solution at 528 nm,
using a 0.1 per cent V/V solution of hydrochloric acid R in
methanol R as the compensation liquid.
Calculate the percentage content of anthocyanins, expressed
Figure 1602.-1. - Illustratwnfor identificatwn test B of the as cyanidin 3-O-glucoside chloride, using the following
herbal drng of fresh bilberry fruit expression:
C. Thin-layer chromatography (2.2.27). Ax 5000

Test solutwn To 5 g of the freshly crushed drug, add 20 mL 718xm
of methanol R. Stir for 15 min and filter. 718 specific absorbance of cyanidin 3-0-glucoside chloride at
528 run;
Reference solution Dissolve 0.10 g of bilberry dry extract HRS A absorbance at 528 nm;
in 25 mL of methanol R. Stir for 15 min and filter. m mass of the substance to be examined in grams.
Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel
F2 54 plate R (2-10 µm)]. STORAGE
Mobile phase anhydrous formic acid R, water R, butanol R When frozen, store at or below -18 °C.
(16:19:65 V/V/V). _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur

Drying In air.
Detection Examine in daylight.
2023 Bilberry Fruit Preparations IV-127
The characteristic anthocyanin peaks (peaks 1-8, 10-15

Refined and Standardised Fresh and 17) in the chromatogram obtained with the test solution
Bilberry Fruit Dry Extract are similar in retention time to those in the chromatogram
obtained with reference solution (b).
PhEw _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ TESTS
Total anthocyanidins
DEFINITION Liquid chromatography (2.2.29). Maintain the solutions at
Refined and standardised dry extract produced from Fresh 4 °C.
bilberry fruit (1602). Solvent mixture hydrochloric acid R, methanol R (2:98 V/V).
Content Test solution Dissolve 0.1250 g of the extract to be
32.4 per cent to 39.6 per cent of anthocyanins, expressed as examined in the solvent mixture and dilute to 25.0 mL with
cyanidin 3-0-glucoside chloride [chrysanthemin the solvent mixture. Dilute 5.0 mL of the solution to
(C21H 21 CI011; M, 484.8)} (dried extract). 20.0 mL with dilute phosphoric acid R.
PRODUCTION Reference solution (a) Dissolve 10.0 mg of cyanidin
The extract is produced from the herbal drug by a suitable chloride CRS in the solvent mixture and dilute to 25.0 mL
procedure using ethanol (70-96 per cent VIV) or methanol with the solvent mixture. Dilute 2.0 mL of the solution to
(minimum 60 per cent V/V). Refinement may be performed 100.0 mL with dilute phosphoric acid R.
by ion-exchange chromatography. Reference solution (b) Dissolve 0.1250 g of bilberry dry
CHARACTERS extract HRS in the solvent mixture and dilute to 25.0 mL
Appearance with the solvent mixture. Dilute 5.0 mL of the solution to
Dark reddish-violet, amorphous, hygroscopic powder. 20.0 mL with dilute phosphoric acid R.
Column:
IDENTIFICATION
First identification: B.
=
- size: l 0.250 m, 0 = 4.6 mm;
Second identification: A. chromatography with extended pH range R (5 µm);
A. Thin-layer chromatography (2.2.27). - temperature: 30 °C.
Test solution Dissolve 0.10 g of the extract to be examined Mobile phase:
in 25 mL of methanol R. Stir for 15 min and filter. - mobile phase A: anhydrous formic acid R, water for
Reference solution Dissolve 0.10 g of bilberry dry extract HRS chromatography R (8.5:91.5 VIV);
in 25 mL of methanol R. Stir for 15 min and filter. - mobile phase B: anhydrous formic acid R, acetonitrile R,
methanol R, water for chromatography R
(8.5:22.5:22.5:41.5 VIV!VIV);
F254 plate R (2-10 µm)].
Mobile phase anhydrous formic acid R, water R, butanol R Time Mobile phase A Mobile phase B
(16:19:65 VIVIV). (min) (per cent VIV) (per cent VIV)
Application 10 µL [or 2 µL] as bands of 15 mm [or 8 mm]. 0 - 35 93 ➔ 75 7 ➔ 25
Development Over a path of 10 cm [or 6 cm]. 35 - 45 75 ➔ 35 25 ➔ 65
45 - 46 35 ➔ 0 65 ➔ 100
Drying In air.
46 - 50 0 100
Flow rate 1.0 mLJmin.
test solution. Furthermore, other faint zones may be present Detection Spectrophotometer at 535 nm.
in the chromatogram obtained with the test solution. Injection 10 µL.
Top of the plate bilberry dry extract HRS and the chromatograms obtained
with reference solutions (a) and (b) to identify the peaks due
-- -- to the anthocyanins and the anthocyanidins.
A violet-red zone A violet-red zone Retention times The retention times and the elution order of
A violet zone A violet zone the peaks are similar to those shown in the chromatogram
(Figure 2394.-1).
A broad unresolved bluish-violet A broad unresolved bluish-violet - peak-to-valley ratio: minimum 2.0, where Hp = height
zone zone above the baseline of the peak due to cyanidin 3-0-
galactoside (peak 3) and Hv = height above the baseline
of the lowest point of the curve separating this peak from
the peak due to delphinidin 3-0-arabinoside (peak 4).
-- -- Calculate the percentage content of total anthocyanidins,
expressed as cyanidin chloride, using the following
expression:
B. Liquid chromatography (2.2.29) as described in the test A I x m 2 x 100 x p

for total anthocyanidins. m 1 xA2 x 1250
IV-128 Bilberry Fruit Preparations 2023
-
2
-
4
3 5
7
-
8
15
-
6 12
11 13
17
10
14
~j
18 19 20
17
L A
16
~ 1 JI. ,-
I I I I I I I I
10 15 20 25 ' ' '

0 5 30 35 40 45 50 55 min
1. delphinidin 3-O-galactoside chloride 11. petunidin 3-0-arabinoside chloride

2. myrtillin (delphinidin 3-0-glucoside chloride) 12. peonidin 3-O-glucoside chloride
3. cyanidin 3-O-galactoside chloride 13. malvidin 3-0-galactoside chloride
4. delphinidin 3-0-arabinoside chloride 14. peonidin 3-0-arabinoside chloride
5. chtysanthetnin (cyanidin 3-O-glucoside chloride) 15. malvidin 3-O-glucoside chloride
6. petunidin 3-0-galactoside chloride 16. cyanidin chloride
7. cyanidin 3-0-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 3-0-galactoside chloride 20. malvidin chloride
Figure 2394.-1. - Chromatogram for the assay of refined and standardised fresh bilbeny fruit dry extract
sum of the areas of the peaks due to the anthocyanidins

(peaks 9, 16, 18-20) in the chromatogram obtained with the
test solution;
area of the peak due to cyanidin chloride (peak 16) in the sum of the areas of the peaks due to the anthocyanins (peaks
chromatogram obtained with reference solution (a); 1-8, l 0-15 and 17) in the chromatogram obtained with the test
mass of the extract to be examined used to prepare the test solution;
solution, in grams; area of the peak due to cyanidin 3-O-glucoside chloride (peak
mass of cyanidin chloride CRS used to prepare reference 5) in the chromatogram obtained with reference solution (b);
solution (a), in grams; mass of the extract to be examined used to prepare the test
p percentage content of cyanidin chloride in cyanidin chloride CRS. solution, in grams;
mass of bilbeny dry extract HRS used to prepare reference
Limit Not more than 1.0 per cent of total anthocyanidins, solution (b), in grams;
expressed as cyanidin chloride. p percentage content of cyanidin 3-0-glucoside chloride in bilbeny
dry extract HRS.
Maximum 4.5 per cent. - - - - - - - - - - - - - - - - - - - - - - - ~ PhEur
Total ash (2.4.16)
ASSAY
Liquid chromatography (2.2.29) as described in the test for
total anthocyanidins with the following modification.
Injection Test solution and reference solution (b).
Calculate the percentage content of total anthocyanins,
expressed as cyanidin 3-O-glucoside chloride, using the
2023 Birch Leaf IV-129
Birch Leaf
~~--------------------
DEFINITION
Whole or fragmented, dried leaves of Benda pendula Roth
and/or Benda pubescens Ehrh. as well as hybrids of both
species.
Content
Minimum 1.5 per cent of flavonoids, expressed as hyperoside
(C21H20012; Mr 464.4) (dried drug).
IDENTIFICATION
A. The leaves of both species are dark green on the adaxial
surface and lighter greenish-grey on the abaxial surface; they
show a characteristic dense reticulate venation. The veins are
light brown or almost white.
The leaves of B. pendula are glabrous and show closely
spaced glandular pits on both surfaces. The leaves of
B. pendula are 3-7 cm long and 2-5 cm wide; the petiole is
long and the doubly dentate lamina is triangular or rhomboid
and broadly cuneate or truncate at the base. The angle on
each side is unrounded or slightly rounded, and the apex is
long and acuminate.
The leaves of B. pubescens show few glandular trichomes and
are slightly pubescent on both surfaces. The abaxial surface
shows small bundles of yellowish-grey trichomes at the
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 Figure 1174. -1. - Illustration for identification test B of powdered
acute rather than acuminate. herbal drug of birch leaf
greenish-grey. Examine under a microscope using chloral Intensity marker Hyperoside.
hydrate solution R. The powder shows the following diagnostic Plate TLC silica gel F254 plate R (2-10 µm).
characters (Figure 117 4. -1): numerous fragments of the Mobile phase anhydrous formic acid R, water R, methyl ethyl
lamina, in surface view, with straight-walled, adaxial ketone R, ethyl acetate R (10:10:30:50 V/V/V/V).
epidermal cells accompanied by underlying palisade Application 4 µL as bands of 8 mm.
parenchyma [E] and cells of the abaxial epidermis
surrounding anomocytic stomata (2.8.3) [G]; large, free,
glandular trichomes usually measuring 100-120 µm [DJ; Drying In a current of air at room temperature for 5 min.
fragments of the lamina (transverse section [BJ), showing Detection Heat at 100-105 °C for 5 min. Spray the warm
glandular trichomes on the epidermises [Ba], heterogeneous, plate with a 10 g/L solution of diphenylboric acid aminoethyl
asymmetrical mesophyll containing cluster crystals [Bb] and ester R in methanol R, then with a 50 g/L solution of macrogol
prisms [Be] of calcium oxalate; fragments of spongy 400 R in methanol R or, alternatively, dip the warm plate in a
parenchyma [A] accompanied by crystal sheaths [Aa] and 5 g/L solution of diphenylboric acid aminoethyl ester R in ethyl
cells containing cluster crystals of calcium oxalate [Ab]; acetate R, then in a 50 g/L solution of macrogol 400 R in
fragments of vessels and sclerenchyma fibres [CJ. methylene chloride R. Allow the plate to dry in air for about
If B. pubescens is present, the powder also contains unicellular 1 min and examine in ultraviolet light at 366 nm.
covering trichomes with very thick walls, about 80-600 µm System suitability Reference solution (c):
long, usually 100-200 µm, numerous on the margin of the - the chromatogram shows 2 distinct zones in the
lamina [F] or on the epidermises (surface view [H]). middle third which may, however, be touching.
C. High-performance thin-layer chromatography (2.8.25). The lower zone (chlorogenic acid) shows a light blue
Test solution To 0.5 g of the powdered herbal drug (355) fluorescence and the upper zone (hyperoside) shows a
(2. 9.12) add 5 .0 mL of methanol R. Sonicate for 15 min, yellow or orange fluorescence.
then filter or centrifuge the solution and use the filtrate or Results See below the sequence of zones present in the
supernatant. chromatograms obtained with reference solution (a) and the
Reference solution (a) Dissolve 2.5 mg of hyperoside Rand test solution. Furthermore, in the chromatogram obtained
3.5 mg of quercitrin R in methanol Rand dilute to 10.0 mL with the test solution, other faint to very faint fluorescent
with the same solvent. zones, which may be yellow or orange, or light blue, may be
present and a light blue fluorescent zone may especially be
present below the zone due to hyperoside.
Reference solution (c) Dissolve 2.5 mg of hyperoside Rand
3 mg of chlorogenic acid R in methanol R and dilute to
IV-130 Bistort Rhizome 2023
Top of the plate over 10 g of anhydrous sodium sulfate R into a 50 mL

volumetric flask and dilute to 50.0 mL with ethyl acetate R.
2 red fluorescent zones, the lower
may be overlapped with an orange Test solution To 10.0 mL of the stock solution add 1 mL of
fluorescent zone aluminium chloride reagent R and dilute to 25.0 mL with a
5 per cent V/V 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 V/V solution of glacial acetic
acid R in methanol R.
A yellow or orange fluorescent zone Measure the absorbance (2.2.25) of the test solution after
or a faint yellow or orange 30 min, by comparison with the compensation liquid at
fluorescent zone 425 nm.
Quercitrin: a yellow or orange A yellow or orange fluorescent zone Calculate the percentage content of flavonoids, expressed as
fluorescent zone or a faint yellow or orange
fluorescent zone (querdtrin)
hyperoside, using the following expression:
Ax 1.25
-- - -
m
A yellow or orange fluorescent zone i.e. taking the specific absorbance ofhyperoside to be 500.
or a faint yellow or orange
fluorescent zone
m mass of the herbal drug to be examined, in grams.
Hyperoside: a yellow or orange An intense yellow or orange

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
fluorescent zone fluorescent zone (hyperoside)
A very faint to intense yellow or

orange fluorescent zone
Bistort Rhizome
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
-- --
DEFINITION
Whole or fragmented, dried rhizome of Persicaria bistorta (L.)
Reference solution (a) Test solution Samp. (syn. Polygonum bistorta L.) without adventitious roots.
Content
TESTS Minimum 3.0 per cent of tannins, expressed as pyrogallol
Maximum 3 per cent of fragments of female catkins and CHARACTERS
maximum 3 per cent of other foreign matter. The whole rhizome is up to 13 cm long and 2.5 cm in
Loss on drying (2.2.32) diameter. The remnants of the roots are not longer than
Maximum 10.0 per cent, determined on 1.000 g of the 1 cm and are about 1 mm in diameter.
powdered herbal drug (355) (2. 9.12) by drying in an oven at IDENTIFICATION
105 °C for 2 h. A. The whole rhizome, reddish-brown or blackish-brown, is
Total ash (2.4.16) thick, twisted, and turned back on itself. Its outer surface
Maximum 6.0 per cent. shows transverse striations and blackish spots. It is flattened
and somewhat depressed on the upper surface, convex on the
ASSAY
lower surface. It shows adventitious root scars on the surface.
Stock solution In a 100 mL round-bottomed flask introduce The fracture, pinkish-beige, shows an elliptical zone of
0.200 g of the powdered herbal drug (355) (2.9.12), 1 mL of
whitish pits corresponding to the vessels. The drug may also
a 5 g/L solution of hexamethylenetetramine R, 20 mL of be obtained as more or less cylindrical fragments about
acetone R and 2 mL of hydrochloric acid Rl. Boil the mixture
0.3 cm in diameter and up to 1 cm long, with a reddish-
under a reflux condenser for 30 min. Filter the liquid
brown outer surface, marked by adventitious root scars and a
through a plug of absorbent cotton into a 100 mL flask.
pinkish-beige fracture.
Add the absorbent cotton to the residue in the round-
bottomed flask and extract with 2 quantities, each of 20 mL, B. Microscopic examination (2.8.23). The powder is reddish-
of acetone R, each time boiling under a reflux condenser for brown. Examine under a microscope using chloral hydrate
10 min. Allow to cool to room temperature, filter the liquid solution R. The powder shows the following diagnostic
through a plug of absorbent cotton then through a filter characters (Figure 2384.-1): very numerous cluster crystals of
paper into the volumetric flask, and dilute to 100.0 mL with calcium oxalate, 15-65 µm in diameter, either free [G] or
acetone R by rinsing the flask and filter. Introduce 20.0 mL of included in parenchyma cells [Da]; rare cork fragments (side
the solution into a separating funnel, add 20 mL of water R view [BJ, surface view [H]); vascular bundles (longitudinal
and extract the mixture with 1 quantity of 15 mL and then section [E], transverse section m)
including small pitted
3 quantities, each of 10 mL, of ethyl acetate R. Combine the vessels [Ea, Ja] accompanied by finely pitted, thick-walled
ethyl acetate extracts in a separating funnel, wash with fibres [Eb, Jb]; free fragments of vessels [C]; free fibres [F];
2 quantities, each of 50 mL, of water R, and filter the extract fragments of parenchyma [DJ with rounded cells with slightly
thickened walls; fragments of collenchyma [K]. Examine
2023 Bitter Apricot Seed IV-131
under a microscope using a 50 per cent V/V solution of Top of the plate
glycerol R. The powder shows rounded or ovoid starch
Catechin: a brown zone A brown zone (catechin)
granules, simple, about 5-12 µm in diameter, free or included
in parenchyma cells [A]. -- --
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
Eb
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.
~- J-1<
~K 105 °C.
Total ash (2.4.16)
p-' """-; ~
ASSAY
Figure 2384.-1. - Illustration for identification test B of powdered Tannins (2.8.14)
herbal drug of biswrt rhizome Use 1.000 g of the powdered herbal drug (180) (2.9.12).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
(2.9.12) add 10 mL of a mixture of equal volumes of
methanol R and water R, heat on a water-bath at about 65 °C Bitter Apricot Seed
for 30 min and filter.
Reference solution Dissolve 5 mg of fructose R and 5 mg of (Ph. Bur. monograph 2935)
catechin R in 5 mL of methanol R. Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Mobile phase water R, anhydrous formic acid R, ethyl acetate R Dried, ripe seed, freed from the shell, of Prunus armeniaca L.,
(5:10:85 VIVIV).
Prunus mandshurica (Maxim.) Koehne or Prunus sibirica L.
Content
Development Over a path of 7 cm. Minimum 3.0 per cent of amygdalin (C20H27NO11;
Drying In air. Mr 457.4) (dried drug).
Detection treat with anisaldehyde solution R and heat at IDENTIFICATION
100-105 °C for 5 min; examine in daylight. A. Ovoid seed 1-2 cm long, 0.8-1.5 cm wide and 0.4-0.8 cm
Results See below the sequence of zones present in the thick. The outer surface is very finely granular and yellowish-
chromatograms obtained with the reference solution and the brown to dark brown; one end is acute, the middle part is
test solution. Furthermore, other faint zones may be present convex, the other end is rounded, sometimes slightly
in the chromatogram obtained with the test solution. cordiform, and frequently asymmetrical. The short and linear
hilum is located at the acute end; the chalaza is rounded to
oval and located at the rounded end. A ramified network of
prominent brown vascular bundles is clearly visible on the
surface of the seed, from the chalaza to the hilum.
A transverse section of the seed shows 2 whitish to pale
yellow, smooth and oily cotyledons.
IV-132 Bitter Apricot Seed 2023
B. Microscopic examination (2.8.23). The powder is System suitability Reference solution (c):
yellowish-white with reddish-brown flecks. Examine under a - the chromatogram shows in the lower third 2 distinct
microscope using chloral hydrate solution R. The powder zones, which may be touching; the lower zone
shows the following diagnostic characters (Figure 2935.-1): (sucrose) and the upper zone (glucose) are brown.
fragments of the testa (surface view [A]) consisting of the Results See below the sequence of zones present in the
outer testa with sclereids, isolated or in groups of 2 or 3 chromatograms obtained with reference solution (a) and the
[Aa], connected to cells with unlignified walls [Ab], several test solution. Furthermore, in the chromatogram obtained
layers of parenchyma with ovoid cells [Ac] and the protein with the test solution, other faint blue fluorescent zones or
layer with polyhedral cells [Ad]; isolated yellow sclereids, up faint brown zones may be present.
to 75 µm long and 150 µm wide at the base, varying in
shape; in surface view [F] the sclereids are rounded to ovoid, Top of the plate
with thick walls, wide channels and numerous rounded or
slit-shaped pits [Fa] in the bottom of the cell walls, whereas A blue fluorescent zone, intense
in transverse section [Ca, Cb] the sclereids are ovoid to A blue fluorescent zone, intense
trapezoid, with a tabular outer surface with very thick pitted
walls [Cc] and a more or Jess flat, thinner, inner surface with A blue fluorescent zone
ramified channels [Cd]; numerous fragments of cotyledons
-- --
with thin-walled polyhedral cells containing oil droplets [Ba]
and small crystals of calcium oxalate, especially in the
outermost layers (transverse section [B]); spiral or annular A blue fluorescent zone
vessels [E]; numerous isolated oil droplets [D].
A blue fluorescent zone, faint
Arnygdalin: a brown zone A brown zone (amygdalin)
-- --
2 blue fluorescent zones, faint
A brown zone
Sucrose: a brown zone A brown zone (sucrose)
TESTS
Peroxide value (2. 5. 5)
Maximum 9.0.
Figure 2935.-1 - Illustration for identification test B of powdered
herbal drug of bitter apricot seed Introduce 35.0 g of the powdered herbal drug (1400)
(2.9.12) into a 250 mL round-bottomed flask. Add 150 mL
C. High-performance thin-layer chromatography (2.8.25). of heptane R and boil under a reflux condenser for 30 min.
Test solution To 0.2 g of the powdered herbal drug (710) Allow to cool, filter through a paper filter and evaporate the
(2.9.12) add 5.0 mL of methanol Rand sonicate for 20 min. filtrate to dryness to obtain the oil. Use the oil to determine
Centrifuge and use the supernatant. the peroxide value.
Reference solution (a) Dissolve 10.0 mg of amygdalin Rand Foreign matter (2.8.2)
5.0 mg of sucrose R in methanol Rand dilute to 5.0 mL with Maximum 2.0 per cent.
the same solvent. Loss on drying (2.2.32)
Reference solution (b) Dilute 2.5 mL of reference solution (a) Maximum 8.0 per cent, determined on 1.000 g of the
to 10.0 mL with methanol R. powdered herbal drug (710) (2.9.12) by drying in an oven at
Reference solutwn (c) Dissolve 5 mg of glucose R and 5 mg of 105 °C for 5 h.
sucrose R in methanol R and dilute to 5.0 mL with the same Total ash (2.4.16)
solvent. Maximum 4.0 per cent.
Intensity marker Amygdalin. ASSAY
Plate TLC silica gel F254 plate R (2-10 µm). Liquid chromatography (2.2.29).
Mobile phase water R, methylene chloride R, methanol R, ethyl Test solution To 0.20 g of the powdered herbal drug (710)
acetate R (10:15:22:40 VIVIVIV). (2.9.12) add 50.0 mL of anhydrous methanol R. Weigh and
Application 5 µL as bands of 8 mm. sonicate for 45 min. Allow to cool and weigh again.
Development 70 mm from the lower edge of the plate. Compensate for the loss of solvent with anhydrous
methanol R. Filter.
Drying In air for 5 min.
Transfer about 5 mL of the filtrate to a test tube containing
Detection Treat with a 10 per cent V/V solution of sulfuric 0.5 g of end-capped octadecylsilyl silica gel for chromatography R.
acid R in ethanol (96 per cent) R and heat at 105 °C for
Shake and allow to stand for 15 min. Filter through a
3 min; examine in ultraviolet light at 365 nm. membrane filter (nominal pore size 0.45 µm).
2023 Black Cohosh IV-133
Reference solutwn (a) Dissolve 5.0 mg of amygdalin CRS in vascular tissue alternating with darker medullary rays and a
metharwl Rand dilute to 50.0 mL with the same solvent. large central pith. Roots attached to the lower surface of the
Reference solutwn (b) Dissolve 1 mg of primeverin R in rhizome are usually broken off, leaving circular scars.
reference solution (a) and dilute to 20 mL with the same The roots are dark brown, 1-3 mm in diameter, brittle,
solution. nearly cylindrical or obtusely quadrangular and longitudinally
wrinkled; the fracture is short; the transverse section shows a
Column:
wide outer bark, a dark brown cylinder, in which the central
- size: l = 0.25 m, 0 = 4.6 mm;
region is composed of 3-6 lighter wedges of vascular tissue
united at the centre and separated by broad, non-lignified
chromatography R (5 µm),
medullary rays.
Mobile phase:
Fragmented drug More or less angular, irregular pieces of
- mobile phase A: water for chromatography R;
the rhizome and cylindrical pieces of the roots. The hard,
- mobile phase B: acetonitrile Rl;
horny rhizome fragments usually show a dark brown surface
Time Mobile phase A Mobile phase B corresponding to the outer surface and several frequently
(min) (per cent VIP) (per cent VIP) striated, light brown surfaces corresponding to the section.
0-3 95 5 The dark brown, more or less cylindrical root fragments are
3 - 13.5 95---. 48 5---. 52 wrinkled longitudinally. The lighter coloured transverse
13.5 - 15 48---. 5 52---. 95 section shows a distinct cambium line separating a thick
15 - 20 5 95 outer bark from a central region composed of 3-6 wedges of
vascular tissue united at the centre and separated by broad
Flow rate 1.2 mUmin. medullary rays.
Injection 10 µL.
Retention time Amygdalin = about 10 min;
primeverin = about 11.6 min.
amygdalin and primeverin.
Calculate the percentage content of amygdalin using the
A1 area of the peak due to amygdalin in the chromatogram

solution, in grams;
m2 mass of amygdalin CRS used to prepare reference solution (a),
in grams;
p percentage content of amygdalin in amygdalin CRS.
Black Cohosh
DEFINITION
Dried, whole or fragmented rhizome and root of Actaea Figure 2069 .-1. - Illustratwn for identificatwn test B of powdered
racemosa L. (syn. Cimicifuga racemosa (L.) Nutt.). herbal drug of black cohosh
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 (C 42 H6sNO16; Mr 840) (dried
solutwn R. The powder shows the following diagnostic
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 [HJ; groups of short vessels with closely
upright or curved branches each terminating in the remains arranged bordered pits [C, TI sometimes accompanied by
of a bud or in a circular, cup-shaped scar. The fracture is finely pitted fibres Ua]; fragments of the parenchyma of the
horny, 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
IV-134 Black Cohosh 2023
Table 2069.-1. -Application scheme

Track I 2 3 4 5 6 7
Application volume (µL) 2 2 2 - 2 2 2
Reference Reference Reference Reference

Solution Test solution Blank Test solution
solution (a) solution (b) solution (a) solution (b)
After development, the plate is cut along track 4 (blank). Tracks 1-3 are used for detection of a substitution by C. amerfrana, C. foetida, C. dahurica or
C. heracleifolia (detection A), tracks 5-7 for Identification C (detection B).
Table 2069.-2. - Application scheme

Track I 2 3 4 5 6 7 8 9
Application
volume (µL)
20 2 2 20 - 20 2 2 20
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).
isolated sclereids [DJ; fragments of the dermal tissue of the TESTS

roots (surface view [E], longitudinal section [BJ), consisting Loss on drying (2.2.32)
of brown cells covered by a dark brown cuticle [Ba]. Maximum 12 per cent, determined on 1.000 g of the
Examine under a microscope using a 50 per cent V/V powdered herbal drug (355) (2.9.12) by drying in an oven at
solution of glycerol R. The powder shows abundant starch 105 °C for 2 h.
granules, spherical or polygonal, simple, 5-10 µm in Foreign matter (2.8.2)
diameter, or 2 or 3 (rarely up to 6) compound; some Maximum 5 per cent.
granules have a slit-shaped hilum [G].
Total ash (2.4.16')
Maximum 10 per cent.
substitution by Cimicijuga americana Michx., C. foetida L., C.
dahurica (Turcz.) Maxim. or C. heracleifolia Korn. Ash insoluble in hydrochloric acid (2.8.1)
Maximum 5 per cent.
Results B Use the chromatograms supplied with Actaea
racemosa HRS and the chromatogram obtained with reference Substitution by Cimicijuga 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) Test solution To 0.50 g of the powdered herbal drug (355)
and the test solution. Furthermore, other faint zones may be (2. 9.12) add 10 mL of ethanol (50 per cent V/V) 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 VIY,) 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.
Mobile phase anhydrous formic acid R, ethyl formate R,
Actein: a brown zone Actein: a brown zone A brown zone (actein)
toluene R (20:30:50 VIVIV).
23-Epi-26-deoxyactein: A brown zone (23-epi- Application 2 µLas bands of 8 mm (see Table 2069.-1).
a brown zone 26-deoxyactein)
Drying In air.
--- ~~
--- System suitability Reference solution (b):
- the RF value of the zone due to actein is between 0.35
and 0.40 (detection B).
A violet zone A violet zone Results A The chromatogram obtained with the test solution
does not show any quenching zones more intense than those
A brown zone A brown zone
in the chromatogram obtained with reference solution (a)
between RF value 0.2 and RF value 0.35.
Reference Reference Detection B Treat with a 10 per cent VIV solution of sulfuric
Test solution
solution (a) solution (h) acid R in methanol R; heat at 100 °C for 5 min; allow to cool
to room temperature and examine in daylight.
2023 Black Cohosh IV-135
Adulteration with Cimicijuga americana Michx,, C. solution indicates adulteration with C. foetida at a level
foetida L., C. dahurica (Turcz.) Maxim. and/or C. greater than 5 per cent.
heracleifolia Kom.
Thin-layer chromatography (2.2.27) as described in the test Top of the plate
for substitution by Cimicifuga americana Michx., C. foetida L.,
C. dahurica (Turcz.) Maxim. or C. heracleifolia Korn., with --- --- --- ---
the following modifications. Actein: a weak Actein: a weak A weak whitish
Reference solution (c) Dissolve 2 mg of cimifugin R in whitish zone whitish zone zone (actein)
methanol R and dilute to 10 mL with the same solvent. -- --- --- ---
Applicatwn 2 µL of reference solutions (b) and (c), 20 µL of

A bluish zone A bluish zone
the test solution and reference solution (a), as bands of
8 mm (see Table 2069.-2).
System suitability Reference solution (b): Cimifugin: a A BRIGHT
- the Rp value of the zone due to actein is between 0.35 bright FLUORESCENT
and 0.40 (detections Band C). fluorescent zone ZONE
(CIMIFUGIN)
A brownish zone A brownish zone
Results A Absence of more than 10 per cent of
C. americana. A bluish zone A bluish zone
Compare the chromatogram supplied with Actaea A FLUORESCENT

racemosa HRS for C. americana and the chromatograms ZO~E
obtained with the test solution and reference solution (a).

The chromatogram obtained with the test solution does not
show any quenching zone at Rp value 0.3 (zone presented in Reference Reference Reference C.foetida
solution (a) solution (b) solution ( c) (5 per cent)
capitals in the chromatogram of C. americana, see below).
The presence of this zone in the chromatogram obtained
with the test solution indicates adulteration with C. americana Detection C Dissolve 8 g of antimony trichloride R in 200 mL
at a level greater than 10 per cent. of methylene chloride R; treat with this solution and heat at
120 °C for 10 min; examine in ultraviolet light at 365 nm.
Top of the plate Results C Absence of more than 5 per cent of C. heracleifolia
and/or C. dahurica.
Compare the chromatogram supplied with Actaea
-- --- racemosa HRS for C. heracleifolia and C. dahurica and the
A weak zone A weak zone chromatograms obtained with the test solution and reference
solutions (a) and (b). The chromatogram obtained with the
2 weak zones 2 weak zones test solution does not show any bright fluorescent zone just
A weak zone A weak zone above the zone due to actein (zone presented in capitals in
the chromatogram of C. heracleifolia or C. dahurica, see
below). The presence of this zone in the chromatogram
--- _ _ _A DARK ZONE 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 A dark zone
Top of the plate
A dark zone A dark zone
Reference solution (a) C. americana (10 per cent) -- --- ---
A BRIGHT FLUORESCENT
ZONE
Detection B Dissolve 4.5 g of boric acid R in 150 mL of
Actein: a weak Actein: a weak A weak brownish zone
anhydrous ethanol R (solution A); dissolve 5 g of oxalic acid R
brownish zone brownish zone (actein)
in 50 mL of anhydrous ethanol R (solution B); combine
solutions A and B and mix well; treat the plate with this --- --- ---
freshly prepared solution and heat at 120 °C for 5 min; A brownish zone A brownish zone
A bluish zone A bluish zone
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
C. heracleilolia
with the test solution and reference solutions (a), (b) and (c). Reference Reference (5 per cent) and/or
The chromatogram obtained with the test solution does not solution (a) solution (b) C. dahurica
show any intense fluorescent zone between Rp value 0.03 (5 per cent)
and Rp value 0.06 or at the same position as the bright
fluorescent zone in the chromatogram obtained with
ASSAY
reference solution (c) (zones presented in capitals in the
chromatogram of C. foetida, see below). The presence of I or Liquid chromatography (2.2.29).
both zones in the chromatogram obtained with the test Test solutwn Introduce 4.00 g of the powdered herbal drug
(355) (2.9.12) into a 200 mL screw-cap bottle. Add 50.0 mL
IV-136 Black Currant 2023
of a mixture of equal volumes of methanol R and water R. logarithm to base 10 of the corresponding peak area as the
Sonicate for 45 min and shake for 15 min. Filter through a ordinate.
membrane filter (nominal pore size 0.45 µm). Calculate the percentage content of each peak using the
Reference solution (a) Dissolve 10.0 mg of Actaea racemosa following expression:
for assay CRS (containing monoammonium glycyrrhizate) in
methanol R with the aid of ultrasound and dilute to 10.0 mL loA x 5
with the same solvent. m
A = logarithm to base 10 of the concentration of each peak in the
to 10.0 mL with methanol R. chromatogram obtained with the test solution, determined from
Reference solution (c) Dilute 2.0 mL of reference solution (a) the calibration curve;
to 10.0 mL with methanol R. m mass of the herbal drug to be examined used to prepare the test
solution, in grams.
Reference solution (d) Dilute 1.0 mL of reference solution (a)
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 - - - - - - - - - - - - - - - - - - - - - - PhEur
10.0 mL with the same solvent; sonicate and filter through a
Column:
- size: l = 0.25 m, 0 = 4.6 mm; Black Currant
(5 µm). Preparation
Black Currant Syrup
Mobile phase:
- 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 Ribes nigrum
- mobile phase B: 0 .1 per cent VIV solution of anhydrous L., together with their pedicels and rachides.
formic acid R in a mixture of equal volumes of
CHARACTERISTICS
acetonitrile R and methanol R;
Odour, strong and characteristic.
Thne Mobile phase A Mobile phase B Macroscopical Berries: globose, ranging in diameter from
(min) (per cent VIP) (per cent VIP) about 7 to 15 mm, occurring in pendulous racemes; epicarp
0 - 40 50--+ 20 50--+ 80 shiny black externally, enclosing a yellowish green translucent
40 - 41 20 -► 5 80--+ 95 pulp containing numerous flattened ovoid seeds, about
41 - 44 5 95 2.5 mm long, 1.25 mm wide and 1 mm thick; berry crowned
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 mIJmin.
Microscopical Epicarp: glands yellow, disc-shaped, roughly
Detection Evaporative light-scattering detector; the following
circular or broadly elliptical, varying in diameter from about
settings have been found to be suitable; if the detector has
140 to 240 µm, each consisting of a single layer of cells
different setting parameters, adjust the detector settings so as
attached in the centre to the epicarp by means of a short,
to comply with the system suitability criterion for the signal-
multiseriate stalk. Calyx: trichomes unicellular, blunt-ended
to-noise ratio:
with thin, crooked walls, about 10 to 14 µm wide and
- carrier gas: nitrogen R;
averaging about 350 µm in length. Seed: testa with pigment
- flow rate: 0.8 mlJmin;
layer composed of small cells with horseshoe-shaped wall
- evaporator temperature: 100 °C;
thickenings as seen in cross section, each cell containing one
- nebuliser temperature: 60 °C.
or two prismatic crystals of calcium oxalate; endosperm cells
Injection 10 µL. with irregularly thickened walls.
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:
- signal-to-noise ratio: minimum 4.0 for the peak due to DEFINITION
monoammonium glycyrrhizate in the chromatogram Black Currant Syrup is prepared either from the clarified
obtained with reference solution (d); juice of Black Currant or from concentrated black currant
- peak-to-valley ratio: minimum 3, where Hp = height above juice of commerce. It contains a suitable antioxidant.
the baseline of peak 4 and Hv = height above the baseline Permitted food grade colours may be added.
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
Establish a calibration curve with the logarithm to base 10 of weight per mL of 1.045 g, or in 560 mL of a solution of the
the concentration (in milligrams per millilitre) of reference same weight per mL prepared from the concentrated juice of
solutions (a), (b), (c) and (d) (corrected by the assigned commerce and Water, and adding to this solution sufficient
percentage content of monoammonium glycyrrhizate in Benzoic Acid to give a final concentration of not more than
Actaea racemosa for assay CRS) as the abscissa and the 800 ppm, or sufficient Sodium Metabisulfite or other suitable
2023 Blackcurrant Leaf IV-13 7
sulfite to give a final concentration of not more than B. Microscopic examination (2.8.23). The powder is
350 ppm of sulfur dioxide. brownish-green. Examine under a microscope using chloral
The syrup complies with the requirements stated under Oral hydrate solution R. The powder shows the following diagnostic
LUJ.uids and with the f oll,owing requirements. characters (Figure 2528.-1): curved, unicellular covering
trichomes, with moderately thickened, slightly verrucose
Content of ascorbic acid 1, C 6 H 8 O6
walls [D]; orange-yellow, globular or ovoid glandular
Not less than 0.055% w/w.
trichomes, lacking a visible stalk, with a multicellular head up
TESTS to 200 µm in diameter (surface view [Al); fragments of the
Sulfur dioxide lower epidermis (surface view [BJ) composed of cells with
Not more than 350 ppm, Appendix IX B. irregularly thickened walls [Ba], numerous anomocytic
Weight per mL stomata (2.8.3) [Bb] accompanied by spongy
1.27 to 1.30 g, Appendix V G. parenchyma [Be]; fragments (surface view [C], transverse
section [El) of the upper epidermis [Ca, Ea], accompanied
ASSAY by palisade parenchyma [Cb, Eb]; cluster crystals of calcium
Mix 5 g with 25 mL of a freshly prepared 20% w/v solution oxalate up to 30 µm in diameter, isolated [F] or included in
of metaphosphoric acid, add 20 mL of acetone and dilute to parenchymatous cells [Bd, Cc, Ee].
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 Ca
equivalent to 0.200 mg of C 6H 8 0 6 •
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.
Blackcurrant Leaf
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Figure 2528.-1. - [ll,ustrationfor identification test B of powdered
DEFINITION
herbal drng of blackcurrant leaf
Dried leaf of Ribes nigrum L.
Content C. Thin-layer chromatography (2.2.27).
Minimum 1.0 per cent of flavonoids, expressed as Test solution To 1 g of the powdered herbal drug (355)
isoquercitroside (C 21 H 200 12; M, 464.4) (dried drug). (2. 9.12) add 10 mL of methanol R. Heat in a water-bath at
IDENTIFICATION 60 °C for 10 min with occasional stirring. Allow to cool.
A. The leaf is simple. The lamina may be up to 10 cm long Filter.
and 12 cm wide and shows 3 (rarely 5) rounded triangular Reference solution Dissolve 5 mg of isoquercitroside R and
lobes, dentate or crenate on the margins, with the median 5 mg of rutoside trihydrate R in 10 mL of methanol R.
lobe being the largest. The light-brown midrib and secondary Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
veins are very visible on the lower surface, and form a plate R (2-10 µm)].
characteristic network through numerous anastomoses. Mobile phase anhydrous formic acid R, water R, ethyl acetate R
The rigid, light-brown petiole shows a very distinct gutter on (10: 10:80 VIVIV).
the upper part and its length is equal to half the length of the
lamina.
Drying At 100-105 °C for 10 min.
Detection Treat with a 10 g/L solution of diphenylboric acid
1 The requirement for Conrenr of ascorbic acid does not apply when Black
aminoethyl ester R in methanol R, then with a 50 g/L solution
Currant Syrup is used as a flavouring agent for pharmaceutical purposes.
IV-138 Black Horehound 2023
of macrogol 400 R in methanol R; allow to dry in air for about Detection Spectrophotometer at 350 nm.
30 min, then examine in ultraviolet light at 365 nm. Injection 10 µL.
Results See below the sequence of zones present in the Identification of peaks Use the chromatogram obtained with
chromatograms obtained with the reference solution and the reference solution (a) to identify the peak due to
test solution. Furthermore, other fluorescent zones may be isoquercitroside and the chromatogram obtained with
present in the chromatogram obtained with the test solution. reference solution (b) to identify the peak due to rutoside.
Retention time Rutoside = about 28 min;
Top of the plate isoquercitroside = about 29 min.
-- -- System suitability Reference solution (c):
A green zone
rutoside and isoquercitroside.
Isoquercitroside: an orange zone An orange zone (mainly Disregard limit The area of the principal peak in the
isoquercitroside) chromatogram obtained with reference solution (d).
A light blue zone
Calculate the percentage content of total flavonoids,
-- --
expressed as isoquercitroside, using the following expression:
Rutoside: an orange-yellow zone An orange-yellow zone (rutoside) A1xmzxp
Reference solution Test solution A 2 x m1 x 5
A, sum of the areas of the peak due to rutoside and all peaks
eluting after the peak due to rutoside in the chromatogram
TESTS obtained with the test solution;
Foreign matter (2.8.2) area of the peak due to isoquercitroside in the chromatogram
Maximum 3 per cent. obtained with reference solution (a);
Loss on drying (2.2.32) solution, in grams;
Maximum 10.0 per cent, determined on 1.000 g of the m2 mass of isoquercitroside CRS used to prepare reference
powdered herbal drug (355) (2.9.12) by drying in an oven at solution (a), in grams;
p percentage content of isoquercitroside in isoquercitroside CRS.
105 °C for 2 h.
Total ash (2.4.16) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
ASSAY
Test solution Disperse 0.200 g of the powdered herbal drug Black Horehound
(355) (2.9.12) in 10 mL of an 80 per cent V/V solution of
methanol R. Heat under a reflux condenser in a water-bath at (Ph. Bur. monograph 1858)
60 °C for 30 min. Sonicate for 15 min. Allow to cool, dilute PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
to 20.0 mL with an 80 per cent V/V solution of methanol R.
Filter through a membrane filter (nominal pore size DEFINITION
0.45 µm). Dried flowering tops of Ballota nigra L.
Reference solution (a) Dissolve 5.0 mg of isoquercitroside CRS Content
in an 80 per cent V/V solution of methanol R and dilute to Minimum 1.5 per cent of total ortho-dihydroxycinnamic acid
100.0 mL with the same solution. derivatives, expressed as acteoside (C 29 H 360 15; Mr 625)
(dried drug).
Reference solution (b) Dissolve 5.0 mg of rutoside trihydrate R
in methanol Rand dilute to 100.0 mL with the same solvent. IDENTIFICATION
Reference solution (c) Dilute 10.0 mL of reference A. The stems are conspicuously 4-angled, longitudinally
solution (a) to 20.0 mL with reference solution (b). striated, dark green or reddish-brown and more or less
pubescent. The leaves are greyish-green, petiolate, the lamina
ovate or orbicular, 2-4 cm wide, the margin irregularly
to 20.0 mL with an 80 per cent V/V solution of methanol R.
crenate, and cuneate or cordate at the base; both surfaces are
Column: covered with abundant whitish hairs; the venation is pinnate,
=
- size: l 0.25 m, 0 4 mm; = prominent on the lower surface, slightly depressed on the
- stationary phase: end-capped octadecylsilyl silica gel for upper. The flowers are sessile or very shortly pedicellate, the
chromatography R (5 µm); calyx is infundibuliform, densely pubescent, with 10
- temperature: 30 °C. prominent ribs and 5 subequal, broadly ovate teeth; the
Mobile phase: corolla, with a tube slightly shorter than the calyx tube, is
- mobile phase A: 0.05 per cent V/V solution of trifiuoroacetic purple and bilabiate, the upper lip pubescent on the outer
acid R; surface and the lower lip with 3 lobes, the middle of which is
- mobile phase B: acetonitrile R; notched.
B. Microscopic examination (2.8.23). The powder is greyish-
green and slightly flocculent. Examine under a microscope
(min) (per cent V/J/) (per cent V/J/)
0 - 45 97--+ 60 3--+ 40
following diagnostic characters (Figure 1858.-1): numerous
long, uniseriate, multicellular covering trichomes consisting of
Flow rate 1.0 mUmin. 4 or more cells, thickened and swollen at the junctions, with
slightly lignified and pitted walls, free [C] or on an epidermis
2023 Black Horehound IV-139
(transverse section [Ea]); fewer glandular trichomes, usually Development Over a path of 15 cm.
on epidermises (transverse section [E, F, G]): some with a Drying In air.
unicellular or multicellular stalk and a globose, uni- or Detection Spray with a solution containing 10 g/L of
bicellular head [Ga], others with a unicellular stalk and a diphenylboric acid aminoethyl ester R and 50 g/L of macrogol
multicellular head (surface view [Ac], transverse section 400 R in methanol R; allow to dry in a current of warm air;
[Eb)), others with a unicellular stalk and an 8-celled head of
examine in ultraviolet light at 365 nm after 30 min.
lamiaceous type (surface view [Ad), transverse section [Fa]);
fragments of the adaxial leaf epidermis [BJ with cells with Results See below the sequence of zones present in the
sinuous walls, accompanied by cells of the palisade chromatograms obtained with the reference solution and the
parenchyma, most containing fine, needle-shaped crystals test solution. Furthermore, other fluorescent zones may be
[Ba]; fragments of the abaxial leaf epidermis [A) bearing present in the chromatogram obtained with the test solution.
numerous stomata, the majority anomocytic (2.8.3) [Aa] but
some diacytic [Ab); fragments of the epidermis of the corolla Top of the plate
composed of polygonal cells, those of the inner epidermis of A reddish fluorescent zone
the lips papillose [HJ and those of the inner epidermis of the
tube bearing uni- or bicellular covering trichomes in a stellate A faint yellow fluorescent zone
arrangement [K]; pollen grains subspherical with 3 pores and A light blue fluorescent zone
a smooth exine [DJ; fragments from the stem (G) with (caffeoylmalic acid)
groups of collenchymatous cells [Gb] and lignified vessels, A greenish-blue fluorescent zone
with annular or spiral thickenings U]. (acteoside)
(luteolin 7-lactate)
Chlorogenic acid: a light blue
fluorescent zone
A greenish-blue fluorescent zone

(forsythoside B)
Rutoside: an orange-yellow 2 greenish-blue fluorescent zones
fluorescent zone (arenarioside)
A yellow fluorescent zone (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 VIV,) 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 Vlli'.) R. Add the
rinsings to the filtrate and dilute to 100.0 mL with ethanol
Figure 1858. -1. - Illustration far identification test B of powdered
(50 per cent VIV,) R.
herbal drug of black horehound
Test solution Into a 10 mL volumetric flask, introduce
C. Tun-layer chromatography (2.2.27). successively, with shaking after each addition, 1.0 mL of the
Test solution To 2 g of the powdered herbal drug (355) stock solution, 2 mL of 0. 5 M hydrochloric acid, 2 mL of a
(2.9.12) add 100 mL of methanol R. Heat on a water-bath solution containing 100 g/L of sodium nitrite R and 100 g/L of
under a reflux condenser for 30 min. Allow to cool. Filter. sodium molybdate R, and 2 mL of dilute sodium hydroxide
Evaporate the filtrate under reduced pressure until a volume solution R, and dilute to 10.0 mL with water R.
of about 10 mL is obtained. Compensation liquid Into a 10 mL volumetric flask,
Reference solution Dissolve 1 mg of chlarogenic acid R and introduce 1.0 mL of the stock solution, 2 mL of 0.5 M
2.5 mg of rutoside trihydrate R in 10 mL of methanol R. hydrochloric acid and 2 mL of dilute sodium hydroxide
solution R, and dilute to 10.0 mL with water R.
Measure immediately the absorbance (2.2.25) of the test
Mobile phase anhydrous formic acid R, glacial acetic acid R,
solution at 525 nm, by comparison with the compensation
water R, ethyl acetate R (7.5:7.5:18:67 VIVIVIV).
liquid.
IV-140 Bogbean Leaf 2023
Calculate the percentage content of total ortho- solutions. Furthermore, other zones are present in the
dihydroxycinnamic acid derivatives, expressed as acteoside, chromatogram obtained with the test solution.
using the following expression:
Top of the plate
Ax 1000
185 X m A violet zone
An intense blue zone

i.e. taking the specific absorbance of acteoside to be 185.
Loganine: a greyish-violet zone A violet to greyish-violet zone
A grey to greyish-blue zone
A brownish zone
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
TESTS
Bogbean Leaf Loss on drying (2.2.32)
(Ph. Bur. monograph 1605) powdered herbal drug (355) (2. 9.12) by drying in an oven at
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 105 °C for 2 h.
DEFINITION Total ash (2.4.16)
Dried, entire or fragmented leaf of Menyanthes trifoliata L. Maximum 10.0 per cent.
Bitterness value (2. 8.15)
CHARACTERS
Minimum 3000.
Very bitter and persistant taste.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
IDENTIFICATION
A. The leaf is long-petiolated, trifoliate, with long sheaths
from the base; the petiole is up to 5 mm in diameter and
strongly striated longitudinally. The lamina is divided into
equal leaflets, sessile, obovate up to 10 cm long and up to
Boldo Leaf
5 cm wide, with an entire, occasionally sinuous margin with (Ph. Bur. monograph 1396)
brownish or reddish hydathodes and a spathulate base; it is
glabrous, dark green on the upper surface and paler green on Preparation
the lower surface, with a wide, whitish, finely striated Boldo Leaf Dry Extract
prominent midrib. PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
B. Microscopic examination (2.8.23). The powder is DEFINITION

yellowish-green. Examine under a microscope using chloral Whole or fragmented dried leaf of Peumus boldus Molina.
hydrate solution R. The powder shows fragments of upper
Content
epidermis with polyhedral cells and thin wavy walls;
Minimum O.1 per cent of total alkaloids, expressed as
fragments of lower epidermis with sinuous walls; anomocytic
boldine (C 19 H 21 NO 4; Mr 327.4) (anhydrous drug).
stomata (2.8.3), on both surfaces, with the subsidiary cells
showing radiating striations; epidermal cells from the veins IDENTIFICATION
straight walled and papillose; fragments of mesophyll A. The leaf is oval or elliptical usually 5 cm long with a short
parenchyma with large intercellular spaces (aerenchyma); petiole, an obtuse or slightly emarginate or mucronate apex
irregular cells with rare sclereids; fragments of spiral or and an equal and rounded base; the margin is entire and
annular vessels. slightly undulate and the thickened edges are more or less
C. Thin-layer chromatography (2.2.27). revolute. The lamina is greyish-green, thick, tough and
Test solution To 1.0 g of the powdered herbal drug (355) brittle. The upper surface is rough with numerous prominent
(2. 9.12) add 10 mL of methanol R. Heat, with stirring, in a small protuberances and a depressed venation. The lower
water-bath at 60 °C for 5 min. Allow to cool and filter. surface is finely pubescent, with the protuberances less well-
Evaporate to dryness under reduced pressure in a water-bath marked, and a prominent, pinnate venation.
at 60 °C. Dissolve the residue in 2.0 mL of methanol R. B. Microscopic examination (2.8.23). The powder is greyish-
Reference solution Dissolve 5 mg of loganin R in 15 mL of green. Examine under a microscope using chloral hydrate
methanol R.
characters (Figure 1396.-1): fragments of the lamina of the
leaf (surface view [C], transverse section [G]) consisting of
Mobile phase water R, methanol R, ethyl acetate R the upper epidermis with rigid, thick-walled cells [Ca, Ga],
(8: 15:77 VIV!V). the hypodermis with cells with straight or slightly sinuous,
Application 30 µL, as bands. thickened and beaded walls [Cb, Gb], and palisade
Development Over a path of 15 cm. parenchyma [Cc] with 2 layers of cells [Ge); fragments of the
Drying In air. lower epidermis with numerous stomata surrounded by
4-7 subsidiary cells (surface view [A, TI); solitary [F],
Detection Spray with vanillin reagent R. Heat in an oven at
bifurcated or stellate clustered [D, H] unicellular covering
100-105 °C for 10 min. Examine in daylight. trichomes with more or less thickened and lignified walls;
Results See below the sequence of the zones present in the debris of the spongy mesophyll (surface view [BJ) including
chromatograms obtained with the reference and test numerous large, rounded oil cells [Ba, Ge]; fragments of
2023 Boldo Leaf IV-141
parenchyma containing fine needle-shaped crystals [Bb, Gd], Top of the plate
thick-walled fibres and Jignified, pitted parenchymatous cells
associated with vascular tissue from the veins [E]. - - --
A yellowish-brown zone
Hyoscine: a pale brown zone
A yellow zone
A brown zone
A brown zone
- - --
Boldine: a brown zone A brown zone (boldine)
Several zones
TESTS
Maximum 40 mUkg (anhydrous drug).
Use 10.0 g of the freshly fragmented herbal 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
Water (2.2.13)
Maximum 100 mUkg, determined by distillation of 20.0 g of
the powdered herbal drug (355) (2. 9.12).
Total ash (2.4.16)
ASSAY
Figure 1396.-1. - Illustration for identification test B of powdered Liquid chromatography (2.2.29).
herbal drug of boldo leaf Test solution Disperse 1.000 g of the powdered herbal drug
(355) (2.9.12) in 50 mL of dilute hydrochloric acid Rand
C. Thin-layer chromatography (2.2.27). shake in a water-bath at 80 °C for 30 min. Filter, take up the
Test solution Mix 1.5 g of the powdered herbal drug (355) residue with 50 mL of dilute hydrochloric acid R and shake in
(2. 9.12) and 5 mL of methanol R and sonicate for 10 min. a water-bath at 80 °C for 30 min. Filter and repeat the
Filter the supernatant through a 3 cm x 0.5 cm column of operation once on the residue obtained. Filter. Combine the
cellulose for chromatography Rl. Use the first 1 mL of the cooled filtrates and shake with 100 mL of a mixture of
eluate. equal volumes of ethyl acetate R and hexane R. Discard the
Reference solution Dissolve 2 mg of boldine R and 10 mg of organic layer. Adjust the aqueous layer to pH 9.5 with
hyoscine hydrobromide R in 5 mL of methanol R. concentrated ammonia R. Shake successively with 100 mL,
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel 50 mL and a further 50 mL of methylene chloride R, taking
plate R (2-10 µm)). care not to form an emulsion. If necessary, centrifuge at
1200 g for 10 min. Combine the lower layers and evaporate
Mobile phase diethylamine R, methanol R, toluene R
to dryness under reduced pressure. Dissolve the residue in
(10:10:80 VIVIV).
the mobile phase and dilute to 10.0 mL with the mobile
Application 40 µL [or 6 µL] of the test solution and 20 µL phase.
[or 2 µL] of the reference solution as bands of 15 mm [or
Reference solution (a) Dissolve 12.0 mg of boldine CRS in the
8mm].
mobile phase and dilute to 100.0 mL with the mobile phase.
Development Over a path of 15 cm [or 6 cm]. Dilute 1.0 mL of the solution to 10.0 mL with the mobile
Drying In air. phase.
Detection Treat with potassium iodobismuthate solution R2, Reference solution (b) Disperse 0.5 g of boldo leaf dry
allow to dry in air for 5 min and treat with sodium nitrite extract HRS in 50 mL of dilute hydrochloric acid R and
solution R; examine in daylight after 30 min. sonicate for 10 min. Transfer to a separating funnel and
Results See below the sequence of zones present in the wash with 10 mL of a mixture of equal volumes of ethyl
chromatograms obtained with the reference solution and the acetate R and hexane R. Discard the organic layer. Adjust the
test solution. aqueous phase to pH 9 .5 with concentrated ammonia R. After
cooling, shake successively with 100 mL, 50 mL and a
further 50 mL of methylene chloride R, taking care not to form
an emulsion. If necessary, centrifuge at 1200 g for 10 min.
Combine the lower layers and evaporate to dryness under
IV-142 Baldo Leaf Preparations 2023
reduced pressure. Dissolve the residue in the mobile phase hydroalcoholic solvent equivalent in strength to ethanol
and dilute to 10.0 mL with the mobile phase. (45-75 per cent V/V).
Column: CHARACTERS
- size: l = 0.25 m, 0 = 4.6 mm; Appearance
- stationary phase: base-deactivated end-capped octadecylsilyl Brown or greenish-brown, hygroscopic powder.
silica gelfcrr chromatography R (5 µm).
IDENTIFICATION
Solution A Mix 0.2 mL of diethylamine Rand 99.8 mL of
acetonitrue R.
Solution B Mix 0.2 mL of diethylamine Rand 99.8 mL of
1 mL of hydrochloric acid R and 20 mL of water R. Sonicate
water R and adjust to pH 3 with anhydrous formic acid R.
for 10 min. Transfer the liquid to a separating funnel and
Mobile phase Solution A, solution B (16:84 V/V). make alkaline with 2 mL of dilute ammonia Rl. Shake with
Flow rate 1.5 mL'min. 2 quantities, each of 20 mL, of methylene chloride R.
Detection Spectrophotometer at 304 nm. Evaporate the combined organic layers to dryness. Dissolve
Injection 20 µL. the residue in 1 mL of methanol R.
Identification of peaks Use the chromatogram supplied with Reference solution Dissolve 2 mg of boldine R and 10 mg of
boldo leaf dry extract HRS and the chromatogram obtained hyoscine hydrobromide R in 5 mL of methanol R.
with reference solution (b) to identify the peaks due to Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
alkaloids 1, 3, 4, 5 and 6; use the chromatogram obtained plate R (2-10 µm)].
with reference solution (a) to identify the peak due to Mobile phase diethylamine R, methanol R, toluene R
boldine. (10:10:80 VIVIV).
Relative retention With reference to boldine (retention Application 20 µL [or 3 µL] as bands of 15 mm [or 8 mm].
time = about 6 min): alkaloid 1 = about 0.9; Development Over a path of 15 cm [or 6 cm].
alkaloid 3 = about 1.8; alkaloid 4 = about 2.0;
Drying In air.
alkaloid 5 = about 2.9; alkaloid 6 = about 3.1.
Additional peaks may be present. Detection Treat with potassium iodobismuthate solution R2,
allow to dry in air for 5 min and treat with sodium nitrite
solution R; examine in daylight after 30 min.
alkaloid 1 and boldine. Results See below the sequence of zones present in the
as boldine, using the following expression:
(~Ai) x m2 xp
A2 x m 1 x 100 Top of the plate
L4 1 sum of the areas of the peaks due to alkaloids I, 3, 4, 5 and 6 -- --

and the peak due to bokline in the chromatogram obtained with A yellowish-brown zone
the test solution;
A2 area of the peak due to boldine in the chromatogram obtained An orange-yellow zone
m, mass of the herbal drug to be examined used to prepare the test Hyoscine: a pale brown zone
solution, in grams;
m2 mass of boldine CRS used to prepare reference solution (a), in An orange zone
grams;
An orange zone
p percentage content of boldine in boldine CRS.
-- --
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Boldine: a brown zone A brown zone (boldine)
Several orange zones

Soldo Leaf Dry Extract
(Ph. Bur. monograph 1816) TESTS
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Water (2.5.12)
Maximum 5.0 per cent, determined on 0.5 g.
DEFINITION
Extract produced from Baldo leaf (1396). ASSAY
Content
- for aqueous extracts: minimum 0.1 per cent of total Test solution Disperse 1.000 g of the extract to be examined
alkaloids, expressed as boldine (C 19H 21N04; Mr 327.4) in 50 mL of dilute hydrochlcrric acid R and sonicate for
(anhydrous extract); 10 min. Transfer to a separating funnel and wash with
- for hydroalcoholic extracts: minimum 0.2 per cent of total 10 mL of a mixture of equal volumes of ethyl acetate R and
alkaloids, expressed as boldine (C 19H 21 N0 4 ; Mr 327.4) hexane R. Discard the organic layer. Adjust the aqueous
(anhydrous extract). phase to pH 9.5 with concentrated ammonia R. After cooling,
shake successively with 100 mL, 50 mL, and a further
PRODUCTION 50 mL of methylene chloride R, taking care not to form an
The extract is produced from the herbal drug by a suitable emulsion. If necessary, centrifuge at 1200 g for 10 min.
procedure using either hot water at not less than 65 °C or a
2023 Buckwheat Herb IV-143
Combine the lower layers and evaporate to dryness under

reduced pressure. Dissolve the residue in the mobile phase
Buckwheat Herb
and dilute to 10.0 mL with the mobile phase. (Ph. Bur. monograph 2184)
Reference solution (a) Dissolve 12.0 mg of boldine CRS in the
Reference soluti.on (b) Disperse 0.5 g of boldo leaf dry DEFINITION
extract HRS in 50 mL of dilute hydrochloric acid R and Whole or fragmented aerial parts of Fagopyrum esculentum
sonicate for 10 min. Transfer to a separating funnel and Moench, collected in the early flowering period prior to
wash with 10 mL of a mixture of equal volumes of ethyl fruiting and dried immediately.
acetate R and hexane R. Discard the organic layer. Adjust the Content
aqueous phase to pH 9.5 with concentrated ammonia R. After Minimum 3.0 per cent ofrutoside (C27H3 0 0 16; Mr 611)
cooling, shake successively with 100 mL, 50 mL, and a (dried drug).
further 50 mL of methylene chloride R, taking care not to form
an emulsion. If necessary, centrifuge at 1200 g for 10 min.
IDENTIFICATION
Combine the lower layers and evaporate to dryness under A. The stem is cylindrical, hollow, finely ridged
reduced pressure. Dissolve the residue in the mobile phase longitudinally, about 2-6 mm in diameter, brownish-green or
and dilute to 10.0 mL with the mobile phase. reddish, with few branches and thickened at the intemodes;
the leaves are arranged spirally and have membranous,
Column:
sheathing stipules; the surface is glabrous except in the region
= =
- size: l 0.25 m, 0 4.6 mm;
of the stipules, where short, white hairs may occur.
- stationary phase: base-deactivated end-capped octadecylsilyl
The leaves are dark green, paler on the lower surface, up to
silica gel for chromatography R (5 µm).
7 cm wide and 11 cm long, saggitate or cordate, almost
Solution A Mix 0.2 mL of diethylamine Rand 99.8 mL of pentagonal with 2 widely rounded lobes; the lower leaves are
acetonitrile R. petiolate, the upper leaves sessile or amplexicaul; the lamina
Solution B Mix 0.2 mL of diethylamine R and 99.8 mL of is glabrous and the margin finely sinuate and fringed with
water R and adjust to pH 3 with anhydrous formic acid R. minute, reddish-brown projections; similar projections occur
Mobile phase Solution A, solution B (16:84 VIV). on the veins on the upper surface. The inflorescence is a
Flow rate 1.5 mIJmin. cymose panicle, the individual flowers 1-2 mm long and
6 mm in diameter with 5 free, white or reddish petals.
B. Microscopic examination (2.8.23). The powder is dark
Injection 20 µL.
Identification of peaks Use the chromatogram supplied with solution R. The powder shows the following diagnostic
boldo leaf dry extract HRS and the chromatogram obtained characters (Figure 2184.-1): fragments of the epidermis of
with reference solution (b) to identify the peaks due to the stem (surface view [DJ) composed of elongated cells
alkaloids 1, 3, 4, 5 and 6; use the chromatogram obtained showing striations on the outer walls [Da] and anomocytic
with reference solution (a) to identify the peak due to stomata (2.8.3) [Db]; fragments of the upper epidermis of
boldine. the lamina (surface view [BJ), consisting of polygonal cells
Relative retention With reference to boldine (retention covered by a striated cuticle [Ba] and anomocytic stomata
= =
time about 6 min): alkaloid 1 about 0.9; [Bb], often accompanied by palisade parenchyma [Be];
alkaloid 3 = about 1.8; alkaloid 4 = about 2.0; fragments of the epidermis of the leaf margins [A] and of the
alkaloid 5 = about 2.9; alkaloid 6 = about 3.1. epidermis covering the veins, often showing ovoid or rounded
Additional peaks may be present. papilla-like projections, often reddish, with thickened and
System suitability Reference solution (b): striated walls; fragments of the lower epidermis of the lamina
- resolution: minimum 1.5 between the peaks due to [C] with thin-walled polygonal cells, numerous stomata [Ca]
alkaloid 1 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
(~A1) xm2 xp cluster crystals of calcium oxalate, varying in diameter
(25-100 µm) [Fb], smaller prismatic crystals of calcium
A 2 xm1xl0
oxalate [Fe], occurring scattered in the mesophyll and also in
L4 1 sum of the areas of the peaks due to alkaloids 1, 3, 4, 5 and 6 the parenchyma of the stem; fragments of lignified tissue [HJ
and the peak due to boldine in the chromatogram obtained with with bordered-pitted [Ha], reticulate or annular [Hb) vessels
the test solution; and thin-walled, pitted fibres [He]; occasional fragments of
A2 area of the peak due to boldine in the chromatogram obtained
the corolla with a papillose epidermis [E]; spherical or ovoid
m1 mass of the extract to be examined used to prepare the test pollen grains, about 50 µm in diameter, with a pitted exine
solution, in grams; and 3 furrows [G).
m2 mass of boldine CRS used to prepare reference solution (a), in
grams;
p percentage content of boldine in boldine CRS. Test solution To 0.5 g of the powdered herbal drug (355)
(2. 9.12) add 5 .0 mL of methanol R and heat in a water-bath
at 60 °C under a reflux condenser for 10 min. Cool and
filter.
Reference solution Dissolve 10 mg of hyperoside R and 10 mg
of rutoside trihydrate R in 10 mL of methanol R.
IV-144 Buckwheat Herb 2023
TESTS
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
(2. 9.12), add 30 mL of an 80 per cent VIV solution of
methanol R. Heat the mixture under a reflux condenser in a
water-bath at 60 °C for 30 min, then extract the mixture in
an ultrasonic bath for 15 min. Allow to cool, dilute to
50.0 mL with an 80 per cent V/V solution of methanol Rand
filter.
Reference solution (a) Dissolve 25.0 mg of rntoside
trihydrate CRS in an 80 per cent V/V solution of methanol R
Reference solution (b) Dissolve 20.0 mg of troxerntin R and
5.0 mg of quercitrin R in an 80 per cent VIV solution of
methanol Rand dilute to 50.0 mL with the same solvent.
Column:
- size: l = 0.125 m, 0 = 4 mm;
(5 µm);
Mobile phase:
Figure 2184.-1. - Illustration for identification test B of powdered - mobile phase A: mix 50 volumes of acetonitrile R and
herbal drng of buckwheat herb 950 volumes of water R adjusted to pH 2 with phosphoric
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel acid R;
plate R (2-10 µm)]. - mobile phase B: mix 95 volumes of water R adjusted to
pH 2 with phosphoric acid R and 905 volumes of
Mobile phase anhydrous formic acid R, water R, ethyl acetate R
acetonitrile R;
(10:10:80 VIVIV).
Application 20 µL [or 5 µL] as bands of 15 mm [or 8 mm]. Time Mobile phase A Mobile phase B
Development Over a path of 10 cm [or 6 cm]. (min) (per cent VIP) (per cent VIV)
Drying At 100-105 °C. 0-6 94 6

6 - 16.5 94-+ 85 6-+ 15
Detection treat with a 10 g/1.. solution of diphenylboric acid
16.5 - 22 85 -+ 76 15 -+ 24
aminoethyl ester R in methanol R, subsequently treat with a
22 - 25 76 -+ 59 24 -+ 41
50 g/1.. solution of macrogol 400 R in methanol R; allow to dry
in air for about 30 min and examine in ultraviolet light at
365 nm. FI.ow rate 1.0 mL/min.
Results See below the sequence of zones present in the Detection Spectrophotometer at 350 nm.
chromatograms obtained with the reference solution and the Injection 10 µL.
test solution. Furthermore, other fluorescent zones may be System suitability Reference solution (b):
present in the chromatogram obtained with the test solution. - elution order: order indicated in the composition of
reference solution (b), when the chromatogram is
Top of the plate recorded in the prescribed conditions;
- resolution: minimum 3 between the peaks due to troxerutin
2 red zones
and quercitrin.
1-2 light blue zones Using the retention times determined from the
chromatogram obtained with reference solution (a), locate
-- --
the peak due to rutoside in the chromatogram obtained with
An orange zone the test solution.
An orange zone Calculate the percentage content of rutoside using the
Hyperoside: an orange zone 2 blue zones
-- --
Rutoside: an orange-yellow zone An orange-yellow zone (rutoside)

AI area of the peak due to rutoside in the chromatogram obtained
2023 Bupleurum Root IV-145
A2 area of the peak due to rutoside in the chromatogram obtained

solution~ in grams;
m2 mass of rutoside trihydrate CRS used to prepare reference
p percentage content of rutoside in rnwsi<k tnhydrare CRS.
- - - - - - - - - - - - - - - - - - - - - - Ph Eur C
\
Bupleurum Root
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
~~-"~;;:::_: ~F
DEFINITION
Dried, whole or fragmented root of Bupleurum chinense DC.
or Bupleurum scorzonerifolium Willd.
Content
Minimum O.16 per cent of saikosaponin A (C 42 H 68 0 13;
Mr 781) (dried drug).
IDENTIFICATION
A. B. chinense. The whole root is cylindrical or elongated
conical, branched in the lower part, up to 23 cm long and Figure 2562.-1. - Illustration for identification test B of powdered
1.2 cm in diameter. The upper part consists of a bulgy root herbal drug of bupleurum root
crown, usually composed of 3-15 stem bases as well as the
fibrous remnants of the leaf bases. The fragmented root C. High-performance thin-layer chromatography (2.8.25).
consists of irregular pieces, 1-5 cm long, 0.2-0.3 cm in Examine the chromatograms obtained in the test for
diameter. The outer surface is blackish-brown or light brown, Bupleurum longiradiatum Turcz.
marked with longitudinal wrinkles and showing rootlet scars Results See below the sequence of zones present in the
and lenticel-like protuberances. The texture is hard and chromatograms obtained with reference solution (a) and the
compact, difficult to break. The fracture shows concentric test solution. Furthermore, other faint zones may be present
fibrous rings in the wood; the bark is thin, light brown or in the chromatogram obtained with the test solution.
orange-brown, while the wood is whitish-yellow.
B. scorzonerifolium The whole root is thinner than that of B. Top of the plate
chinense, elongated conical, usually unbranched or very
slightly branched in the lower part, up to 15 cm long and
0.3-0.5 cm in diameter. The root crown bears numerous A violet zone
fibres from the bases of wilted leaves in a brush-like shape.
The fragmented root consists of irregular pieces, 1-5 cm
long. The outer surface is blackish-brown or reddish-brown
-- --
and shows numerous annular striations near the root crown.
The texture is slightly soft and the root breaks easily.
The fracture is even and non-fibrous. An orange or faint orange zone
B. Microscopic examination (2.8.23). The powder is reddish-
brown or yellowish-brown. Examine under a microscope
using chloral hydrate solution R. The powder shows the Saikosaponin D: an orange-brown An orange-brown zone
following diagnostic characters (Figure 2562.-1): fragments of zone (saikosaponin D)
yellowish-brown to reddish-brown cork (surface view [A]) Saikosaponin A: a grey zone A grey zone (saikosaponin A)
with rectangular and flattened cells (transverse section [H]),
some cork cells containing small prism crystals visible mainly -- - -
in polarised light [Ha]; fragments of vessels (5-80 µm in

diameter) with various thickenings [G], most of which are
A reddish-violet zone
reticulate, accompanied by xylem parenchyma with
(saikosaponins C, H)
elongated, thin-walled cells or by fibres with thick (2-8 µm)
and channelled walls [C]; xylem fibres, isolated [F] or in
bundles [E], long, fusiform, about 7-25 µm in diameter; Reference solution (a) Test solution
fragments of secretary canals ( 10-15 µm up to 40 µm in
diameter) with inconspicuous secretary cells and yellowish to
orange contents [D]; fragments of parenchyma with TESTS
conspicuous secretory canals visible as yellowish to orange- Bupleurum longiradiatum Turcz
red ribbon-like secretions [BJ. High-performance thin-layer chromatography (2. 8. 25).
(2.9.12) add 10.0 mL of methanol Rand sonicate for 10 min.
Centrifuge and use the supernatant.
IV-146 Greater Burnet Root 2023
Reference solution (a) Dissolve 1.0 mg of saikosaponin A R System suitability Reference solution (b):
and 1.0 mg of saikosaponin D R in methanol R and dilute to - resolution: minimum 3.0 between the peaks due to propyl
5.0 mL with the same solvent. parahydroxybenzoate and saikosaponin A.
Reference solution (b) Dilute 2.5 mL of reference solution (a) Calculate the percentage content of saikosaponin A using the
to 10.0 mL with methanol R. following expression:
Intensity marker Saikosaponin D.
A 1 x m2 xpx0.5
Plate TLC silica gel F 254 plate R (2-10 µm).
A2 xm 1
Mobile phase water R, 2-propanol R, ethyl acetate R
(10:30:80 VIVIV). area of the peak due to saikosaponin A in the chromatogram
Application 5 µL as bands of 8 mm. obtained with the test solution;
area of the peak due to saikosaponin A in the chromatogram
Development 70 mm from the lower edge of the plate. obtained with reference solution (a);
Drying In air. mass of the herbal drug to be examined used to prepare the test
solution, in grams;
Detection Treat with anisaldehyde solution R and heat at mass of saikosaponin A CRS used to prepare reference
100 °C for 3 min; examine in daylight. solution (a), in grams;
p percentage content of saikosaponin A in saikosaponin A CRS.
System suitability Reference solution (a):
- the chromatogram shows 2 distinct zones in the lower _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEw
part of the middle third; the lower zone (saikosaponin A)
is grey and the upper zone (saikosaponin D) is orange-
brown.
Results In the chromatogram obtained with the test
solution, the presence of a greyish zone directly above the
Greater Burnet Root
zone due to saikosaponin H (which co-migrates with
(Sanguisorba Root, Ph. Bur. monograph 2385)
saikosaponin C) indicates adulteration by B. longiradiatum.
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 Whole or fragmented, dried underground parts of
105 °C for 2 h. Sanguisorba officinalis L. without rootlets.
Total ash (2.4.16) Content
Maximum 9.0 per cent. Minimum 5.0 per cent of tannins, expressed as pyrogallol
Ash insoluble in hydrochloric acid (2.8.1) (C6H6O3; Mr 126.1) (dried drug).
Maximum 3.5 per cent. IDENTIFICATION
ASSAY A. Whole drug. The whole root is irregular, fusiform or
Liquid chromatography (2.2.29). cylindrical, slightly curved or twisted, 5-25 cm long and
0.5-2 cm in diameter. The outer surface is greyish-brown or
dark brown, rough with longitudinal wrinkles and sometimes
(355) (2.9.12) in a mixture of 3 mL of concentrated
bears the remains of short, hard rootlets. The texture is hard
ammonia Rand 12 mL of methanol R. Sonicate for 30 min
and brittle. The fracture is relatively even, pinkish or pale
and centrifuge for 10 min. Repeat the extraction twice,
yellow, dense and granular. The xylem has a slightly radiate
combine the supernatants and evaporate to dryness under
structure.
reduced pressure. Dissolve the residue in methanol R and
dilute to 5.0 mL with the same solvent. Filter through a Fragmented drug The fragmented root, 0.5-2 cm in
membrane filter (nominal pore size 0.45 µm). diameter, occurs as short, more or less cylindrical pieces or as
oblique, subrounded or irregular slices. The outer surface is
greyish-brown or dark brown, rough with longitudinal
saikosaponin A CRS in methanol R and dilute to 10.0 mL
wrinkles and sometimes bears the remains of short, hard
rootlets. The texture is hard and brittle. The fracture is
Reference solution (b) Dissolve 1 mg of propyl relatively even, dense and granular; the cut surface is pinkish,
parahydroxybenzoate R in 1 mL of reference solution (a) and pale yellow or yellowish-brown and shows the xylem which is
dilute to 10.0 mL with methanol R. sometimes lighter in colour, and has a slightly radiate
Column: structure.
=
- size: l 0.25 m, 0 = 4.6 mm; B. Microscopic examination (2.8.23). The powder is
- stationary phase: base-deactivated end-capped octadecylsilyl yellowish-brown or dark brown. Examine under a microscope
silica gel for chromatography R (5 µm). using chloral hydrate solution R. The powder shows the
Mobile phase acetonitrile Rl, water for chromatography R following diagnostic characters (Figure 2385.-1): abundant
(36:64 V/V). cluster crystals of calcium oxalate 11-65 µm in diameter, free
Flow rate 1.0 mUmin. [L] or included in parenchyma cells sometimes forming
Detection Spectrophotometer at 210 nm. crystal sheaths [Ca]; prisms of calcium oxalate; rare, whole or
fragmented phloem fibres, usually isolated [B, F, HJ;
Injection 20 µL.
reticulate or pitted lignified vessels [E, K], 13-60 µm in
Run time 4 times the retention time of saikosaponin A. diameter, sometimes up to 70 µm in diameter; fragments of
Relative retention With reference to saikosaponin A yellowish-brown cork with cells that are polygonal in surface
(retention time = about 16 min): propyl view U] or rectangular in transverse section [A]. Examine
parahydroxybenzoate = about 0.9. under a microscope using a 50 per cent V/V solution of
glycerol R. The powder shows subrounded or ovoid starch
2023 Butcher's Broom IV-147
granules, free or included in parenchyma cells or in cells of Top of the plate

the medullary rays [DJ; the granules are mostly single,
A quenching zone
3-9 µm in diameter, sometimes up to 30 µm in diameter; 2-
to 4-compound starch granules are also present [G]. - - --
Resorcinol: a quenching zone
A quenching zone
-- --
Gallic acid: a quenching zone A quenching zone (gallic acid)
A quenching zone
A quenching zone
Detection B Spray with a 10 g/L solution of ferric chloride R

in anhydrous ethanol Rand heat at 100-105 °C for 15 min;
examine in daylight.
Results B See below the sequence of the zones present in
the chromatograms obtained with the reference solution and
the test solution. Furthermore, other faint zones may be
present in the chromatogram obtained with the test solution.
Top of the plate
-- --
Resorcinol: a brown zone
A blackish-blue zone
- - --
Gallic acid: a blackish-blue zone A blackish-blue zone (gallic acid)
A blackish-blue zone

herbal drug of sanguisorba root
TESTS
C. Thin-layer chromatography (2.2.27). Loss on drying (2.2.32)
Test solution To 2.0 g of the powdered herbal drug (355) Maximum 12.0 per cent, determined on 1.000 g of the
(2. 9.12) add 50 mL of water R and boil under a reflux powdered herbal drug (355) (2.9.12) by drying in an oven at
condenser for 30 min. Cool the solution and centrifuge for 105 °C.
10 min. Shake the supernatant with 2 quantities, each of Total ash (2.4.16)
15 mL, of di-isopropyl ether R saturated with hydrochloric Maximum 10.0 per cent.
acid R. Combine the ether layers. Evaporate to dryness and Ash insoluble in hydrochloric acid (2.8.1)
dissolve the residue in 1.0 mL of methanol R. Filter through a Maximum 2.0 per cent.
polypropylene syringe filter (nominal pore size 0.45 µm).
ASSAY
Reference solution Dissolve 5 mg of gallic acid R and 20 mg Tannins (2.8.14)
of resorcinol R in 20 mL of methanol R. Use 0.500 g of the powdered herbal drug (180) (2.9.12).
Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel - - - - - - - - - - - - - - - - - - - - - ~ PhEur
F2 54 plate R (2-10 µm)].
Mobile phase anhydrous formic acid R, ethyl acetate R,
toluene R (10:30:60 VIVIV).
Butcher's Broom
Development Over a path of 10 cm [or 6 cm] . (Ph. Bur. monograph 1847)
Drying In air. PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Results A See below the sequence of quenching zones Dried, whole or fragmented underground parts of Ruscus
present in the chromatograms obtained with the reference aculeatus L.
solution and the test solution. Furthermore, other faint
Content
quenching zones may be present in the chromatogram
Minimum 1.0 per cent of total sapogenins, expressed as
ruscogenins [mixture of neoruscogenin (C21H4 0 0 4;
M, 428.6) and ruscogenin (C 27 H 42 0 4; Mr 430.6)] (dried
drug).
IV-148 Butcher's Broom 2023
IDENTIFICATION extract the unfiltered mixture with 3 quantities, each of

A. The rhizome consists of yellowish, branched, articulated, 25 mL, of methylene chloride R. Combine the organic
somewhat knotty pieces, cylindrical or subconical, about solutions and dry over anhydrous sodium sulfate R. Filter and
5-10 cm long and about 5 mm thick. The surface is marked evaporate to dryness. Dissolve the residue in 5 mL of
with thin annulations about 1-3 mm wide, separated from methanol R.
one another; rounded scars of the aerial stems are present on Reference solutwn Dissolve 1 mg of ruscogenins CRS and
the upper surface. On the lower surface numerous roots, or 1 mg of stigmasterol R in methanol R and dilute to 5 mL with
their scars, occur; the roots are about 2 mm in diameter and the same solvent.
similar in colour to the rhizome. The outer layer is easily Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
detached, revealing a yellowish-white, very hard central plate R (2-10 µm)].
cylinder.
Mobile phase methanol R, methylene chloride R (7:93 V/V).
Applicatwn 10 µL [or 4 µL] as bands.
yellowish. Examine under a microscope using chloral hydrate
solutwn R. The powder shows the following diagnostic Development Over a path of 15 cm [or 6 cm].
characters (Figure 1847.-1): groups of sclereids of the Drying In air.
rhizome, with variously-shaped cells, rounded, elongated or Detection Spray with vanillin reagent R, dry in an oven at
rectangular; the walls are moderately thickened and distinctly 100-105 °C for 1 min and examine in daylight.
beaded, with large, rounded or oval pits [F, G, L, P, Q]; Results See below the sequence of zones present in the
fragments of the endodermis composed of a single layer of chromatograms obtained with the reference solution and the
irregularly thickened cells [K]; groups of rounded test solution. Furthermore, other weak zones may be present
parenchymatous cells, thickened at the comers, with small, in the chromatogram obtained with the test solution.
triangular intercellular spaces [D, E, N]; thin-walled
parenchyma W with some cells containing raphides of
Top of the plate
calcium oxalate [C]; groups [HJ of thick-walled fibres [Ha]
and small vessels, up to about 50 µm in diameter, the walls Several zones of various colours
showing numerous small, slit-shaped pits [A, Hb]; rare
Srigmasterol: a violet zone A violet zone
fragments of dermal tissue of the root [BJ; raphides of
calcium oxalate, isolated [M]. -- --
A violet zone
Ruscogenins: a yellow zone A yellow zone (ruscogenins)
Several zones of various colours
-- --
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
Figure 184 7.-1. - Illustratwn for identificatwn test B of powdered flask. Dilute to 100.0 mL with anhydrous ethanol R.
herbal drug of butcher's broom Evaporate 25.0 mL of this solution to dryness under reduced
pressure. Dissolve the residue in 10 mL of butanol R and add
C. Thin-layer chromatography (2.2.27). 3 mL of hydrochloric acid Rl and 8 mL of water R. Heat on a
Test solutwn Introduce 1.0 g of the powdered herbal drug water-bath under a reflux condenser for 1 h. Allow to cool
(355) (2.9.12) and 50 mL of dilute hydrochloric acid R into a and transfer to a 100 mL volumetric flask. Rinse the round-
100 mL flask with a ground-glass neck. Heat on a water-bath bottomed flask with 3 quantities, each of 20 mL, of
under a reflux condenser for 40 min. Allow to cool and
2023 Calendula Flower IV-149
methanol R. Add the rinsings to the volumetric flask and IDENTIFICATION

dilute to 100.0 mL with methanol R. A. The ligulate florets consist of a yellow or orange-yellow
Reference solution Dissolve 5.0 mg of ruscogenins CRS in ligule, about 3-5 mm wide and about 7 mm in the middle
100 mL of methanol R. part, with a 3-toothed apex and a hairy, partly sickle-shaped,
Column: yellowish-brown or orange-brown tube with a projecting style
- size: l = 0.25 m, 0 = 4.6 mm; and a bifid stigma occasionally with a partly bent yellowish-
- stationary phase: octadecylsi"/yl silica gel for chromatography R brown or orange-brown ovary. The tubular florets, about
(5 µm). 5 mm long, are present and consist of the yellow, orange-red
or reddish-violet 5-lobed corolla and the yellowish-brown or
Mobile phase:
orange-brown tube, hairy in its lower part, mostly with a
- mobile phase A: water R;
partly bent yellowish-brown or orange-brown ovary.
- mobile phase B: acewnitrile Rl;
'
Time Mobile phase A Mobile phase B A

(min)
0 - 25
25 - 27
(per cent VIV)
40
40--> 0
(per cent VIV)
60
60--> 100
d .
.
27 - 37 0 100 Da

Injection 20 µL.
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
time= about 16 min): ruscogenin = about 1.2.
neoruscogenin and ruscogenin.
Calculate the percentage content of sapogenins, expressed as
ruscogenins (neoruscogenin and ruscogenin), using the
A 1 xm 2 x4x~ A 3 xm 2 x4x~
------+------
A2 xm1 A4 xm1
AI area of the peak due to ruscogenin in the chromatogram
A2 area of the peak due to ruscogenin in the chromatogram
obtained with the reference solution;
A3 area of the peak due to neoruscogenin in the chromatogram Figure 1297.-1. - Illustration for identification test B of powdered
obtained with the test solution; herbal drug of calendula flower
A4 area of the peak due to neoruscogenin in the chromatogram
obtained with the reference solution; B. Microscopic examination (2.8.23). The powder is
solution, in grams;
yellowish-brown. Examine under a microscope using chloral
m2 mass of ruscogenins CRS used to prepare the reference solution, hydrate solution R. The powder shows the following diagnostic
in grams; characters (Figure 1297.-1): fragments of epidermises of the
p1 percentage content of ruscogenin in ruscogenins CRS; corolla [C, F, K] containing light yellow oil droplets, some
p2 percentage content of neoruscogenin in ruscogenins CRS.
with fairly large anomocytic stomata (2.8.3) [Fa, Ka];
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur covering trichomes biseriate, multicellular and conical [G],
usually fragmented, and glandular trichomes with a
multicellular stalk [E], very abundant on the base of the
corolla [D]; fragments of parenchyma of the corolla [BJ
Calendula Flower containing prisms and very small cluster crystals of calcium
oxalate [Ba, Da] and small vessels [Bb]; spherical pollen
(Ph. Bur. monograph 1297) grains up to about 40 µm in diameter with a sharply spiny
exine and 3 germinal pores [A, TI; occasional fragments of
Ph Eur - - - - - - - - - - - - - - - - - - - - - ~
the stigmas with short, bulbous papillae [H] .
DEFINITION C. High-performance thin-layer chromatography (2.8.25).
Whole or cut, dried, and fully opened flowers that have been Test solution To 0.5 g of the powdered herbal drug (355)
detached from the receptacle of the cultivated, double- (2.9.12) add 5.0 mL of methanol R. Sonicate for 15 min,
flowered varieties of Calendula ofjicinalis L. filter or centrifuge and use the filtrate or supernatant.
Content Reference solution (a) Dissolve 3.0 mg of chlorogenic acid R
Minimum 0.4 per cent offlavonoids, expressed as hyperoside and 4.0 mg of isorhamnetin-3-0-rutinoside R in methanol R
(C 2 1H20012; Mr 464.4) (dried drug). and dilute to 10.0 mL with the same solvent.
IV-150 Calendula Flower 2023
Reference solution (b) Dilute 2.5 mL of reference solution (a) TESTS

to 10.0 mL with methanol R. Foreign matter (2.8.2)
Reference solution (c) Dissolve 2.5 mg of hyperoside R and Maximum 5 per cent of bracts and maximum 2 per cent of
3 mg of chlorogenic acid R in methanol R and dilute to 10 mL other foreign matter.
with the same solvent. Loss on drying (2.2.32)
Intensity marker Isorhamnetin-3-O-rutinoside. Maximum 12.0 per cent, determined on 1.000 g of the
Plate TLC silica gel F254 plate R (2-10 µm). powdered herbal drug (500) (2. 9. 12) by drying in an oven at
105 °C for 2 h.
(10:10:80 V/V/V). Total ash (2.4.16)
Application 4 µL as bands of 8 mm. Maximum 10.0 per cent.
Development 70 mm from the lower edge of the plate. ASSAY
Drying In a current of air at room temperature for 5 min. Stock solution Into a 100 mL round-bottomed flask
introduce 0.800 g of the powdered herbal drug (500)
(2.9.12), 1 mL ofa 5 g/L solution of
hexamethylenetetramine R, 7 mL of hydrochloric acid Rl and
ester R in methanol R, then with a 50 g/L solution of macrogol
20 mL of acetone R. Boil the mixture under a reflux
400 R in methanol R or, alternatively, dip the warm plate in a
condenser for 30 min. Filter the liquid through a plug of
5 g/L solution of diphenylboric acid aminoethyl ester R in ethyl
absorbent cotton into a 100 mL volumetric flask. Add the
acetate R and then in a 50 g/L solution of macrogol 400 R in
absorbent cotton to the residue in the round-bottomed flask
methylene chloride R; allow to dry in air for about 1 min and
and extract with 2 quantities, each of 20 mL, of acetone R,
each time boiling under a reflux condenser for 10 min. Allow
System suitability Reference solution (c): to cool to room temperature, filter the liquid through a plug
- the chromatogram shows in the middle third 2 distinct of absorbent cotton, then filter the combined acetone
zones, which may be touching; the lower zone solution through a filter-paper into the volumetric flask, and
(chlorogenic acid) shows a light blue fluorescence and dilute to 100.0 mL with acetone R by rinsing the flask and
the upper zone (hyperoside) shows a yellow or orange filter. Introduce 20.0 mL of this solution into a separating
fluorescence. funnel, add 20 mL of water R and extract the mixture with
Results See below the sequence of fluorescent zones present 1 quantity of 15 mL and then with 3 quantities, each of
in the chromatograms obtained with reference solution (a) 10 mL, of ethyl acetate R. Combine the ethyl acetate extracts
and the test solution. Furthermore, in the chromatogram in a separating funnel, rinse with 2 quantities, each of
obtained with the test solution, other faint to very faint blue, 50 mL, of water R, filter the extract over 10 g of anhydrous
brown or orange fluorescent zones may be present. sodium sulfate R into a 50 mL volumetric flask and dilute to
50.0 mL with ethyl acetate R.
Top of the plate Test solution To 10.0 mL of the stock solution add 1 mL of
aluminium chloride reagent Rand dilute to 25.0 mL with a
Compensation liquid Dilute 10.0 mL of the stock solution to
2 blue zones, faint to equivalent 25.0 mL with a 5 per cent V/V solution of glacial acetic
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
A greenish-yellow zone, faint
Ax 1.25
m
Chlorogenic acid: a light blue zone A light blue zone, faint to equivalent i.e. taking the specific absorbance of hyperoside to be 500.

-- -- _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Isorhamnetin-3-0-rutinoside: a A greenish-yellow zone

greenish-yellow zone (isorhamnetin-3-0-rutinoside)
A brown or orange zone, faint to

equivalent
A greenish-yellow zone
A brownish-orange zone, very faint

to faint

2023 Cape Jasmine Fruit IV-151
380 µm) or more rarely longer, with smooth, thick walls

Cape Jasmine Fruit [Fa]; fragments ofmesocarp [A, BJ consisting of thin-walled
(Ph. Bur. monograph 2565) parenchymatous cells [Aa, Ba] associated with fibres and
yellowish spiral or annular xylem vessels, up to 12 µm in
diameter [Bb]; parenchyma, sometimes containing oil
DEFINITION droplets [De] or cluster crystals [Ab, Db, Bf] or prisms [Ac,
Whole or fragmented, ripe fruit of Gardenia jasminoides J. Be] of calcium oxalate; fibres, fusiform [Bd] or rectangular
Ellis, with stalk removed, steamed or treated with boiling [Be], with thickened, pitted walls about 10 µm in diameter
water, then dried. and up to 100 µm long; parenchyma may also be associated
with small groups of subrounded, polygonal or
Content
subrectangular sclereids with regularly thickened, pitted walls,
Minimum 2.0 per cent of geniposide (C 17 H 24 O 10; M, 388.4)
15-57 µm long and 11-30 µm in diameter [Ad]; fragments of
(dried drug).
endocarp [C] consisting of compact masses of mosaic-
IDENTIFICATION arranged fibres overlaid or cross-embedded with sclereids
A. Whole drug. The fruit is obovate or elliptical and may be containing calcium oxalate prisms, 4-14 µm long [Ca, Cb],
elongated or more rounded; it is about 1-5 cm long especially visible in polarised light; endocarp fibres, long and
(including the sepal remains) and 0.8-1.7 cm in diameter. thin, about 10 µm in diameter and up to 100 µm long [Cc];
The smooth or slightly rough outer surface is yellowish, fragments of testa [HJ consisting of large irregularly shaped
reddish-yellow or brownish-red and usually has 6 sclereids, 58-150 µm in diameter and up to 260 µm long,
antesepalous raised, longitudinal winged ribs; between the with irregularly thickened yellowish walls, wide pits and
winged ribs, a well-marked, longitudinal and sometimes containing a reddish-brown substance; fragments of
branched vein is clearly visible; raised, yellowish-brown, dark endosperm with cells containing aleurone grains [K]; prisms
brown or black papillae are also commonly present on the or scattered clusters of calcium oxalate [E].
fruit surface between the winged ribs. The remains of the
basally gamosepalous calyx are visible at the upper end of the
fruit (inferior ovary); the tips of the sepals are free and are
either tapering and pointed or broken. At the lower end of
the fruit, the 6 winged ribs join together at the stalk. In the
centre of the fused sepals, a stylar scar is visible, surrounded
by a disc-shaped nectary. A transverse section of the fruit
shows a single locule resulting from the incomplete fusion of
both carpels; remnants of the incomplete septa are present on
the inner surface of the fruit wall; the locule contains
numerous seeds aggregated into a mass in a yellowish,
reddish-yellow or, more rarely, dark red placenta, which
protrudes between the seeds; the seed shape is variable and
may be rounded, triangular or irregularly angular, and the
seeds themselves are flat, brownish-red or yellowish-red,
measuring up to 0.5 cm in length; the outer surface is
densely and finely verrucose. The pericarp is brittle with a
smooth, lustrous pale yellow or brownish-yellow inner
surface.
Fragmented drug The fruit fragments consist of thin, brittle
pieces of pericarp, with a smooth or slightly rough outer
surface that is yellowish, reddish-yellow or brownish-red,
sometimes with raised yellowish-brown, dark brown or black
papillae or crossed by a fragment of winged rib and/or vein;
the inner surface is smooth, lustrous, and pale yellow or
brownish-yellow. The remains of the calyx or the stalk may
be present. Numerous seeds, often aggregated, are visible,
they are near circular, triangular or irregularly angular in
shape, flat and measure to 0.5 cm in the long axis; their
brownish-red or yellowish-red outer surface is densely and
finely verrucose. The remains of the yellowish, yellowish-red
or, more rarely, dark red placenta are present together with Figure 2565.-1. - Illustration for identification test B of powdered
the seeds. herbal drug of cape jasmine fruit
B. Microscopic examination (2.8.23). The powder is orange-
brown or orange-red. Examine under a microscope using C. Thin-layer chromatography (2.2.27).
chloral hydrate solution R. The powder shows the following Test solution To 1 g of the powdered herbal drug (355)
diagnostic characters (Figure 2565.-1): fragments of the (2. 9.12) add 10 mL of a 50 per cent V/V solution of
epicarp (surface view [G]) consisting of polyhedral cells; methanol R. Sonicate for 10 min. Centrifuge and use the
fragments of pericarp (transverse section [DJ) consisting of supernatant.
thin-walled parenchymatous cells [Da]; fragments of epicarp Reference solution Dissolve 6 mg of aescin R and 5 mg of
from the winged ribs or the calyx (surface view [F, J]) geniposide R in 1 mL of methanol R.
consisting of polyhedral cells, scars of covering trichomes Ua] Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
and rare, pointed, unicellular trichomes either short (up to plate R (2-10 µm)].
IV-152 Capsicum 2023
Mobile phase anhydrous formic acid R, water R, acetone R, Time Mobile phase A Mobile phase B
(min) (per cent V/Jl) (per cent VIJ!)
ethyl acetate R (8:8:42:42 V!VIV!v').
0-4 80 20
4 - 40 80---> 20 20---> 80
Drying In air.
Detection Treat with anisaldehyde solution R and heat at Detection Spectrophotometer at 240 nm.
100-105 °C for 2 min; examine in daylight.
Injection 20 µL.
chromatograms obtained with the reference solution and the Identification of peaks Use the chromatogram obtained with
test solution. Furthermore, other faint zones may be present reference solution (a) to identify the peak due to geniposide;
in the chromatogram obtained with the test solution. use the chromatogram supplied with cape jasmine fruit for
system suitability HRS and the chromatogram obtained with
Top of the plate reference solution (b) to identify the peak due to genipin
gentiobioside.
--
- -
Retention time Genipin gentiobioside = about 14 min;
geniposide = about 16 min.
Geniposide: a prominent brown zone A prominent brown zone System suitabuity Reference solution (b):
(geniposide) - resolution: minimum 5.0 between the peaks due to genipin
gentiobioside and geniposide.
Calculate the percentage content of geniposide using the
A blue zone
A reddish zone
A1 x m 2 xpx 8
-- --
A2 xm1
Aescin: a violet zone
A1 area of the peak due to geniposide in the chromatogram
A brown zone obtained with the test solution;
A2 area of the peak due to geniposide in the chromatogram
A blue zone
solution, in grams;
m2 mass of geniposide CRS used to prepare reference solution (a), in
grams;
p percentage content of geniposide in geniposide CRS.
TESTS _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
105 °C for 2 h.
Total ash (2.4.16)
Capsicum
Maximum 6.0 per cent. (Ph. Eur. monograph 1859)
ASSAY Preparations
Liquid chromatography (2.2.29). Refined and Quantified Capsicum Oleoresin
Test solution Disperse 0.500 g of the powdered herbal drug Standardised Capsicum Oleoresin
(355) (2. 9.12) in 50 mL of a 50 per cent V/V solution of
Capsicum Tincture
methanol R and sonicate for 40 min. Centrifuge and transfer
the supernatant to a 200 mL volumetric flask. Repeat the Standardised Capsicum Tincture
procedure and dilute to 200.0 mL with a 50 per cent V/V Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
solution of methanol R. DEFINITION
Reference solution (a) Dissolve 5.0 mg of geniposide CRS in Dried ripe fruits of Capsicum annuum L. var. minimum
methanol R and dilute to 25.0 mL with the same solvent. (Miller) Heiser and small-fruited varieties of Capsicum
Reference solution (b) Disperse 0.250 g of cape jasmine fruit frutescens L.
for system suitability HRS in 25 mL of a 50 per cent V/V Content
solution of methanol R and sonicate for 40 min. Centrifuge Minimum 0.4 per cent of total capsaicinoids, expressed as
and transfer the supernatant to a 100 mL volumetric flask. capsaicin (C 18 H 27N03 ; Mr 305.4) (dried drug).
Repeat the procedure and dilute to 100.0 mL with a
50 per cent V/V solution of methanol R. CHARACTERS
Column: Extremely pungent taste.
- size: l =0.25 m, 0 =4.6 mm; IDENTIFICATION
- stationary phase: end-capped octadecylsilyl silica gel for A. The fruit is yellowish-orange or reddish-brown, oblong
chromatography R (5 µm). conical with an obtuse apex, about 1-3 cm long and up to
Mobue phase: 1 cm in diameter at the widest part, occasionally attached to
- mobile phase A: water R adjusted to pH 3.2 with anhydrous a 5-toothed inferior calyx and a straight peduncle. Pericarp
formic acid R; somewhat shrivelled, glabrous, enclosing about 10-20 flat,
- mobile phase B: methanol R;
2023 Capsicum IV-153
reniform seeds 3-4 mm long, either loose or attached to a Plate TLC octadecylsilyl silica gel plate R.
reddish dissepiment. Mobile phase water R, methanol R (20:80 V/V).
B. Microscopic examination (2.8.23). The powder is orange. Application 20 µL as bands.
Examine under a microscope using chloral hydrate solution R.
The powder shows the following diagnostic characters
(Figure 1859.-1): fragments of the epicarp, in surface view, Drying In air.
with cells often arranged in rows of 5 to 7 [E], thick-walled Detection Treat with a 5 g/L solution of
when close to the peduncle [BJ and with a cuticle uniformly dichloroquinonechlorimide R in methanol R, and expose to
striated [A]; fragments of the pericarp (transverse section ammonia vapour until blue zones appear. Examine in
[DJ) showing the epicarp covered by a thick cuticle [Da] and daylight.
parenchymatous cells frequently containing droplets of red Results See below the sequence of zones present in the
oil, occasionally containing microsphenoidal crystals of chromatograms obtained with the reference solution and the
calcium oxalate [Db]; fragments of endocarp [CJ with test solution. Furthermore, other zones may be present in the
characteristic island groups of sclerenchymatous cells [Ca], chromatogram obtained with the test solution.
the groups being separated by thin-walled parenchymatous
cells [Cb]; fragments of the seeds having an episperm Top of the plate
composed of large, greenish-yellow, sinuous-walled sclereids
with thin outer walls and strongly and unevenly thickened -- --
radial and inner walls which are conspicuously pitted [G]; Capsaicin: a blue zone A blue zone (capsaicin)
endosperm parenchymatous cells with drops of oil and
aleurone grains, 3-6 µm in diameter [HJ; occasional Dihydrocapsaicin: a blue zone A blue zone (dihydrocapsaicin)
fragments from the calyx having an outer epidermis with - - - -
anisocytic stomata (2.8.3) W, an inner epidermis with no

stomata and many glandular trichomes with uniseriate stalks Reference solution Test solution
and multicellular heads [N], and a mesophyll [L] with many

idioblasts containing prisms of calcium oxalate [La] or TESTS
microsphenoidal crystals of calcium oxalate [Lb]; prisms [K]
Nonivamide
or clusters [M] of calcium oxalate, isolated; annularly and Liquid chromatography (2.2.29).
spirally thickened vessels [F].
(2. 9.12) add 100 mL of methanol R. Allow to macerate for
30 min. Place in an ultrasonic bath for 15 min. Filter into a
100 mL volumetric flask, rinse the flask and filter with
methanol R, then dilute to 100.0 mL with the same solvent.
Reference solution (a) Dissolve 10.0 mg of capsaicin CRS and
2.0 mg of nonivamide CRS in methanol Rand dilute to
Reference solution (b) Dissolve 4.0 mg of nonivamide CRS in
Column:
- size: l = 0.25 m, 0 = 4.6 mm;
- stationary phase: base-deactivated end-capped phenylsilyl silica
gel for chromatography R (5 µm);
Mobile phase acetonitrile Rl, 1 g/L solution of phosphoric
acid R (40:60 V/V).
ln.fection 10 µL.
Run time 1.2 times the retention time of dihydrocapsaicin.
Elution order Nordihydrocapsaicin, nonivamide, capsaicin,
dihydrocapsaicin.
Kola System suitability Reference solution (a):

nonivamide and capsaicin.
25 µm
Calculate the percentage content of nonivamide with
reference to the total capsaicinoid content, using the
Figure 1859.-1. - Illustration for identification test B of powdered following expression:
herbal drug of capsicum
A 1 xm 2 xp 1 x 100
A2 xm 1 xC
(2. 9.12) add 5.0 mL of ether R, shake for 5 min and filter. area of the peak due to nonivamide in the chromatogram
Reference solution Dissolve 2 mg of capsaicin R and 2 mg of area of the peak due to nonivamide in the chromatogram
dihydrocapsaicin R in 5.0 mL of ether R. obtained with reference solution (b);
IV-154 Capsicum Oleoresin 2023
mass of the herbal drug to be examined used to prepare the test Test solution Dissolve 50 mg of the oleoresin to be examined
solution, in grams;
mass of nonivamide CRS used to prepare reference solution (b),
in 5 mL of ether R.
in grams; Reference solution Dissolve 2 mg of capsaicin R and 2 mg of
percentage content of nonivamide in nonivamide CRS; dihydrocapsaicin R in 5 mL of ether R.
percentage content of total capsaicinoids, as determined in the
assay. Plate TLC octadecylsilyl silica gel plate R (5-40 µm) [or
TLC octadecylsilyl silica gel plate R (2-10 µm)].
Limit: Mobile phase water R, methanol R (20:80 V/V).
- nonivamide: maximum 5.0 per cent of the total Application 20 µL [or 2 µL] as bands of 15 mm [or 8 mm].
capsaicinoid content. Development Over a path of 12 cm [or 6 cm].
Foreign matter (2.8.2) Drying In air.
Fruits of C. annuum L. var. kmgum (Sendtn.) are absent.
Detection Treat with a 0.25 g/L solution of
Loss on drying (2.2.32) dichloroquinonechlorimide R in ethyl acetate R, expose to
Maximum 11.0 per cent, determined on 1.000 g of the ammonia vapour until blue zones appear. Examine in
powdered herbal drug (500) (2. 9. 12) by drying in an oven at daylight.
105 °C for 2 h.
Total ash (2.4.16) chromatograms obtained with the reference solution and the
Maximum 10.0 per cent. test solution. Furthermore, other zones may be present in the
ASSAY chromatogram obtained with the test solution.
nonivamide. Top of the plate
Calculate the percentage content of total capsaicinoids (C), -- --

expressed as capsaicin, using the following expression:
Capsaicin: a blue zone A blue zone (capsaicin)
(A3 +As +A6) x m4 xpz x 2 Dihydrocapsaicin: a blue zone A faint blue zone (dihydrocapsaicin)
A4 xm3
-- --
area of the peak due to capsaicin in the chromatogram obtained
area of the peak due to dihydrocapsaicin in the chromatogram TESTS
obtained with the test solution; Nonivamide
area of the peak due to nordihydrocapsaicin in the Liquid chromatography (2.2.29).
mass of the herbal drug to be examined used to prepare the test Test solution Dissolve 0.300 g of the oleoresin to be
solution, in grams; examined in 60 mL of methanol Rand dilute to 100.0 mL
mass of capsaicin CRS used to prepare reference solution (a), in with the same solvent.
grams;
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.
Column:
Refined and Standardised - size: l = 0.25 m, 0 = 4.6 mm;
Capsicum Oleoresin gel for chromatography R (5 µm);
(Ph. Bur. monograph 2336) - temperature: 30 °C.
Mobile phase acetonitrile RJ, 1 g/L solution of phosphoric
acid R (40:60 V/V).
DEFINITION
Refined and standardised oleoresin produced from
Capsicum (1859). Detection Spectrophotometer at 225 nm.
Content Injection 10 µL.
12.0 per cent to 18.0 per cent m/m of total capsaicinoids, Run time 1.2 times the retention time of dihydrocapsaicin.
expressed as capsaicin (C 18H 27NO3 ; M, 305.4). Elution order Nordihydrocapsaicin, nonivamide, capsaicin,
PRODUCTION dihydrocapsaicin.
The oleoresin is produced from the herbal drug by an Relative retention With reference to capsaicin (retention
appropriate procedure, using ethanol (minimum time= about 19 min): nordihydrocapsaicin = about 0.9;
90 per cent V/V). nonivamide = about 0.95; dihydrocapsaicin = about 1.3.
CHARACTERS
Appearance
nonivamide and capsaicin.
Red or brown mobile extract.
IDENTIFICATION reference to the total capsaicinoid content, using the
Thin-layer chromatography (2.2.27). following expression:
2023 Capsicum Preparations IV-155
A1 x m2 x P1 x 100 CHROMATOGRAPHIC CONDITIONS

A2 xm1 xC (a) Use TLC octadecylsilyl silica gel plate (5-40 µm) (Merck
area of the peak due to nonivamide in the chromatogram
silica gel plates are suitable) [or TLC octadecylsilyl silica gel
obtained with the test solution; plate (2-10 µm)].
area of the peak due to nonivamide in the chromatogram (b) Use the mobile phase as described below.
mass of the oleoresin to be examined used to prepare the test (c) Apply 20 µL [or 2 µL] of solutions as bands of 15 mm
solution, in grams; [or 8 mm].
in grams;
(d) Develop the plate to 12 cm (6 cm].
P1 percentage content of nonivantide in nonivamide CRS; (e) Remove the plate and allow to dry in air. Treat with a
C percentage content of total capsaicinoids, as determined in the 0.25 g/L solution of dichloroquinonechlorimide in ethyl acetate
assay.
and expose to ammonia vapour until blue zones appear.
Examine in daylight.
Limit:
- nonivamide: maximum 5.0 per cent of the total MOBILE PHASE
capsaicinoid content. 20 volumes of water and 80 volumes of methanol.
Water (2.5.12) CONFIRMATION
Maximum 8.0 per cent, determined on 5.00 g. See below the sequence of zones present in the
ASSAY chromatograms obtained with the solutions (1) and (2).
Liquid chromatography (2.2.29) as described in the test for Furthermore, other faint zones may be present in the
nonivamide. chromatogram obtained with solution (1).
Calculate the percentage content of total capsaicinoids (C),
expressed as capsaicin, using the following expression: Top of the plate
(A3 +As +A6) x m3 xp2 x 2 A blue zone (capsaicin} capsaicin: A blue zone
A 4 xm1 A faint blue zone (Dihydrocapsaicin) Dihydrocapsaicin: A blue zone
A3 area of the peak due to capsaicin in the chromatogram obtained

with the test solution; Solution (1) Solution (2)
A, area of the peak due to dihydrocapsaicin in the chromatogram
obtained with the test solution; TESTS
area of the peak due to nordihydrocapsaicin in the Nonivamide
mass of the oleoresin to be examined used to prepare the test
Not more than 5% of the total capsaicinoids content.
solution, in grams; Carry out the method for liquid chromatography,
mass of capsaicin CRS used to prepare reference solution (a), in Appendix III D, using the following solutions.
grams;
P2 percentage content of capsaicin in capsaicin CRS. (1) Dissolve 0.3 g of the preparation being examined in
60 mL of methanol and dilute to 100 mL with the same
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur solvent.
(2) 0.02% w/v of capsaicin EPCRS and 0.004% w/v of
nonivamide EPCRSin methanol.
(3) 0.004% w/v of nonivamide EPCRS in methanol.
Standardised Capsicum Oleoresin CHROMATOGRAPHIC CONDITIONS
DEFINITION (a) Use a stainless steel column (25 cm x 4.6 mm) packed
Standardised Capsicum Oleoresin is produced from with base-deactivated end-capped phenylsilyl silica gel for
Capsicum. chromatography (5 µm) (GL Sciences Inertsil Ph-3 is
Content suitable).
8.0 to 8.8% w/w of total capsaicinoids, expressed as (b) Use isocratic elution and the mobile phase described
capsaicin, C1sH21NO3. below.
PRODUCTION (c) Use a flow rate of 1.0 mL per minute.
The oleoresin is produced from whole or cut capsicums by (d) Use a column temperature of 30°.
an appropriate procedure, using ethanol (minimum 90% v/v) (e) Use a detection wavelength of 225 nm.
as the extraction solvent. (f) Inject 10 µL of each solution.
Characteristics (g) Allow the chromatography to proceed for 1. 7 times the
Dark red-brown to orange-brown mobile oily extract. retention time of dihydrocapsaicin.
IDENTIFICATION MOBILE PHASE
Carry out the method for thin-layer chromatography, 40 volumes of acetonitrile Rl and 60 volumes of 1 g/L
Appendix III A, using the following solutions in ether. solution of orthophosphoric acid.
(1) Dissolve 50 mg of the preparation being examined in The order of elution of the peaks is nordihydrocapsaicin,
10 mL. nonivamide, capsaicin and dihydrocapsaicin.
(2) 0.02% w/v each of capsaicin and dihydrocapsaicin. The relative retentions with reference to capsaicin (retention
time is about 19 minutes) are nordihydrocapsaicin, about
0.9; nonivamide, about 0.95 and dihydrocapsaicin, about 1.3.
IV-156 Capsicum Preparations 2023
SYSTEM SUITABILITY
Standardised Capsicum Soft
with solution (2), the resolution between the peaks due to Extract
capsaicin and nonivamide is at least 1.5. (Ph. Bur. monograph 2529)
reference to the total capsaicinoid content, using the DEFINITION
following expression: Standardised soft extract produced from Capsicum (1859).
Content
2.0 per cent to 2.4 per cent of total capsaicinoids, expressed
A1 xPi xPi xlOO as capsaicin (C 18H 27NO 3 ; Mr 305.4).
A2 xm1 xC PRODUCTION
The extract is produced from the herbal drug by a suitable
procedure using ethanol (80 per cent V/V).
area of the peak due to nonivamide in the chromatogram The content of total capsaicinoids in the extract is
obtained with solution (l); determined and adjusted, if necessary, to the value specified
obtained with solution (3);
by adding a suitable inert excipient, for example liquid
weight of standardised oleoresin to be examined used to prepare glucose.
solution (1), in grams;
content of nonivamide EPCRS in solution (3), in percentage CHARACTERS
w/v; Appearance
declared content of nonivamide EPCRS; Reddish-brown, glutinous matter.
percentage content of total capsaicinoids, as determined in the
C IDENTIFICATION
assay.
Water Test solutwn To 0.25 g of the extract to be examined add
Maximum 8% w/w, Appendix IX C. Use 5 g. 10 mL of a mixture of water R and propanol R (40:60 V/V).
ASSAY Shake for 5 min. Filter, if necessary.
Carry out the method for liquid chromatography, Reference solutwn Dissolve 2 mg of capsaicin R and 1 mg of
Appendix III D, using the following solutions. dihydrocapsaicin R in 5 mL of methanol R.
( 1) Dissolve O.3 g of the preparation being examined in Plate TLC octadecylsilyl silica gel plate R (5-40 µm) [or
60 mL of methanol and dilute to 100 mL with the same TLC octadecylsilyl silica gel plate R (2-10 µm)].
solvent. Mobile phase water R, methanol R (20:80 V/V).
(2) 0.02% w/v of capsaicin EPCRS and 0.004% w/v of Applicatwn 20 µL [or 2 µL] as bands of 15 mm [or 8 mm].
nonivamule EPCRS in methanol. Development Over a path of 12 cm [or 6 cm].
CHROMATOGRAPHIC CONDITIONS Drying In air.
The chromatographic conditions described under the test for Detectwn Treat with a 0.25 g/L solution of
Nonivamide may be used. dichloroquinonechlorimule R in ethyl acetate R, expose to
DETERMINATION OF CONTENT ammonia vapour until blue zones appear. Examine in
daylight.
expressed as capsaicin, using the following expression: Results See below the sequence of zones present in the
( A3 + A5 + A6 ) x ~ x ~ chromatogram obtained with the test solution.
A4 xm1
Top of the plate
- - --
with solution (l);
area of the peak due to capsaicin in the chromatogram obtained Dihydrocapsaicin: a blue zone A blue zone (dihydrocapsaicin)
with solution (2);
area of the peak due to dihydrocapsaicin in the chromatogram - - --
obtained with solution (l);
area of the peak due to nordihydrocapsaicin in the
chromatogram obtained with solution (l);
weight of the standardised oleoresin to be examined used to Reference solution Test solution
prepare solution (1), in grams;
content of capsaicin EPCRS in solution (2) in percentage w/v;
declared content of capsaicin EPCRS. TESTS
Nonivamide
Test solutwn Stir the extract to be examined until
homogeneous, heating, if necessary, to not more than 60 °C.
Disperse 0.350 g of the homogeneous extract in 35 mL of a
mixture of water Rand propanol R (40:60 V/V). Shake for
2023 Capsicum Preparations IV-157
30 min and dilute to 50.0 mL with propanol R. Dilute = area of the peak due to capsaicin in the chromatogram obtained
25.0 mL of the solution to 50.0 mL with the mobile phase
area of the peak due to dihydrocapsaicin in the chromatogram
and filter through a membrane filter (nominal pore size obtained with the test solution;
0.45 µm). area of the peak due to nordihydrocapsaicin in the
Reference solution (a) Dissolve 2.0 mg of nonivamide CRS in
mass of the extract to be examined used to prepare the test
the mobile phase and dilute to 25.0 mL with the mobile solution, in grams;
phase (solution A). Dissolve 8.0 mg of capsaicin CRS in a mass of capsaicin CRS used to prepare reference solution (a), in
mixture of 5.0 mL of solution A and 45 mL of the mobile grams;
phase. Dilute to 100.0 mL with the mobile phase. Pz percentage content of capsaicin in capsaicin CRS.
Reference solution (b) Dissolve 8.0 mg of nonivamide CRS in _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur

the mobile phase and dilute to 100.0 mL with the mobile
phase. Dilute 5.0 mL of the solution to 100.0 mL with the
mobile phase.
Column:
- size: l = 0.25 m, 0 = 4.6 mm;
Capsicum Tincture
- stationary phase: base-deactivated end-capped phenylsilyl silica DEFINITION
gel for chromatography R (5 µm); Capsicum Tincture is a tincture produced from Standardised
- temperature: 30 °C. Capsicum Oleoresin.
Mobile phase acetonitrile RJ, 1 g!L solution of phosphoric Content
acid R (40:60 V/V). 0.036 to 0.044 % w/v of total capsaicinoids, expressed as
Flow rate 1.0 mUmin. capsaicin, C1sH21N03.
Detection Spectrophotometer at 225 nm. PRODUCTION
Injection 10 µL. The tincture is produced by dissolving Standardised
Run time 1.2 times the retention time of dihydrocapsaicin. Capsicum Oleoresin in a suitable quantity of ethanol
Elution order N ordihydrocapsaicin, nonivamide, capsaicin, (96 % v/v) and water by an appropriate procedure.
dihydrocapsaicin. Characteristics
Relative retention With reference to capsaicin (retention Yellow-orange to red-orange ethanolic liquid.
time= about 19 min): nordihydrocapsaicin = about 0.9; IDENTIFICATION
nonivamide = about 0.95; dihydrocapsaicin = about 1.3. Carry out the method for thin-layer chromatography,
System suitability Reference solution (a): Appendix III A, using the following solutions.
- resolution: minimum 1.5 between the peaks due to ( 1) Shake 10 mL of the preparation being examined with
nonivamide and capsaicin. 10 mL of hexane. Allow to separate and use the lower layer.
Calculate the percentage content of nonivamide with (2) 0.02% w/v each of capsaicin and dihydrocapsaicin in ether.
following expression: CHROMATOGRAPHIC CONDITIONS
(a) Use a TLC octadecylsiryl silica gel plate (5-40 µm) (Merck
A1 x m2 xp1 x 5 silica gel plates are suitable) [or TLC octadecylsiryl silica gel
A2 xm1 xC plate (2-10 µm)].
area of the peak due to nonivarnide in the chromatogram (b) Use the mobile phase described below.
obtained with the test solution; (c) Apply 20 µL [or 2 µL] of solution (1) and (2) as bands of
area of the peak due to nonivarnide in the chromatogram
15 mm [or 8 mm].
mass of the extract to be examined used to prepare the test (d) Develop the plate to 12 cm [6 cm].
solution, in grams; (e) Remove the plate and allow to dry in air. Treat with a
in grams; 0.25 g/L solution of dichloroquinonechlorimide in ethyl acetate
P1 percentage content of nonivarnide in nonwamide CRS; and expose to ammonia vapour until blue zones appear.
C percentage content of total capsaicinoids, as determined in the Examine in daylight.
assay.
MOBILE PHASE
Limit: 20 volumes of water and 80 volumes of methanol.
- nonivamide: maximum 5.0 per cent of the total CONFIRMATION
capsaicinoid content.
See below the sequence of zones present in the
Dry residue (2.8.16) chromatograms obtained with solutions (1) and (2).
Minimum 70.0 per cent, determined on 2.00 g. Furthermore other faint zones may be present in the
ASSAY chromatogram obtained with solution (1).
nonivamide. Top of the plate
expressed as capsaicin, using the following expression: A blue zone (capsaicin) capsaicin: A blue zone
A faint blue zone (Dihydrocapsaicin) Dihydrocapsaicin: A blue zone

(A3 +As +A6) x m3 XP2
A4 xm1
IV-158 Capsicum Preparations 2023
TESTS (1) Dilute 50 mL of the preparation being examined to

Nonivamide 100 mL with methanol.
Not more than 5% of the total capsaicinoids content. (2) 0.02% w/v of capsaicin EPCRS and 0.004% w/v of
Carry out the method for liquid chromawgraphy, nonivamide EPCRS in metharwl.
Appendix III D, using the following solutions in methanol. CHROMATOGRAPHIC CONDITIONS
(1) Dilute 50 mL of the preparation being examined to The chromatographic conditions described under the test for
100 mL. Nonivamide may be used.
(2) 0.02% w/v of capsaicin EPCRS and 0.004% w/v of Calculate the percentage content of total capsaicinoids (C),
nonivamide EPCRS. expressed as capsaicin, using the following expression:
(3) 0.004% w/v of nonivamide EPCRS.
CHROMATO GRAPHIC CONDITIONS (A3 +A5 +A6 )x~ x~
(a) Use a stainless steel column (25 cm x 4.6 mm) packed A4 x50
with base-deactivated end-capped phenyl,silyl silica gel for
chromatography R (5 µm) (GL Sciences Inertsil Ph-3 is
suitable).
(b) Use isocratic elution and the mobile phase described with solution(]);
below. area of the peak due to capsaicin in the chromatogram obtained
with solution (2);
(c) Use a flow rate of 1.0 mL per minute. area of the peak due to dihydrocapsaicin in the chromatogram
(d) Use a column temperature of 30°. obtained with solution (]);
(e) Use a detection wavelength of 225 nm. chromatogram obtained with solution (]);
(f) Inject 10 µL of each solution. content of capsaicin EPCRS in solution (2), in percentage w/v;
declared content of capsaicin EPCRS.
(g) Allow the chromatography to proceed for 1. 7 times the
retention time of dihydrocapsaicin.
MOBILE PHASE
40 volumes of acetonitrile RJ and 60 volumes of 1 g/L
solution of orthophosphoric acid. Standardised Capsicum Tincture
The order of elution of the peaks is nordihydrocapsaicin,
nonivamide, capsaicin and dihydrocapsaicin.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
The relative retentions with reference to capsaicin (retention
time is about 19 minutes) are nordihydrocapsaicin about 0.9; DEFINITION
nonivamide about 0.95 and dihydrocapsaicin about 1.3. Standardised tincture produced from Capsicum (1859) or
SYSTEM SUITABILITY Refined and standardised capsicum oleoresin (2336).
The test is not valid unless, in the chromatogram obtained Content
with solution (2), the resolution between the peaks due to 90 per cent to 110 per cent of the nominal content of total
nonivamide and capsaicin is at least 1.5. capsaicinoids, expressed as capsaicin (C 18H 27NO3;
DETERMINATION OF CONTENT Mr 305.4), stated on the label, which is between
0.020 per cent m/m and 0.060 per cent mlm.
reference to the total capsaicinoid content, using the PRODUCTION
following expression: The tincture is produced from the herbal drug or oleoresin
and ethanol (70 per cent V/V to 85 per cent V/V) by an
appropriate procedure.
CHARACTERS
Appearance
Yellowish-orange or reddish-orange liquid.
area of the peak due to nonivamide in the chromatogram IDENTIFICATION
obtained with solution(!); Thin-layer chromatography (2.2.27).
obtained with solution (3); Test solution Shake 10 mL of the tincture to be examined
content of nonivamide EPCRS in solution (3), in percentage with 10 mL of hexane R. Allow to separate and use the lower
w/v; layer.
declared content of nonivamide EPCRS;
percentage content of total capsaicinoids, as determined in the Reference solution Dissolve 1 mg of capsaicin R and 1 mg of
C dihydrocapsaicin R in 5 mL of ether R.
assay.
Plate TLC octadecy'-silyl silica gel plate R (5-40 µm) [or
Ethanol TLC octadecylsilyl silica gel plate R (2-10 µm)].
83 to 88 % v/v, Appendix VIII F. Mobile phase water R, methanol R (20:80 V/V).
Methanol Application 20 µL [or 2 µL] as bands of 15 mm [or 8 mm].
Maximum 0.05 % v/v, Appendix VIII G, method II.
ASSAY Drying In air.
Carry out the method for liquid chromawgraphy, Detection Treat with a 0.25 g/L solution of
Appendix III D, using the following solutions. dichloroquinonechlorimide R in ethyl acetate R, expose to
2023 Caraway IV-159
ammonia vapour until blue zones appear. Examine in Ethanol (2. 9.10)
daylight. 95 per cent to 105 per cent of the content stated on the
Results See below the sequence of zones present in the label.
chromatograms obtained with the reference solution and the Methanol and 2-propanol (2.9.11)
test solution. Furthermore, other zones may be present in the Maximum 0.05 per cent V/V of methanol and maximum
chromatogram obtained with the test solution. 0.05 per cent V/V of 2-propanol.
ASSAY
Top of the plate
--- --
nonivamide.
expressed as capsaicin, using the following expression:
Dihydrocapsaicin: a blue zone A faint blue zone (dihydrocapsaicin)
(A3 +As +A6) x m4 xp2 x 2
--- ---
A4 xm3
A, area of the peak due to capsaicin in the chromatogram obtained
TESTS
Nonivamide A, area of the peak due to dihydrocapsaicin in the chromatogram
Liquid chromatography (2.2.29). obtained with the test solution;
Test solution Dilute 50.0 g of the tincture to be examined to
100.0 mL with methanol R. mass of the tincture to be examined used to prepare the test
Reference solution (a) Dissolve 10.0 mg of capsaicin CRS and solution, in grams;
mass of capsaicin CRS used to prepare reference solution (a), in
2.0 mg of nonivamide CRS in methanol R and dilute to
grams;
50.0 mL with the same solvent. P, percentage content of capsaicin in capsaicin CRS.
methanol Rand dilute to 100.0 mL with the same solvent. - - - - - - - - - - - - - - - - - - - - - - - Ph Eur
Column:
- size: l = 0.25 m, 0 = 4.6 mm;
gel for chromatography R (5 µm); Caraway
Mobile phase acetonitrile Rl, 1 glL solution of phosphoric (Caraway Fruit, Ph. Bur. monograph 1080)
acid R (40:60 V/V). When Powdered Caraway is prescribed or demanded,
Flow rate 1.0 mLJmin. material complying with the appropriate requirements below
Detection Spectrophotometer at 225 nm. and containing not less than 2.5% v/w (25 mUkg) of
essential oil shall be dispensed or supplied.
Injection 10 µL.
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Run time 1.2 times the rentention time of dihydrocapsaicin.
Elution order Nordihydrocapsaicin, nonivamide, capsaicin, DEFINITION
dihydrocapsaicin. Whole, dry mericarp of Carum carvi L.
System suitability Reference solution (a): Content
- resolution: minimum 1.5 between the peaks due to Minimum 30 mLJkg of essential oil (anhydrous drug).
nonivamide and capsaicin. CHARACTERS
Calculate the percentage content of nonivamide with Odour reminiscent of carvone.
following expression: IDENTIFICATION
A. The fruit is a cremocarp of almost cylindrical shape. It is
A1 xm2 xpi x 100 generally 3-6.5 mm long and 1-1.5 mm wide. The mericarps,
A2 xm 1 xC usually free, are greyish-brown or brown, glabrous, mostly
sickle-shaped, with both ends sharply terminated. Each bears
AI area of the peak due to nonivamide in the chromatogram 5 prominent narrow ridges. When cut transversely the profile
obtained with the test solution; shows an almost regular pentagon and 4 vittae on the dorsal
A2 area of the peak due to nonivamide in the chromatogram
surface and 2 on the commissural surface may be seen with a
m1 mass of the tincture to be examined used to prepare the test lens.
solution, in grams; B. Microscopic examination (2.8.23). The powder is
m2 mass of nonivamide CRS used to prepare reference solution (b),
in grams;
p1 percentage content of nonivamide in nonivamide CRS; hydrate solution R. The powder shows the following diagnostic
C percentage content of total capsaicinoids, as determined in the characters (Figure 1080.-1): fragments of broad secretory
assay. canals (surface view (C, DJ, transverse section [F])
composed of yellowish-brown, thin-walled, polygonal
Limit: secretory cells [Ca, Da, Fa], frequently associated with a
- nonivamide: maximum 5.0 per cent of the total layer of thin-walled, transversely elongated endocarp
capsaicinoid content. cells [Cb, Db], 8-12 µm wide and more or less rectangular in
transverse section [Fb]; fragments of the epicarp covered by a
IV-160 Caraway Oil 2023
striated cuticle (surface view [B, L]), with thick-walled cells Results B The zones due to carvone are dark orange-brown;
[Ba] and occasional anomocytic stomata (2.8.3) [Bb]; the chromatogram obtained with the test solution shows
numerous endosperm fragments with more or less thickened above the zone due to carvone a violet zone similar in
polygonal cells [A, H] containing aleurone grains, droplets of position and colour to the zone due to triglycerides of olive
fatty oil [Aa, Ha] and microcrystals of calcium oxalate in oil in the chromatogram obtained with the reference solution;
microrosette formation [Ab, Hb]; spiral vessels accompanied the chromatogram obtained with the test solution shows
by sclerenchymatous fibres [G]; rarely, fibre bundles from the close to the solvent front a weak violet zone due to terpene
carpophore; rectangular or subrectangular sclereids from the hydrocarbons and in the lower part some weak, mostly violet-
mesocarp [E, J, K] with moderately thickened and pitted grey and brownish zones.
walls, sometimes accompanied by thin-walled parenchyma TESTS
cells [Ja].
Water (2.2.13)
Total ash (2.4.16)
ASSAY
B Essential oil (2. 8.12)
.et_ Use 10.0 g of the herbal drug reduced to a powder (710)
E {;.;{J (2. 9.12) immediately before the determination, 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 tube. Distil
at a rate of 2-3 mUmin for 90 min.
---------------------~&
Caraway Oil
Fb F PhEw _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
~
Fa
DEFINITION
Oil obtained by steam distillation from the dry fruits of
Camm carviL.
CHARACTERS
Appearance
Clear, colourless or yellow liquid.
K
IDENTIFICATION
Figure 1080.-1. - Illustration for identification test B of powdered Second identification: A.
herbal drug of caraway fruit A. Thin-layer chromatography (2.2.27).
C. Thin-layer chromatography (2.2.27). Test solution Dissolve 40 µL of the substance to be
Test solution Shake 0.5 g of the powdered herbal drug (710) examined in 1.0 mL of toluene R.
(2.9.12) with 5.0 mL of ethyl acetate R for 2-3 min. Filter Reference solution Dissolve 10 µL of carvone R and 5 µL of
over 2 g of anhydrous sodium sulfate R. carveol R in 1.0 mL of toluene R.
Reference solution Dissolve 2 µL of carvone R and 5 µL of Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel
olive oil R in 1.0 mL of ethyl acetate R. plate R (2-10 µm)].
Plate TLC silica gel F254 plate R. Mobile phase ethyl acetate R, toluene R (5:95 V!Jl).
Mobile phase ethyl acetate R, toluene R (5:95 V!Jl). Application 10 µL [or 2 µL] as bands.
Application 20 µL of the test solution and 10 µL of the Development Over a path of 10 cm [or 5 cm].
reference solution, as bands. Drying In air.
Development Over a path of 10 cm. Detection A Examine in ultraviolet light at 254 nm.
Drying In air. Results A See below the sequence of zones present in the
Detection A Examine in ultraviolet light at 254 nm. chromatograms obtained with the reference solution and the
Results A The chromatograms obtained with the test test solution. Furthermore, other zones may be present in the
solution and with the reference solution show a quenching
zone (carvone) in the central part against a light background.
Detection B Spray with anisaldehyde solution R and, while
observing, heat at 100-105 °C for 2-4 min; examine in
daylight.
2023 Caraway Oil IV-161
Top of the plate Cam'er gas helium for chromatography R.

-- --
Split ratio l :50.
Carvone: a quenching zone A quenching zone (carvone)
Temperature:
-- --
Time Temperature
(min) CC)
Column 0- 5 60
Detection B Spray with anisaldehyde solution R and heat at S - 68 60---+ 250
100-105 °C for 5-10 min. Examine immediately in daylight. 68 - 75 250
Results B See below the sequence of zones present in the Injection port 250
chromatograms obtained with the reference solution and the Detector 260
test solution. Furthermore, several zones of weak intensity are
present, particularly in the lower third, in the chromatogram Detection Flame ionisation.
obtained with the test solution. Injection 1.0 µL.
Elution order Order indicated in the composition of
Top of the plate reference solution (a). Record the retention times of these
A reddish-violet zone substances.
-- --
A reddish-violet zone ~-myrcene and limonene.
Carvone: a red to orange-brown An intense red to orange-brown zone Using the retention times determined from the
zone (carvone) chromatogram obtained with the reference solution, locate
the components of the reference solution in the
-- --
Carveol: a reddish-violet zone A reddish-violet zone ( carveol) Limits:
A violet-blue zone - /3-myrcene: 0.1 per cent to 1.0 per cent,
- limonene: 30.0 per cent to 45.0 per cent,
Reference solution Test solution - trans-dihydrocarvone: maximum 2.5 per cent,
- carvone: 50.0 per cent to 65.0 per cent,
B. Examine the chromatograms obtained in the test for - trans-carveol: maximum 2.5 per cent.
chromatographic profile. - disregard limit: the area of the peak in the chromatogram
Results The characteristic peaks in the chromatogram obtained with reference solution (b).
obtained with the test solution are similar in retention time to Chiral purity
those in the chromatogram obtained with the reference Gas chromatography (2.2.28).
solution. Test solution Dissolve 20 mg of the substance to be
TESTS examined in heptane Rand dilute to 10.0 mL with the same
Relative density (2.2.5) solvent.
0.904 to 0.920. Reference solution Dissolve 10 mg of (-)-carvone Rand
10 mg of carvone Rl in heptane Rand dilute to 10.0 mL with
the same solvent.
1.484 to 1.490.
Column:
Optical rotation (2.2. 7) - material: fused silica,
+ 65° to+ 81°. - size: l = 30 m, 0 = 0.25 mm,
Acid value (2.5.1) - stationary phase: modified ~-cyclodextrin for chiral
Maximum 1.0, determined on 5.00 g. chromatography Rl (film thickness 0.25 µm).
Chromatographic profile Carrier gas helium for chromatography R.
Gas chromatography (2.2.28): use the normalisation Flow rate 2.0 mIJmin.
procedure.
Split ratio 1:30.
Test solution Dissolve 0.200 g of the substance to be
Temperature:
examined in heptane Rand dilute to 10.0 mL with the same
solvent.
Time Temperature
Reference solution (a) Dissolve 5 µL of /3-myrcene R, 80 µL (min) CC)
of limonene R, 5 µL of dihydrocarvone R, 100 µL of carvone R Column 0 - 80 so ---+ 170
and 5 µL of carveol R in heptane Rand dilute to 10.0 mL Injection port 230
with the same solvent. Detector 230
Reference solution (b) Dissolve 10 µL of carvone R in
heptane Rand dilute to 10 mL with the same solvent. Dilute Detection Flame ionisation.
0.1 mL of this solution to 10 mL with heptane R.
Injection 1 µL.
Column:
- material: fused silica,
- resolution: minimum 2.4 between the peaks due to (-)-
=
- size: l 30 m, 0 =
0.53 mm,
carvone (1 st peak) and carvone Rl (2nd peak).
- stationary phase: macrogol 20 000 R, (film thickness 1 µm).
IV-162 Cardamom Fruit 2023
Calculate the percentage content of the (-)-carvone from the Volatile oil
following expression: In the seeds, not less than 4.0% v/w, Appendix XI E,
Method I. Use 20 g of the unground seeds and distil for
A1 5 hours.
- A xlOO
A l + 2 Acid-insoluble ash
area of the peak due to (-)-carvone,
Of the seeds, not more than 3.5%, Appendix XI K.
area of the peak due to carvone R!. Ash
Of the seeds, not more than 6.0%, Appendix XI J.
Limit:
- (-)-carvone: maximum 1 per cent.
STORAGE
At a temperature not exceeding 25 °C. Cardamom Oil
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur Preparations
Aromatic Cardamom Tincture
Compound Cardamom Tincture
DEFINITION
Cardamom Fruit Cardamom Oil is obtained by distillation from crushed
In making preparations of Cardamom, only the seed is used. Cardamom Fruit.
The seed is removed from the fruit, immediately powdered
CHARACTERISTICS
or bruised and used immediately in making the preparation.
Cardamom seed, after removal from the fruit, should not be A clear, colourless or pale yellow liquid, visibly free from
water; odour, that of Cardamom Fruit.
stored.
DEFINITION TESTS
Ester value
Cardamom Fruit consists of the dried, nearly ripe fruit of
90 to 156, Appendix X C.
Elettaria cardamomum Maton var. minuscula Burkill.
Optical rotation
CHARACTERISTICS
+20° to +40°, Appendix VF.
Odour and taste of the seeds, strongly aromatic.
Refractive index
Macroscopical Fruit: a trilocular inferior capsule, up to about
1.461 to 1.467, Appendix VE.
2 cm long, ovoid or oblong, dull green to pale buff, plump or
slightly shrunken, obtusely triangular in cross section, nearly Solubility in ethanol
smooth or longitudinally striated. Seeds in each loculus in Soluble, at 20°, in 6 volumes of ethanol (70%),
two rows, forming an adherent mass attached to the axile Appendix X M.
placenta. Seed: pale to dark reddish brown, about 4 mm long Weight per mL
and 3 mm broad, irregularly angular, marked with six to 0.917 to 0.940 g, Appendix VG.
eight transverse wrinkles, with a longitudinal channel
STORAGE
containing the raphe, each seed enveloped by a colourless,
Cardamom Oil should be kept in a well-filled container and
membranous aril. Transversely cut surface of seed showing a
protected from light.
brown testa, white starchy perisperm, grooved on one side,
yellowish endosperm and a paler embryo.
Microscopical Seed: aril composed of flattened, thin-walled,
parenchymatous cells. Testa composed of the following
layers: (i) outer epidermis of thick-walled, narrow, axially Aromatic Cardamom Tincture
elongated cells; (ii) a layer of collapsed parenchyma subjacent DEFINITION
to the outer epidermis; (iii) a single layer (two or three layers
near the raphe) of large, thin-walled, rectangular cells Cardamom Oil 3mL
containing volatile oil; (iv) two or three layers of parenchyma; Caraway Oil !0mL
Cinnamon Oil JOmL
(v) layers of thin-walled, flattened cells; (vi) distinctive Clove Oil !0mL
sclerenchymatous layer of closely packed brown, thick-walled Strong Ginger Tincture 60mL
cells, each with a bowl-shaped cavity in the upper part Ethanol (90 per cent) Sufficient to produce I 000 mL
containing a warty silica body; (vii) inner layer consisting of
flattened cells. Perisperm: cells thin-walled, packed with The tincture complies with the requirements for Tinctures stated
numerous starch granules up to 6 µm in diameter and, in a under Extracts and with the following requirements.
small cavity, one to seven prisms of calcium oxalate about TESTS
10 to 30 µm long. Endosperm parenchymatous, thin-walled, Ethanol content
with a granular hyaline mass of protein in each cell. Embryo: 84 to 87% vlv, Appendix VITI F, Method III.
cells small, containing aleurone grains.
Relative density
TESTS 0.825 to 0.845, Appendix V G.
Foreign matter
Of the fruit, not more than 1.0%; of the separated seeds, not
more than 3.0%, Appendix XI D.
2023 Cascara IV-163
IDENTIFICATION
Compound Cardamom Tincture A. The bark occurs in slightly channelled or nearly flat
DEFINITION pieces, usually 1-5 mm in thickness, usually varying greatly in
length and width. The outer surface is grey or dark greyish-
Cardamom Oil 0.450 mL brown and shows occasional lenticels that are orientated
Caraway Oil 0.400 mL transversally. It is usually more or less completely covered by
Cinnamon Oil 0.225 mL
Cochineal, in moderately coarse powder 7g a whitish coat of lichens, epiphytic moss and foliaceous
Glycerol 50 mL liverwort. The inner surface is yellow or reddish-brown or
Ethanol (60 per cent) Sufficient to produce I 000 mL almost black with fine longitudinal striations; it turns red
when treated with alkali. The yellow fracture is short and
Extemporaneous preparation granular in the outer part and somewhat fibrous in the inner
The following directions apply. part.
Moisten the Cochineal with a sufficient quantity of Ethanol B. Microscopic examination (2.8.23). The powder is
(60 per cent) and prepare 900 mL of tincture by percolation, yellowish-brown. Examine under a microscope using chloral
Appendix XI F. Add the Cardamom Oil, the Caraway Oil, hydrate solution R. The powder shows the following diagnostic
the Cinnamon Oil and the Glycerol and sufficient Ethanol characters (Figure 0105.-1): bundles [A] of partly lignified
(60 per cent) to produce 1000 mL; mix. Filter, if necessary. phloem fibres [Aa], accompanied by crystal sheaths
The tincture complies with the requirements for Tinctures stated containing prisms of calcium oxalate [Ab] and sometimes
under Extracts and with the following requirements. including medullary rays [Ac]; isolated sclereids [G] or
groups of sclereids [BJ accompanied by crystal sheaths [Ba];
TESTS
isolated cluster crystals [CJ or prisms [E] of calcium oxalate;
Ethanol content parenchymatous cells [F, HJ containing a yellow substance
52 to 57% v/v, Appendix VIII F, Method III. that becomes deep red when treated with alkali, sometimes
Glycerol accompanied by cells containing cluster crystals of calcium
4.5 to 5.5% v/v when determined by the following method. oxalate [Ha]; cork cells (surface view [DJ, transverse
Dilute 20 mL to 100 mL with water. To 20 mL of this section [J]), associated with parenchyma, some cells of which
solution add 100 mL of water and 1 g of activated charcoal contain cluster crystals of calcium oxalate Ua]; frequently
and boil under a reflux condenser for 15 minutes. Filter and epiphytes [Kl, which may be liverworts, entire or in
wash the filter and charcoal with sufficient water to produce fragments, having a lamina 1 cell thick without a midrib and
150 mL. Add 0.25 mL of bromocresol purple solution and composed of isodiametric cells, or leaves of mosses, having a
neutralise with 0.lM sodium hydroxide or 0.05M sulfuric acid to lamina 1 cell thick composed of elongated cells and
the blue colour of the indicator. Add 1.4 g of sodium periodate possessing a midrib several cells thick.
and allow to stand for 15 minutes. Add 3 mL of propane-1,2-
diol, shake and allow to stand for 5 minutes. Add 0.25 mL of
bromocresol purple solution and titrate with 0.1 M sodium
hydroxide VS to the same blue colour. Each mL of 0.lM
sodium hydroxide VS is equivalent to 9 .210 mg of glycerol.
Calculate the percentage v/v of glycerol, taking its weight per
mL to be 1.260 g.
Relative density
0.925 to 0.937, Appendix VG.
Cascara
Preparation
Standardised Cascara Dry Extract
,;
When Powdered Cascara is prescribed or demanded, r1••,,,.,.,-~.....,."--Ac
material complying with the requirements below with the
exception ofldentification test A and the test for Foreign H
matter shall be dispensed or supplied.
Ph Eur - - - - - - - - - - - - - - - - - - - - ~
Ha
DEFINITION
Dried, whole or fragmented bark of Rhamnus purshiana DC.
(syn. Frangula purshiana (DC.) A.Gray).
Content
Minimum 8.0 per cent of hydroxyanthracene glycosides of
which minimum 60 per cent consists of cascarosides, both 25µm
expressed as cascaroside A (C 27 H 32 0 14; Mr 580.5) (dried Ja
drug).
Figure 0 105.-1. - Illustration for identification test B of powdered
herbal drug of cascara
IV-164 Cascara 2023
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) (2.9.12) 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
several zones with the same fluorescence, situated above and ASSAY
particularly below (cascarosides) that due to barbaloin in the Cany out the assay in 24 h, protected from bright light.
chromatogram obtained with the reference solution. Stir 1.00 g of the powdered herbal drug (180) (2. 9.12) into
D. Heat 0.2 g of the powdered herbal drug (180) (2.9.12) 100 mL of boiling water R and continue boiling and stirring
with 50 mL of water Ron a water-bath for 15 min. Allow to for 5 min. Allow to cool, dilute to 100.0 mL with water R,
cool and filter. To 10 mL of the filtrate add 20 mL of shake, filter and discard the first 20 mL of filtrate. Transfer
hydrochloric acid Rl and heat on a water-bath for 15 min. 10.0 mL of the filtrate to a separating funnel, add 0.1 mL of
Allow to cool, transfer to a separating funnel and shake with 1 M hydrochloric acid and shake with 2 quantities, each of
3 quantities, each of 20 mL, of ether R. Reserve the aqueous 20 mL, of a mixture of 1 volume of ether R and 3 volumes of
layer (solution A). Combine the 3 ether extracts and shake hexane R. Wash the combined organic extracts with 5 mL of
with 10 mL of dilute ammonia R2. The aqueous layer water R, discard the organic layer and return the rinsings to
becomes reddish-violet. Transfer solution A to a small flask, the aqueous layer. Shake the combined aqueous layers with
add 5 g of ferric chloride R and heat on a water-bath for 4 quantities, each of 30 mL, of ethyl acetate R freshly
30 min. Allow to cool, transfer to a separating funnel and saturated with water R (to 150 mL of ethyl acetate R add
shake with 15 mL of ether R. Wash the ether layer with 15 mL of water R, shake for 3 min and allow to stand) on
10 mL of water R, discard the aqueous layer and shake the each occasion allowing separation to take place until the
ether layer with 5 mL of dilute ammonia R2. A red colour organic layer is clear. Combine the ethyl acetate extracts.
develops in the aqueous layer. Use the aqueous layer for the assay for cascarosides and the
organic layer for the assay for hydroxyanthracene glycosides
TESTS
other than cascarosides.
Other species of Rhamnus; anthrones
Thin-layer chromatography (2.2.27). Hydro:xyanthracene glycosides other than cascarosides
Transfer the organic layer to a suitable flask and remove the
solvent by distillation, evaporating almost to dryness.
(2.9.12) add 5 mL of ethanol (70 per cent VIV) Rand heat to
Dissolve the residue in 0.3-0.5 mL of methanol Rand transfer
boiling. Cool and centrifuge. Decant the supernatant
to a volumetric flask, rinsing the 1st flask with warm water R
immediately and use within 30 min.
and adding the rinsings to the methanolic solution. Allow to
Reference solution Dissolve 20 mg of barbaloin R in ethanol cool and dilute to 50.0 mL with water R. Transfer 20.0 mL
(70 per cent VIV) Rand dilute to 10 mL with the same of this solution to a 100 mL round-bottomed flask with a
solvent. ground-glass neck and containing 2 g of ferric chloride R and
Plates TLC silica gel plate R (2 plates). 12 mL of hydrochloric acid R. Attach a reflux condenser and
Mobile phase water R, methanol R, ethyl acetate R place the flask in a water-bath so that the level of the water is
(13:17:100 VIVIV). above that of the liquid in the flask and heat for 4 h. Allow
A. Application: 10 µL as bands. to cool, transfer the solution to a separating funnel and rinse
the flask successively with 3-4 mL of 1 M sodium hydroxide
and 3-4 mL of water R, adding the rinsings to the separating
Drying In air for 5 min. funnel. Shake the contents of the separating funnel with
Detection Spray with about 10 mL of a 50 g/L solution of 3 quantities, each of 30 mL, of a mixture of 1 volume of
potassium hydroxide R in ethanol (50 per cent VIV) R and heat ether Rand 3 volumes of hexane R. Wash the combined
at 100-105 °C for 15 min; examine immediately after organic layers with 2 quantities, each of 10 mL, of water R
heating. and discard the rinsings. Dilute the organic layer to
Results The chromatogram obtained with the reference 100.0 mL with the mixture of ether and hexane. Take
solution shows, in the central part, a reddish-brown zone due 20.0 mL, evaporate carefully to dryness on a water-bath and
to barbaloin; examine in ultraviolet light at 365 nm; the zone dissolve the residue in 10.0 mL of a 5 g/L solution of
due to barbaloin shows intense yellowish-brown fluorescence; magnesium acetate R in methanol R. Measure the absorbance
in the chromatogram with the test solution, no zone with (2.2.25) at 440 nm and 515 nm using methanol Ras the
orange-brown fluorescence is seen between the zone due to compensation liquid. If the ratio of the absorbance at
barbaloin and the zones due to cascarosides. 515 nm to that at 440 nm is less than 2.4, the assay is
B. Application: 10 µL of the test solution, as a band. invalid.
Calculate the percentage content of hydroxyanthracene
glycosides other than cascarosides, expressed as
Drying In air for not more than 5 min.
cascaroside A, using the following expression:
Detection Spray immediately with a 5 g/L solution of
nitrotetrazolium blue R in methanol R and examine Ax 6.95
immediately. m
Results No violet or greyish-blue zones appear.
i.e. taking the specific absorbance to be 180.
2023 Cascara Preparations IV-16 5
A absorbance at 515 nm; Development Over a path of 10 cm [or 6 cm].

Cascarosides Detection Treat with a 50 glL solution of potassium
Dilute the aqueous layer to 50.0 mL with water R. Treat hydroxide R in ethanol (50 per cent VIY,) R and heat to
20.0 mL of this solution as described above in the assay of 100-105 °C for 15 min; examine in ultraviolet light at
hydroxyanthracene glycosides other than cascarosides. 365 nm.
Measure the absorbance (2. 2. 25) of the test solution at Results See below the sequence of zones present in the
440 nm and 515 nm. If the ratio of the absorbance at chromatograms obtained with the reference solution and the
515 nm to that at 440 nm is less than 2. 7, the assay is test solution. Furthermore, other zones may be present in the
invalid. chromatogram obtained with the test solution.
Calculate the percentage content of cascarosides, expressed
as cascaroside A, using the following expression: Top of the plate
Ax 6.95 Emodin: a red fluorescent zone A faint red fluorescent zone
m -- --

Barbaloin: a yellowish-brown A yellowish-brown fluorescent zone
fluorescent zone
- - - - - - - - - - - - - - - - - - - - - ~ Ph Eur
-- --
An intense yellowish-brown
Standardised Cascara Dry Extract fluorescent zone
3 yellowish-brown fluorescent zones
Preparation
Cascara Tablets Reference solution Test solution
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION TESTS
Standardised dry extract obtained from Cascara (0105). Loss on drying (2.8.17)
Content Maximum 5.0 per cent.
- hydroxyanthracene glycosides expressed as cascaroside A ASSAY
(C21H32O14; M, 580.5): 8.0 per cent to Carry out the assay within 24 h, protected from bright light.
25.0 per cent m/m (dried extract) and 90 per cent to To 0.500 g of the extract to be examined add 90 mL of
110 per cent of the nominal content stated on the label; water R and sonicate at 40 °C for 15 min. Shake, cool and
- cascarosides expressed as cascaroside A (C 27 H 320 14; dilute to 100.0 mL with water R. Shake and filter, discarding
M, 580.5): minimum 60 per cent of the the first 20 mL of filtrate. Transfer 10.0 mL of the filtrate to
hydroxyanthracene glycosides. a separating funnel, add 0.1 mL of JM hydrochloric acid and
PRODUCTION shake with 2 quantities, each of 20 mL, of a mixture of
The extract is produced from the herbal drug by an 1 volume of ether Rand 3 volumes of hexane R. Wash the
appropriate procedure using either boiling water or a combined organic extracts with 5 mL of water R. Discard the
hydroalcoholic solvent at least equivalent in strength to organic layer and return the rinsings to the hydroalcoholic
ethanol (60 per cent V/V). layer. Shake with 4 quantities, each of 30 mL, of ethyl
acetate R freshly saturated with water R (prepared as follows:
CHARACTERS
to 150 mL of ethyl acetate R add 15 mL of water R, shake for
Appearance 3 min and allow to stand), on each occasion allowing the
Brown, free-flowing powder. layers to separate until the organic layer is clear. Combine
IDENTIFICATION the ethyl acetate extracts. Use the aqueous layer for the assay
Thin-layer chromatography (2.2.27). of cascarosides and the organic layer for the assay of
Test solution To 0.2 g of the extract to be examined add hydroxyanthracene glycosides other than cascarosides.
5 mL of ethanol (70 per cent V/V) R and heat to boiling. Cool Hydroxyanthracene glycosides other than cascarosides
and centrifuge. Decant the supernatant solution immediately Transfer the organic layer to a round-bottomed flask and
and use within 30 min. remove the solvent by distillation, evaporating almost to
Reference solution Dissolve 20 mg of barbawin R and 2 mg of dryness. Dissolve the residue in 0.5 mL of methanol R, add
emodin R in ethanol (70 per cent V/V) R and dilute to 10 mL 10 mL of water R at 40 °C and transfer to a 50 mL
with the same solvent. volumetric flask, rinsing the round-bottomed flask with
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel water R at 40 °C and adding the rinsings to the
plate R (2-10 µm)]. hydromethanolic solution. Allow to cool and dilute to
50.0 mL with water R. Transfer 20.0 mL of the solution to a
100 mL round-bottomed flask with a ground-glass neck
(13:17: 100 VIVIV).
containing 2 g offerric chwride R and 12 mL of hydrochwric
IV-166 Cascara Preparations 2023
acid R. Attach a reflux condenser and place the flask in a IDENTIFICATION

water-bath so that the level of the water is above that of the Carry out the method for thin-layer chromatography,
liquid in the flask and heat for 4 h. Allow to cool, transfer Appendix III A, using the following solutions.
the solution to a separating funnel and rinse the flask (1) Boil a quantity of the powdered tablets containing the
successively with 4 mL of 1 M sodium hydroxide and 4 mL of equivalent of 32 mg of total hydroxyanthracene derivatives
water R, adding the rinsings to the separating funnel. Shake with 5 mL of 70% v/v of ethanol, cool and centrifuge. Decant
the contents of the separating funnel with 3 quantities, each the supernatant liquid immediately and use within
of 30 mL, of a mixture of 1 volume of ether R and 3 volumes 30 minutes.
of hexane R. Wash the combined organic layers with
(2) Dissolve 20 mg of barbaloin and 2 mg of emodin in
2 quantities, each of 10 mL, of water Rand discard the
70% v/v of ethanol and dilute to 10 mL with the same
rinsings. Dilute the organic layer to 100.0 mL with a mixture
solvent.
of 1 volume of ether R and 3 volumes of hexane R. Take
20.0 mL of the solution, evaporate carefully to dryness on a CHROMATOGRAPHIC CONDITIONS
water-bath and dissolve the residue in 10.0 mL of a 5 g/L (a) Use silica gel F254 precoated plates or high-performance
solution of magnesium acetate R in methanol R. Measure the silica gel F2s4 (Merck silica gel F 254 HPTLC plates are
absorbance (2.2.25) at 440 nm and 515 nm, using suitable).
methanol R as the compensation liquid. (b) Use the mobile phase as described below.
System suitability: (c) Apply 10 µL [or 2 µL] of each solution, as bands.
- absorbance ratio: A 515 /A 440 is not less than 2.4.
(d) Develop the plate to 10 cm [or 6 cm].
(e) After removal of the plate, dry in air, spray with a 5% w/v
glycosides other than cascarosides, expressed as
solution of potassium hydroxide in 50% v/v ethanol, heat at
cascaroside A, using the following expression:
100 to 105° for 15 minutes and examine under ultraviolet
Ax 6.95 light (365 nm).
m MOBILE PHASE
13 volumes of water, 17 volumes of methanol and
i.e. taking the specific absorbance to be 180. 100 volumes of ethyl acetate.
CONFIRMATION
m mass of the substance to be examined, in grams. The chromatogram obtained with solution (1) show
yellowish-brown fluorescent bands with Rf values of between
Cascarosides 0.2 and 0.25, an intense yellowish-brown fluorescent band
Dilute the aqueous layer to 50.0 mL with water R. Treat with an Rf value of about 0.3, a blue fluorescent band with
20.0 mL of this solution as described above in the assay of an Rf value of about 0.6, a yellowish-brown fluorescent band
hydroxyanthracene glycosides other than cascarosides. with an Rf value of about 0. 7 corresponding in colour and
Measure the absorbance (2.2.25) at 440 nm and 515 nm. position to the band obtained with barbaloin in solution (2)
System suitability: and a faint reddish fluorescent band with an Rf value of
- absorbance ratw: A 515/A 440 is not less than 2.7. about 0.9 corresponding in position to emodin in
Calculate the percentage content of cascarosides, expressed solution (2).
as cascaroside A, using the following expression:
Top of the plate
Ax 6.95
A faint red fluorescent band Emodin a red fluorescent band
m

A yellow-brown fluorescent band Barbaloin: a yellow-brown
fluorescent band
A blue fluorescent band
LABELLING
The label states the nominal content of hydroxyanthracene
glycosides, expressed as cascaroside A. An intense yellow-brown
fluorescent band
3 yellow-brown fluorescent bands
Cascara Tablets Solution (1) Solution (2)
DEFINITION
Cascara Tablets contain Standardised Cascara Dry Extract. TESTS
They are coated. Disintegration
The tablets comply with the requirements stated under Tablets and Comply with the requirements stated under Tablets but for
with the following requirements. sugar-coated tablets the maximum time is 120 minutes.
Content of total hydroxyanthracene derivatives ASSAY
17.0 to 23.0 mg, of which not less than 60% consists of Carry out the assay within 24 hours, protected from bright light.
cascarosides, both expressed as cascaroside A.
2023 Cassia Oil IV-167
Add 80 mL of 70% v/v ethanol to a quantity of the powdered and at 515 nm, Appendix II B, using methanol in the
tablets containing 7 5 mg of total hydroxyanthracene reference cell. The assay is not valid if the ratio of the
derivatives. Shake and allow to stand in the dark for at least absorbance at 515 nm to that at 440 nm is less than 2.7.
8 hours. Dilute to 100.0 mL with 70% v/v of ethanol. Shake Calculate the percentage content of cascarosides, expressed
and filter, discarding the first 20 mL of filtrate. Transfer as cascaroside A, using the following expression:
10.0 mL of the filtrate to a separating funnel, add 0.1 mL of
lM hydrochloric acid and shake with 2-quantities, each of Ax 6.95
20 mL, of a mixture of 1 volume of ether and 3 volumes of m
hexane. Wash the combined organic extracts with 5 mL of
water. Discard the organic layer and return the rinsings to the i.e. taking the specific absorbance to be 180.
hydroalcoholic layer. Shake with 4-quantities, each of 30 mL,
of ethyl acetate freshly saturated with water prepared by A absorbance at 515 nm;
shaking 150 mL of ethyl acetate with 15 mL of water for m = weight of the substance being examined, in grams.
3 minutes and allowing to stand until the layers have
separated and the organic layer is clear. Combine the ethyl LABELLING
acetate extracts and use the aqueous layer for the assay of The label states the nominal content of hydroxyanthracene
cascarosides and the organic layer for the assay of glycosides, expressed as cascarosides A.
hydroxyanthracene glycosides other than cascarosides.
Hydroxyanthracene glycosides other than cascarosides
Transfer the organic layer to a round-bottomed flask and
remove the solvent by distillation, evaporating almost to Cassia Oil
dryness. Dissolve the residue in 0.5 mL of methanol, add
10 mL of water at 40° and transfer to a 50 mL volumetric (Ph. Bur. monograph 1496)
flask, rinsing the round-bottomed flask with water at 40° and PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
adding the rinsings to the hydromethanolic solution. Allow to
cool and dilute to 50.0 mL with water. Transfer 20.0 mL of DEFINITION
the solution to a 100 mL round-bottomed flask with a Essential oil obtained by steam distillation of the leaves and
ground-glass neck containing 2 g of iron (III) chloride young branches of Cinnamomum cassia (L.) J.Presl (syn.
hexahydrate and 12 mL of 7M hydrochlonc acid. Attach a Cinnamomum aromaticum Nees).
reflux condenser and place the flask in a water-bath so that CHARACTERS
the level of the water is above that of the liquid in the flask Appearance
and heat for 4 hours. Allow to cool, transfer the solution to a Clear, mobile, yellow or reddish-brown liquid.
separating funnel and rinse the flask successively with 4 mL
Characteristic odour reminiscent of cinnamic aldehyde.
of lM sodium hydroxide and 4 mL of water, adding the
rinsings to the separating funnel. Shake the contents of the IDENTIFICATION
separating funnel with 3-quantities, each of 30 mL, of a First identification: B.
mixture of 1 volume of ether and 3 volumes of hexane. Wash Second identification: A.
the combined organic layers with 2-quantities, each of A. Thin-layer chromatography (2.2.27).
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 Test solution Dissolve 0.5 mL of the essential essential oil to
3 volumes of hexane. Take 20.0 mL of the solution, be examined in acetone R and dilute to 10 mL with the same
evaporate carefully to dryness on a water-bath and dissolve solvent.
the residue in 10.0 mL of a 0.5% w/v solution of magnesium Reference solution Dissolve 50 µL of trans-cinnamic
acetate in methanol. Measure the absorbance of the resulting aldehyde R, 10 µL of eugenol Rand 50 mg of coumarin R in
solution at 440 nm and at 515 nm, Appendix II B, using acetone R and dilute to 10 mL with the same solvent.
methanol in the reference cell. The assay is not valid if the Plate TLC silica gel plate R.
ratio of the absorbance at 515 nm to that at 440 nm is less Mobile phase methanol R, toluene R (10:90 V!V).
than 2.4. Application 10 µL as bands.
glycosides other than cascarosides, expressed as cascaroside
A, using the following expression: Drying In air.
Ax 6.95 Results A The zone of blue fluorescence in the
m chromatogram obtained with the test solution is similar in
position and colour to the zone in the chromatogram
i.e. taking the specific absorbance to be 180. obtained with the reference solution (coumarin).
Detection B Spray with anisaldehyde solution R; examine in
A absorbance at 5 I 5 nm; daylight while heating at 100-105 °C for 5-10 min.
m = weight of the substance being examined, in grams.
Results B The chromatogram obtained with the reference
solution shows in its upper part a violet zone (eugenol) and
Cascarosides above this zone a greenish-blue zone (trans-cinnamic
To the aqueous solution reserved from the preliminary
aldehyde). The chromatogram obtained with the test solution
extraction add sufficient water to produce 50.0 mL. Carry
shows a zone similar in position and colour to the zone due
out the Assay for hydroxyanthracene gycosides other than
to trans-cinnamic aldehyde in the chromatogram obtained
cascarosides, beginning at the words, 'Transfer 20 mL ... '.
with the reference solution and may show a very faint zone
Measure the absorbance of the resulting solution at 440 nm
due to eugenol. Other faint zones are present.
IV-168 Greater Celandine 2023
B. Examine the chromatograms obtained in the test for STORAGE

chromatographic profile. Protected from heat.
Results The principal peaks in the chromatogram obtained
with the test solution are similar in retention time to those in
the chromatogram obtained with the reference solution.
Eugenol may be absent from the chromatogram obtained
with the test solution. Greater Celandine
TESTS
Relative density (2.2.5) (Ph. Bur. monograph 1861)
1.052 to 1.070.
Refractive index (2.2.6) DEFINITION
1.600 to 1.614. Dried, whole or cut aerial parts of Chelidonium majus L.
Optical rotation (2. 2. 7) collected during flowering.
-1 ° to + 1°. Content
Chromatographic profile Minimum 0.6 per cent of total alkaloids, expressed as
Gas chromatography (2.2.28): use the normalisation chelidonine (C 20H 19N0 5; Mr 353.4) (dried drug).
procedure.
IDENTIFICATION
Test solution The essential oil to be examined. A. The stems are rounded, ribbed, yellowish or greenish-
Reference solution Dissolve 100 µL of trans-cinnamic brown, somewhat pubescent, about 3-7 mm in diameter,
aldehyde R, 10 µL of cinnamyl acetate R, 10 µL of eugenol R, hollow and mostly collapsed. The leaves are thin, irregularly
10 µL of trans-2-methoxycinnamaldehyde Rand 20 mg of pinnate, the leaflets ovate to oblong with coarsely dentate
coumarin R in 1 mL of acetone R. margins, the terminal leaflet often 3-lobed; the adaxial
Column: surface is bluish-green and glabrous, the abaxial surface paler
- material: fused silica; and pubescent, especially on the veins. The flowers have
=
- size: l 60 m, 0 = about 0.25 mm; 2 deeply concavo-convex sepals, readily removed, and
- stationary phase: macrogol 20 000 R. 4 yellow, broadly ovate, spreading petals about 8-10 mm
Carrier gas helium for chromatography R. long; the stamens are numerous, yellow, and a short style
arises from a superior ovary; long, capsular, immature fruits
are rarely present.
Split ratio 1: 100.
Temperature: greyish-green or brownish-green. Examine under a
Time Temperature
(min) (°C)
numerous fragments of upper epidermis, composed of cells
Column 0 - 10 60
with sinuous walls (surface view [BJ), accompanied by
10 - 75 60 -, 190
underlying palisade parenchyma [Ba]; numerous fragments of
75 - 160 190
lower epidermis in surface view [A, E] bearing anomocytic
Injection port 200
stomata (2.8.3) [Aa] and bases of covering trichomes [Ab],
Detector 240
sometimes accompanied by underlying spongy parenchyma
[Ea]; long, uniseriate, multicellular covering trichomes,
Detection Flame ionisation. usually fragmented, with thin-walled cells, sometimes
Injection 0.2 µL. collapsed [G]; vascular tissue from the leaves and stems
Elution order Order indicated in the composition of the consisting of pitted and spirally thickened vessels [DJ; groups
reference solution, depending on the operating conditions of fibres [CJ; articulated latex tubes with yellowish-brown
and the state of the column, coumarin may elute before or contents [F]; occasional fragments of the corolla [HJ
after trans-2-methoxycinnamaldehyde; record the consisting of thin-walled cells containing numerous pale
retention times of these substances. yellow droplets of oil [Ha]; spherical pollen grains about
30-40 µm in diameter with 3 pores and a finely pitted
- resolution: minimum 1.5 between the peaks due to trans-2- exine m.
methoxycinnamaldehyde and coumarin. C. Thin-layer chromatography (2.2.27).
Identification of components Using the retention times Test solution To 0.4 g of the powdered herbal drug (710)
determined from the chromatogram obtained with the (2. 9.12) add 50 mL of dilute acetic acid R. Boil in a water-
reference solution, locate the components of the reference bath under a reflux condenser for 30 min. Cool and filter.
solution in the chromatogram obtained with the test solution. To the filtrate add concentrated ammonia R until a strong
alkaline reaction is produced. Shake with 30 mL of methylene
Determine the percentage content of each of these
chloride R. Dry the organic layer over anhydrous sodium
components. The percentages are within the following
sulfate R, filter and evaporate in vacua to dryness. Dissolve
ranges:
the residue in 1.0 mL of methanol R.
- trans-cinnamic aldehyde: 70 per cent to 90 per cent;
- cinnamyl acetate: 1.0 per cent to 6.0 per cent; Reference solution Dissolve 2 mg of methyl red R and 2 mg of
- eugenol: maximum 0.5 per cent; papaverine hydrochloride R in 10 mL of ethanol (96 per cent) R.
- trans-2-methoxycinnamaldehyde: 3.0 per cent to Plate TLC silica gel plate R.
15 per cent; Mobile phase anhydrous formic acid R, water R, propanol R
- coumarin: 1.5 per cent to 4.0 per cent. (1:9:90 V!VIV).
2023 Centaury IV-169
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 Rand 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
with dilute sulfuric acid Rand dilute to 25.0 mL with the
same solvent. To 5.0 mL of the solution add 5.0 mL of a
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 Rand stopper
the flask.
Compensation liquid Prepare at the same time and in the
same manner as for the test solution: place in a 25 mL
\~•® \U
volumetric flask 5.0 mL of dilute sulfuric acid Rand 5.0 mL
0 of a 10 g/L solution of chromotropic acid, sodium salt R in

sulfuric acid R, stopper the flask and mix carefully. Dilute to
25.0 mL with suljuric acid R and stopper the flask.
-- Place both solutions on a water-bath for 10 min. Cool to
about 20 °C and dilute if necessary to 25.0 mL with sulfuric
Figure 1861.-1. - Illustration for identification test B of powdered acid R. Measure the absorbance (2.2.25) of the test solution
herbal drng of greater celandine at 570 nm by comparison with the compensation liquid.
Application 10 µL as bands. as chelidonine, using the following expression:
Ax 2.23
Drying In air.
m
Detection Spray with potassium iodobismuthate solution R and
dry in air; spray with sodium nitrite solution R and allow to dry i.e. taking the specific absorbance of chelidonine to be 933.
in air; examine in daylight.
Results See below the sequence of zones present in the A absorbance at 570 nm;
test solution. Furthermore, other weaker zones may be
Top of the plate

Centaury
-- --
Methyl red: a red zone A brown zone
A brown zone
DEFINITION
Papaverine: a greyish-brown zone A greyish-brown zone
Whole or fragmented dried flowering aerial parts of
- - -- Centaurium erythraea Rafu s. I. including C. maJus (H. et L.)
Zeltner and C. suffruticosum (Griseb.) Ronn. (syn.: Erythraea
centaurium Persoon; C. umbellatum Gilibert; C. minus Gars.).
2 brown zones
CHARACTERS
Reference solution Test solution Bitter taste.
IDENTIFICATION
TESTS A. The hollow cylindrical, light green to dark brown stem has
Foreign matter (2.8.2) longitudinal ridges, and is branched only in its upper part.
Maximum 10.0 per cent. The sessile leaves are entire, decussately arranged, and have
an ovate to lanceolate lamina, up to about 3 cm long. Both
Loss on drying (2.2.32) surfaces are glabrous and green to brownish-green.
Maximum 10.0 per cent, determined on 1.000 g of the The inflorescence is diaxially branched. The tubular calyx is
powdered herbal drug (355) (2. 9.12) by drying in an oven at green and has 5 lanceolate, acuminate teeth. The corolla
105 °C for 2 h. consists of a whitish tube divided into 5 elongated lanceolate
IV-170 Centella 2023
pink to reddish lobes, about 5-8 mm long. 5 stamens are Detection B Spray with anisaldehyde solution R and heat at
present attached to the top of the corolla tube. The ovary is 100-105 °C for 5-10 min. Examine in daylight.
superior and has a short style, a broad bifid stigma and Results B See below the sequence of the zones present in
numerous ovules. Cylindrical capsules, about 7-10 mm long, the chromatograms obtained with the reference solution and
with small brown markedly rough seeds are frequently the test solution. Furthermore, other less intense coloured
present zones may be present in the chromatogram obtained with the
B. Microscopic examination (2.8.23). The powder is test solution.
greenish-yellow or brownish. Examine under a microscope,
using chloral hydrate solution R. The powder shows the Top of the plate
following diagnostic characters: fragments from the stem with
lignified groups of fibres associated with narrow vessels, -- --
tracheidal vessels occasional vessels with spiral thickening;
-- --
pitted parenchyma of the pith and medullary rays; fragments
of leaf lamina with sinuous epidermal cells and striated Swertiamarin: a brown zone A brown zone (swertiamarin)
cuticle, especially over the margins and surrounding the Rutoside: a yellow zone
stomata; numerous stomata, mainly anisocytic (2.8.3);
fragments of the palisade mesophyll, each cell containing a A brownish-grey zone
single prism crystal or, less frequently, a cluster crystal of A yellow 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 A grey zone
striated cuticle; parts of the endothecium with reticulate or
ridge-shaped wall thickenings; triangularly rounded or Reference solution Test solution
elliptical, yellow pollen grains, about 30 µm in diameter, with
a distinctly pitted exine and 3 germinal pores; fragments of TESTS
the wall of the fruit capsule composed of crossed layers of Foreign matter (2.8.2)
fusiform cells; oil droplets from the seeds, fragments of the Maximum 3 per cent.
epidermis of the testa showing large, brown reticulations and
a pitted surface. Bitterness value (2. 8. 15)
Minimum 2000.
Test solution To 1.0 g of the powdered herbal drug (355) Loss on drying (2.2.32)
(2.9.12) add 25 mL of methanol R, shake for 15 min and Maximum 10.0 per cent, determined on 1.000 g of the
filter. Evaporate the filtrate to dryness under reduced powdered herbal drug (355) (2.9.12) by drying in an oven at
pressure and at a temperature not exceeding 50 °C. Take up 105 °C for 2 h.
the residue with small quantities of metharwl R so as to Total ash (2.4.16)
obtain 5 mL of solution, which may contain a sediment. Maximum 6.0 per cent.
Reference solution Dissolve 1 mg of rutoside trihydrate R and _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
1 mg of swertiamarin R in methanol R and dilute to 1 mL
F2s 4 Plate R (2-10 µm)]. Centella
Mobile phase water R, anhydrous formic acid R, ethyl
formate R (4:8:88 VIVIV). (Ph. Bur. monograph 1498)
Application 10 µL [or 5 µL] as bands. Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Development In an unsaturated tank over a path of 12 cm
[or6cm]. DEFINITION
Drying In air. Dried, fragmented aerial parts of Gentelia asiatica (L.) Urb.
Detection A Examine in ultraviolet light at 254 nm. Content
Results A See below the sequence of the zones present in Minimum 6.0 per cent of total triterpenoid derivatives,
the chromatograms obtained with the reference solution and expressed as asiaticoside (C 48 H 78 0 19 ; Mr 959) (dried drug).
the test solution. Furthermore, other less intense quenching IDENTIFICATION
zones may be present in the chromatogram obtained with the A. The leaves are alternate, sometimes grouped together at
test solution. the nodes, reniform or orbicular or oblong-elliptic and have
palmate nervation, usually with 7 veins, and a crenate
Top of the plate
margin. The leaves are very variable in size; the petiole is
-- -- usually 5-10, sometimes 15, times longer than the lamina,
which is 10-40 mm long and 20-40 mm, sometimes up to
-- -- 70 mm, wide. Young leaves show a few trichomes on the
Swertiamarin: a quenching zone A prominent quenching zone lower surface while adult leaves are glabrous.
(swertiamarin) The inflorescence, if present, is a single umbel which usually
Rutoside: a quenching zone consists of 3 flowers, rarely 2 or 4; the flowers are very small
(about 2 mm), pentamerous and have an inferior ovary; the
fruit, a brownish-grey, orbicular cremocarp, up to 5 mm
Reference solution Test solution long, is very flattened laterally and has 7-9 prominent curved
ridges per mericarp.
2023 Centella IV- I 71
B. Microscopic examination (2.8.23). The powder is Top of the plate

greenish-grey. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic A violet zone A violet zone
characters (Figure 1498.-1): numerous fragments of upper
epidermis of the leaf [D] composed of polygonal cells having -- --
an irregularly striated cuticle, accompanied by underlying
-- --
palisade parenchyma [Da], with some cells containing cluster
crystals of calcium oxalate [Db]; fragments of the lower A violet zone A violet zone
epidermis of the leaf with usually paracytic stomata
(2.8.3) [C]; fragments of petiole epidermis with elongated Reference solution Test solution
cells covered by a striated cuticle m;whole [A] or, usually,
fragmented [BJ, uniseriate, long, flexuous, unicellular or
occasionally multicellular covering trichomes of young leaves; TESTS
secretory canals [M]; small prisms [L] and cluster Bacopa monnieri L.
crystals [E] of calcium oxalate up to 40 µm in diameter; Thin-layer chromatography (2.2.27).
vascular bundles from the stem [F] containing vessels [Fa] Test solution To 0.5 g of the powdered herbal drug (355)
and narrow septate fibres [Fb]; fragments of the epicarp of (2. 9.12) add 5 mL of ethanol (96 per cent) R, sonicate for
the fruit [H] with polygonal cells having a markedly striated 10 min and centrifuge. Use the supernatant.
cuticle; fragments of the fruit [K] usually containing 2 layers Reference solution Dissolve 50 mg of centella dry extract HRS
of short fibres in a perpendicular parquetry in 2 mL of ethanol (96 per cent) R.
arrangement [Ka], annular vessels, parenchymatous cells
containing prisms of calcium oxalate [Kb] and numerous oil
droplets [Kc]; short fibres, free or in small groups [G];
fragments of the endosperm with oily contents [N]. Mobile phase water R, methanol R, methylene chloride R
(1:6:14 VIVIV).
ff B
Drying In air.
Detection Treat with a 10 per cent V/V solution of sulfuric
acid R in methanol R, heat at 120 °C for 3 min and examine
in daylight.
Results No brown zone is present in the middle third of the
Maximum 5 per cent of underground organs and maximum
2 per cent of other foreign matter.
E~ ,IJ F:
~g powdered herbal drug (355) (2.9.12) by drying in an oven at
105 °C for 2 h.
Fa Total ash (2.4.16)
H Ash insoluble in hydrochloric acid (2.8.1)
ASSAY
Kb Liquid chromatography (2.2.29).
Test solution Place 1.000 g of the powdered herbal drug
(355) (2.9.12) in a cellulose fingerstall in a continuous
M
extraction apparatus (Soxhlet type). Add 100 mL of
methanol R and heat for 8 h. Cool and dilute the extract to
100.0 mL with methanol R. Filter through a membrane filter
(nominal pore size 0.45 µm). Dilute 5.0 mL of the filtrate to
10.0 mL with methanol R.
Figure 1498.-1. - Illustration for identification test B of powdered Reference solution (a) Dissolve 6.0 mg of asiaticoside CRS in
herbal drug of centella methanol R and dilute to 20.0 mL with the same solvent.
C. Examine the chromatograms obtained in the test for Reference solution (b) Dissolve 50.0 mg of centella dry
Bacopa monnieri L. extract HRS in methanol R, using sonication if necessary, and
dilute to 10.0 mL with the same solvent. Filter through a
Results See below the sequence of zones present in the membrane filter (nominal pore size 0.45 µm).
test solution. Furthermore, several brown zones may be Column:
present in the lower third of the chromatograms obtained - size: l =0.25 m, 0 =4.6 mm;
with the test solution and the reference solution. - stationary phase: octadecylsilyl silica gel for chromatography R
(5 µm).
IV-172 Chaenomeles Fruit 2023
Mobile phase: Content

- mobile phase A: 0.3 per cent V/V solution of phosphoric Minimum 0.50 per cent for the sum of oleanolic acid
acid R; (C 30H4sO 3 ; Mr 456.7) and ursolic acid (C30 H4sO 3 ;
- mobile phase B: acetonitrile RI; Mr 456.7), expressed as ursolic acid (dried drug).
IDENTIFICATION
(min) (per cent V/Jl) (per cent V/JI)
A. Whole drug. The fruit, split longitudinally into 2 halves, is
oblong, 4-9 cm long, 2-5 cm wide and 1-2.5 cm thick.
0 - 65 78 ➔ 45 22 ➔ 55
The outer surface is violet-red or reddish-brown, shiny, with
65 - 66 45 ➔ 5 55 ➔ 95
deep, irregular wrinkles and an in-rolled outer margin; it is
66 - 75 5 95
sometimes covered by a dull, purplish-grey wax-like frosting;
the surface is shiny when the frosting is removed.
Flow rate 1.0 mUmin. The internal (cut) surface is reddish-brown, dull and finely
Detection Spectrophotometer at 200 nm. granular in the region of the septum or slightly lustrous,
Injection 10 µL. reddish- or yellowish-brown, finely wrinkled or smooth in the
Identification of peaks Use the chromatogram supplied with region of the exposed locules. Seeds, if remaining, are brown
centella dry extract HRS and the chromatogram obtained with and flattened, broadly rounded at one end and pointed at the
reference solution (b) to identify the peaks due to other. The texture is hard and woody, difficult to break.
asiaticoside B, madecassoside, asiaticoside, madecassic acid, Fragmented drug Narrow sub-crescent-shaped slices with an
terminolic acid and asiatic acid. irregular, convex outer margin, sometimes with in-rolled
System suitability Reference solution (b): ends, or irregularly-shaped slices; slices 0.1-0.6 cm thick.
- peak-to-valley ratw: minimum 10, where Hp = height The outer surface is violet-red or brownish-red, with deep
above the baseline of the peak due to terminolic acid and irregular wrinkles. The mostly concave inner surface is
Hv = height above the baseline of the lowest point of the slightly lustrous and reddish- or yellowish-brown in the
curve separating this peak from the peak due to region of the exposed locules or dull and granular. The cut
madecassic acid. surface is brownish-red, yellowish-brown or blackish; sclereid
clusters may be visible as 2 lighter-coloured layers, one
Calculate the percentage content of total triterpenoid
directly beneath the epicarp and the other close to the inner
derivatives, expressed as asiaticoside, using the following
surface. The texture is hard and fragile, easily broken.
expression:
brownish-red to brownish-yellow. Examine under a
[A 1 + (A 2 x 1.017) + (A 3 x 0.526) + (A 4 x 0.509)] x m2 xp x 10
microscope using chloral hydrate solutwn R. The powder
Asxm1 shows the following diagnostic characters (Figure 2713.-1):
numerous colourless or pale yellow or orange, rounded,
AI area of the peak due to asiaticoside in the chromatogram elliptical or polygonal, mostly thick-walled sclereids,
A2 sum of the areas of the peaks due to asiaticoside B and
isolated [D] or in groups [A, E], about 20-80 µm in
madecassoside in the chromatogram obtained with the test diameter, with distinctly striated and finely channelled walls
solution; and a lumen that sometimes contains orange-brown
A3 sum of the areas of the peaks due to madecassic acid and substances; fragments of the epicarp consisting of polygonal
terminolic acid in the chromatogram obtained with the test
cells, up to 35 µm in diameter, containing brown or reddish-
solution;
A4 area of the peak due to asiatic acid in the chromatogram brown substances [BJ; cells of the mesocarp [F], more or less
obtained with the test solution; polygonal or rounded, usually collenchymatous; rare
A, area of the peak due to asiaticoside in the chromatogram fragments of vascular bundles [G] with narrow reticulate or
annular vessels [Ga]; rare fibres [C], faintly channelled;
m1 mass of the herbal drug to be examined used to prepare the
test solution, in grams; parenchymatous cells [Aa] occasionally containing small
m2 mass of asiaticoside CRS used to prepare reference solution (a), prisms of calcium oxalate [Ab, C, Gb] associated with
in grams; sclereid clusters [A], fibres [C] or vascular bundles [G].
p percentage content of asiaticoside in asiaticoside CRS;
1.017 molecular mass conversion factor for asiaticoside B and
C. High-performance thin-layer chromatography (2.8.25).
madecassoside; Test solutwn To 0.5 g of the powdered herbal drug (355)
0.526 molecular mass conversion factor for madecassic acid and (2.9.12) add 5.0 mL of methanol R. Sonicate for 10 min,
terminolic acid;
0.509 molecular mass conversion factor for asiatic acid.
centrifuge and use the supernatant.
Reference solutwn (a) Dissolve 10.0 mg of oleanolic acid R in
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur methanol Rand dilute to 10.0 mL with the same solvent.
Reference solutwn (b) Dilute 2.5 mL of reference solution (a)
Reference solutwn (c) Dissolve 2.5 mg of {3-sitosterol R and
Chaenomeles Fruit 2.5 mg of oleanolic acid R in methanol Rand dilute to 10 mL
(Ph. Bur. monograph 2713) Intensity marker Oleanolic acid.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION Mobile phase anhydrous formic acid R, methylene chloride R,
Nearly ripe fruit of Chaenomeles lagenaria (Loisel.) Koidz. ethyl acetate R, cyclohexane R (1:5:8:20 V/V/V/V).
(syn. Chaenomeles speciosa (Sweet) Nakai), collected when it Application 5 µL as bands of 8 mm.
becomes greenish-yellow, split longitudinally into halves or Development 70 mm from the lower edge of the plate.
cut into slices, and dried.
2023 Chaenomeles Fruit IV-173
Total ash (2.4.16)

ASSAY
&, liquid chromatography (2.2.29).
(355) (2.9.12) in 80.0 mL of methanol R, stopper the flask
tightly and weigh. Sonicate for 30 min, weigh again and
compensate for the loss of solvent with methanol R.
Add 20.0 mL of a 10 per cent VIV solution of anhydrous
fomiic acid R in methanol R and mix thoroughly. Filter
through a membrane filter (nominal pore size 0.45 µm) and
dilute the filtrate to 100.0 mL with a 2 per cent VIV solution
· ,-, Ga Gb of anhydrous fomiic acid R in methanol R.
c:.... ---~-'-- 1/, : ,-.... 1
Solution A Dissolve 10.0 mg of ursolic acid CRS in
Reference solution (a) Dilute 1.0 mL of solution A to
10.0 mL with a 2 per cent VIV solution of anhydrous formic
\ /J '· i acid R in methanol R.
. ✓
' >< ~
Reference solution (b) Dissolve 10 mg of oleanolic acid R in
· • ~ ~ __) G
methanol R and dilute to 10 mL with the same solvent.
Mix 1 mL of the solution with 1 mL of solution A and dilute
~ ' \:J to 10 mL with a 2 per cent VIV solution of anhydrous formic
Column:
herbal drug of chaenomeles fruit - size: l = 0.15 m, 0 = 4.6 mm;
Drying In a current of cold air. chromatography R (5 µm);
Detection Treat with anisaldehyde solution R2 and heat at - temperature: 25 °C.
100 °C for 3 min; examine in daylight. Mobile phase Mix 25 volumes of a 4.6 g/L solution of
System suitability Reference solution (c): ammonium dihydrogen phosphate R previously adjusted to
- the chromatogram shows in the middle third 2 distinct pH 6.0 with strong sodium hydroxide solution R, 35 volumes of
zones; the upper zone (~-sitosterol) and the lower
methanol R2 and 40 volumes of acetonitrile Rl.
zone (oleanolic acid) are reddish-violet. Flow rate 1.0 mUmin.
chromatograms obtained with reference solution (a) and the Injection 20 µL of the test solution and reference
test solution. Furthermore, in the chromatogram obtained solutions (a) and (b).
with the test solution, other faint zones may be present. Run time 1.1 times the retention time of ursolic acid.
Top of the plate
Relative retention With reference to ursolic acid (retention
time= about 42 min): oleanolic acid= about 0.95.
A reddish-violet zone, faint to System suitability Reference solution (b):
equivalent
- peak-to-'IJalley ratio: minimum 13.0, where Hp = height
above the baseline of the peak due to ursolic acid and
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to oleanolic
-- -- acid.
A reddish-violet zone, very faint to Calculate the sum of the percentage contents of ursolic acid
equivalent and oleanolic acid, expressed as ursolic acid, using the
Oleanolic acid: a reddish-violet zone A reddish-violet zone, equivalent
A1xmzxp A3xmzxp
A reddish-violet zone, very faint to
-----+----
A2 xm1 A2 xm1
equivalent
area of the peak due to ursolic acid in the chromatogram
-- --
area of the peak due to ursolic acid in the chromatogram
area of the peak due to oleanolic acid in the chromatogram
Reference solution (a) Test solution mass of the herbal drug to be examined used to prepare the test
solution, in grams;
mass of urso/ic acid CRS used to prepare solution A, in grams;
TESTS assigned percentage content of ursolic acid in urso/ic acid CRS.
Maximum 14.0 per cent, determined on 1.000 g of the - - - - - - - - - - - - - - - - - - - - - ~ Ph Eur
105 °C for 2 h.
IV-17 4 Chamomile Flowers 2023
Chamomile Flowers B
(Roman Chamomile Flower, Ph. Bur. monograph "··. , "·;,·:.,:,:,,:,:.•.-:,,:,.',iii.:; ·-~~.:.Ba

0380)
. ' ' '. ' •. ,:. ,' ~' ' ' .
D
DEFINITION
Dried flower-head of the cultivated double variety of
Chamaemelum nobile (L.) All. (Anthemis nobilis L.).
Content
Minimum 7 mIJkg of essential oil (dried drug).
CHARACTERS
The flower-heads are white or yellowish-grey, composed of
solitary hemispherical capitula, made up of a solid conical
receptacle bearing the florets, each subtended by a
transparent small palea.
IDENTIFICATION
A. The capitula have a diameter of 8-20 mm; the receptacle
is solid; the base of the receptacle is surrounded by an
involucre consisting of 2-3 rows of compact and imbricated
bracts with scarious margins. Most florets are ligulate, but a
few pale yellow tubular florets occur in the central region.
Ligulate florets are white, dull, lanceolate and reflexed with a
dark brown, inferior ovary, a filiform style and a bifid stigma;
tubular florets have a five-toothed corolla tube,
5 syngenesious, epipetalous stamens and a gynoecium similar
to that of the ligulate florets.
B. Microscopic examination (2.8.23). The powder is pale
yellowish-green. Examine under a microscope using chloral Figure 0380.-1. - Illustration for identijicatwn test B of powdered
hydrate solution R. The powder shows the following diagnostic herbal drug of Roman chamomile flower
characters (Figure 0380.-1): numerous glandular trichomes,
Reference solution (c) Dissolve 2.5 mg of hyperoside R and
free (side view [D, G]) or on an epidermis (surface
3 mg of chlorogenic acid R in methanol R and dilute to
view [Hal), short, biseriate, with a stalk consisting of 2-4
cells and a head usually consisting of 2 cells covered by a
swollen cuticle; numerous conical covering trichomes, free or Intensity marker Chlorogenic acid.
on an epidermis [M], up to 900 µm long, each consisting of Plate TLC silica gel F254 plate R (2-10 µm).
3-4 very short basal cells and a long, thin-walled, terminal Mobile phase anhydrous formic acid R, water R, ethyl acetate R
cell, about 20 µm wide; all epidermises bear glandular (10:10:80 VIVIV).
trichomes and whole or fragmented covering trichomes [Ka]; Applicatwn 4 µL as bands of 8 mm.
fragments of the involucral bracts (F] with fine spiral vessels
[Fb] and cells, some of which are thick-walled, in
longitudinal rows [Fa]; bases of the bracts [K] show Drying In a current of air at room temperature for 5 min.
anomocytic stomata; fragments of paleae [BJ, with elongated Detection Heat at 100-105 °C for 5 min. Spray the warm
cells and finely pitted sclereids at the base [Ba], the margins plate with a 10 g/L solution of diphenylboric acid aminoethyl
of the paleae consist of a single layer of elongated thin-walled ester R in methanol R, then with a 50 g/L solution of
cells [E]; fragments of the corolla of the Iigulate flowers macrogol 400 R in methanol R or, alternatively, dip the warm
whose epidermis is covered by a striated cuticle (surface view plate in a 5 g/L solution of diphenylboric acid aminoethyl
[H, M]); papillose cells m at the apex of the florets; ester R in ethyl acetate R, then in a 50 g/L solution of macrogol
fragments of the ovary [A] which have at their base a 400 R in methylene chloride R. Allow the plate to dry in air for
sclerous ring [Ab]; cells of the parenchyma of the ovary and about 1 min and examine in ultraviolet light at 366 nm.
the involucre containing small cluster crystals of calcium System suitability Reference solution (c):
oxalate [Aa, Fe]; fragments of papillose stigmas [L]; rare - the chromatogram shows 2 distinct zones in the
rounded or triangular pollen grains about 35 µm in diameter, middle third which may, however, be touching.
with 3 germinal pores and a spiny exine [C]. The lower zone (chlorogenic acid) shows a light blue
C. High-performance thin-layer chromatography (2.8.25). fluorescence and the upper zone (hyperoside) shows a
Test solution To 0.5 g of the powdered herbal drug (355) yellow or orange fluorescence.
(2.9.12) add 5.0 mL of methanol R. Sonicate for 15 min, Results See below the sequence of zones present in the
then filter or centrifuge the solution and use the filtrate or chromatograms obtained with reference solution (a) and the
supernatant. test solution. Furthermore, in the chromatogram obtained
Reference solutwn (a) Dissolve 3 mg of chlorogenic acid R and with the test solution, other faint to very faint blue and
5 mg of apigenin 7-glucoside R in methanol R and dilute to greenish-blue fluorescent zones may be present, and the
10.0 mL with the same solvent. greenish-blue fluorescent zone and the brownish-yellow or
orange fluorescent to faint fluorescent zone near the solvent
front may be overlapped.
2023 Chinese Goldthread Rhizome IV-175
Top of the plate IDENTIFICATION

A. Whole drug. Whole rhizome with roots and rootlets
A greenish-blue fluorescent zone or a
faint greenish-blue fluorescent zone removed. The outer surface, yellowish-grey or yellowish-
brown, is rough, with irregular nodular protuberances,
A brownish-yellow or orange
fluorescent zone or a faint brownish-
sometimes with fine, smooth intemodes; it is shiny in some
yellow or orange fluorescent zone parts or covered with a fine powder. Rootlet remains are
sometimes present. The apex bears the remains of brown
scale leaves and sometimes the remains of stems and petioles.
2 light blue fluorescent zones or 2 The texture is hard, the fracture uneven with reddish-orange,
faint light blue fluorescent zones
dark reddish-brown or dark brown bark in transverse section;
A usually intense light blue the wood is bright yellow or orange-yellow, radially arranged
fluorescent zone
and clefts may be present. The pith is often of similar colour
-- - - to the bark and sometimes hollowed. The rhizome of C.
chinensis, 3-6 cm long and up to 0.8 cm in diameter (below
the crown), may be curved, usually with a few to several
rhizomes coming together at the crown; that of C. delwidea is
Apigenin-7-glucoside: a greenish- A greenish-blue fluorescent zone or usually singular, somewhat cylindrical and curved with
blue fluorescent zone an intense greenish-blue fluorescent smooth and relatively long intemodes, about 4-8 cm long
zone (apigenin-7-glucoside) and up to 1 cm in diameter; that of C. teeta is singular,
A brownish-yellow or orange distinctly hook-shaped and narrow.
fluorescent zone or a faint to very
Fragmented drug The rhizome is fragmented in transverse or
faint brownish-yellow or orange
fluorescent zone longitudinal slices, 1.2-4.2 cm long, up to 1.1 cm in diameter
and up to 4 mm thick. The slices show reddish-orange, dark
A light blue fluorescent zone or a
faint light blue fluorescent zone reddish-brown or dark brown bark, with bright yellow or
orange-yellow wood. The wood is radially arranged in
fluorescent zone transverse section and clefts may be present. The pith is
often of similar colour to the bark and sometimes hollowed.
- - -- The outer surface is yellowish-grey or yellowish-brown,
Reference solution (a) Test solution rough, with irregular nodular protuberances; rootlet remains
are sometimes present. The slices are easily broken.
B. Microscopic examination (2.8.23). The powder is orange
TESTS or orange-brown. Examine under a microscope using chloral
Diameter of the flower-heads hydrate solution R. The powder shows the following diagnostic
Maximum 3 per cent of flower-heads have a diameter smaller characters (Figure 2715.-1): yellowish-brown or reddish-
than 8 mm. brown fragments of cork (surface view [B], transverse section
Deteriorated flower-heads [FJ); groups of long, often narrow-elliptical, slightly lignified
Brown or darkened flower-heads are absent. pericyclic fibres with thick channelled walls [C] and a narrow
Loss on drying (2.2.32) lumen, fibres up to 26 µm in diameter; numerous sclereids
Maximum 11.0 per cent, determined on 1.000 g of the (C. chinensis or C. deltoidea) isolated [D, TT or in groups [E],
powdered herbal drug (355) (2. 9.12) by drying in an oven at or also included in parenchyma; sclereids, up to 90 µm in
105 °C for 2 h. diameter, with thick, yellow lignified walls with pits, channels
and striations, cell lumen sometimes with reddish-brown
Total ash (2.4.16) contents; occasional xylem bundles [G]; groups of lignified
Maximum 8.0 per cent. xylem fibres [A] with slightly thickened walls, 7-28 µm in
ASSAY diameter; the remains of leaves [H] showing an epidermis
Essential oil (2. 8.12) consisting of cells with beaded walls [Ha] and parenchyma
Use 20.0 g of whole herbal drug, a 500 mL round-bottomed cells with smooth walls [Hb].
flask, 250 mL of water R as the distillation liquid and C. Thin-layer chromatography (2.2.27).
0.50 mL of xylene R in the graduated tube. Distil at a rate of Test solution To 0.5 g of the powdered herbal drug (355)
3-3.5 mUmin for 3 h. (2. 9.12) add 5 mL of methanol R. Sonicate for 15 min.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Centrifuge the solution and use the supernatant.
Reference solution Dissolve 5 mg of berberine chloride R and
5 mg of palmatine R in methanol R and dilute to 100 mL with
the same solvent.
Chinese Goldthread Rhizome
(Ph. Bur. monograph 2715) Mobile phase water R, methanol R, 2-propanol R, ethyl
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ acetate R, toluene R (1:5:5:10:20 VIVIVIVIV).
DEFINITION
Whole or fragmented, dried rhizome of Coptis chinensis
Development In a twin trough tank previously saturated for
20 min without filter paper, over a path of 6 cm.
Franch., Coptis delwidea C.Y. Cheng & P.K. Hsiao and/or
Coptis teeta Wall., with roots removed, collected in autumn. For saturation, add mobile phase to one trough and
concentrated ammonia R to the other.
Content
Minimum 5.0 per cent of berberine (C 20H 18NO 4 T; Drying In a current of cold air for 10 min.
Mr 336.4) (dried drug). Detection Examine in ultraviolet light at 365 nm.
IV-176 Chinese Goldthread Rhizome 2023

ASSAY
(355) (2.9.12) in 100.0 mL ofa mixture of hydrochloric
acid Rand methanol R (1:100 V!V). Sonicate for 20 min.
Filter 1.5 mL of the solution through a membrane filter
Reference solution (a) Dissolve 10.0 mg of berberine
chloride CRS in a mixture of hydrochloric acid R and
methanol R (1:100 VIV) and dilute to 100.0 mL with the
same mixture.
Reference solution (b) Dissolve 1 mg of palmatine R in
reference solution (a) and dilute to 10 mL with reference
solution (a).
Column:
- size: l= 0.25 m, 0 = 4 mm;
- stationary phase: end-capped octadecylsi"/yl silica gel for
chromatography Rl (5 µm).
Mobile phase:
- mobile phase A: 0.5 per cent V!V solution of phosphoric
acid R;
- mobile phase B: 0.5 per cent V/V solution of phosphoric
acid R in acetonitrile R;

Figure 2715.-1. - Illustration for identification test B of powdered (min) (per cent V/J/) (per cent V/J/)
herbal drug of chinese goldthread rhizome 0 - JO 80 20
10 - 20 80-, 79 20 -, 21
20 - 35 79--, 65 21-> 35
35 - 40 65 35
and the test solution. Furthermore, other fluorescent zones
solution. Flow rate 0.7 mUmin.
Top of the plate Injection 10 µL.
Retention time Palmatine = about 25 min;
berberine = about 28 min.
A yellow fluoresceot zone (may be - resolution: minimum 1.5 between the peaks due to
missing) palmatine and berberine.
-- -- Calculate the percentage content of berberine using the
Berberine: a greenish-yellow A greenish-yellow fluorescent zone
fluorescent zone (berberine)
A1Xmzxp
Palmatine: a greenish-yellow A greenish-yellow fluorescent zone
fluorescent zone (palmatine)
A2 xm1
AI area of the peak due to berberine in the chromatogram obtained
-- -- with the test solution;
A2 area of the peak due to berberine in the chromatogram obtained
Reference solution Test solution m1 mass of the herbal drug to be examined used to prepare the test
solution, in grams;
mass of berberine chloride CRS used to prepare reference
TESTS solution (a), in grams;
p percentage content of berberine in berberine chloride CRS.
Maximum 5 per cent of fragments of roots and leaves and
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
maximum 3 per cent of other foreign matter.
105 °C for 2 h.
Total ash (2.4.16)
2023 Chinese Motherwort IV-1 77
walls and rounded tips, often associated with the vasculature

Chinese Motherwort of the calyx; pollen grains, sub-rounded to sub-ovate, about
(Ph. Bur. monograph 2785) 25 µm in diameter, with 3 pores and a smooth exine [BJ;
rare fragments of nutlets consisting of cells with thick,
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
strongly lobed walls, each containing a single prism crystal of
DEFINITION calcium oxalate [J].
Whole or cut, dried aerial parts of Leonurus japonicus Houtt.,
harvested during the flowering season.
Content
(C2,H20O,2; Mr 464.4) (dried drug).
IDENTIFICATION
A. Fragments of stems, externally greenish-grey or greenish-
yellow, square in transverse section and furrowed
longitudinally on 4 sides, 2-10 mm in diameter (more rarely
up to 20 mm), with a large, whitish, spongy central pith.
Leaves are opposite, mostly fragmented and of 2 types: stem
leaves that are deeply lobed and floral leaves that are long,
narrow and unlobed. Leaf fragments with a pubescent
lamina, dark green on the adaxial surface and paler green,
with prominent veins, on the abaxial surface, some fragments
include part of the petiole with a decurrent lamina.
Verticillaster inflorescence with 5-15 sessile axial flowers.
Flowers with a tubular, campanulate calyx, pubescent, with 5
triangular lobes, each ending in a spiny tip; corolla
gamopetalous, 2-lipped, reddish-brown or purple, hairy and
about 1 cm long; fruit is split into 4 brownish nutlets,
oblong, trigonal, with a truncate apex, about 2.5 mm long.
green to brownish-green. Examine under a microscope using
diagnostic characters (Figure 2785.-1): stem, leaf and flower
fragments with abundant covering trichomes and glandular
trichomes (covering trichomes 1-4-celled, mostly thick-walled
with fine warty protuberances [Cb, Ef, F, Kc]; glandular
trichomes (with a unicellular stalk and a multicellular head,
either tetracellular [Cc, Ed, Kb] or of the lamiaceous type, Figure 2785.-1. - Illustration for identification test B of p<nVdered
with a 4, 6 or 8-celled head and a swollen cuticle [De, Eel); herbal drug of Chinese motherwort
fragments of the abaxial epidermis of the lamina [El with
wavy-walled cells [Ea], abundant diacytic stomata [Eb] and C. High-performance thin-layer chromatography (2.8.25).
anomocytic [Ee] stomata (2.8.3) and numerous, mostly 2-4- Test solution To 1.0 g of the powdered herbal drug (355)
celled, covering trichomes [Ef] and glandular trichomes [Cc, (2.9.12) add 10.0 mL of methanol R. Sonicate for 10 min,
Ed, Kb; lamiaceous type: De, Ee]; fragments of the adaxial centrifuge and use the supernatant.
epidermis of the lamina [C] consisting of cells with sinuous Reference solution (a) Dissolve 1.0 mg of hyperoside R,
walls [Ca], numerous, mostly unicellular, curved covering 1.0 mg of isoquercitrin R and 1.0 mg of rutoside trihydrate R in
trichomes [Cb] or their scars [Ce] and glandular trichomes; methanol Rand dilute to 10.0 mL with the same solvent.
fragments of leaf palisade parenchyma with small rounded Reference solution (b) Dilute 2.5 mL of reference solution (a)
cells (surface view) containing numerous small needles of to 10.0 mL with methanol R.
calcium oxalate [Cd]; epidermal fragments of the stems or
Intensity marker (reference solutions (a) and (b)) Rutoside.
veins of the leaves [Kl, with elongated cells [Ka], curved,
multicellular covering trichomes [Kc] and glandular Plate TLC silica gel F254 plate R (2-10 µm).
trichomes; small groups of stem pericyclic fibres, rigid, with Mobile phase formic acid R, water R, methyl ethyl ketone R,
thick, sparsely pitted walls [G]; large bordered-pitted or ethyl acetate R (10:10:30:50 V/V/V/V).
spirally thickened xylem vessels from the stems; pith Application 10 µL, as bands of 8 mm.
parenchyma colourless and large-celled, some cells with Development 70 mm from the lower edge of the plate.
irregularly-pitted walls; fragments of the corolla containing
Detection Heat at 100 °C for 3 min; spray the warm plate
minute clusters of calcium oxalate; the outer epidermis [D]
with a 5 g/L solution of diphenylboric acid aminoethyl ester R in
with polygonal cells [Da] and mostly bicellular, relatively
ethyl acetate R, then with a 50 g/L solution of macrogol 400 R
thin-walled covering trichomes, with a short basal cell and a
in methylene chloride R; examine in ultraviolet light at 366 nm.
very elongated, sinuous, smooth-walled distal cell [Db] plus
glandular trichomes; some fragments of the inner epidermis System suitability Reference solution (a):
of the corolla with small papillose cells [H]; fragments of the - the chromatogram shows an orange fluorescent zone
epidermis of the calyx [A] with polyhedral cells [Aa], and due to rutoside in the lower half and 2 distinct zones
glandular trichomes and covering trichomes [Ab] or their in the middle third; the lower zone (hyperoside) and,
scars [Ac]; groups of short parenchyma fibres [Ad] with thick directly above it, the upper zone (isoquercitrin) show
an orange fluorescence.
IV-178 Cinchona Bark 2023
Results See below the sequence of fluorescent zones present Test solution To 10.0 mL of the stock solution add 1 mL of
in the chromatograms obtained with reference solution (a) aluminium chloride reagent Rand dilute to 25.0 mL with a
and the test solution. Furthermore, in the chromatogram 5 per cent V/V solution of glacial acetic acid R in methanol R.
obtained with the test solution, other faint zones may be Compensation liquid Dilute 10.0 mL of the stock solution to
present. 25.0 mL with a 5 per cent V/V solution of glacial acetic
Top of the plate Measure the absorbance (2. 2. 25) of the test solution after
2 red zones, intense 30 min, by comparison with the compensation liquid at
425 nm. Calculate the percentage content of flavonoids,
expressed as hyperoside, using the following expression:
Ax 1.25
-- -- m
Isoquercitrin: an orange zone 2 or 3 overlapping orange or bluish
zones, faint to equivalent
i.e. taking the specific absorbance of hyperoside to be 500.
Hyperoside: an orange zone An orange zone, faint to equivalent
A = absorbance at 425 nm;
(hyperoside), may be absent
A blue zone, faint to equivalent
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
_ _ Rutoside: an orange zone _ _ An orange zone, faint to

equivalent (rutoside)
A blue zone, faint to equivalent, may
be absent
Cinchona Bark
Cinchona
Red Cinchona Bark
Preparation
Cinchona Liquid Extract, Standardised
When Powdered Cinchona is prescribed or demanded,
TESTS material complying with the requirements below with the
Foreign matter (2.8.2) exception of Identification test A and the test for Foreign
Maximum 5 per cent. matter shall be dispensed or supplied.
Loss on drying (2.2.32) PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Whole or cut, dried bark of Cinchona pubescens Vah!
105 °C for 2 h.
(Cinchona succirubra Pav.), of Cinchona calisaya Wedd., of
Total ash (2.4.16) Cinchona ledgeriana Moens ex Trimen, or of their varieties or
Maximum 11.0 per cent. hybrids.
ASSAY Content
Stock solution In a 100 mL round-bottomed flask place Minimum 6.5 per cent of total alkaloids, of which
1.00 g of the powdered herbal drug (355) (2.9.12), add 30 per cent to 60 per cent consists of quinine-type alkaloids
1 mL of a 5 g/L solution of hexamethylenetetramine R, 2 mL (dried drug).
of hydrochloric acid Rl and 20 mL of acetone R. Boil the
CHARACTERS
mixture under a reflux condenser for 30 min. Filter the
Intense bitter, somewhat astringent taste.
liquid through a plug of absorbent cotton into a flask.
Add the absorbent cotton to the residue in the round- IDENTIFICATION
bottomed flask and extract with 2 quantities, each of 20 mL, A. The stem and branch bark is supplied in quilled or curved
of acetone R, each time boiling under a reflux condenser for pieces 2-6 mm thick. The outer surface is dull brownish-grey
10 min. Allow. to cool and filter each extract through the or grey and frequently bears lichens; it is usually rough,
plug of absorbent cotton into the flask. After cooling, filter marked with transverse fissures and longitudinally furrowed
the combined acetone extracts through a paper filter into a or wrinkled; exfoliation of the outer surface occurs in some
volumetric flask and dilute to 100.0 mL with acetone R by varieties. The inner surface is striated and deep reddish-
rinsing the flask and the paper filter. Introduce 20.0 mL of brown; the fracture is short in the outer part and fibrous in
the solution into a separating funnel, add 20 mL of water R the inner part.
and shake the mixture with 1 quantity of 15 mL and then B. Microscopic examination (2.8.23). The powder is reddish-
3 quantities, each of 10 mL, of ethyl acetate R. Combine the brown. Examine under a microscope using chloral hydrate
ethyl acetate extracts in a separating funnel, wash with solution R. The powder shows the following diagnostic
2 quantities, each of 50 mL, of water R, filter the extracts characters (Figure 0 17 4. -1): thin-walled cork cells filled with
over 10 g of anhydrous sodium sulfate R into a volumetric flask reddish-brown contents (surface view [K], transverse section
and dilute to 50.0 mL with ethyl acetate R. [H]); yellow, spindle-shaped striated phloem fibres up to
90 µm in diameter and up to 1300 µm in length, very thick-
walled with an uneven lumen and with conspicuous, funnel-
shaped pits, whole [A] or fragmented [F, TI; parenchymatous
2023 Cinchona Bark IV-179
idioblasts filled with microprisms of calcium oxalate [E, G]; Top of the plate
clusters of thin-walled phloem parenchyma cells [L]
accompanied by medullacy rays (tangential section [DJ). -- --
Examine under a microscope using a 50 per cent V/V Quinidine: a distinct blue fluorescent A distinct blue fluorescent zone
solution of glycerol R. The powder shows a few starch zone (quinidine)
granules 6-10 µm in diameter, mostly simple but occasionally
-- --
with 2 or 3 components, free [BJ or included in
parenchymatous cells [CJ. Quinine: a distinct blue fluorescent A distinct blue fluorescent zone
zone (quinine)
Detection B Spray with iodoplatinate reagent R.

test solution. Furthermore, other zones are present in the
Top of the plate
-- --
Cinchonine: a violet zone that A violet zone that becomes violet-
becomes violet-grey grey (cinchonine)
Quinidine: a violet zone that A violet zone that becomes violet-
becomes violet-grey grey (quinidine)
Cinchonidine: an intense dark blue An intense dark blue zone
zone (cinchonidine)
-- --
Quinine: a violet zone that becomes A violet zone that becomes violet-
violet-grey grey (quinine)
TESTS
Total ash (2.4.16)
Maximum 10 per cent, determined on 1.000 g of the
Figure O17 4.-1. - Illustrati.on for identificati.on test B of powdered 105 °C for 2 h.
herbal drug of cinchona bark ASSAY
Test soluti.on In a 250 mL conical flask mix 1.000 g of the
powdered herbal drug (180) (2.9.12) with 10 mL of water R
Test soluti.on To 0.10 g of the powdered herbal drug (180) and 7 mL of d11ute hydrochloric acid R. Heat in a water-bath
(2. 9.12) in a test-tube add 0.1 mL of concentrated ammonia R for 30 min, allow to cool and add 25 mL of methylene
and 5 mL of methylene chloride R. Shake vigorously chloride R, 50 mL of ether R and 5 mL of a 200 g/L solution
occasionally during 30 min and filter. Evaporate the filtrate of sodium hydroxide R. Shake the mixture repeatedly for
to dryness on a water-bath and dissolve the residue in 1 mL 30 min, add 3 g of powdered tragacanth R and shake until
of anhydrous ethanol R. the mixture becomes clear. Filter through a plug of absorbent
Reference soluti.on Dissolve 17.5 mg of quinine R, 2.5 mg of cotton and rinse the flask and the cotton with 5 quantities,
quinidine R, 10 mg of cinchonine R and 10 mg of each of 20 mL, of a mixture of 1 volume of methylene
cinchonidine R in 5 mL of anhydrous ethanol R. chloride R and 2 volumes of ether R. Combine the filtrate and
Plate TLC silica gel plate R. washings, evaporate to dryness and dissolve the residue in
Mobile phase diethylamine R, ethyl acetate R, toluene R 10.0 mL of anhydrous ethanol R. Evaporate 5.0 mL of this
(10:20:70 VIVIV). solution to dryness, dissolve the residue in 0.1 M hydrochloric
acid and dilute to 1000.0 mL with the same acid.
Applicati.on 10 µL as bands.
Reference soluti.ons Dissolve separately 30.0 mg of quinine R
Development Twice over a path of 15 cm.
and 30.0 mg of cinchonine R in 0.1 M hydrochloric aci,d and
Drying At 100-105 °C, then allow to cool. dilute each solution to 1000.0 mL with the same acid.
Detection A Spray with anhydrous formic acid R and allow to Measure the absorbances (2. 2. 25) of the 3 solutions at
dry in air; examine in ultraviolet light at 365 nm. 316 nm and 348 nm using 0.1 M hydrochloric acid as the
Results A See below the sequence of zones present in the compensation liquid.
chromatograms obtained with the reference solution and the Calculate the percentage content of alkaloids using the
test solution. Furthermore, other fluorescent zones are following equations:
IV-180 Cinchona Preparations 2023
Drying At 100-105 °C then allow to cool.

Detection A Spray with a 50 g/L solution of anhydrous formic
acid R and allow to dry in air; examine in ultraviolet light at
365 nm.
Results A See below the sequence of the zones present in
m mass of the herbal drug used, in grams; the test solution. Furthermore, other fluorescent zones may
x percentage content of quinine-type alkaloids; be present in the chromatogram obtained with the test
y percentage content of cinchonine-type alkaloids;
solution.
A 316 absorbance of the test solution at 316 nm;
A 348 absorbance of the test solution at 348 nm;
A 316 c absorbance of the reference solution containing cinchonine at Top of the plate
3 I 6 nm, corrected to a concentration of I mg/ 1000 mL;
A 316q absorbance of the reference solution containing quinine at -- ---
316 nm, corrected to a concentration of I mg/ I 000 mL;
A 348 c absorbance of the reference solution containing cinchonine at Quinidine: a distinct blue fluorescent A distinct blue fluorescent zone
348 nm, corrected to a concentration of I mg/ 1000 mL; zone (quinidine)
A 348 q absorbance of the reference solution containing quinine at
348 nm, corrected to a concentration of I mg/ 1000 mL. --- ---
Quinine: a distinct blue fluorescent A distinct blue fluorescent zone

Calculate the content of total alkaloids (x + y), and calculate zone (quinine)
the relative content of quinine-type alkaloids using the
l00x Detection B Spray with iodoplatinate reagent R.
x+y
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur the chromatograms obtained with the reference solution and
the test solution. Furthermore, other zones may be present in
the chromatogram obtained with the test solution.
Standardised Cinchona Liquid Top of the plate
Extract --- ---

Cinchonine: a violet-grey zone A violet-grey zone (cinchonine)
Quinidine: a violet-grey zone A violet-grey zone (quinidine)
Ph Eur - - - - - - - - - - - - - - - - - - - - - - -
DEFINITION Cinchonidine: an intense dark blue An intense dark blue zone

zone (cinchonidine)
Liquid extract produced from Cinchona bark (0174).
Content --- ---
Minimum 4.0 per cent and maximum 5.0 per cent of total Quinine: a violet-grey zone A violet-grey zone (quinine)
alkaloids, of which 30 per cent to 60 per cent are alkaloids of
the quinine type (C 20H 24N202; Mr 324.4).
PRODUCTION
Standardised cinchona liquid extract is produced from the TESTS
herbal drug by an appropriate procedure using: Ethanol (2. 9.10)
- ethanol (30 per cent V/V to 90 per cent VIV), or; 95 per cent to 105 per cent of the content stated on the
- a mixture of diluted hydrochloric acid, ethanol label.
(96 per cent VIV), glycerol, water (1:2:5:20 V/V). Methanol and 2-propanol (2. 9.11)
CHARACTERS Maximum 0.05 per cent V/V of methanol and maximum
Appearance 0.05 per cent V/V of 2-propanol.
Brownish-red liquid. Dry residue (2.8.16)
It has a bitter, astringent taste. Minimum 12.0 per cent for glycerol-free standardised
cinchona liquid extract and minimum 30.0 per cent for
IDENTIFICATION
glycerol-containing standardised cinchona extract, determined
Thin-layer chromatography (2.2.27). on 2.0 g.
Test solution Dilute 1 mL of the extract to be examined in
ASSAY
1 mL of anhydrous ethanol R.
Test solution In a 250 mL conical flask, mix about 1.000 g
Reference solution Dissolve 2.5 mg of quinidine R, 10 mg of
of the extract to be examined with 10 mL of water R and
cinchonidine R, 10 mg of cinchonine R and 17.5 mg of
7 mL of dilute hydrochloric acid R. Heat in a water-bath for
quinine R in 5 mL of anhydrous ethanol R.
30 min, allow to cool and add 25 mL of methylene chloride R,
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel 50 mL of ether R and 5 mL of a 200 g/L solution of sodium
plate R (2-10 µm)]. hydroxide R. Shake the mixture frequently for 30 min, add
Mobile phase diethylamine R, ethyl acetate R, toluene R 3 g of powdered tragacanth R and shake until the mixture
(10:20:70 V/V/V). becomes clear. Filter through a plug of absorbent cotton,
Application 10 µL [or 2 µL] as bands. rinse the flask and the cotton with 5 quantities, each of
Development Twice over a path of 15 cm [or 6 cm]. 20 mL, of a mixture of 1 volume of methylene chloride R and
2023 Cinnamon IV-181
2 volumes of ether R. Combine the filtrate and washings, Content

evaporate to dryness and dissolve the residue in 10.0 mL of Minimum 12 mUkg of essential oil.
ethanol (96 per cent) R. Evaporate 5.0 mL of this solution to
CHARACTERS
dryness, dissolve the residue in 0.1 M hydrochloric acid and
Characteristic, aromatic odour.
dilute to 1000.0 mL with the same acid.
Reference solutwn (a) Dissolve 30.0 mg of cinchonine R in IDENTIFICATION
0.1 M hydrochloric acid and dilute to 1000.0 mL with the A. The bark is about 0.2-0.8 mm thick and occurs in closely
same acid. packed compound quills made up of single or double quills.
The outer surface is smooth, yellowish-brown with faint scars
Reference solutwn (b) Dissolve 30.0 mg of quinine R in
marking the position of leaves and axillary buds and has fine,
0.1 i\-1 hydrochloric acid and dilute to 1000. 0 mL with the
whitish and wavy longitudinal striations. The inner surface is
same acid.
slightly darker and longitudinally striated. The fracture is
Measure the absorbances (2. 2. 25) of the 3 solutions at short and fibrous.
316 nm and 348 nm, using 0.1 M hydrochloric acid as the
compensation liquid.
yellowish or reddish-brown. Examine under a microscope
Calculate the percentage content of alkaloids from the using chloral hydrate solution R. The powder shows the
following equations: following diagnostic characters (Figure 0387.-1): rounded
sclereids with pitted, channelled and moderately thickened
[A1 xA2a] - [A1a xA2] 100 2
n1 = --------- X - X -- walls, single [E, FJ or in groups [CJ; numerous colourless,
[Atb xA2a] - [A1a xA2b] m 1000 single fibres, often whole [AJ, or fragmented [DJ, with a
narrow lumen, thickened, lignified walls and few pits; small
acicular crystals of calcium oxalate in parenchymatous
cells m; very numerous oil droplets [BJ. Cork fragments [GJ
are absent or very rare. Examine under a microscope using a
50 per cent V/V solution of glycerol R. The powder shows
m mass of the liquid extract to be examined in grams;
n1 percentage content of quinine-type alkaloids; abundant starch granules [HJ.
n2 percentage content of cinchonine-type alkaloids;
A1 absorbance of the test solution at 316 nm;
A2 absorbance of the test solution at 348 nm;
A,a absorbance of reference solution (a) at 316 nm, corrected to a
concentration of 1 mg/1000 mL;
A" absorbance ofreference solution (b) at 316 nm, corrected to a
concentration of 1 mg/1000 mL;
A 2a absorbance of reference solution (a) at 348 nm, corrected to a
concentration of I mg/1000 mL;
A 2, absorbance of reference solution (b) at 348 nm, corrected to a
concentration of I mg/1000 mL.
Calculate the content of total alkaloids (n 1 + n2), and the

relative content of quinine-type alkaloids, from the following
expression:
LABELLING
The label states the solvent composition used for the
production.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Cinnamon
Cinnamon Bark
Ceylon Cinnamon
When Powdered Cinnamon is prescribed or demanded, J
exception of Identification test A and containing not less than
1.0% v/w (10 mUkg) of essential oil shall be dispensed or Figure 0387.-1. - Illustratwnfor identification test B of powdered
supplied. herbal drug of cinnamon
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION Test solutwn 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.
IV-182 Ceylon Cinnamon Bark Oil 2023
Reference solution Dissolve 50 µL of cinnamic aldehyde R and Reference solution Dissolve 50 µL of trans-cinnamic
10 µL of eugenol R in toluene R and dilute to 10 mL with the aldehyde R, 10 µL of eugenol R, 10 µL of linalol R and 10 µL
same solvent. of /3-caryophyllene R in ethanol (96 per cent) R and dilute to
Plate TLC silica gel GF2s4 plate R. 10 mL with the same solvent.
Mobile phase methylene chloride R. Plate TLC silica gel plate R.
Application 10 µL as bands of 20 mm by 3 mm. Mobile phase methanol R, toluene R (10:90 V/V).
Development Over a path of 10 cm. Application 10 µL as bands.
Drying In air. Development Over a path of 15 cm.
Detection A Examine in ultraviolet light at 254 nm and Drying In air.
mark the quenching zones, then examine in ultraviolet light Detection Spray with anisaldehyde solution R; heat at
at 365 nm and mark the fluorescent zones. 100-105 °C for 5-10 min and examine in daylight.
Results A Examined in ultraviolet light at 254 nm, the Results The zones in the chromatogram obtained with the
chromatograms obtained with the test solution and the test solution are similar in position and colour to those in the
reference solution show a quenching zone due to chromatogram obtained with the reference solution.
cinnamaldehyde in the median part and, just above it, a B. Examine the chromatograms obtained in the test for
weaker quenching zone due to eugenol; examined in chromatographic profile.
ultraviolet light at 365 nm, the chromatogram obtained with Results The principal peaks in the chromatogram obtained
the test solution shows a fluorescent light blue zone due to with the test solution are similar in retention time to those in
o-methoxycinnamaldehyde just below the zone due to the chromatogram obtained with the reference solution.
cinnamaldehyde. Safrole, coumarin and cineole may be absent from the
Detection B Spray with phloroglucinol solution R. chromatogram obtained with the test solution.
Results B The zone due to cinnamaldehyde is yellowish- TESTS
brown and the zone due to o-methoxycinnamaldehyde is
Relative density (2.2.5)
violet.
1.000 to 1.030.
TESTS Refractive index (2.2.6)
Total ash (2.4.16) 1.572 to 1.591.
Optical rotation (2.2. 7)
ASSAY -2° to+ 1°.
Essential oil (2.8.12) Chromatographic profile
Use 20.0 g of drug reduced to a powder (710) (2. 9.12) Gas chromatography (2.2.28): use the normalisation
immediately before the determination, a 500 mL flask, procedure.
200 mL of 0.1 M hydrochloric acid as the distillation liquid,
and 0.50 mL of xylene R in the graduated tube. Distil at a Test solution The essential oil to be examined.
rate of 2.5-3.5 mlJmin for 3 h. Reference solution Dissolve 10 µL of cineole R, 10 µL of
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur linalol R, 10 µL of /3-caryophyllene R, 10 µL of safrole R,
100 µL of trans-cinnamic aldehyde R, l O µL of eugenol R,
20 mg of coumarin R, 10 µL of trans-2-
methoxycinnamaldehyde R and 10 µL of benzyl benzoate R in
1 mL of acetone R.
Ceylon Cinnamon Bark Oil Column:
Cinnamon Oil - material: fused silica;
= =
- size: l 60 m, 0 0.25 mm;
- stationary phase: macrogol 20 000 R.
Preparation Carrier gas helium for chromatography R.
Concentrated Cinnamon Water
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Flow rate 1.5 mlJmin.
Split ratio 1: 100.
DEFINITION Temperature:
Essential oil obtained by steam distillation of the bark of the
shoots of Cinnamomum verum J.Presl. Time Temperature
(min) CC)
CHARACTERS Column 0 - 10 60
Appearance 10 - 75 60 ➔ 190
Clear, mobile, light yellow liquid becoming reddish over 75 - 200 190
time. Injection port 200
Characteristic odour reminiscent of cinnamic aldehyde. Detector 240
IDENTIFICATION
First identification: B. Detection Flame ionisation.
Second identification: A. Injection 0.2 µL.
A. Thin-layer chromatography (2.2.27). Elution order Order indicated in the composition of the
Test solution Dissolve 1 mL of the essential oil to be reference solution; depending on the operating conditions
examined in acetone R and dilute to 10 mL with the same and the state of the column, coumarin may elute before or
solvent. after trans-2-methoxycinnamaldehyde; record the
retention times of these substances.
2023 Ceylon Cinnamon Leaf Oil IV-183
System suitability Reference solution: A. Thin-layer chromatography (2.2.27).

- resolution: minimum 1.5 between the peaks due to linalol Test solutwn Dilute 1 mL of the substance to be examined
and ~-caryophyllene. in acetone R and dilute to 10 mL with the same solvent.
Identification of components Using the retention times Reference solutwn Dilute about 50 µL of trans-cinnamic
determined from the chromatogram obtained with the aldehyde R, 10 µL of eugenol R, 10 µL of linalol R and 10 µL
reference solution, locate the components of the reference of fJ-caryophyllene R in alcohol R and dilute to 10 mL with the
solution in the chromatogram obtained with the test solution. same solvent.
Determine the percentage content of each of these Plate TLC silica gel plate R.
components. The percentages are within the following Mobile phase methanol R, toluene R (10:90 V/V).
ranges:
- cineole: maximum 3.0 per cent; Applicatwn 10 µL, as bands.
- linalol: 1.0 per cent to 6.0 per cent; Development Over a path of 15 cm.
- fJ-caryophyllene: 1.0 per cent to 4.0 per cent; Drying In air.
- safrole: maximum 0.5 per cent; Detection Spray with anisaldehyde solution R. Examine in day
- trans-cinnamic aldehyde: 55 per cent to 75 per cent; light while heating at 100-105 °C for 5-10 min.
- eugenol: maximum 7.5 per cent; Results The zones in the chromatogram obtained with the
- coumarin: maximum 0.5 per cent; test solution are similar in position and colour to those in the
- trans-2-methoxycinnamaldehyde: 0.1 per cent to chromatogram obtained with the reference solution.
1.0 per cent; The zone due to trans-cinnamic aldehyde may be very faint
- benzyl benzoate: maximum 1.0 per cent.
or absent.
STORAGE B. Examine the chromatogram obtained in the test for
Protected from heat. chromatographic profile.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhE~
Results The characteristic peaks in the chromatogram
obtained with the test solution are similar in retention time to
those in the chromatogram obtained with the reference
Concentrated Cinnamon Water solution. The peaks corresponding to cineole, safrole, trans-
cinnamic aldehyde, cinnamyl acetate and coumarin may be
DEFINITION
absent in the chromatogram obtained with the test solution.
Cinnamon Oil 20mL TESTS
Ethanol (90 per cent) 600mL Relative density (2.2.5)
1.030 to 1.059.
Extemporaneous preparation Refractive index (2.2.6)

The following directions apply. 1.527 to 1.540.
Dissolve the Cinnamon Oil in the Ethanol (90 per cent) and Optical rotation (2. 2. 7)
add gradually, with vigorous shaking after each addition, -2.5° to + 2.0°.
sufficient Water to produce 1000 mL. Add 50 g of previously Chromatographic profile
sterilised Purified Talc, or other suitable filtering aid, allow to Gas chromatography (2.2.28): use the normalisation
stand for a few hours, shaking occasionally, and filter. procedure.
The water complies with the requirements stated under Aromatic Test solutwn The substance to be examined.
Waters and with the fol!O?Ving requirements. Reference solution Dissolve 10 µL of cineole R, 10 µL of
TESTS linalol R, 10 µL of fJ-caryophyllene R, 10 µL of safrole R,
Ethanol content 10 µL of trans-cinnamic aldehyde R, 10 µL of cinnamyl
52 to 56% v/v, Appendix VIII F. acetate R, 100 µL of eugenol R and 10 mg of coumarin R in
1 mL of acetone R.
Weight per mL
0.914 to 0.922 g, Appendix VG. Column:
=
- size: l 60 m, 0 =
0.25 mm,
Ceylon Cinnamon Leaf Oil Carrier gas helium for chromatography R.
(Ph. Bur. monograph 1608) Flow rate 1.5 mIJmin.
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Split ratw 1/100.
Temperature:
DEFINITION
Oil obtained by steam distillation of the leaves of Time Temperature
Cinnamomum verum J.S. Presl. (min) ('C)
CHARACTERS Column 0 - 10 45
10 - 78 45---> 180
Appearance 78 - 88 180
Clear, mobile, reddish-brown or dark brown liquid. Injection port 200
Characteristic odour reminiscent of eugenol. Detector 240
IDENTIFICATION
First identification: B. Detection Flame ionisation.
Second identificatwn: A.
IV-184 Citronella Oil 2023
Injection 0.2 µL. Top of the plate

Elution order The order indicated in the composition of the Citronella!: a violet zone A zone similar in colour to the
reference solution. Record the retention times of these citronella! zone
substances. An orange zone (citronellol-geraniol)
- resolution: minimum of 1.5 between the peaks due to Reference solution Test solution
linalol and ~-caryophyllene.

Using the retention times determined from the B. Examine the chromatograms obtained in the test for
chromatogram obtained with the reference solution, locate chromatographic profile.
the components of the reference solution in the Results The characteristic peaks in the chromatogram
chromatogram obtained with the test solution. obtained with the test solution are similar in retention time to
Determine the percentage content of these components. those in the chromatogram obtained with the reference
The percentages are within the following ranges: solution. Neral and geranial may be absent in the
- cineole: maximum 1.0 per cent, chromatogram obtained with the test solution.
- linalol: 1.5 per cent to 3.5 per cent, TESTS
- /J-caryophyllene: 1.5 per cent to 7.0 per cent, Relative density (2.2.5)
- safrole: maximum 3.0 per cent, 0.881 to 0.895.
- trans-cinnamic aldehyde: maximum 3.0 per cent,
- cinnamyl acetate: maximum 2.0 per cent,
1.463 to 1.475.
- eugenol: 70 per cent to 85 per cent,
- coumarin: maximum 1.0 per cent. Optical rotation (2.2. 7)
-4° to+ 1.5°.
STORAGE
Protected from heat. Chromatographic profile
Gas chromatography (2.2.28): use the normalisation
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
procedure.
Reference solutwn Dilute 25 µL of limonene R, 100 µL of
citronella! R, 25 µL of citronellyl acetate R, 25 µL of citral R,
Citronella Oil 25 µL of geranyl acetate R, 25 µL of citronellol R and 100 µL
(Ph. Bur. monograph 1609) of geraniol R in 5 mL of hexane R.
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Column:
DEFINITION =
- size: l 60 m, 0 0.25 mm, =
Oil obtained by steam distillation from the fresh or partially - statwnary phase: macrogol 20 000 R (0.2 µm).
dried aerial parts of Cymbopogon winterianus Jowitt. Carrier gas helium for chromatography R.
CHARACTERS Flow rate 1.0 mUmin.
Appearance Split ratw 1:100.
Pale yellow or brown-yellow liquid. Temperature:
Very strong odour of citronellal.
Time Temperature
IDENTIFICATION
(min) CC)
First identijicatwn: B.
Column 0-2 80
Second identijicatwn: A. 2 - 26 80-, 150
A. Thin-layer chromatography (2.2.27). 26 - 42 150--> 185
Test solutwn Dilute 0.1 g of citronella oil in 10.0 mL of 42 - 49 185 --> 250
alcohol R. Injection port 260
Reference solutwn Dilute 20 µL of citronella! R in 10.0 mL of Detector 260
alcohol R.
Plate TLC silica gel plate R. Detection Flame ionisation.
Mobile phase ethyl acetate R, toluene R (10:90 V/V). Injection 1 µL of the reference solution, 0.2 µL of the test
Applicatwn 5 µL, as bands. solution.
Development Over a path of 15 cm. Elution order The order indicated in the composition of the
reference solution. Record the retention times of these
Drying In air. substances.
Detection Spray with anisaldehyde solution R and heat at System suitability Reference solution:
100-105 °C for 10 min. Examine in ultraviolet light at - resolution: minimum of 1.2 between the peaks due to
365 nm. geranyl acetate and citronellol.
Result See below the sequence of the zones present in the Using the retention times determined from the
chromatograms obtained with the reference and test chromatogram obtained with the reference solution, locate
solutions. Furthermore, other zones are present in the the components of the reference solution in the
chromatogram obtained with the test solution. chromatogram obtained with the test solution.
2023 Clematis Armandii Stem IV-185
Determine the percentage content of each of these lignified walls [H]; sub-rectangular or fusiform sclereids,
components. about 100 µm long and 35 µm wide, with thick and pitted
The percentages are within the following values: walls [C]; rare fragments of orange-brown cork covered by a
- limonene: 1.0 per cent to 5.0 per cent, cuticle showing striations [A). Examine under a microscope
- citronella!: 30.0 per cent to 45.0 per cent, using a 50 per cent VIV solution of glycerol R. The powder
- citronellyl acetate: 2.0 per cent to 4.0 per cent, shows rare starch granules, simple or 2-3 compound,
- neral: maximum 2.0 per cent, spherical or ovate; individual granules up to 17 µm in
- geranial: maximum 2.0 per cent, diameter, with a punctiform or slit-shaped hilum [D].
- geranyl acetate: 3.0 per cent to 8.0 per cent,
- citronella!: 9.0 per cent to 15.0 per cent,
- geraniol: 20.0 per cent to 25.0 per cent. --~
.-,:,,=-.=l~=--
-
--
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
- ~
Clematis Armandii Stem

~ ~~0 B

Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
'
DEFINITION
Whole or fragmented, dried stem of Clematis armandii
Franch., with cork removed, collected in spring or autumn.
Content
Minimum 0.30 per cent of oleanolic acid (C 30H 48 03;
Mr 456.7) (dried drug). F ,,
IDENTIFICATION
.1s!;
A. The whole stem is long and cylindrical, slightly twisted on
itself, about 1-6.5 cm in diameter. It shows nodes, usually
>!
swollen, with leaf and branch scars. The outer surface is Fa ;~--
brownish-yellow or dull brownish-yellow, showing \~'
longitudinal grooves and striations corresponding to the ends
of the medullary rays. Rare cork remnants are easily removed .:1:!1
as longitudinal strips. The texture is hard. The fracture is --~
- \ 25 µm ~--·-· ~~
difficult. >---i
j
The fragmented stem occurs in thick or thin slices, about
1-5 mm thick and 1.2-4.6 cm in diameter, with uneven Figure 2463.-1. - Illustration for identification test B of powdered
margins; most of the transverse section consists of the pale herbal drug of Clematis armandii stem
yellow or slightly brownish-yellow wood and shows numerous
radial striations and cracks corresponding to the medullary C. Thin-layer chromatography (2.2.27).
rays; the vessels are clearly visible in transverse section Test solution To 1 g of the powdered herbal drug (1400)
arranged in groups in more or less discontinuous concentric (2.9.12) add 5 mL of methanol Rand heat on a water-bath at
rings. Narrow, convex-shaped bark remnants are sometimes 60 °C for 5 min. Filter.
present with an enclosed lacuna between the bark and wood Reference solution Dissolve 4 mg of hederagenin R and 4 mg
parts. The bark is easily removed. The pale yellow or whitish, of oleanolic acid R in 10 mL of methanol R.
rarely blackened, reduced pith, is sometimes replaced by a
hollow.
F2s 4 plate R (2-10 µm)].
B. Microscopic examination (2.8.23). The powder is whitish-
Mobile phase acetic acid R, acetone R, toluene R
yellow to brownish-yellow. Examine under a microscope
(2:8:32 V/V/V).
following diagnostic characters (Figure 2463.-1): very Application 40 µL [or 10 µL] as bands of 10 mm [or
numerous fragments of pitted vessels [B, G] up to 250 µm in 8 mm].
diameter, isolated or associated with elongated tracheids Development Over a path of 13 cm [or 6 cm] .
about 15-25 µm in diameter with lignified, thickened and Drying In air.
pitted walls [Ba], some groups of vessels [F] show oblique Detection Treat with vanillin reagent R, heat at 100 °C for
striations [Fa], sometimes associated with xylem parenchyma 5 min and examine in daylight.
cells with lignified and pitted walls [Fb]; groups of fusiform
pericyclic fibres 25-30 µm in diameter, with thick, pitted
walls U]; thin-walled parenchymatous cells of the phloem and chromatograms obtained with the reference solution and the
test solution. Furthermore, other mainly grey zones may be
of outer parts of the medullary rays [E]; parenchymatous
cells of the secondary xylem and the inner parts of the
medullary rays and pith, with slightly thickened, pitted and
IV-186 Clivers 2023
Top of the plate System suitability Reference solution (b):

oleanolic acid and ursolic acid.
-- -- Calculate the percentage content of oleanolic acid using the
A bluish-violet zone following expression:
Oleanolic acid: a reddish-violet zone A reddish-violet zone
A weak light blue or grey zone
A, area of the peak due to oleanolic acid in the chromatogram

-- --
Hederagenin: a greenish-brown zone A2 area of the peak due to oleanolic acid in the chromatogram
An orange zone m, mass of the herbal drug to be examined used to prepare the test
solution, in grams;
Reference solution Test solution m2 mass of o/eanolic acid CRS used to prepare reference solution (a),
in grams;
p percentage content of oleanolic acid in o/eanolic acid CRS.
TESTS
Aristolochia manshuriemis Kom. and other species of - - - - - - - - - - - - - - - - - - - - - - PhEur
Aristolochia
Examine the powdered herbal drug (355) (2.9.12) under a
microscope using chloral hydrate solution R; no cluster crystals
are visible. Clivers
Aristolochic acids (2.8.21, Method A) Cleavers
DEFINITION
Maximum 12.0 per cent, determined on 1.000 g of the Clivers contains the whole herb, or parts of, Galium apari.ne
powdered herbal drug (1400) (2.9.12) by drying in an oven L. collected during the flowering and fruiting period.
at 105 °C for 2 h. IDENTIFICATION
Total ash (2.4.16) A. Stems, which comprise the bulk of the cut herb, are green
Maximum 3.0 per cent. to brown, branched at nodes, hollow, quadrangular, with a
distinct ridge at each corner and up to about 3 mm wide.
ASSAY Remains of leaf whorls may be visible and younger stems
Liquid chromatography (2.2.29). have stiff, downward- projecting bristles. Leaves are linear or
Test solution In a 100 mL flask, disperse 1. 00 g of the lanceolate, up to 6 cm long and 5 mm wide, arranged in
powdered herbal drug (355) (2. 9.12) in methanol R, add whorls of 6-8. The upper surface is dark green, with stiff
3 mL of 6 M hydrochloric acid R and dilute to 30 mL with hairs along the entire, revolute margins, and the lower
methanol R. Shake for 2 h. Filter, add to the filtrate 10 mL of surface pale with a matt, felted appearance and a prominent
water R by rinsing the flask and the filter, and extract with midrib. Flowers, if present, are small, white, about 3 mm in
3 quantities, each of 30 mL, of methylene chloride R. Combine diameter, consisting of 4 elliptical petals, either solitary or in
the methylene chloride extracts and evaporate to dryness. small cymes of 2-3. At the base of each flower is a pair of
Dissolve the residue in 10.0 mL of methanol R, shake and carpels, covered with stiff hooked hairs, and at the base of
filter through a membrane filter (nominal pore size 0.45 µm). each short peduncle are 1-4 secondary leafy bracts. The fruit
Reference solution (a) Dissolve 10.0 mg of oleanolic acid CRS is a purplish double achene, covered in stiff, hooked bristles.
in methanol Rand dilute to 20.0 mL with the same solvent. B. Reduce to a powder, Appendix XVIl A. Examine under a
Reference solution (b) Dissolve 5.0 mg of ursolic acid R in microscope using chloral hydrate solution. The powder shows
reference solution (a) and dilute to 10.0 mL with the same the following characteristics: fragments of stem dominate and
solution. are very abundant; thin-walled, elongated epidermal cells,
Column: layers of collenchyma from the ridges, parenchyma from the
=
- size: l 0.25 m, 0 =
4.6 mm; cortex, and lignified xylem vessels and tracheids.
- stationary phase: end-capped octadecylsilyl silica gel for The numerous unicellular covering trichomes are
chromatography R (5 µm); characteristic, 70-100 µm long, faintly striated longitudinally
- temperature: 30 °C. and tapering to an acute, hooked apex. Leaf fragments show
wavy-walled upper epidermal cells and more sinuous lower
Mobile phase 0.4 per cent VIV solution of acetic acid R,
epidermal cells, with paracytic stomata on both surfaces but
methanol R (15:85 V/V).
more numerous on the lower. Hooked unicellular trichomes,
Flow rate 1.0 mUmin. similar to those on the stem, occur near the margins and
Detection Spectrophotometer at 210 nm. midrib on the lower surface of the leaf and covering, usually
Injection 20 µL. non-hooked, trichomes occur randomly on the upper surface.
Run time 1.2 times the retention time of ursolic acid. Large idioblasts, up to 250 µm long and 30 µm wide,
containing bundles of needle crystals of calcium oxalate,
Retention time Oleanolic acid = about 21 min; ursolic
occur in the spongy mesophyll. Flower fragments infrequent,
acid = about 22 min.
outer epidermis of corolla thin-walled and faintly striated,
2023 Clivers IV-187
inner epidermis papillose, and calcium oxalate idioblasts Table 1: Visualisation under 366 nm
similar to those from the leaf. Pollen grains spherical,
Top of the plate
30-35 µm in diameter, with up to 8 pores and a faintly warty
exine. The fruit pericarp, if present, shows a dense mass of One or two equivalent
the characteristic hooked trichomes. to very faint red zones
C. Carry out the method for high performance thin-layer

chromatography, Appendix XI W, using the following -
solutions.
An orange to yellow
(1) To 0.5 g of the powdered herbal drug add 5.0 mL of zone (hyperoside)
methanol and mix with the aid of ultrasound for 15 minutes;
Centrifuge or filter and use the supernatant liquid or filtrate A blue zone A blue zone An intense to faint blue
(chlorogenic acid) (chlorogenic acid) zone (chlorogenic acid)
as the test solution.
(2) 0.020% w/v of chlorogenic acid, 0.025% w/v of rutin, and An orange to yellow A faint to very faint
0.05% w/v of fructose in methanol. zone (rutin) orange to yellow zone
(rutin)
(3) Dilute 1 volume of solution (2) to 4 volumes with -
methanol.
(4) 0.020% w/v of chlorogenic acid and 0.030% w/v of
hyperoside in methanol. An equivalent to very
CHROMATOGRAPHIC CONDITIONS faint orange to yellow
zone
(a) Use as the coating high performance silica gel F254 (Merck
silica gel 60 F 254 plates are suitable). Solution (4) Solution (2) and (3) Solution (1)
System Suitability Intensity marker Test solution
(c) Apply 5 µL of each solution, as 8 mm bands.
The chromatogram obtained with solution (1) in white light
shows an intense to faint blue zone above two intense to faint
(e) After removal of the plate, dry in a current of air. brown zones in the bottom third of the plate. Additional
(t) Heat the plate at 100° for 3 minutes, dip the warm plate bands to very faint zones may be present.
in a 0.5% w/v solution of 2-aminoethyl diphenylborinate in ethyl
acetate and examine under ultraviolet light (366 nm). Table 2: Visualisation under white light
(g) Dip the plate in anisaldehyde solution, heat at 100° for Top of the plate
3 minutes and examine under white light.
MOBILE PHASE
11 volumes of acetic acid, 11 volumes of formic acid,
26 volumes of water and 100 volumes of ethyl acetate. -
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with -
solution (4) shows two clearly separated zones under
366 nm. An intense to faint blue
zone
CONFIRMATION
A brown zone An intense to faint
The chromatogram obtained with solution (1) under 366 nm (fructose) brown zone (fructose)
shows one or two equivalent to very faint red zones at the
An intense to faint
top of the plate, an intense to faint blue zone above a faint to
brown zone
very faint orange to yellow zone in the middle third of the
plate, and an equivalent to very faint orange to yellow zone Solution ( 4) Solution (2) and (3) Solution (1)
at the bottom of the plate. Additional faint zones may be System Suitability Intensity marker Test solution
present.
There should be no yellow to orange zone above the band TESTS
corresponding to cholorgenic acid. This would indicate the Foreign matter
presence of Galium verum. Not more than 2.0%, Appendix XI D.
Acid-insoluble ash
ANNEX
This section is non-mandatory.
A DNA reference sequence for the identity of Galium aparine
is published in Supplementary Chapter VII D.
IV-188 Clove 2023
Clove Cb

When Powdered Clove is prescribed or demanded, material
complying with the requirements below with the exception of
Identification test A and the test for Foreign matter and
containing not less than 12.0% v/w (120 mUkg) of essential
oil shall be dispensed or supplied.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Ca
DEFINITION
Whole flower buds of Syzygium aromaticum (L.) Merr. et L.
M.Perry (syn. Eugenia caryophyllus (Spreng.) Bullock et S.G.
(\
Harrison) dried until they become reddish-brown. G
'' \ \
Content Fa ~\
Minimum 150 mUkg of essential oil. '\
IDENTIFICATION
.
'
A. The flower bud is reddish-brown and consists of a

quadrangular stalked portion, the hypanthium, 10-12 mm
long and 2-3 mm in diameter, surmounted by 4 divergent
'
J ·,{~.
,·.' . '
•
V
K
lobes of sepals which surround a globular head 4-6 mm in - ::::

diameter. A bilocular ovary containing numerous ovules is
situated in the upper part of the hypanthium. The head is ...
~
globular and dome-shaped, composed of 4 imbricated petals L
25 µm
that enclose numerous incurved stamens and a short, erect
style with a nectary disc at the base. The hypanthium exudes
essential oil when indented with the finger-nail.
Figure 0376.-1. - Illustratwn for identification test B of powdered
B. Microscopic examination (2.8.23). The powder is dark herbal drug of clove
brown. Examine under a microscope using chloral, hydrate
solutwn R. The powder shows the following diagnostic Development Twice, in an unsaturated tank over a path of
characters (Figure 0376.-1): fragments of the epidermis of 10 cm; allow the plate to stand for 5 min between the
the hypanthium (surface view [A]) consisting of polygonal 2 developments.
cells [Aa] and anomocytic stomata (2.8.3) [Ab]; fragments of Drying In air.
the hypanthium (surface view [DJ, transverse section W),
showing the epidermis [Da, Ja] covered by a thick Detectwn A Examine in ultraviolet light at 254 nm and
mark the quenching zones.
cuticle [Jb] and an underlying parenchyma [Db, Jc]
containing large oil glands [Dd, Jd]; numerous fragments of Results A In the chromatogram obtained with the test
the parenchyma of the hypanthium [C, Ca] with oil glands solution there is in the median part a quenching zone due to
[Cd] and with some cells containing one [Cb, De] or more eugenol similar in position to the quenching zone in the
cluster crystals of calcium oxalate [Cc]; short fibres occurring chromatogram obtained with the reference solution and there
singly or in small groups, with thickened, lignified and may be a weak quenching zone due to acetyleugenol just
slightly pitted walls [F] sometimes accompanied by below the zone due to eugenol.
parenchyma with some cells in rows containing cluster Detection B Spray with anisaldehyde solution R using 10 mL
crystals of calcium oxalate [Fa]; vascular bundles [G] for a plate 200 mm square and heat at 100-105 °C for
sometimes included in parenchyma [K]; fragments of the 5-10 min. Examine in daylight.
filaments of the stamens with an epidermis consisting of Results B The zones due to eugenol in the chromatograms
small, elongated cells covered by a finely striated cuticle [HJ; obtained with the test and reference solutions are strong
fragments of the endothecium of the stamens (surface view brownish-violet and the zone due to acetyleugenol in the
[L]); isolated sclereids from the peduncle [BJ; numerous chromatogram obtained with the test solution is faint violet-
triangular pollen grains about 20 µm in diameter with 3 blue. In the chromatogram obtained with the test solution
pores in the angles [E] . there are other coloured zones, particularly a faint red zone
C. Thin-layer chromatography (2.2.27). in the lower part and a reddish-violet zone due to
Test solutwn Shake 0.1 g of the powdered herbal drug (500) caryophyllene in the upper part.
(2. 9.12) with 2 mL of methylene chloride R for 15 min. Filter TESTS
and carefully evaporate the filtrate to dryness on a water- Foreign matter (2.8.2)
bath. Dissolve the residue in 2 mL of toluene R. Maximum 6 per cent of peduncles, petioles and fruits,
Reference solution Dissolve 20 µL of eugenol R in 2 mL of maximum 2 per cent of deteriorated cloves and maximum
toluene R. 0.5 per cent of other foreign matter.
Plate TLC silica gel GF254 plate R. Total ash (2.4.16)
Mobile phase toluene R. Maximum 7.0 per cent.
Application 10 µL of the reference solution and 20 µL of ASSAY
the test solution, as bands of 20 mm by 3 mm. Essential oil (2. 8.12)
Use a 250 mL flask, 100 mL of water Ras the distillation
liquid and 0.50 mL of xylene R in the graduated tube. Grind
2023 Clove Oil IV-189
5.0 g of the herbal drug with 5.0 g of diawmaceous earth R to 3 principal peaks in the chromatogram obtained with the
form a fine, homogeneous powder and proceed immediately reference solution.
with the determination using 4.0 g of the mixture. Distil at a
TESTS
rate of 2.5-3.5 mIJmin for 2 h.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur 1.030 to 1.063.
1.528 to 1.537.
Clove Oil -2° to 0°.
(Ph. Bur. monograph 1091) Fatty oils and resinified essential oils (2. 8. 7)
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ It complies with the test.
Solubility in alcohol (2. 8.10)
DEFINITION 1.0 mL is soluble in 2.0 mL and more of ethanol
Essential oil obtained by steam distillation from the dried (70 per cent VIV) R.
flower buds of Syzygium aromaticum (L.) Merr. et L.M.Perry
(syn. Eugenia caryophyllus (Spreng.) Bullock et S.G.Harrison). Chromatographic profile
CHARACTERS procedure.
Appearance Test solution Dissolve 0.2 g of the substance to be examined
Clear, yellow liquid, which becomes brown when exposed to in 10 g of hexane R.
air.
Reference solution Dissolve 7 mg of {3-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
Column:
oils.
IDENTIFICATION - size: l =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 mIJmin.
Test solution Dissolve 20 µL of the substance to be Split ratio 1:100.
examined in 2.0 mL of toluene R. Temperature:
Reference solution Dissolve 15 µL of eugenol R and 15 µL of
acetyleugenol R in 2.0 mL of toluene R. Time Temperature
(min) (C)
Plate TLC silica gel F 254 plate R.
Column 0-8 60
Mobile phase toluene R.
8 - 48 60 ➔ 180
Application 20 µL of the test solution and 15 µL of the 48 - 53 180
reference solution, as bands. Injection port 270
Development Twice in an unsaturated tank over a path of Detector 270
10 cm; allow to stand for 5 min between the 2 developments.
Drying In air. Detection Flame ionisation.
Detection A Examine in ultraviolet light at 254 nm and Injection 1.0 µL.
mark the quenching zones. Elution order Order indicated in the composition of the
Results A The chromatogram obtained with the test solution reference solution. Record the retention times of these
shows in the middle part a quenching zone (eugenol) that is substances.
similar in position to the quenching zone in the
chromatogram obtained with the reference solution; just - resolution: minimum 1.5 between the peaks due to eugenol
below, there is a weak quenching zone (acetyleugenol) that is
and acetyleugenol;
similar in position to the zone of acetyleugenol in the - number of theoretical plates: minimum 30 000, calculated
chromatogram obtained with the reference solution. for the peak due to ~-caryophyllene at 110 °C.
Detection B Spray with anisaldehyde solution R and examine Identification of components Using the retention times
in daylight while heating at 100-105 °C for 5-10 min.
determined from the chromatogram obtained with the
Results B The zone due to eugenol in the chromatograms reference solution, locate the components of the reference
obtained with the test and reference solutions is strong solution on the chromatogram obtained with the test
brownish-violet and the zone due to acetyleugenol in the solution.
chromatogram obtained with the test solution is faint violet- Determine the percentage content of each of these
blue; in the chromatogram obtained with the test solution
components. The limits are within the following ranges:
there are other coloured zones, particularly a faint red zone
- {3-caryophyllene: 5.0 per cent to 14.0 per cent;
in the lower part and a reddish-violet zone (~-caryophyllene) - eugenol: 75.0 per cent to 88.0 per cent;
in the upper part.
- acetyleugenol: 4.0 per cent to 15.0 per cent.
chromatographic profile. STORAGE
Protected from heat.
Results The 3 principal peaks in chromatogram obtained
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
with the test solution are similar in retention time to the
IV-190 Cocoa Butter 2023
Column:
Cocoa Butter - material: fused silica, glass or quartz;
Theobroma Oil =
- size: l 30 m, 0 =0.32 mm;
(Ph. Bur. monograph 2607) 0.25 µm).
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Carrier gas helium for chromatography R.
DEFINITION Flow rate 1.3 mUmin.
Solid fat obtained from the roasted seeds of Theobroma Split ratio 1:50.
cacao L.
Temperature:
CHARACTERS
Appearance Time Temperature
Yellowish-white, solid mass. (min) (°C)
Column 0 - 15 70➔ 205
Solubility
15 - 25 205
Freely soluble in boiling anhydrous ethanol and in light 25 - 27.5 205 ➔ 230
petroleum, slightly soluble in ethanol (96 per cent). 27.5 - 50 230
Relative density Injection port 250
About 0.895 at 40 °C. Detector 250
Refractive index
About 1.457 at 40 °C. Detection Flame ionisation.
Injection 0.5 µL.
IDENTIFICATION
A. Melting point (2.2.15): 31 °C to 35 °c. Composition of the fatty-acid fraction of the substance:
- [auric acid: maximum 0.5 per cent;
Introduce 10 g of the substance to be examined into a beaker
- myristic acid: maximum 0.5 per cent;
and melt at 55 °C. Cool in a water-bath to 25 °C and stir
- palmitic acid: 24.0 per cent to 31.0 per cent;
continuously until it assumes a paste-like consistency, taking
- stearic acid: 30.0 per cent to 38.0 per cent;
care to avoid the introduction of air bubbles. Place the
- oleic acid: 31.0 per cent to 38.0 per cent;
beaker in a water-bath maintained at 32-33 °C. Continue
- linoleic acid: maximum 4.5 per cent;
stirring for about 30 min until the substance reaches the
- arachidic acid: maximum 1.5 per cent.
temperature of the water-bath and changes to a liquid cream.
Pour into another beaker, and allow to solidify at room STORAGE
temperature for at least 2 h. In an airtight container, protected from light, at a
Introduce the substance into the capillary tubes and allow to temperature not exceeding 25 °C.
stand at 2-8 °C for at least 48 h. - - - - - - - - - - - - - - - - - - - - - PhEur
B. Composition of fatty acids (see Tests).
TESTS
Acid value (2.5.1)
Maximum 4.0. Codonopsis Root
Peroxide value (2.5.5, Method A) (Ph. Bur. monograph 2714)
Maximum 3.0. PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
The starch solution R must be introduced before starting the
titration. DEFINITION
Whole or fragmented, dried root of Codonopsis pilosula
Saponification value (2. 5. 6)
(Franch.) Nannf., collected in autumn.
188 to 198, determined on 2.5 g.
Alkaline impurities IDENTIFICATION
Solvent mixture Dilute 15 mL of water R to 500 mL with A. The whole root is cylindrical, slightly curved and may be
acetone R and mix. Add 2.5 mL of a 1 g/L solution of up to 35 cm long and 2 cm in diameter, whereas the
bromophenol blue R in ethanol (50 per cent V/V) R and mix fragmented root occurs in pieces about 1-2 cm long.
again. If the solution is blue or yellow instead of green, The root crown, which is broader than the root, forms an
neutralise with 0.01 M hydrochloric acid or 0.01 M sodium irregular head with a convex centre and consisting of buds
hydroxide, respectively, to obtain a green solution. and numerous, prominent, rounded verrucose stem scars.
The root shows numerous transverse annulations below the
Melt 50 g of the substance to be examined at about 50 °C
root crown, occurring further and further apart until half the
and mix thoroughly. In a 150 mL conical flask, introduce
length of the root is reached. These transverse annulations
10.0 g of the melted substance and add 50 mL of the solvent
may be absent in the cultivated drug. The root shows deep
mixture. Stir vigorously and allow the 2 layers to separate.
longitudinal wrinkles and prominent scattered protuberances,
Not more than 2 mL of 0.01 M hydrochloric acid is required
resembling lenticels, over its entire length; the fracture of the
to change the colour of the upper layer to yellow; this
secondary roots is sometimes covered by a blackish
coloration must persist after vigorous stirring.
gelatinous secretion. The outer surface is brownish-yellow or
Composition of fatty acids brownish-grey. The bark adheres tenaciously to the wood
Gas chromatography (2.4.22, Method C) with the following and is difficult to remove. The texture is compact and the
modifications. fracture is relatively short.
Use the mixture of calibrating substances in Table 2.4.22.-1. B. Microscopic examination (2.8.23). The powder is pale
yellow or light brown. Examine under a microscope using
2023 Codonopsis Root IV- I 91
lactic reagent R. The powder shows the following diagnostic Top of the plate
characters (Figure 2714.-1): cork fragments (smface
view [F]), consisting of numerous layers of rectangular or
polyhedral cells; very numerous fragments of parenchyma A brownish zone
[A, H, TI containing ramified laticiferous vessels (longitudinal
section [Aa], transverse section [Hal), with brown granular -- --
contents; numerous rectangular sclereids, with distinctly Xylose: a yellowish-brown zone A weak violet zone
thickened walls and conspicuous channels, isolated [C] or in
-- A yellowish-brown zone _ _
groups [BJ; granular secretions of the laticiferous vessels, as
more or less coiled strands [E]; reticulate or scalariform Glucose: a yellowish-brown zone
vessels about 100 µm in diameter [D, G]; violet-blue
rounded or ovoid starch granules, simple, with a diameter of
about 2-10 µm, or 2- to 3-compound, either free or included Reference solution Test solution
in colourless parenchyma cells [Jb]; colourless pieces of inulin
in parenchyma cells Ua], either fan-shaped or as angular,
polyhedral fragments. TESTS
Platycodon grandiflorus
(2.9.12) add 5.0 mL of ethanol (70 per cent VIV,) R. Sonicate
for 10 min and centrifuge or filter. Evaporate the supernatant
or filtrate to dryness under reduced pressure. Dissolve the
residue in 1.0 mL of water R. Prepare a ready-to-use sample
preparation cartridge containing 50 mg of octadecylsilyl silica
gel (55 µm) using 3 mL of methanol R followed by 3 mL of
water R. Apply 1.0 mL of the solution to be analysed to the
top of the cartridge. Elute the cartridge with 3 mL of
water R; collect the eluate. Under reduced pressure,
evaporate the eluate to dryness. Dissolve the residue in 1 mL
of methanol R.
Reference solutwn Dissolve 1 mg of glucose R and 1 mg of
xylose R in 2 mL of methanol R.
Mobile phase water R, methanol R, glacial acetic acid R,
methylene chloride R (2:3:8:15 VIVIVIV).
Applicatwn 10 µL as bands of 8 mm.
Drying In air.
Detection Treat with anisaldehyde solutwn R and heat at
100 °C for 3 min; examine in daylight.
solution, the presence of a yellowish-brown zone at, directly
above, or directly below the zone due to glucose in the
chromatogram obtained with the reference solution indicates
adulteration with Platycodon grandifiorus.
Figure 2714.-1. - Illustratwn for identificatwn test B of powdered Loss on drying (2.2.32)
herbal drug of codonopsis root Maximum 12.0 per cent, determined on 1.000 g of the
C. Examine the chromatograms obtained in the test for 105 °C for 2 h.
Platycodon grandifiorus.
Total ash (2.4.16)
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 2.5 per cent.
Extractable matter
100 g of ethanol (70 per cent V/V) Rand allow to macerate
for 6 h, shaking frequently. Allow to stand for 18 h. Filter,
evaporate 20 g of the filtrate to dryness on a water-bath and
dry the residue in an oven at 105 °C for 3 h. The residue
weighs a minimum of O.168 g.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
IV-192 Coix Seed 2023
Test solutwn To 1 g of the powdered herbal drug (710)

Coix Seed (2. 9.12) add 10 mL of light petroleum Rl and sonicate for
(Ph. Bur. monograph 2454) 30 min. Filter and reduce in vacuo to 1 mL.
Reference solutwn Dissolve 2 mg of oleic acid R and 2 mg of
Ph Eur - - - - - - - - - - - - - - - - - - - - ~
triolein R in methanol R and dilute to 1 mL with the same
DEFINITION solvent.
Dried, ripe, caryopsis, freed from the shell, of Coix lacryma- Plate TLC octadecylsilyl silica gel plate R (5-40 µm) [or
jobi L. subsp. ma-yuen (Rom. Caill.) T.Koyama. TLC octadecylsilyl silica gel plate R (2-10 µm)].
Content Mobile phase methylene chloride R, glacial acetic acid R,
Minimum 0.50 per cent of triolein (C 57 H 104O 6; Mr 885) acetone R (20:40:50 VIVIV).
(dried drug). Application 10 µL [or 2 µL] as bands of 10 mm [or 8 mm].
IDENTIFICATION Development Over a path of 7 cm.
A. The white or pale yellow caryopsis freed from the shell is Drying In air.
roughly ovoid or elongated-elliptical, about 4-8 mm long and Detection Treat with a 100 g/L solution of phosphomolybdic
3-6 mm wide. The dorsal surface is rounded, milky white acid R in ethanol (96 per cent) R, heat at 120 °C for about
and smooth; the ventral surface shows a deep longitudinal 3 min and examine in daylight.
furrow; yellowish-brown remnants of the membranous floral
parts may be present. One end is obtusely rounded, the other
end is relatively flat and slightly dented with an indistinct,
pale brown hilum.
B. Microscopic examination (2.8.23). The powder is light
grey or light brown. Examine under a microscope using
Top of the plate
endosperm with oval to elongated [B] or rounded [E] thin-
walled cells and fragments consisting of elongated cells with
thicker walls [F]; fragments of tissue, with light yellow walls, A purple zone
with large, elongated, slightly sinuous cells [A]; cells of the
Oleic acid: a purple zone A purple zone (oleic acid)
middle layer of the mesocarp are brownish-yellow, irregularly
tube-like, slightly curved and irregularly crossed [D]. -- --
Examine under a microscope using a 50 per cent V/V
A faint purple zone
solution of glycerol R. The powder shows numerous starch
granules, simple or 2-3 compound, spherical or slightly A purple zone
polyhedral, 3-20 µm in diameter, with a stellate, Y-shaped,
A purple zone
cleft-like or point-like hilum [C] and fragments of
parenchyma containing starch granules [G]. -- --
Triolein: a purple zone A purple zone (triolein)
TESTS
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
(2. 9.12) add 50 mL of the mobile phase and stir with a
magnetic stirrer for 2 h. Sonicate for 30 min. Allow to cool,
dilute to 50.0 mL with the mobile phase and filter.
Reference solution (a) Dissolve 10.0 mg of triolein CRS in the
Reference solution (b) To 0.600 g of cove seed HRS add
50 mL of the mobile phase and stir with a magnetic stirrer
Figure 2454. -1. - Illustratwn for identificatwn test B of powdered for 2 h. Sonicate for 30 min. Allow to cool, dilute to
herbal drug of coix seed 50.0 mL with the mobile phase and filter.
Reference solutions (c), (d), (e), (fJ, (g), (h) Dilute reference
C. Thin-layer chromatography (2.2.27). solution (a) to obtain 6 reference solutions of triolein, the
2023 Cola IV-193
concentrations of which span the expected value in the test pressure inside the fruit; they vary in size and mass, ranging
solution. from 5-15 g; the outside is hard, smooth and very dark
Column: brown, the inside is more reddish-brown. In C. nitida and its
=
- size: l 0.25 m, 0 4.6 mm;= varieties, the kernels are divided in 2 parts, almost plano-
- stationary phase: end-capped octadecylsilyl silica gel for convex, corresponding to the cotyledons and usually
chromatography R (5 µm). occurring separated in the commercial drug; the cotyledons
Mobile phase methylene chloride R, acewnitrile R (35:65 V/V). are 3-4 cm long, 2-2.5 cm wide and 1-2 cm thick.
In C. acuminata, the cotyledons are smaller and divided into
Flow rate 2.0 mUmin. 4-6 irregular parts.
Detection Evaporative light-scattering detector; the following B. Microscopic examination (2.8.23). The powder is reddish-
settings have been found to be suitable; if the detector has brown. Examine under a microscope using a 50 per cent V/V
different setting parameters, adjust the detector settings so as solution of glycerol R. The powder shows the following
to comply with the system suitability criterion for signal-to- diagnostic characters: numerous ovoid or reniform starch
noise ratio: granules, 5-25 µm in size, with concentric striations and a
- carrier gas: nitrogen R; stellate, slightly eccentric hilum; fragments of cotyledon tissue
- flow rate: 0.8 mUmin; showing large, thick-walled, reddish polygonal cells filled with
- evaporawr temperature: 100 °C. starch granules; occasional fragments of the external
Injection 10 µL. epidermis of the cotyledons.
Run time 30 min. C. Thin-layer chromatography (2.2.27).
=
Retention time Triolein about 18 min; peak 2 about = Test solution To 1.0 g of the powdered herbal drug (355)
19 min. (2. 9.12) add 5 mL of ethanol (60 per cent Vlv? R. Shake
System suitability: mechanically at 40 °C for 30 min and filter.
- resolution: minimum 1.5 between the peak due to triolein Reference solution (a) Dissolve 25 mg of caffeine R in 10 mL
and peak 2 in the chromatogram obtained with reference of ethanol (60 per cent V/V) R.
solution (b); use the chromatogram supplied with coix Reference solution (b) Dissolve 50 mg of theobromine R in
seed HRS to identify peak 2; 10 mL of the mobile phase. Filter.
- signal-w-noise ratio: minimum 30 for the peak due to
triolein in the chromatogram obtained with reference Plate TLC silica gel F254 plate R.
solution (a). Mobile phase water R, methanol R, ethyl acetate R
Establish a calibration curve with the logarithm of the mass (10:13:77 VIVIV).
of triolein (in milligrams) per 50 mL of reference Application 20 µL, as bands.
solutions (c), (d), (e), (t), (g) and (h) (corrected by the Development Over a path of 10 cm.
assigned percentage content of triolein CRS) as the abscissa Drying In air for 5 min.
and the logarithm of the corresponding peak area as the Detection A Examine in ultraviolet light at 254 nm.
ordinate.
Calculate the percentage content of triolein using the shows 2 principal quenching zones which are similar in
following expression: position to the zones in the chromatograms obtained with
reference solutions (a) and (b).
10"1
Detection B Spray with a mixture of equal volumes of
mx 10
ethanol (96 per cent) R and hydrochloric acid R and then with a
A logarithm of the mass of triolein in the test solution, determined solution prepared immediately before use by dissolving 1 g of
from the calibration curve and the area of the corresponding iodine R and 1 g of potassium iodide R in 100 mL of ethanol
peak in the chromatogram obtained with the test solution; (96 per cent) R.
solution, in grams. Results B The chromatogram obtained with the test solution
shows a reddish-brown principal zone similar in position and
colour to the zone in the chromatogram obtained with
reference solution (a).
TESTS
Cola Maximum 12.0 per cent, determined on 2.00 g of the
(Ph. Bur. monograph 1504) 105 °C for 2 h.
Total ash (2.4.16)
DEFINTilON Maximum 9.0 per cent.
Whole or fragmented dried seeds, freed from the testa, of ASSAY
Cola nitida (Vent.) Schott et End!. (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 (m 1) of the powdered herbal drug
et End!. (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
IDENflFICATION the residue with 50 mL of methanol R. Proceed as before.
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
IV-194 Colophony 2023
Transfer 20.0 mL of this solution into a round-bottomed Detection Spray with anisaldehyde solution R and heat at
flask and evaporate to dryness under reduced pressure. Take 100-105 °C for 10 min; examine in daylight.
up the residue with the mobile phase, transfer to a 50.0 mL Results See below the sequence of the zones present in the
volumetric flask and dilute to 50.0 mL with the mobile chromatograms obtained with the reference solution and the
phase. test solution. Furthermore, other coloured zones are present
Reference solutwn In a 100.0 mL volumetric flask, dissolve in the chromatogram obtained with the test solution.
30.0 mg (m 2 ) of caffeine CRS and 15.0 mg of theobromine R
in the mobile phase and dilute to 100.0 mL with the mobile Top of the plate
phase. Transfer 10.0 mL ofthis solution to a 100.0 mL
volumetric flask and dilute to 100.0 mL with the mobile A purple band
phase. A purple band
Column:
- size: l = 0.25 m, 0 = 4.6 mm; -- --
- stationary phase: octadecylsilyl silica gel for chromatography R 2 purple bands
(5 µm).
Thymol: an orange band
Mobile phase methanol R, water R (25:75 V/V).
Flow rate 1 mUmin. -- --
Detection Spectrophotometer at 272 nm. Linalol: a purple band Sequence of narrow purple bands
Injection The chosen volume of each solution; loop injector. Purple extended baseline band
System suitability Reference solution: Reference solution Test solution
- resolutwn: minimum 2.5 between the peaks due to caffeine
and theobromine. If necessary, adjust the volume of
water R in the mobile phase. TESTS
Calculate the caffeine content using the following expression: Acid value (2.5.1)
Total ash (2.4.16)
area of the peak due to caffeine in the chromatogram obtained STORAGE
with the test solution; Do not reduce to a powder.
area of the peak due to caffeine in the chromatogram obtained
m1 mass of the herbal drug to be examined in the test 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
(Ph. Bur. monograph 1862) dispensed or supplied.
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Preparation
Flexible Collodion DEFINITION
Dried cremocarp of Coriandrum sativum L.
DEFINITION Content
Residue remaining after distillation of the volatile oil from the Minimum 3 mUkg of essential oil (dried drug).
oleoresin obtained from various species of Pinus. IDENTIFICATION
IDENTIFICATION A. The fruit is brown or light brown, more or Jess spherical,
A. Translucent, pale yellow to brownish-yellow, angular, about 1.5-5 mm in diameter, or oval and 2-6 mm Jong.
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,
slightly raised primary ridges and 8 straight, more prominent
secondary ridges. The mericarps are concave on the internal
Test solutwn Dissolve 1 g in 10 mL of methanol R by gently surface. The stylopod crowns the apex and a small fragment
warming. of the pedicel may be present.
Reference solutwn Dissolve 10 mg of thymol R and 10 mg of B. Microscopic examination (2.8.23). The powder is brown.
linalol R in 10 mL of methanol R. Examine under a microscope using chloral hydrate solution R.
Plate TLC silica gel plate R. The powder shows the following diagnostic characters
Mobile phase methylene chloride R. (Figure 1304.-1): numerous oil droplets [B]; fragments of
Applicatwn 10 µL, as bands. endosperm [A] with small, thick-walled, regular cells
containing microrosettes [Aa] and microcrystals of calcium
oxalate and oil droplets [Ab]; fragments of endocarp (surface
Drying In air. view [C, TI, transverse section [H]), with very narrow cells
2023 Coriander Oil IV-195
having a parquetry arrangement [Ca, Ha] and usually Top of the plate
associated with a layer of thin-walled [Cb, Hb] or thicker-
A bluish-violet zone
walled Ua] rectangular sclereids of the mesocarp; fragments
from the sclerenchymatous layer of the mesocarp [G] with - - - -
short, strongly thickened, pitted, fusiform cells occurring in

Geranyl acetate: a violet-blue 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

Linalol: an intense violet zone A violet zone (linalol)
slightly thickened walls [Ea], the remains of secretory canals
[Eb] and sclereids [Ee]; fragments of epicarp (surface view A violet-blue zone
[F]) with thin-walled polyhedral cells, some of which contain
small prisms of calcium oxalate [Fa]; rare fragments of
secretory canals with brown cells, (surface view [DJ);
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
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
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Essential oil obtained by steam distillation from the fruits of
Coriandrum sativum L.
Figure 1304. -1. - Illustration for identification test B of powdered CHARACTERS

herbal drug of coriander Appearance
Clear, colourless or pale yellow liquid.
C. Thin-layer chromatography (2.2.27). IDENTIFICATION
Test solution To 0.5 g of the freshly powdered herbal drug First identification: B.
(355) (2.9.12) add 5 mL of methanol R. Sonicate for 10 min Second identification: A.
and centrifuge or filter; use the supernatant or filtrate.
A. Examine by thin-layer chromatography (2.2.27).
Reference solution Dissolve 10 µL of linalol R and 2 µL of
geranyl acetate R in 1.0 mL of toluene R. Test solution Dissolve 10 µL of the substance to be
examined in 1.0 mL of toluene R.
F254 plate R (2-10 µm)]. Reference solution Dissolve 10 µL of linalol R and 2 µL of
geranyl acetate R in 1.0 mL of toluene R.
Mobile phase ethyl acetate R, toluene R (5:95 V/V).
Application 10 µL [or 2 µL] as bands of 15 mm [or 8 mm]. F2 54 plate R (2-10 µm)].
Development Over a path of 10 cm [or 6 cm]. Mobile phase ethyl acetate R, toluene R (5:95 V/V).
Drying In air for 5 min. Application 10 µL [or 2 µL] as bands of 15 mm [or 8 mm).
Detection Treat with anisaldehyde solution R and heat at Development Over a path of 10 cm [or 6 cm].
IV-1 96 Coriander Oil 2023
Results See below the sequence of zones present in the Detection Flame ionisation.
chromatograms obtained with the reference solution and the Injection 0.2 µL.
reference solution (a). Record the retention times of these
substances.
Top of the plate
A bluish-violet zone - resolution: minimum 1.5 between the peaks due to linalol
and camphor.
- - --
Using the retention times determined from the
Geranyl acetate: a violet-blue zone chromatogram obtained with reference solution (a), locate
the components of reference solution (a) in the
- - --
Llnalol: an intense violet zone An intense violet zone (linalol)
A violet-blue zone components. The percentages are within the following
ranges:
- rx-pinene: 3.0 per cent to 7.0 per cent;
- limonene: 1.5 per cent to 5.0 per cent;
B. Examine the chromatograms obtained in the test for - y-terpinene: 1.5 per cent to 8.0 per cent;
chromatographic profile. - p-cymene: 0.5 per cent to 4.0 per cent;
Results The characteristic peaks in the chromatogram - camphor. 3.0 per cent to 6.0 per cent;
obtained with the test solution are similar in retention time to - linalol: 65.0 per cent to 78.0 per cent;
those in the chromatogram obtained with the reference - rx-terpineol: 0.1 per cent to 1.5 per cent;
solution. - geranyl acetate: 0.5 per cent to 4.0 per cent;
- geraniol: 0.5 per cent to 3.0 per cent;
TESTS - disregard limit: area of the peak in the chromatogram
Relative density (2.2.5) obtained with reference solution (b) (0.05 per cent).
0.860 to 0.880.
Chiral purity
Refractive index (2.2.6) Gas chromatography (2.2.28).
1.462 to 1.470.
Optical rotation (2.2. 7) examined in pentane R and dilute to 10 mL with the same
+ 7cto + 13°. solvent.
Acid value (2.5.1) Reference solution Dissolve 10 µL of linalol R and 5 mg of
Maximum 3.0, determined on 5.00 g of the substance to be borneol R in pentane R and dilute to 10 mL with the same
examined. solvent.
Chromatographic profile Column:
Gas chromatography (2.2.28): use the normalisation - material: fused silica;
procedure. - size: l = 25 m, 0 = 0.25 mm;
Test solution The substance to be examined. - stationary phase: modified /3-cyclodextrin for chiral
Reference solution (a) Dissolve 10 µL of rx-pinene R, 10 µL of chromatography R (film thickness 0.25 µm).
limonene R, 10 µL of y-terpinene R, I O µL of p-cymene R, Carrier gas helium for chromatography R.
10 mg of camphor R, 20 µL of linalol R, 10 µL of Flow rate 1.3 mUmin.
rx-terpineol R, 10 µL of geranyl acetate R and 10 µL of Split ratio 1:30.
geraniol R in 1 mL of heptane R.
Temperature:
Reference solution (b) Dissolve 5 µL of geraniol R in
heptane R and dilute to 10 mL with the same solvent. Time Temperature
Column: (min) CC)
- material: fused silica; Column 0 - 65 50---+ 180
- size: l = 60 m, 0 = 0.25 mm; Injection pon 230
- stationary phase: macrogol 20 000 R (film thickness Detector 230
0.25 µm).
Carrier gas helium for chromatography R. Detection Flame ionisation.
Flow rate 1 mUmin. /refection l µL.
Split ratio 1:65. System suitability Reference solution:
Temperature: - resolution: minimum 5.5 between the peaks due to
(R)-linalol (1 st peak) and (S)-linalol (2 nd peak) and
Time Temperature minimum 2.9 between the peaks due to (S)-linalol and
(min) CC) borneol (3 rd peak).
Column 0 - 10 60 Limit Calculate the percentage content of (R)-linalol using
10 - 75 60---+ 190 the expression:
75 - 120 190
Injection port 220
AR
A
+A R x 100
Detector 240
S
2023 Corydalis Rhizome IV-197
As area of the peak due to (S)-linalol;

AR area of the peak due to (R)-linalol.
- (R)-linalol: maximum 14 per cent.

STORAGE
At a temperature not exceeding 25 "C.
Corydalis Rhizome
DEFINITION
Whole or fragmented tuber of Corydalis yanhusuo (Y.H.Chou
& Chun C.Hsu) W.T.Wang ex Z.Y.Su & C.Y.Wu, with
roots removed, treated with boiling water until no white core
is visible and then dried.
Content
Minimum 0.20 per cent for the sum of tetrahydropalmatine
and corydaline, expressed as tetrahydropalmatine
(C21H2sNO4; Mr 355.4) (dried drug).
IDENTIFICATION
.,
A. Whole drug. The whole tuber is irregularly oblate, . .~ ., . ,
" ., '
"
,,. , ~ ;
0.5-1.8 cm in diameter and 0.5-1.3 cm (rarely 1.7 cm) thick.
Occasionally, 2-3 tubers are grouped together. The apex
F
usually shows a slightly depressed stem scar and occasional
remains of aerial parts. The base is tuberculate or with a
slight navel-like depression from which shallow furrows may
herbal drug of corydalis rhizome
radiate. The external surface is yellow or yellowish-brown to
greenish-yellow with coarse or fine reticulate wrinkles. B. Microscopic examination (2.8.23). The powder is
The underlying cortex, when exposed, is blackish-green or greenish-yellow to brownish-yellow. Examine under a
brownish-yellow. The texture is hard, mostly difficult to microscope using chloral hydrate solution R. The powder
break. The fracture is horny, slightly shiny and translucent shows the following diagnostic characters (Figure 2976.-1):
with a yellow, yellowish-brown or dark brown surface. greenish-yellow to brownish-yellow fragments of cortical
The pith, if present, is whitish-yellow. sclerenchyma [F], consisting of elongated, polygonal, sub-
Fragmented drug The fragmented tuber occurs as thick or square or irregularly shaped cells 35-321 µm long and
thin, sub-rounded or sub-oval transversal or longitudinal 15-12 7 µm in diameter with lignified, often sinuous and
slices or as irregular pieces. The slices are 0.5-1.9 cm in beaded cell walls having fine, sparse pits and large cell
diameter and 0.1-0.5 cm thick. Occasionally, slices consist of lumens; some fragments [AJ of cortical parenchyma [AbJ are
2-3 tubers grouped together. Rare slices from the tuber apex covered by dermal tissue consisting of a layer of rectangular
or base are present, with those from the apex having a or polygonal thin-walled cells [AaJ; sclereids [DJ isolated or
slightly depressed stem scar and occasional remains of aerial in groups [DbJ, sub-square to sub-rounded, sub-polygonal,
parts, while those from the base have tuberculate protrusions sub-triangular or irregular in shape, 32-290 µm Jong and
or have a slight navel-like depression from which shallow 22-103 µm in diameter, with yellowish-green to brownish-
furrows may radiate. The external surface is yellow or yellow cell walls; sclereids with lignified cell walls having
yellowish-brown with coarse or fine wrinkles. The underlying dense, fine pits and occasional striations; some sdereids have
cortex, when exposed, is blackish-green or brownish-yellow. irregularly thickened cell walls, with one thinner wall [Da];
The texture is hard and fragile. The fracture is horny and rare, inconspicuous fragments of xylem vessels, often
slightly shiny. The cut surface is horny, slightly shiny and embedded in parenchyma tissue, colourless or pale yellow,
translucent with occasional fissures; the surface is yellow, irregularly spiral, annular or reticulate and 10-43 µm in
yellowish-brown, reddish-brown or dark blackish-brown and diameter [BJ. Examine under a microscope using a
occasionally shows a gradient of colours. The pith, if present, 50 per cent VIV solution of glycerol R. The powder shows the
is a lighter colour than the surrounding tissue, or if absent, it following diagnostic characters: abundant large and small
is replaced by a central cavity. colourless or pale yellow masses of gelatinised starch
granules [CJ, varying in shape, but often polygonal, elongate
oval or rectangular (up to 530 µm long and 332 µm in
diameter) and surrounded by colourless parenchyma [EJ; rare
free starch granules, single or 2-5 compound, with single
granules being sub-rounded or irregular with a distinct hilum.
C. High-performance thin-layer chromatography (2. 8. 25).
(2.9.12) add 10.0 mL of ethanol (50 per cent V/V) Rand
IV-198 Corydalis Rhizome 2023
sonicate for 15 min. Centrifuge or filter the solution and use Total ash (2.4. 16)
the supernatant or filtrate. Maximum 3.0 per cent.
Reference solutwn (a) Dissolve 2.5 mg of corydaline Rand Ash insoluble in hydrochloric acid (2. 8.1)
2.5 mg of tetrahydropalmatine R in methanol Rand dilute to Maximum 0.5 per cent.
20.0 mL with the same solvent. Aflatoxins (2.8.18)
Reference solutwn (b) Dilute 2.5 mL ofreference solution (a) Maximum 2 µg/kg (aflatoxin B1) and maximum 4 µg/kg
to 10.0 mL with methanol R. (sum of aflatoxins B 1, B 2 , G 1 and G 2).
Intensity marker Tetrahydropalmatine.
ASSAY
Plate TLC silica gel F 254 plate R (2-10 µm). Liquid chromatography (2.2.29).
Mobile phase glacial acetic acid R, water R, butanol R, ethyl Test solutwn To 1.00 g of the powdered herbal drug (355)
acetate R (10:10:50:50 VIVIVIV). (2.9.12) add 25.0 mL of ethanol (50 per cent V/V) R. Weigh
Application 2 µL as bands of 8 mm. and sonicate for 30 min. Allow to cool, weigh and
Development 70 mm from the lower edge of the plate. compensate for the loss of solvent with ethanol
Drying In a current of cold air for 5 min. (50 per cent V/V) R; shake thoroughly. Filter through a
Detection Expose the plate for 3 min to iodine vapour using
a chromatographic tank previously saturated with iodine for Reference solutwn (a) Dissolve 5.0 mg of
30 min; remove the excess of absorbed iodine in air for tetrahydropalmatine CRS in ethanol (50 per cent Vlv] R and
10 min and examine in ultraviolet light at 366 nm. dilute to 25.0 mL with the same solvent. Sonicate for 1 h.
Dilute 2.0 mL of the solution to 10.0 mL with ethanol
(50 per cent V/v] R.
- the chromatogram shows near the border between the
lower and middle thirds 2 distinct zones, which may Reference solutwn (b) To 0.10 g of corydalis rhizome dry
be touching; the lower zone (tetrahydropalmatine) extract HRS add 5.0 mL of ethanol (50 per cent Vlv] R.
shows a green fluorescence and the upper zone Weigh and sonicate for 10 min. Allow to cool, weigh and
(corydaline) shows a light blue fluorescence. compensate for the loss of solvent with ethanol
(50 per cent Vlv] R; shake thoroughly. Centrifuge and filter
through a membrane filter (nominal pore size 0.45 µm).
test solution. Furthermore, other faint blue, yellow or green Column:
fluorescent zones may be present in the chromatogram - size: l = 0.25 m, 0 = 4.6 mm;
obtained with the test solution. - statwnary phase: end-capped octadecylsilyl silica gel for
chromatography R (5 µm);
Top of the plate
Mobile phase:
- mobile phase A: mix 0.05 volumes of anhydrous acetic
acid Rand 99.95 volumes of water for chromatography R
-- --
and adjust to pH 6 with triethylamine R;

A green fluorescent zone, faint to (min) (per cent V/J/) (per cent V/J/)
equivalent
0-3 60 40
A green fluorescent zone, faint 3 - 31 60 ➔ 38 40 ➔ 62
31 - 32 38 ➔ 5 62 ➔ 95
32 - 37 5 95
Corydaline: a light blue fluorescent A light blue fluorescent zone
zone (corydaline)
How rate 1.0 mLJmin.
-- -- Detection Spectrophotometer at 282 nm.
Tetrahydropalmatine: a green A green fluorescent zone Injection 20 µL.
fluorescent zone (tetrahydropalmatine)
corydalis rhizome dry extract HRS and the chromatogram
obtained with reference solution (b) to identify the peaks due
to corydaline and peak 2; use the chromatogram obtained
A green fluorescent zone, faint with reference solution (a) to identify the peak due to
A green or yellow fluorescent zone,
tetrahydropalmatine.
equivalent to intense Relative retention With reference to tetrahydropalmatine
(retention time = about 16.4 min): peak 2
(unknown) = about 1.54; corydaline = about 1.65.
- resolution: minimum 2.0 between peak 2 and the peak due
TESTS to corydaline.
Loss on drying (2.2.32) Calculate the percentage content of the sum of
Maximum 12.0 per cent, determined on 1.000 g of the tetrahydropalmatine and corydaline, expressed as
powdered herbal drug (355) (2. 9.12) by drying in an oven at tetrahydropalmatine, using the following expression:
105 °C for 5 h.
2023 Cyathula Root IV-199
Ba
sum of the areas of the peaks due to tetrahydropahnatine and
B
corydaline in the chromatogram obtained with the test solution;
A, area of the peak due to tetrahydropalmatine in the
chromatogram obtained with reference solution (a);
solution, in grams;
m2 mass of tetrahydropalmatine CRS used to prepare reference
p percentage content of tetrahydropahnatine in
tetrahydropalmatine CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Couch Grass Rhizome

Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or cut, washed and dried rhizome of Agropyron
repens (L.) P.Beauv. (Blymus repens (L.) Gould); the
E_.~ ~ L--::;-· ..;~
adventitious roots are removed. I
IDENTIFICATION
A. The shiny yellowish, light brown or yellowish-brown ,___..,
25µm
pieces of the rhizome are 2-3 mm thick and longitudinally
Gb
furrowed. At the nodes are the remains of very thin, more or
less branched roots and whitish or brownish scale-like leaves;
the intemodes, up to 6 cm long, are furrowed and hollow
herbal drug of couch grass rhizome
inside. The transverse section of the nodes shows a yellowish
medulla.
B. Microscopic examination (2.8.23). The powder is whitish- Maximum 12.0 per cent, determined on 1.000 g of the
yellow. Examine under a microscope using chloral hydrate powdered herbal drug (355) (2. 9.12) by drying in an oven at
solution R. The powder shows the following diagnostic 105 °C for 2 h.
characters (Figure 1306.-1): fragments of the epidermis
Total ash (2.4.16)
(surface view [A]) covered with a thick cuticle and composed
of rectangular and elongated, thick-walled cells with pitted,
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 Maximum 1.5 per cent.
(transverse section [B]) showing the epidermis [Ba] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhE~
associated 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 Cyathula Root
thickened fibres [CJ; groups of vessels [D, G] with slit-
shaped pits [Da] or with spiral and annular thickening [Ga], (Ph. Bur. monograph 2998)
accompanied by fibres [Db, Gb]; numerous fragments of the
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
cortical parenchyma and the pith with slightly thickened and
pitted cells [E]. DEFINITION
TESTS Whole or fragmented, dried root of Cyathula officinalis K.C.
Cynodon dactylon, Imperata cylindrica Kuan, with rootlets removed.
Examine under a microscope using iodine solution Rl. Content
No blue starch grains are visible. Minimum 0.065 per cent of cyasterone (C 29 H 44 O 8 ;
Foreign matter (2.8.2) M, 520.7) (dried drug).
Maximum 15 per cent of blackish-grey pieces of rhizome in IDENTIFICATION
the cut herbal drug. A. The whole root is cylindrical, straight or slightly curved,
Water-soluble extractive somewhat twisted, tapering slightly from the upper part to
Minimum 25 per cent. the lower part, sometimes branched, 14-60 cm long and
0.5-5 cm in diameter. The fragmented root occurs as
To 5.0 g of the powdered herbal drug (355) (2. 9.12) add
transverse or oblique slices, rounded to elliptical, 0.5-1.8 cm
200 mL of boiling water R. Allow to stand for 10 min,
shaking occasionally. Allow to cool, dilute to 200.0 mL with in diameter. The outer surface is yellowish-brown or greyish-
brown, with distinct fine longitudinal wrinkles, frequent
water R and filter. Evaporate 20.0 mL of the filtrate to
dryness on a water-bath. Dry the residue in an oven at transverse lenticel-like protuberances and a few rootlet scars.
The texture is firm; the whole root is difficult to break.
100-105 °C. The residue weighs a minimum of 0.125 g.
IV-200 Cyathula Root 2023
The fracture is horny and slightly uneven. The cut surface Top of the plate
(slices) and fracture are brownish-yellow and show numerous
vascular bundles, mostly appearing as yellowish-white dots
arranged in several concentric rings; the central bundles are A reddish zone, faint
larger than the surrounding bundles. The cortex is narrow
A reddish zone, faint
and dark brown.
B. Microscopic examination (2.8.23). The powder is greyish- Cyasterone: a bright blue fluorescent A bright blue fluorescent zone
zone (cyasterone)
brown to brown. Examine under a microscope using ch/,oral
hydrate solution R. The powder shows the following diagnostic -- --
characters (Figure 2998.-1): abundant microcrystals of
A bright blue fluorescent zone
calcium oxalate, 1-18 µm in diameter, triangular, pointed,
sub-square or irregularly shaped; the crystals are free [E] or
are scattered in or completely fill parenchyma cells [F, Ha] ;
vessels, 12-73 µm in diameter, usually with bordered pits
[C]; fibres commonly in bundles, whole [B] or fragmented A green zone, faint
[D], elongated, with curved or tapering ends, 6-26 µm in
A green zone, faint
diameter, with mostly weakly lignified, thickened walls and
oblique-slit-shaped, cross-shaped or V-shaped pits; the pit -- --
canals are distinct and vary in size [Ba]; numerous fragments
Ginsenoside Ro: a green zone A green zone, faint
of colourless parenchyma with ovoid or rectangular cells [H];
fragments of brownish-yellow cork appearing as sub-square, A green zone, faint
polyhedral or rectangular cells (surface view [Al) or
superimposed, flattened, rectangular cells (side view [G)).
B ,'
, Reference solution (a) Test solution
1/ /
I
TESTS
Cyathula capitata Moq. and Achyranthes bidentata
,!
Blume
High-performance thin-layer chromatography (2. 8. 25).
(2. 9.12) add 5 mL of a mixture of 2 volumes of water R and
8 volumes of methanol R. Sonicate for 30 min and centrifuge.
Decant the supernatant. Take up the residue with 8 mL of
water R. Sonicate (or shake) for 1 min, centrifuge and
combine the supernatants. Condition a 4 mL solid phase
extraction column containing 0.200 g of octadecylsilyl siUca gel
for chromatography R with 5 mL of methanol R and then with
::?,
.
-': ·.. . ~
F 3 mL of water R at a rate of 1 drop per second, ensuring that
. ;::::: -:: the column does not dry out. Transfer the combined
supernatants to the column. Allow to drain ensuring that the
column does not dry out and discard the eluate. Wash the
H column with 2 mL of water R and discard the washings.
Elute the column with 1 mL of methanol R and collect the
eluate.
Reference solution (a) Dissolve 1.0 mg of cyasterone R and
25 µm 1.0 mg of ginsenoside Ro R in 2.0 mL of methanol R.
f-------j
Reference solution (b) Mix 1.0 mL of reference solution (a)
and 3.0 mL of methanol R.
Reference solution (c) Dissolve 1 mg of /3-ecdysterone R and
1 mg of cyasterone R in 2 mL of methanol R.
Figure 2998.-1. - Illustration for identification test B of powdered Intensity marker Cyasterone.
herbal drug of cyathula root Plate TLC silica gel F 254 plate R (2-10 µm).
Mobile phase anhydrous formic acid R, water R, methanol R,
C. High-performance thin-layer chromatography (2.8.25) as
methylene chloride R (5:5:25:70 VIVIVIV).
described in the test for Cyathula capitata Moq.
and Achyranthes bidentata Blume with the following Application 3 µL as bands of 8 mm.
modification. Development 10 mm from the lower edge of the plate.
Results See below the sequence of zones present in the Drying In a current of cold air for 5 min.
chromatograms obtained with reference solution (a) and the Detection Treat with a 100 g/L solution of sulfuric acid R in
test solution. Furthermore, in the chromatogram obtained ethanol (96 per cent) R and heat at 100 °C for 5 min; examine
with the test solution, other faint blue, faint green or faint in ultraviolet light at 366 nm.
reddish zones may be present.
2023 Dandelion Herb with Root IV-201
System suitability Reference solution (c):

- the chromatogram shows in the middle and upper thirds
2 distinct zones, which may be touching; the lower zone
A1 area of the peak due to cyasterone in the chromatogram
(~-ecdysterone) shows a blue fluorescence and the upper obtained with the test solution;
zone (cyasterone) shows a bright blue fluorescence. A2 area of the peak due to cyasterone in the chromatogram
shows no intense reddish zones in the lower half or just solution, in grams;
below the zone due to cyasterone in the chromatogram m2 mass of cyasterone CRS used to prepare the reference solution,
obtained with reference solution (a). in grams;
p percentage content of cyasterone in cyasterone CRS.
Maximum 12.0 per cent, determined on 1.000 g of the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
105 °C for 2 h.
Total ash (2.4.16)
Maximum 8.0 per cent. Dandelion Herb with Root
Maximum 2.0 per cent. (Ph. Bur. monograph 1851)
ASSAY PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Liquid chromatography (2.2.29). DEFINITION

Test solutwn To 1.00 g of the powdered herbal drug (710) Mixture of whole or fragmented, dried aerial and
(2.9.12) add 20.0 mL of an 80 per cent VIV solution of underground parts of Taraxacum officinale F.H. Wigg.
methanol R. Weigh and sonicate for 60 min. Allow to cool
and weigh again. Compensate for the loss of solvent with an CHARACTERS
80 per cent V/V solution of methanol R and shake thoroughly. Bitter taste.
Filter through a membrane filter (nominal pore size IDENTIFICATION
0.45 µm). A. The underground parts consist of dark brown or blackish
Reference solutwn Dissolve 4.0 mg of cyasterone CRS in an fragments 2-3 cm long, deeply wrinkled longitudinally on the
80 per cent V/V solution of methanol R and dilute to outer surface. The thickened crown shows many scars left by
10.0 mL with the same solvent. Dilute 1.0 mL of the the rosette of leaves. The fracture is short. A transverse
solution to 10.0 mL with an 80 per cent V/V solution of section shows a greyish-white or brownish cortex containing
methanol R. concentric layers of brownish laticiferous vessels and a
Column: porous, pale yellow, non-radiate wood. Leaf fragments are
- size: l = 0.15 m, 0 = 4.6 mm; green, glabrous or densely pilose. They are crumpled and
- stationary phase: solid core octylsilyl silica gel for usually show a clearly visible midrib on the inner surface.
chromatography R (2. 7 µm); The lamina, with deeply dentate margins, is crumpled.
- temperature: 35 °C. The solitary flower heads, on hollow stems, consist of an
involucre of green, foliaceous bracts surrounding the yellow
Mobile phase:
florets, all of which are ligulate; a few achenes bearing a
- mobile phase A: phosphoric acid R, water for
white, silky, outspread pappus may be present.
chromatography R (0.1:99.9 V/V);
- mobile phase B: methanol Rl; B. Microscopic examination (2.8.23). The powder is
Time Mobile phase A Mobile phase B hydrate solutwn R. The powder shows the following diagnostic
(min) (per cent V/J,') (per cent V/J,') characters (Figure 1851.-1): fragments of cork [G] with
0-3 88 12 flattened, thin-walled cells; reticulate lignified vessels [H]
3- 5 88 --, 67 12--, 33 from the roots; fragments of parenchyma containing
5 - 35 67--, 64 33--, 36 branched laticiferous vessels [F]; fragments ofleaves, in
35 - 39 64..., 5 36--, 95 surface view, showing upper [E] and lower [C] epidermises
consisting of interlocking lobed cells and anomocytic stomata
(2.8.3) [Ca, Ea]; elongated, multicellular covering trichomes
with constrictions, which are more or less abundant
Detection Spectrophotometer at 248 nm. depending on the variety or sub-variety [B, D]; fragments of
Injection 10 µL. the upper [E) epidermis usually accompanied by underlying
Identification of peaks Use the chromatogram supplied with palisade parenchyma [Eb] and fragments of the lower [C]
cyasterone CRS and the chromatogram obtained with the epidermis accompanied by underlying spongy parenchyma
reference solution to identify the peak due to cyasterone and [Cb]; lignified, spirally or annularly thickened vessels;
peak 2. fragments of flower-stem epidermis with stomata and rigid-
Retention time Cyasterone = about 32.6 min; peak 2 walled, elongated cells [A); pollen grains with a pitted exine
(unknown) = about 34 min. IJ]. Examine under a microscope using glycerol R.
The powder shows angular, irregular inulin fragments, free or
included in the parenchyma cells.
- resolution: minimum 1. 7 between the peak due to
cyasterone and peak 2.
Calculate the percentage content of cyasterone using the
IV-202 Dandelion Root 2023
Top of the plate
A faint red zone
A faint yellow zone
-- --
Chlorogenic acid: a blue zone 2 light blue zones
- -
--
Rutoside: a yellowish-brown zone
A light blue zone
TESTS
Ea-,-(t--c--£:;;:\ Loss on drying (2.2.32)
,.,.~._..,,,,....,,.,""\\ Maximum 10.0 per cent, determined on 1.000 g of the
E ,..._,,~=""' powdered herbal drug (355) (2.9.12) by drying in an oven at
Eb 105 °C for 2 h.
Total ash (2.4.16)
Extractable matter
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
Figure 1851.-1. - Illustratwn for identificatwn test B of powdered 100-105 °C for 2 h. The residue weighs a minimum of
herbal drug of dandelion herb with root 0.15 g.
C. Thin-layer chromatography (2.2.27). Bitterness value (2. 8.15)
Minimum 100.
(2. 9.12) add 10 mL of methanol R. Heat in a water-bath at ---------------------~~
60 °C or sonicate for 10 min. Cool and filter.
Reference solution Dissolve 2 mg of chlorogenic acid R and
2 mg of rutoside trihydrate R in methanol R and dilute to
20 mL with the same solvent. Dandelion Root
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
(10:10:80 VIVIV). DEFINITION
Applicatwn 20 µL [or 5 µL] as bands of 10 mm [or 8 mm]. Whole or cut, dried underground parts of Taraxacum
Development Over a path of 12 cm [or 7 cm]. officinale F.H.Wigg.
Drying In air. CHARACTERS
Detection Heat at 100 °C for 5 min; spray with or dip Bitter taste.
briefly into a 10 g/L solution of diphenylboric acid aminoethyl IDENTIFICATION
ester R in methanol R and dry at 100 °C for 5 min; spray with A. The dark brown or blackish taproot shows little branching
or dip briefly into a 50 g/L solution of macrogol 400 R in and is deeply wrinkled longitudinally on the outer surface.
methanol R; heat at 100 °C for 5 min and examine in The thickened crown shows many scars left by the rosette of
ultraviolet light at 365 nm. leaves. The fracture is short. A transverse section shows a
Results See below the sequence of zones present in the greyish-white or brownish cortex containing concentric layers
chromatograms obtained with the reference solution and the of brownish laticiferous vessels and a porous, pale yellow,
test solution. Furthermore, other faint zones may be present non-radiate wood.
in the chromatogram obtained with the test solution. B. Microscopic examination (2.8.23). The powder is
characters (Figure 1852.-1): fragments of brown or reddish-
brown cork (surface view [G], transverse section [Cl) with
flattened, thin-walled cells [Ca], sometimes accompanied by
parenchyma [Cb]; reticulate lignified vessels [E, J, M];
fragments of parenchyma [A, D, K, L], some containing
branched laticiferous vessels (longitudinal section [Ka],
2023 Devil's Claw IV-203
transverse section [Da]); granular contents of laticiferous Top of the plate

vessels [B, HJ. Examine under a microscope using glycerol R.
A light blue zone
The powder shows numerous irregular, angular inulin
fragments, free [F] or included in the parenchyma cells [La]. -- --
Chlorogenic acid: a blue zone A blue zone (chlorogenic acid)
-- --
Rutoside: 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. 12) 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.
Bitterness value (2.8.15)
Minimum 100.
- - - - - - - - - - - - - - - - - - - - ~ PhEvr
Figure 1852.-1. -Illustration for identification test B of powdered

herbal drug of dandelion root Devil's Claw
C. Thin-layer chromatography (2.2.27). Harpagophytum
Test solution To 2.0 g of the powdered herbal drug (355) (Devil's Claw Root, Ph. Bur. monograph 1095)
(2. 9.12) add 10 mL of methanol R. Heat in a water-bath at
Preparation
60 °C or sonicate for 10 min. Cool and filter.
Devil's Claw Dry Extract
Reference solution Dissolve 2 mg of chlorogenic acid R and PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
2 mg of rutoside trihydrate R in methanol R and dilute to
20 mL with the same solvent. DEFINITION
Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel Cut and dried, tuberous secondary roots of Harpagophytum
F2s4 plate R (2-10 µm)]. procumbens DC. and/or Harpagophytum zeyheri Decne.
Mobile phase anhydrous formic acid R, water R, ethyl acetate R Content
(10:10:80 VIVIV). Minimum 1.2 per cent of harpagoside (C 24 H 30 0 11 ;
Application 20 µL [or 5 µL] as bands of 10 mm [or 8 mm]. Mr 494.5) (dried drug).
Development Over a path of 12 cm [or 7 cm]. CHARACTERS
Drying In air. The root is greyish-brown or dark brown.
Detection Heat at 100 °C for 5 min; spray with or dip IDENTIFICATION
briefly into a 10 g/L solution of diphenylboric acid aminoethyl A. It consists of thick, fan-shaped or rounded slices or of
ester R in methanol R and dry at 100 °C for 5 min; spray with roughly crushed discs. The darker outer surface is traversed
or dip briefly into a 50 g/L solution of macrogol 400 R in by tortuous longitudinal wrinkles. The paler cut surface
methanol R; heat at 100 °C for 5 min and examine in shows a dark cambial zone and xylem bundles distinctly
ultraviolet light at 365 nm. aligned in radial rows. The central cylinder shows fine
Results See below the sequence of zones present in the concentric striations. Seen under a lens, the cut surface
chromatograms obtained with the reference solution and the presents yellow or brownish-red granules.
test solution. Furthermore, other faint zones may be present B. Microscopic examination (2.8.23). The powder is
in the chromatogram obtained with the test solution. brownish-yellow. Examine under a microscope using chloral
characters (Figure 1095.-1): fragments of cork consisting of
yellowish-brown, thin-walled cells (surface view [BJ,
IV-204 Devil's Claw 2023
transverse section [Cl); fragments of cortical parenchyma solution shows other distinct zones, mainly above the zone
consisting of large, thin-walled cells [E, K, N, P], sometimes due to harpagoside. Furthermore, other faint zones may be
containing reddish-brown granular inclusions and isolated present in the chromatogram obtained with the test solution.
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 [Al and rare small needles of -- --
calcium oxalate in the parenchyma. The powder may also Harpagoside: a quenching zone A quenching zone: harpagoside
show rectangular or polygonal sclereids with dark reddish-
brown contents [H, TI- With a solution of phloroglucinol in -- --
hydrochloric acid, the parenchyma turns green. Reference solution Test solution
Detection B Spray with a 10 g/L solution of phloroglucinol R

in ethanol (96 per cent) R and then with hydrochloric acid R;
heat at 80 °C for 5-10 min and examine in daylight.
test solution; the chromatogram obtained with the test
solution also shows several yellow or brown zones above the
zone due to harpagoside. Furthermore, other faint zones may
solution.
Top of the plate
-- --
Harpagoside: a green zone A green zone (harpagoside)
-- --
A yellow zone
A light green zone
Fructose: a yellowish-grey zone A yellowish-grey zone may be
~
present (fructose)
A brown zone
p TESTS
Starch
Examine the powdered herbal drug (355) (2.9.12) under a
microscope using water R. Add iodine solution Rl. No blue
Figure 1095.-1. - Illustration for identification test B of powdered colour develops.
herbal drng of devil's claw root Loss on drying (2.2.32)
C. Thin-layer chromatography (2.2.27). powdered herbal drug (355) (2.9.12) by drying in an oven at
Test solution Heat 1.0 g of the powdered herbal drug (355) 105 °C.
(2. 9.12) with 10 mL of methanol R on a water-bath at 60 °C
Total ash (2.4.16)
for 10 min. Filter and reduce the filtrate to about 2 mL
under reduced pressure at a temperature not exceeding
40 °C. ASSAY
Reference solution Dissolve 1 mg of harpagoside R and 2.5 mg Liquid chromatography (2.2.29).
of fructose R in 1 mL of methanol R. Test solution To 0.500 g of the powdered herbal drug (355)
Plate TLC silica gel F 254 plate R (5-40 µm) [or TLC silica gel (2. 9.12) add 100.0 mL of methanol R. Shake for 4 h and
F254 plate R (2-10 µm)]. filter through a membrane filter (nominal pore size 0.45 µm).
Mobile phase water R, methanol R, ethyl acetate R Reference solution Dissolve the contents of a vial of
(8:15:77 VIVIV). harpagoside CRS in methanol Rand dilute to 10.0 mL with
the same solvent.
Application 20 µL [or 5 µL) as bands.
Column:
Development Over a path of 10 cm [or 7.5 cm].
Drying In a current of warm air.
=
- size: l 0.10 m, 0 4.0 mm; =
Detection A Examine in ultraviolet light at 254 nm. chromatography R (5 µm).
Results A See below the sequence of zones present in the Mobile phase methanol R, water for chromatography R
chromatograms obtained with the reference solution and the (50:50 V/V).
test solution; the chromatogram obtained with the test
2023 Digitalis Leaf IV-205
Flow rate 1.5 m!Jmin. Top of the plate

Detection Spectrophotometer at 278 run.
- - --
Injection 10 µL.
Harpagoside: a green zone A green zone (harpagoside)
Run time 3 times the retention time of harpagoside.
Retention time Harpagoside = about 7 min. - - --
Calculate the percentage content of harpagoside using the A yellow zone

A light green zone
Fructose: a yellowish-grey zone A yellowish-grey zone may be

present (fructose)
A brown zone
AI area of the peak due to harpagoside in the chromatogram
obtained with the test solution; Reference solution Test solution
area of the peak due to harpagoside in the chromatogram
mass of the herbal drug to be examined used to prepare the test ASSAY
solution, in grams; Liquid chromatography (2.2.29).
mass of harpagoszde CRS in the reference solution, in grams.
Test solution Introduce 0.350 g of the extract to be
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
examined into a 100 mL volumetric flask, add 90 mL of
methanol R and sonicate for 20 min. Cool to room
temperature, dilute to 100.0 mL with methanol Rand filter
Reference solution Dissolve the contents of 1 vial of
Devil's Claw Dry Extract harpagosiae CRS in methanol Rand dilute to 10.0 mL with
the same solvent.
Column:
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
- size: l = 0.10 m, 0 = 4.0 mm;
DEFINITION - stationary phase: octadecylsilyl silica gel for chromatography R
Dry extract obtained from Devil's claw root (1095). (5 µm).
Content Mobile phase methanol R, water R (50:50 V/V).
Minimum 1.5 per cent of harpagoside (C 24 H 300 11 ; Flow rate 1.5 mIJmin.
Mr 494.5) (dried extract). Detection Spectrophotometer at 278 run.
PRODUCTION Injection 10 µL.
The extract is produced from the herbal drug by an Run time 3 times the retention time of harpagoside.
appropriate procedure using either water or a hydroalcoholic Retention time Harpagoside = about 7 min.
solvent that is at most equivalent in strength to ethanol
Calculate the percentage content of harpagoside using the
(95 per cent V/V).
CHARACTERS
Appearance
Light brown powder.
IDENTIFICATION area of the peak due to harpagoside in the chromatogram
Thin-layer chromatography (2.2.27). obtained with the test solution;
area of the peak due to harpagoside in the chromatogram
Test solution To 1.0 g of the extract to be examined add obtained with the reference solution;
10 mL of methanol R and heat in a water-bath at 60 °C for mass of the extract to be examined used to prepare the test
10 min. Cool and filter. solution, in grams;
mass of harpagoside contained in I vial of harpagoside CRS, in
Reference solution Dissolve 1.0 mg of harpagoside R and grams.
2.5 mg of fructose R in 1.0 mL of methanol R. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Plate TLC si1ica gel plate R.
(8:15:77 VIVIV).
Digitalis Leaf
Development Over a path of 10 cm. (Ph. Bur. monograph O11 7)
Drying In a current of warm air. When Powdered Digitalis is prescribed or demanded,
Detection Spray with a 10 g/L solution of phloroglucinol R in material complying with the requirements below with the
ethanol (96 per cent) R and then with hydrochloric acid R; heat exception ofldentification test A and the test for Foreign
at 80 °C for 5-10 min and examine in daylight. maner shall be dispensed or supplied.
Results See below the sequence of zones present in the PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried leaf of Digitalis purpurea L.
Content
Minimum 0.3 per cent of cardenolic glycosides, expressed as
digitoxin (Mr 765) (dried drug).
IV-206 Digitalis Leaf 2023
CHARACTERS
Faint but characteristic odour.
The whole leaf is about 10-40 cm long and 4-15 cm wide.
The lamina is ovate lanceolate or broadly ovate. The winged
petiole is from 1/4 as long as to equal in length to the
lamina.
IDENTIFICATION
A. The leaf is brittle and often occurs broken. The upper
surface is green and the lower surface is greyish-green.
The apex is subacute and the margin is irregularly crenate,
dentate or serrate. The base is decurrent. The venation is
pinnate, the lateral veins being prominent especially on the
lower surface, leaving the midrib at about 45° and
anastomosing near the margin; a veinlet terminates in each
tooth of the margin and the lower veins run down the winged
petiole. The upper surface is rugose and pubescent; the lower
$
surface shows a network of raised veinlets and is densely
pubescent.
B. Microscopic examination (2.8.23). Examine under a
fragments of the upper epidermis (surface view [K, L]), with Ka
cells with a smooth cuticle and anticlinal walls that are
slightly thickened, are straight or slightly sinuous, and may
show slight beading and pitting [La] and sometimes scars of
covering trichomes [Ka], accompanied by underlying palisade
parenchyma [Lb]; fragments of the lower epidermis (surface
view [G]), with markedly sinuous cells and anomocytic
stomata (2.8.3) [Ga]; trichomes are of 2 types: a) uniseriate
Figure 0 117 .-1. - Illustration for identification test B of powdered
covering trichomes with blunt apex, usually consisting of 3-5
herbal drug of digitalis leaf
cells [H, TI, often with 1 or more collapsed cells Ua], walls
mostly finely warty or faintly striated; b) glandular trichomes Results The chromatogram obtained with the reference
usually with a unicellular [C, D], sometimes a multicellular, solution shows a zone of light blue fluorescence in the lower
uniseriate [A, B, E] stalk and a unicellular head [A, B, C, E] part of the chromatogram, due to purpureaglycoside B, and,
or bicellular head (side view [D], surface view [F]) or just above it, a zone of brownish-yellow fluorescence due to
exceptionally a tetracellular head. purpureaglycoside A; a zone of light blue fluorescence, due to
C. Thin-layer chromatography (2.2.27). gitoxin, appears in the middle of the chromatogram and
Test solution To 1.0 g of the powdered herbal drug (180) above it a zone of brownish-yellow fluorescence, due to
(2. 9.12) add a mixture of 20 mL of ethanol digitoxin; the zones in the chromatogram obtained with the
(50 per cent V/V) R and 10 mL of lead acetate solution R. Boil test solution are similar in position, colour and size to the
for 2 min, allow to cool and centrifuge. Shake the zones in the chromatogram obtained with the reference
supernatant solution with 2 quantities, each of 15 mL, of solution. Other zones of fluorescence may also appear in the
chloroform R; separate the 2 layers by centrifugation if chromatogram obtained with the test solution.
necessary. Dry the chloroform layers over anhydrous sodium D. Evaporate 5 mL of the chloroformic solution obtained in
sulfate R and filter. Evaporate 10 mL of the solution to identification test C to dryness on a water-bath. To the
dryness on a water-bath and dissolve the residue in 1 mL of residue add 2 mL of dinitrobenzoic acid solution R and 1 mL
a mixture of equal volumes of chloroform R and methanol R. of 1 M sodium hydroxide. A reddish-violet colour develops
Reference solution Dissolve 5 mg of purpureaglycoside A CRS, within 5 min.
2 mg of purpureaglycoside B CRS, 5 mg of digitoxin R and E. Evaporate 5 mL of the chloroformic solution obtained in
2 mg of gitoxin R in a mixture of equal volumes of identification test C to dryness on a water-bath. To the
chloroform R and methanol R, then dilute to 10 mL with the residue add 3 mL of xanthydrol solution R and heat on a
same mixture of solvents. water-bath for 3 min. A red colour develops.
Plate TLC silica gel G plate R. TESTS
Mobile phase water R, methanol R, ethyl acetate R Digitalis lanata Ehrh.
(7.5:10:75 VIVIV). The presence of leaves with few or no trichomes and with
Application 20 µLas bands of 2 cm by 0.3 cm. parallel venation or the presence of cells of the abaxial
Development Over a path of 10 cm. epidermis with beaded anticlinal walls and of cells of the
adaxial epidermis with numerous stomata indicates
Drying Until the solvents have evaporated.
adulteration by Digitalis lanata Ehrh.
Detection Treat with a mixture of 2 volumes of a 10 g/L
solution of chloramine R and 8 volumes of a 250 g/L solution Loss on drying (2.2.32)
of trichloroacetic acid R in ethanol (96 per cent) R, then heat at Maximum 6.0 per cent, determined on 1.000 g of the
100-105 °C for 10 min; examine in ultraviolet light at powdered herbal drug (355) (2.9.12) by drying in an oven at
365 nm. 105 °C.
2023 Dioscorea Nipponica Rhizome IV-207
Total ash (2.4.16) Content of carvone

Maximum 12.0 per cent. 43.0 to 63.0% w/w when determined by the following
Ash insoluble in hydrochloric acid (2.8.1) method. To 1.5 gin a glass-stoppered tube (approximately
Maximum 5.0 per cent. 150 mm x 25 mm) add 10 mL of a solution prepared in the
following manner. Dissolve 7.0 g of hydroxylamine
ASSAY hydrochloride in 90 mL of ethanol (90%), warming gently if
Shake 0.250 g of the powdered herbal drug (180) (2.9.12) necessary, add 1.6 mL of dimethyl yelww solution and
with 50.0 mL of water R for 1 h. Add 5.0 mL of a 150 g/L sufficient lM potassium hydroxide in ethanol (90%) to produce
solution of lead acetate R, shake, and after a few minutes add a pure yellow colour and dilute to 100 mL with ethanol
7 .5 mL of a 40 g/L solution of disodium hydrogen phosphate (90%). Titrate with lM potassium hydroxide in ethanol (90%)
dodecahydrate R. Filter through a pleated paper filter. Heat VS until the red colour changes to yellow. Place the tube in a
50.0 mL of the filtrate with 5 mL of hydrochloric acid water bath at 75° to 80° and, at 5-minute intervals, neutralise
(150 g/L HCl) under a reflux condenser on a water-bath for with 1M potassium hydroxide in ethanol (90%) VS; after
1 h. Transfer to a separating funnel, rinse the flask with 40 minutes complete the titration to the full yellow colour of
2 quantities, each of 5 mL, of water R and shake with the indicator. This procedure gives an approximate value for
3 quantities, each of 25 mL, of chloroform R. Dry the the carvone content of the oil. Repeat the procedure, using as
combined chloroform layers over anhydrous sodium sulfate R the colour standard for the end point of the titration the
and dilute to 100.0 mL with chloroform R. Evaporate titrated liquid of the first determination with the addition of
40.0 mL of the chloroformic solution to dryness, dissolve the 0.5 mL of lM potassium hydroxide in ethanol (90%) VS.
residue in 7 mL of ethanol (50 per cent Vlv,) R, add 2 mL of Calculate the content of carvone from the second
dinitrobenzoic acid solution R and 1 mL of 1 M sodium determination. Each mL of 1M potassium hydroxide in ethanol
hydroxide. At the same time prepare a reference solution as (90%) VS is equivalent to 151.4 mg of carvone, C 10H140.
follows. Dissolve 50.0 mg of digi,toxin CRS in ethanol
(96 per cent) R and dilute to 50.0 mL with the same solvent. STORAGE
Dilute 5.0 mL of the solution to 50.0 mL with ethanol Dill Oil should be kept in a well-filled container and
(96 per cent) R. To 5.0 mL of the resulting solution add protected from light. It darkens in colour on storage.
25 mL of water Rand 3 mL of hydrochloric acid (150 g/L
HCl). Heat the 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 Dioscorea Nipponica Rhizome
540 nm several times during the first 12 min until the
maximum is reached, using as the compensation liquid a (Ph. Bur. monograph 2890)
mixture of 7 mL of ethanol (50 per cent V!v,) R, 2 mL of Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
dinitrobenzoic acid solution R and 1 mL of 1 M sodium
hydroxide. DEFINITION
Dried, whole or fragmented, scraped rhizome of Di.oscorea
From the absorbances measured and the concentrations of
nipponica Makino, with roots removed.
the solutions, calculate the content of cardenolic glycosides,
expressed as digitoxin. Content
Minimum 1.0 per cent of diosgenin (C 27 H 42 0 3; Mr 414.6)
STORAGE (dried drug).
Protected from moisture.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
IDENTIFICATION
A. Whole drug. The rhizome is subcylindrical, slightly curved,
up to 15-20 cm long and 1.0-1.5 cm in diameter. The outer
surface is yellowish-white or brownish-yellow, irregularly and
longitudinally furrowed, bearing spinous remains of roots and
Dill Oil protuberant stem scars.
DEFINITION Fragmented drug Transverse slices of the rhizome, oblique or
Dill Oil is obtained by distillation from the dried ripe fruits of more or less longitudinal, circular, oval or elongated, whole
Anethum graveolens L. or broken, up to 0.7 cm thick. The outer surface is light
CHARACTERISTICS brown or yellowish-brown, irregularly and longitudinally
furrowed; spinous remains of roots and protuberant stem
A clear, colourless or pale yellow liquid, visibly free from
scars may be visible. A transverse section is yellowish-white,
water; odour, characteristic of the crushed fruit.
can easily be imprinted with one's nail due to an abundance
TESTS of starch, and shows numerous light yellowish-brown pits
Optical rotation corresponding to the vascular bundles.
+ 70° to +80°, Appendix V F. B. Microscopic examination (2.8.23). The powder is whitish
Refractive index or light yellow. Examine under a microscope using chloral
1.481 to 1.492, Appendix VE. hydrate soluti.on R. The powder shows the following diagnostic
Solubility in ethanol characters (Figure 2890.-1): vessels up to 50 µm in diameter
Soluble, at 20°, in 1 volume or more of ethanol (90%) and in with numerous fine, dense and elliptical pits [G]; numerous
10 volumes or more of ethanol (80%), Appendix X M. fragments of parenchyma with polyhedral or ovoid cells with
slightly thickened and pitted walls (transverse section [BJ,
Weight per mL longitudinal section [CJ); cells containing raphides of calcium
0.895 to 0.910 g, Appendix VG. oxalate up to 110 µm long [D]; free needles of calcium
oxalate [A); rare cork fragments with polyhedral cells (surface
view [F]). Examine under a microscope using a
IV-208 Dioscorea Nipponica Rhizome 2023
50 per cent V/V solution of glycerol R. The powder shows Top of the plate
extremely abundant starch granules, simple, irregular, ovoid,
3 olive-green zones
oblong, sub-triangular, with a maximum dimension of up to
30 µm; the hilum is usually eccentric and slit-shaped [E]. -- --
C. Thin-layer chromatography (2.2.27). Aescin: a violet zone
A green zone
(2.9.12) add 5 mL of methanol R. Sonicate for 10 min.
Centrifuge and use the supernatant. A faint to strong green zone
Reference solutwn Dissolve 1 mg of aescin R and 1 mg of Glucose: a yellow or brown zone A yellowish-brown zone
glucose R in 1 mL of methanol R.
A faint brown zone
Mobile phase water R, methanol R, methylene chloride R -- --
(10:50:64 VIVIV). A yellow zone
Drying In air.
Detection Treat with anisaldehyde solution R, heat at 100 °C TESTS
for 3 min, allow to cool for 10 min and examine in daylight. Loss on drying (2.2.32)
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
(2. 9.12) in a round-bottomed flask add 40 mL of a
15 per cent V/V solution of sulfuric acid R. Heat in a water-
bath under a reflux condenser for 3 h. Allow to cool and
filter. Wash the residue with water R until the filtrate is
neutral. Sonicate the residue for 30 min in 80 mL of
methanol Rand filter. Wash the residue with 20 mL of
methanol R, combine the filtrate and the washing and dilute
Reference solutwn (a) Dissolve 5.0 mg of dwsgenin CRS in
Reference solutwn (b) Dissolve 2 mg of (25R)-spirost-5-en-3-
one CRS in reference solution (a) and dilute to 10.0 mL with
the same solution.
Column:
=
- size: l 0.15 m, 0 =
4.6 mm;
- statwnary phase: end-capped octadecylsilyl silica gel for
Mobile phase water for chromatography R, acetonitrile for
Figure 2890.-1. - Illustration for identification test B of powdered chromatography R (15:85 V/V).
herbal drug of Dwscorea nipponica rhizome Flow rate 1.5 mUmin.
chromatograms obtained with the reference solution and the Injection 5 µL.
test solution. Furthermore, other faint zones may be present Run time 20 min.
in the chromatogram obtained with the test solution. Retention time Diosgenin = about 8 min; (25R)-spirost-5-en-
3-one = about 10 min.
- resolution: minimum 1. 9 between the peaks due to
diosgenin and (25R)-spirost-5-en-3-one.
Calculate the percentage content of diosgenin using the
2023 Dioscorea Oppositifolia Rhizome IV-209
A, area of the peak due to diosgenin in the chromatogram obtained

area of the peak due to diosgenin in the chromatogram obtained
m, mass of the herbal drug to be examined used to prepare the test
solution, in grams;
mass of dwsgenin CRS used to prepare reference solution (a), in
grams;
p percentage content of diosgenin in dwsgenin CRS.
Dioscorea Oppositifolia Rhizome

(Ph. Eur. mMograph 2473)
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried, scraped, whole or fragmented rhizome of Dwscorea
oppositifolia L. (syn. Dwscorea opposita Thunb.), with roots
removed, collected in winter when the stem and leaves are
withered.
IDENTIFICATION
A. The rhizome occurs in subcylindrical pieces, sometimes
flattened, about 15-30 cm long and 1.5-6 cm thick; the outer
surface is yellowish-white or pale yellow, longitudinally
furrowed and wrinkled, and bearing slit-shaped root scars Figure 2473.-1. - lllustratwnfor identificatwn test B of powdered
with occasional patches of brownish cork. The fragmented herbal drug of Dwscorea oppositifolia rhizome
rhizome occurs in whole or fragmented slices, about
0.2-0.5 cm thick; some slices still bear slit-shaped root scars.
The texture is heavy, compact and tough; the fracture is Top of the plate
white and starchy.
3-5 violet zones
B. Microscopic examination (2.8.23). The powder is whitish
or yellowish-white. Examine under a microscope using chloral -- --
hydrate solutwn R. The powder shows the following diagnostic
Aescin: a violet zone A violet zone
characters (Figure 2473.-1): reticulate or pitted vessels
(longitudinal section [C, E]); fragments of xylem (transverse Glucose: a yellow or green zone A yellow or brown zone
section [G]) consisting of vessels up to 80 µm in diameter
--- --
[Ga] and ligneous parenchyma with distinctly pitted cells
[Gb]; numerous fragments ofparenchyma consisting of A violet zone
polyhedral or ovoid cells with thin walls [BJ; cells containing Reference solution Test solution
calcium oxalate raphides up to 250 µm long and up to 5 µm
in diameter [F]; free needles of calcium oxalate [A]. Examine
under a microscope using a 50 per cent V/V solution of TESTS
glycerol R. The powder shows extremely abundant starch Dioscorea bulbifera L
granules [D], simple, ovoid or oblong, flattened with Thin-layer chromatography (2.2.27).
rounded extremities, with a maximum dimension of up to Test solutwn To 0.5 g of the powdered herbal drug (355)
50 µm; the hilum is usually eccentric and punctiform; rare 2- (2. 9.12) add 5 mL of methanol R. Sonicate for 10 min.
to 4-compound starch granules may be present. Centrifuge and use the supernatant.
C. Examine the chromatograms obtained in the test for Reference solutwn Dissolve 1 mg of aescin R and 3 mg of
Dwscorea bulbifera L. glucose R in 7 mL of methanol R.
Results See below the sequence of zones present in the Plate TLC silica gel plate R (2-10 µm).
Mobile phase water R, methanol R, methylene chloride R
(8:40:52 VIVIV).
Drying In air.
Detection Treat with anisaldehyde solutwn R, heat at 100 °C
for 3 min, allow to cool for 10 min and examine in daylight.
shows no brownish-green or orange-brown zones in the
upper third of the chromatogram.
IV-210 Dog Rose 2023
Loss on drying (2.2.32) cluster crystal of calcium oxalate Ub]; scattered cluster
Maximum 12.0 per cent, determined on 1.000 g of the crystals of calcium oxalate [F].
105 °C for 2 h.
Total ash (2.4.16)
Ash insoluble in hydrochloric acid (2.8. 1)
Extractable matter
Minimum 7.0 per cent (dried drug).
25 mL of water R, shake for 6 h and allow to macerate for
18 h. Filter, evaporate the filtrate to dryness on a water-bath
under reduced pressure and dry in an oven at 100-105 °C for
2 h. The residue weighs a minimum of 70 mg.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Dog Rose
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Rose hips made up of the receptacle and the remains of the
dried sepals of Rosa canina L., Rosa pendulina L. and other
Rosa species, with the achenes removed.
Content
Minimum 0.3 per cent of ascorbic acid (C 6 H 8 0 6 ; Mr 176.1)
(dried drug).
Figure 1510. -1. - Illustration for identification test B of powdered
IDENTIFICATION herbal drug of dog rose
A. It consists of fragments of the fleshy, hollow, urceolate
receptacle, bearing the remains of the reduced sepals, light C. Thin-layer chromatography (2.2.27).
pink or orange-pink, the convex outer surface shiny and Test solution To 5 g of the powdered herbal drug (355)
strongly wrinkled; bearing on its lighter inner surface (2. 9.12) add 25 mL of ethanol (96 per cent) R, shake for
abundant bristle-like hairs. 30 min and filter.
B. Microscopic examination (2.8.23). The powder is orange- Reference solution Dissolve 10 mg of ascorbic acid R in 5 mL
yellow. Examine under a microscope using chloral hydrate of ethanol (60 per cent V/V) R.
solution R. The powder shows the following diagnostic Plate TLC silica gel F254 plate R.
characters (Figure 1510.-1): numerous fragments of the outer Mobile phase acetone R, glacial acetic acid R, methanol R,
surface of the receptacle (transverse section [BJ, surface toluene R (5:5:20:70 V/V/V/V).
view [DJ) consisting of the outer epidermis with irregularly
Application 20 µL of the test solution and 2 µL of the
thickened polyhedral cells [Ba] and a thick cuticle, sometimes
reference solution.
accompanied by parenchyma [Bb]; fragments of the inner
epidermis of the receptacle (transverse section [C], surface Development Over a path of 15 cm.
view [G]) with slightly wavy cells [Ca, Ga] and lignified Drying In air.
bases of the covering trichomes [Cc, Gb], covered in a finely Detection A Examine in ultraviolet light at 254 nm.
wrinkled cuticle around the basal cell of the trichomes, Results A The chromatogram obtained with the test solution
usually accompanied by the inner layers of the shows a quenching zone similar in position to the principal
parenchyma [Cd, Ge]; fragments of the inner layers of the zone in the chromatogram obtained with the reference
parenchyma [HJ, whose cells almost all contain crystals of solution.
calcium oxalate (mostly cluster crystals [Ce, Gd, Ha] but
Detection B Spray with a 0.2 g/L solution of
also prisms [Cb, Ge, Hb]); scattered lignified cells,
dichlorophenolindophenol, sodium salt R in ethanol
isodiametric or oval, with thickened and pitted walls
(96 per cent) R and examine in daylight.
corresponding to the trichome bases [E]; abundant
unicellular trichomes [A], up to 2 mm long and 30-45 µm Results B The chromatogram obtained with the test solution
thick, tapering towards each end (apical end [Aa], distal end shows a white zone on a pink background (ascorbic acid)
[Ab, K]), with walls heavily thickened and with a waxy similar in position and colour to the principal zone in the
cuticle that may show fissures in a spiral arrangement [Ac, chromatogram obtained with the reference solution; the
Ka]; fragments of parenchyma [J] consisting of large ovoid chromatogram also shows an intense orange-yellow zone near
cells with irregularly thickened walls and granular contents the solvent front and a yellow zone in the upper third
with small oil droplets Ua], and small cells each containing a (carotenoids).
2023 Drynaria Rhizome IV-211
TESTS
Drynaria Rhizome
Maximum 1 per cent. (Ph. Bur. monograph 2563)
Loss on drying (2.2.32) ~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
powdered herbal drug (355) (2. 9.12) by drying in an oven at DEFINITION
105 cc. Dried rhizome of Drynaria fortunei (Kunze) J. Sm. The
Total ash (2.4.16) ramenta may be removed.
Maximum 7.0 per cent. Content
Minimum 0.5 per cent of naringin (C 27 H 32 0 14; Af, 580.5)
ASSAY
(dried drug).
Test solution In a round-bottomed flask, weigh 0.500 g of
the freshly powdered herbal drug (710) (2. 9.12). Add a IDENTIFICATION
solution of 1.0 g of oxalic acid R in 50.0 mL of methanol R. A. Long, flattened, slat-shaped rhizome, often curved and
Boil under a reflux condenser for 10 min, and cool in iced branched, 5-15 cm long and 1-1.5 cm thick. The surface is
water until the temperature reaches 15-20 °C. Filter. either completely covered in scaly, dark brown hairs (rhizome
Transfer 2.0 mL of the filtrate to a 50 mL conical flask. with ramenta) or glabrous with dark brown dots (rhizome
Add successively, with gentle shaking after each addition, without ramenta). The upper surface and both sides show
2.0 mL of dichlorophenolindophenol standard solution Rand circular frond scars, rarely the frond bases. The lower surface
then, exactly 60 s later, 0.5 mL of a 100 g/L solution of shows scars or the remains of fibrous roots. The texture is
thiourea R in ethanol (50 per cent VIJ,) Rand 0.7 mL of light, fragile, easily broken. The section is reddish-brown; the
dinitrophenylhydrazine-sulfuric acid solution R. Heat under a steles form a ring of small yellow dots.
reflux condenser at 50 °C for 75 min, and place immediately B. Microscopic examination (2.8.23). The powder is reddish-
in iced water for 5 min. Add dropwise 5.0 mL of a mixture brown. Examine under a microscope using chloral hydrate
of 12 mL of water R and 50 mL of su[furic acid R, taking care solution R. The powder shows the following diagnostic
to carry out the addition over a period of minimum 90 s and characters (Figure 2563.-1): numerous polyhedral
maximum 120 s while maintaining vigorous stirring in iced parenchymatous cells [A] or fragments of parenchymatous
water. Allow to stand for 30 min at room temperature and cells [E, F, G] with slightly and regularly thickened pitted
measure the absorbance (2.2.25) at 520 nm using solution A walls; scalariform vessels of variable diameter up to 60 µm
as compensation liquid. [D]; fragments of reddish-brown scaly hairs forming a tissue
Solution A Treat 2.0 mL of the filtrate obtained during the consisting of cells with thickened, somewhat sinuous walls
preparation of the test solution as described but adding the [BJ; the margins of the scaly hairs [C] have elongated cells
dinitrophenylhydrazine-sulfuric acid solution R just before the with rigid walls [Ca] and expansions, usually bicellular [Cb].
absorbance is measured.
Reference solution Dissolve 40.0 mg of ascorbic acid Rina
freshly prepared 20 g/L solution of oxalic acid R in
Dilute 5.0 mL of this solution to 100.0 mL with a freshly
prepared 20 g/L solution of oxalic acid R in methanol R. Treat
2.0 mL of the solution as described above for the filtrate
obtained during the preparation of the test solution. Measure
the absorbance (2.2.25) at 520 nm using solution B as the
compensation liquid.
Solution B Treat 2.0 mL of the reference solution as
described above for solution A.
Calculate the percentage content of ascorbic acid from the
A1 absorbance of the test solution;

A2 absorbance of the reference solution;
m1 mass of the substance to be examined, in grams;
m2 mass of ascorbic acid used, in grams.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

herbal drug of drynaria rhizome

IV-212 Dwarf Lilyturf Tuber 2023
Test solution To 0.5 g of the powdered herbal drug (355) Mobile phase acetonitrile R, 0.4 per cent VIV solution of
(2.9.12) add 5 mL of methanol Rand sonicate for 10 min. acetic acid R (18:82 V/V).
Cool, centrifuge and use the supernatant. Fww rate 1.0 mUmin.
Reference solution Dissolve 1 mg of naringin R and I mg of Detection Spectrophotometer at 283 nm.
hyperoside R in 2 mL of methanol R. Injection 20 µL of the test solution and reference
Plate TLC silica gel plate R (2-10 µm). solutions (b) and (c).
Mobile phase acetic add R, anhydrous formic acid R, water R, Run time Twice the retention time of naringin.
ethyl acetate R (11:11:26:100 VIVIVIV).
Retention time Naringin = about 9 min;
Application 10 µL as bands of 8 mm. neohesperidin = about 12 min.
Development Over a path of 6 cm. System suitability Reference solution (b):
Drying In air. - resolution: minimum 5.0 between the peaks due to
Detection Treat with aluminium chloride reagent R; examine naringin and neohesperidin.
in ultraviolet light at 365 nm. Calculate the percentage content of naringin using the
Results See below the sequence of zones present in the following expression:
A1 area of the peak due to naringin in the chromatogram obtained
Top of the plate with the test solution;
A2 area of the peak due to naringin in the chromatogram obtained
with reference solution (c);
-- -- solution, in grams;
Hyperoside: a yellow zone m2 mass of naringin CRS used to prepare reference solution (a), in
grams;
Naringin: a bluish-white zone A bluish-white zone (naringin) p percentage content of naringin in naringin CRS.
A bluish-white zone _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
-- --
Reference solution Test solution Dwarf Lilyturf Tuber

TESTS
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
powdered herbal drug (355) (2.9.12) by drying in an oven at Dried root tuber of Ophiopogon japonicus (Thunb.) Ker
105 °C for 2 h. Gaw!., with the spindly roots removed.
Maximum 7.0 per cent. Minimum O.12 per cent of total saponins, expressed as
Ash insoluble in hydrochloric acid (2.8.1) ruscogenin (C 27H 42 0 4 ; Mr 430.6) (dried drug).
Maximum 2.0 per cent. IDENTIFICATION
ASSAY A. It is fusiform with both ends slightly tapered, 1.5-3 cm
Liquid chromatography (2.2.29). long and 3-6 mm in diameter. The outer surface is whitish-
Test solution Disperse O.100 g of the powdered herbal drug yellow, fine and longitudinally wrinkled. The texture is hard,
(355) (2. 9.12) in a 50 per cent V/V solution of methanol R the fracture is yellowish-white and translucent, and the
and dilute to 10.0 mL with the same solvent. Weigh, vascular system appears as a small ring in the centre.
sonicate for 45 min. Allow to cool, weigh and compensate B. Microscopic examination (2.8.23). The powder is
the loss of solvent with a 50 per cent VIV solution of yellowish-white. Examine under a microscope using chloral
methanol R, shake well. Filter through a membrane filter hydrate solution R. The powder shows the following diagnostic
(nominal pore size 0.45 µm). characters (Figure 3000.-1): numerous fragments of
Reference solution (a) Dissolve 10.0 mg of naringin CRS in parenchyma consisting of suborbicular or elliptical cells [A];
methanol Rand dilute to 20.0 mL with the same solvent. different sized raphides of calcium oxalate, free [F] or
included in parenchymatous cells [Aa], either fine, 12-65 µm
Reference solution (b) Dissolve 5.0 mg of neohesperidin R in
long [Aa], or thicker, up to 130 µm long [F]; sclereids,
reference solution (a) and dilute to 10.0 mL with reference
usually in groups (surface view [E]), subsquare, rectangular
solution (a).
or polygonal (20-57 µm in diameter) with distinct, dense
Reference solution (c) Dilute 1.0 mL of reference solution (a) channels; fragments of the endodermis with rectangular or
to 10.0 mL with methanol R. elongated cells, with channelled and pitted walls (surface
Column: view [C]); fragments (transverse section [BJ) showing the
- size: l = 0.15 m, 0 = 4.6 mm; endodermis [Ba], sclereids with unevenly thickened
- stationary phase: octadecylsilyl silica gel for chromatography R walls [Bb] and parenchymatous cells [Be]; thin xylem
(5 µm). fibres [G], with oblique or square ends, slightly thickened
walls, and oblique, cross-shaped or V-shaped pits; fragments
2023 Dwarf Lilyturf Tuber IV-213
of xylem [D] consisting of spiral or reticulate vessels [Da] Results See below the sequence of zones present in the
and fibres [Db, De]. chromatograms obtained with reference solution (a) and the
with the test solution, other faint fluorescent zones may be
present.
Top of the plate
2 violet-red zones
-- --
Methylophiopogonanone A: a A brownish-yellow zone, faint to

brownish-yellow zone equivalent
-- --
I or 2 violet-red or reddish zones,
faint to equivalent (possibly
overlapping)
~-sitosterol: a violet-red zone A violet-red zone
A broad, intense violet-red zone
TESTS
Figure 3000.-1. - Illustratwn for identificatwn test B of powdered Maximum 15.0 per cent, determined on 1.000 g of the
herbal, drug of dwarf lilyturf tuber powdered herbal drug (355) (2.9.12) by drying in an oven at
105 °C for 2 h.
Total ash (2.4.16)
Test solutwn To 2.0 g of the powdered herbal drug (355) Maximum 2.5 per cent.
(2. 9. 12) add 20 mL of anhydrous ethanol R. Sonicate for
30 min, filter and evaporate to dryness. Dissolve the residue
in 0.5 mL of anhydrous ethanol R, centrifuge and use the
supernatant. ASSAY
Reference solutwn ( a) Dissolve 1 mg of fJ-sitosterol R and Test solutwn Introduce 1.20 g of the powdered herbal
1 mg of methylophiopogonanone AR in 2.0 mL of anhydrous drug (355) (2.9.12) into a 50 mL round-bottomed flask, add
ethanol R. 20.0 mL of methanol Rand weigh. Boil under a reflux
Reference solutwn (b) Mix 1.0 mL of reference solution (a) condenser for 2 h. Allow to cool to room temperature, weigh
and 3.0 mL of anhydrous ethanol R. again and adjust to the original mass with methanol R.
Mix thoroughly and filter through a membrane filter
Reference solutwn (c) Dilute 3 µL of isoeugenol R and 6 µL of (nominal pore size 0.22 µm). Evaporate 12.5 mL of the
methyleugenol R in 20 mL of toluene R.
filtrate to dryness under a current of nitrogen R. Dissolve the
Intensity markers ~-sitosterol and residue in 5 mL of water R and extract with 5 quantities,
methylophiopogonanone A. each of 5 mL, of butanol R saturated with water R using a
Plate TLC silica gel F254 plate R (2-10 µm). separating funnel. Combine the butanol extracts in a
Mobile phase ethyl acetate R, toluene R (10:90 V/V). separating funnel and wash with 2 quantities, each of
Applicatwn 5 µL as bands of 8 mm. 2.5 mL, of dilute ammonia RI. Evaporate to dryness under a
current of nitrogen R. Dissolve the residue in a mixture of
Development 70 mm from the lower edge of the plate. 20 volumes of water R and 80 volumes of methanol R and
Drying In a current of cold air for 5 min. dilute to 25.0 mL with the same mixture of solvents.
Detection Dip the plate in a 10 per cent V/V solution of Evaporate 2.5 mL of the solution to dryness under a current
sulfuric acid R in anhydrous ethanol R; heat the plate at 105 °C of nitrogen R. Dissolve the residue in perchloric acid R and
for 10 min and examine under white light. dilute to 10.0 mL with the same solvent. Transfer the
System suitability Reference solution (c): solution to a test tube, stopper and heat at 80 °C in a water-
- the chromatogram shows 2 distinct zones in the bath for 15 min. Cool to room temperature.
middle third; the lower zone (isoeugenol) and the Measure the absorbance (2. 2. 25) of the test solution at
upper zone (methyleugenol) show a violet-red 397 nm using perchloric acid R as the compensation liquid.
fluorescence. Calculate the percentage content of total saponins, expressed
as ruscogenin, using the following expression:
IV-214 Echinacea 2023
Ax 160
m X 196
i.e. taking the specific absorbance of ruscogenin to be 196.

solution, in grams.
Echinacea Angustifolia Root

(Narrow-leaved Coneflower Root, Ph. Bur. monograph
1821)
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried, whole or cut underground parts of Echinacea
angustifolia DC.
Content
Minimum 0.5 per cent of echinacoside (C 35H 46 O 20; Mr 787)
(dried drug).
IDENTIFICATION
First identification: A, B, C.
Second identification: A, B, D.
A. The root crown is up to about 30 mm in diameter and
shows only a few stem bases. The roots are not very
numerous, up to about 15 mm in diameter, cylindrical or Figure 1821. -1. - Illustration for identification test B of powdered
slightly tapering and sometimes spirally twisted; the outer herbal drug of narrow-leaved coneflower root
surface is pale brown to yellowish-brown, longitudinally
wrinkled or deeply furrowed. The fracture is short, dark
Top of the plate
brown with a radiate structure.
B. Microscopic examination (2.8.23). The powder is greyish- Caffeic acid: an intense blue zone
characters (Figure 1821.-1): narrow lignified fibres up to Cynarin: an intense greenish zone A greenish zone (cynarin)
about 800 µm in length and 50 µm in diameter joined
together in long bundles surrounded by black phytomelanin -- --
deposits [BJ; lignified vessels up to about 60 µm in diameter, -- --
with reticulate, scalariform or bordered-pitted thickenings [J, Echinacoside: an intense greenish An intense greenish zone
L]; abundant sclereids occurring singly [D, H] or, more zone (echinacoside)
usually, in groups of 2-10, mostly elongated to rectangular,
up to about 150 µm in length and 40 µm wide, with
intercellular spaces filled with black phytomelanin deposits Reference solution Test solution
[E, F]; fragments of oleoresin canals 80-150 µm in diameter,
with orange-yellow to reddish-brown contents [A]; fragments
of the outer layers of the roots with groups of squarish to D. Examine the chromatograms obtained in the assay.
rectangular cells, about 30-45 µm [C]; abundant fine-walled Results The chromatogram obtained with the test solution
pitted parenchyma (transverse section [K], longitudinal shows a principal peak due to echinacoside and a minor peak
section [G]). due to cynarin. Peaks due to caffeic acid, caftaric acid,
C. Examine the chromatograms obtained in the test for chlorogenic acid and cichoric acid are minor or may be
Echinacea purpurea. absent.
Results See below the sequence of fluorescent zones present TESTS
in the chromatograms obtained with the reference solution Foreign matter (2.8.2)
and the test solution. Furthermore, other faint dark blue Maximum 3 per cent.
fluorescent zones may be present between the zones of Echinacea purpurea
echinacoside and cynarin in the chromatogram obtained with Thin-layer chromatography (2.2.27).
the test solution. Test solution To 1.0 g of the powdered herbal drug (355)
(2.9.12) add 10 mL of methanol Rand sonicate for 5 min.
2023 Echinacea IV-215
2
6
3
' ' I I ' I I I I I I I I

2 4 6 8 10 12 14 16 18 min
I. caftaric acid 3. caffeic acid 5. echinacoside

2. chlorogenic acid 4. cynarin 6. cichoric acid
Figure 1821.-2. - Chromatogram for the assay of echinacoside in narrow-leaved coneflower root
Reference solution Dissolve 1 mg of echinacoside R, 1 mg of Test solution In a 200 mL volumetric flask place 0.500 g of
cynarin Rand 0.5 mg of cafjeic acid R in methanol R and the powdered herbal drug (355) (2.9.12) and add 80 mL of
dilute to 5 mL with the same solvent. ethanol (70 per cent V/V) R. Sonicate for 15 min and dilute to
Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel 200.0 mL with the solvent mixture. Mix the suspension and
F2s4 plate R (2-10 µm)]. allow to stand for a few minutes to allow visible solids to
Mobile phase anhydrous formic acid R, water R, methyl ethyl settle. Filter a suitable proportion of the solution through a
ketone R, ethyl acetate R (10:10:30:50 VIVIVIV). membrane filter (nominal pore size 0.45 µm) before
injection.
Application 25 µL [or 5 µL] of the test solution and 10 µL
[or 2 µL] of the reference solution, as bands. Reference solution Dissolve 10.0 mg of chlorogenic acid CRS
and 10.0 mg of cafjeic acid R in the solvent mixture, sonicate
Development Over a path of 15 cm [or 5 cm]. for 15 min and dilute to 10.0 mL with the solvent mixture.
Drying In a stream of cold air for about 10 min, then at Dilute 2.0 mL of the solution to 50.0 mL with the solvent
100-105 °C for 2 min. mixture.
Detection Treat the hot plate using a 5 g/L solution of Column:
diphenylboric acid aminoethyl ester R in ethyl acetate R; examine - size: l = 0.25 m, 0 = 4.6 mm;
in ultraviolet light at 365 nm after 30 min. - stationary phase: octadecylsilyl silica gel for chromatography R
Results The chromatogram obtained with the test solution (5 µm);
shows no greenish fluorescent zone just below the zone due - temperature: 35 °C.
to caffeic acid in the chromatogram obtained with the Mobile phase:
reference solution and no greenish fluorescent zone below the - mobile phase A: 0.1 per cent V/V solution of phosphoric
zone due to cynarin in the chromatogram obtained with the acid R;
reference solution. In the chromatogram obtained with the - mobile phase B: acetonitrile R;
test solution no zones apart from faint dark blue fluorescent
zones are visible between the zones due to echinacoside and Time Mobile phase A Mobile phase B
cynarin. (min) (per cent V/J!) (per cent V/J/)
Maximum 12.0 per cent, determined on 1.000 g of the 0 - 13 90 ➔ 78 10 ➔ 22
powdered herbal drug (355) (2.9.12) by drying in an oven at 13 - 14 78 ➔ 60 22 ➔ 40
105 °C for 2 h. 14 - 20 60 40
Total ash (2.4.16)
Maximum 9.0 per cent. Flow rate 1.5 mUmin.
Ash insoluble in hydrochloric acid (2.8.1) Detection Spectrophotometer at 330 nm.
Maximum 3.0 per cent. Injection 10 µL.
ASSAY Identification of peaks Use the chromatogram obtained with
Liquid chromatography (2.2.29). the reference solution to identify the peaks due to caffeic acid
and chlorogenic acid; use the chromatogram shown in Figure
Solvent mixture acetonitrile R, 0.1 per cent V/V solution of
1821.-2 to identify the peaks due to caftaric acid, cichoric
phosphoric acid R (10:90 V/V).
acid, cynarin and echinacoside.
Relative retention With reference to chlorogenic acid

(retention time= about 7 min): caftaric acid= about 0.8;
caffeic acid = about 1.5; cynarin = about 1.6;
echinacoside = about 1.7; cichoric acid= about 2.3.
chlorogenic acid and caffeic acid.
Calculate the percentage content of echinacoside using the
A1 X C2 X 100 X 2.221
A2 xC1
A1 area of the peak due to echinacoside in the chromatogram
C1 concentration of the test solution, in milligrams per millilitre;
C2 concentration of chlorogenic acid in the reference solution, in
milligrams per millilitre;
2.221 peak correlation factor between chlorogenic acid and
echinacoside.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Echinacea Pallida Root

(Pale Coneflower Root, Ph. Bur. monograph 1822)
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION Figure 1822.-1. - Illustration for identification test B of powdered

Dried, whole or cut underground parts of Echina,eea pallida herbal drug of pale coneflower root
(Nutt.) Nutt.
Content chromatograms obtained with the reference solution and the
Minimum 0.2 per cent of echinacoside (C 35 H 460 20; Mr 787) test solution. Furthermore, a faint zone close to the solvent
(dried drug). front may be present in the chromatogram obtained with the
IDENTIFICATION test solution.
A. The rhizome and roots are 4-20 mm in diameter,
cylindrical and sometimes spirally twisted; the outer surface is Top of the plate
reddish-brown to greyish-brown, longitudinally wrinkled or
deeply furrowed.
~~
~~
B. Microscopic examination (2.8.23). The powder is greyish- A greenish-brown to brown zone

brown to light yellow. Examine under a microscope using A yellow zone
diagnostic characters (Figure 1822.-1): short lignified fibres, A violet zone
100-300 µm in length, occurring singly or joined together in

long bundles, sometimes with black phytomelanin deposits
l3-Sitosterol: a violet to pink zone A violet to pink zone (P-sitosterol)
[G]; xylem vessels (longitudinal section [C, M], transverse
section [Kb]) up to about 50 µm in diameter with reticulate, N-lsobutyldodecatetraenarnide: a
scalariform or areolate thickenings sometimes accompanied greyish-blue zone
by ligneous fibres (longitudinal section [Ca], transverse
section [Ka]); abundant sclereids, occurring singly or in small
~~
~~
groups of not more than 10 with black phytomelanin A dark greyish-blue zone
deposits, varying considerably in shape from rounded [E] to
rectangular or irregular [D, TI, sometimes very elongated and
fibre-like and measuring up to 400 µm in length [F]; Reference solution Test solution
fragments of oleoresin canals up to 240 µm in diameter, with
orange-yellow contents [BJ; groups of squarish to rectangular D. Examine the chromatograms obtained in the assay.
cells of the outer layers [A]; abundant thin-walled and pitted
Results The principal peak in the chromatogram obtained
parenchyma [H]; small isolated fragments of phytomelanin
with the test solution is due to echinacoside. Peaks due to
[L].
caftaric acid, caffeic acid, cynarin, chlorogenic acid and
C. Examine the chromatograms obtained in the test for other cichoric acid are minor or may be absent.
Echinacea species and Parthenium integrifolium.
2023 Echinacea IV-21 7
2 3 4
' I I ' ' I ' I I I I I

2 4 6 8 10 12 14 16 18
I. caftaric acid 2. chlorogenic acid 3. caffeic acid 4. cynarin S. echinacoside 6. cichoric acid
Figure 1822.-2. - Chromatogram for the assay of echinacoside in pale coneflower root
TESTS ASSAY
Foreign matter (2.8.2) Liquid chromatography (2.2.29).
Maximum 3 per cent. Solvent mixture acetonitn?e R, 0.1 per cent V/V solution of
Other Echinacea species and Parthenium integrifolium phosphoric acid R (10:90 V/V).
Thin-layer chromatography (2.2.27). Test solution In a 200 mL volumetric flask place 0.500 g of
Test solution To 1.0 g of the powdered herbal drug (355) the powdered herbal drug (355) (2. 9.12) and add 80 mL of
(2. 9.12) add 10 mL of methylene chloride R and sonicate for ethanol (70 per cent Vlv,) R. Sonicate for 15 min and dilute to
5 min. Centrifuge and use the supernatant. 200.0 mL with the solvent mixture. Mix the suspension and
Reference solution Dissolve 1 mg of P-sitosterol R and a allow to stand for a few minutes to allow visible solids to
volume of N-isoburyldodecatetraenamide solution R settle. Filter a suitable proportion of the solution through a
corresponding to 1 mg of N-isobutyldodecatetraenamide R in membrane filter (nominal pore size 0.45 µm) before
methanol R and dilute to 5 mL with the same solvent. injection.
Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel Reference solution Dissolve 10.0 mg of chlorogenic acid CRS
F254 plate R (2-10 µm)]. and 10.0 mg of caffeic acid R in the solvent mixture, sonicate
for 15 min and dilute to 10.0 mL with the solvent mixture.
Mobile phase anhydrous formic acid R, cyclohexane R, ethyl
Dilute 2.0 mL of the solution to 50.0 mL with the solvent
acetate R, toluene R (3: 10:20:80 VIVIVIV).
mixture.
Application 25 µL [or 5 µL] of the test solution and 10 µL
Column:
[or 4 µL] of the reference solution, as bands.
- size: l =0.25 m, 0 =4.6 mm;
Development Over a path of 15 cm [or 5 cm]. - stationary phase: octadecylsilyl silica gel for chromatography R
Drying In a stream of cold air for about 10 min. (5 µm);
Detection Treat with anisaldehyde solution R and heat at - temperature: 35 °C.
105 °C for 3 min; examine in daylight. Mobile phase:
Results The chromatogram obtained with the test solution - mobile phase A: 0.1 per cent V/V solution of phosphoric
shows no greyish-blue zone at the position of acid R;
N-isobutyldodecatetraenamide in the chromatogram obtained - mobi7e phase B: acetonitrile R;
with the reference solution, and no blue zone at the position
of the violet zone due to ~-sitosterol in the chromatogram Time Mobile phase A Mobile phase B
obtained with the reference solution. (min) (per cent V/Jl) (per cent V/Jl)

0 - 13 90..., 78 10..., 22
13 - 14 78..., 60 22..., 40
14 - 20 60 40
105 °C for 2 h.
Total ash (2.4.16)
Maximum 2.0 per cent. Injection 10 µL.
Identification of peaks Use the chromatogram obtained with sometimes accompanied by vascular bundles; fragments of
the reference solution to identify the peaks due to caffeic acid leaf epidermis (surface view [A, C, N]) with more or less
and chlorogenic acid; use the chromatogram shown in wavy cells in the area of the lamina [Ac, Ne] or elongated
Figure 1822.-2 to identify the peaks due to caftaric acid, cells with beaded walls near the veins [Nd], with
cichoric acid, cynarin and echinacoside. anomocytic [Ab, Nb] or anisocytic [Aa, Na] stomata (2.8.3);
Relative retention With reference to chlorogenic acid some of these fragments are accompanied by palisade
(retention time = about 7 min): caftaric acid = about 0.8; parenchyma [Ad]; uniseriate covering trichomes [Ca] or
caffeic acid = about 1.5; cynarin = about 1.6; fragments [OJ thereof, consisting mainly of 3 or 4 thick-
echinacoside = about 1.7; cichoric acid= about 2.3. walled cells of which the apical cell is markedly longer than
the others; fragments of leaves with epidermal cells in a
- resolutwn: minimum 5.0 between the peaks due to rosette arrangement around the base of the covering
chlorogenic acid and caffeic acid. trichomes [C]; uniseriate glandular trichomes [Ne] composed
of very thin-walled cells; pitted parenchymatous cells from
Calculate the percentage content of echinacoside using the the pith of the stem O]; fragments of the epidermis of ligulate
following expression: florets composed of red to violet papillose cells [BJ; finely
A1 X C2 X 100 X 2.221 pined, elongated cells [M] and fibres surrounded by black
phytomelanin deposits [L], from the mesocarp of the
A2 xC1
achenes; fragments of the cotyledons of the seeds with oil
A1 area of the peak due to echinacoside in the chromatogram droplets [K]; spheroidal pollen grains about 30 µm in
obtained with the test solution; diameter each with 3 pores and a spiny exine [F, H].
C1 concentration of the test solution, in milligrams per millilitre;
C2 concentration of chlorogenic add in the reference solution, in
2.221 peak correlation factor between chlorogenic acid and
echinacoside.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Echinacea Purpurea Herb

(Purple Coneflower Herb, Ph. Bur. monograph 1823)
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried, whole or cut flowering aerial parts of Echinacea
purpurea (L.) Moench.
Content
Minimum 0.1 per cent for the sum of caftaric acid
(C 13H 120 9 ; Mr 312.2) and cichoric acid (C 22Hl80 12;
IDENTIFICATION
First identification: A, B, C.
Second identification: A, B, D.
A. The herbaceous perennial plant is 60-150 cm, rarely up to
180 cm high. The stem is green to red, upright and slightly
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 Figure 1823.-1. - Illustratwnfor identificatwn test B of powdered
2 or 3 rows. The solid receptacle is slightly convex. Each of herbal drug of purple coneflower herb
the outer violet ligulate florets (4-6 cm) and of the inner
violet-pink tubular florets is attached to a reddish acute and
coriaceous bract, which overtops the tubular florets. Test solutwn To 1.0 g of the powdered herbal drug (355)
The calyx is reduced to a very short crown. (2.9.12) add 10 mL of methanol Rand sonicate for 5 min.
B. Microscopic examination (2.8.23). The powder is green to
brownish-green. Examine under a microscope using chloral Reference solution Dissolve 0.5 mg of caffeic acid Rand
hydrate solutwn R. The powder shows the following diagnostic 0.5 mg of chlorogenic acid R in methanol Rand dilute to 5 mL
characters (Figure 1823.-1): groups of fibres, 150-200 µm in with the same solvent.
length, 10-20 µm in diameter (longitudinal section [G]), Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel
sometimes with black phytomelanin deposits [L], from the F2 54 plate R (2-10 µm)].
stems; numerous fragments of vascular tissue [E]; rare Mobz1e phase anhydrous formic acid R, water R, methyl ethyl
fragments of parenchyma [D] containing secretory canals ketone R, ethyl acetate R (10:10:30:50 VIVIVIV).
(longitudinal section [Da]) with yellow granular contents,
• • I
2 4 6 8 10 12 14 16 18 min
1. caftaric acid 2. chlorogenic acid 3. cichoric acid
Figure 1823.-2. - Chromatogram for the assay of caftaric acid and cichoric acid in purple coneflower herb
Application 25 µL [or 5 µL] of the test solution and 10 µL TESTS

[or 2 µL] of the reference solution, as bands. Loss on drying (2.2.32)
Development Over a path of 15 cm [or 5 cm] . Maximum 10.0 per cent, determined on 1.000 g of the
Drying In a stream of cold air for about 10 min, then at powdered herbal drug (355) (2.9.12) by drying in an oven at
100 °C for 2 min. 105 °C for 2 h.
Detection Treat the warm plate with a 5 g/L solution of Total ash (2.4.16)
diphenylboric acid aminoethyl ester R in ethyl acetate R; after Maximum 12.0 per cent.
30 min, examine in ultraviolet light at 365 nm. ASSAY
Results See below the sequence of fluorescent zones present Liquid chromatography (2.2.29).
in the chromatograms obtained with the reference solution Solvent mixture acetonitrile R, 0.1 per cent V/V solution of
and the test solution. Furthermore, other faint blue phosphoric acid R (I0:90 V/V).
fluorescent zones may be present in the chromatogram
Test solution In a 200 mL volumetric flask place 0.500 g of
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
Top of the plate 200.0 mL with the solvent mixture. Mix the suspension and
An intense red zone allow to stand for a few minutes to allow visible solids to
settle. Filter a suitable proportion of the solution through a
Caffeic acid: an intense blue zone A blue zone membrane filter (nominal pore size 0.45 µm) before
injection.
-- --
Reference solution Dissolve 10.0 mg of chlorogenic acid CRS
and 10.0 mg of caffeic acid R in the solvent mixture, sonicate
A blue zone for 15 min and dilute to 10.0 mL with the solvent mixture.
Dilute 2.0 mL of the solution to 50.0 mL with the solvent
Chlorogenic acid: an intense blue
zone mixture.
Column:
A faint yellow-orange zone
- size: l = 0.25 m, 0 = 4.6 mm;
-- -- - stationary phase: octadecylsilyl silica gel for chromatography R
(5 µm);
Reference solution Test solution Mobile phase:
- mobile phase A: 0.1 per cent VIV solution of phosphoric
D. Examine the chromatograms obtained in the assay. acid R;
with the test solution is due to cichoric acid and a smaller
peak is due to caftaric acid. Peaks due to caffeic acid and
chlorogenic acid are minor or may be absent.
Time Mobile phase A Mobile phase B bases; the inside is fibrous and white. The numerous roots
are spirally twisted, light to dark brown and show a fine cross
structuring on the surface.
0 - 13 90 ➔ 78 10 ➔ 22
13 - 14 78 ➔ 60 22 ➔ 40
yellow to light pinkish-brown. Examine under a microscope
14 - 20 60 40
using chloral hydrate solutwn R. The powder shows the
following diagnostic characters (Figure 1824.-1): numerous
Flow rate 1.5 mL'min. spindle-shaped fibres that are joined together in long bundles
Detection Spectrophotometer at 330 run. without black phytomelanin deposits [F]; sclereids from the
Injectwn 10 µL. rhizomes and roots, usually occurring singly, those from the
rhizomes being isodiametric, about 60 µm in diameter, with
Identificatwn of peaks Use the chromatogram obtained with
black phytomelanin deposits [B, DJ, those from the roots
the reference solution to identify the peaks due to caffeic acid
being about 50-120 µm in length with no black phytomelanin
and chlorogenic acid; use the chromatogram shown in
deposits [HJ; secretory canals up to 180 µm in diameter with
Figure 1823.-2 to identify the peaks due to caftaric acid and
yellow oil droplets (transverse section [El); fragments of cork
cichoric acid.
with squarish to rectangular cells, some with reddish walls
Relative retention With reference to chlorogenic acid (surface view ill, transverse section [Al); reticulate [CJ or
(retention time = about 7 min): caftaric acid = about 0.8; pitted [GJ vessels with a diameter up to 30-40 µm.
caffeic acid = about 1.5; cichoric acid = about 2.3.
chlorogenic acid and caffeic acid.
Calculate the percentage content of caftaric acid using the
A1 X C2 X 100 X 0.881
A2 xC1
Calculate the percentage content of cichoric acid using the
A3 X C2 X 100 X 0.695
A 2 xC 1
area of the peak due to caftaric acid in the chromatogram
area of the peak due to chlorogenic acid in the chromatogram
area of the peak due to cichoric acid in the chromatogram
concentration of the test solution, in milligrams per millilitre;
concentration of chlorogenic acid in the reference solution, in
0.695 peak correlation factor between cichoric acid and chlorogenic
acid, based upon the liquid chromatography response observed;
0.881 peak correlation factor between caftaric acid and chlorogenic
acid.
Figure 1824.-1. - Illustratwn for identification test B of powdered

Echinacea Purpurea Root herbal drug of purple coneflower root
(Purple Coneflower Root, Ph. Bur. monograph 1824) C. Examine the chromatogram obtained in the test for other
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Echinacea species and Parthenium integrifolium.
DEFINITION Results See below the sequence of fluorescent zones present
Dried, whole or cut underground parts of Echinacea in the chromatograms obtained with the reference solution
purpurea (L.) Moench. and the test solution. Furthermore, faint greenish fluorescent
zones may be present just below the zone situated in the
Content middle of the chromatogram obtained with the test solution.
Minimum 0.5 per cent for the sum of caftaric acid
(C 13H 12 0 9 ; Mr 312.2) and cichoric acid (C 22H 180 12;
IDENTIFICATION
First identificatwn: A, B, C, E.
Second identificatwn: A, B, D, E.
A. The rhizome is up to 15 cm long, branched, reddish-
brown to dark brown on the surface and carries many stem
I I ' I I ' I I I I ! j I I I I ' I ' I

2 4 6 8 10 12 14 16 18 min
I. caftaric acid 2. chlorogenic acid 3. cichoric acid
Figure 1824.-2. - Chromatogram for the assay of caftaric acid and cichoric acid in purple coneflower root
Top of the plate Drying In a stream of cold air for about 10 min.
Caffeic acid: an intense blue zone An intense blue zone
Detection Dip the plate into anisaldehyde solution R for 1 s
and heat at 100-105 °C for 3 min; examine in daylight.
Cynarin: an intense greenish zone
-- -- chromatograms obtained with the reference solution and the
A blue zone
Top of the plate
-- --
Echinacoside: an intense greenish

zone
-- --
!3-Sitosterol: a violet or pink zone A violet or pink zone (P-sitosterol)
N-Isobutyldodecatetraenamide: a A greyish-blue zone (N-

D. Examine the chromatograms obtained in the assay. greyish-blue zone isobutyldodecatetraenamide)
with the test solution is due to cichoric acid and a smaller
~-
--
peak is due to caftaric acid. Peaks due to caffeic acid and A dark greyish-blue zone
chlorogenic acid are minor or may be absent.
E. Thin-layer chromatography (2.2.27).
(2. 9.12) add 10 mL of methylene chloride R and sonicate for TESTS
5 min. Centrifuge and use the supernatant. Other Echinacea species and Parthenium integrifolium
Reference solution Dissolve 1 mg of {3-sitosterol R and a
volume of N-isobutyldodecatetraenamide solution R Test solution To 1.0 g of the powdered herbal drug (355)
corresponding to 1 mg of N-isobutyldodecatetraenamide R in (2. 9.12) add 10 mL of methanol R and sonicate for 5 min.
methanol R, and dilute to 5 mL with the same solvent. Centrifuge and use the supernatant.
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel Reference solution Dissolve 1 mg of echinacoside R, 1 mg of
plate R (2-10 µm)]. cynarin R and 0.5 mg of caffeic acid R in methanol Rand
dilute to 5 mL with the same solvent.
Mobile phase anhydrous formic acid R, cycwhexane R, ethyl
acetate R, toluene R (3:10:20:80 VIVIVIV). Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel
F254 plate R (2-10 µm)].
Application 25 µL [or 5 µLl, as bands.
Mobile phase anhydrous formic acid R, water R, methyl ethyl
Development Over a path of 15 cm [or 5 cm).
ketone R, ethyl acetate R (10:10:30:50 VIVIVIV).
IV-222 Eclipta Herb 2023
Application 10 µL [or 5 µL] of the test solution and 5 µL Injection l O µL.

[or 2 µL] of the reference solution, as bands. Identification of peaks Use the chromatogram obtained with
Development Over a path of 10 cm [or 5 cm]. the reference solution to identify the peaks due to caffeic acid
Drying In a stream of cold air for about 10 min, then at and chlorogenic acid; use the chromatogram shown in
105 °C for 2 min. Figure 1824.-2 to identify the peaks due to caftaric acid and
Detection Treat the warm plate with a 5 g/L solution of cichoric acid.
diphenylboric acid aminoethyl ester R in ethyl acetate R; after Relative retention With reference to chlorogenic acid
30 min, examine in ultraviolet light at 365 nm. (retention time = about 7 min): caftaric acid= about 0.8;
Results The chromatogram obtained with the test solution caffeic acid = about 1.5; cichoric acid = about 2.3.
shows no greenish fluorescent zone corresponding to the System suitability Reference solution:
zone due to echinacoside in the chromatogram obtained with - resolution: minimum 5.0 between the peaks due to
the reference solution, and no greenish fluorescent zone chlorogenic acid and caffeic acid.
corresponding to the zone due to cynarin in the Calculate the percentage content of caftaric acid using the
chromatogram obtained with the reference solution. No other following expression:
zones apart from very faint dark blue fluorescent zones are
A1 X C2 X 100 X 0.881
seen in the lower half of the chromatogram of the test
solution. A2 xC1
Foreign matter (2.8.2) Calculate the percentage content of cichoric acid using the
Maximum 3 per cent. following expression:
Loss on drying (2.2.32) A3 X C2 X 100 X 0.695
A2 xC1
105 °C for 2 h. A1 area of the peak due to caftaric acid in the chromatogram
Total ash (2.4.16) A2 area of the peak due to chlorogenic acid in the chromatogram
Maximum 9.0 per cent. obtained with the reference solution;
A, area of the peak due to cichoric acid in the chromatogram
Ash insoluble in hydrochloric acid (2.8.1) obtained with the test solution;
Maximum 2.0 per cent. C1 concentration of the test solution, in milligrams per millilitre;
C2 concentration of chlorogenic acid in the reference solution, in
ASSAY milligrams per millilitre;
Liquid chromatography (2.2.29). 0.695 peak correlation factor between cichoric acid and chlorogenic
acid based upon the liquid chromatography response observed;
Solvent mixture acetonitrile R, 0.1 per cent V/V solution of
0.881 peak correlation factor between caftaric acid and chlorogenic
phosphoric acid R (10:90 VIV). acid.
Test solution In a 200 mL volumetric flask place 0.500 g of
the powdered herbal drug (355) (2.9.12) and add 80 mL of
ethanol (70 per cent Viii? R. Sonicate for 15 min and dilute to
200.0 mL with the solvent mixture. Mix the suspension and
allow to stand for a few minutes to allow visible solids to
settle. Filter a suitable proportion of the solution through a
membrane filter (nominal pore size 0.45 µm) before
Eclipta Herb
injection. (Ph. Bur. monograph 2564)
Reference solution Dissolve 10.0 mg of chlorogenic acid CRS Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
and 10.0 mg of caffeic acid R in the solvent mixture, sonicate
for 15 min and dilute to 10.0 mL with the solvent mixture. DEFINITION
Dilute 2.0 mL of the solution to 50.0 mL with the solvent Dried, whole or fragmented, flowering aerial parts of Eclipta
mixture. prostrata (L.) L.
Column: Content
- size: l = 0.25 m, 0 = 4.6 mm; Minimum 0.04 per cent of wedelolactone (C 16H 10 0 7 ;
- statumary phase: octadecylsilyl silica gel for chromatography R Mr 314.2) (dried drug).
(5 µm); IDENTIFICATION
A. The cylindrical stems are striated longitudinally and are
Mobile phase: 2-5 mm in diameter. The external surface is brownish-green
- mobile phase A: 0.1 per cent V/V solution of phosphoric or dark green and bears covering trichomes, flattened against
acid R; the stem and all pointing upwards. The hairy, dark green
- mobile phase B: acetonitrile R; leaves are opposite, always sessile, elongate, lanceolate, with
entire or slightly dentate margins. The capitula are 2-6 mm
Time Mobile phase A Mobile phase B in diameter, with whitish flowers that do not extend beyond
the bracts of the involucre. The fruits are elliptical, flattened
achenes, brown or pale brown, 2-3 mm long.
0 - 13 90 ➔ 78 10 ➔ 22
13 - 14 78 ➔ 60 22 ➔ 40
greenish-brown. Examine under a microscope using chloral
14 - 20 60 40
characters (Figure 2564.-1): numerous isolated, whole [HJ or
Flow rate 1.5 mL/min. broken [D] covering trichomes, usually tricellular, up to
Detection Spectrophotometer at 330 nm. 700 µm long, with a broad basal cell, a relatively long
2023 Eclipta Herb IV-223
median cell with thick warty walls, and a very short, pointed, Top of the plate
sub-triangular distal cell; fragments of lamina [B, C] with
sinuous epidermal cells [Ca, Ba] and anomocytic stomata
(2.8.3) with 3-4 subsidiary cells [Cb, Bb]; some fragments of A diffuse pale blue fluorescent zone
epidermis showing covering trichomes similar to those
A greenish-white fluorescent zone
previously described, whole [Be) or broken [Cc], and
subsidiary cells with slightly thickened walls [Bd, Cd]; -- --
fragments of the upper epidermis usually accompanied by
A pa]e blue fluorescent zone
palisade parenchyma [Ce]; fragments of stem with different
types of vascular bundles [A]; rare fragments of parenchyma
(longitudinal section [F]) containing secretory canals with
orange-brown contents [Fa]; bundles of fibres with thickened
walls [E]; pollen grains [G] about 20-25 µm in diameter, Wedelolactone: a bluish-white A bluish-white fluorescent zone
with 3 pores and a spiny exine. fluorescent zone (wedelolactone)
B .h,• -- --
Rosmarinic acid: a bluish-white

fluorescent zone
TESTS
\ ·1:1 ... 105 °C for 2 h.
Total ash (2.4.16)
D~o': 0 H •.
2~m ~-::-:~ ~ · ~~~€::IDQ ASSAY
~ Liquid chromatography (2.2.29).
Figure 2564.-1. - Illustration for identification test B of powdered (355) (2. 9.12) in 10 mL of ethanol (70 per cent VIV) R in a
herbal drng of eclipta herb conical flask and weigh. Heat under a reflux condenser for
1 h, cool and weigh again. Compensate the Joss of solvent
C. Thin-layer chromatography (2.2.27). with ethanol (70 per cent V/V) R, mix well and allow to stand.
Test solution To 0.5 g of the powdered herbal drug (355) Filter through a membrane filter (nominal pore size
(2. 9. 12) add 5 mL of methanol R and sonicate at 60 °C for 0.22 µm).
10 min. Allow to cool, centrifuge and use the supernatant. Reference solution (a) Dissolve 4.0 mg of wedelolactone CRS
Reference solution Dissolve 1 mg of wedelolactone R and 1 mg in methanol Rand dilute to 100.0 mL with the same solvent.
of rosmarinic acid R in 1 mL of methanol R. Reference solution (b) Dissolve 2 mg of ethyl
Plate TLC silica gel F254 plate R (2-10 µm). parahydroxybenzoate R in reference solution (a) and dilute to
Mobile phase anhydrous formic acid R, acetone R, toluene R 50 mL with reference solution (a).
(1:6:11 V/V/V). Column:
Application 10 µL as bands of 8 mm. - size: l = 0.15 m, 0 = 4.6 mm;
Drying In air.
Mobile phase acetonitrile R, 0.2 per cent V/V solution of
Detection Heat at 100 °C for 5 min and treat the plate phosphoric acid R (24:76 V/V).
whilst still hot with a 0.5 per cent V/V solution of
diphenylboric acid aminoethyl ester R in ethyl acetate R; examine
in ultraviolet light at 365 nm. Detection Spectrophotometer at 249 nm.
Results See below the sequence of zones present in the Injection 20 µL.
chromatograms obtained with the reference solution and the Run time 1.5 times the retention time of wedelolactone.
test solution. Furthermore, other faint fluorescent zones may Relative retention With reference to wedelolactone (retention
be present in the chromatogram obtained with the test time = about 17 min): ethyl
solution. parahydroxybenzoate = about 1. 1.
IV-224 Eclipta Prostrata 2023
System suitability Reference solution (b): lignified and pitted. Fragments of root, if present, show
- resolution: minimum 2.0 between the peaks due to poorly developed cork, consisting of 3-5 rows of thin-walled
wedelolactone and ethyl parahydroxybenzoate. elongated cells, a secondary cortex of parenchyma, with
Calculate the percentage content of wedelolactone using the scattered stone cells and fibres either singly or in groups,
following expression: xylem vessels and tracheids, and fibres with peg-like
projections. Pollen grains with spiny exine and 3 pores.
(1) Reduce to a powder (355). To 2.0 g of powdered sample
area of the peak due to wedelolactone in the chromatogram
obtained with the test solution; add 40 mL of methanol. Mix with the aid of ultrasound for
area of the peak due to wedelolactone in the chromatogram 2 hours at 50° with occasional shaking and allow to cool.
obtained with reference solution (a); Dilute to 50 mL with methanol and filter.
solution, in grams; (2) 0.05% w/v each of wedelolactone EPCRS and rosmarinic
mass of wedewlacwne CRS used to prepare reference acid in methanol.
p percentage content of wedelolactone in wedelolactone CRS.
(a) Use as the coating silica gel F254 (Merck silica gel
- - - - - - - - - - - - - - - - - - - - - - Ph Eur HPTLC plates are suitable).
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% ofwedelolactone MOBILE PHASE
(C 16H 100 7 ), calculated with reference to the dried material. 1 volume of anhydrous formic acid, 6 volumes of acetone and
IDENTIFICATION 11 volumes of toluene.
A. Stems cylindrical, four sided or flattened, 2 to 5 mm in SYSTEM SUIT ABILITY
diameter, greyish, with appressed, whitish hairs pointing The test is not valid unless the chromatogram obtained with
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
Top of the plate
2.5 cm wide with an entire or slightly dentate margin and
A red zone
appressed trichomes on both surfaces. Flowerheads 2 to
A diffuse pale blue zone
6 mm in diameter, greenish-brown, solitary or in pairs on A greenish-white zone
unequal axillary peduncles, up to 8 involucral bracts, ovate,
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;
Rosmarinic acid: A blue-white zone
unfertilised achenes pale yellow, flattened and smooth; A blue-white zone
fertilised achenes pale to dark brown, 3 to 4 angled, Solution (1) Solution (2)
tuberculate and bulbous. Root, if present, cylindrical, greyish,
main root up to about 7 mm in diameter, with secondary
branching. CONFIRMATION
B. Reduce to a powder (355). The powder is greenish The chromatogram obtained with solution (1) shows a
brown. Examine under a microscope using chloral hydrate fluorescent band corresponding to wedelolactone and several
solution. The main diagnostic characters include numerous other fluorescent bands as shown in the table. Other
free, whole or broken, large covering trichomes, with warty or fluorescent bands may be present.
spiny walls, up to 7 00 µm long, uniseriate, usually tricellular, TESTS
with a broad basal cell, a long median cell and a short, Loss on drying
pointed sub-triangular apical cell; less frequently, smaller, When dried at 100° to 105° for 2 hours, loses not more than
unicellular, pointed covering trichomes from the midrib and 11.0% of its weight. Use 1 g.
stem. Fragments of leaf show sinuous walled epidermal cells,
underlying palisade, anomocytic stomata, cuticular striations Total Ash
and covering trichomes on both surfaces. Stem fragments Not more than 22.0%, Appendix XI J, Method II.
with unicellular and multicellular trichomes, epidermis of Acid-insoluble Ash
elongated cells, or in mature stem, poorly developed Not more than 11.0%, Appendix XI K.
rectangular cork cells; secondary cortex of parenchyma with ASSAY
numerous air-spaces, pericyclic fibres thick-walled, lignified, Carry out the method for liquid chromatography,
simple pitted; secretary canals may be visible; xylem vessels Appendix III D, using the following solutions.
usually simple pitted or spirally thickened, xylem parenchyma
2023 Elder Flower IV-225
(1) Reduce to a powder (355). To 2.0 g of powdered sample 5 broadly oval petals fused at their bases into a tube.
add 40 mL of methanol. Mix with the aid of ultrasound for The filaments of the 5 yellow stamens alternate with the
2 hours at 50° with occasional shaking and allow to cool. petals. The corolla is often isolated or attached to the
Dilute to 50 mL with methanol and filter. stamens, to which it is fused at the base. The ovary is inferior
(2) 0.005% w/v each of wedelolactone EPCRS and coumestrol and it bears a short style with 3 obtuse stigmata.
in methanol. B. Microscopic examination (2.8.23). The powder is
CHROMATOGRAPHIC CONDITIONS greenish-yellow. Examine under a microscope using chloral
(a) Use a stainless steel column (15 cm x 4.6 mm) packed
characters (Figure 1217.-1): numerous spherical, sometimes
with octadecylsilyl silica gel for chromatography (5µm) (Waters
ellipsoidal, pollen grains about 30 µm in diameter, with
Symmetry Cl8 is suitable).
3 germinal pores and very finely pitted exine [GJ; cells of the
(b) Use isocratic elution and the mobile phase described lower epidermis of the sepals often containing oil globules
below. and covered by a striated cuticle (surface view [A]); rare
(c) Use a flow rate of 1.0 mL per minute. fragments of the rim of the sepals showing unicellular
(d) Use an ambient column temperature. marginal teeth (transverse section [EJ); petal fragments with
(e) Use a detection wavelength of 249 nm. numerous small globules of essential oil [HJ; fragments of
upper epidermis of the sepals [BJ or petals [F], in surface
(t) Inject 10 µL of each solution.
view, with slightly and irregularly thickened walls [Ba, Fa],
MOBILE PHASE anomocytic stomata (2.8.3) [Bb, Fb] and a striated cuticle;
24 volumes of acetonitril£ and 76 volumes of a 0.2% v/v mesophyll cells of petals and sepals with idioblasts containing
solution of orthophosphoric acid. numerous microsphenoid crystals of calcium oxalate [Be];
fragments of anthers (transverse section [CJ, surface view
SYSTEM SUITABILITY
[DJ) showing the outer layer [Ca] and the cells of the fibrous
The assay is not valid unless, in the chromatogram obtained layer [Cb, Cc, DJ.
with solution (2):
the symmetry factor of the peak due to wedelolactone is less
than 1.2;
the symmetry factor of the peak due to coumestrol is less than
1.1.
Calculate the content of wedelolactone in the sample using
the declared content of wedelolactone (C 16H 100 7 ) in
wedelolactone EPCRS and the following expression:
A1 1Tu V, 100
-x-x-xpx
A2 V 2 mi 100 - d

obtained with solution (I);
obtained with solution (2);
weight of the herbal drug being examined in mg;
weight of wedewlactone EPCRS in mg;
dilution volume of solution (I) in mL;
dilution volume of solution (2) in ml.;
percentage content of wedelolactone (C 16 H 10 O 7 ) in
wedelolactone EPCRS;
d percentage loss on drying of the herbal drug being examined.
Elder Flower
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Figure 121 7. -1. - Illustratwn for identijicatwn B of powdered
herbal drug of elder flower
DEFINITION
Dried flowers of Sambucus nigra L. C. Thin-layer chromatography (2.2.27).
Content Test solutwn To 0.5 g of the powdered herbal drug (355)
Minimum 0.80 per cent of flavonoids, expressed as (2. 9. 12) add 5 mL of methanol R and sonicate for 10 min.
isoquercitroside (C 21 H 200 12; Mr 464.4) (dried drug). Centrifuge for 5 min.
IDENTIFICATION Reference solutwn Dissolve 1 mg of cafjeic acid R, 1 mg of
A. The flower, about 5 mm in diameter, has 3 small bracts, chlorogenic acid R, 2.5 mg of hyperoside R and 2.5 mg of
visible under a lens, and may have a peduncle. rutoside trihydrate R in 10 mL of methanol R.
The 5-toothed calyx is small; the corolla is light yellow, with Plate TLC silica gel plate R (2-10 µm).
IV-226 Elder Flower 2023
Mobile phase anhydrous Jonnie acid R, water R, methyl ethyl TESTS

ketone R, ethyl acetate R (10:10:30:50 VIVIVIV). Foreign matter (2.8.2)
Application 4 µL as bands of 8 mm. Maximum 8 per cent of fragments of coarse pedicels and
Development Over a path of 6 cm. other foreign matter and maximum 15 per cent of
discoloured, brown flowers. Carry out the determination on
Drying In air.
10 g.
Detection Heat the plate for 5 min at 100 °C and treat with
Sambucus ebulus L
a 1 g/L solution of diphenylboric acid aminoethyl ester R in ethyl
Examine the chromatograms obtained in identification C.
acetate R, then treat with a 5 g/L solution of macrogol 400 R
in methylene chloride R; allow to dry in air for 30 min. Results B The chromatogram obtained with the test solution
Examine in daylight (results A) and in ultraviolet light at does not show a greenish-white zone above the zone due to
365 nm (results B). caffeic acid in the chromatogram obtained with the reference
solution; in the chromatogram obtained with the test
solution, no green fluorescent zone is seen just below the
orange fluorescent zone due to rutoside in the chromatogram
obtained with the reference solution.
Top of the plate
105 °C for 2 h.
-- -- Total ash (2.4.16)
An orange zone
ASSAY
Hyperoside: a dark yellow zone
Stock solution In a 100 mL round-bottomed flask, introduce
0.600 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 through a plug of absorbent cotton into a flask.
Rutoside: a dark yellow zone A dark yellow zone
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, filter each extract through the plug of
absorbent cotton into the flask. After cooling, filter the
Results B See below the sequence of zones present in the combined acetone extracts through a filter paper into a
chromatograms obtained with the reference solution and the volumetric flask and dilute to 100.0 mL with acetone R by
test solution. Furthermore, other faint zones may be present rinsing the flask and the filter paper. Introduce 20.0 mL of
in the chromatogram obtained with the test solution. this solution into a separating funnel, add 20 mL of water R
and shake the mixture with 1 quantity of 15 mL and then
Top of the plate 3 quantities, each of 10 mL, of ethyl acetate R. Combine the
ethyl acetate extracts in a separating funnel, wash with
Caffeic acid: a blue fluorescent zone
2 quantities, each of 50 mL, of water R, and filter the
An intense, light blue fluorescent extracts over 10 g of anhydrous sodium sulfate R into a
zone volumetric flask and dilute to 50.0 mL with ethyl acetate R.
2 light blue fluorescent zones Test solution To 10.0 mL of the stock solution add 1 mL of
aluminium chloride reagent Rand dilute to 25.0 mL with a
-- --
Compensation liquid Dilute 10. 0 mL of the stock solution to
Hyperoside: an orange fluorescent 25.0 mL with a 5 per cent VIV solution of glacial acetic
zone acid R in methanol R.
Chlorogenic acid: a light blue An intense, light blue fluorescent After 30 min, measure the absorbance (2.2.25) of the test
fluorescent zone zone solution at 425 nm, by comparison with the compensation
liquid.
-- -- isoquercitroside, using the following expression:
Ax 1.25
. m
i.e. taking the specific absorbance of isoquercitroside to

Reference solution Test solution be 500.

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
2023 Eleutherococcus IV-227
the test solution. Furthermore, other faint zones are present

Eleutherococcus in the chromatogram obtained with the test solution.
Siberian Ginseng
Top of the plate
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ A brown zone (eleutheroside B)
DEFINITION Esculin: a blue fluorescent zone

Dried, whole or cut underground organs of Eleutherococcus (marked at 365 nm)
senticosus (Rupr. et Maxim.) Maxim. A reddish-brown zone
Content (eleutheroside E)
Minimum 0.08 per cent for the sum of eleutheroside B -- --
(Mr 372.4) and eleutheroside E (Mr 742.7). Catalpol: a violet-brown zone
IDENTIFICATION
-- --
A. The rhizome is knotty, of irregular cylindrical shape,
1.5 cm to 4.0 cm in diameter; the surface is rugged, 2 brown zones
longitudinally wrinkled and greyish-brown to blackish-brown; Reference solution Test solution
the bark, about 2 mm thick, closely adheres to the xylem; the
heartwood is light brown and the sapwood is pale yellow; the
fracture shows short thin fibres in the bark and is coarsely TESTS
fibrous, especially in the internal part of the xylem. Foreign matter (2.8.2)
The lower surface bears numerous cylindrical and knotty Maximum 3 per cent.
roots, 3.5 cm to 15 cm long and 0.3 cm to 1.5 cm in
diameter; with a smooth, greyish-brown to blackish-brown mAU
surface; the bark is about 0.5 mm thick, closely adhering to
the pale yellow xylem; the fracture is slightly fibrous;
in places where the outer layer has been removed, the outer
surface is yellowish-brown.
B. Microscopic examination (2.8.23). The powder is 60
yellowish-brown. Examine under a microscope, using ch/,oral
hydrate solution R. The powder shows numerous groups of
thick-walled, lignified fibres; fragments of reticulate and
bordered pitted vessels with a wide lumen; groups of
secretory canals, up to 20 µm in diameter with brown
50
contents; parenchymatous cells containing cluster crystals of
calcium oxalate 10 µm to 50 µm in diameter. Examine under
a microscope, using a 50 per cent V/V solution of glycerol R.
The powder shows small starch granules, rounded to slightly
angular in outline, single compounds or with 2 or
3 components. 40
(2. 9.12) add 10 mL of alcohol (50 per cent V/V) R and boil
under reflux for 1 h. Cool and filter. Evaporate the filtrate to
dryness on a water-bath. Dissolve the residue in 2.5 mL of a 30
mixture of 5 volumes of water R and 20 volumes of alcohol
(50 per cent V/V) R and filter.
Reference solution Dissolve 2.0 mg of esculin Rand 2.0 mg of
catalpol R in 20 mL of a mixture of 2 volumes of water R and
8 volumes of alcohol (50 per cent V/V) R. 20

Mobz7e phase water R, methanol R, methylene chloride R
(4:30:70 V/V/V).
Application 20 µL, as bands. 10
Drying In air.
Results A The chromatogram obtained with the reference
solution shows in the upper half a blue fluorescent zone 0
(esculin).
Detection B Spray with anisaldehyde solution R and examine 220 240 260 280 300 320 340 360 380 nm
in daylight while heating at 100-105 °C for 5-10 min.
Results B See below the sequence of the zones present in Figure 141 9. -1. - UV spectrum of eleutheroside B for the assay
the chromatograms obtained with the reference solution and of eleutherococcus
IV-228 Eleutherococcus 2023
mAU 250 mL round-bottomed flask. Repeat this operation twice,

using the residue obtained in the filtration step instead of the
powdered herbal drug. Add both fractions of supernatant to
the 250 mL round-bottomed flask. Evaporate under reduced
2500 pressure until about 10 mL of supernatant is left in the flask.
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 µm).
Reference solution (a) Dissolve 10 mg offernlic acid Rina
mixture of equal volumes of methanol R and water R and
dilute to 20.0 mL with the same mixture of solvents.
2000
Reference solution (b) Dissolve 10 mg of caffeic acid R in a
mixture of equal volumes of methanol R and water R and
dilute to 20.0 mL with the same mixture of solvents.
Reference solution (c) Transfer 1 mL of reference solution (a)
to a 25 mL volumetric flask and dilute to 25.0 mL with a
mixture of equal volumes of methanol R and water R. Filter
through a nylon filter (pore size 0.45 µm).
Reference solution (d) Transfer 1 mL of reference
solution (a) and 1 mL of reference solution (b) in a mixture
of equal volumes of methanol R and water R and dilute to
25.0 mL with the same mixture of solvents. Filter through a
nylon filter (pore size 0.45 µm).
Precolumn:
- size: l = 4 mm, 0 = 4.6 mm,
(5 µm).
Column:
=
- size: l 0.25 m, 0 = 4.6 mm,
(5 µm).
Mobile phase:
- mobile phase A: phosphoric acid R, water R (0.5:99.5 VIV),
- mobile phase B: acetonitrile for chromatography R,

(min) (per cent V/J!) (per cent V/J!)
0-5 90 10
5 - 27 90 ➔ 80 10 ➔ 20
27 - 30 80 ➔ 50 20 ➔ 50
30 - 35 so so

Injection 20 µL of the test solution and reference
220 240 260 280 300 320 340 360 380 nm
solutions (c) and (d).
Retention time Eleutheroside B = about 10 min;
Figure 1419.-2. - UV spectrnm of eleutheroside E for the assay
eleutheroside E = about 22 min.
of eleutherococcus
Locate the peaks due to eleutheroside B and eleutheroside E
Loss on drying (2.2.32) using the UV spectra shown in Figures 1419.-1 and 1419.-2.
Maximum 10.0 per cent, determined on 1.000 g of the System suitability Reference solution (d):
powdered herbal drug (355) (2.9.12) by drying in an oven at - resolution: minimum 15 between the peaks due to caffeic
105 °C for 2 h. acid and ferulic acid.
Total ash (2.4.16) Calculate the total percentage content of eleutheroside B and
Maximum 8.0 per cent. eleutheroside E from the expression:
ASSAY
(ABxCx0.73x2) (AExCxl.90x2)
Liquid chromatography (2.2.29). -------+-------
(AR X m) (AR X m)
(2. 9.12) in a 100 mL round-bottomed flask, add 30 mL of a
mixture of equal volumes of alcohol R and water R. Heat in a
water-bath at 60 °C for 30 min. Allow to cool and filter
through a sintered-glass filter (2.J.2). Collect the liquid in a
2023 Ephedra Herb IV-229
As area of the peak due to eleutheroside B in the chromatogram

obtained with the test solution,
AE area of the peak due to eleutheroside E in the chromatogram
obtained with the test solution,
AR area of the peak due to ferulic acid in the chromatogram
obtained with reference solution (c),
C concentration of ferulic acid in reference solution (c), in
micrograms per millilitre,
m mass of the herbal drug to be examined, in milligrams.
- - - - - - - - - - - - - - - - - - - - - - PhEur
Ephedra Herb
~~----------------------
DEFINITION
Whole or fragmented, dried sterile aerial part of Ephedra
sinica Stapf, Ephedra intermedia Schrenk and C.A.Mey.
or Ephedra equisetina Bunge or a mixture of these.
Content
Minimum 1.0 per cent of ephedrine (C 10H 15NO; Mr 165.2)
(dried drug).
IDENTIFICATION
A. The stems are pale green, yellowish green or light brown
in colour, thin, cylindrical, branched or unbranched, and up Figure 2451. -1. - Illustration for identification test B of powdered
to 30 cm long (whole drug) or in fragments up to 5 cm long herbal dn.tg of ephedra herb
(fragmented drug) and 1-3 mm in diameter; they are
longitudinally striated and slightly rough, with intemodes C. Thin-layer chromatography (2.2.27).
varying in length from 1-6 cm. The leaves, opposite and Test solution To 0.2 g of the powdered herbal drug (355)
decussate, are reduced to sheaths surrounding the stem at the (2.9.12) add 0.5 mL of concentrated ammonia Rand 10 mL of
nodes, carrying diminutive laminae 1.5-4 mm long with 2 methylene chloride R. Boil in a water-bath under a reflux
(rarely 3) acutely triangular lobes with a greyish-white apex condenser for 1 h. Allow to cool, filter and evaporate the
and a reddish-brown or blackish-brown tubular base (laminae filtrate to dryness; dissolve the residue in 2 mL of
can also be shorter, with a more rounded apex). The fracture methanol R.
is fibrous with a light or dark reddish-brown pith. Reference solution Dissolve 1 mg of ephedrine
B. Microscopic examination (2.8.23). The powder is hydrochloride CRS and 1 mg of 2-indanamine hydrochloride R
greenish-yellow, greenish-brown or light brown. Examine in 2 mL of methanol R.
under a microscope using chloral hydrate solution R. Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
The powder shows the following diagnostic characters plate R (2-10 µm)].
(Figure 2451.-1): fragments of the epidermis (surface
Mobile phase concentrated ammonia R, methanol R, methylene
view [A, F]) consisting of elongated, more or less thick-
chloride R (1:10:40 VIVIV).
walled cells [Aa] and stomata partially covered by an
occasionally speckle-patterned cuticle [Ab] so that only the Application 10 µL [or 1 µL] as spots with a diameter of
pore is visible; anomocytic stomata clearly visible below the 5 mm [or 2 mm].
cuticle (surface view [F]), with guard cells with thickened Development Over a path of 10 cm [or 6 cm].
end walls [Fa]; fragments of the stem (transverse section [DJ) Drying In air.
showing the epidermis covered by a thick cuticle [Da] with Detection Spray with a 2 g/L solution of ninhydrin R in
papillose thickenings above some cells [Db] and composed of ethanol (96 per cent) R; heat at 110 °C for 10 min and
thick-walled epidermal cells [De] and sunken stomata [Dd] examine immediately in daylight.
sometimes accompanied by cortical parenchyma with
Results See below the sequence of spots present in the
palisade cells containing small prisms of calcium oxalate [Df]
and groups of fibres [De]; fibres, isolated or in groups [BJ,
test solution. Furthermore, other faint spots may be present
long, thick-walled and with a reduced lumen; fragments of
vascular tissue [C, E] composed of pitted tracheids [Ca, Ea]
and annular, spiral [Cb] or rarely reticulate vessels; groups of
more or less rectangular parenchymatous cells from the pith
with slightly thickened walls and often with orange or brown
contents [Eb]; numerous fragments of the orange or brown
contents of parenchymatous cells [G]; numerous small
crystals of calcium oxalate occurring as prisms or cluster
crystals [Bal, frequently agglutinated along fibres or in
parenchymatous cells [HJ or isolated.
IV-230 Eucalyptus Leaf 2023
Top of the plate mass of ephedrine hydrochloride CRS used to prepare reference
-- --
p percentage content of ephedrine hydrochloride in ephedrine
hydrochloride CRS.
2-Indanamine: a purple spot
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
A purple spot may be present
-- --
Ephedrine: a purple spot at the A purple spot (ephedrine) at the

border between the middle and
lower thirds
border between the middle and
lower thirds
Eucalyptus Leaf
Reference solution Test solution (Ph. Bur. monograph 1320)
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
TESTS DEFINITION
Loss on drying (2.2.32) Whole or cut, dried leaves of older branches of Eucalyptus
Maximum 10.0 per cent, determined on 1.000 g of the globulus Labill.
powdered herbal drug (355) (2.9.12) by drying in an oven at Essential oil content:
105 °C for 2 h. - for the whole drug, minimum 20 mUkg (anhydrous
Total ash (2.4.16) drug);
Maximum 9.0 per cent. - for the cut drug, minimum 15 mUkg (anhydrous drug).
ASSAY CHARACTERS
Liquid chromatography (2.2.29). Aromatic odour of cineole.
Test solution To 0.200 g of the powdered herbal drug (355) IDENTIFICATION
(2. 9.12) add 25.0 mL of methanol R, weigh and sonicate for A. The leaves, which are mainly greyish-green and relatively
45 min. Allow to cool, weigh and adjust to the original mass thick, are elongated, elliptical and slightly sickle-shaped and
with methanol R, shake well and filter. Transfer 1.0 mL of the usually up to 25 cm in length and up to 5 cm in width.
filtrate to a small column (1 cm in diameter) packed with The petiole is twisted, strongly wrinkled and is 2-3 cm, rarely
1.50 g of neutral aluminium oxide R (60-210 µm). Elute with 5 cm, in length. The coriaceous, stiff leaves are entire and
a mixture of equal volumes of methanol R and water R. glabrous and have a yellowish-green midrib. Lateral veins
Collect about 9 mL of the eluate, add 0.5 mL of phosphoric anastomose near the margin to a continuous line.
acid Rand dilute to 10.0 mL with a mixture of equal The margin is even and somewhat thickened. On both
volumes of methanol R and water R. surfaces there are minute, irregularly distributed, warty, dark
Reference solution (a) Dissolve 10.0 mg of ephedrine brown spots. Small oil glands may be seen in transmitted
hydrochloride CRS in methanol Rand dilute to 100.0 mL with light.
the same solvent. Dilute 2.0 mL of the solution to 25.0 mL B. Microscopic examination (2.8.23). The powder is greyish-
with the mobile phase. green. Examine under a microscope using chloral hydrate
Reference solution (b) Dissolve 1 mg of ephedrine solution R. The powder shows the following diagnostic
hydrochloride CRS and 1 mg of terbutaline sulfate CRS in characters (Figure 1320.-1): fragments of glabrous lamina
methanol R and dilute to 10 mL with the same solvent. (surface view [A, L], transverse section [F, HJ), with small,
Dilute 2 mL of the solution to 25 mL with the mobile phase. thick-walled epidermal cells bearing a thick cuticle [Fa, Ha],
Column: numerous anomocytic stomata (2.8.3) greater than 80 µm in
=
- size: l 0.25 m, 0 4.6 mm; = diameter [Aa, La] with occasional groups of brown cork cells,
300 µm in diameter and brownish-black in their centre, and
(5 µm). underlying palisade parenchyma [Ab, Fb]; fragments of
bilateral mesophyll (side view [G]), with 2-3 layers of
Mobile phase acetonitrile Rl, 0.1 per cent V/V solution of
palisade parenchyma [Ga] on each side and in the centre
phosphoric acid R (15:85 VIV).
several layers of spongy mesophyll [Gb] with elongated cells
Flow rate 2.0 mUmin. having the same orientation as the palisade cells and
Detection Spectrophotometer at 207 nm. containing prisms [B, Gd] and cluster crystals of calcium
Injection 10 µL. oxalate [Ge, K]; large schizogenous oil glands, whole [E] or
Run time 3 times the retention time of ephedrine. usually broken, accompanied by palisade parenchyma [Ea];
fragments of vessels [J] and thick-walled and slightly
channelled fibres [CJ accompanied by crystal sheaths [Ca,
Ja]; crystal sheaths containing prisms of calcium oxalate [DJ.
terbutaline and ephedrine.
Calculate the percentage content of ephedrine using the
following expression: Test solution Shake 0.5 g of the freshly powdered herbal
drug (355) (2.9.12) with 5 mL of toluene R for 2-3 min and
A1 xm2 xpx 165.2 filter over about 2 g of anhydrous sodium sulfate R.
A2xm1x5x201.7 Reference solution Dissolve 50 µL of cineole R in toluene R
and dilute to 5 mL with the same solvent.
= area of the peak due to ephedrine in the chromatogram
obtained with the test solution; Plate TLC silica gel plate R.
area of the peak due to ephedrine in the chromatogram Mobile phase ethyl acetate R, toluene R (10:90 VIV).
mass of the herbal drug to be examined used to prepare the test Application 10 µL as bands.
solution, in grams;
2023 Eucalyptus Oil IV-231
Eucalyptus Oil
DEFINITION
Essential oil obtained by steam distillation and rectification
from the fresh leaves or the fresh terminal branchlets of
various species of Eucalyptus rich in 1,8-cineole. The species
mainly used are Eucalyptus globulus Labill., Euca(Vj)tus
polybractea RT.Baker and Eucalyptus smithii RT.Baker.
CHARACTERS
Appearance
Colourless or pale yellow liquid.
Odour. reminiscent of 1,8-cineole.
IDENTIFICATION
Second identification: A.
Test solutwn Dissolve O.1 g of the essential oil to be
examined in toluene R and dilute to 10 mL with the same
solvent.
Reference solutwn Dissolve 20 µL of rx-terpineol R and 50 µL
of cineole R in toluene R and dilute to 5 mL with the same
solvent.
Figure 1320.-1. - Illustration for identification test B of powdered plate R (2-10 µm)].
herbal drug of eucalyptus leaf Mobile phase ethyl acetate R, toluene R (10:90 V!V).
Drying In air.
Drying In air.
Results The chromatogram obtained with the reference
solution shows in the middle a zone due to cineole.
The chromatogram obtained with the test solution shows a
principal zone similar in position and colour to the zone due test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution, near the
to cineole in the chromatogram obtained with the reference
solvent front and at the level of o:-terpineol.
solution, it also shows an intense violet zone (hydrocarbons)
near the solvent front and there may also be other fainter
zones. Top of the plate
TESTS -- --
Foreign matter (2.8.2) 1,8-Cineole: a violet-brown zone An intense violet-brovm zone (1,8-
Maximum 3 per cent of dark and brown leaves, maximum cineole)
5 per cent of stems and maximum 2 per cent of other foreign
-- --
matter. Cordate or ovate sessile leaves of young branches,
with numerous glands on both sides, visible as points in cx-Terpineol: a violet-brown zone
transmitted light, are not present. Carry out the Reference solution Test solution
determination using 30 g of the herbal drug to be examined.
Water (2.2.13)
Maximum 100 mlJkg, determined on 20.0 g of the
chromatographic profile.
Results The characteristic peaks due to o:-pinene, ~-pinene,
Total ash (2.4.16) o:-phellandrene, limonene and 1,8-cineole in the
Maximum 6.0 per cent. chromatogram obtained with the test solution are similar in
ASSAY retention time to those in the chromatogram obtained with
Essential oil (2.8.12) reference solution (a). Sabinene and camphor may be present
Use 10.0 g of the herbal drug, cut immediately before in the chromatogram obtained with the test solution.
determination, a 500 mL round-bottomed flask, 200 mL of TESTS
water R and 100 mL of glycerol R as the distillation liquid and Relative density (2. 2. 5)
0.5 mL of xylene R in the graduated tube. Distil at a rate of 0.906 to 0.927.
2-3 mIJmin for 2 h.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
IV-232 Eucommia Bark 2023
Refractive index (2.2.6) Elution order Order indicated in the composition of

1.458 to 1.470. reference solution (a). Record the retention times of these
Optical rotation (2.2.7) substances.
0° to+ 10°. System suitabi7ity Reference solution (a):
Solubility in alcohol (2. 8.10) - resolution: minimum 1.5 between the peaks due to
- eucalyptus oil containing 70-79 per cent of 1,8-cineole is limonene and cineole.
soluble in 10 volumes of ethanol (70 per cent VIV,) R; Identification of components Using the retention times
- eucalyptus oil containing minimum 80 per cent of determined from the chromatogram obtained with reference
1,8-cineole is soluble in 5 volumes of ethanol solution (a), locate the components of reference solution (a)
(70 per cent V/V) R. in the chromatogram obtained with the test solution.
Aldehydes Determine the percentage content of each of these
To 10 mL in a ground-glass-stoppered tube 25 mm in components. The percentages are within the following
diameter and 150 mm long, add 5 mL of toluene R and 4 mL ranges:
of alcoholic hydroxylamine solution R. Shake vigorously and - rx-pinene: 0.05 per cent to 10.0 per cent;
titrate immediately with 0. 5 M potassium hydroxide in alcohol - fJ-pinene: 0.05 per cent to 1.5 per cent;
(60 per cent V/V) until the red colour changes to yellow. - sabinene: maximum 0.3 per cent;
Continue the titration with shaking; the end-point is reached - rx-phellandrene: 0.05 per cent to 1.5 per cent;
when the pure yellow colour of the indicator is permanent in - limonene: 0.05 per cent to 15.0 per cent;
the lower layer after shaking vigorously for 2 min and - 1,8-cineole: minimum 70.0 per cent;
allowing separation to take place. The reaction is complete in - camphor. maximum 0.1 per cent;
about 15 min. Repeat the titration using a further 10 mL of - disregard limit: the area of the principal peak in the
the substance to be examined and, as a reference solution for chromatogram obtained with reference solution (b)
the end-point, the titrated liquid from the 1st determination (0.05 per cent).
to which has been added 0.5 mL of 0.5 M potassium STORAGE
hydroxide in alcohol (60 per cent VIV). Not more than 2.0 mL At a temperature not exceeding 25 °C.
of 0.5 M potassium hydroxide in alcohol (60 per cent V/V,) is _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
required in the 2nd titration.
procedure.
Eucommia Bark
Test solution Dissolve 200 µL of the essential oil to be
examined in heptane Rand dilute to 10.0 mL with the same (Ph. Eur. monograph 2412)
solvent. Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Reference solution (a) Dissolve 10 µL of rx-pinene R, 5 µL of
fJ-pinene R, 5 µL of sabinene R, 5 µL of rx-phellandrene R, DEFINITION
10 µL of limonene R, 50 µL of cineole Rand 5 mg of Whole or fragmented, scraped, dried bark of the stem of
camphor R in heptane R and dilute to 10 mL with the same Eucommia ulmoides Oliv.
solvent. Content
Reference solution (b) Dissolve 5 µL of limonene R in Minimum 0.10 per cent of pinoresinol diglucoside
heptane R and dilute to 50.0 mL with the same solvent. (C32H42016; Mr 683) (dried drug).
Dilute 0.5 mL of the solution to 5.0 mL with heptane R. IDENTIFICATION
Column: A. Pieces are flat, curved or channelled, varying in size, about
- material: fused silica; 3-7 mm thick. The outer surface is pale brown or greenish-
= =
- size: l 60 m, 0 about 0.25 mm; brown, markedly wrinkled or fissured, sometimes with
- stationary phase: macrogol 20 000 R (film thickness intentional scarring in a rhombus shape; some barks show
0.25 µm). lenticels. The inner surface is dark reddish-brown or dark
Carrier gas helium for chromatography R. purplish-brown, smooth to the touch. The texture is fragile,
F0w rate 1.5 mL/min. easily broken, with the edges of the fracture connected by
fine, dense, silvery and elastic rubber threads.
Split ratio 1:50.
Temperature:
brownish. Examine under a microscope using chloral hydrate
Time Temperature
(min) CC) characters (Figure 2412.-1): many ribbon-shaped latex
Column 0-5 60
fragments with a granular surface, twisted or folded back on
5 - 33 60 ➔ 200
themselves [B]; numerous sclereids up to 180 µm long and
33 - 38 200
20-80 µm in diameter, isolated [F] or mostly in groups [C],
Injection port 220
with very thick and markedly channelled walls, some sclereids
Detector 220
having masses of latex in their lumen [Ca]; fibres with very
narrow lumens, usually in groups associated with
sclereids [D]; fragments of cork (transverse section [A])
Detection Flame ionisation. consisting of layers of cells with slightly and regularly
Injection 1 µL. thickened walls [Aa] and layers of hard cork with rectangular
cells whose walls are irregularly thickened with fine pits on 3
sides and thin on the upper side [Ab]; fragments of cork
2023 Eucommia Bark IV-233
consisting of polygonal cells with slightly thickened walls Drying In air.

(surface view [G]) and fragments of hard cork consisting of Detection Treat with anisaldehyde solution R and heat at
polygonal cells 15-40 µm in diameter (surface view [E]); 100-105 °C for 5 min; examine in daylight.
numerous isolated hard cork cells ([K), surface view [Ka],
transverse section [Kb]); ovoid parenchyma cells m;
rare
fragments of phloem parenchyma (transverse section [H])
consisting of medullary rays [Ha], phloem parenchyma [Hb]
and laticiferous vessels [He].
Top of the plate
A violet zone
-- --
A violet zone
~-Sitosterol: a blue zone A violet zone
-- --
A violet zone
5, 7-Dihydroxy-4-methylcoumarin: an
D *F?i
~#
orange zone
Several zones
Several zones
~
TESTS
10s 0 c.
~ JJ Total ash (2.4.16)

ASSAY
t:Ptft-25µm
Hb -
Test solution Treat 2.00 g of the freshly powdered herbal
drug (355) (2.9.12) with 75 mL of methylene chloride Rina
continuous extraction apparatus (Soxhlet type) for 1 h. Cool.
Figure 2412.-1. - Illustration for identification test B of powdered Discard the organic solution and replace with 75 mL of
herbal drng of eucommia bark methanol R. Extract for 6 h in the same apparatus. Filter and
evaporate the filtrate to dryness. Take up the residue with
C. To 1.0 g of the powdered herbal drug (355) (2.9.12) add 10. 0 mL of a mixture of methanol R and water R
10 mL of methylene chloride R and allow to stand for 2 h. (30:70 V/V). Centrifuge. Filter through a membrane filter
Filter and evaporate the filtrate to dryness. Take up the (nominal pore size 0.45 µm).
residue with 1.0 mL of anhydrous ethanol R; an elastic film is Reference solution (a) Treat 2.00 g of eucommia bark HRS
formed. with 75 mL of methylene chloride Rina continuous extraction
D. Thin-layer chromatography (2.2.27). apparatus (Soxhlet type) for 1 h. Cool. Discard the organic
Test solution To 1 g of the powdered herbal drug (355) solution and replace with 75 mL of methanol R. Extract for
(2. 9.12) add 10 mL of methylene chloride R. Sonicate for 6 h in the same apparatus. Filter and evaporate the filtrate to
10 min. Discard the liquid phase and repeat the extraction dryness. Take up the residue with 10.0 mL of a mixture of
with another 10 mL of methylene chloride R. Discard the methanol Rand water R (30:70 V/V). Centrifuge. Filter
liquid phase again. Dry the residue in air. Add 7 mL of through a membrane filter (nominal pore size 0.45 µm).
methanol R. Sonicate in a centrifuge tube at 60 °C for Reference solution (b) Dissolve 20.0 mg of caffeine CRS in
20 min. Centrifuge; use the supernatant. mobile phase A and dilute to 20.0 mL with mobile phase A.
Reference solution Dissolve 2 mg of 5,7-dihydrox:y-4- Dilute 1.0 mL of the solution to 25.0 mL with mobile
methylcoumarin R and 20 mg of /J-sitosterol R in 10 mL of phase A.
methanol R. Column:
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel - size: l = 0.25 m, 0 = 4.0 mm;
plate R (2-10 µm)]. - stationary phase: end-capped octadecylsilyl silica gel for
Mobile phase anhydrous formic acid R, ethyl acetate R, chromatography R.
toluene R (1:35:65 V/V/V). Mobile phase:
Application 20 µL [or 10 µL] as bands of 10 mm [or - mobile phase A: 1.0 g/L solution of phosphoric acid R;
8 mm]. - mobile phase B: acetonitrile R;
IV-234 Evodia Fruit 2023
Time Mobile phase A Mobile phase B finely grained cuticle [Aa, Ba], bearing unicellular [Ac] or
(min) (per cent V/JI) (per cent V/JI)
multicellular [Ab] covering trichomes (with fine internal
0 - 35 87---+ 75 13---+ 25 separations between the cells) with spiny outer walls,
35 - 40 75---+ 0 25 _. 100 occasionally remaining only as circular scars [Ae], and
glandular trichomes with a multicellular stalk (2-6 cells) with
Flow rate 1.0 mL'min. slightly thickened walls and a globular, ovoid, multicellular
Detection Spectrophotometer at 278 nm. head [Ad]; anomocytic stomata (2.8.3) [Bb] and acicular
crystals in dark brown bundles [Be] are present; isolated
InJection 20 µL.
covering trichomes, often fragmented, with a sharp [C, L] or
Identification of peaks Use the chromatogram supplied with sometimes rounded [E] tip; numerous isolated glandular
eucommia bark HRS and the chromatogram obtained with trichomes [HJ; fragments of the outer part of the pericarp
reference solution (a) to identify the peak due to pinoresinol (transverse section [F]), consisting of the epicarp [Fa] with
diglucoside and peak 2 (unknown). small cells, the mesocarp [Fe] and fragments of
Retention time Caffeine = about 8 min; pinoresinol schizolysigenous oil glands [Fb]; fragments from the inner
= =
diglucoside about 10 min; peak 2 about 11 min. part of the mesocarp (surface view [G], transverse
System suitability Reference solution (a): section [Kl) consisting of small vascular bundles with spiral
- resolution: minimum 2.0 between the peak due to vessels [Kc] and parenchyma containing very numerous
pinoresinol diglucoside and peak 2. cluster crystals of calcium oxalate [Ga, Kb], sometimes
Calculate the percentage content of pinoresinol diglucoside associated with the endocarp composed of fine elongated
using the following expression: cells [Gb, Ka]; fragments ofparenchyma from the base of
the fruit [D] containing prisms of calcium oxalate [Db] and a
A1 xm2x5.6xp few sclereids [Da]; sclereids, isolated or in small groups W-
A2 x m1 x 50
A1 area of the peak due to pinoresinol diglucoside in the
A2 area of the peak due to caffeine in the chromatogram obtained
with reference solution (b );
solution, in grams;
m2 mass of caffeine CRS used to prepare reference solution (b), in
grams;
p percentage content of caffeine in caffeine CRS;
5.6 correction factor for caffeine with respect to pinoresinol
diglucoside.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Evodia Fruit
(Ph. Bur. monograph 2718) Fe
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
y ~K
~~
DEFINffiON
Dried, whole unopened fruit of Tetradium ruticarpum (A.
Juss.) T.G.Hartley (syn. Evodia ruticarpa (A.Juss.) Hook.f. &
Thomson) collected just before ripening.
Content
Minimum 0.15 per cent for the sum of evodiamine and
rutecarpine, expressed as evodiamine (C 19H 17N 3 O;
IDENTIFICATION Figure 2718.-1. - Illustration for identification test B of powdered
A. The whole fruit is pentagonal to spheroidal, flattened at herbal drug of evodia fruit
the apex, about 2-5 mm in diameter. It consists of 5 follicles
more or less fused together, with a dark greenish-brown or C. Thin-layer chromatography (2.2.27).
brown external surface, rough, with numerous protuberances Test solution To 1 g of the powdered herbal drug (355)
due to the presence of oil cavities; the flattened upper part is (2. 9.12) add 10 mL of ethanol (96 per cent) R, sonicate for
more or less regularly stellate whereas the conical lower 10 min, centrifuge and use the supernatant.
surface bears the hairy remnants of the sepals and peduncle. Reference solution Dissolve 1 mg of evodiamine R and 1 mg
The texture is tough. A transverse section of the fruit shows of rutecarpine R in 1 mL of ethanol (96 per cent) R.
5 loculi, each containing a tiny yellowish seed.
B. Microscopic examination (2.8.23). The powder is brown.
Mobile phase ethanol (96 per cent) R, triethylamine R, ethyl
acetate R, cyclohexane R (1:1:5:19 VIVIVIV).
(Figure 2718.-1): fragments of epicarp (surface view [A, BJ) Application 5 µL as bands of 8 mm.
with cells whose walls are usually rigid and covered in a Development Over a path of 6 cm.
2023 Evodia Fruit IV-235
Drying In air. Test solutwn To 0.200 g of the powdered herbal drug (355)
Detection A Examine in ultraviolet light at 254 nm. (2.9.12) add 10.0 mL of ethanol (50 per cent VIV) Rand
Results A See below the sequence of zones present in the allow to macerate for 1 h. Sonicate for 30 min, centrifuge for
5 min and transfer the supernatant to a 50.0 mL volumetric
flask. Add 10.0 mL of ethanol (50 per cent Vlv,) R to the
test solution. Furthermore, other faint quenching zones may
residue and sonicate for 30 min. Centrifuge for 5 min and
transfer the supernatant to the same volumetric flask. Repeat
solution.
this operation once more. Dilute the 3 fractions of
supernatant in the volumetric flask to 50.0 mL with ethanol
Top of the plate
(50 per cent V/V) R and filter through a membrane filter
--- ---
Reference solutwn (a) Dissolve 5.0 mg of evodiamine CRS in
anhydrous ethanol Rand dilute to 100.0 mL with the same
Rutecarpine: a quenching zone A quenching zone (rutecarpine) solvent.
Reference solutwn (b) Dilute 10.0 mL of reference
solution (a) to 100.0 mL with anhydrous ethanol R.
Evodiamine: a quenching zone A quenching zone (evodiamine)
Reference solutwn (c) Dissolve 2.5 mg of rutecarpine R in
--- --- 50.0 mL ofreference solution (a) and dilute to 100.0 mL
A quenching zone
with anhydrous ethanol R.
Column:
=
- size: l 0.25 m, 0 4.6 mm; =
Reference solution Test solution - statwnary phase: end-capped octadecylsilyl silica gel for
Detection B Treat with a mixture of 10 volumes of sulfuric Mobile phase:
acid R and 90 volumes of ethanol (96 per cent) R, heat at
100 °C for 3 min and examine in daylight.
Results B 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/J') (per cent V/J')
test solution. Furthermore, other faint zones may be present 0 - 15 45 55
in the chromatogram obtained with the test solution. 15 - 17 45--+ 10 55--+ 90
17 - 30 10 90
Top of the plate 30 - 32 10--+ 45 90---. 55
32 - 45 45 55
3 faint brown to violet zones
A faint brownish zone

--- --- Detection Spectrophotometer at 225 nm.
Injection 10 µL.
Retention time Evodiamine = about 8 min;
Rutecarpine: a very faint brownish A faint brownish zone (rutecarpine)
zone rutecarpine = about 10 min.
- resolutwn: minimum 5.0 between the peaks due to
Evodiamine: a brownish zone A brownish zone (evodiamine) evodiamine and rutecarpine.
Calculate the percentage content of evodiamine and
--- ---
rutecarpine, expressed as evodiamine, using the following
A brownish zone expression:
A brownish-grey zone
[A1 + (A3 x4)] xm2 xp
A2 xm1 x20
area of the peak due to evodiamine in the chromatogram
A, area of the peak due to evodiamine in the chromatogram
TESTS obtained with reference solution (b);
Loss on drying (2.2.32) area of the peak due to rutecarpine in the chromatogram
Maximum 12.0 per cent, determined on 1.000 g of the obtained with the test solution;
solution, in grams;
105 °C for 2 h. mass of evodiamine CRS used to prepare reference solution (a),
Total ash (2.4.16) in grams;
p percentage content of evodiamine in evodiamine CRS.
Ash insoluble in hydrochloric acid (2.8.1) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
ASSAY
IV-236 Fennel 2023
Bitter Fennel
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINIDON
Dry cremocarps and mericarps of Foeniculum vulgare
Mill. ssp. vulgare var. vulgare.
Content
- essential oil: minimum 40 mllkg (anhydrous drug);
- anethole: minimum 60.0 per cent in the essential oil;
- fenchone: minimum 15.0 per cent in the essential oil.
CHARACTERS
Bitter fennel is greenish-brown, brown or green.
Fb
IDENTIFICATION J
Q°oo
Qi
A. The fruit of bitter fennel is a cremocarp, of almost
cylindrical shape with a rounded base and a narrower summit
0
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 ridges. When cut K
transversely, 4 vittae on the dorsal surface and 2 on the
commissural surface may be seen with a lens.
brown or greyish-yellow. Examine under a microscope using
diagnostic characters (Figure 0824.-1): yellow fragments of Figure 0824.-1. - Illustration for identification test B of powdered
wide secretory canals, often made up of yellowish-brown- herbal drug of bitter fennel
walled polygonal secretory cells [D, HJ; reticulate
parenchyma of the mesocarp [BJ; numerous fibre bundles TESTS
[G] from the ridges [Ga], often accompanied by narrow Estragole
spiral vessels [Gb]; very numerous endosperm fragments [F] Gas chromatography (2.2.28): use the normalisation
containing aleurone grains [Fb] and very small cluster procedure.
crystals of calcium oxalate [Fa]; some fibre bundles from the Test solution Dilute the mixture of essential oil and xylene R
carpophore [E]; fragments of the endocarp (surface view [A, obtained in the determination of essential oil to 5.0 mL with
K]) consisting of thin-walled, transversely elongated cells, xylene R, by rinsing the apparatus.
2-9 µm wide, having a parquetry arrangement, sometimes Reference solution Dissolve 5 mg of estragole R in 0.5 mL of
accompanied by the inner layer of the mesocarp [Aa]; xylene R.
fragments of the epicarp with stomata accompanied by oil Column:
droplets [C]; very numerous oil droplets [J]. =
- size: l 30-60 m, 0 = 0.3 mm;
C. Thin-layer chromatography (2.2.27). - stationary phase: macrogol 20 000 R.
Test solution Shake 0.3 g of the freshly powdered herbal Carrier gas nitrogen for chromatography R.
drug (1400) (2.9.12) with 5.0 mL of methylene chloride R for Flow rate 0.40 mllmin.
15 min. Filter and carefully evaporate the filtrate to dryness
Split ratio 1:200.
on a water-bath at 60 °C. Dissolve the residue in 0.5 mL of
toluene R. Temperature:
Reference solution Dissolve 50 µL of anethole R and 10 µL of
Time Temperature
fenchone R in 5.0 mL of hexane R. (min) (°C)
Plate TLC silica gel GF254 plate R. Column 0-4 60
Mobile phase hexane R, toluene R (20:80 V/V). 4 - 26 60---+ 170
Application 10 µLas bands of 20 mm by 3 mm. 26 - 41 170

Injection port 220
Detector 270
Drying In air.
Results A The chromatograms show in the central part a
Injection 1 µL.
quenching zone due to anethole.
Limit:
Detection B Treat with sulfuric acid R and heat at 140 °C for
- estragole: maximum 5.0 per cent in the essential oil
5-10 min until a yellow zone due to fenchone appears in the
obtained in the assay.
lower third of the chromatograms.
Results B Anethole appears as a violet band in the central
Maximum 1.5 per cent of peduncles and maximum
part; the chromatogram obtained with the test solution also
1.5 per cent of other foreign matter.
shows a reddish-brown zone in its upper third (terpenes).
2023 Bitter-Fennel Fruit Oil IV-237
Water (2.2.13) Top of the plate

Anethole: a dark blue to dark violet A dark blue to dark violet zone
powdered herbal drug (710) (2. 9.12). zone (anethole)
Total ash (2.4.16)
Maximum 10.0 per cent. -- --
Fenchone: a blue or bluish-grey A blue or bluish-grey zone (fenchone)

ASSAY zone
Use 5.0 g of the herbal drug reduced to a powder (1400) - - - -
(2. 9.12) immediately before the assay, 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 tube. Distil at a
rate of 2-3 mUmin for 2 h. B. Examine the chromatograms obtained in the test for
Anethole and fenchone
Gas chromatography (2.2.28) as described in the test for Results The characteristic peaks in the chromatogram
estragole with the following modifications. obtained with the test solution are similar in retention time to
Reference solution Dissolve 5 mg of fenchone R and 5 mg of
solution.
anethole R in 0.5 mL of xylene R.
Elution order The order indicated in the composition of the TESTS
reference solution; record the retention times of these Relative density (2.2.5)
substances. 0.961 to 0.975.
STORAGE Refractive index (2.2.6)
Protected from moisture. 1.528 to 1.539.
+ 10.0° to+ 24.0°.
Bitter-Fennel Fruit Oil procedure.
Test solutwn Dissolve 0.20 mL of the essential oil to be
(Ph. Eur. monograph 1826) examined in heptane Rand dilute to 10.0 mL with the same
solvent.
Reference solutwn Dissolve 20 µL of a-pinene R, 20 µL of
DEFINITION limonene R, 50 µL offenchone R, 20 µL of estragole R, 100 µL
Essential oil obtained by steam distillation from the ripe fruits of anethole R and 20 µL of anisaldehyde R in heptane R and
of Foeniculum vulgare Miller, ssp. vulgare var. vulgare. dilute to 10.0 mL with the same solvent.
Content Column:
- fenchone: 12.0 per cent to 25.0 per cent, - material: fused silica,
- trans-anethole: 55.0 per cent to 75.0 per cent. =
- size: l 60 m, 0 =
0.25 mm,
CHARACTERS - stationary phase: macrogol 20 000 R (film thickness
Appearance 0.25 µm).
Clear, colourless or pale yellow liquid. Carrier gas helium for chromatography R.
Characteristic odour. Flow rate l mUmin.
IDENTIFICATION Split ratw 1:200.
First identification: B. Temperature:
Second identijicatwn: A.
Time Temperature
A. Thin-layer chromatography (2.2.27). (min) CC)
Test solutwn Dissolve O.1 mL of the essential oil to be Column 0-4 60
examined in 5 mL of toluene R. 4 - 26 60 - 170
Reference solution Dissolve 10 µL offenchone R and 80 µL of 26 - 41 170
anethole R in 5 mL of toluene R. Injection port 220
Plate TLC silica gel plate R. Detector 270
Applicatwn 10 µL as bands. Detection Flame ionisation.
Development Over a path of 15 cm. Injection 1.0 µL.
Drying In air. Elution order Order indicated in the composition of the
Detection Spray with a freshly prepared 200 g/L solution of
substances.
phosphomolybdic acid R in ethanol (96 per cent) R and heat at
150 °C for 15 min; examine in daylight. System suitability Reference solution:
estragole and trans-anethole.
test solution. Furthermore, other zones may be present in the Using the retention times determined from the
chromatogram obtained with the test solution. chromatogram obtained with the reference solution, locate
IV-238 Bitter-Fennel Herb Oil 2023
"J
1 3 6
-
2
~
~
]
- 4
-·
j
·-
7
J I
5
I
i I I J A A
•I• •I••'! c 1 •I,• r I, , 1,,, I,,, I,,, I,,, l,,, I,,,! '' l t. I l' t 'I''' j J'' I I ('I''' I''' I''
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 min
I. IX-pinene 3. fenchone 5. cis-anethole 7. anisaldehyde

2. limonene 4. estragole 6. trans-anethole
Figure 1826.-1. - Chromatogram for the test for chromatographi.c profile of bitter-fennel fruit oil
the components of the reference solution on the

chromatogram obtained with the test solution and locate cis-
Bitter-Fennel Herb Oil
anethole using Figure 1826.-1. (Disregard the peak due to (Ph. Bur. monograph 2380)
heptane).
PhEur - - - - - - - - - - - - - - - - - - - - ~
components. The percentages are within the following DEFINITION
ranges: Essential oil obtained by steam distillation of the aerial parts
- rx-pinene: 1.0 per cent to 10.0 per cent, of Foeniculum vulgare Mill. ssp. vulgare, var. vulgare collected
- limonene: 0.9 per cent to 5.0 per cent, during fruiting.
- fenchone: 12.0 per cent to 25.0 per cent,
CHARACTERS
- estragole: maximum 6.0 per cent,
- cis-anethole: maximum 0.5 per cent, Appearance
Clear, pale or intense yellow liquid.
- trans-anethole: 55.0 per cent to 75.0 per cent,
- anisaldehyde: maximum 2.0 per cent. Anise-like odour.
The ratio of a-pinene content to limonene content is greater IDENTIFICATION
than 1.0. First identification: B.
STORAGE Second identification: A.
At a temperature not exceeding 25 "C. A. Thin-layer chromatography (2.2.27).
---------------------~fu Test solutwn Dissolve 0.1 mL of the oil to be examined in
5 mL of toluene R.
Reference solution Dissolve 10 µL of fenchone R and 40 µL of
anethole R in 5 mL of toluene R.
Mobile phase ethyl acetate R, toluene R (5:95 VIV).
2023 Bitter-Fennel Herb Oil IV-239
5 6
7
2
8 10 11
0 5 10 15 20 25 30 35 40 min
I. o:-pinene 4. o:-phellandrene 7. estragole 10. anisaldehyde

2. P-pinene 5. limonene 8. cis-anethole 11. anise ketone
3. P-myrcene 6. fenchone 9. trans-anethole
Figure 2380.-1. - Chromatogram for the test for chromatographic profile of Spanish-type
bitter-fennel herb oil
2 3 5
I
I. ,l 11 •
I
I
I
l,
I
I 6
I I
8
I
9
0 5 10 15 20 25 30 35 40 min
1. ct-pinene 4. fenchone 7. trans-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-l;Ype
bitter-fennel herb oil
IV-240 Bitter-Fennel Herb Oil 2023
Development Over a path of 8 cm [or 6 cm]. Reference solution (b) Dissolve 5 µL of anethole R in
Drying In air. 25.0 mL of acetone R. Dilute 0.5 mL of this solution to
20.0 mL with acetone R.
Detection Spray with a freshly prepared 200 g/L solution of
phosphomolybdic acid R in ethanol (96 per cenr) R and heat at Column:
150 °C for 15 min; examine in daylight. - material: fused silica;
- size: l = 60 m, 0 = 0.25 mm;
0.25 µm).
in the chromatogram obtained with the test solution. Carrier gas helium for chromatography R.
Flow rate l mUmin.
Top of the plate Split ratio l :50.
Anethole: a dark blue or dark violet A dark blue or dark violet zone Temperature:
zone (anethole)
Time Temperature
-- - - (rnin) ('C)
Fenchone: a blue or bluish-grey zone A sometimes faint blue or bluish- Column 0 - 35 70 ➔ 210
grey zone (fenchone) 35 - 42 210
-- Injection port 250

--
Detector 270

B. Examine the chromatograms obtained in the test for Injection 1 µL.
Elution order Order indicated in the composition of the
Results: reference solution; record the retention times of these
- Spanish type: the characteristic peaks due to ll-pinene, substances.
~-pinene, ~-myrcene, ll-phellandrene, limonene,
fenchone, estragole and trans-anethole in the
chromatogram obtained with the test solution are
~-myrcene and ll-phellandrene.
similar in retention time to those in the chromatogram
obtained with reference solution (a); Using the chromatogram obtained with the reference
- Tasmanian type: the characteristic peaks due to solution, locate the relevant components for the type of the
ll-pinene, ll-phellandrene, limonene, fenchone, essential oil to be examined in the chromatogram obtained
estragole and trans-anethole in the chromatogram with the test solution, and locate cis-anethole using
obtained with the test solution are similar in retention Figures 2380.-1 and 2380.-2.
time to those in the chromatogram obtained with Determine the percentage content of each of these
reference solution (a). components.
TESTS For Spanish-type bitter-fennel herb oil, the percentages are
Relative density (2.2.5) within the following ranges:
- Spanish type: 0.877 to 0.921; - rx-pinene: 2.0 to 8.0 per cent;
- Tasmanian type: 0.940 to 0.973. - [3-pinene: 1.0 to 4.0 per cent;
- [3-myrcene: 1.0 to 12.0 per cent;
Refractive index (2.2.6) - rx-phellandrene: 1.0 to 25.0 per cent;
- Spanish type: 1.487 to 1.501; - limonene: 8.0 to 30.0 per cent;
- Tasmanian type: 1.512 to 1.538. - fenchone: 7.0 to 16.0 per cent;
Optical rotation (2.2. 7) - estragole: 2.0 to 7.0 per cent;
- Spanish type: + 42° to + 68°; - cis-anethole: maximum 0.5 per cent;
- Tasmanian type: + 11 ° to + 35°. - trans-anethole: 15.0 to 40.0 per cent;
Solubility in alcohol (2. 8.10) - anisaldehyde: maximum 1.0 per cent;
- Spanish type: l volume is soluble in 2 volumes and more - anise ketone: maximum 0.05 per cent;
of ethanol (90 per cent V/V) R; - disregard limit: the area of the principal peak in the
- Tasmanian type: 1 volume is soluble in 10 volumes and chromatogram obtained with reference solution (b)
more of ethanol (85 per cent V/V) R. (0.025 per cent).
Chromatographic profile For Tasmanian-type bitter-fennel herb oil, the percentages
Gas chromatography (2.2.28): use the normalisation are within the following ranges:
procedure. - rx-pinene: 2.0 to 11.0 per cent;
- rx-phellandrene: 1.0 to 8.5 per cent;
Test solution Dissolve 0.20 mL of the oil to be examined in
- limonene: 1.0 to 6.0 per cent;
acetone Rand dilute to 10.0 mL with the same solvent.
- fenchone: 10.0 to 25.0 per cent;
Reference solution (a) Dissolve 20 µL of rx-pinene R, 10 µL of - estragole: 1.5 to 6.0 per cent;
[3-pinene R, 20 µL of [3-myrcene R, 20 µL of rx-phellandrene R, - cis-anethole: maximum 0.5 per cent;
20 µL of limonene R, 40 µL of fenchone R, 10 µL of - trans-anethole: 45.0 to 78.0 per cent;
estragole R, 40 µL of anethole R, 10 µL of anisaldehyde R and - anisaldehyde: maximum 1.0 per cent;
10 µL of anise ketone R in acetone Rand dilute to 10.0 mL - anise ketone: maximum 0.05 per cent;
2023 Sweet Fennel IV-241
- disregard limit: the area of the principal peak in the

chromatogram obtained with reference solution (b)
(0.025 per cent).
STORAGE
At a temperature not exceeding 25 °C.
LABELLING
The label states that the content is Spanish-type or
Tasmanian-type.
- - - - - - - - - - - - - - - - - - - - - Ph Eur
•. G
:~ ·11
/
~o
•c
D
.. .~
Sweet Fennel -~ :;~

(Ph. Eur. monograph 0825) =· ·~
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dry cremocarps and mericarps of Foeniculum vulgare Mill.
subsp. vulgare var. dulce (Mill.) Batt. & Trab.
Content
- essential oil: minimum 20 mUkg (anhydrous drug);
- anethole: minimum 80.0 per cent in the essential oil.
CHARACTERS
Sweet fennel is pale green or pale yellowish-brown. Figure 0825.-1. - Illustration for identification test B of powdered
herbal drug of sweet fennel
IDENTIFICATION
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 Rand 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.): 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
narrow spiral vessels [Gb]; very numerous endosperm
procedure.
fragments [F] containing aleurone grains [Fb] and very small
calcium oxalate cluster crystals [Fa]; some fibre bundles from Test solution Dilute the mixture of essential oil and :xylene R
the carpophore [E]; fragments of the endocarp (surface obtained in the assay of essential oil to 5.0 mL with xylene R,
view [K, A]) consisting of thin-walled, transversely elongated by rinsing the apparatus.
cells 2-9 µm wide, having a parquetry arrangement, Reference solution Dissolve 5 mg of estragole R and 5 mg of
sometimes accompanied by the inner layer of the fenchone R in 0.5 mL of xylene R.
mesocarp [Aa]; fragments of the epicarp with stomata Column:
accompanied by oil droplets [C]; very numerous oil =
- size: l 30-60 m, 0 = 0.3 mm;
droplets m. - stationary phase: macrogol 20 000 R.
C. Thin-layer chromatography (2.2.27). Carrier gas nitrogen for chromatography R.
Test solution Shake 0.3 g of the freshly powdered herbal Flow rate 0.40 mUmin.
drug (1400) (2.9.12) with 5.0 mL of methylene chwride R for Split ratio 1:200.
15 min. Filter and carefully evaporate the filtrate to dryness
on a water-bath at 60 °C. Dissolve the residue in 0.5 mL of Time Temperature
toluene R. (min) CC)
Reference solution Dissolve 60 µL of anethole R in 5.0 mL of Column 0-4 60
hexane R. 4 - 26 60 ➔ 170
Plate TLC silica gel GF254 plate R. 26 - 41 170
Injection port 220
Mobile phase hexane R, toluene R (20:80 V/V).
Detector 270
Application 10 µL as bands of 20 mm by 3 mm.
IV-242 Fenugreek 2023
Detection Flame ionisation. loosely arranged cells leaving numerous spaces [E); fragments
Injection 1 µL. of endosperm with cells that are rounded [F] or elongated
Limits:
[D] depending on the orientation, associated with
- estragole: maximum 10.0 per cent in the essential oil;
mucilage [Fa] and sometimes with small spiral or annular
- fenchone: maximum 7.5 per cent in the essential oil.
vessels [J].
Maximum 1.5 per cent of peduncles and maximum
1.5 per cent of other foreign matter.
Water (2.2.13)
Maximum 80 mllkg, determined on 20.0 g of the powdered
herbal drug (710) (2.9.12).
Total ash (2.4.16)
ASSAY
Use 10.0 g of the herbal drug reduced to a powder (1400)
(2.9.12) immediately before the assay, 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 tube. Distil at a
rate of 2-3 mUmin for 2 h.
Anethole
Gas chromatography (2.2.28) as described in the test for
estragole and fenchone with the following modification.
Reference solution Dissolve 5 mg of anethole R in 0.5 mL of Figure 1323.-1. - Illustration for identification test B of powdered
xylene R. herbal drug of fenugreek
STORAGE
Protected from moisture. C. Thin-layer chromatography (2.2.27).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur Test solutwn Place 1.0 g of the powdered herbal drug (710)
(2.9.12) in a 25 mL conical flask and add 5.0 mL of
methanol R. Heat in a water-bath at 65 °C for 5 min. Cool
and filter.
Reference solutwn Dissolve 3.0 mg of trigonelline
Fenugreek hydrochloride R in 1.0 mL of methanol R.
(Ph. Bur. monograph 1323) Plate TLC silica gel F254 plate R.
Mobile phase water R, methanol R (30:70 VIV).
DEFINITION
reference solution, as bands.
Dried, ripe seeds of Trigonella foenum-graecum L.
IDENTIFICATION Drying In air.
A. The seed is hard, flattened, brown or reddish-brown and
more or less rhomboidal with rounded edges. It is 3-5 mm
long, 2-3 mm wide and 1.5-2 mm thick. The widest surfaces Results A The chromatogram obtained with the test solution
are marked by a groove that divides the seed into 2 unequal shows in its lower half a quenching zone similar in position
parts. The smaller part contains the radicle; the larger part and fluorescence to the zone in the chromatogram obtained
contains the cotyledons. with the reference solution.
B. Microscopic examination (2.8.23). The powder is Detection B Spray with potassium iodobismuthate solution R2.
yellowish-brown. Examine under a microscope using chloral Results B The chromatogram obtained with the test solution
hydrate solution R. The powder shows the following diagnostic shows an intense orange-red zone similar in position and
characters (Figure 1323.-1): fragments of the testa colour to the zone in the chromatogram obtained with the
(transverse section [BJ) with lageniform epidermal cells [Bb] reference solution. It also shows in its upper half, a broad
covered by a thick cuticle [Ba], a hypodermis consisting of light brownish-yellow zone (triglycerides).
large cells, narrower at the upper end and constricted in the TESTS
middle, with bar-like thickenings of the radial walls [Be] and Loss on drying (2.2.32)
parenchyma with flattened cells [Bd]; fragments of the Maximum 12.0 per cent, determined on 1.000 g of the
epidermis in surface view, yellowish-brown, consisting of powdered herbal drug by drying in an oven at 105 °C
small polygonal cells, either with thick, channelled walls and for 2 h.
a narrow lumen (view from above [G]) or with smooth walls
and a larger lumen (view from below [Cl); fragments of the Total ash (2.4.16)
hypodermis in surface view, with cells having either a circular Maximum 5.0 per cent.
outline and thickened walls, closely beaded (view from Swelling index (2.8.4)
above [HJ), or having a polyhedral outline whose bar-like Minimum 6, determined on the powdered herbal drug (710)
thickenings extend from the lower to the upper walls (view (2.9.12).
from below [Al); parenchyma of the testa consisting of _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
2023 Feverfew IV-243
fragments of the ovary [E] with parenchymatous cells

Feverfew containing small crystals of calcium oxalate [Ea] occurring as
(Ph. Bur. monograph 1516) cluster crystals or occasionally as prisms [Eb], accompanied
by small spiral vessels [Ee]; spherical pollen grains about
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
25 µm in diameter, with 3 pores and a spiny exine [D, G].
DEFINITION
Dried, whole or fragmented aerial parts of Tanacetum
parthenium (L.) Sch. Bip.
Content
Minimum 0.20 per cent ofparthenolide (C 15 H 20 0 3 ;
CHARACTERS
Camphoraceous odour.
IDENTIFICATION
A. The leafy, more or less branched stem has a diameter of
up to 5 mm; it is almost quadrangular, channelled
longitudinally and slightly pubescent. The leaves are ovate,
2-5 cm long, sometimes up to 10 cm, yellowish-green,
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.
When present, the flowering heads are 12-22 mm in
diameter with long pedicels; they are clustered into broad
corymbs consisting of 5-30 flower-heads. The hemispherical
involucre is 6-8 mm wide and consists of many overlapping
bracts, which are rather narrow, obtuse and scarious and
have membranous margins. The central flowers are yellow,
hermaphrodite, tube-shaped with 5 teeth and have 5 stamens
inserted in the corolla; the filaments of the stamens are
separate from each other but the anthers are fused into a
tube through which passes the style, bearing 2 stigmatic
branches. The peripheral flowers are female and have a
white, three-toothed ligule, 2-7 mm long. The fruit is an
achene, 1.2-1.5 mm long, brown when ripe, with 5-10 white
herbal drng offeverfew
longitudinal ribs. It is glandular and bears a short, crenate,
membranous crown. C. Thin-layer chromatography (2.2.27).
B. Microscopic examination (2.8.23). The powder is Test solution To 1 g of the powdered herbal drug (355)
yellowish-green. Examine under a microscope using ch/,oral (2. 9.12) add 20 mL of methanol R. Heat in a water-bath at
hydrate solution R. The powder shows the following diagnostic 60 °C for 15 min. Allow to cool and filter. Evaporate to
characters (Figure 1516.-1): numerous isolated, large, dryness under reduced pressure and dissolve the residue in
multicellular, uniseriate covering trichomes m consisting of a 2 mL of methanol R.
rhomboidal basal cell, 3-5 smaller, thick-walled rectangular Reference solution Dissolve 5 mg of parthenolide R in
cells, and a very long, flat, slender terminal cell, often curved methanol R and dilute to 5 mL with the same solvent.
at a right angle to the axis of the basal cell; isolated glandular
trichomes [C, F] with a short, biseriate, 2- to 4-celled stalk
and a biseriate head of 4-6 cells around which the cuticle Mobile phase acetone R, toluene R (15:85 VIV).
forms a bladder-like covering; fragments of the epidermis of Application 20 µL, as bands.
the leaves [A) consisting of cells with sinuous anticlinal walls, Development Over a path of 10 cm.
with a striated cuticle [Aa], anomocytic stomata (2.8.3) [Ab], Drying In air.
covering trichomes [Ac] or their basal part [Ad] and
Detection Spray with a 5 g/L solution of vanillin R in a
glandular trichomes; numerous fragments of the stems
mixture of 20 volumes of anhydrous ethanol R and
(longitudinal section [H, L)) showing pitted or reticulate
80 volumes of sulfuric acid R; examine in daylight after 5 min.
vessels [Ha] accompanied by cells of the pith with pitted
walls [Hb] and groups of fibres [La]. If flowers are present, Results The chromatogram obtained with the test solution
the powder also shows: fragments of the outer epidermis of shows in its central part a blue principal zone that is similar
the bracts of the involucre (surface view [Ml), consisting, on in position, colour and size to the principal zone in the
the margin, of thin-walled cells [Ma) and occasional covering chromatogram obtained with the reference solution, and
trichomes [Mb], and a central region composed of elongated somewhat below the principal zone a 2nd blue zone may be
sclereids with strongly thickened and channelled walls [Mc]; present; 1 or 2 blue zones are also present in its lower third;
fragments of the inner epidermis of the corolla [K] consisting other violet zones may be present.
of polygonal cells, papillose, with finely striated walls; TESTS
fragments of the outer epidermis of the corolla (surface Foreign matter (2.8.2)
view [BJ) consisting of sinuous cells, covered by a striated Maximum 10.0 per cent of stem with a diameter greater than
cuticle, bearing numerous glandular trichomes [Ba]; 5 mm and maximum 2.0 per cent of other foreign matter.
IV-244 Fig 2023
Loss on drying (2.2.32) receptacle bearing on the inner surface numerous drupelets,
Maximum 10.0 per cent, determined on 1.000 g of the each containing a stone about 1.5 to 2.0 mm long; seed
powdered herbal drug (355) (2.9.12) by drying in an oven at containing endosperm and a curved embryo.
105 °C for 2 h. Microscopical Receptacle: epidermal cells polyhedral,
Total ash (2.4.16) stomata raised, trichomes unicellular, thick walled, of varying
Maximum 12.0 per cent. length up to about 300 µm; hypodermis composed of
rounded polyhedral cells, some containing small rosette
ASSAY
crystals of calcium oxalate; parenchyma made up of large,
irregular cells, forming the greater part of the receptacle,
Test solution Completely reduce about 50 g of the herbal containing large rosette crystals of calcium oxalate and
drug to be examined to a powder (355) (2.9.12). After interspersed with numerous latex tubes, about 30 to 50 µm
homogenisation, introduce 1.00 g of the powdered herbal wide, and slender vascular bundles. Pericarp: epicarp
drug into a flask and add 40 mL of methanol R. Heat in a consisting of radially elongated cells with mucilaginous outer
water-bath at 60 °C for 10 min. Allow to cool and filter. walls; mesocarp of delicate, often disorganised cells; endocarp
Rinse the filter with 15 mL of methanol R. Take up the of radially elongated sclereids with pitted walls. Endosperm
residue with 40 mL of methanol R. Repeat the operation. and embryo: small cells containing aleurone grains and fixed
Collect the filtrates and rinsings and evaporate to dryness oil; starch absent.
under reduced pressure. Take up the residue with methanol R
Water-soluble extractive
and dilute to 20.0 mL with the same solvent. Dilute 10.0 mL
Not less than 60.0% when determined by the following
of this solution to 50.0 mL with the mobile phase. Filter
method. To 25 g, minced, add 500 mL of water, boil under
a reflux condenser for 1 hour, cool and filter. To 20 mL of
Reference solution Dissolve 5.0 mg of panhenolide CRS in the filtrate add 20 g of washed and ignited sand, evaporate to
methanol Rand dilute to 10.0 mL with the same solvent. dryness in a tared, flat bottomed shallow dish and dry the
Dilute 2.0 mL of the solution to 50.0 mL with the mobile residue to constant weight at 100°. Calculate the water-
phase. soluble extractive by subtracting the weight of sand from the
Column: weight of the residue obtained.
= =
- size: l 0.25 m, 0 4.6 mm;
STORAGE
- stationary phase: octadecylsilyl silica gel for chromawgraphy R
(5 µm). Figs should be stored in a dry place.
Mobile phase acewnitrile for chromawgraphy R, water for
chromawgraphy R (40:60 V/V).
Flow rate 1 mUmin. Fleeceflower Root
Injection 20 µL. (Ph. Bur. monograph 2433)
Retention time Parthenolide = about 11.5 min. PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Calculate the percentage content of parthenolide using the DEFINITION

following expression: Whole or fragmented dried tuberous root of Fallopia
A 1 xm2 xpx0.4 multifiora (Thunb.) Haraldson (syn. Polygonum multijlorum
Thunb.).
A2xm1
Content
A1 area of the peak due to parthenolide in the chromatogram Minimum 1.0 per cent of 2,3,5,4 '-tetrahydroxystilbene-2-O-
obtained with the test solution; ~-D-glucoside (C 20 H 22O 9; Mr 406.4) (dried drug).
A2 area of the peak due to parthenolide in the chromatogram
obtained with the reference solution; IDENTIFICATION
m1 mass of the herbal drug to be examined used to prepare the test A. The whole drug consists of an irregular, fusiform,
solution, in grams;
m2 mass of parthenolide CRS used to prepare the reference solution,
tuberous, root 6-15 cm long and 4-12 cm in diameter; the
in grams; fragmented drug consists of slices or irregular pieces.
p assigned percentage content of parthenolide in parthenolide CRS. The external surface of the root is reddish-brown with
irregular wrinkles, resembling transversely elongated lenticels,
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
and with fine rootlet scars. The texture is dense, compact
and granular. The fracture is pale yellowish-brown or
reddish-brown. The drug is powdery when it is fractured.
In the cortex there are 4-11 bundles giving rise to a cloud-
Fig like appearance. The central xylem is large, sometimes
DEFINITION distinguishable as a central lignified part.
The sun-dried succulent fruit of Ficus carica L. B. Microscopic examination (2.8.23). The powder is
CHARACTERISTICS
Odour, pleasantly fruity; taste, sweet.
characters: cluster crystals of calcium oxalate 10-80 µm,
Macroscopical Fruit compound: soft, fleshy, brown or sometimes up to 160 µm, in diameter, with obtuse angles;
yellowish brown, sometimes covered with a saccharine rare, relatively large, isolated tetragonal prism crystals;
efflorescence; at the summit a small opening surrounded by fragments of parenchyma consisting of thin-walled, sub-
scales and at the base a short, stalk-like prolongation; fruit up rounded or rectangular cells, sometimes containing brown,
to about 5 cm in length and breadth, consisting of a hollow yellowish-brown or reddish-brown inclusions; rare fragments
2023 Fleeceflower Root IV-245
of cork consisting of several layers of regular cells filled with Reference solution (b) Weigh 0.250 g ofjleecefiower root HRS
brown contents; fragments of pitted vessels 15-180 µm in in a 100 mL glass vial with a screw cap. Add 50.0 mL of a
diameter; few groups of xylem fibres; scattered brown 50 per cent V/V solution of methanol R, close and extract for
masses, varying in shape, size and colour. Examine under a 1 h using ultrasound. Filter the solution through a membrane
microscope using a 50 per cent VIV solution of glycerol R. filter (nominal pore size 0.45 µm).
The powder shows simple or 2-9 compound starch granules, Column:
the simple granules are sub-rounded, 4-50 µm in diameter, - size: l = 0.125 m, 0 =4.6 mm;
with a V-shaped, stellate or Y-shaped hilum, the large - stationary phase: octadecylsilyl silica gel for chromatography R
granules show clearly visible layers. (5 µm);
C. Thin-layer chromatography (2.2.27). - temperature: 30 °C.
Test solution To 0.500 g of the powdered herbal drug (355) Mobile phase:
(2. 9.12) add 5 mL of methanol R. Heat in a water-bath at - mobile phase A: 0.1 per cent VIV solution of anhydrous
60 °C for 15 min and filter. formic acid R;
Reference solution Dissolve 1 mg of emodin R and 1 mg of - mobile phase B: acewnitrile R;
resveratrol R in 2 mL of methanol R.
Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel (min) (per cent V/J') (per cent V/J')
F2s4 plate R (2-10 µm)].
0 - 15 90--+ 70 10--+ 30
Mobile phase glacial acetic acid R, anhydrous ethanol R, 15 - 16 70--+ 20 30--+ 80
wluene R (1:4:16 VIV/V). 16 - 21 20 80
Development Over a path of 10 cm [or 6 cm]. Flow rate 1.0 mUmin.
Drying In air. Detection Spectrophotometer at 320 nm.
Detection Examine in ultraviolet light at 365 nm. Injection 10 µL.
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 fieecefiower root HRS and the chromatogram obtained with
test solution. Furthermore, other faint zones may be present reference solution (b) to identify the peak due to 2,3,5,4'-
in the chromatogram obtained with the test solution. tetrahydroxystilbene-2-0-~-D-glucoside and peak 2
(unknown).
Top of the plate Retention time 2,3,5,4 '-tetrahydroxystilbene-2-0-~-D-
A yellow fluorescent zone =
glucoside about 12 min; peak 2 about 13 min; =
resveratrol = about 17 min.
Emodin: a yellow fluorescent zone A yellow fluorescent zone (emodin)
-- -- - resolution: minimum 2.0 between the peak due to 2,3,5,4'-
tetrahydroxystilbene-2-0-~-n-glucoside and peak 2.
Resveratrol: a light blue fluorescent
zone Calculate the percentage content of 2,3,5,4'-
tetrahydroxystilbene-2-0-~-D-glucoside using the following
- - -- expression:
area of the peak due to 2,3,5,4 '-tetrahydroxystilbene-2-0-~-D-

glucoside in the chromatogram obtained with the test solution;
TESTS
area of the peak due to resveratrol in the chromatogram
Loss on drying (2.2.32) obtained with reference solution (a);
Maximum 10.0 per cent, determined on 1.000 g of the mass of the herbal drug to be examined used to prepare the test
powdered herbal drug (355) (2.9.12) by drying in an oven at solution, in grams;
mass of resveratrol CRS used to prepare reference solution (a),
105 °C for 2 h.
in grams;
Total ash (2.4.16) p percentage content of resveratrol in resveratrol CRS.
Maximum 5.0 per cent. 0.5 correction factor for resveratrol with respect to 2,3,5,4 ' -
tetrahydroxystilbene-2-0-P-o-glucoside.
Maximum 2.0 per cent. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
ASSAY
Test solution Weigh 0.250 g of the powdered herbal drug
(355) (2.9.12) in a 100 mL glass vial with a screw cap.
Add 50.0 mL of a 50 per cent VIV solution of methanol R,
close and extract for 1 h using ultrasound. Filter the solution
Reference solution (a) Dissolve 10.0 mg of resveratrol CRS in
IV-246 Forsythia Fruit 2023
Forsythia Fruit
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried ripe or steamed and dried unripe fruit of Forsythia
suspensa (Thunb.) Vahl.
Content
Minimum 0.15 per cent offorsythoside A (C 29 H 36 01s;
IDENTIFICATION
A. Loculicidal woody capsule which opens apically into 2
brittle, boat-shaped valves; the capsule is slightly compressed,
ovoid to elongated-ellipsoid. Valves are 1.2-2.5 cm long and
0.5-1.2 cm in diameter. The valve apex is acute and the
rounded base bears the peduncle scar or the peduncle, which
may be as long as the fruit. The outer surface of each valve
bears numerous verrucose, pale brown lenticels, irregular
longitudinal wrinkles and a deep median, longitudinal furrow.
The inner surface of the valve is mostly pale yellowish-brown,
smooth, slightly lustrous and is divided in 2 by a longitudinal
septum. Each capsule contains numerous seeds (around
11-20 in smaller fruits, up to 30 in larger fruits) which are
elongated, 0.5-0.9 cm long, flattened, slightly curved and
noticeably winged on one side along their entire length.
The unripe capsule is closed, yellowish-, brownish- or ~ Lb Le
blackish-green or dark brown. Seeds are greenish-yellow or
pale brownish-yellow.
Figure 2720.-1. - Illustratwnfor identificatwn test B of powdered
The ripe light brown or orange-brown fruit capsule may be
herbal drug of forsythia fruit
present as 2, free, fully dehisced valves or partially open, the
valves connected at the base and the apices recurved. Seeds Reference solutwn (a) Dissolve 5.0 mg of ursolic acid Rand
are rare, light- or dark brown. The inner surface of the valve 50.0 mg offorsythoside AR in methanol Rand dilute to
may bear the remains of the apical placenta, with or without 5.0 mL with the same solvent.
seeds. Reference solutwn (b) Dilute 2.5 mL of reference solution (a)
B. Microscopic examination (2.8.23). The powder is typically to 10.0 mL with methanol R.
greenish-brown (unripe fruits) or orange-brown (ripe fruits). Reference solution (c) Dissolve 30 mg of arbutin R and
Examine under a microscope using chloral hydrate solution R. 50 mg of forsythoside A R in methanol R and dilute to 5 mL
(Figure 2720.-1): fragments of the pericarp (surface view [A],
transverse section [F]) showing a layer of more or less Intensity marker Ursolic acid.
polygonal epicarp cells (surface view [Ab]) with slightly Plate TLC silica gel F 254 plate R (2-10 µm).
thickened walls [Ab, Fb] covered by a thick, fissured cuticle Mobile phase formic acid R, water R, wluene R, acewne R,
[Aa, Fa], the cuticle is 9-26 µm thick; fragments of the outer ethyl acetate R (6:6:40:50:60 VIVIVIVIV).
layers of the mesocarp consisting of cells with irregularly Applicatwn 2 µL as bands of 8 mm.
thickened walls, some with small rounded outgrowths
(surface view m, transverse section [Fe]); sclerenchymatous
Drying In a current of cold air for 5 min.
fibres in numerous bundles [DJ, isolated [G] or in groups
[CJ as well as sclereids of various shapes and sizes, isolated Detection Treat with anisaldehyde solutwn R2 and heat at
[HJ or in groups [BJ, from the inner layers of the mesocarp, 100 °C for 3 min; examine in daylight.
sclerenchymatous fibres sometimes in a parquetry System suitability Reference solution (c):
arrangement. Xylem vessels are narrow, 6-15 µm in - the chromatogram shows in the lower third 2 distinct
diameter. If seeds are present, the powder also shows zones; the upper zone (arbutin) and the lower zone
fragments of the outer testa (surface view [Kl), consisting of (forsythoside A) show a brownish colour.
large cells, often containing oil droplets; fragments of the Results See below the sequence of zones present in the
testa (transverse section [L]), consisting of the outer testa chromatograms obtained with reference solution (a) and the
with large cells [La] and several layers of more or less test solution. Furthermore, in the chromatogram obtained
flattened cells of the inner testa [Lb], some of which contain with the test solution, other faint zones may be present.
small prisms of calcium oxalate [Le]; small fragments of the
endosperm [E]; isolated oil droplets are also present [Ld].
2023 Frangula Bark IV-24 7
Top of the plate Flow rate 0.6 mUmin.

Injection 10 µL.
2 violet zones, faint to intense
Ursolic acid: a reddish-violet zone A reddish-violet zone, faint to reference solution (a) to identify the peak due to
equivalent forsythoside A; use the chromatogram obtained with
reference solution (b) to identify the peak due to rutoside.
--
Retention time Forsythoside A = about 16 min;
--
rutoside = about 17 min.
A greyish-reddish-violet zone, faint
forsythoside A and rutoside.
Calculate the percentage content of forsythoside A using the
-- -- following expression:
A reddish-brown zone, faint to
equivalent
Forsythoside A: a brownish zone A brownish or brownish-yellow zone, area of the peak due to forsythoside A in the chromatogram
faint to equivalent obtained with the test solution;
area of the peak due to forsythoside A in the chromatogram
A brownish-yellow zone, faint to
equivalent
Reference solution (a) Test solution solution, in grams;
mass of forsythosi.de A CRS used to prepare reference
p percentage content of forsythoside A in forsythosi.de A CRS.
TESTS
Foreign matter (2.8.2) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
105 °C for 2 h.
Frangula Bark
Total ash (2.4.16) (Ph. Bur. monograph 0025)
Preparation
ASSAY Standardised Frangula Bark Dry Extract
Liquid chromatography (2.2.29). When Powdered Frangula Bark is prescribed or demanded,
Test solution Place 0.500 g of the powdered herbal drug material complying with the requirements below, with the
(355) (2.9.12) in a 250 mL round-bottomed flask, add exception ofldentification test A and the test for Foreign
100.0 mL of methanol Rand weigh. Heat under a reflux matter, shall be dispensed or supplied.
condenser at 70 °C for 30 min, cool and weigh again. PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Compensate for the loss of solvent with methanol R. Filter
1.5 mL of the solution through a membrane filter (nominal DEFINITION
pore size 0.45 µm). Dried, whole or fragmented bark of the stems and branches
of Rhamnus frangula L. (Frangula alnus Miller).
Reference solution (a) Dissolve 6.0 mg offorsythoside A CRS
in methanol Rand dilute to 200.0 mL with the same solvent. Content
Minimum 7.0 per cent of glucofrangulins, expressed as
Reference solution (b) Dissolve 1.5 mg of rutoside trihydrate R
glucofrangulin A (C 27 H 30 O 14; Mr 578.5) (dried drug).
in 50 mL of reference solution (a).
Column: IDENTIFICATION
=
- size: l 0.25 m, 0 4.0 mm; = A. The bark occurs in curved, almost flat or rolled fragments
- stationary phase: end-capped octadecylsilyl silica gel for or in single or double quilled pieces usually 0.5-2 mm thick
chromatography Rl (5 µm); and variable in length and width. The greyish-brown or dark
- temperature: 20 °C. brown outer surface is wrinkled longitudinally and covered
Mobile phase: with numerous greyish, transversely elongated lenticels; when
- mobile phase A: formic acid R, water for chromatography R the outer layers are removed, a dark red layer is exposed.
(0.1:99.9 V/V); The orange-brown or reddish-brown inner surface is smooth
- mobile phase B: formic acid R, acetonitrile R (0.1 :99. 9 V/V); and bears fine longitudinal striations; it becomes red when
treated with alkali. The fracture is short, fibrous in the inner
part.
(min) (per cent V/J/) (per cent V/J/) B. Microscopic examination (2.8.23). The powder is
0 - 20 83 ---+ 77.5 17 ---+ 22.5 yellowish or reddish-brown. Examine under a microscope
20 - 25 77.5 ---+ 10 22.5 ---+ 90 using chloral hydrate solution R. The powder shows the
25 - 28 10 90 following diagnostic characters (Figure 0025.-1): numerous
phloem fibres (tangential section [D], longitudinal
section [Kl), partially lignified, in groups [Da, Ka] with
IV-248 Frangula Bark 2023
crystal sheaths containing calcium oxalate prisms [Db, Kb], Mobile phase water R, methanol R, ethyl acetate R
sometimes including medullary rays [De]; reddish-brown (13:17:100 VIVIV).
fragments of cork [H]; fragments of phloem parenchyma, in A. Applicatwn: 10 µL as bands.
longitudinal section [G] containing calcium oxalate cluster Development Over a path of 10 cm.
crystals [A, E] or in tangential section [C] including
medullary rays [Ca] and cells containing calcium oxalate Drying In air for 5 min.
cluster crystals [Cb]; a few fragments of collenchyma [F]; Detection Spray with a 50 g/L solution of potassium
isolated calcium oxalate cluster crystals [B] and prisms m. hydroxide R in ethanol (50 per cent VIV,) R, and heat at
100-105 °C for 15 min; examine in ultraviolet light at
365 nm.
A Results The chromatogram obtained with the reference
solution shows a brownish-yellow zone due to barbaloin in
the central part; the chromatogram obtained with the test
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.
B. Application: 10 µL of the test solution as a band.
Drying In air for maximum 5 min.
Detection Spray immediately with a 5 g/L solution of
nitrotetrazolium blue R in methanol R; examine immediately.
Results No violet or greyish-blue zones appear.
Maximum 1 per cent.
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
In a tared, round-bottomed flask with a ground-glass neck,
weigh 0.250 g of the powdered herbal drug (180) (2.9.12).
Figure 0025.-1. - Illustratwn for identificatwn test B of powdered
Add 25.0 mL of a 70 per cent V/V solution of methanol R;
herbal drng of frangula bark mix and weigh. Heat in a water-bath under a reflux
C. Examine the chromatogram obtained in test A for other condenser for 15 min. Allow to cool, weigh and adjust to the
species of Rhamnus; anthrones in ultraviolet light at 365 nm. 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 O.1 mL of hydrochloric
shows 2 orange-brown zones (glucofrangu]ins) in the lower
acid R. Shake with 5 quantities, each of 20 mL, of light
third and 2-4 red zones (frangu]ins, not always clearly
petroleum R. Allow the layers to separate and transfer the
separated, and above them frangula-emodin) in the upper
aqueous layer to a 100 mL volumetric flask. Combine the
third. light petroleum layers and wash with 2 quantities, each of
D. To about 50 mg of the powdered herbal drug (180) 15 mL, of water R. Use this water for washing the separating
(2. 9.12) add 25 mL of dilute hydrochloric acid R and heat the funnel and add it to the aqueous solution in the volumetric
mixture on a water-bath for 15 min. Allow to cool, shake flask. Add 5 mL of a 50 g/L solution of sodium carbonate R
with 20 mL of ether R and discard the aqueous layer. Shake and dilute to 100.0 mL with water R. Discard the light
the ether layer with 10 mL of dilute ammonia Rl. petroleum layer. Transfer 40.0 mL of the aqueous solution to
The aqueous layer becomes reddish-violet. a 200 mL round-bottomed flask with a ground-glass neck.
TESTS Add 20 mL of a 200 g/L solution of ferric chloride R and heat
Other species of Rhamnus; anthrones under a reflux condenser for 20 min in a water-bath with the
Thin-layer chromatography (2.2.27). water level above that of the liquid in the flask. Add 2 mL of
hydrochloric acid R and continue heating for 20 min, shaking
frequently, until the precipitate is dissolved. Allow to cool,
(2. 9.12) add 5 mL of ethanol (70 per cent V/v,) R and heat to
transfer the mixture to a separating funnel and shake with
boiling. Cool and centrifuge. Decant the supernatant
3 quantities, each of 25 mL, of ether R, previously used to
immediately and use within 30 min.
rinse the flask. Combine the ether extracts and wash with
Reference solutwn Dissolve 20 mg of barbaloin R in ethanol 2 quantities, each of 15 mL, of water R. Transfer the ether
(70 per cent Vlv,) Rand dilute to 10 mL with the same layer to a volumetric flask and dilute to 100.0 mL with
solvent. ether R. Evaporate 20.0 mL carefully to dryness and dissolve
Plates TLC silica gel plate R (2 plates). the residue in 10.0 mL of a 5 g/L solution of magnesium
2023 Frangula Preparations IV-249
acetate R in methanol R. Measure the absorbance (2.2.25) at B. To about 25 mg add 25 mL of dilute hydrochloric acid R
515 nm using methanol Ras the compensation liquid. and heat the mixture on a water-bath for 15 min. Allow to
Calculate the percentage content of glucofrangulins, cool, shake with 20 mL of ether R and discard the aqueous
expressed as glucofrangulin A, using the following expression: layer. Shake the ether layer with 10 mL of dilute ammonia Rl.
The aqueous layer becomes reddish-violet.
Ax 3.06 TESTS
m Loss on drying (2. 8.17)
i.e. taking the specific absorbance of glucofrangulin A to be
204. ASSAY
A absorbance at 515 nm; Into a tared round-bottomed flask with a ground-glass neck,
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
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
water-bath under a reflux condenser at 70 °C 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
Standardised Frangula Bark Dry water Rand 0.1 mL of hydrochloric acid R. Shake with
Extract 5 quantities, each of 20 mL, of light petroleum Rl. Allow the
layers to separate and transfer the aqueous layer to a 100 mL
(Ph. Bur. monograph 1214) volumetric flask. Combine the light petroleum layers and
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ wash with 2 quantities, each of 15 mL, of water R. Use this
water for washing the separating funnel and add it to the
DEFINITION aqueous solution in the volumetric flask. Add 5 mL of a
Standardised dry extract obtained from Frangula bark (0025). 50 g/L solution of sodium carbonate Rand dilute to 100.0 mL
Content with water R. Discard the light petroleum layer. Transfer
15.0 per cent to 30.0 per cent of glucofrangulins, expressed 40.0 mL of the aqueous solution to a 200 mL round-
as glucofrangulin A (C 27 H 30 O 14; Mr 578.5) (dried extract). bottomed flask with a ground-glass neck. Add 20 mL of a
The measured content does not deviate from that stated on 200 g/L solution of ferric chloride R and heat under a reflux
the label by more than ± 10 per cent. condenser for 20 min in a water-bath with the water level
PRODUCTION above that of the liquid in the flask. Add 2 mL of hydrochloric
acid R and continue heating for 20 min, shaking frequently,
procedure using ethanol (50-90 per cent VIV). until the precipitate is dissolved. Allow to cool, transfer the
mixture to a separating funnel and shake with 3 quantities,
CHARACTERS each of 25 mL, of ether R, previously used to rinse the flask.
Appearance Combine the ether extracts and wash with 2 quantities, each
Yellowish-brown, fine powder. of 15 mL, of water R. Transfer the ether layer to a volumetric
IDENTIFICATION flask and dilute to 100.0 mL with ether R. Evaporate
A. Thin-layer chromatography (2.2.27). 20.0 mL carefully to dryness and dissolve the residue in
10. 0 mL of a 5 g/L solution of magnesium acetate R in
5 mL of ethanol (70 per cent V/V) R and heat to boiling. Cool using methanol R as the compensation liquid.
and centrifuge. Decant the supernatant immediately and use
within 30 min. Calculate the percentage content of glucofrangulins,
expressed as glucofrangulin A, using the following expression:
Reference solution Dissolve 20 mg of barbaloin R in ethanol
(70 per cent Vlv,J R and dilute to 10 mL with the same Ax 3.06
solvent. m
Mobile phase water R, methanol R, ethyl acetate R i.e. taking the specific absorbance of glucofrangulin A to be
(13:17:100 VIVIV). 204, calculated on the basis of the specific absorbance of
barbaloin.
Development Over a path of 10 cm. A absorbance at 515 nm;
Drying In air for 5 min. m mass of the extract to be examined, in grams.
Detection Treat with a 50 g/L solution of potassium
hydroxide R in ethanol (50 per cent V/V) R and heat at LABELLING
100-105 °C for 15 min; examine immediately after heating. The label states the content of glucofrangulins.
Results The chromatogram obtained with the reference _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
solution shows in the middle third a reddish-brown zone due
to barbaloin. The chromatogram obtained with the test
solution shows 2 orange-brown zones (glucofrangulins) in the
lower third and 2-4 red zones (frangulins, not always clearly
separated, and above them frangula-emodin) in the upper
third.
IV-250 Frankincense 2023

Indian Frankincense (2. 9.12) add 90 mL of methanol R and sonicate for 10 min.
(Ph. Bur. monograph 2310) Shake the mixture vigorously 3 or 4 times during this
procedure. Dilute to 100.0 mL with methanol R. Centrifuge
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
for 5 min. Dilute 1.0 mL of the clear solution to 10.0 mL
DEFINITION with a mixture of 16 volumes of mobile phase A and
Air-dried gum-resin exudate, obtained by incision in the stem 84 volumes of mobile phase B.
or branches of Boswellia serrata Roxb. ex Colebr. Reference solution Dissolve 1.0 mg of 11-keto-/3-boswellic
Content acid R and 1.0 mg of acetyl-11-keto-/3-boswellic acid R in
- ll-keto-/3-boswellic a.cul, (C 3 olf 460 4; Mr 470.7): minimum 20.0 mL of methanol R. Dilute 1.0 mL of this solution to
1.0 per cent (dried drug); 10.0 mL with a mixture of 16 volumes of mobile phase A
- acetyl-11-keto-/3-boswellic acid (C 32H 48 0 5; Mr 512.7): and 84 volumes of mobile phase B.
minimum 1.0 per cent (dried drug). Column:
- size: l = 0.25 m, 0 = 4.6 mm;
IDENTIFICATION - stationary phase: octadecylsilyl silica gel for chromatography R
A. Indian frankincense consists of translucent, roundish or (5 µm).
irregularly shaped, variable size pieces of up to 3 cm. They
Mobile phase:
are yellowish or reddish-brown. Their surface is covered with
- mobile phase A: phosphoric a.cul, R, water R (0.1:99.9 V/V);
grey dust. The fracture is dull or slightly glossy.
- mobile phase B: phosphoric acid R, acetonitrile R
B. Thin-layer chromatography (2.2.27). (0.1:99.9 V/V);
(2.9.12) add 90 mL of methanol Rand sonicate for 10 min. Time Mobile phase A Mobile phase B
Shake the mixture vigorously 3 or 4 times during this (min) (per cent V/J/) (per cent V/J/)
procedure. Dilute to 100 mL with methanol R. Centrifuge 0 - 12.5 16--+ 6 84--+ 94
and use the clear supernatant solution. 12.5-13.5 6--+ 0 94 --+ 100
Reference solution Dissolve 2 mg of 11-keto-/3-boswellic acid R 13.5 - 28 0 100
and 2 mg of acetyl-11-keto-/3-boswellic acid R in 20 mL of
methanol R. Flow rate 1.0 mIJmin.
Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel Detection Spectrophotometer at 250 nm.
F254 plate R (2-10 µm)]. Injection 20 µL.
Mobile phase anhydrous formic acid R, heptane R, ethyl Retention time 11-keto-~-boswellic acid = about 8 min;
acetate R, toluene R (3: 10:20:80 V/V/VIV). acetyl-11-keto-~-boswellic acid = about 12 min.
Application 10 µL [or 3 µL] as bands. System suitability Reference solution:
Development Over a path of 8 cm [or 6 cm]. - resolution: minimum 6.0 between the peaks due to
Drying In air. 11-keto-~-boswellic acid and acetyl-11-keto-~-boswellic
Detection Examine in ultraviolet light at 254 nm. acid.
Results See below the sequence of zones present in the Calculate the percentage content of ll-keto-!3-boswellic acid
chromatograms obtained with the reference solution and the using the following expression:
test solution. The zones due to 11-keto-~-boswellic acid and
acetyl-11-keto-~-boswellic acid in the test solution are of
approximately equivalent intensity. Furthermore, other weak
quenching zones may be present in the chromatogram A1 area of the peak due to 11-keto-P-boswellic acid in the
obtained with the test solution. chromatogram obtained with the test solution;
A2 area of the peak due to l l-keto-!3-boswellic acid in the
Top of the plate chromatogram obtained with the reference solution;
m mass of the substance to be examined, in grams;
-- -- m1 mass of 11-keto-P-boswellic acid R in the reference solution, in
grams;
- - -- p1 percentage content of l l-keto-!3-boswellic acid in 11-keto-P-
boswellic aci.d R.
Acetyl-11-keto-P-boswellic acid: a A quenching zone (acetyl-11-keto-P-
quenching zone boswellic acid)
Calculate the percentage content of acetyl-11-keto-~-
11-Keto-P-boswellic acid: a A quenching zone ( 11-keto-P- boswellic acid using the following expression:
quenching zone boswellic acid)
TESTS area of the peak due to acetyl-11-keto-P-boswellic acid in the

Loss on drying (2.2.32) chromatogram obtained with the test solution;
Maximum 8.0 per cent, determined on 1.000 g of the area of the peak due to acetyl-11-keto-P-boswellic acid in the
chromatogram obtained with the reference solution;
powdered herbal drug (355) (2.9.12) by drying in an oven at m mass of the substance to be examined, in grams;
105 °C for 3 h. m2 = mass of acetyl-11-keto-P-boswellic acid R in the reference solution,
Total ash (2.4.16) in grams;
percentage content of acetyl-11-keto-Jl-boswellic acid in acetyl-
11-keto-P-boswellic acid R.
ASSAY
Liquid chromatography (2.2.29). _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
2023 Fraxinus Chinensis Bark IV-251
slightly thickened and pitted walls (surface view [Al) and of

Fraxinus Chinensis Bark superimposed layers of cells with slightly thickened and
Fraxinus Rhynchophylla Bark channelled walls (transverse section [DJ), sometimes
associated with one or more layers of collenchyma [Da]; long
(Ph. Eur. monograph 2452) fibres, usually fragmented, with very thick walls and a greatly
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
reduced lumen, slightly channelled, isolated [B, G] or in
DEFINITION groups [C, F, HJ, sometimes accompanied by sclereids [Ca,
Whole or fragmented, dried branch or trunk bark of Fraxinus Ha] or medullary rays with rectangular cells containing tiny
chinensis subsp. rhynchophylla (Hance) A.E.Murray (syn. acicular crystals of calcium oxalate [Fa]; groups of
Fraxinus rhynchophylla Hance), collected in spring or autumn. parenchymatous cells [Kl, some containing tiny crystals of
calcium oxalate [Ka].
Content
C. To 0.1 g of the powdered herbal drug (355) (2.9.12) add
Minimum 1.0 per cent for the sum of esculetin (C 9 H 6 0 4 ;
10 mL of water R previously heated to 60 °C. Allow to stand
M, 178.1) and esculin (C 15 H 16 0 9; M, 340.3) (dried drug).
for 2 min and filter. Examined in ultraviolet light at 365 nm,
IDENfIFICATION the solution shows an intense blue fluorescence that fades
A. The branch bark occurs as flexible, curved or channelled, considerably after the addition of 2 mL of hydrochloric acid R.
rolled or folded pieces up to 60 cm long and 3 mm thick; the D. Thin-layer chromatography (2.2.27).
outer surface is whitish-grey to dark brownish-grey,
sometimes in patches, and is smooth or slightly rough, dotted
with whitish-grey, rounded lenticels; the inner surface is
60 °C for 1 min. Centrifuge and use the supernatant; filter, if
smooth, soft to the touch, yellowish-white or brown.
necessary.
The fracture is fibrous.
Reference solution Dissolve 1 mg of esculetin R and 1 mg of
The trunk bark occurs as compact, rigid, slat-shaped pieces,
esculin R in 2 mL of methanol R.
up to 6 mm thick; the outer surface is brownish-grey, with
fine longitudinal furrows and many reddish-brown lenticels, Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel
rounded or slightly split transversally; the inner surface is F2s4plate R (2-10 µm)].
smooth, orange-brown. The fracture is fibrous. Mobue phase anhydrous formic acid R, water R, ethyl acetate R
(10:10:80 V/V/V).
Application 10 µLas bands of 15 mm [or 8 mm].
Drying In air.
Detection Treat with a solution containing 10 g/L of
diphenylboric acid aminoethyl ester R and 50 g/L of macrogol
400 R in methanol R; examine in ultraviolet light at 365 nm.
test solution. Furthermore, other fluorescent zones may be
Top of the plate
Esculetin: a greenish-yellow A greenish-yellow fluorescent zone

fluorescent zone (esculetin)
' '
-- --
A green fluorescent zone may be

present
G
A blue fluorescent zone may be
present
-- .\', -- --
·:::.,- :_; ;
·-,_ -. ___.,/., Esculin: an intense blue fluorescent An intense blue fluorescent zone
zone (esculin)
A whitish-blue fluorescent zone
Figure 2452.-1. - Illustratwnfor identification B of powdered

herbal drug of fraxinus chinensis bark TESTS
B. Microscopic examination (2.8.23). The powder is Maximum 12.0 per cent, determined on 1.000 g of the
brownish. Examine under a microscope using chloral hydrate powdered herbal drug (355) (2. 9.12) by drying in an oven at
solution R. The powder shows the following diagnostic 105 °C.
characters (Figure 2452.-1): large sclereids up to 300 µm in Total ash (2.4.16)
diameter, single ill or in groups [E], with a very narrow Maximum 5.0 per cent.
lumen and channelled walls with concentric striations;
fragments of brownish cork consisting of polygonal cells with
IV-252 Fumitory 2023

Maximwn 2.0 per cent.
Fumitory
Liquid chromatography (2.2.29). PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
(2.9.12) add 50.0 mL of methanol Rand weigh. Heat on a
DEFINITION
water-bath under a reflux condenser for 1 h. Cool and weigh Whole or fragmented, dried aerial parts of Fumaria
again. Compensate for the loss of solvent with methanol R officinalis L. harvested in full bloom.
and mix. Filter through a membrane filter (nominal pore size Content
0.45 µm). Minimum 0.40 per cent of total alkaloids, expressed as
Reference solutwn (a) Dissolve 10.0 mg of esculin CRS in the protopine (C20H 1 9N0 5; Mr 353.4) (dried drug).
mobile phase and dilute to 50.0 mL with the mobile phase. IDENTIFICATION
Reference solution (b) Dissolve 10.0 mg of esculetin CRS in A. The hollow, angular stem is light green or greenish-
10 mL of acetonitrile Rand dilute to 50.0 mL with the brown. The leaves are alternate, bipinnatisect with 2 or 3 leaf
mobile phase. segments, the ultimate lobes lanceolate or obovate; they are
Reference solutwn (c) Mix 3.0 mL ofreference solution (b) greenish-blue and glabrous on both surfaces. The flowers are
and 5.0 mL of reference solution (a) and dilute to 10.0 mL small and occur in loose racemes; each has a short pedicel
with the mobile phase. and is subtended by a leafy bract; they are pink or purplish-
Column: red, dark purple or brown at the apex; the calyx is short,
=
- size: l 0.15 m, 0 = 4.0 mm; composed of 2 petalloid sepals and the corolla is tubular with
- statwnary phase: end-capped octadecylsilyl silica gel for 4 petals, the upper petal slightly spurred; there are 6 stamens
chromatography R (5 µm). united by their filaments into 2 groups of 3. The greenish-
brown, indehiscent fruits are globular or keel-shaped,
Mobile phase acetonitrile R, 0.1 per cent V/V solution of truncated or slightly emarginate at the apex, and each
phosphon·c acid R (12:88 V/V). contains a small brown seed.
Fk>w rate 0.75 mllmin. B. Microscopic examination (2.8.23). The powder is green.
Detection Spectrophotometer at 334 nm. Examine under a microscope using chloral hydrate solution R.
Injection 10 µL. The powder shows the following diagnostic characters
Run time 1.5 times the retention time of esculetin. (Figure 1869.-1): fragments of the leaf lamina (surface view
[D]) with the upper epidermis composed of irregularly
=
Retention time Esculin about 4.5 min; esculetin about = polygonal cells [Da], some of which contain microcrystals of
8.5 min.
calciwn oxalate [Db], and underlying palisade parenchyma
Identification of peaks Use the chromatogram obtained with [De]; marginal cells at the apex of the lamina elongated to
reference solution (a) to identify the peak due to esculin and form blunt papillae [Dd), and with the lower epidermis [A]
the chromatogram obtained with reference solution (b) to composed of cells having wavier walls [Aa] and underlying
identify the peak due to esculetin. spongy parenchyma [Ac]; anomocytic stomata (2.8.3) [Ab,
System suitability Reference solution (c): De] on both surfaces; groups [G] of lignified fibres [Ga] and
- resolution: minimwn 5.0 between the peaks due to esculin spiral [Gb], reticulate or bordered-pitted [BJ vessels from the
and esculetin. stem; fragments of the epidermis of the petals [F] composed
Calculate the percentage content of the swn of esculetin and of polygonal cells with sinuous or wavy anticlinal walls and
esculin using the following expression: no papillae; spherical pollen grains [E], about 30 µm 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 [HJ, and sinuous sclereids with thick and
channelled walls, from the endocarp [C].
area of the peak due to esculin in the chromatogram
area of the peak due to esculin in the chromatogram Test solutwn To 2 g of the powdered herbal drug (355)
obtained with reference solution (c); (2. 9.12) add 15 mL of dilute sulfuric acid Rl and stir for
area of the peak due to esculetin in the chromatogram
15 min. Filter. Dilute the filtrate to 20 mL with dilute sulfuric
area of the peak due to esculetin in the chromatogram acid Rl. Add 1 mL of concentrated ammonia R and 10 mL of
obtained with reference solution (c); ethyl acetate R. Stir and centrifuge. Collect the upper organic
mass of the herbal drug to be examined used to prepare the layer. Repeat the extraction in the same manner. Collect the
organic layers and dry over anhydrous sodium sulfate R.
mass of esculin CRS used to prepare reference solution (a),
in grams; Evaporate to dryness under reduced pressure. Take up the
mass of esculetin CRS used to prepare reference solution (b), residue with 0.5 mL of methanol R.
in grams;
Reference solutwn Dissolve 5 mg of protopine hydrochloride R
P1 percentage content of esculin in esculin CRS;
P2 percentage content of esculetin in esculetin CRS. and 5 mg of quinine R in 10 mL of methanol R.
- - - - - - - - - - - - - - - - - - - - - - Ph Eur Mobile phase concentrated ammonia R, ethanol (96 per cent) R,
acetone R, toluene R (2:6:40:52 V/VIVIV).
Applicatwn 30 µL as bands.
Drying In air.
2023 Ganoderrna IV-253
Top of the plate
Protopine: an orange zone An orange zone (protopine)
2 orange zones
-- --
Quinine: an orange zone A faint orange zone
-- --
TESTS
Cadmium (2. 4. 27)
Maximum 1.5 ppm.
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
To 5.000 g of the powdered herbal drug (355) (2. 9. 12) add
5 mL of dilute ammonia Rl and 50 mL of ethyl acetate R.
Shake for 15 min. Filter. Repeat the procedure in the same
manner and combine the filtrates. Evaporate the filtrates to
dryness under reduced pressure. Dissolve the residue by
sonication for 10 min in 50 mL of dilute suljuric acid Rl.
Figure 1869.-1. - Illustration for identification test B of powdered Filter. Dilute the filtrate to 100 mL with dilute suljuric
herbal drug of fumitory acid Rl. Adjust to pH 9-10 with concentrated ammonia Rand
then add 50 mL of ethyl acetate R. Shake gently. Collect the
Detection A Examine in ultraviolet light at 365 nm. upper organic layer, after centrifugation if necessary. Repeat
Results A See below the sequence of zones present in the the procedure in the same manner. Combine the organic
chromatograms obtained with the reference solution and the layers and dry over anhydrous sodium sulfate R. Evaporate to
test solution. Furthermore, other blue fluorescent zones are dryness under reduced pressure. Take up the residue with
present in the chromatogram obtained with the test solution. 100 mL of anhydrous acetic acid R. Titrate with 0. 02 M
perchloric acid, determining the end-point potentiometrically
(2.2.20).
Top of the plate
1 mL of 0.02 M perchloric acid is equivalent to 7.068 mg of
protopine.
4 blue fluorescent zones Calculate the percentage content of total alkaloids, expressed
as protopine, using the following expression:
-- --
Quinine: a blue fluorescent zone
n X 706.8
m
A greenish-blue fluorescent zone
n volume of 0. 02 M perchloric acid used, in millilitres;
-- -- m mass of the herbal drug to be examined, in milligrams.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Detection B Treat with a mixture of potassium iodobismuthate

solution R2, acetic acid Rand water R (1:2:10 VIVIV) until
orange zones appear against a yellow background.
Ganoderma
chromatograms obtained with the reference solution and the (Ph. Bur. monograph 3001)
test solution. Furthermore, other less intense orange zones
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
are present in the chromatogram obtained with the test
solution. DEFINITION
Whole or fragmented sporophore (reproductive body) of
Ganoderma lucidum (Curtis) P.Karst., cultivated or wild,
dried in the shade or in an oven at 40-50 °C.
Content
Minimum 0.3 per cent of total triterpene acids, expressed as
ganoderic acid A (C 30H 44 0 7; Mr 516.7) (dried drug).
IV-254 Ganoderma 2023
IDENTIFICATION Intensity marker Oleanolic acid.

A. Whole drng. The sporophore consists of a stipe (stalk) and Plate TLC silica gel F254 plate R (2-10 µm).
a cap (pileus), with a woody appearance. At maturity, it is Mobile phase anhydrous formic acid R, ethyl formate R,
covered in a hard, glossy, violet-brown to reddish-brown toluene R (2:50:50 VIVIV).
varnish, except for the under surface of the cap. The stipe,
Application l O µL as bands of 8 mm.
about 7-15 cm high and 1-5 cm in diameter, is cylindrical,
uneven and somewhat bent. The cap is usually eccentric, Development 70 mm from the lower edge of the plate.
reniform or subrounded, 10-18 cm in diameter and 1-2 cm Drying In a current of cold air for 5 min.
thick. The upper surface is hard and glossy and shows Detection Treat with a 10 per cent V/V solution of sulfuric
concentric furrows and radial striations. The margin is either acid R in ethanol (96 per cent) R; heat at 105 °C for 5 min
fine and slightly curved inwards, or thick, obtuse, and examine in ultraviolet light at 366 nm.
protuberant, matt and whitish. The under surface is whitish System suitabi7ity Reference solution (c):
to brownish and shows small, round, orange-brown pores less - the chromatogram shows 2 distinct zones in the
than 0.2 mm in diameter. Cultivated ganoderma often has a middle third, which may, however, be touching; the
larger cap (10-22 cm in diameter and 1.5-4 cm thick), with a upper zone (oleanolic acid) is reddish-brown and the
less glossy outer surface and is often covered by abundant lower zone (diosgenin) shows a bright blue
spores appearing as a yellowish-brown powder. fluorescence.
Fragmented drng Slices of the cap. The outer surface is Results See below the sequence of zones present in the
reddish-brown to blackish-brown; the fracture, spongy, shows chromatograms obtained with reference solution (a) and the
several brown to pale brown zones. A darker brown zone, test solution. Furthermore, other faint zones of various
consisting of numerous parallel tubes 0.5-2 mm long, is colours may be present in the chromatogram obtained with
visible near the under surface. the test solution.
B. Microscopic examination (2.8.23). The powder is brown,
blackish-brown or yellowish-brown. Examine under a Top of the plate
microscope using lactic reagent R. The powder shows the
following diagnostic characters (Figure 3001. -1): thin-walled, A pale yellow zone
colourless or pale brown hyphae [A], 2.5-6.5 µm in diameter,
curved, twisted or branched, isolated or in groups; orange-
brown spores (8-12 µm long and 5-8 µm wide), oval, An intense dark red zone
truncated, with a germinal pore at the apex [BJ; the outer
walls of the spores are colourless and the inner walls show
protuberances; the orange-yellow contents are granular and
sometimes show droplets.
-- --
Oleanolic acid: an intense reddish- An intense reddish-brown zone

brown zone overlapping with a yellow zone
1-3 yellow zones of variable intensity
A faint to intense yellow zone
-- --
Ganoderic acid A: a greenish-blue A very faint to faint greenish-blue

fluorescent zone fluorescent zone (ganoderic acid A)
A very faint or intense yellow zone

herbal drng of ganoderma
TESTS
C. High-performance thin-layer chromatography (2.8.25). Loss on drying (2.2.32)
Test solution To 0.5 g of the powdered herbal drug (4000) Maximum 17.0 per cent, determined on 1.000 g of the
(2.9.12) add 5.0 mL of ethanol (70 per cent V/V) R. Sonicate powdered herbal drug (4000) (2. 9.12) by drying in an oven
for 15 min, centrifuge and use the supernatant. at 105 °C for 4 h.
Reference solution (a) Dissolve 2.5 mg of oleanolic acid Rand Total ash (2.4.16)
5.0 mg of ganoderic acid A R in ethanol (70 per cent V/"V,) R Maximum 2.0 per cent.
ASSAY
Reference solution (b) Dilute 2.5 mL of reference solution (a) Liquid chromatography (2.2.29).
to 10.0 mL with ethanol (70 per cent V/V) R.
Stock solution To 2.0 g of the powdered herbal drug (4000)
Reference solution (c) Dissolve 2.5 mg of oleanolic acid Rand (2. 9.12) add 75 mL of ethanol (96 per cent) R and heat on a
5 mg of diosgenin R in ethanol (70 per cent V/V) R and dilute water-bath under a reflux condenser for 45 min. Allow to
to 10 mL with the same solvent.
2023 Garlic Powder IV-255
cool and filter. Evaporate the filtrate to dryness under triterpene 7 =about 1.05; triterpene 8 =about 1.25;
reduced pressure. Dissolve the residue in ethanol triterpene 9 =about 1.33; triterpene 10 =about 1.54.
(96 per cent) R and dilute to 25.0 mL with the same solvent. System suitability Reference solution (b):
Filter through a membrane filter (nominal pore size - peak-to-valley ratio: minimum 10.0, where Hp = height
0.45 µm). above the baseline of the peak due to ganoderic acid A
Test solution To 2.0 mL of the stock solution add 18 mL of and Hv = height above the baseline of the lowest point of
water R and mix thoroughly. Apply the solution to a 4 mL the curve separating this peak from the peak due to
solid phase extraction column, containing 0.200 g of triterpene 7.
octadecylsilyl silica gel for chromatography R, previously Calculate the percentage content of total triterpene acids,
conditioned with 5 mL of methanol R and then with 3 mL of expressed as ganoderic acid A, using the following
water R ensuring that the column does not dry out. Wash the expression:
column with 3 mL of water R and discard the washings.
Elute the column with 2 mL of methanol R, collect the eluate
and mix thoroughly. Filter through a membrane filter
Reference solution (a) Dissolve 5.0 mg of ganoderic A1 sum of the areas of the peaks due to triterpenes I, 2, 3, 4, S, 7,
8, 9, I 0 and to ganoderic acid A in the chromatogram obtained
acid A CRS in methanol Rand dilute to 5.0 mL with the with the test solution;
same solvent. Dilute 1.0 mL of the solution to 10.0 mL with A2 area of the peak due to ganoderic acid A in the chromatogram
methanol R. obtained with reference solution (a);
m1 mass of the herbal drug to be examined used to prepare the
Reference solution (b) To 0.200 g of ganoderma dry extract for stock solution, in grams;
system suitability HRS add 25 mL of ethanol (96 per cent) R. m2 mass of ganoderic acid A CRS used to prepare reference
Sonicate for 10 min and centrifuge. To 2 mL of the solution (a), in grams;
supernatant add 18 mL of water Rand mix thoroughly. p percentage content of ganoderic acid A in ganoderic acid A CRS.
Apply the solution to a 4 mL solid phase extraction column,
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
containing 0.200 g of octadecylsilyl silica gel for
chromatography R, previously conditioned with 5 mL of
methanol R and then with 3 mL of water R ensuring that the
column does not dry out. Wash the column with 3 mL of
water R and discard the washings. Elute the column with Garlic Powder
2 mL of methanol R, collect the eluate and mix thoroughly.
Filter through a membrane filter (nominal pore size (Ph. Bur. monograph 1216)
0.22 µm). ~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Column:
DEFINITION
=
- size: l 0.15 m, 0 = 2.1 mm;
Bulb of Allium sativum L., with the outer comeous layer
removed, cut, freeze-dried or dried at a temperature not
chromatography compatible with 100 per cent aqueous mobile
exceeding 65 °C and powdered.
phases R (1.8 µm);
- temperature: 25 °C. Content
Mobile phase: Minimum 0.45 per cent of allicin (C 6H 10OS 2 ; Mr 162.3)
- mobile phase A: 0.075 per cent V/V solution of phosphoric (dried drug).
acid R; CHARACTERS
- mobile phase B: acetonitrile R; Appearance
Light yellowish powder.
(min) (per cent VIV) (per cent VIV) IDENTIFICATION
0-3 80 ➔ 73.5 20 ➔ 26.5
A. Microscopic examination (2.8.23). Examine under a
3 - 35 73.5 26.5
35 - 53 73.5 ➔ 61.S 26.5 ➔ 38.S shows the following diagnostic characters (Figure 1216.-1):
53 - 58 61.S 38.5
numerous fragments of parenchyma (surface view [A],
transverse section [BJ) and groups of spiral or annular
vessels, sometimes with a large diameter [C, DJ,
Flow rate 0.4 mLJmin. accompanied by thin-walled parenchymatous cells.
Injection 5 µL.
Test solution To 1.0 g of garlic powder add 5.0 mL of
Identification of peaks Use the chromatogram supplied with methanol R, shake for 60 s and filter.
ganoderma dry extract for system suitability HRS and the
Reference solution Dissolve 5 mg of alanine R in 10 mL of
chromatogram obtained with reference solution (b) to
water R and dilute to 20 mL with methanol R.
identify the peaks due to triterpenes 1, 2, 3, 4, 5, 6
(ganoderic acid A), 7, 8, 9 and 10; use the chromatogram Plate TLC silica gel plate R.
obtained with reference solution (a) to identify the peak due Mobile phase glacial acetic acid R, propanol R, water R,
to ganoderic acid A. anhydrous ethanol R (20:20:20:40 VIVIVIV).
Relative retention With reference to ganoderic acid A Application 20 µL of the test solution and 10 µL of the
(retention time = about 34.4 min): triterpene 1 = about 0.36; reference solution, as bands.
triterpene 2 = about 0.41; triterpene 3 = about 0.56; Development Over a path of 10 cm.
triterpene 4 = about 0.60; triterpene 5 = about 0.66; Drying In air.
IV-256 Gastrodia Elata Rhizome 2023
Precolumn:
=
- size: l 20 mm, 0 =
4 mm;
- stationary phase: silanised octadecylsilyl silica gel for
Column:
- size: l = 0.25 m, 0 = 4 mm;
- stationary phase: silanised octadecylsilyl silica gel for
Mobile phase Mix 40 volumes of a 1 per cent V/V solution
of anhydrous formic acid R and 60 volumes of methanol R.
Injection 1 µL of the internal standard solution and 10 µL
of the test solution.
Calculate the percentage content of allicin using the following
expression:
S1 Xm2 X 22.75
S2 xm 1
= area of the peak due to allicin (principal peak) in the

area of the peak due to butyl parahydroxybenzoate in the
mass of the herbal drug to be examined, in grams;
mass of butyl parahydroxybenzoate in 100.0 mL of the internal
Figure 1216.-1. - Illustration for identification test A of garlic standard solution, in grams. 1 mg of butylparahydroxybenzoate
powder corresponds to 8.65 mg of allicin.
Detection Spray with a 2 giL solution of ninhydrin R in a - - - - - - - - - - - - - - - - - - - - - - Ph Eur

mixture of 5 volumes of glacial acetic acid R and 95 volumes
of butanol Rand heat at 105-110 °C for 5-10 min; examine
in daylight.
Results The chromatogram obtained with the reference Gastrodia Elata Rhizome
solution shows a violet zone (alanine) in the middle third.
The chromatogram obtained with the test solution shows a (Gastrodia Rhizome, Ph. Bur. monograph 2721)
violet or brownish-red zone similar in position to that in the Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
chromatogram obtained with the reference solution and
corresponding to alliin; above and below this zone are other, DEFINITION
generally fainter, violet zones. Steamed, fragmented and dried tuber of Gastrodia elata
Blume.
TESTS
Starch Content
Examine the powdered herbal drug under a microscope using Minimum 0.20 per cent of gastrodin (C 13 H 180 7 ; Mr 286.3)
water R. Add iodine solution Rl. No blue colour develops. (dried drug).
Loss on drying (2.2.32) IDENTIFICATION
Maximum 7.0 per cent, determined on 1.000 g of the A. Whole or fragmented slices up to 3 cm in diameter and
powdered herbal drug by drying in an oven at 105 °C. 0.1-0.2 cm thick, uniformly yellow or brownish-yellow,
Total ash (2.4.16) translucent and vitreous. The texture is corneous.
Maximum 5.0 per cent. The fracture is hard.
ASSAY brown. Examine under a microscope using chloral hydrate
Liquid chromatography (2.2.29). Carry out the assay as quickly solution R. The powder shows the following diagnostic
as possible. characters (Figure 2721.-1): fragments of internal
Internal standard solution Dissolve 20.0 mg of butyl parenchyma consisting of large spheroidal cells, up to
parahydroxybenzoate CRS in 100.0 mL of a mixture of 350 µm in diameter, with slightly thickened and pitted walls
equal volumes of methanol R and water R. [E]; numerous fragments of parenchyma with shrivelled and
Test solution To 0.800 g of garlic powder add 20.0 mL of often ripped cells caused by the treatment of the herbal drug
water R and homogenise the mixture in an ultrasonic bath at [BJ; rare fragments of dermal tissue [A, C] with cells that are
4 °C for 5 min. Allow to stand at room temperature for polyhedral (surface view [Al) and elongated (transverse
30 min, then centrifuge for 30 min. Dilute 10.0 mL of the section [Ca]) accompanied by a few layers of parenchyma
supernatant to 25.0 mL with a mixture of 40 volumes of a cells of the same shape [Cb]; rare fragments of parenchyma
1 per cent V/V solution of anhydrous formic acid R and containing idioblasts with raphides of calcium oxalate, the
60 volumes of methanol R (stock solution). Shake and needles of which are 50-70 µm long [D]; fragments of
centrifuge for 5 min. Place 0.50 mL of the internal standard annular or scalariform vessels up to 35 µm in diameter [F].
solution in a volumetric flask and dilute to 10.0 mL with the Examine under a microscope using a 50 per cent V/V
stock solution. solution of glycerol R. The powder mainly shows colourless or
2023 Gastrodia Elata Rhizome IV-257
pale yellow, gelatinised masses, that turn uniformly violet or Top of the plate
brownish-violet upon addition of iodine solutum Rl.
~-Sitosterol: a reddish-violet zone A faint reddish-violet zone
A prominent brown zone
-- --
A faint reddish-violet zone
-- --
Gastrodin: a brown zone A brown zone (gastrodin)
A prominent dark brown zone
TESTS
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Solvent mixture acetonitrile R, water R (3:97 V/V).
(2.9.12) in a round-bottomed flask add 30 mL of ethanol
(50 per cent V/V) R. Heat under a reflux condenser in a
water-bath at 90 °C for 3 h. Allow to cool. Filter through a
paper filter and rinse the round-bottomed flask and the filter
Figure 2721.-1. - Illustration for identification test B of powdered with ethanol (50 per cent Vlv,) R. Combine the filtrate and the
herbal drug of gastrodia rhizome rinsings and dilute to 50.0 mL with ethanol
(50 per cent Vlv,) R. Evaporate 10.0 mL of the solution to
dryness. Dissolve the residue in the solvent mixture and
Test solution To 1.0 g of the powdered herbal drug (355) dilute to 25.0 mL with the solvent mixture. Filter through a
(2.9.12) add 5.0 mL of methanol R. Sonicate for 10 min. membrane filter (nominal pore size 0.45 µm).
Reference solution (a) Dissolve 5.0 mg of gastrodin CRS in
Reference solution Dissolve 1.5 mg of gastrodin Rand 1.5 mg the solvent mixture and dilute to 10.0 mL with the solvent
of P-sitosterol R in methanol Rand dilute to 5.0 mL with the mixture.
same solvent.
Plate TLC silica gel plate R (2-10 µm). to 10.0 mL with the solvent mixture.
Mobile phase water R, ethyl acetate R, methanol R, methylene Reference solution (c) Dissolve 1 mg of arbutin R in 2 mL of
chloride R (2:20:20:58 VIVIVIV). reference solution (a) and dilute to 20 mL with the solvent
Application 15 µL as bands of 8 mm. mixture.
Development Over a path of 6 cm. Column:
Drying In air. - size: l = 0.15 m, 0 = 4.6 mm;
Detection Treat with a 10 per cent V/V solution of sulfuric - stationary phase: end-capped octadecylsil,yl silica gel for
acid R in methanol R, heat at 120 °C for 3 min and examine chromatography R (3 µm).
in daylight. Mobile phase:
Results See below the sequence of zones present in the - mobile phase A: phosphoric acid R, water for
chromatograms obtained with the reference solution and the chromatography R (0.1:99.9 V/V);
test solution. Furthermore, other faint zones may be present - mobile phase B: acetonitrile Rl;
(min) (per cent V/f') (per cent Vlf')
0 - 18 97 3
18 - 22 97 -> 0 3 -> 100
Flow rate O.5 mUmin.

IV-258 Gentian 2023
Injection 10 µL of the test solution and reference (phelloderm) [Cb]; fragments of parenchyma (longitudinal
solutions (b) and (c). section [BJ, transverse section [D]) with moderately thick-
Retention time Arbutin = about 9 min; gastrodin = about walled cells containing droplets of oil [Ba, Da], small
14 min. prisms [Bb, Db] and minute needles of calcium
oxalate [Be, De]; isolated fragments of lignified vessels with
- resolution: minimum 3.0 between the peaks due to arbutin
spiral [HJ or reticulate [G] thickening and up to 80 µm in
diameter; fragments of xylem (longitudinal section [A],
and gastrodin.
transverse section [F]) consisting of vessels [Aa, Fa] and of
Calculate the percentage content of gastrodin using the moderately thick-walled parenchymatous cells containing
following expression: droplets of oil [Ab, Fb].
Ai xm2 xpx 1.25
A2xm1
area of the peak due to gastrodin in the chromatogram obtained

area of the peak due to gastrodin in the chromatogram obtained
with reference solution (b);
solution, in grams;
m2 mass of gastrodin CRS used to prepare reference solution (a), in
grams;
p percentage content of gastrodin in gastrodin CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Gentian
(Gentian Root, Ph. Bur. monograph 0392)
When Powdered Gentian is prescribed or demanded,
exception of Identification test A shall be dispensed or
supplied.
Preparations
Compound Gentian Infusion
Gentian Tincture
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
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
A. Gentian root occurs as single or branched subcylindrical 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
short fracture but they absorb moisture readily to become Plate TLC silica gel F 25 4 plate R.
flexible. The smoothed, transversely cut surface shows a Mobile phase water R, anhydrous formic acid R, ethyl
bark, occupying about one-quarter of the radius, separated by formate R (4:8:88 VIVIV).
the well-marked cambium from an indistinctly radiate and Application 20 µL as bands.
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
following diagnostic characters (Figure 0392.-1): fragments of Results A See below the sequence of zones present in the
cork with polyhedral, thin-walled, yellowish-brown cells chromatograms obtained with the reference solution and the
(surface view [E]); fragments of dermal tissue (transverse test solution. Furthermore, other zones may be present in the
section [C]) consisting of thin-walled, yellowish-brown cork chromatogram obtained with the test solution.
cells [Ca] and thick-walled collenchymatous cells
2023 Gentian Preparations IV-259
Top of the plate

Compound Gentian Infusion
A prominent quenching zone
DEFINITION
Phenazone: a quenching zone
Concentrated Compound Gentian Infusion 100ml
A weak quenching zone Water Sufficient to produce 1000 ml
(amarogentin)
- - - - The infuswn complies with the requirements stated under Infusions.
-- --
CONCENTRATED COMPOUND GENTIAN
Hypcroside: a quenching zone A prominent quenching zone
(gentiopicroside) INFUSION
DEFINITION
Gentian, cut small and bruised 125 g

Detection B Treat with a 100 g/L solution of potassium Dried Bitter-orange Peel, cut small 125 g
hydroxide R in methanol R and then with a freshly prepared Dried Lemon Peel, cut small 125 g
Ethanol (25 per cent) 1200 ml
2 g/L solution of fast blue B salt R in a mixture of 50 volumes
of anhydrous ethanol R and 50 volumes of water R; examine in
daylight.
Macerate the Gentian, the Dried Bitter-orange Peel and the
Dried Lemon Peel in a covered vessel for 48 hours with
1000 ml of the Ethanol (25 per cent); express the liquid.
To the pressed mare add 200 ml of the Ethanol
(25 per cent), macerate for 24 hours, press and add the
Top of the plate
liquid to the product of the first pressing. Allow to stand for
A prominent dark violet zone not less than 14 days; filter.
A violet-red zone (amarogentin) TESTS
Ethanol content
-- -- 20 to 24% v/v, Appendix VIII F.
-- -- Total solids
Hyperoside: a brownish-red zone A weak light brown zone Not less than 9.5% w/v, Appendix XIA.
(gentiopicroside)
Gentian Tincture
TESTS
Other species of Gentiana (Ph. Bur. monograph 1870)
Examine the chromatograms obtained in identification PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
test C, detection B.
DEFINITION
Tincture produced from Gentian root (0392).
does not show violet zones immediately above the zone due
to amarogentin. PRODUCTION
Total ash (2.4.16) The tincture is produced from 1 part of the comminuted
Maximum 6.0 per cent. drug and 5 parts of ethanol (70 per cent VIV) by a suitable
procedure.
Bitterness value (2.8. 15)
Minimum 10 000. CHARACTERS
Appearance
Yellowish-brown or reddish-brown liquid.
Minimum 33 per cent.
It has a strong bitter taste.
200 mL of boiling water R. Allow to stand for 10 min, IDENTIFICATION
shaking occasionally. Allow to cool, dilute to 200.0 mL with Thin-layer chromatography (2.2.27).
water R and filter. Evaporate 20.0 mL of the filtrate to Test solutwn The tincture to be examined.
dryness on a water-bath. Dry the residue in an oven at Reference solutwn Dissolve 5 mg of phenazone R and 5 mg of
100-105 "C. The residue weighs a minimum of0.165 g. hyperoside R in 10 mL of methanol R.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Plate TLC silica gel F 254 plate R.
Mobile phase water R, anhydrous formic acid R, ethyl
formate R (4:8:88 VIVIV).
Application 20 µL, as bands.
Development Over a path of 8 cm, in an unsaturated tank.
Drying In air.
IV-260 Gentian Preparations 2023
Results A See below the sequence of the zones present in ASSAY

the chromatograms obtained with the reference solution and To 10 mL add 10 mL of water, adjust the pH to between 5.0
the test solution. Furthermore, other zones may be present in and 6.0 with 2M sodium hydroxide and dilute to 25 mL with
the chromatogram obtained with the test solution. water. Add 75 mL of acetate buffer pH 5.0 and titrate with
0.1M silver nitrate VS determining the end point
Top of the plate 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
Phenazone: a quenching zone of HCI.
A weak quenching zone
(amarogentin)
--- --
Alkaline Gentian Mixture
-- -- Alkaline Gentian Oral Solution
Hyperoside: a quenching zone A prominent quenching zone DEFINITION
(gentiopicroside)
Alkaline Gentian Mixture is an oral solution containing
Reference solution Test solution 10% v/v of Concentrated Compound Gentian Infusion and
5% w/v of Sodium Bicarbonate in a suitable vehicle.
Detection B Spray with a 10 per cent VIV solution of The mixture complies with the requirements stated under Oral
potassium hydroxide R in methanol R and then with a freshly Li.quids and with the following requirements.
prepared 2 giL solution offast blue B salt R in a mixture of Content of sodium bicarbonate, NaHCO 3
ethanol Rand water R (50:50 V/V). Examine in daylight. 4.75 to 5.25% w/v.
Results B See below the sequence of the zones present in ASSAY
the chromatograms obtained with the reference solution and To 10 mL of the mixture add 100 mL of water and 25 mL
the test solution. Furthermore, other zones may be present in of 0. 5M hydrochloric acid VS, boil for 10 minutes and titrate
the chromatogram obtained with the test solution. the excess of hydrochloric acid with 0.5M sodium hydroxide
VS using 0.5 mL of methyl red solution as indicator. Each mL
Top of the plate of 0.5M hydrochloric acid VS is equivalent to 42.00 mg of
A prominent dark violet zone
NaHCO 3 •
A violet-red zone (amarogentin)
-- --
Ginger
-- --
Hyperoside: a brownish-red zone A weak light brown zone (Ph. Bur. monograph 1522)
(gentiopicroside)
Preparation
Reference solution Test solution Strong Ginger Tincture
Ginger may be known in commerce as unbleached ginger.
When Powdered Ginger is prescribed or demanded, material
TESTS
complying with the appropriate requirements below shall be
Ethanol content (2. 9. 10) dispensed or supplied.
62 per cent VIV to 67 per cent V/V.
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Bitterness value (2.8. 15)
Minimum 1000. DEFINITION
Dried, whole or cut rhizome of Zingiber officinale Roscoe,
with the cork removed, either completely or from the wide,
Minimum 5.0 per cent, determined on 3.00 g.
flat surfaces only.
- - - - - - - - - - - - - - - - - - - - - ~ PhEur
Content
Minimum 15 mUkg of essential oil (anhydrous drug).
CHARACTERS
Acid Gentian Mixture Characteristic aromatic odour.
Acid Gentian Oral Solution Spicy and burning taste.
Acid Gentian Mixture is an oral solution containing 10% v/v A. The rhizome is laterally compressed, bearing short,
of Concentrated Compound Gentian Infusion and 5% v/v of flattened, obovate oblique branches on the upper side, each
Dilute Hydrochloric Acid in a suitable vehicle. sometimes having a depressed scar at the apex; the whole
The mixture complies with the requirements stated under Oral rhizomes are about 5-10 cm long, 1.5-3 cm or 4 cm wide
Li.quids and with the following requirements. and 1-1.5 cm thick, sometimes split longitudinally.
The scraped rhizome with a light-brown external surface
Content of hydrochloric acid, HCI shows longitudinal striations and occasional loose fibres; the
0.48 to 0.56% w/v. 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
2023 Ginger Preparations IV-261
cork readily exfoliates from the lateral surfaces but persists Reference solutwn Dissolve 10 µL of citral R and 10 mg of
between the branches. The fracture is short and starchy with resorcinol R in 10 mL of methanol R. Prepare the solution
projecting fibres. The smoothed transversely cut surface immediately before use.
exhibits a narrow cortex separated by an endodermis from a Plate TLC silica gel plate R.
much wider stele; it shows numerous, scattered, fibrovascular Mobile phase hexane R, ether R (40:60 V/V).
bundles and abundant scattered oleoresin cells with yellow
contents. The unscraped rhizome shows, in addition, an
outer layer of dark brown cork. Development In an unsaturated tank, over a path of 15 cm.
B. Microscopic examination (2.8.23). The powder is pale Drying In air.
yellow or brownish. Examine under a microscope using Detection Spray with a 10 g/L solution of vanillin R in
chloral hydrate solutwn R. The powder shows the following sulfuric add R and examine in daylight while heating at
diagnostic characters (Figure 1522.-1): groups of large, thin- 100-105 °C for 10 min.
walled, septate fibres, with one wall frequently dentate [C, D, Results The chromatogram obtained with the reference
G]; fragments [K] containing vessels with reticulate solution shows in the lower half an intense red zone
thickening [Ka] often accompanied by narrow, thin-walled (resorcinol) and in the upper half 2 violet zones (citral); the
cells containing brown pigment [Kb] and amyliferous chromatogram obtained with the test solution shows below
parenchyma [Kc]; abundant reticulate vessels, fairly large, the zone due to resorcinol in the chromatogram obtained
isolated [H, L]; abundant thin-walled parenchyma of the with the reference solution 2 intense violet zones (gingerols)
ground tissue U, M], some cells containing brown and in the middle, between the zones due to resorcinol and
oleoresin Ua]; fragments of brown cork, usually seen in citral in the chromatogram obtained with the reference
surface view [F] but sometimes in transverse section [E] . solution, 2 other less intense violet zones (shogaols); other
Examine under a microscope using a 50 per cent V/V zones may be present.
solution of glycerol R. The powder shows abundant starch
granules, simple, flattened, oblong or oval or irregular, up to TESTS
about 50 µm long and 25 µm wide, with a small point hilum Water (2.2.13)
situated at the narrower end; sometimes, granules show faint, Maximum 100 mUkg, determined by distillation on 20.0 g
transverse striations, and may be free (A], agglomerated (BJ of the powdered herbal drug (710) (2.9.12).
or included in parenchymatous cells (Kc). Total ash (2.4.16)
ASSAY
Use 20.0 g of the freshly, coarsely powdered herbal drug, a
1000 mL round-bottomed flask, 10 drops of liquid paraffin R
or other antifoam, 500 mL of water R as distillation liquid
and 0.5 mL of xylene R in the graduated tube. Distil at a rate
of 2-3 mUmin for 4 h.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Strong Ginger Tincture

Ginger Essence
DEFINITION
Ginger, in moderately coarse powder 500 g

Ethanol (90 per cent) Sufficient to produce
1000 ml
Prepare by percolatwn, Appendix XI F.
under Extracts and with the following requirements.
TESTS
M Ethanol content
Dry residue
Figure 1522.-1. - Illustration for identificatwn test B of powdered 2.0 to 3.0% w/v.
herbal drug of ginger Relative density
C. Thin-layer chromatography (2.2.27). 0.832 to 0.846, Appendix V G.
(2. 9.12) add 5 mL of methanol R. Shake for 15 min and
filter.
IV-262 Ginger Preparations 2023
Weak Ginger Tincture

DEFINITION
Strong Ginger Tincture 200ml

Ethanol (90 per cent) Sufficient to produce 1000 ml

TESTS
Ethanol content
Dry residue
Not less than 0.4% w/v. Use 10 ml.
Relative density
0.825 to 0.835, Appendix V G.
Ginkgo Leaf
Preparation
Ginkgo Leaf Dry Extract, Refined and Quantified
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Cb
DEFINITION
Whole or fragmented, dried leaf of Ginkgo biloba L. 25 µm
Content
Not less than 0.5 per cent of flavonoids, expressed as flavone Figure 1828.-1. - Illustration for identification test B of powdered
glycosides (Mr 757) (dried drug). herbal drug of ginkgo leaf
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 rutoside trihydrate 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
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
dichotomous, the veins appearing to radiate from the base; Application 20 µL as bands.
they are equally prominent on both surfaces. The distal Development Over a path of 17 cm.
margin is incised, irregularly and to different degrees, and Drying At 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. Microscopic examination (2.8.23). The powder is greyish the same volume of a 50 g/L solution of macrogol 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 following 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 (surface view [D], transverse test solution. Furthermore, other weak fluorescent zones may
section [El) 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 (surface view [A),
transverse section [B]), consisting of small cells, with a finely
striated cuticle and each cell shortly papillose [Aa), and
stomata [Ab] about 60 µm, wide, deeply sunken with 6-8
subsidiary cells; fragments of vascular tissue from the petiole
and veins [C] with xylem [Ca] and parenchyma, some cells
containing abundant cluster crystals of calcium oxalate of
various sizes [Cb].
65 °C for 10 min. Shake frequently. Allow to cool to room
temperature and filter.
2023 Ginkgo Preparations IV-263
Top of the plate Time Mobile phase A Mobile phase B

A yellowish-brown fluorescent zone 0- l 60 40
1 - 20 60 ➔ 45 40 ➔ 55
A green fluorescent zone
20 - 21 45 ➔ 0 55 ➔ 100
2 yellowish-brown fluorescent zones 21 - 25 0 100
An intense light blue fluorescent
zone sometimes overlapped by a Flow rate 1.0 mLJmin.
greenish-brown fluorescent zone
fluorescent zone Injection 10 µL.
Relative retention With reference to quercetin (retention
time = about 12.5 min): kaempferol = about 1.4;
Rutoside: a yellowish-brown 2 yellowish-brown fluorescent zones isorhamnetin = about 1.5.
fluorescent zone
System suitability:
A green fluorescent zone - resolution: minimum 1.5 between the peaks due to
kaempferol and isorhamnetin.
Do not take into account peaks eluting before the quercetin
Reference solution Test solution peak or after the isorhamnetin peak in the chromatogram
TESTS Calculate the percentage content of flavonoids, expressed as
Foreign matter (2.8.2) flavone glycosides, using the following expression:
Maximum 5 per cent of stems and 2 per cent of other
F1 xm 1 x2.514xp
foreign matter. 2x-------
F2xm2
Maximum 11.0 per cent, determined on 1.000 g of the F1 sum of the areas of all the considered peaks in the
powdered herbal drug (355) (2. 9.12) by drying in an oven at chromatogram obtained with the test solution;
F2 area of the peak due to quercetin in the chromatogram obtained
105 °C for 2 h. with the reference solution;
Total ash (2.4.16) m1 mass of quercetin used to prepare the reference solution, in
Maximum 11.0 per cent. grams;
ASSAY solution, in grams;
p percentage content of anhydrous quercetin in quercetin
Flavonoids
dihydrate R.
Test solution Heat 2.500 g of the powdered herbal drug - - - - - - - - - - - - - - - - - - - - - ~ PhEur
(710) (2. 9. 12) in 50 mL of a 60 per cent V/V solution of
acetone R under a reflux condenser for 30 min. Filter and
collect the filtrate. Extract the drug residue a 2nd time in the
same manner, using 40 mL of a 60 per cent V/V solution of
acetone R and filter. Collect the filtrates and dilute to
Refined and Quantified Ginkgo
100.0 mL with a 60 per cent V/V solution of acetone R. Dry Extract
Evaporate 50.0 mL of the solution to eliminate the acetone
and transfer to a 50.0 mL vial, rinsing with 30 mL of
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
methanol R. Add 4.4 mL of hydrochwric acid Rl, dilute to
50.0 mL with water R and centrifuge. Place 10 mL of the DEFINITION
supernatant in a 10 mL brown-glass vial. Close with a rubber Refined and quantified dry extract produced from Ginkgo leaf
seal and an aluminium cap and heat on a water-bath for (1828).
25 min. Allow to cool to room temperature.
Content
Reference solution Dissolve 10.0 mg of quercetin dihydrate R
- fiavonoids, expressed as fiavone glycosides (Mr 756.7):
in 20 mL of methanol R. Add 15. 0 mL of dilute hydrochloric
22.0 per cent to 27.0 per cent (dried extract);
acid R and 5 mL of water R and dilute to 50.0 mL with
- bilobalide: 2.6 per cent to 3.2 per cent (dried extract);
methanol R.
- ginkgolides A, Band C: 2.8 per cent to 3.4 per cent (dried
Column: extract);
=
- size: l 0.125 m, 0 4 mm; = - ginkgolic acids: maximum 5 ppm (dried extract).
(5 µm); PRODUCTION
- temperature: 25 °C. The extract is produced from the herbal drug by an
appropriate procedure using organic solvents and their
Mobile phase:
mixtures with water, physical separation steps as well as other
- mobile phase A: 0.3 g/L solution of phosphoric acid R
suitable processes.
adjusted to pH 2.0;
- mobile phase B: methanol R; CHARACTERS
Appearance
Bright yellow-brown, powder or friable mass.
IDENTIFICATION
IV-264 Ginkgo Preparations 2023
Test solutwn Dissolve 20.0 mg of the extract to be examined - statwnary phase: octadecylsilyl silica gel for chromatography R
in 10 mL of a mixture of 2 volumes of water R and (5 µm);
8 volumes of methanol R. - temperature: 25 °C.
Reference solutwn Dissolve 1.0 mg of chlorogenic acid R and Mobile phase:
3.0 mg of rutoside trihydrate R in 20 mL of methanol R. - mobile phase A: 0.3 g/L solution of phosphoric acid R
Plate TLC silica gel plate R (5-40 µm) or [TLC silica gel adjusted to pH 2.0;
plate R (2-10 µm)]. - mobile phase B: methanol R;
water R, ethyl acetate R (7.5:7.5:17.5:67.5 V/V/V/V).
(min) (per cent V/Jl) (per cent V/V)
Applicatwn 20 µL [or 5 µL], as bands. 0- l 60 40
Development Over a path of 17 cm [or 6 cm] . 1 - 20 60---> 45 40---> 55
Drying At 100-105 °C. 20 - 21 45--+ 0 55 ---> 100
Detection Spray the plate whilst still hot with a 10 g/L 21 - 25 0 100
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 R; allow to dry in air for about 30 min and examine Detector Spectrophotometer at 370 nm.
in ultraviolet light at 365 nm. Injection 10 µL.
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
Several faint coloured zones
the chromatogram obtained with the test solution (see Figure
1827.-1).
--- --- Calculate the percentage content of flavonoids, expressed as
A brown fluorescent zone flavone glycosides, using the following expression:
A green fluorescent zone F 1 x m1 x 2.514 xp
An intense light blue fluorescent F2xm2
zone sometimes overlapped by a
greenish-brown fluorescent zone F1 sum of the areas of all the peaks from the peak due to quercetin
to the peak due to isorhamnetin in the chromatogram obtained
fluorescent zone
F2 area of the peak due to quercetin in the chromatogram obtained
One or two green fluorescent zones with the reference solution;
m1 mass of quercetin dihydrate CRS in the reference solution, in
Rutoside: a yellowish-brown One or two yellowish-brown grams;
fluorescent zone fluorescent zones m2 mass of the extract to be examined used to prepare the test
solution, in grams;
--- --- p percentage content of anhydrous quercetin in quercetin
dihydrate CRS.
Several green and yellowish-brown
fluorescent zones
Terpene lactones
Reference solution Test solution Test solutwn Place 0.120 g of the extract to be examined in
a 25 mL beaker and dissolve it in 10 mL of phosphate buffer
solutwn pH 5. 8 R by stirring. Transfer the solution into a
ASSAY
chromatography column, about 0.15 m long and about
Flavonoid.s 30 mm in internal diameter, containing 15 g of kieselguhr for
Liquid chromatography (2.2.29). chromatography R. Wash the beaker with 2 quantities, each of
Test solutwn Dissolve 0.200 g of the extract to be examined 5 mL, of phosphate buffer solutwn pH 5.8 R and transfer the
in 20 mL of methanol R. Add 15.0 mL of dilute hydrochloric washings to the chromatography column. Allow to stand for
acid Rand 5 mL of water Rand dilute to 50.0 mL with 15 min. Elute with 100 mL of ethyl acetate R. Evaporate the
methanol R. Transfer 10.0 mL of this solution into a 10 mL eluate to dryness at a pressure not exceeding 4 kPa in a
brown-glass vial. Close the vial with a tight rubber membrane water-bath at 50 °C. The residue of solvent is eliminated by
stopper and secure with an aluminium crimped cap. Heat on an air-current. Take up the residue in 2.5 mL of the mobile
a water-bath for 25 min. Allow to cool to 20 °C. phase.
Reference solutwn Dissolve 10.0 mg of quercetin Reference solution (a) Dissolve 30.0 mg of benzyl alcohol CRS
dihydrate CRS in 20 mL of methanol R. Add 15. 0 mL of in the mobile phase and dilute to 100.0 mL with the mobile
dilute hydrochloric acid R and 5 mL of water R and dilute to phase.
50.0 mL with methanol R. Reference solution (b) Place 0.120 g of the ginkgo dry extract
Column: for peak identificatwn CRS in a 25 mL beaker and dissolve it
- size: l = 0.125 m, 0 = 4 mm;
2023 Ginkgo Preparations IV-265
0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 min
I. quercetin 2. kaempferol 3. isorhamnetin
Figure 1827.-1. - Chromatogram for the assay of fiavonoids in refined and quantified ginkgo dry extract
F 1 x m 1 xp x 0.025 x 1.20
in 10 mL of phosphate buffer solution pH 5. 8 R by stirring,
then proceed as described for the test solution. Fs xm2
Column: Calculate the percentage content of ginkgolide A, using the
=
- size: l 0.25 m, 0 =
4 mm; following expression:
- stationary phase: octylsilyl silica gel for chromatography R
(5 µrn); F2 x m1 xp x 0.025 x 1.22
- temperature: 25 °C. Fs xm2
Mobile phase tetrahydrofuran R, methanol R, water R
(10:20:75 VIVIV). Calculate the percentage content of ginkgolide B, using the
Detection Refractometer maintained at 35 °C. F3 x m 1 xp x 0.025 x 1.19
Injection 100 µL. Fs xm2
Identification of peaks Use the chromatogram supplied with Calculate the percentage content of ginkgolide C, using the
ginkgo dry extract for peak identification CRS and the following expression:
chromatogram obtained with the reference solution (b) to
identify the peaks due to bilobalide and ginkgolides A, B F4 x m 1 x p x 0.025 x 1.27
and C. Fs x m2
System suitability:
area of the peak due to bilobalide in the chromatogram
- the chromatogram obtained with reference solution (b) is obtained with the test solution;
similar to the chromatogram supplied with ginkgo dry area of the peak due to ginkgolide A in the chromatogram
extract for peak identification CRS. obtained with the test solution;
F, area of the peak due to ginkgolide B in the chromatogram
Calculate the percentage content of bilobalide, using the obtained with the test solution;
following expression: area of the peak due to ginkgolide C in the chromatogram
F, area of the peak due to benzyl alcohol in the chromatogram
IV-266 Ginseng 2023
mass of benzyl alcohol CRS in reference solution (a), in grams; mass of ginkgolic acids CRS used to prepare the reference
mass of the extract to be examined used to prepare the test solution, in grams;
solution, in grams; p percentage content of ginkgolic acid C 17 in ginkgolic
p percentage content of benzyl alcohol in benzyl alcohol CRS. acids CRS.
Calculate the percentage content of the sum of ginkgolides _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur

A, B and C, using the following expression:
percentage content of ginkgolide A; Ginseng

percentage content of ginkgolide B;
percentage content of ginkgolide C. (Ph. Bur. monograph 1523)
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Ginkgolic acids
Test solution Dissolve 0.500 g of the powdered extract to be Whole or cut dried root, designated white ginseng; treated
examined in 8 mL of methanol R, sonicating if necessary, and with steam and then dried, designated red ginseng, of Panax
dilute to 10.0 mL with the same solvent. Centrifuge if ginseng C.A.Mey.
necessary. Content
Reference solution Dissolve 10.0 mg of ginkgolic acids CRS in Minimum 0.40 per cent for the sum of ginsenosides Rgl
8 mL of methanol R, sonicating if necessary, and dilute to (C42Hn014,2H20; Mr 837) and Rbl (Cs4H92023,3H20;
10.0 mL with the same solvent. Dilute 2.0 mL ofthis Mr 1163) (dried drug).
solution to 10.0 mL with methanol R. IDENTIFICATION
Column: A. The principal root is fusiform or cylindrical, sometimes
=
- size: l 0.25 m, 0 =4.6 mm; branched, up to about 20 cm long and 2.5 cm in diameter,
- stationary phase: octylsilyl silica gel for chromatography R and may be curved or markedly re-curved. The surface is
(5 µm); pale yellow to cream in white ginseng, brownish-red in red
- temperature: 35 °C. ginseng and shows longitudinal ridges. Stem scars may be
Mobile phase: seen at the crown. The fracture is short. The transversely cut
- mobile phase A: dilute 0.1 mL of trifiuoroacetic acid R to surface shows a wide outer zone with scattered orange-red
1000 mL with water R; resin canals and a finely radiate inner region. The rootlets,
- mobile phase B: dilute 0.1 mL of trifiuoroacetic acid R to numerous in the lower part of white ginseng, are normally
1000 mL with acetonitrile R; absent in red ginseng.
Time Mobile phase A Mobile phase B yellow. Examine under a microscope using chloral hydrate
(min) (per cent V/J,') (per cent V/Jl) solution R. The powder shows the following diagnostic
0 - 30 25 ➔ 10 75 ➔ 90 characters (Figure 1523.-1): abundant fragments of
30 - 35 10 90 parenchymatous cells [A, E] with thin [Ea] or slightly
35 - 36 10 ➔ 25 90 ➔ 75 thickened [Aa] walls, some of which contain cluster crystals
36 - 45 25 75 of calcium oxalate [Ab]; fragments of large secretory canals
[Eb] containing yellowish-brown resin in granular masses
Flow rate 1.0 mUmin. [Ee]; non-lignified tracheids and partially lignified vessels
Detection Spectrophotometer at 210 nm. with spiral or reticulate thickening, isolated m;
fragments of
xylem (longitudinal section [C], transverse section [F])
Injection 50 µL. consisting of vessels [Ca, Fa] and thin-walled
Identification of components Use the chromatogram supplied parenchymatous cells [Cb, Fb]; isolated cluster crystals of
with ginkgolic acids CRS and the chromatogram obtained with calcium oxalate [D, G]; fragments of cork (surface view [B],
the test solution to identify the peaks due to ginkgolic acids transverse section [H]) often associated with phelloderm
C13, C15 and Cl 7. having slightly thickened cells [Ha] and with the outer layers
System suitability Reference solution: of the cortical parenchyma [Hb]. Examine under a
- resolution: minimum 2.0 between the peaks due to microscope using a 50 per cent V/V solution of glycerol R.
ginkgolic acids C13 and C15; The starch granules [K] are very abundant, simple or
- symmetry factor. 0.8 to 2.0 for the peaks due to ginkgolic compound, and range from 1-10 µm in diameter. In red
acids C13, C15 and C17. ginseng, the starch granules are deformed and often
Calculate the content in parts per million of ginkgolic acids destroyed by treating with steam, or may be absent.
expressed as ginkgolic acid C 17, using the following C. Thin-layer chromatography (2.2.27).
expression: Test solution Boil 1.0 g of the powdered herbal drug (355)
(2. 9.12) under a reflux condenser with 10 mL of a
A1 x m2 xpx 2000 70 per cent V/V solution of methanol R for 15 min. Filter
A2xm1 after cooling and dilute to 10.0 mL with methanol R.
sum of the areas of the peaks due to the ginkgolic acids Reference solution Dissolve 5.0 mg of aescin R and 5.0 mg of
C 13, C 15 and C 17 in the chromatogram obtained with arbutin R in 1 mL of methanol R.
the test solution;
area of the peak due to ginkgolic acid Cl 7 in the
mass of the extract to be examined used to prepare the
2023 Ginseng IV-267
TESTS
Panax quinquefolium
Examine the chromatograms obtained in the assay.
The chromatogram obtained with the test solution shows a
peak due to ginsenoside Rf (see Figure 1523.-2). In the case
of a substitution by Panax quinquefolium, no peak due to
ginsenoside Rf is present.
105 °C.
Total ash (2.4.16)
ASSAY
Test solution Reduce about 50 g to a powder (355) (2.9.12).
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
a water-bath under a reflux condenser for 1 h. After cooling,
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
exceeding 60 °C. Take up the residue with 20.0 mL of a
~
mixture of 20 volumes of acetonitrile R and 80 volumes of
water R. Dilute 2.0 mL of the solution to 10.0 mL with a
mixture of 20 volumes of acetonitrile R and 80 volumes of
Figure 1523.-1. - Illustration for identification test B of powdered water R. Filter through a suitable membrane filter (nominal
herbal drug of ginseng pore size 0.45 µm) before injection.
Mobile phase ethyl acetate R, water R, butanol R Reference solution Dissolve 3.0 mg of ginsenoside Rgl CRS,
(25:50:100 V/V/V); allow the mixture to separate for 10 min 3.0 mg of ginsenoside Re R, 3.0 mg of ginsenoside Rf Rand
and use the upper layer. 3.0 mg of ginsenoside Rbl CRS in methanol Rand dilute to
Application 20 µL [or 4 µL] as bands. 10.0 mL with the same solvent.
Development Over 10 cm [or 5 cm] in an unsaturated tank. Column:
Drying In air. - size: l = 0.125 m, 0 = 4.6 mm;
Detection Treat with anisaldehyde solution R and heat at - stationary phase: octadecylsilyl silica gel for chromatography R
(5 µm);
chromatograms obtained with the reference solution and the Mobile phase:
test solution. - mobile phase A: water for chromatography R adjusted to
pH 2 with phosphoric acid R;
Top of the plate - mobile phase B: acetonitrile Rl;
Arbutin: a brown zone
-- --
0-8 80 20
A violet zone (ginsenosides Rgl
8 - 40 80 ➔ 60 20 ➔ 40
+ Rg2)
40 - 45 60 ➔ 40 40 ➔ 60
A faint violet zone (ginsenoside Rf)
45 - 47 40 ➔ 0 60 ➔ 100
A violet zone (ginsenoside Re) 47 - 52 0 100

A violet zone (ginsenoside Rd)
A faint violet zone Equilibration 20 min.
-- --
Injection 20 µL.
A violet zone (ginsenoside Re)
reference solution; record the retention times of these
Aescin: a grey zone A violet zone (ginsenosides Rb I substances.
+ Rb2)
IV-268 Ginseng Preparations 2023
0.35
4 5
0.30
0.25 6
0.20
7
0.15
2
0.10
3
0.05
0.00
I I
0 5 10 15 20 25 30 35 40 45 min
I. ginsenoside Rg I 3. ginsenoside Rf 5. ginsenoside Re 7. ginsenoside Rd

2. ginsenoside Re 4. ginsenoside Rbl 6. ginsenoside Rb2
Figure 1523.-2. - Chromatogram for the assay of ginseng: test solution
System suitability Reference solution: PRODUCTION

- resolution: minimum 1.0 between the peaks due to The extract is produced from the herbal drug by a suitable
ginsenoside Rgl and ginsenoside Re. procedure using a hydroalcoholic solvent equivalent in
Locate the peaks due to ginsenoside Rbl and strength to ethanol (35-90 per cent V/V).
ginsenoside Rgl in the chromatogram obtained with the test CHARACTERS
solution. Appearance
Calculate the percentage content of ginsenosides Rbl and Light brownish-yellow, hygroscopic powder or brittle mass.
Rgl using the following expression:
IDENTIFICATION
A 1 xmzXh
A2xm3Xh Thin-layer chromatography (2.2.27).
-----+------
A3 x m1 x 100 A4xm1xl00 Test solution Dissolve O.15 g of the extract to be examined
in 10 mL of a 70 per cent V/V solution of methanol R.
area of the peak due to ginsenoside Rb I in the chromatogram
Reference solution Dissolve 0.15 g of ginseng dry extract HRS
area of the peak due to ginsenoside Rgl in the chromatogram in 10 mL of a 70 per cent V/V solution of methanol R.
obtained with the test solution; Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
area of the peak due to ginsenoside Rb I in the chromatogram plate R (2-10 µm)].
area of the peak due to ginsenoside Rgl in the chromatogram Mobile phase ethyl acetate R, water R, butanol R
obtained with the reference solution; (25:50: 100 VIVIV); allow the phases to separate for 10 min
mass of the herbal drug to be examined used to prepare the test and use the upper layer.
solution, in grams;
mass of ginserwside Rb! CRS used to prepare the reference Application 20 µL [or 4 µL] as bands of 10 mm [or 8 mm).
solution, in milligrams; Development Over a path of 10 cm [or 5 cm] in an
mass of ginsenoside Rg 1 CRS used to prepare the reference
solution, in milligrams;
unsaturated tank.
P1 percentage content of ginsenoside Rb! in ginsenoside Rbl CRS; Drying In air.
P2 percentage content of ginsenoside Rgl in ginsenoside Rgl CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
in the chromatograms obtained with the test solution and the
Ginseng Dry Extract reference solution.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dry extract produced from Ginseng (1523).
Content
Minimum 4.0 per cent of the sum of ginsenosides Rbl, Rb2,
Re, Rd, Re, Rf, Rgl and Rg2, expressed as ginsenoside Rbl
(Cs4H92023; Mr 1109) (dried extract).
2023 Ginseng Preparations IV-269
Top of the plate methanol R. Filter through a suitable membrane filter

Reference solution (b) Dissolve 3.0 mg of ginsenoside
-- -- Rbl CRS in methanol R and dilute to 5.0 mL with the same
A violet zone (ginsenosides Rgl A violet zone (ginsenosides solvent.
+ Rg2) Rgl + Rg2) Reference solution (c) Dissolve 3.0 mg of ginsenoside Rg2 R in
A faint violet zone (ginsenoside Rf) A faint violet zone (ginsenoside Rf)
Reference solution (d) Dilute 1.0 mL of reference
A violet zone (ginsenoside Re) A violet zone (ginsenoside Re) solution (b) to 2.0 mL with reference solution (c).
Column:
- size: l = 0.125 m, 0 = 4.6 mm;
A violet zone (ginsenoside Rd) A violet zone (ginsenoside Rd)
A faint violet zone A faint violet zone (5 µm);
A violet zone (ginsenoside Re) A violet zone (ginsenoside Re)
Mobile phase:
A faint violet zone A faint violet zone - mobile phase A: water R adjusted to pH 2 with phosphoric
-- --
acid R;
A violet zone (ginsenosides Rb I A violet zone (ginsenosides
+ Rb2) Rb!+ Rb2)
Several unresolved violet and Several unresolved violet and (min) (per cent V/J!) (per cent V/J!)
greenish zones greenish zones 0-8 80 20
Reference solution Test solution 8 - 40 80-> 60 20-> 40
40 - 45 60-, 40 40-> 60
45 - 47 40-> 0 60-, 100
TESTS
Loss on drying (2.8.17) Flow rate 1.0 mL/min.
ASSAY Injection 20 µL.
Liquid chromatography (2.2.29). Elution order Ginsenoside Rgl, ginsenoside Re,
Buffer solution Dissolve 3.5 g of disodium hydrogen phosphate ginsenoside Rf, ginsenoside Rbl, ginsenoside Rg2,
dihydrate R and 7.2 g of potassium dihydrogen phosphate R in ginsenoside Re, ginsenoside Rb2, ginsenoside Rd; depending
water R and dilute to 1000 mL with the same solvent. on the operating conditions and the state of the column,
Test solution Dissolve 0.100 g of the extract to be examined ginsenoside Rbl may elute before or after ginsenoside Rg2.
in the buffer solution and dilute to 10.0 mL with the buffer Identification of peaks Use the chromatogram supplied with
solution. Prepare a ready-to-use sample-preparation cartridge ginseng dry extract HRS and the chromatogram obtained with
containing 0.50 g of octadecylsilyl silica gel (45 µm), using reference solution (a) to identify the peaks due to
5 mL of methanol R followed by 20 mL of water R. Apply ginsenosides Rgl, Re, Rf, Re, Rb2 and Rd; use the
5.0 mL of the solution to be analysed to the top of the chromatogram obtained with reference solution (b) to
cartridge. Wash the cartridge with 20 mL of water R followed identify the peak due to ginsenoside Rbl; use the
by 15 mL of a 30 per cent V/V solution of methanol R. chromatogram obtained with reference solution (c) to identify
Discard the eluates after confirming that no ginsenosides are the peak due to ginsenoside Rg2.
present, otherwise repeat the preparation of the solution with Relative retention With reference to ginsenoside Rb 1
another brand of cartridge where no ginsenosides are eluted (retention time = about 33 min): ginsenoside Rgl = about
with a 30 per cent V/V solution of methanol R. Elute the 0.53; ginsenoside Re = about 0.54;
cartridge with 20 mL of methanol R; collect the eluate. Under ginsenoside Rf= about 0.88; ginsenoside Rg2 = about 0.98;
reduced pressure, evaporate the eluate to dryness. Dissolve ginsenoside Re = about 1.04; ginsenoside Rb2 = about 1.08;
the residue in 2.0 mL of methanol R. Filter through a suitable ginsenoside Rd = about 1.1 7.
System suitability Reference solution (d):
Reference solution (a) Dissolve O.1 00 g of ginseng dry - resolution: minimum 1.5 between the peaks due to
extract HRS in the buffer solution and dilute to 10.0 mL with ginsenosides Rg2 and Rb 1.
the buffer solution. Prepare a ready-to-use sample-
preparation cartridge containing 0.50 g of octadecylsilyl silica Calculate the percentage content of the sum of
gel (45 µm), using 5 mL of methanol R followed by 20 mL of ginsenosides Rbl, Rb2, Re, Rd, Re, Rf, Rgl and Rg2,
water R. Apply 5 .0 mL of the solution to be analysed to the expressed as ginsenoside Rb 1, using the following expression:
top of the cartridge. Wash the cartridge with 20 mL of A 1 x mz xpx 0.8
water R followed by 15 mL of a 30 per cent V/V solution of
methanol R. Discard the eluates after confirming that no A 2 xm1
ginsenosides are present, otherwise repeat the preparation of sum of the areas of the peaks due to ginsenosides Rb!, Rb2,
the solution with another brand of cartridge where no Re, Rd, Re, Rf, Rgl and Rg2 in the chromatogram obtained
ginsenosides are eluted with a 30 per cent V/V solution of with the test solution;
methanol R. Elute the cartridge with 20 mL of methanol R; area of the peak due to ginsenoside Rb I in the chromatogram
obtained with reference solution (b );
collect the eluate. Under reduced pressure, evaporate the mass of the extract to be examined used to prepare the test
eluate to dryness. Dissolve the residue in 2.0 mL of solution, in grams;
IV-270 Glehnia Littoralis Root 2023
mass of ginsenoside Rbl CRS used to prepare reference (d) Develop the plate to 7 cm.
solution (b), in grams;
p percentage content of ginsenoside Rb! in ginsenoside Rbl CRS. (e) After removal of the plate, dry in a current of air and
examine under ultraviolet light (366 nm).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur (t) Dip the plate in freshly-prepared anisaldehyde solution, heat
at 100° for 5 minutes and examine under white light.
MOBILE PHASE
1 volume of ethyl acetate and 2 volumes of cycl.ohexane.
Glehnia Littoralis Root SYSTEM SUITABILITY
DEFINITION
Glehnia Littoralis Root is the dried and peeled root of The test is not valid unless the chromatogram obtained with
Glehnia littoralis F. Schmidt ex Miq. solution (4) shows two clearly separated bands under white
light.
It is collected in summer and autumn, removed from rootlets
and dried. CONFIRMATION
The chromatogram obtained with solution (1) under
IDENTIFICATION
366 nm, should show no bands above the band for bergapten
A. The root is slender-cylindrical, 9 cm to 30 cm in length
in the chromatogram obtained with solution (5). Bands
and 2 mm to 15 mm in diameter. The outer surface is pale
above bergapten indicate the presence of Adenophora root.
buflish-white to buff, somewhat rough with occasional
fragments of brown cork; fine longitudinal wrinkles and The chromatogram obtained with solution (1) when observed
grooves and brownish yellow punctiform scars of rootlets. in white light shows the presence of all bands described for
The top is slender, sometimes marked with the yellowish solution (1) as shown in the table. In the chromatogram
brown rhizome base; the greater part of the root is thick, obtained with solution (1) no orange band should be seen
narrowing towards the distal end. The root is often cut above the orange band for thymol seen in solution (4) and
transversely to give discs 6 mm to 15 mm in diameter and would indicate the presence of Adenophora root.
1 mm thick, or short cylinders 3 mm to 9 mm in diameter
and 10 mm to 20 mm long, or short sticks 2 mm in diameter Top of the plate
and 15 mm to 60 mm long. The cut surface (a transverse

section of the root) shows concentric rings of buff and golden An intense purple band
brown, especially on the larger diameter roots, which also A faint purple-brown band
sometimes have a small circular cavity in the centre.
A faint to equivalent A diffuse orange band
pale buff to buff. Examine under a microscope using chloral greenish- brown band A purple band (beta- (thymol)
hydrate solution. The powder shows the following characters: amyrin) A diffuse brown band
A very faint to equivalent (linalool)
abundant parenchyma of several types including large purple-brown band
rounded rectangular, oval or bottle shaped thin walled cells
A blue band
often arranged in rows or groups; smaller sub-rectangular or
A very faint to equivalent
irregular cells with slightly thickened walls and elongated greenish-brown band A blue band (ursolic acid)
rectangular thin walled cells; fragments of xylem vessels and
An intense to equivalent
tracheids with reticulate or scalariform thickening, singly or purple band
in groups; bundles of narrow, thin walled fibres; secretory
canals with brownish-yellow contents. Examine under a
microscope using water. Many of the parenchyma cells show
uniformly granular contents and abundant fragments of a
similar granular material occur throughout the slide. Solution (1) Solution (2) and (J) Solution (4)
C. Carry out the method for high-performance thin-layer

solutions.
(1) Mix with the aid of ultrasound 1.0 g of the powdered TESTS
herbal drug (425) with 5 mL of methanol, centrifuge and use Foreign matter
the supernatant liquid. Not more than 2%, Appendix XI D.
(2) 0.0125% w/v of ursolic acid and 0.0125% w/v of beta- Loss on drying
amyrin in methanol. When dried for 2 hours at 105°, loses not more than 11.0%
of its weight, Appendix IX D. Use 1 g.
(3) Dilute 1 volume of solution (2) to 4 volumes with
methanol. Total ash
(4) 0.025% w/v of thymol and 0.125% v/v of linalool in Not more than 5.0%, Appendix XI J.
methanol. Acid-insoluble ash
(5) 0.05% w/v of bergapten in methanol. Not more than 0.5%, Appendix XI K.
CHROMATOGRAPHIC CONDITIONS ANNEX
(a) Use as the coating high-performance silica gel F254 (Merck This section is non-mandatory.
silica gel 60 F 254 HPTLC plates are suitable). DNA reference sequence
(b) Use the mobile phase as described below. A DNA reference sequence for the identity of Glehnia
Littoralis Root is published in Supplementary Chapter VII D.
(c) Apply 18 µL of solution (1) and 2 µL of solutions (2) to
(5), as 8 mm bands.
2023 Goldenrod IV-271
;; . ., -
Goldenrod /' ,---- _,

t'
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or cut, dried, flowering aerial parts of Solidago gigantea
Aiton or Solidago canadensis L., their varieties or hybrids
and/or mixtures of these.
Fb
Content
(C21H20012; Mr 464.4) (dried drug).
IDENTIFICATION
A. The stems are greenish-yellow or greenish-brown, partly
tinted reddish, roundish, more or less conspicuously grooved,
glabrous and smooth in the lower part, slightly or densely
pubescent in the upper part. They are solid with a whitish
pith. G
The leaves are green, sessile, lanceolate, with a serrate
margin, 8-12 cm long and about 1-3 cm wide, the upper
surface is green and more or less glabrous, the lower surface
25µm
is greyish-green and pubescent, especially on the veins. >--t
The inflorescence consists of a number of unilateral, curved

J
racemes which together form a pyramidal panicle at the end
of the stems. ~
Each capitulum has an involucre composed of linear-
lanceolate, imbricated yellowish-green bracts, surrounding a Figure 1892.-1. - Illustration for identification test B of powdered
single row of yellow ligulate florets about the same length as herbal drng of goldenrod
the involucre; yellow, radially arranged tubular florets, as
long as, or longer, than the ligulate florets; a brownish Application 20 µL of the test solution and 10 µL of the
inferior ovary surmounted by a white pappus of silky hairs. reference solution, as bands.
B. Microscopic examination (2.8.23). The powder is green or Development Over a path of 10 cm.
greyish-green. Examine under a microscope using chloral Drying At 100-105 °C.
hydrate solution R. The powder shows the following diagnostic Detection Treat with a 10 g/L solution of diphenylboric acid
characters (Figure 1892.-1): pappus bristles, usually broken, aminoethyl ester R in methanol R and then with a 50 g/L
consisting of multiseriate trichomes composed of elongated solution of macrogol 400 R in methanol R. Allow to stand for
cells with the tips free from the surface and forming pointed 30 min. Examine in ultraviolet light at 365 nm.
projections over the entire length OJ; fragments of the
mesophyll [F] with vascular bundles [Fa], oil glands [Fb]
and palisade parenchyma [Fe]; fragments of the leaf
epidermises [E, HJ with sinuous or wavy-walled cells
accompanied by palisade parenchyma [Ea] (upper
epidermis), with whip-like, multicellular covering trichomes Top of the plate
[Eb] and anomocytic stomata (mainly on the lower
epidermis) (2.8.3) [Ha]; multicellular uniseriate covering
trichomes from the stems and the margins of the leaves with A bluish-green fluorescent zone
up to 5 or 6 thick-walled cells [C]; in Solidago canadensis L.,
covering trichomes with a terminal cell that may be bent at a Quercitrin: a yellowish-brown A faint to intense yellowish-brown
fluorescent zone fluorescent zone (quercitrin)
right angle [G]; fragments of the style [A] with short papillae
[Aa] and long, slender papillae with wrinkled walls [Ab]; -- --
pollen grains with 3 germinal pores and a spiny exine [BJ;
A more or less intense yellowish-
a few rare fragments of the ovary with twin covering brown zone
trichomes [DJ.
C. Thin-layer chromatography (2.2.27). fluorescent zone ( chlorogenic acid) and/or a yellow
Test solution To 0.75 g of the powdered herbal drug (355) fluorescent zone
(2. 9.12) add 5 mL of methanol R and boil in a water-bath Rutoside: an orange fluorescent zone A faint to intense yellowish-brown
under a reflux condenser for 10 min. Cool and filter. fluorescent zone (rutoside)
Reference solution Dissolve 1.0 mg of chlorogenic acid R, - - - -
2.5 mg of quercitrin Rand 2.5 mg of rutoside trihydrate R in Reference solution Test solution
10 mL of methanol R.
Plate TLC silica gel plate R. TESTS
Mobile phase anhydrous formic acid R, water R, methyl ethyl Foreign matter (2.8.2)
ketone R, ethyl acetate R (6:6:18:30 VIVIVIV). Maximum 5 per cent of brownish parts and maximum
IV-272 Goldenrod 2023

Maximum 10 per cent, determined on 0.500 g of the A. The stem is cylindrical, striated, the lower part often
powdered herbal drug (355) (2.9.12) by drying in an oven at reddish-violet, sometimes entirely glabrous or pubescent with
105 °C for 2 h. short, bent, apically directed hairs. The basal leaves are
Total ash (2.4.16) obovate or oblanceolate, with a serrate margin, and taper at
Maximum 7 .0 per cent. the base into a long, winged petiole; the cauline leaves are
Ash insoluble in hydrochloric acid (2.8.1) alternate, smaller than the basal leaves and more elliptical in
Maximum 1.0 per cent. outline, with an entire or slightly toothed margin; they are
sessile or with only a short petiole. Both surfaces of the leaves
ASSAY are glabrous or only slightly pubescent with a prominent
Stock solution In a 100 mL round-bottomed flask, introduce reticulate venation on the lower surface. The capitula form a
0.200 g of the powdered herbal drug (250) (2.9.12), add tightly packed panicle. At the base of the pedicels there are 2
1 mL of a 5 g/L solution of hexamethylenetetramine R, 20 mL small, linear bracts with scarious margins. The involucre
of acetone Rand 2 mL of hydrochloric acid Rl. Boil the consists of 2-4 rows of loosely arranged, imbricate bracts,
mixture under a reflux condenser for 30 min. Filter the each bract greenish-yellow with a smooth and shiny inner
liquid through a small plug of absorbent cotton into a surface, the outer surface hairy or glabrous, with a scarious
100 mL flask. Add the absorbent cotton to the residue in the margin. Each capitulum contains 6-12 widely separated
round-bottomed flask, extract with 2 quantities, each of female ray florets, about twice as long as the bracts, and
20 mL of acetone R, each time boiling under a reflux about 10-30 hermaphrodite, tubular florets. All florets are
condenser for I O min. Allow to cool. Filter the combined yellow. The brown, inferior ovary tapers towards the base
acetone extracts through a filter paper into a volumetric flask. and has a ribbed surface, covered with scattered hairs; it is
Rinse the flask and the filter paper and dilute to 100.0 mL surmounted by a whitish pappus composed of smooth or
with acetone R. Introduce 20.0 mL of the solution into a rough, bristly hairs.
separating funnel, add 20 mL of water R and shake the
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
characters (Figures 1893.-1 and 1893.-2): fragments of the
water R and filter the extracts over 10 g of anhydrous sodium
upper epidermis of the leaf (surface view [B, H, M]), covered
sulfate R into a volumetric flask. Dilute to 50.0 mL with ethyl
by a distinctly striated cuticle, composed of polygonal cells
acetate R, rinsing the separating funnel and the sodium
with straight, beaded, thickened walls [Ba, Ma], uniseriate,
sulfate.
multicellular covering trichomes [Ha] or rounded, thick-
Test solution To 10.0 mL of the stock solution add 1.0 mL walled, covering trichome scars with a pitted lumen [Mb],
of aluminium chloride reagent Rand dilute to 25.0 mL with a and a few anomocytic stomata (2. 8. 3) [Bb] sometimes
5 per cent V/V solution of glacial acetic acid R in methanol R. accompanied by underlying palisade parenchyma [Be];
Compensation solution Dilute 10.0 mL of the stock solution fragments of the lower epidermis of the leaf (surface view [A,
to 25.0 mL with a 5 per cent V/V solution of glacial acetic K, N]) covered by a slightly striated cuticle composed of cells
acid R in methanol R. with sinuous walls in the area of the lamina [Aa] or with
Measure the absorbance of the test solution (2.2.25) at more rigid walls near the veins [N], numerous anomocytic
425 nm after 30 min by comparison with the compensation stomata (2.8.3) [Ab], occasional glandular trichomes with a
solution. unicellular stalk and a unicellular head [Ka, Na], covering
Calculate the percentage content of flavonoids, expressed as trichomes some of which are pennant-like [Ac, F], uniseriate,
hyperoside, using the following expression: multicellular, with 1-3 thin-walled basal cells [Fa], a flagella-
like distal cell [Fb], and an enlarged, more or less rounded
Ax 1.25
cell [Fe] between them, others are uniseriate, multicellular
m (up to about 10 cells), with thick, finely wrinkled walls and a
i.e. taking the value of the specific absorbance of hyperoside rigid conical distal cell (side view [El); rare fragments from
to be 500. the ovary [G] bearing paired, covering trichomes with a
distinctly pitted central wall and a bifid apex (surface
A absorbance measured at 425 run; view [Ga], side view [Gb]); vascular tissue from the
m mass of the herbal drug to be examined, in grams. stems [L] composed of vessels [La] and groups of
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur fibres [Lb]; fragments of the epidermis of the petals with a
striated cuticle, through which run fine spiral vessels [S], and
bearing biseriate glandular trichomes (side view [P]);
spherical pollen grains, with 3 germinal pores and a spiny
European Goldenrod exine m; abundant pappus hairs and their fragments [C, D],
multiseriate with the marginal cells overlapping outwards;
fragments of parenchyma [Q], some showing cells containing
small, isolated cluster crystals of calcium oxalate [Qa];
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
fragments of bracts [R] with a finely striated cuticle,
DEFINITION polygonal cells [Raj, bearing pennant-like covering
Whole or fragmented, dried, flowering aerial parts of Solidago trichomes [Rb] and whose margin bears uniseriate,
virgaurea L. multicellular covering trichomes [Re].
C. Thin-layer chromatography (2.2.27) as described in the
Content
test for Solidago gigantea Aiton and Solidago canadensis L.
Minimum 0.5 per cent and maximum 1.5 per cent of
flavonoids, expressed as hyperoside (C 21 H200,2; Mr 464.4)
(dried drug).
2023 Goldenrod IV-273

Top of the plate
Quercitrin: an orange fluorescent

zone
G -- --

(rutoside)
-- --
TESTS
Maximum 5 per cent of brown coloured matter and
maximum 5 per cent of other foreign matter.
Ga
Solidago gigantea Aiton and Solidago canadensis L
Test solution To 0. 75 g of the powdered herbal drug (355)
(2. 9.12) add 5 mL of methanol R and heat on a water-bath
under a reflux condenser for 10 min. Cool and filter.
Reference solution Dissolve 1.0 mg of chlorogenic acid R,
2.5 mg of quercitrin Rand 2.5 mg of rutoside trihydrate R in
herbal drug of European goldenrod
ketone R, ethyl acetate R (6:6: 18:30 V/V/V/V).
Drying In air.
Ka
Detection Treat the plate with a 10 g/L solution of
Na diphenylboric acid aminoethyl ester R in methanol R and then
Mb
I , , with a 50 g/L solution of macrogol 400 R in methanol R.
-
-- ~_I,.,
N Examine in ultraviolet light at 365 nm after 30 min.
,, "'" '0' Results The chromatogram obtained with the test solution
\
. !
.
shows no strong orange fluorescent zone similar in position
to the zone of quercitrin in the chromatogram obtained with
the reference solution.
Q }: '. -:
-= . Loss on drying (2.2.32)
Qa Maximum 12.0 per cent, determined on 1.000 g of the
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Stock solution In a 100 mL round-bottomed flask, place
0.200 g of the powdered herbal drug (355) (2. 9.12), add
of acetone Rand 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
in the round-bottomed flask and extract with 2 quantities,
each of 20 ml., of acetone R, each time boiling under a reflux
herbal drug of European goldenrod
condenser for 10 min. Allow to cool. Filter the combined
Results See below the sequence of the zones present in the acetone extracts through filter paper, dilute to 100.0 mL with
chromatograms obtained with the reference solution and the acetone R, rinsing the volumetric flask and the filter paper
IV-274 Goldenseal Root 2023
with acetone. Introduce 20.0 mL of the solution into a found associated with the vessels; numerous ovoid or
suitable separating funnel, add 20 mL of water R and shake spherical, orange-brown granular masses. Examine under a
the mixture with 1 quantity of 15 mL and then with microscope using a 50 per cent V/V solution of glycerol R.
3 quantities, each of 10 mL, of ethyl acetate R. Combine the The powder shows abundant starch granules [C], mostly
ethyl acetate extracts in a separating funnel, wash twice with simple but sometimes compound with up to 4 components;
50 mL of water R and filter the extracts over 10 g of the granules are small, spherical or ovoid, up to about 10 µm
anhydrous sodium sulfate R into a volumetric flask. Dilute to in diameter, occasionally with a small, rounded or slit-shaped
50.0 mL with ethyl acetate R, rinsing the separating funnel hilum.
and the sodium sulfate.
Test solution To 10.0 mL of the stock solution add 1.0 mL
of aluminium chloride reagent Rand dilute to 25.0 mL with a
Compensatwn liq_uid Dilute 10.0 mL of the stock solution to
25.0 mL with a 5 per cent VIV solution of glacial acetic 0 0 0
acid R in methanol R. (8
CP
After 30 min, measure the absorbance (2.2.25) of the test
@i C
solution at 425 nm by comparison with the compensation
(l)
liquid.
~
Ax 1.25
m
i.e. taking the specific absorbance of hyperoside to be 500. F
A measured absorbance at 425 nm;

mass of the herbal drug to be examined, in grams.
{8
m
- - - - - - - - - - - - - - - - - - - - - - PhEur
H
Goldenseal Root
(Goldenseal Rhizome, Ph. Bur. monograph 1831)
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
-
50 µm
DEFINITION Figure 1831. -1. - Illustratwn for identificatwn test B of powdered

Whole or cut, dried rhizome and root of Hydrastis herbal drug of goldenseal rhizome
canadensis L.
Content
- hydrastine (C21H21NO 6; Mr 383.4): minimum Test solutwn To 250 mg of the powdered herbal drug (180)
2.5 per cent (dried drug); (2. 9.12) add 4 mL of a mixture of 20 volumes of water R and
- berberine (C 2oH 18NO4 ; Mr 336.4): minimum 3.0 per cent 80 volumes of methanol R. Sonicate for 10 min and filter.
(dried drug). Wash the residue with 2 quantities, each of 2 mL, of
methanol R. Combine the solutions and dilute to 20 mL with
IDENTIFICATION methanol R.
A. The rhizome is tortuous and knotty, about 5 cm long and
Reference solution Immediately before use, dissolve 5 mg of
5-10 mm thick. The surface is yellowish or brownish-grey,
hydrastine hydrochloride R and 5 mg of berberine chloride R in
irregularly wrinkled, and bears the remains of numerous
slender, wiry roots; stem bases and scale leaves occur on the
upper surface. The fracture is short and resinous. Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
The transversely cut surface is yellowish-brown and shows a plate R (2-10 µm)].
fairly wide bark, a ring of 12-20 widely separated xylem Mobile phase anhydrous formic acid R, water R, ethyl acetate R
bundles and a large, central pith. (10:10:80 VIVIV).
B. Microscopic examination (2.8.23). Reduce to a powder Applicatwn 20 µL [or 2 µL] as bands.
(180) (2.9.12). The powder is greenish-yellow. Examine Development Over a path of 15 cm [or 6 cm].
under a microscope using chloral hydrate solution R. Drying In air.
(Figure 1831.-1): abundant thin-walled fragments of
parenchyma [A, G, K]; occasional fragments ofyellowish- Results See below the sequence of zones present in the
brown cork from the rhizome and roots (surface view m, chromatograms obtained with the reference solution and the
transverse section [F]); groups of small vessels with
conspicuous perforations in the oblique end walls [L) and present in the chromatogram obtained with the test solution.
with simple or bordered, slit-shaped pits [B, D, E];
infrequent groups of thin-walled, pitted fibres [H], usually
2023 Green Tea IV-275
hydrastine and berberine.
-- --
Berberine: a bright yellow A bright yellow fluorescent zone chromatogram obtained with the reference solution, locate in
the chromatogram obtained with the test solution the
Hydrastine: a deep blue fluorescent A deep blue fluorescent zone components of the reference solution.
zone (bydrastine) Calculate the percentage content of each alkaloid (hydrastine
-- --
and berberine) using the following expression:
A bright light blue fluorescent zone Ai x m2 xp _

(hydrastinine) ----x2.':>
A2 xm1
A deep blue fluorescent zone
area of the peak due to hydrastine or berberine in the
Reference solution Test solution chromatogram obtained with the test solution;
area of the peak due to hydrastine or berberine in the
TESTS mass of the herbal drug to be examined used to prepare the test
mass of hydrastine hydrochloride CRS or berberine chloride CRS
used to prepare the reference solution, in grams;
powdered herbal drug (180) (2.9.12) by drying in an oven at p percentage content of hydrastine in hydrastine hydrochloride CRS
105 °C for 2 h. or berberine in berberine chlon"de CRS.
Total ash (2.4.16)
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
ASSAY Green Tea
Test solution To 1.000 g of the powdered herbal drug (355) (Ph. Bur. monograph 2668)
(2. 9.12) in a 100 mL round-bottomed flask, add 50 mL of a Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
1 per cent V/V solution of concentrated ammonia R in ethanol
(96 per cent) R and boil the mixture under a reflux condenser DEFINITION
for 30 min. Allow to cool to room temperature and filter the Young, unfermented leaf of Camellia sinensis (L.) Kuntze
liquid through a plug of absorbent cotton into a flask. rapidly stabilised by short time heating and then dried.
Add the plug of absorbent cotton to the residue in the Content
round-bottomed flask and repeat the extraction with a further - caffeine (C 8 H 10N 4 0 2; Mr 194.2); minimum 1.5 per cent
2 quantities, each of 30 mL, of a 1 per cent V/V solution of (dried drug);
concentrated ammonia R in ethanol (96 per cent) R, each time - total catechins, expressed as (- )-epigallocatechin-3-0-gallate
boiling under a reflux condenser for 10 min and filtering (C 22H 18011; M, 458.4): minimum 8.0 per cent (dried
through a plug of absorbent cotton in the same flask as drug).
previously. Filter the combined filtrates through a filter paper
CHARACTERS
into a 250 mL round-bottomed flask, and rinse the flask and
Greyish-green leaf with petiole removed, rolled and often
the filter with 20 mL of a 1 per cent VIV solution of
folded or twisted, whole or cut before being rolled.
concentrated ammonia R in ethanol (96 per cent) R. Evaporate
the filtrate to dryness in vacua in a water-bath at 55 °C. IDENTIFICATION
Dissolve the residue in 50.0 mL of the mobile phase. Dilute A. Examine after allowing the leaf to unroll for a few minutes
10.0 mL of this solution to 50.0 mL with the mobile phase. in warm water. The leaf is oval, elongated and acuminate.
Reference solution Immediately before use, dissolve 10.0 mg Starting at a quarter of the distance from the base, the
of hydrastine hydrochloride CRS and 10.0 mg of berberine margins are serrate with peculiarly shaped teeth bearing a
chloride CRS in methanol Rand dilute to 100.0 mL with the small blackish point curved in a claw shape. The secondary
same solvent. veins branch from the midrib, curving near the margins of
Column: the lamina so that they anastomose in an arc. Flexuous,
- size: l = 0.125 m, 0 = 4 mm; conical, covering trichomes are abundant on the lower
- stationary phase: end-capped octadecylsilyl silica gel for surface of the leaves.
chromatography R (5 µm). B. Microscopic examination (2.8.23). The powder is
Mobile phase Dissolve 9.93 g of potassium dihydrogen greenish-grey. Examine under a microscope using chloral
phosphate R in 730 mL of water R, add 270 mL of hydrate solution R. The powder shows the following diagnostic
acetonitrile R and mix. characters (Figure 2668.-1): numerous fragments of
unicellular, flexuous, thick-walled covering trichomes [E];
Flow rate 1.2 mUmin. fragments of abaxial epidermis (surface view [A]) consisting
Detection Spectrophotometer at 235 nm. of cells with slightly wavy walls [Aa], numerous anomocytic
Injection 10 µL. stomata (2.8.3) surrounded by 3 or 4 narrow subsidiary
System suitability Reference solution: cells [Ab] and long, flexuous, thick-walled, unicellular
- elution order: order indicated in the composition of the covering trichomes, pointed at the apex [Ac]; fragments of
reference solution; record the retention times of these adaxial epidermis (surface view [F]) consisting of cells with
substances; rigid, slightly thickened walls [Fa], accompanied by cells of
the underlying palisade parenchyma [Fb] and sometimes by
IV-276 Green Tea 2023
the end of a sclereid [Fe]; numerous isolated sclereids, often Top of the plate
very ramified, with thick-walls and distinct channels [CJ;
fragments of lamina (transverse section [BJ), showing the
adaxial epidermis covered by a smooth cuticle [Ba], palisade ·-- --
parenchyma usually arranged in 2 layers [Bb], spongy
(-)-Epicatechin: a reddish-brown A reddish-brown zone ( (-)-
parenchyma [Be] including cells containing cluster crystals of zone epicatechin)
calcium oxalate [Bd], and the abaxial epidermis [Be] with
stomata; fragments of spiral vessels (longitudinal section [DJ) l or 2 reddish-brown zone(s)
accompanied by spongy parenchyma [Db] with some cells (-)-Epigallocatechin-3-0-gallate: a A reddish-brown zone ((-)-
containing cluster crystals of calcium oxalate [Da]; reddish-brown zone epigallocatechin-3-0-gallate)
vessels [G] accompanied by lignified fibres [Ga] from the
principal veins. -- --··
TESTS
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Stabilising solution Dissolve 250 mg of (ethylenedinitrilo)tetra-
acetic acid R and 250 mg of ascorbic acid R in 500 mL of
water R, add 100 mL of acetonitrile R and dilute to 1000 mL
with water R.
Extraction solution Place a sealed container filled with a
mixture of water R and methanol R (30:70 V/V) in a water-
bath at 70 °C for at least 30 min in order to reach the
temperature.
(355) (2.9.12) into a test tube, place in a water-bath at 70 °C
and add 5 mL of the extraction solution (pre-warmed to
70 °C). Stopper the tube and carefully mix using a vortex
mixer. Continue heating the test tube in the water-bath for
1O min, mixing on the vortex mixer after 5 min and 10 min.
Remove the test tube from the water-bath, allow to cool and
centrifuge at 2350 g for 10 min. Decant the supernatant
Figure 2668.-1. - Illustrarion for identification test B of powdered liquid into a 10 mL volumetric flask. Repeat the extraction
herbal drug of green tea procedure again with 5 mL of extraction solution, combine
the extracts in the same volumetric flask and dilute to
10.0 mL with a cold mixture of water R and methanol R
Test solurion To 0.1 g of the powdered herbal drug (355) (30:70 V/V). Dilute 1.0 mL of this solution to 20.0 mL with
(2. 9.12) add 10 mL of a mixture of water R and ethanol the stabilising solution. Filter through a membrane filter
(96 per cent) R (20:80 VIV). Sonicate for 10 min and filter. (nominal pore size 0.45 µm).
Reference solution Dissolve 2 mg of (-)-epicatechin Rand Reference solution (a) Dissolve 20.0 mg of(-)-
5 mg of (-)-epigallocatechin-3-0-gallate R in methanol Rand
epigall-Ocatechin-3-0-gallate CRS and 20.0 mg of caffeine CRS
dilute to 10 mL with the same solvent. in water R and dilute to 25.0 mL with the same solvent.
Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel Dilute 2.0 mL of the solution to 50.0 mL with the stabilising
F2 54 plate R (2-10 µm)]. solution. Filter through a membrane filter (nominal pore size
Mobile phase anhydrous formic acid R, acetone R, toluene R 0.45 µm).
(10:45:45 V/V/V). Reference solution (b) Dissolve 0.100 g of green tea dry
Application 30 µL [or 5 µL] as bands of 15 mm [or 8 mm]. extract HRS in 5 mL of water R using sonication, add 1.0 mL
Development Over a path of 10 cm [or 6 cm]. of acetonitrile R and dilute to 10.0 mL with water R. Dilute
2.0 mL of the solution to 50.0 mL with the stabilising
Drying In air.
solution. Filter through a membrane filter (nominal pore size
Detection Treat with a freshly prepared 5 g/L solution offast 0.45 µm).
blue B salt R. Examine in daylight.
Column:
Results See below the sequence of zones present in the =
- size: l 0.15 m, 0 4.6 mm; =
chromatograms obtained with the reference solution and the - stationary phase: end-capped octadecylsilyl silica gel for
test solution. Furthermore, other zones may be present in the chromatography R (3 µm).
2023 Guarana IV-277
Mobile phase:
- mobile phase A: 0.05 per cent V/V solution of anhydrous
Guarana
Jonnie acid R; (Ph. Bur. monograph 2669)
- mobile phase B: 0.05 per cent V/V solution of anhydrous
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
formic acid R in methanol R2;
- mobile phase C: acetonitrile Rl; DEFINITION
Dried seed of Paullinia cupana Kunth (syn. Paullinia sorbilis
Time Mobile phase A Mobile phase B Mobile phase C Mart.).
(min) (per cent VIP) (per cent VIP) (per cent VIP)
0-5 97 0 3
Content
5 - 23 97 ➔ 67 0 ➔ 30 3
Minimum 3.5 per cent of caffeine (C 8 H 10N 4 O 2 ; M, 194.2)
23 - 29 67 30 3
(dried drug).
29 - 30 67 ➔ 30 30 ➔ 67 3 IDENTIFICATION
30 - 31 30 67 3 A. The seed is more or less spherical, about 12 mm in
diameter, shiny, glabrous, dark brown with a large, light-
Flow rate 1 mUmin. coloured spot corresponding to the hilum.
Detection Spectrophotometer at 210 nm. B. Microscopic examination (2.8.23). Reduce to a powder
Injection 10 µL. without sieving. The powder is brownish and oily. Examine
under a microscope using chloral hydrate solution R.
green tea dry extract HRS and the chromatogram obtained
(Figure 2669.-1): polyhedral endosperm cells with thick
with reference solution (b) to identify the peaks due to (+)-
cellulose walls; fragments of episperm cells with lobed and
gallocatechin, (-)-epigallocatechin, (+)-catechin, (-)-
regularly thickened walls (surface view [D]) and forming a
epigallocatechin-3-O-gallate, (-)- epicatechin, (-)-
palisade layer (transverse section [B, El); numerous
gallocatechin-3-0-gallate and (-)-epicatechin-3-O-gallate.
fragments of cotyledons with rounded to ovoid cells, with
System suitability Reference solution (b): spaces between them [HJ and filled with starch; more or less
- resolution: minimum 1.5 between the peaks due to (-)- polyhedral sclereids with channelled walls, free [F] or in
epigallocatechin-3-O-gallate and (-)-epicatechin. groups [A]; rare spiral vessels [G]; fragments of the embryo
Calculate the percentage content of total catechins, expressed with thin-walled elongated cells (surface view [C]). Examine
as (-)-epigallocatechin-3-O-gallate, using the following under a microscope using a 50 per cent V/V solution of
expression: glycerol R. The powder shows numerous rounded or oval
starch granules [J], up to 30 µm long and 18 µm wide,
A1 x m2 x 0.32 xp simple or sometimes 2-3 compound, either free or included
A 2 xm1 in the cells of the cotyledons.
sum of the area of the peaks due to catechins [ (+)-

gallocatechin, (-)-epigallocatechin, (+)-catechin, (-)-
epigallocatechin-3-0-gallate, (-)-epicatechin, (-)-gallocatechin-
3-0-gallate and (-)-epicatechin-3-O-gallate] in the
area of the peak due to (-)-epigallocatechin-3-O-gallate in the
solution, in grams;
m2 mass of (- )-epigallocatechin-3-0-gallate CRS used to prepare
reference solution (a), in grams;
p percentage content of (-)-epigallocatechin-3-O-gallate in (- )-
epigallocatechin-3-0-gallate CRS.
Calculate the percentage content of caffeine using the

Ai x m2 x 0.32 xp
A 2 xm 1

solution, in grams;
m2 mass of caffeine CRS used to prepare reference solution (a), in
grams;
p percentage content of caffeine in caffeine CRS.
J
STORAGE
G
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
herbal drug of guarana
IV-278 Guarana 2023
C. Thin-layer chromatography (2.2.27). ASSAY

Test solution To 1 g of the powdered herbal drug (355) Liquid chromatography (2.2.29).
(2. 9.12) add 10 mL of methanol R, sonicate for 10 min, then Solution A Dissolve 30.0 mg of caffeine CRS in the mobile
centrifuge for 10 min and use the supernatant. phase and dilute to 100.0 mL with the mobile phase.
Reference solution Dissolve 2.0 mg of caffeine R and 1.0 mg Test solution Introduce 0.500 g of the powdered herbal drug
of catechin R in 2 mL of methanol R. (355) (2. 9.12) into a 100 mL round-bottomed flask and add
Plate TLC silica gel F254 plate R (2-10 µm). 40 mL of methanol R. Heat in a water-bath at 70 °C under a
Mobile phase anhydrous formic acid R, toluene R, acetone R reflux condenser for 30 min. Allow to cool and filter through
(10:45:45 VIVIV). a plug of absorbent cotton into a 100 mL volumetric flask.
Transfer the absorbent cotton with the drug residue into the
round-bottomed flask. Add 40 mL of methanol R, proceed as
Development In an unsaturated tank over a path of 6 cm. before and filter into the same volumetric flask. Rinse the
Drying In a current of air at room temperature. round-bottomed flask and the filter with methanol R, add the
Detection A Examine in ultraviolet light at 254 nm. rinsings to the volumetric flask and dilute to 100.0 mL with
Results A See below the sequence of zones present in the methanol R. Shake to homogenise the solution. Transfer
chromatograms obtained with the reference solution and the 10.0 mL of this solution into a volumetric flask, add 30 mL
test solution. Furthermore, other faint quenching zones may of water R and dilute to 50.0 mL with the mobile phase.
be present in the chromatogram obtained with the test Filter through a membrane filter (nominal pore size
solution. 0.45 µm).
Top of the plate 50.0 mL with the mobile phase.
Reference solution (b) Dissolve 15.0 mg of (-)-epicatechin R
Caffeine: an intense quenching zone An intense quenching zone (caffeine)
in the mobile phase and dilute to 25.0 mL with the mobile
-- --
phase. To 10.0 mL of the solution add 5 mL of solution A
and dilute to 50.0 mL with the mobile phase.
Column:
-- -- - size: l =0.25 m, 0 =4.6 mm;
Reference solution Test solution Mobile phase methanol R, water for chromatography R
(25:75 VIV).
Detection B Heat at 100 °C for 2 min, then treat the still- Flow rate 1 mUmin.
hot plate with fast blue B salt solution R and examine in Detection Spectrophotometer at 272 nm.
daylight.
Injection 10 µL.
Run time 3 times the retention time of caffeine.
test solution. Furthermore, other faint zones may be present Retention time Caffeine = about 11 min.
in the chromatogram obtained with the test solution. System suitability Reference solution (b):
- resolution: minimum 2.0 between the peaks due to caffeine
Top of the plate and (-)-epicatechin.
-- --
Catechin: a reddish-brown zone An intense reddish-brown zone

(catechin)
AI area of the peak due 10 caffeine in the chromatogram obtained
2 red or brown zones of varying
intensity
-- -- with reference solution (a);
1 or 2 reddish-brown zones solution, in grams;
m2 mass of caffeine CRS used to prepare solution A, in grams;
A reddish-brown zone p percentage content of caffeine in caffeine CRS.
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
TESTS
105 °C.
Total ash (2.4.16)
2023 Hamamelis Bark IV-279
Hamamelis Bark
DEFINITION
Cut, dried bark from the trunk and branches of Hamamelis
virginiana L.
Content
IDENTIFICATION
A. Pieces of different lengths, channelled, sometimes quilled
or in strips, up to 2 cm long and 3 mm thick. The outer
surface is ochre-brown or reddish-brown and has a thin,
whitish or greyish-brown cork with numerous lenticels.
The inner surface is yellowish-brown or reddish-brown and is
longitudinally striated. The fracture is splintery and fibrous.
B. Microscopic examination (2.8.23). The powder is brown. DaDb~H
.
. Ha
(Figure 2532.-1): very abundant sclereids of 2 types: one Hb .
type (more abundant) rounded, oval or subrectangular,
isolated [C] but more often in groups [A], with heavily
thickened and striated walls and numerous and branched
channels, sometimes accompanied by cells containing prisms Figure 2532.-1. - Illustration for identification test B of powdered
of calcium oxalate [Aa]; the other type much more regular in herbal drug of hamamelis bark
size and shape, polygonal, with walls only moderately
thickened and with simple channels, occurring in dense
Top of the plate
groups with no intercellular spaces [El, sometimes
accompanied by cells containing prisms of calcium Gallic acid: a blue fluorescent zone
oxalate [Ea]; abundant groups of fibres [G], usually
surrounded by a sheath of prisms of calcium oxalate [Gb]; -- --
fibres, very thick-walled and lignified, with an indistinct Hamamelitannin: a blue fluorescent An intense blue fluorescent zone
lumen [Ga]; thin-walled, parenchymatous cells of the zone (hamamelitannin)
phloem [DJ, sometimes filled with dark brown A blue fluorescent zone
contents [Db]; uniseriate medullary rays, composed of cells
with slightly thickened walls, somewhat rectangular -- - -
(longitudinal section [Gel) and rounded (tangential 2 or 3 blue fluorescent zones

section [Da]); fragments of cork [HJ consisting of thin-walled
cells, polyhedral (surface view [B, Hal), sometimes Reference solution Test solution
accompanied by an underlying layer of sclereids with slightly
thickened and channelled walls [Hb]; abundant isolated TESTS
prisms of calcium oxalate [F]. Corylus avellana L. bark and twigs and Hamamelis
C. Examine the chromatogram obtained in the test for virginiana L. twigs
Corylus avellana L. bark and twigs and Hamamelis Thin-layer chromatography (2.2.27).
virginiana L. twigs. Test solution To 1.0 g of the powdered herbal drug (355)
Results See below the sequence of zones present in the (2.9.12) add 10.0 mL of methanol R, shake for 15 min and
chromatogram obtained with the reference solution and the filter.
test solution. Furthermore, other faint zones may be present Reference solution Dissolve 5.0 mg of gallic acid Rand
in the upper third of the chromatogram obtained with the 8.0 mg of hamamelitannin R in 5.0 mL of ethanol
test solution. (60 per cent V/V) R.
Plate TLC silica gel F 254 plate R (5-40 µm) [or TLC silica
gel F 254 plate R (2-10 µm)].
Mobile phase anhydrous formic acid R, water R, ethyl
formate R (10:10:80 VIVIV).
Drying In air.
Detection Heat at 100 °C for 3 min; treat the plate whilst
still hot with a 5 g/L solution of diphenylboric acid aminoethyl
ester R in ethyl acetate R, allow to dry in air and examine in
IV-280 Hamamelis Leaf 2023
Results Toe chromatogram obtained with the test solution broken [A, D, M], composed of 4-12 unicellular branches
shows no yellow or orange zones. that are united by their bases, elongated, conical and curved,
Foreign matter (2.8.2) usually up to 250 µm long, thick-walled and with a clearly
Maximum 3 per cent of branches and twigs and maximum visible lumen whose contents are often brown; fibres are
2 per cent of other foreign matter. lignified and thick-walled, isolated or in groups, and
accompanied by a sheath of prismatic calcium oxalate crystals
[N, P]; sclereids, frequently enlarged at 1 or both ends,
150-180 µm long, whole or fragmented [HJ; fragments of
annular or spiral vessels [E]; isolated prisms of calcium
105 °C for 2 h.
oxalate [G].
Total ash (2.4.16)
ASSAY
Tannins (2.8.14)
Carry out the determination with the following modifications.
Preparation of the test solution Introduce 0 .150 g of the
powdered herbal drug (355) (2. 9.12) into a 250 mL round-
bottomed flask, add 150 mL of water R and heat on a water-
bath for 30 min. Cool under running water and transfer
quantitatively to a 250 mL volumetric flask. Rinse the round-
bottomed flask and collect the washings in the volumetric
flask, dilute to 250.0 mL with water Rand shake. Centrifuge
25 mL at 1000 g for 10 min.
Total polyphenols Use the supernatant instead of the filtrate
described in the general method. I . E
Polyphenols not adsorbed by hide powder Use the supernatant !
instead of the filtrate described in the general method. ID .·
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Hamamelis Leaf
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
( t<
DEFINITION ~- ··•· ;· L.
Whole or cut, dried leaf of Hamamelis virginiana L.
Content
Minimum 3 per cent of tannins, expressed as pyrogallol
100µm
(C 6H603; M, 126.1) (dried drug).
IDENTIFICATION Figure 0909. -1. - Illustration for identification test B of powdered
A. The leaf is green or greenish-brown, often broken, herbal drug of hamamelis leaf
crumpled and compressed into more or less compact masses.
The lamina is broadly ovate or obovate; the base is oblique C. Thin-layer chromatography (2.2.27).
and asymmetric and the apex is acute or, rarely, obtuse. Test solution To 1.0 g of the powdered herbal drug (355)
Toe margins of the lamina are roughly crenate or dentate. (2.9.12) add 10 mL of ethanol (60 per cent V/V) R, shake for
The venation is pinnate and prominent on the abaxial 15 min and filter.
surface. Usually, 4-6 pairs of secondary veins are attached to Reference solution ( a) Dissolve 30 mg of tannic acid R in
the main vein, emerging at an acute angle and curving gently 5 mL of ethanol (60 per cent V/V) R.
to the marginal points where there are fine veins often at
Reference solution (b) Dissolve 5 mg of gallic acid R in 5 mL
right angles to the secondary veins.
of ethanol (60 per cent VIV) R.
B. Microscopic examination (2.8.23). Toe powder is
brownish-green. Examine under a microscope using chloral
hydrate solution R. Toe powder shows the following diagnostic Mobile phase anhydrous formic acid R, water R, ethyl
characters (Figure 0909.-1): fragments of adaxial epidermis formate R (10:10:80 VIVIV).
with wavy anticlinal walls (surface view [C, TI), often Application 10 µL, as bands.
accompanied by small, cylindrical cells of the palisade Development Over a path of 10 cm.
parenchyma (surface view Ua]), or elongated (transverse Drying At 100-105 °C for 10 min, then allow to cool.
section [F]); fragments of abaxial epidermis with stomata
Detection Spray with ferric chloride solution R2 until bluish-
mainly paracytic (2.8.3) (surface view [BJ), which may be
grey zones (phenolic compounds) appear.
accompanied by irregular-shaped cells of spongy mesophyll
[K, L]; star-shaped covering trichomes, either entire or Results Toe chromatogram obtained with the test solution
shows in its lower third a principal zone similar in position to
2023 Hawthorn Berries IV-281
the principal zone in the chromatogram obtained with view [G]); fragments of the inner layers of the receptacle [A],
reference solution (a) and, in its upper part, a narrow zone some cells containing cluster crystals [Aa] or prisms [Ab] of
similar in position to the principal zone in the chromatogram calcium oxalate; occasional fragments IT, K] including groups
obtained with reference solution (b); the chromatogram of sclereids [Ka] and vascular bundles Ua, Kb] associated
obtained with the test solution shows, in addition, several with rows of cells containing prisms of calcium oxalate Ub,
slightly coloured zones in the central part. Kc]; fragments of the pericarp [BJ consisting of parenchyma
including some cells containing cluster crystals of calcium
TESTS
oxalate [Ba] and groups of sclereids of various sizes with
numerous pits [Bb]; thick-walled sclereids [E, HJ, channelled
[E], some with conspicuously branched channels [H]; a few
other foreign matter, determined on 50 g.
fragments of the testa [C] having an outer layer composed of
Loss on drying (2.2.32) hexagonal, mucilaginous cells [Ca] beneath which is a
Maximum 10.0 per cent, determined on 2.000 g of the yellowish-brown pigment layer containing numerous prisms
powdered herbal drug (355) (2.9.12) by drying in an oven at of calcium oxalate [Cb]; parenchyma of the endosperm and
105 °C for 4 h. cotyledons consisting of cells containing aleurone grains and
Total ash (2.4.16) globules of fixed oil [D].
ASSAY
Tannins (2.8.14)
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Hawthorn Berries
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried false fruits of Crataegus monogyna Jacq. 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 (C 15H 11 ClO 6 ; 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 Figure 1220.-1. - Illustration for identification test B of powdered
or, more frequently, a small, pale, circular scar where the herbal drug of hawthorn berries
pedicel was attached. The receptacle is fleshy and encloses a
yellowish-brown, ovoid fruit with a hard, thick wall C. High-performance thin-layer chromatography (2.8.25).
containing a single, elongated, pale brown, smooth and shiny Test solution To 0.5 g of the powdered herbal drug (355)
seed. (2.9.12) add 5.0 mL of methanol R. Sonicate for 15 min,
The false fruit of C. laevigata is up to 13 mm long. filter or centrifuge and use the filtrate or supernatant.
It contains 2-3 stony fruits, ventrally flattened, with short Reference solution (a) Dissolve 2.5 mg of hyperoside Rand
hairs at the top. The centre of the disc of the false fruit 3.5 mg of rutoside trihydrate R in methanol R and dilute to
frequently bears the remains of the 2 styles. 10.0 mL with the same solvent.
B. Microscopic examination (2.8.23). The powder is greyish- Reference solution (b) Dilute 2.5 mL of reference solution (a)
red. Examine under a microscope using chloral hydrate to 10.0 mL with methanol R.
solution R. The powder shows the following diagnostic Reference solution (c) Dissolve 2.5 mg of hyperoside Rand
characters (Figure 1220.-1): covering trichomes [F] from 3 mg of chlorogenic acid R in methanol R and dilute to 10 mL
inside the disc that are Jong, unicellular, frequently bent, with the same solvent.
tapering to a point, with heavily thickened and lignified walls; Intensity marker Hyperoside.
fragments of the red outer layer of the receptacle (surface
IV-282 Hawthorn Leaf and Flower 2023
Plate TLC silica gel F 254 plate R (2-10 µm). 15.0 mL of hydrochloric acid Rl and 10.0 mL of water R.
Mobile phase anhydrous formic acid R, water R, ethyl acetate R Heat under a reflux condenser for 80 min. Allow to cool,
(10:10:80 VIVIV). filter and wash the residue with ethanol (70 per cent V/V) 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
Development 70 mm from the lower edge of the plate. solution in a round-bottomed flask to about 3 mL and
Drying In a current of air at room temperature for 5 min. transfer to a separating funnel. Rinse the round-bottomed
Detection Heat at 100-105 °C for 5 min; spray the warm flask sequentially with 10 mL and 5 mL of water R and
plate with a 10 g/L solution of diphenylboric acid aminoethyl transfer to the separating funnel. Shake the combined
ester R in methanol R, then with a 50 g/L solution of macrogol solution with 3 quantities, each of 15 mL, of butanol R.
400 R in methanol R or, alternatively, dip the warm plate in a Combine the organic layers and dilute to 100.0 mL with
5 g/L solution of diphenylboric acid aminoethyl ester R in ethyl butanol R.
acetate R, then in a 50 g/L solution of macrogol 400 R in Measure the absorbance (2.2.25) of the solution at 555 nm.
methylene chloride R; allow to dry in air for about 1 min and Calculate the percentage content of procyanidins, expressed
examine in ultraviolet light at 366 nm. as cyanidin chloride, using the following expression:
- the chromatogram shows in the middle third 2 distinct
zones, which may be touching; the lower zone Ax500
(chlorogenic acid) shows a light blue fluorescence and ]200 X m
the upper zone (hyperoside) shows a yellow or orange
fluorescence. i.e. taking the specific absorbance of cyanidin chloride to be
Results See below the sequence of fluorescent zones present 1200.
in the chromatograms obtained with reference solution (a)
and the test solution. Furthermore, in the chromatogram A absorbance at 555 nm;
obtained with the test solution, other faint blue, greenish and m mass of the substance to be examined, in grams.
orange or yellow fluorescent zones may be present.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Top of the plate
1-2 blue zones, very faint to faint
-- --
Hawthorn Leaf and Flower
A yellow or orange zone, very faint (Ph. Eur. monograph 1432)
to faint
Hyperoside: a yellow or orange zone A yellow or orange zone, faint to
Preparations
equivalent (hyperoside) Hawthorn Leaf and Flower Dry Extract
A light blue zone, faint (chlorogenic Hawthorn Leaf and Flower Liquid Extract
acid) PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
-- --
DEFINITION
Whole or fragmented, dried flower-bearing branches of
Rutoside: a yellow or orange zone A yellow or orange zone, very faint
Crataegus monogyna Jacq. (Lindm.), C. laevigata (Poir.) DC.
(rutoside) or their hybrids or, more rarely, C. pentagyna Waldst. et Kit.
ex Willd. or C. azarolus L. These species may be mixed.
Content
Minimum 0.2 per cent of total vitexin-2 "-O-rhamnoside
TESTS derivatives, expressed as vitexin-2 11 -0-rhamnoside
Foreign matter (2.8.2) (C21H30O14; Mr 578.5) (dried drug).
Maximum 5 per cent of deteriorated false fruit and IDENTIFICATION
maximum 2 per cent of other foreign matter. It does not A. The stems are dark brown, woody, 1-2.5 mm in diameter,
contain false fruits of other Crataegus species (C. nigra bearing alternate, petiolate leaves with small, often deciduous
Waldst. et Kit., C. pentagyna Waldst. et Kit. ex Willd. stipules and corymbs of numerous small white flowers.
and C. azarolus L.), which are characterised by the presence The leaves are more or less deeply lobed with slightly serrate
of more than 3 hard stones. or almost entire margins; those of C. azarolus are the
Loss on drying (2.2.32) narrowest of all species, pinnatisect, with 3, 5 or 7 acute to
Maximum 12.0 per cent, determined on 1.000 g of the cuspidate, long, narrow lobes and almost entire margins;
powdered herbal drug (355) (2.9.12) by drying in an oven at those of C. laevigata are broad, pinnately lobed or pinnatifid
105 °C for 2 h. with 3, 5 or 7 obtuse to acute lobes and finely crenate-serrate
Total ash (2.4.16) margins; those of C. monogyna are pinnatisect with 3, 5 or
Maximum 5.0 per cent. 7 acute lobes and entire or irregularly serrate margins; those
of C. pentagyna are pinnatisect with 3, 5, 7 or 9 acute or
ASSAY obtuse lobes and irregularly serrate margins; the adaxial
To 2.50 g of the powdered herbal drug (355) (2.9.12) add surface is dark green or brownish-green, the abaxial surface is
30 mL of ethanol (70 per cent V/V) R. Heat under a reflux lighter greyish-green and shows a prominent, dense,
condenser for 30 min and filter. Wash the residue with reticulate venation. The leaves of C. laevigata, C. monogyna
10.0 mL of ethanol (70 per cent V/V) R. Add to the filtrate and C. azarolus are more or less glabrous or with few
2023 Hawthorn Leaf and Flower IV-283
trichomes, those of C. pentagyna are mostly densely C. pentagyna The lower epidermis [P] is covered by a
pubescent. The flowers have a brownish-green tubular calyx smooth cuticle and comprises cells with sinuous anticlinal
composed of 5 free, reflexed sepals, a corolla composed of walls, stomata [Pb] and rolled unicellular covering trichomes
5 free, yellowish-white or brownish, rounded or broadly ovate [Pc]. The upper epidermis [R] comprises polygonal cells
and shortly unguiculate petals and numerous stamens. covered by a fine striated cuticle, with slightly sinuous
The hypanthium and calyx of C. azarolus, C. laevigata and unicellular covering trichomes [Ra]; it is accompanied by
C. monogyna are glabrous or with few trichomes; those of palisade parenchyma [Rb], with some cells containing a
C. pentagyna are mostly densely pubescent. The ovary is cluster crystal of calcium oxalate.
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, 3, or
sometimes only 1, in C. pentagyna 5 or, rarely, 3 or 4.
yellowish-green to brownish-green. Examine under a
microscope using chloral hydrate solution R. Irrespective of the
species, the powder shows the following diagnostic characters
(Figures 1432.-1 and 1432.-2): unicellular covering
trichomes, usually with a thick wall and wide lumen, almost
straight or slightly curved, isolated or attached to the
epidermis of the leaves by a rosette of cells [A]; fragments of
petals showing rounded or polygonal epidermal cells, strongly
papillose, covered by a cuticle which clearly shows wavy
striations (surface view [M]); fragments of the epidermis of
the leaves showing anomocytic stomata (2.8.3) surrounded
by 4-7 subsidiary cells [Da, La, Pa, Qa, Ta]; fragments of
the endothecium of the anthers [G]; fragments of stems [B,
TI containing bordered-pitted vessels [Ba] and groups of
lignified fibres with narrow lumens Ua] associated with
calcium oxalate prism sheaths Ubl, sclereids [Bb, Jc] and
sometimes cells of the pith with thick, pitted walls [Be];
unicellular covering trichomes from the sepals, with sinuous
walls, up to 400 µm long [HJ; fragments of the leaf
mesophyll [F] showing parenchymatous cells usually
measuring 10-20 µm and containing calcium oxalate cluster
crystals [Fa], and veins with fibres attached [Fb] surrounded
by a calcium oxalate prism sheath [Fe]; isolated cluster
crystals and prisms [E, S, U]; numerous tricolporate pollen
grains, spherical to elliptical or triangular, up to 45 µm in
diameter [N].
The species can be distinguished by their leaf epidermis as Figure 1432.-1. - Illustration for identification test B of powdered
follows: herbal drug of hawthorn leaf and flower
C. laevigata The lower epidermis [DJ is covered by a
smooth cuticle and comprises polygonal cells or cells with
sinuous anticlinal walls; stomata [Db]; frequently, scars of Test solution To 0.5 g of the powdered herbal drug (355)
covering trichomes [De] surrounded by a rosette of cells with (2.9.12) add 5.0 mL of methanol R. Sonicate for 15 min,
thickened walls [Dd]; along the veins, elongated cells [De]. then filter or centrifuge the solution and use the filtrate or
The upper epidermis [C] comprises polygonal cells covered supernatant.
by a smooth cuticle and is accompanied by palisade Reference solution (a) Dissolve 2.5 mg of hyperoside Rand
parenchyma [Ca]. 4.0 mg of vitexin-2' '-O-rhamnoside R in methanol R and dilute
C. monogyna The lower epidermis [L] is covered by a to 10.0 mL with the same solvent.
striated cuticle and comprises polygonal cells or cells with Reference solution (b) Dilute 2.5 mL of reference solution (a)
sinuous anticlinal walls; stomata [Lb]; frequently, scars of to 10.0 mL with methanol R.
covering trichomes surrounded by a rosette of cells with Reference solution (c) Dissolve 2.5 mg of hyperoside R and
thickened walls; along the veins, elongated cells. The upper 3 mg of chlorogenic acid R in methanol R and dilute to 10 mL
epidermis [K] comprises polygonal cells covered by a striated with the same solvent.
cuticle and is accompanied by palisade parenchyma [Ka]. Intensity markers Reference solutions (a) and (b):
C. azarolus The lower epidermis (view of the cuticle [OJ, - hyperoside for yellow, orange or red fluorescent zones;
view of epidermal cells [Q]) is covered by a thick and deeply - vitexin-2 "-0-rhamnoside for green, greenish-blue or
wrinkled cuticle [Oa] and comprises polygonal cells [Qb], blue fluorescent zones.
stomata [Qc] and unicellular covering trichomes. The upper Plate TLC silica gel F254 plate R (2-10 µm).
epidermis [T] comprises polygonal cells covered by a striated
cuticle, and stomata; it is accompanied by palisade
ketone R, ethyl acetate R (10:10:30:50 V/V/V/V).
parenchyma [Tb], with some cells containing a cluster crystal
or a prism crystal of calcium oxalate. Application 4 µL as bands of 8 mm.
IV-284 Hawthorn Leaf and Flower 2023
Top of the plate
1 or 2 red zones, very I or 2 red zones, very

faint to equivalent faint to equivalent
I or 2 light blue zones, I or 2 light blue zones,

very faint to equivalent very faint to equivalent
--- -- ---
A light blue zone, very A light blue zone, very

faint to equivalent faint to equivalent
A yellow or orange A yellow or orange

zone, very faint to zone, faint to intense
equivalent
Hyperoside: a yellow or A yellow or orange A yellow or orange

orange zone zone, faint to intense zone, very faint to
intense
A light blue zone, very A light blue zone, faint
faint to intense to intense (chlorogenic
(chlorogenic acid) acid)
A yellow zone, faint to

equivalent

herbal drug of hawthorn leaf and flower
--- -- ---
Detection Heat at 100-105 °C for 5 min; spray the warm Vitexin-2' '-0- A green or greenish- A green or greenish-
plate with a 10 g/L solution of diphenylboric acid aminoethyl rhamnoside: a green or blue zone, faint to blue zone, very faint to
greenish-blue zone equivalent (vitexin-2' '- faint (vitexin-2 "-0-
O-rhamnoside) rhamnoside)
5 g/L solution of diphenylboric acid aminoethyl ester R in ethyl A yellow or orange A yellow or orange
acetate R, then in a 50 g/L solution of macrogol 400 R in zone, very faint to faint zone, faint to
equivalent
examine in ultraviolet light at 366 nm. Reference Test solution ( C. Test solution
solution (a) monogyna, (C. pentagyna and
System suitabi/,ity Reference solution (c):
C. laevigata, mixtures with other
- the chromatogram shows in the middle third 2 distinct C. azarolus) allowed species)
(chlorogenic acid) shows a light blue fluorescence and
the upper zone (hyperoside) shows a yellow or orange TESTS
fluorescence. Foreign matter (2.8.2)
Results See below the sequence of fluorescent zones present Maximum 8 per cent of lignified branches with a diameter
in the chromatograms obtained with reference solution (a) greater than 2.5 mm and maximum 2 per cent of other
and the test solution. Furthermore, in the chromatogram foreign matter.
obtained with the test solution, other very faint to faint blue, Loss on drying (2.2.32)
greenish-blue, brownish, yellow or orange fluorescent zones Maximum 10.0 per cent, determined on 1.000 g of the
may be present. powdered herbal drug (355) (2.9.12) by drying in an oven at
Development 70 mm from the lower edge of the plate. 105 °c for 2 h.
Drying In a current of air at room temperature for 5 min. Total ash (2.4.16)
ASSAY
Test solution To about 0.35 g of the powdered herbal drug
(710) (2. 9.12) add 10 mL of a 50 per cent V/V solution of
methanol R and 10 mL of a 2 g/L solution of sodium
hydroxide R. Sonicate for 20 min at about 25-35 °C. Allow to
cool and neutralise with 50 µL of anhydrous formic acid R.
Dilute to 25.0 mL with a 50 per cent V/V solution of
methanol R. Filter through a membrane filter (nominal pore
size 0.2 µm).
Reference solution (a) Dissolve 2.0 mg of vitexin-2"-O-
rhamnoside CRS in a 50 per cent V/V solution of methanol R
and dilute to 10.0 mL with the same solution.
2023 Hawthorn Preparations IV-285
Reference solution (b) To about 0.1 g of hawthorn leaf and

flower dry extract HRS add 10 mL of a 50 per cent V/V
Hawthorn Leaf and Flower Dry
solution of methanol R and 10 mL of a 2 g/L solution of Extract
sodium hydroxide R. Sonicate for 20 min at about 25-35 °C.
Allow to cool and neutralise with 50 µL of anhydrous formic
acid R. Dilute to 25 mL with a 50 per cent V/V solution of Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
methanol R. Filter through a membrane filter (nominal pore DEFINITION

size 0.2 µm). Dry extract produced from Hawthorn leaf and flower (1432).
Column:
Content
- size: l = 0.125 m, 0 = 4.0 mm;
- aqueous extracts: minimum 1.0 per cent of total vitexin-2"-
0-rhamnoside derivatives, expressed as vitexin-2 "-0-
rhamnoside (C 27H 300 14; Mr 578.5) (dried extract);
- hydroalcoholic extracts: minimum 2.0 per cent of total
Mobile phase: vitexin-2"-0-rhamnoside derivatives, expressed as vitexin-
- mobile phase A: mix 1.0 g of phosphoric acid R, 2.6 g of 2"-0-rhamnoside (C 27 H 30 0 14; Mr 578.5) (dried extract).
acetonitrile R, 3.3 g of methanol Rand 82.1 g of
tetrahydrofuran Rand dilute to 1000.0 g with water for PRODUCTION
chromatography R; The extract is produced from the herbal drug by a suitable
- mobile phase B: methanol R; procedure using either water or a hydroalcoholic solvent at
least equivalent in strength to ethanol (45 per cent VIV).
Time Mobile phase A Mobile phase B CHARACTERS
(min) (per cent V/V) (per cent V/JI)
Appearance
0 - 20 100 0 Light brown or greenish-brown powder.
20 - 22 100 ➔ 10 0 ➔ 90
22 - 26 10 90
IDENTIFICATION
Flow rate 1.0 mL'min. Test solution Suspend 0.2 g of the extract to be examined in
20.0 mL of methanol R. Sonicate for 5 min, then filter or
centrifuge. Use the filtrate or supernatant.
Injection 10 µL.
Reference solution (a) Dissolve 2.5 mg of hyperoside Rand
Identification of peaks Use the chromatogram obtained with 4.0 mg of vitexin-2 11 -0-rhamnoside R in methanol R and dilute
reference solution (a) to identify the peak due to vitexin-2 11- to 10.0 mL with the same solvent.
0-rhamnoside; use the chromatogram supplied with hawthorn
leaf and flower dry extract HRS and the chromatogram
obtained with reference solution (b) to identify peak 2
(unknown). Reference solution (c) Dissolve 2.5 mg of hyperoside Rand
3 mg of chlorogenic acid R in methanol R and dilute to 10 mL
- peak-to-valley ratio: minimum 1.5, where Hp = height
above the baseline of peak 2 (eluted immediately after Intensity markers Reference solutions (a) and (b):
vitexin-2 11 -0-rhamnoside) and Hv = height above the - hyperoside for yellow, orange or red fluorescent zones,
baseline of the lowest point of the curve separating this - vitexin-2"-0-rhamnoside for green, greenish-blue or
peak from the peak due to vitexin-2"-0-rhamnoside. blue fluorescent zones.
Calculate the percentage content of total vitexin-2 11 -0- Plate TLC silica gel F 254 plate R (2-10 µm).
rhamnoside derivatives, expressed as vitexin-2 11 -0- Mobile phase anhydrous formic acid R, water R, methyl ethyl
rhamnoside resulting from alkaline hydrolysis, using the ketone R, ethyl acetate R (10:10:30:50 VIVIVIV).
following expression: Application 4 µL as bands of 8 mm.
A1 xm 2 xpx2.5
A2 xm1
area of the peak due to vitexin-2"-O-rhamnoside in the plate with a 10 g/L solution of diphenylboric acid aminoethyl
chromatogram obtained with the test solution; ester R in methanol R, then with a 50 g/L solution of macrogol
area of the peak due to vitexin-2' '-O-rhamnoside in the
mass of the herbal drug to be examined used to prepare the test 5 g/L solution of diphenylboric acid aminoethyl ester R in ethyl
solution, in grams; acetate R, then in a 50 g/L solution of macrogol 400 R in
mass of vitexin-2"-0-rhamnoside CRS used to prepare reference methyl.ene chloride R; allow to dry in air for about 1 min and
p percentage content of vitexin-2 1 '-O-rhamnoside in vitexin-2"-0-
rhamnoside CRS. System suitability Reference solution (c):
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur zones, which may be touching; the lower zone
(chlorogenic acid) shows a light blue fluorescence and the
upper zone (hyperoside) shows a yellow or orange
fluorescence.
Results (aqueous extracts) See below the sequence of
fluorescent zones present in the chromatograms obtained
with reference solution (a) and the test solution.
IV-286 Hawthorn Preparations 2023
Furthermore, in the chromatogram obtained with the test TESTS

solution, other very faint or faint blue or brownish Loss on drying (2.2.32)
fluorescent zones may be present. Maximum 6.0 per cent, determined on 0.500 g of the extract
to be examined by drying in an oven at 105 °C for 2 h.
Top of the plate ASSAY
A blue zone, very faint to faint Liquid chromatography (2.2.29).
Test solution To about 0 .1 g of the extract to be examined
add 10 mL of a 50 per cent V/V solution of methanol R and
-- --- 10 mL of a 2 g/L solution of sodium hydroxide R. Sonicate for
A green or greenish-blue zone, very
20 min at about 25-35 °C. Allow to cool and neutralise with
faint to faint 50 µL of anhydrous formic acid R. Dilute to 25.0 mL with a
50 per cent V/V solution of methanol R. Filter through a
Hyperoside: a yellow or orange zone A green or greenish-blue zone, faint Reference solution (a) Dissolve 2.0 mg of vitexin-2 11 -O-
to equivalent
rhamnoside CRS in a 50 per cent V/V solution of methanol R
A blue zone, very faint to faint
and dilute to 10.0 mL with the same solution.
Reference solution (b) To about 0.1 g of hawthorn leaf and
flower dry extract HRS add 10 mL of a 50 per cent VIV
-- ---
solution of methanol R and 10 mL of a 2 g/L solution of
Vitexin-2 "-O-rhamnoside: a green or A green or greenish-blue zone, faint sodium hydroxide R. Sonicate for 20 min at about 25-35 °C.
greenish-blue zone to equivalent (vitexin-2 11 -O- Allow to cool and neutralise with 50 µL of anhydrous formic
rhamnoside)
acid R. Dilute to 25 mL with a 50 per cent VIV solution of
size 0.2 µm).
Column:
= =
- size: l 0.125 m, 0 4.0 mm;
Reference solution (a) Test solution (aqueous extracts) - stationary phase: end-capped octadecylsilyl silica gel for
Results (hydroalcoholic extracts) See below the sequence of
fluorescent zones present in the chromatograms obtained Mobile phase:
with reference solution (a) and the test solution. - mobile phase A: mix 1.0 g of phosphoric acid R, 2.6 g of
Furthermore, in the chromatogram obtained with the test acetonitrile R, 3.3 g of methanol R and 82.1 g of
solution, other very faint or faint blue, greenish-blue, green, tetrahydrofuran Rand dilute to 1000.0 g with water for
brownish, yellow or orange fluorescent zones may be present. chromatography R;
Top of the plate
A red zone, very faint to faint (min) (per cent V/JI) (per cent V/JI)
0 - 20 100 0
A wide blue zone, very faint to faint
20 - 22 100 ➔ 10 0 ➔ 90
--- --- 22 - 26 10 90
A green or greenish-blue zone, very

faint Flow rate 1.0 mL/min.
A yellow or orange zone, faint to Injection 10 µL.
equivalent Identification of peaks Use the chromatogram obtained with
Hyperoside: a yellow or orange zone A yellow or orange zone (hyperoside) reference solution (a) to identify the peak due to vitexin-2 11 -
O-rhamnoside; use the chromatogram supplied with hawthorn
A light blue zone (ch!orogenic acid)
leaf and flower dry extract HRS and the chromatogram
obtained with reference solution (b) to identify peak 2
(unknown).
--- ---
Vitexin-2"-O-rhamnoside: a green or A green or greenish-blue zone - peak-to-valley ratio: minimum 1.5, where Hp = height
greenish-blue zone (vitexin-2 11 -O-rhamnoside)
above the baseline of peak 2 (eluted immediately after
A yellow or orange zone, very faint vitexin-2"-O-rhamnoside) and Hv = height above the
to faint baseline of the lowest point of the curve separating this
peak from the peak due to vitexin-2 11 -0-rhamnoside.
Calculate the percentage content of total vitexin-2 11 -O-
rhamnoside derivatives, expressed as vitexin-2 11-O-
Reference solution (a) Test solution (hydroalcoholic rhamnoside resulting from alkaline hydrolysis, using the
extracts)
2023 Hawthorn Preparations IV-287
A1 xm2 xpx 2.5 methylene chloride R; allow to dry in air for about 1 min and
A2 xm1 examine in ultraviolet light at 366 nm.
area of the peak due to vitexin-2 "-0-rhamnoside in the System suitability Reference solution (c):
chromatogram obtained with the test solution; - the chromatogram shows in the middle third 2 distinct
area of the peak due to vitexin-2 "-O-rhamnoside in the zones, which may be touching; the lower zone
chromatogram obtained with reference solution (a); (chlorogenic acid) shows a light blue fluorescence and the
solution, in grams; upper zone (hyperoside) shows a yellow or orange
mass of vitexin-2" -O-rhamnoside CRS used to prepare reference fluorescence.
solution (a), in grams; Results See below the sequence of fluorescent zones present
p percentage content of vitexin-2 11 -0-rhamnoside in vitexin-2 11 -O-
rharnnoside CRS.
and the test solution. Furthermore, in the chromatogram
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur obtained with the test solution, other very faint or faint blue
or red fluorescent zones may be present.
Top of the plate
Hawthorn Leaf and Flower Liquid 1 or 2 blue zones, very faint to faint
Extract
Quantified Hawthorn Leaf and Flower Liquid - - - -
Extract
A green or greenish-blue zone, faint
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
A yellow or orange zone

DEFINITION
Liquid extract produced from Hawthorn leaf and flower Hyperoside: a yellow or orange zone A yellow or orange zone, intense
(1432).
A blue zone
Content
Minimum 0.4 per cent of total vitexin-2 11 -O-rhamnoside
derivatives, expressed as vitexin-2 11 -O-rhamnoside
(C21H30O14; Mr 578.5).
-- --
PRODUCTION
The extract is produced from the herbal drug by a suitable Vitexin-2' '-O-rhamnoside: a green A green or greenish-blue zone
or greenish-blue zone (vitexin-2' '-0-rhamnoside)
procedure using ethanol (30-70 per cent V/V).
An orange zone, very faint to faint
IDENTIFICATION
Test solution Dilute 2.0 g of the extract to be examined in
10.0 mL of methanol R then filter or centrifuge. Use the
filtrate or supernatant. Reference solution (a) Test solution

4.0 mg of vitexin-2' '-O-rhamnoside R in methanol R and dilute TESTS
to 10.0 mL with the same solvent. Ethanol (2.9.10)
Reference solution (b) Dilute 2.5 mL of reference solution (a) 95 per cent to 105 per cent of the content stated on the
to 10.0 mL with methanol R. label.
Reference solution (c) Dissolve 2.5 mg of hyperoside R and ASSAY
3 mg of chlorogenic acid R in methanol R and dilute to 10 mL Liquid chromatography (2.2.29).
Test solution To about 1.0 g of the extract to be examined
Intensity markers Reference solutions (a) and (b):
add 10 mL of a 50 per cent V/V solution of methanol R and
- hyperoside for yellow, orange or red fluorescent zones, 10 mL of a 2 g/L solution of sodium hydroxide R. Sonicate for
- vitexin-2"-O-rhamnoside for green, greenish-blue or 20 min at about 25-35 °C. Allow to cool and neutralise with
blue fluorescent zones. 50 µL of anhydrous formic acid R. Dilute to 25.0 mL with a
Plate TLC silica gel F254 plate R (2-10 µm). 50 per cent V/V solution of methanol R. Filter through a
Mobue phase anhydrous formic acid R, water R, methyl ethyl membrane filter (nominal pore size 0.2 µm).
ketone R, ethyl acetate R (10:10:30:50 VIVIVIV). Reference solution (a) Dissolve 2.0 mg of vitexin-2 11 -O-
Application 4 µL as bands of 8 mm. rhamnoside CRS in a 50 per cent VIV solution of methanol R
Development 70 mm from the lower edge of the plate. and dilute to 10.0 mL with the same solution.
Drying In a current of air at room temperature for 5 min. Reference solution (b) To about 0.1 g of hawthorn leaf and
flower dry extract HRS add 10 mL of a 50 per cent V/V
plate with a 10 g/L solution of diphenylboric acid aminoethyl solution of methanol Rand 10 mL of a 2 g/L solution of
sodium hydroxide R. Sonicate for 20 min at about 25-35 °C.
Allow to cool and neutralise with 50 µL of anhydrous formic
acid R. Dilute to 25 mL with a 50 per cent VIV solution of
acetate R, then in a 50 g/L solution of macrogol 400 R in
size 0.2 µm).
IV-288 Hop Strobile 2023
=
- size: l 0.125 m, 0 =4.0 mm; A. Hop strobiles are generally isolated and 2-5 cm long,
- stationary phase: end-capped octadecylsilyl silica gel for petiolate, ovoid, made up of many oval, greenish-yellow,
chromatography R (3 µm); sessile, membranous, overlapping bracts. The external bracts
- temperature: 40 °C. are flattened and symmetrical. The internal bracts are longer
Mobile phase: and asymmetrical at the base because of a fold generally
- mobile phase A: mix 1.0 g of phosphoric acid R, 2.6 g of encircling an induviate fruit (achene). The ovary or rarely the
acetonitrile R, 3.3 g of methanol Rand 82.1 g of fruit, the base of the bracts and especially the induvial fold,
tetrahydrofuran Rand dilute to 1000.0 g with water for are covered with small orange-yellow glands.
chromatography R; B. Microscopic examination (2.8.23). The powder is
- mobile phase B: methanol R; greenish-yellow. Examine under a microscope using chloral
Time Mobile phase A Mobile phase B characters (Figure 1222.-1): fragments of bracts and
(min) (per cent V/JI) (per cent V/J/) bracteoles covered by polygonal, irregular or wavy-walled
0 - 20 100 0 epidermal cells [D, L, M]; unicellular, conical, straight or
20 - 22 100---+ 10 0---+ 90 curved covering trichomes with thin, smooth walls,
22 - 26 10 90 fragmented [E, G] or attached to an epidermis [A]; rare
anomocytic stomata (2.8.3) [K]; glandular trichomes, usually
Flow rate 1.0 mIJmin. free, with bicellular biseriate stalks and heads consisting of
Detection Spectrophotometer at 340 nm. 8 small cells [H, N], rarely attached to an epidermis [La];
fragments of mesophyll containing small calcium oxalate
Infection 10 µL.
cluster crystals m; many characteristic orange-yellow
glandular trichomes with short, bicellular biseriate stalks,
reference solution (a) to identify the peak due to vitexin-2 11 - bearing a part widening into a cup, 150-250 µm in diameter,
0-rhamnoside; use the chromatogram supplied with hawthorn made up of a hemispherical layer of secretory cells with a
leaf and flower dry extract HRS and the chromatogram cuticle that has been detached and distended by the
obtained with reference solution (b) to identify peak 2 accumulation of oleoresinous secretions (surface view [B],
(unknown). side view [C]); fragments of elongated sclerenchymatous cells
System suitability Reference solution (b): of the testa with thick walls showing striations and numerous
- peak-to-valley ratio: minimum 1.5, where Hp = height pits [F].
above the baseline of peak 2 (eluted immediately after
vitexin-2 11 -0-rhamnoside) and Hv = height above the
baseline of the lowest point of the curve separating this
C
peak from the peak due to vitexin-2 11 -0-rhamnoside.
Calculate the percentage content of total vitexin-2 11 -0-
rhamnoside derivatives, expressed as vitexin-2 11 -0-
rhamnoside resulting from alkaline hydrolysis, using the
A1 x m2 x p x 2.5
A2xm 1
A, area of the peak due to vitexin-2 11-0-rhamnoside in the

area of the peak due to vitexin-2' '-O-rhamnoside in the G
solution, in grams;
m2 mass of vitexi.n-2"-0-rhamnoside CRS used to prepare reference
p percentage content of vitexin-2 11 -O-rhamnoside in vitexin-2 "-0-
rhamnoside CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
ff? N
Hop Strobile ,__....,
100 µm

~~----------------------
DEFINITION
Dried, generally whole, female inflorescence of Humulus Figure 1222. -1. - Illustration for identification test B of powdered
herbal drug of hop strobile
lupulus L.
CHARACTERS C. Thin-layer chromatography (2.2.27).
Characteristic, aromatic odour. Test solution To 1.0 g of the freshly powdered herbal drug
(355) (2.9.12) add 10 mL of a mixture of 3 volumes of
2023 White Horehound IV-289
water R and 7 volumes of metharwl R; shake for 15 min and

filter.
White Horehound
Reference solutwn Dissolve 1.0 mg of Sudan orange R, (Ph. Bur. monograph 1835)
2.0 mg of curcumin R and 2.0 mg of
dimethylaminobenzaldehyde R in 20 mL of metharwl R.
Plate TLC silica gel F 254 plate R. DEFINITION
Mobile phase anhydrous acetic acid R, ethyl acetate R, Whole or fragmented dried flowering aerial parts of
cyclohexane R (2:38:60 V/V/V). Marrubium vulgare L.
Application 20 µL as bands. Content
Minimum 0.7 per cent ofmarrubiin (C 20 H 28 O 4; Mr 332.4)
(dried drug).
Drying In air.
IDENTIFICATION
A. The stems are up to 50 cm long, quadrangular, up to
Results A The chromatogram obtained with the reference
7 mm wide, young stems are densely covered with whitish
solution shows 3 quenching zones; in the lower quarter is the downy hairs, older stems are greenish-grey and less hairy.
faint zone due to curcurnin, somewhat below the middle is The lower leaves are broadly ovate to almost orbicular, upper
the zone due to dimethylaminobenzaldehyde, and above, the leaves less broadly ovate, both petiolate; lamina 1.5-4 cm
zone due to Sudan orange. The chromatogram obtained with long, 1-3.5 cm wide, apex sub-acute, base tapering or
the test solution shows a number of quenching zones similar somewhat cordate, margin dentate to crenate, petiole up to
in position to the zones in the chromatogram obtained with 3 cm long; venation pinnate, prominent on the lower surface,
the reference solution: at about the level of the zone due to distinctly depressed on the upper surface. Both leaf surfaces
curcumin is a faint zone due to xanthohumol, near the level are densely covered with fine, white, woolly hairs, older
of the zone due to dimethylaminobenzaldehyde are zones due leaves having fewer hairs on the dark greyish-green upper
to humulones, and near the level of the zone due to Sudan surface. The flowers are small, sessile in dense axillary
orange are zones due to lupulones. clusters. The calyx is 5 mm long, persistent, with 5 long and
Detection B Examine in ultraviolet light at 365 nm. 5 short, alternating, hooked, recurved fringing spines; throat
Results B In the chromatogram obtained with the test of calyx with an internal ring of long silky hairs; corolla
solution the zones due to lupulones show blue fluorescence, 7 mm long, dull white, 4-lobed, upper lobe 2-lipped, lower-
the zones due to humulones show brown fluorescence and lobe 3-lipped; 4 short stamens; style with bifid stigma.
the zone due to xanthohumol shows dark brown B. Microscopic examination (2.8.23). The powder is greyish-
fluorescence. green. Examine under a microscope using chloral hydrate
Detection C Spray with dilute phosphomolybdotungstic solution R. The powder shows the following diagnostic
reagent R; expose to ammonia vapour and examine in characters (Figure 1835.-1): numerous covering trichomes of
daylight. different types, isolated or associated with fragments of the
Results C In the chromatogram obtained with the test epidermis of the leaves: a) covering trichomes in tufts [B, H]
solution the zones due to humulones and to lupulones are with 3-20 rigid branches of various sizes with smooth,
bluish-grey and the zone due to xanthohumol is greenish- regularly thickened walls, sometimes sessile [B], sometimes
grey; in the chromatogram obtained with the reference arising from a multicellular stalk [H], some branches are
solution the zones are bluish-grey or brownish-grey. unicellular [Ba, Ha], others are multicellular (2-6 cells),
100-200 µm long, thick-walled and swollen at the junctions
TESTS [Bb, Hb]; b) unicellular covering trichomes; c) multicellular
Matter extractable by ethanol (70 per cent V/V) covering trichomes similar to the branches of the trichomes
Minimum 25.0 per cent. in tufts; glandular trichomes of different types: a) glandular
To 10.0 g of the powdered herbal drug (355) (2.9.12) add trichomes with a unicellular stalk and a unicellular [D],
300 mL of ethanol (70 per cent V/V) R and heat for 10 min bicellular [He] or quadricellular head (surface view [Gb]); b)
on a water-bath under a reflux condenser. Allow to cool, glandular trichomes with a multicellular stalk and a
filter, and discard the first 10 mL of the filtrate. Evaporate unicellular head [A]; c) glandular trichomes of lamiaceous
30.0 mL of the filtrate to dryness on a water-bath and dry in type with a unicellular stalk and an 8-celled head covered
an oven at 100-105 °C for 2 h. The residue weighs a with a cuticle (surface view [Gd]); fragments of the epidermis
minimum of 0.250 g. of the leaves [G] consisting of polygonal cells with slightly
Loss on drying (2.2.32) sinuous walls [Ga], stomata of the diacytic type (2.8.3) [Ge],
Maximum 10.0 per cent, determined on 1.000 g of the covering trichomes and glandular trichomes; covering
powdered herbal drug (355) (2.9.12) by drying in an oven at trichomes from the inner surface of the calyx, twisted or
105 °C for 2 h. coiled, up to 1000 µm long, bi- or tricellular, thickened at
the cell junctions, with a flexuous, very elongated distal
Total ash (2. 4.16)
cell [C]; fragments of palisade parenchyma (surface
view [Be]) containing small needle-shaped crystals of calcium
oxalate; fragments of vascular tissue from the stems and veins
[F]; fragments of petals with papillose cells [J); spherical
pollen grains, about 25 µm in diameter with a smooth exine
[E].
IV-290 White Horehound 2023
Top of the plate
Guaiazulene: a reddish- A bluish-violet zone A bluish-violet zone

violet zone
-- --
A bluish-violet zone A bluish-violet zone
-- --
Cholesterol: a bluish- An intense bluish-violet A bluish-violet zone

violet zone zone (marrubiin) (marrubiin)
Reference solntion Test solution (a) Test solution (b)
TESTS
Maximum 10. 0 per cent, determined on 1.000 g of the
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Test solution Reduce 50 g of the herbal drug to a
Figure 1835.-1. - Illustration for identification test B of powdered powder (250) (2.9.12) and homogenise. To 1.00 g of the
herbal drug of white horehound powdered herbal drug in a 50 mL round-bottomed flask add
15 mL of a mixture of 2 volumes of dilute hydrochloric acid R
and 8 volumes of methanol R. Heat in a water bath at 80 °C
Test solution (a) To 1.0 g of the powdered herbal drug under a reflux condenser for 30 min. Allow to cool to room
(710) (2.9.12) add 2 mL of dilute hydrochloric acid Rand temperature and filter through a plug of adsorbent cotton
8 mL of methanol R. Heat under a reflux condenser for into a 25 mL volumetric flask. Dilute to 25.0 mL with
30 min, cool and filter. methanol R by rinsing the round-bottomed flask and the filter.
Test solution (b) To 1.0 g of the powdered herbal drug Reference solution Dissolve 2.0 mg of marrubiin CRS in
(710) (2. 9.12) add 10 mL of methanol R. Heat under a reflux methanol Rand dilute to 10.0 mL with the same solvent.
condenser for 30 min, cool and filter.
Column:
Reference solution Dissolve 10 mg of cholesterol R and 10 mg - size: l = 0.25 m, 0 = 4 mm;
of guaiazulene R in 10 mL of methanol R. - stationary phase: end-capped octadecylsilyl silica gel for
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel chromatography R (5 µm).
plate R (2-10 µm)]. Mobile phase:
Mobile phase methanol R, toluene R (5:95 V/V). - mobile phase A: acetonitrile RI;
Application 20 µL [or 5 µL] of test solutions (a) and (b) - mobile phase B: dilute 0.5 mL of phosphoric acid R to
and 10 µL [or 2 µL] of the reference solution, as bands. 1000 mL with water for chromatography R;
Drying In air. (min) (per cent V/J/) (per cent V/J/)
Detection Treat with a 5 g/L solution of vanillin R in a 0 - 15 40 -, 90 60-, 10
mixture of 20 volumes of ethanol (96 per cent) R and 15 - 20 90-, 40 10 -, 60
80 volumes of suljuric acid R and examine in daylight 20 - 25 40 60
immediately after heating at 130 °C for 5-10 min.
Results See below the sequence of zones present in the Flow rate 1.5 mUmin.
chromatograms obtained with the reference solution and test
Detection Spectrophotometer at 21 7 nm.
solutions (a) and (b). Further zones in the chromatograms
obtained with test solutions (a) and (b) may be present. Injection 20 µL.
The zone due to marrubiin in the chromatogram obtained Locate the peak due to marrubiin by comparison with the
with test solution (a) is more intense than that in the chromatogram obtained with the reference solution.
chromatogram obtained with test solution (b). During Calculate the percentage content of marrubiin from the
extraction with hydrochloric acid and methanol, conversion following expression:
of pre-marrubiin to marrubiin takes place which leads to an
increase in intensity of the zone. A 1 xmzxpx2.5
A2 xm1
2023 Horse-chestnut Fruit IV-291
AI area of the peak due to marrubiin in the chromatogram

A2 area of the peak due to marrubiin in the chromatogram
solution, in milligrams;
m2 mass of mamtbiin CRS used to prepare the reference solution,
in milligrams;
p percentage content of marrubiin in marrubiin CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Horse-chestnut Fruit
(Horse-chestnut, Ph. Bur. monograph 1830)
Preparation
Standardised Horse-chestnut Dry Extract
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or fragmented, dried, ripe seeds of Aesculus
hippocastanum L.
Content
protoaescigenin (C 30H 500 6 ; Mr 506.7) (dried drug).
IDENTIFICATION
A. The whole, spherical or oval, slightly flattened seed is 1-----i
2-4 cm in diameter. It has a shiny, dark-brown testa with a 25 µm
broad, round, matt, light-brown spot (hilum); particularly on
larger seeds, a short, narrow v-shaped ridge marks the
position of the radicle, with the point terminating close to the
hilum. Figure 1830.-1. - Illustratwn for identificatwn test B of powdered
herbal drug of horse-chestnut
The fragmented seed occurs as more or less polyhedral
pieces, about 1-2 cm in diameter, or as slices. The surfaces Reference solutwn Dissolve 5 mg of aescin for LG assay HRS
corresponding to the cotyledons are matt, light brown, with a and 5 mg of sucrose R in 1.0 mL of ethanol
clean fracture. Those corresponding to the testa are shiny, (70 per cent V/J;? R.
dark brown, except at the hilum, where they are matt, light
brown. The testa is weakly bound to the cotyledons and is
often detached.
Mobile phase acetic acid R, ethyl acetate R, water R,
B. Microscopic examination (2.8.23). The powder is propanol R (1.5:30:30:40 VIVIVIV).
characters (Figure 1830.-1): numerous different-sized
droplets of fatty oil that are either free [D] or inside the thin- Drying In air.
walled colourless cells of the cotyledons [E, G]; yellowish- Detection Treat with anisaldehyde solution R, heat at
brown fragments of the outer testa consisting of 100-105 °C for 10 min and examine in daylight.
sclerenchymatous cells with thick walls (surface view [C], Results See below the sequence of zones present in the
transverse section [A]); fragments from the inner testa chromatogram obtained with the reference solution and the
consisting of thick-walled, colourless parenchymatous cells test solution. Furthermore, other zones may be present in the
varying in shape [B, H, TI with poorly visible pits and chromatogram obtained with the test solution.
occasional annular or spiral vessels [F]. Examine under a
microscope using a 50 per cent V/V solution of glycerol R. Top of the plate
Starch [K] is present in 3 forms: pyriform or reniform simple

granules, often with verruciforrn excrescences, about
A yellowish-violet zone
15-25 µm in size, sometimes up to 30 µm; roundish simple
granules 5-10 µm in diameter; and a few granules that form -- --
rows consisting of 2-4 simple granules that are up to about
Aescin: a violet-blue zone An intense violet-blue zone (aescin)
35 µm in length and occasionally up to about 45 µm; most
of the granules have either a stellate (2 or more rays) or more
rarely a punctiform hilum.
Sucrose: a brownish-green zone A brol'nish-green zone (sucrose)
2 brownish-green zones
(2. 9.12) add 10 mL of ethanol (70 per cent V/V) R and heat -- --
under a reflux condenser for 15 min. Cool and filter.
IV-292 Horse-chestnut Preparations 2023
TESTS - signal-to-noise ratio: minimum 10 for the peak due to

Loss on drying (2.2.32) methyl salicylate in the chromatogram obtained with
Maximum 10.0 per cent, determined on 1.000 g of the reference solution (b).
powdered herbal drug (355) (2.9.12) by drying in an oven at Integration:
105 °C for 2 h. - test solution: integrate the peaks eluting between the peak
Total ash (2.4.16) due to methyl salicylate and the peak due to ibuprofen;
Maximum 4.0 per cent. integrate the area of the individual peaks or integrate
groups of peaks in case of incomplete separation of the
ASSAY individual peaks;
Liquid chromatography (2.2.29). - reference solution (a): for quantification, integrate as
Solvent mixture acetonitrile Rl, 0.05 per cent V/V solution of 1 integral, valley-to-valley, starting from the 1st peak after
trifiuoroacetic acid R (40:60 V/V). the peak due to methyl salicylate and ending with the last
Internal marker solution Dissolve 25.0 mg of methyl peak before the peak due to ibuprofen; if a peak is poorly
salicylate Rand 75.0 mg of ibuprofen R in the solvent mixture separated from the peaks due to methyl salicylate or
and dilute to 50.0 mL with the solvent mixture. Dilute ibuprofen, integrate it separately; use the sum of the peak
5.0 mL of the solution to 25.0 mL with the solvent mixture. areas for the calculation.
Test solution To 2.00 g of the powdered herbal drug (355) Calculate the percentage content of triterpene glycosides,
(2.9.12) add 10.0 mL of the internal marker solution and expressed as protoaescigenin, using the following expression:
10. 0 mL of the solvent mixture and sonicate for 10 min. A1 xmzxpx2
Centrifuge and filter 2.0 mL of the supernatant through a
A2 xm1
membrane filter (0.45 µm). Use the filtrate as the test
solution. total area (integrated as described above) of the peaks eluting
between the peaks due to methyl salicylate and ibuprofen in
Reference solution (a) Dissolve 50.0 mg of aescin for LG the chromatogram obtained with the test solution;
assay HRS in the internal marker solution and dilute to total area (integrated as described above) of the peaks eluting
10.0 mL with the internal marker solution. Sonicate for between the peaks due to methyl salicylate and ibuprofen in
10 min and filter through a membrane filter (0.45 µm). the chromatogram obtained with reference solution (a);
mass of the herbal drug to be examined used to prepare the
Reference solution (b) Dilute 1.0 mL of the internal marker test solution, in grams;
solution to 50.0 mL with the solvent mixture. Dilute 1.0 mL mass of aescin for LG assay HRS used to prepare reference
of this solution to 10.0 mL with the solvent mixture. solution (a), in grams;
p percentage content of aescin, expressed as protoaescigenin, in
Column: aescin for LG assay HRS.
=
- size: l 0.25 m, 0 = 4.6 mm;
- stationary phase: end-capped octadecylsilyl silica gel for STORAGE
chromatography R (5 µm) with a pore size of 30 nm; In an airtight container.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Mobile phase:
- mobile phase A: 0.05 per cent V/V solution of trifiuoroacetic
acid R;
Standardised Horse-chestnut Dry
Time
(rnin)
Mobile phase A
(per cent V/J!)
Mobile phase B
(per cent Vil')
Extract
0 - 15 70 30 (Ph. Bur. monograph 1829)
15 - 25 70 ➔ 65 30 ➔ 35 Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
25 - 35 65 35
DEFINITION
35 - 65 65 ➔ 50 35 ➔ 50
65 - 70 50 ➔ 10 50 ➔ 90
Standardised dry extract produced from Horse-chestnut
(1830).
Flow rate 1.0 mUmin. Content

6.5 per cent to 10.0 per cent of total triterpene glycosides,
expressed as protoaescigenin (C 30H 50O 6 ; Mr 506.7) (dried
Injection 20 µL. extract).
PRODUCTION
aescin for LG assay HRS and the chromatogram obtained with
reference solution (a) to identify peaks A and B due to
procedure using a hydroalcoholic solvent equivalent in
aescm.
strength to ethanol (40-80 per cent VIV).
System suitability:
- the chromatogram obtained with reference solution (a) is CHARACTERS
similar to the chromatogram supplied with aescin for LG Appearance
assay HRS regarding the peaks between those due to Yellowish or yellowish-brown, hygroscopic powder or
methyl salicylate and ibuprofen; agglomerate.
- retention time: methyl salicylate = 11. 5 min to 15. 5 min; IDENTIFICATION
ibuprofen = 34.0 min to 46.0 min; Thin-layer chromatography (2.2.21).
- resolution: minimum 2.0 between peaks A and B due to
aescin in the chromatogram obtained with reference
10 mL of ethanol (70 per cent V/V) R, shake for 2 min and
solution (a);
filter.
2023 Horse-chestnut Preparations IV-293
Reference solution Dissolve 5 mg of aescin for LC assay HRS Time Mobile phase A Mobile phase B
(min) (per cent V/Jl) (per cent V/V)
and 5 mg of sucrose R in 1.0 mL of ethanol
(70 per cent V/V) R. 0 - 15 70 30
15 - 25 70 ➔ 65 30 ➔ 35
25 - 35 65 35
35 - 65 65 ➔ 50 35 ➔ 50
Mobile phase acetic acid R, ethyl acetate R, water R, 65 - 70 50 ➔ 10 50 ➔ 90
propanol R (1.5:30:30:40 VIVIVIV).
Drying In air.
Injection 20 µL.
Detection Treat with anisaldehyde solution R, heat at
100-105 °C for 10 min and examine in daylight.
aescin for LC assay HRS and the chromatogram obtained with
Results See below the sequence of zones present in the reference solution (a) to identify peaks A and B due to
chromatogram obtained with the reference solution and the aescin.
System suitability:
- the chromatogram obtained with reference solution (a) is
similar to the chromatogram supplied with aescin for LC
Top of the plate assay HRS regarding the peaks between those due to
methyl salicylate and ibuprofen;
- retention time: methyl salicylate = 11. 5 min to 15. 5 min;
I or 2 yellowish-violet zones ibuprofen = 34.0 min to 46.0 min;
--- ---
- resolution: minimum 2.0 between peaks A and B due to
aescin in the chromatogram obtained with reference
Aescin: an intense violet-blue zone An intense violet-blue zone (aescin) solution (a);
Sucrose: a brownish-green zone A brownish-green zone (sucrose) - signal-to-noise ratio: minimum 10 for the peak due to
methyl salicylate in the chromatogram obtained with
--- --- reference solution (b).
Reference solution Test solution Integration:
- test solution: integrate the peaks eluting between the peak
due to methyl salicylate and the peak due to ibuprofen;
TESTS integrate the area of the individual peaks or integrate
Loss on drying (2. 8.17) groups of peaks in case of incomplete separation of the
Maximum 6.0 per cent. individual peaks;
ASSAY - reference solution (a): for quantification, integrate as
Liquid chromatography (2.2.29). 1 integral, valley-to-valley, starting from the 1st peak after
Solvent mixture acetonitrile Rl, 0.05 per cent V/V solution of the peak due to methyl salicylate and ending with the last
trifiuoroacetic acid R (40:60 V/V). peak before the peak due to ibuprofen; if a peak is poorly
separated from the peaks due to methyl salicylate or
internal marker solution Dissolve 25.0 mg of methyl
ibuprofen, integrate it separately; use the sum of the peak
salicylate Rand 75.0 mg of ibuprofen R in the solvent mixture
areas for the calculation.
and dilute to 50.0 mL with the solvent mixture. Dilute
5.0 mL of the solution to 25.0 mL with the solvent mixture. Calculate the percentage content of triterpene glycosides,
expressed as protoaescigenin, using the following expression:
Test solution Dissolve O.1000 g of the extract to be
examined in the internal marker solution and dilute to
10.0 mL with the internal marker solution. Sonicate for
10 min and filter through a membrane filter (0.45 µm).
Reference solution (a) Dissolve 20.0 mg of aescin for LC total area (integrated as described above) of the peaks eluting
assay HRS in the internal marker solution and dilute to between the peaks due to methyl salicylate and ibuprofen in the
10.0 mL with the internal marker solution. Sonicate for total area (integrated as described above) of the peaks eluting
10 min and filter through a membrane filter (0.45 µm). between the peaks due to methyl salicylate and ibuprofen in the
Reference solution (b) Dilute 1.0 mL of the internal marker chromatogram obtained with reference solution (a);
solution to 50.0 mL with the solvent mixture. Dilute 1.0 mL solution, in grams;
of this solution to 10.0 mL with the solvent mixture. mass of aescin for LG assay HRS used to prepare reference
Column: solution (a), in grams;
- size: l = 0.25 m, 0 =4.6 mm; p percentage content of aescin, expressed as protoaescigenin, in
aescin for LG assay HRS.
chromatography R (5 µm) with a pore size of 30 nm;
- - - - - - - - - - - - - - - - - - - - - - - Ph Eur
Mobile phase:
- mobile phase A: 0.05 per cent V/V solution of trifiuoroacetic
acid R;
IV-294 Horsetail 2023
Horsetail
(Equisetum Stem, Ph. Bur. monograph 1825)
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or cut, dried sterile aerial parts of Equisetum arvense L.
Content
Minimum 0.3 per cent of total flavonoids, expressed as
isoquercitroside (C 21 H 200 12; M, 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
the base into a sheath, light green or greenish-grey.
The fragments are rough to the touch, brittle and crunchy
when crushed. The main stems are about 1-4.5 mm in
diameter, hollow, jointed at the nodes, which occur at
intervals of about 1.5-4.5 cm; distinct vertical grooves are
present on the intemodes, ranging in number from 4 to 14
or more. The central hollow is less than 50 per cent but
more than 25 per cent of the diameter of the main stem.
Verticils of widely spaced and erect branches, usually simple,
each about 1 mm thick with 3-5 longitudinal, sharp ridges,
50 µm
occur at the nodes; at the end of each ridge is a protruding,
distinct collenchymatic bundle under the epidermis.
The branches are not hollow. The leaves are small, linear, Figure 1825.-1. - Illustration for identification test B of powdered
verticillate at each node, concrescent at the base; they form a herbal drug of equisetum stem
toothed sheath around the stem with the number of teeth
corresponding to the number of grooves on the stem. Each
tooth, often brown, is lanceolate-triangular. The lowest Top of the plate
intemode of each branch is longer than the sheath of the
2 red fluorescent zones
stem to which it belongs.
B. Microscopic examination (2.8.23). The powder is Caffeic acid: a greenish-blue
fluorescent zone
hydrate solution R. The powder shows the following diagnostic 2 greenish-blue fluorescent zones
characters (Figure 1825.-1): fragments of the epidermis
(surface view [B, Cl) composed of rectangular cells with -- --
wavy walls and paracytic stomata (2.8.3) in 2-4 rows, the An orange fluorescent zone
2 subsidiary cells are in the same plane as the epidermis,
Hyperoside: an orange fluorescent
cover the guard cells and show radial ridges; small silica zone
pilulae are scattered on the surface of the subsidiary cells and
appear more frequent at the margin forming a distinct ring 2 greenish-blue fluorescent zones
surrounding the subsidiary cells [C]; 2-celled papillae on the - - --
ridges, less distinct on the main stem [A) but large and
rectangular on the branches, oriented longitudinally [F]; Rutoside: an orange fluorescent zone
in surface view, the epidermis of the main stems consists of Reference solution (b) Test solution
elongated cells [G], the epidermis of the secondary branches
shows the 2-celled papillae which resemble pairs of small
cells separated by a larger cell [DJ; fragments of large-celled TESTS
parenchyma [HJ and groups of long unlignified fibres with Foreign matter (2.8.2)
narrow lumens; small vessels with spiral or annular Maximum 5 per cent.
thickening [E]. Equisetum palustre
C. Examine the chromatograms obtained in the test for Thin-layer chromatography (2.2.27).
Equisetum palustre. Test solution To 1.0 g of the powdered herbal drug (355)
Results See below the sequence of zones present in the (2.9.12) add 10 mL of methanol R. Heat in a water-bath at
chromatograms obtained with reference solution (b) and the 60 °C for 10 min with occasional shaking. Allow to cool.
test solution. Furthermore, other weak fluorescent zones may Filter.
be present in the chromatogram obtained with the test Reference solution (a) To 100.0 mg of Equisetum
solution. palustre HRS add 10 mL of methanol R. Heat in a water-bath
at 60 °C for 10 min with occasional shaking. Allow to cool.
Filter.
Reference solution (b) Dissolve 1.0 mg of caffeic acid R,
2.5 mg of hyperoside Rand 2.5 mg of rutoside trihydrate R in
2023 Houttuynia Herb IV-295
Plate TLC silica gel plate R (2-10 µm). i.e. taking the specific absorbance of isoquercitroside to be
Mobi?e phase anhydrous fomiic acid R, glacial acetic acid R, 500.
water R, ethyl acetate R (7.5:7.5: 18:67 VIVIVIV).
Application 5 µL as bands of 8 mm. m mass of the substance to be examined, in grams.
Drying In a current of cold air for 5 min. - - - - - - - - - - - - - - - - - - - - - - Ph Eur
Detection Heat at I 00 °C for 3 min and treat the still-warm
ester R in methanol R, then treat with a 50 g/L solution of
macrogol 400 R in methanol R; allow to dry in a current of Houttuynia Herb
cold air and examine after 10 min in ultraviolet light at
365 nm. (Ph. Bur. Monograph 2722)
System suitability The chromatogram obtained with PhEw - - - - - - - - - - - - - - - - - - - - - -
reference solution (a) shows 2 greenish fluorescent zones just
DEFINITION
above the line of application.
Whole or fragmented, dried, flowering aerial parts of
Results In the chromatogram obtained with the test Houttuynia cordata Thunb.
solution, any greenish fluorescent zones just above the line of
application are not more intense than the corresponding Content
zones (characteristic of E. palustre L.) in the chromatogram Minimum 0.10 per cent of quercitrin (C 21 H 200 11 ; Mr 448.4)
obtained with reference solution (a). (dried drug).
Maximum 10.0 per cent, determined on 1.000 g of the A. Whole drug. It comprises stems, abundant leaves and
powdered herbal drug (355) (2. 9.12) by drying in an oven at flowering spikes. Stems are compressed-cylindrical, externally
105 °C for 2 h. yellowish-brown, with several deep longitudinal furrows and
distinctly jointed nodes. The leaves (if still attached to stems)
are alternate, with petioles shorter than the leaf blade and
Minimum 3.0 per cent and maximum 15.0 per cent.
enclosed by a downy membranous sheath (stipule) about
Total ash (2.4.16) 1/4 to 1/2 the length of the petiole; leaf blades are rolled or
Minimum 12.0 per cent and maximum 27.0 per cent. crumpled and densely gland-dotted; when intact and
ASSAY expanded, the leaf blades are broadly ovate (3-10 cm long,
Stock solution In a 100 mL round-bottomed flask, introduce 3-5 cm wide) with a cordate base and a narrowly pointed tip
0.800 g of the powdered herbal drug (355) (2.9.12) and add and entire margins; the upper surface is dark yellowish-green
1 mL of a 5 g/L solution of hexamethylenetetramine R, 20 mL or dark brown, the lower surface greyish-green or greyish-
of acetone R and 2 mL of hydrochloric acid Rl. Boil the brown. The flowers are densely packed in yellowish-brown
mixture under a reflux condenser for 30 min. Filter the cylindrical heads (about 1 cm long, 0.3 cm in diameter),
liquid through a plug of absorbent cotton into a flask. occasionally with 4 (rarely 6 or 8) shrivelled brown bracts
Add the absorbent cotton to the residue in the round- (about 1 cm long), persistent at the base. Individual flowers
bottomed flask and extract with 2 quantities, each of 20 mL, are very small (about 1 mm in diameter), always petal-less,
of acetone R, each time boiling under a reflux condenser for comprising of 3 stamens and 3 carpels, each with a recurved
10 min. Allow to cool and filter each extract through a plug style and a semi-inferior ovary. A few fruiting spikes may be
of absorbent cotton into the flask. After cooling, filter the present. Individual fruits are very small (about 2-3 mm long,
combined acetone extracts through a filter paper into a 1 mm in diameter), dry, subglobose capsules with persistent
volumetric flask and dilute to 100.0 mL with acetone R by styles, and are apically dehiscent. Once dehisced, fruits
rinsing the flask and the filter paper. Introduce 20.0 mL of resemble tiny goblets with a 3-toothed lip.
the solution into a separating funnel, add 20 mL of water R Fragmented drug It consists of fragments (up to about 4 cm
and shake the mixture with 1 quantity of 15 mL and then long, 2.5-5 mm in diameter) of stems, leaves and the spike,
3 quantities, each of 10 mL, of ethyl acetate R. Combine the similar to those described above.
ethyl acetate extracts in a separating funnel, wash with B. Microscopic examination (2.8.23). The powder is dark
2 quantities, each of 50 mL, of water R, and filter the brownish-green. Examine under a microscope using chloral
extracts over 10 g of anhydrous sodium sulfate R into a hydrate solution R. The powder shows the following diagnostic
volumetric flask. Dilute to 50.0 mL with ethyl acetate R. characters (Figure 2722.-1): fragments of the upper
Test solution To 10.0 mL of the stock solution add 1 mL of epidermis of the leaf (surface view [BJ), covered by a strongly
aluminium chloride reagent Rand dilute to 25.0 mL with a striated cuticle, composed of polygonal cells [Ba] usually
5 per cent V/V solution of glacial acetic acid R in methanol R. associated with a hypodermis consisting of a layer of large
Compensation solution Dilute 10.0 mL of the stock solution polygonal cells with irregularly thickened walls [Bb];
to 25.0 mL with a 5 per cent V/V solution of glacial acetic fragments of the lower epidermis of the leaf (surface
acid R in methanol R. view [Al), covered by a striated cuticle, with polygonal
cells [Aa], anomocytic stomata [Ab] (2.8.3) and uniseriate
Measure the absorbance (2. 2. 25) of the test solution after
glandular trichomes, with a multicellular stalk and a
30 min, by comparison with the compensation solution at
unicellular head, whole (rare) [Ac] or broken
(frequent) [Cb]; epidermal cells located above the oil cells
expressed as isoquercitroside, using the following expression:
are arranged in a rosette and the cuticle shows radial
Ax 1.25 striations [Ad]; fragments of palisade parenchyma (surface
view [El) with each cell containing a small cluster crystal of
m
calcium oxalate [Ea], sometimes associated with spiral vessels
IV-296 Houttuynia Herb 2023
of the veins [Eb]; fragments of the epidermis of the veins in methanol R or, alternatively, dip the warm plate in a 5 g/L
with elongated cells [C], uniseriate and multicellular covering solution of diphenylboric acid aminoethyl ester R in ethyl
trichomes, with rounded ends and strongly striated acetate R, then in a 50 g/L solution of macrogol 400 R in
walls [Ca], and glandular trichomes; scattered covering methylene ch/.oride R. Allow the plate to dry in air for about
trichomes of the veins [D]; fragments of parenchyma [G] 1 min and examine in ultraviolet light at 366 nm.
consisting of oil cells with orange-yellow contents [Ga]; System suitability Reference solution (c):
fragments of vascular tissue from the stems [F] with large - the chromatogram shows 2 distinct zones in the
vessels [Fb] and pitted fibres [Fa]. middle third; the lower zone (chlorogenic acid) shows
a light blue fluorescence and the upper zone
(hyperoside) shows a yellow fluorescence.
Top of the plate
2 red zones
Quercitrin: a yellow A yellow zone

zone (quercitrin)
-- -- --
A faint yellow zone
Hyperoside: a yellow Hyperoside: a yellow A yellow zone

zone zone (hyperoside)
A blue zone
Chlorogenic acid: a A blue zone

bluish zone
A faint yellow zone
- - -- --
Reference Reference Test solution

solution (c) solution (a)
Figure 2722.-1. - Illustration for identification test B of powdered TESTS

herbal drug of houttuynia herb
C. High performance thin-layer chromatography (2.8.25). Maximum 12.0 per cent, determined on 1.000 g of the
105 °C for 2 h.
Centrifuge and use the supernatant. If necessary, filter Total ash (2.4.16)
through a membrane filter (nominal pore size 0.45 µm). Maximum 15.0 per cent.
Reference solutwn (a) Dissolve 2.0 mg of hyperoside Rand Ash insoluble in hydrochloric acid (2.8.1)
5.0 mg of quercitrin R in methanol Rand dilute to 20.0 mL Maximum 3.0 per cent.
with the same solvent. ASSAY
Reference solutwn (b) Dilute 2.5 mL of reference solution (a) Liquid chromatography (2.2.29).
to 10. 0 mL with methanol R. Test solution To 1.000 g of the powdered herbal drug (355)
Reference solution (c) Dissolve 1 mg of ch/.orogenic acid R and (2.9.12) add 50.0 mL of a mixture of equal volumes of
2 mg of hyperoside R in methanol Rand dilute to 20.0 mL methanol R and water R. Weigh and sonicate for 1 h. Allow
with the same solvent. to cool and weigh again. Compensate for the loss of solvent
Intensity marker Quercitrin. with a mixture of equal volumes of methanol R and water R.
Plate TLC silica gel F254 plate R (2-10 µm). Filter through a membrane filter (nominal pore size
0.45 µm).
Mobile phase acetic acid R, anhydrous formic acid R, water R,
ethyl acetate R (11:11:27:100 VIVIV/V). Reference solutwn (a) Dissolve 5.0 mg of quercitrin CRS in
methanol R and dilute to 10.0 mL with the same solvent.
Reference solution (b) Dissolve 2.5 mg of isoquercitrin R in
Drying In air. reference solution (a) and dilute to 5.0 mL with reference
Detection Heat at 100 °C for 5 min. Spray the warm plate solution (a). Dilute 1.0 mL of this solution to 10.0 mL with
with a 10 g/L solution of diphenylboric acid aminoethyl ester R methanol R.
in methanol R, then with a 50 g/L solution of macrogol 400 R
2023 Iceland Moss IV-297
Column: medullary zone, greenish or brownish algae cells up to 15 µm

- size: l = 0.25 m, 0 = 4.0 mm; in diameter, are embedded; occasionally marginal fragments
- stationary phase: end-capped octadecylsilyl silica gel for of the thallus with tube-like or cylindrical spermogonia, up to
chromatography R (5 µm). about 160 µm wide and up to about 400 µm long.
Mobile phase: C. To 1.0 g of the powdered herbal drug (355) (2.9.12) add
- mobile phase A: water for chromatography R; 10 mL of water R and boil for 2-3 min. The greyish-brown
- mobile phase B: acetonitn"le R; solution forms a gel after cooling which gives a blue colour
with iodine solution Rl.
Time Mobile phase A Mobile phase B D. Thin-layer chromatography (2.2.27).
0 - 10 80--, 70 20--, 30
(2. 9.12) add 5 mL of acetone R and heat in a water-bath
10 - 10.1 70--, 5 30--, 95
under a reflux condenser for 2-3 min. Cool and filter.
JO.I - 20 5 95
Reference solutwn Dissolve 5 mg of anethole R and 5 mg of
caffeu: acid R in 2 mL of acetone R.
Detection Spectrophotometer at 258 run. plate R (2-10 µm)].
Injection 20 µL. Mobile phase acetone R, methanol R, anhydrous formic acid R,
Retention time Isoquercitrin = about 6.5 min; toluene R (5:5:10:80 VIVIV/V).
quercitrin = about 8 min. Application 20 µL [or 4 µL] of the test solution and 10 µL
System suitability Reference solution (b): [or 2 µL] of the reference solution, as bands.
- resolution: minimum 5 between the peaks due to Development Over a path of 10 cm [or 6 cm] .
isoquercitrin and quercitrin.
Drying In air.
Calculate the percentage content of quercitrin using the
following expression: Detection Spray with anisaldehyde solution R. Heat at
100-105 °C for 5-10 min and examine in daylight.
A1xm2xp Results See below the sequence of zones present in the
A2 xm1 x 2 chromatograms obtained with the reference solution and the
area of the peak due to quercitrin in the chromatogram
area of the peak due to quercitrin in the chromatogram
obtained with reference solution (a); Top of the plate
solution, in grams; A greyish-blue zone
m2 mass of quercitrin CRS used to prepare reference solution (a), in
grams; Anethole: a blue or bluish-violet
p percentage content of quercitrin in quercitrin CRS. zone
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur -- --
2 weak greyish-blue zones
A weak greyish-brown or grey zone
Iceland Moss -- - -
A greyish-violet zone
Caffeic acid: a greyish-blue zone
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or cut, dried thallus of Cetraria islandica (L.) Reference solution Test solution
Acharius s.l.
IDENTIFICATION TESTS
A. The thallus, up to 15 cm long, is irregularly dichotomous Foreign matter (2.8.2)
and consists of glabrous, groove-shaped or almost flat, stiff, Maximum 5 per cent.
brittle bands, 0.3-1.5 cm wide and about 0.5 mm thick, Lead (2.4.27)
sometimes serrated with the margin appearing ciliated Maximum 10.0 ppm.
(pycnidia). The upper surface is greenish or greenish-brown,
the lower surface is greyish-white or light brownish and Loss on drying (2.2.32)
shows whitish, depressed spots (so-called respiratory cavities). Maximum 12.0 per cent, determined on 1.000 g of the
On the apices of the terminal lobes, very rarely, there are powdered herbal drug (355) (2. 9.12) by drying in an oven at
brown, discoid apothecia. 105 °C for 2 h.
B. Microscopic examination (2.8.23). The powder is greyish- Total ash (2.4.16)
brown. Examine under a microscope, using chloral hydrate Maximum 3.0 per cent.
solution R. The powder shows the following diagnostic Swelling index (2.8.4)
characters: numerous fragments of the pseudoparenchyma Minimum 4.5, determined on the powdered herbal drug
consisting of narrow-lumened, thick-walled hyphae from the (355) (2.9.12).
marginal layer and wide-lumened hyphae from the adjacent _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
layer consisting of loosely entwined hyphae, in which, in the
IV-298 Indian Sandalwood Oil 2023
Indian Sandalwood Oil Top of the plate
A faint purple zone
DEFINITION
Indian Sandalwood Oil is the non-rectified or rectified
essential oil of the heartwood of Santa/um album L. trees, not
younger than 12 years of age.
The essential oil compHes with the requirements stated under - A purple-brown zone A pink zone
Essential Oils and with the following requirements. (linalyl acetate)
PRODUCTION
Non-rectified Indian Sandalwood Oil is produced by steam A faint purple-brown
distillation. Rectified Indian Sandalwood Oil is obtained by zone (isoeugenyl
sequential steam and vacuum distillation. acetate)
A faint purple-brown
CHARACTERISTICS zone (isoeugenol)
A clear to yellow-coloured liquid with a characteristic sweet - A faint purple-brown

woody odour. zone
A faint purple-brown
IDENTIFICATION
zone
A. Carry out the method for high-performance thin-layer
A purple-brown zone An intense purple-
chromatography, Appendix XI W, using the following (linalool) brown zone
solutions.
(1) Dilute the preparation being examined with sufficient
toluene to produce a solution containing 0.7% v/v of Indian Solution (4) Solution (2) and (3) Solution (1)
Sandalwood Oil. SST Intensity marker Test solution
(2) 0.1 % v/v of linalool and 0.1 % v/v oflinalyl acetate in
toluene. B. In the test for Chromatographic profile the characteristic
(3) Dilute 1 volume of solution (2) to 4 volumes with toluene peaks in the chromatogram obtained with solution (1) are
and use linalyl acetate as the intensity marker. similar in retention times to those in the chromatogram
(4) 0.025% v/v of isoeugenol and 0.05% v/v of isoeugenyl obtained with solution (2).
acetate in toluene. TESTS
CHROMATOGRAPHIC CONDITIONS Santalum spicatum Oil
(a) Use as the coating high performance silica gel 254 (Merck In the test for Chromatographic profile, the amount of (E,E)-
silica gel 60 F 254 plates are suitable). farnesol present is not greater than 1.0%.
(b) Use the mobile phase as described below. The presence of (E,E)-famesol at levels of greater than 1.0%
may suggest the presence of the oil of Santa/um spicatum (oil
(c) Apply 2 µL of each solution, as 8-mm bands.
of Santa/um spicatum typically has an (E,E)-farnesol content
(d) Develop the plate to 7 cm. of around 10% to 12%).
(e) After removal of the plate, dry in a current of air.
Relative density
(f) Dip the plate in freshly prepared anisaldehyde solution, heat 0.968 to 0.983, Appendix VG.
at 100° for 3 minutes and examine under white light.
Refractive index
MOBILE PHASE 1.503 to 1.509, Appendix V E.
5 volumes of ethyl acetate and 95 volumes of toluene. Optical rotation
SYSTEM SUITABILITY - 21° to -12°, Appendix VF.
The test is not valid unless the chromatogram obtained with Solubility in ethanol
solution (4) shows two clearly separated zones under white One volume of the neat oil is soluble, sometimes with
light. opalescence, in 5 volumes of ethanol (70%), Appendix X M.
CONFIRMATION Ester value
The chromatogram obtained with solution (1) shows a faint Not more than 10, Appendix X C.
purple zone at the top of the plate. There is a pink zone in Chromatographic profile
the middle of the plate. There are two faint purple-brown Carry out the method for gas chromatography, Appendix III B
zones in the bottom third of the plate, below which is an using the following solutions. Use the normalisation
intense purple-brown zone. Other faint zones may be procedure.
present. ( 1) Dissolve a quantity of the substance to be examined with
sufficient dichloromethane to produce a solution containing
approximately 0.5% w/v of Indian Sandalwood Oil.
(2) 0.5% w/v of indian sandalwood oil BPCRS in
dichloromethane.
(3) 0.005% w/v of (E,E)-farnesol in dichloromethane.
(4) 0.00025% of (E,E)-farnesol in dichloromethane.
2023 Indigo Plant Leaf IV-299
(a) Use a fused silica column (30 m x 0.25 mm) bonded
Indigo Plant Leaf
with a film (0.25 µm) of polydimethylsiloxane (RTX-1 is (Ph. Bur. monograph 2 72 7)
suitable).
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
(b) Use hydrogen for chromatography as the carrier gas at
2.7 mL per minute. DEFINITION
(c) Use the gradient conditions described below. Whole or fragmented, dried leaf of Persicaria tinctoria (Aiton)
Spach (syn. Polygonum tinctorium Aiton) collected in summer
(d) Use an inlet temperature of 250°.
and autumn.
(e) Use a flame ionisation detector at a temperature of 250°.
Content
(e) Inject 1.0 µL of each solution.
Minimum 0.55 per cent of indigo (C 16H 1oN 2 02; Mr 262.3)
(g) Use a split ratio of 1:50. (dried drug).
(h) Collect the data up to 90 minutes. The re-equilibration
IDENTIFICATION
time may be adjusted depending on equipment.
A. The leaf is usually fragmented into small crumpled pieces.
The whole leaf is elliptical, about 3-8 cm long and 2-5 cm
Time (Minutes) Temperature Comment
wide, bluish-green or dark bluish-green, with an obtuse or
0-10 7Qc isocratic
slightly acute apex and attenuate base, and with entire
10-85 70° ➔ 220' linear gradient
margins, which are slightly ciliated. The adaxial surface is
85-90 220° isocratic
glabrous and the abaxial surface is slightly pubescent along
90-120 220' ➔ 70° linear gradient
the veins. The veins, slightly prominent on the abaxial
surface, are yellow or pale brown. The petiole is short, about
When recorded under the prescribed conditions, the relative 5-10 mm long, flattened, occasionally with membranous
retentions with reference to (E,E)-famesol (retention time ochrea with long ciliate extensions.
about 48.8 minutes) are: sesquiterpene olefins, between 0.11 B. Microscopic examination (2.8.23). The powder is dark
and 0.74; (Z)-o:-santalol, about 0.94; (Z)-exo-o:-bergamotol, bluish-green. Examine under a microscope using chloral
about 0.96; (Z)-epi-~-santalol, about 0.97; (Z)-~-santalol, hydrate solutwn R. The powder shows the following diagnostic
about 0.99 and (Z)-lanceol, about 1.04. characters (Figure 2727.-1): fragments of epidermis covered
SYSTEM SUITABILITY by a fine striated cuticle (surface view [C, E, F]) consisting
The test is not valid unless, in the chromatogram obtained of polygonal cells with straight [Ea, Fa] or slightly sinuous
with solution (2): [Ca] anticlinal walls, glandular trichomes with a bi- or
multicellular stalk and a 2- to 8-celled head [Fe], or their
the resolutwn between the peaks due to (Z)-o:-santalol and
scars [Cb], paracytic stomata (2.8.3), rare on the adaxial
(Z)-exo-o:-bergamotol is at least 3.0;
surface [Fd] and frequent on the abaxial surface [Cc];
the column efficiency calculated using the peak due to (Z)-o:- fragments of mesophyll [A] consisting of palisade
santalol is at least 44,000. parenchyma (surface view [Fb]) and spongy parenchyma
DETERMINATION OF CONTENT (surface view [Ab]) with granular bluish or bluish-black
Use the chromatogram obtained with solution (2) to identify pigmented contents including some idioblastic cells
the peaks in the chromatogram obtained with solution ( 1). containing calcium oxalate cluster crystals [Aa] or calcium
Use the chromatogram obtained with solution (3) to identify oxalate spherocrystals [Ac] of various sizes (10-80 µm);
any peak due to (E,E)-famesol in the chromatogram cluster crystals [BJ or spherocrystals, whole [H] or
obtained with solution (1), as (E,E)-famesol may co-elute fragmented [DJ, isolated; multicellular, multiseriate covering
with another peak. Locate all sesquiterpene olefins between trichomes, 500 µm long or more, sometimes whole [Eb] or,
the solvent peak and 34 minutes. most often, occurring as isolated fragments [G], with cells
with lignified walls; fragments of the vascular bundles of the
LIMITS
petioles or veinsm with spiral or annular vessels Ua]
For non-rectified Indian Sandalwood Oil: accompanied by fibres Ub].
- Total sesquiterpene olefins not more than 7.0%,
- (Z)-o:-santalol, 41.0% to 55.0%,
- (Z)-~-santalol, 16.0% to 24.0% Solution A 20 g/L solution of chloral hydrate R in methylene
- (Z)-lanceol, not more than 5.0% chloride R.
- (E,E)-famesol, not more than 1.0%. Test solutwn To 0.1 g of the powdered herbal drug (355)
For rectified Indian Sandalwood Oil: (2. 9.12) add 45 mL of solution A and sonicate at 30 °C for
- Total sesquiterpene olefins not more than 3.5%, 30 min. Allow to cool, centrifuge, dilute the supernatant to
- (Z)-o:-santalol, 41.0% to 55.0%, 50.0 mL with solution A and mix thoroughly. Filter through
- (Z)-~-santalol, 16.0% to 24.0% a membrane filter (nominal pore size 0.45 µm).
- (Z)-lanceol, not more than 5.0% Reference solutwn To 1 mg of indigo Rand 1 mg of
- (E,E)-famesol, not more than 1.0%. indirubin R add 9.5 mL of solution A and sonicate at 30 °C
Disregard peaks with a percentage area less than 0.05%. for 30 min. Allow to cool, dilute to 10.0 mL with solution A
and mix thoroughly. Filter through a membrane filter
LABELLING (nominal pore size 0.45 µm).
The labelling states whether the oil is rectified or non- Plate TLC silica gel F254 plate R (2-10 µm).
rectified.
Mobile phase acetone R, methylene chloride R (5:95 V/V).
Development Immediately, over a path of 6 cm.
IV-300 Ipecacuanha 2023
Solution A 20 g/L solution of chloral hydrate R in methylene

chloride R.
Test solution To 50.0 mg of the powdered herbal drug (355)
(2. 9.12) add 45 mL of solution A and sonicate at 30 °C for
30 min. Allow to cool, centrifuge, dilute the supernatant to
50.0 mL with solution A and mix thoroughly. Filter through
a membrane filter (nominal pore size 0.45 µm).
Reference solution To 5.0 mg of indigo CRS and 5.0 mg of
indirubin R add 950 mL of solution A and sonicate at 30 °C
for 30 min. Allow to cool, dilute to 1000.0 mL with
solution A and mix thoroughly. Filter through a membrane
filter (nominal pore size 0.45 µm).
Column:
- size: l = 0.25 m, 0 = 4.0 mm;
Mobile phase 70 per cent V/V solution of methanol R.
Injection 5 µL.
Run time 2.5 times the retention time of indigo.
Relative retention With reference to indigo (retention
time= about 10 min): indirubin = about 1.7.
- resolution: minimum 4.0 between the peaks due to indigo
and indirubin.
Calculate the percentage content of indigo using the
Figure 2727 .-1. - Illustration for identification test B of powdered
A 1 xm 2 xpx0.05
herbal drug of indigo plant leaf
A 2 xm 1
Drying Immediately, in air.

Detection Examine immediately in daylight. area of the peak due to indigo in the chromatogram obtained
Results See below the sequence of zones present in the with the test solution;
area of the peak due to indigo in the chromatogram obtained
test solution. Furthermore, other faint zones may be present mass of the herbal drug to be examined used to prepare the test
in the chromatogram obtained with the test solution. solution, in grams;
mass of indigo CRS used to prepare the reference solution, in
grams;
Top of the plate
p percentage content of indigo in indigo CRS.
A light blue zone _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
-- --
Indigo: a blue zone A blue zone (indigo)
-- -- lpecacuanha
Indirubin: a red zone A red zone (indirubin)
(Ipecacuanha Root, Ph. Bur. monograph 0094)
Preparations
Reference solution Test solution Prepared lpecacuanha
lpecacuanha Liquid Extract
TESTS Standardised lpecacuanha Liquid Extract
Loss on drying (2.2.32) Standardised lpecacuanha Tincture
Maximum 7 .0 per cent, determined on 1.000 g of the Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
105 °C for 2 h. DEFINITION
Fragmented and dried underground organs of Carapichea
Total ash (2.4.16) ipecacuanha (Brot.) L. Andersson (syn. Cephaelis ipecacuanha
Maximum 15.0 per cent. (Brot.) A. Rich.; Cephaelis acuminata H. Karst.) from Mato
Ash insoluble in hydrochloric acid (2.8.1) Grosso or Costa Rica. The principal alkaloids are emetine
Maximum 7 .0 per cent. and cephaeline.
ASSAY Content
Liquid chromatography (2.2.29). Minimum 2.0 per cent of total alkaloids, expressed as
emetine (C 29 H 4 oN2 0 4 ; Mr 480.6) (dried drug).
2023 lpecacuanha IV-301
IDENTIFICATION
A. Mato Grosso ipecacuanha: the root occurs as somewhat
tortuous pieces, dark reddish-brown or very dark brown,
seldom more than 15 cm long or 6 mm thick, closely
annulated externally, having rounded ridges completely
encircling the root; the fracture is short in the bark and
splintery in the wood. The transversely cut surface shows a
wide greyish bark and a small uniformly dense wood.
The rhizome occurs as short lengths usually attached to
roots, cylindrical, up to 2 mm in diameter, finely wrinkled
longitudinally and with pith occupying approximately one-
sixth of the whole diameter.
Costa Rica ipecacuanha The root in general resembles the
root of Maw Grosso ipecacuanha, but differs in the following
particulars: it is often up to 9 mm thick; the external surface
is greyish-brown or reddish-brown with transverse ridges at
intervals of usually 1-3 mm, the ridges being about 0.5-1 mm
wide, extending about half-way round the circumference and
fading at the extremities into the general surface level.
grey or yellowish-brown. Examine under a microscope using
diagnostic characters (Figure 0094.-1): fragments of
parenchyma [G] with ovoid cells [Ga]; raphides of calcium
oxalate up to 80 µm in length either in bundles [Be] or
scattered throughout the powder [Gb]; fragments [E] of
tracheids and vessels usually 10-20 µm in diameter, with
bordered pits [Eb] accompanied by ligneous parenchyma
with longitudinally elongated rectangular cells [Ea]; larger
vessels and sclereids from the rhizome [D]; fragments of
dermal tissue (transverse section [BJ) with reddish-brown Figure 0094. -1. - Illustratwn for identificatwn test B of powdered
cork [Ba] and phelloderm [Bb], with some cells containing herbal drng of ipecacuanha root
raphides of calcium oxalate [Be]; large parenchymatous cells
with slightly thickened and pitted walls, from the pith of the
rhizome [C]; a few fragments of fibres with moderately Top of the plate
thickened and slightly pitted walls from the xylem, isolated
-- -- --
[HJ or associated with vessels [F]. Examine under a
microscope using a 50 per cent V/V solution of glycerol R. Emetine: a blue A blue fluorescent zone A blue fluorescent zone
fluorescent zone (emetine) (emetine)
The powder shows simple [Aa] or 2- to 10-compound [Ab]
starch granules contained in parenchymatous cells [A], the Cephaeline: a blue A blue fluorescent zone A faint blue fluorescent
simple granules being up to 15 µm in diameter in Costa Rica fluorescent zone (cephaeline) zone (cephaeline)
ipecacuanha and up to 22 µm in diameter in Mato Grosso
-- -- - -
ipecacuanha.
C. Thin-layer chromatography (2.2.27). Test solution (Costa Test solution (Mato
Reference solution
Ricaipecacuanha) Grosso ipecacuanha)
(2.9.12) in a test-tube add 0.05 mL of concentrated
ammonia R and 5 mL of ether R and stir the mixture Detection B Treat with a 5 g/L solution of iodine R in ethanol
vigorously with a glass rod. Allow to stand for 30 min and (96 per cent) R, heat at 60 °C for 10 min and examine in
filter. ultraviolet light at 365 nm.
Reference solutwn Dissolve 2.5 mg of emetine System suitability Reference solution:
hydrochloride CRS and 3 mg of cephaeline hydrochloride CRS in - the blue fluorescent zone due to cephaeline and the
methanol R and dilute to 20 mL with the same solvent. yellow fluorescent zone due to emetine are clearly
Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel separated.
F2 54 plate R (2-10 µm)]. Results B See below the sequence of zones present in the
Mobile phase concentrated ammonia R, methanol R, ethyl chromatograms obtained with the reference solution and the
acetate R, toluene R (2:15:18:65 VIVIVIV). test solution. Furthermore, other faint fluorescent zones may
Application 10 µL [or 5 µL] as bands of 10 mm [or 8 mm]. be present in the chromatogram obtained with the test
solution.
Drying In air.
solution.
IV-302 lpecacuanha Preparations 2023
Top of the plate diagnostic characters (Figure 0093.-1): fragments of

parenchyma [G] with ovoid cells [Ga]; raphides of calcium
-- -- -- oxalate up to 80 µm in length either in bundles [Be] or
Emetine: a yellow A yellow fluorescent A yellow fluorescent scattered throughout the powder [Gb]; fragments [E] of
fluorescent zone zone (emetine) zone (emetine) tracheids and vessels usually 10-20 µm in diameter, with
Cephaeline: a blue A blue fluorescent zone A blue fluorescent zone
bordered pits [Eb] accompanied by ligneous parenchyma
fluorescent zone (cephaeline) (cephaeline) with longitudinally elongated rectangular cells [Ea]; larger
vessels and sclereids from the rhizome [DJ; fragments of
-- -- -- dermal tissue (transverse section [BJ) with reddish-brown
Test solution (Costa Test solution (Mato cork [Ba] and phelloderm [Bb], with some cells containing
Reference solution
Ricaipecacuanha) Grosso ipecacuanha) raphides of calcium oxalate [Be]; large parenchymatous cells
with slightly thickened and pitted walls, from the pith of the
rhizome [CJ; a few fragments of fibres with moderately
TESTS thickened and slightly pitted walls from the xylem, isolated
Loss on drying (2.2.32) [HJ or associated with vessels [F] . Examine under a
Maximum 10.0 per cent, determined on 1.000 g of the microscope using a 50 per cent V/V solution of glycerol R.
powdered herbal drug (180) (2.9.12) by drying in an oven at The powder shows simple [Aa] or 2- to 10-compound [Ab]
105 °C. starch granules contained in parenchymatous cells [A], the
Total ash (2.4.16) simple granules being up to 15 µm in diameter in Costa Rica
Maximum 5.0 per cent. ipecacuanha and up to 22 µm in diameter in Mato Grosso
Ash insoluble in hydrochloric acid (2.8.1) ipecacuanha.
ASSAY
Place 7 .5 g of the powdered herbal drug (180) (2. 9.12) in a
dry flask, add 100 mL of ether R and shake for 5 min.
Add 5 mL of dilute ammonia Rl, shake for 1 h, add 5 mL of
water Rand shake vigorously. Decant the ether layer into a
flask through a plug of cotton. Wash the residue in the flask
with 2 quantities, each of 25 mL, of ether R, decanting each
portion through the same plug of cotton. Combine the ether
solutions and eliminate the ether by distillation. Dissolve the
residue in 2 mL of ethanol (90 per cent V/V) R, evaporate to
dryness and heat at 100 °C for 5 min. Dissolve the residue in
5 mL of previously neutralised ethanol (90 per cent V/V) R,
warming on a water-bath. Add 15.0 mL of 0.1 M hydrochloric
acid and titrate the excess acid with 0.1 M sodium hydroxide
using 0.5 mL of methyl red mixed solution Ras indicator.
1 mL of 0.1 M hydrochloric acid is equivalent to 24.03 mg
of total alkaloids, expressed as emetine.
STORAGE
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Prepared lpecacuanha
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
lpecacuanha root (0094) powdered (180) (2.9.12) and Figure 0093.-1. - Illustration for identification test A of prepared
adjusted, if necessary, by the addition of powdered lactose or ipecacuanha
ipecacuanha root powder with a lower alkaloidal content.
Content
1. 9 per cent to 2.1 per cent of total alkaloids, expressed as Test solution To 0.1 g of the herbal drug to be examined in
emetine (C 29 H 40 N 2 O4; Mr 480.6) (dried drug). a test-tube add 0.05 mL of concentrated ammonia Rand 5 mL
of ether R and stir the mixture vigorously with a glass rod.
CHARACTERS Allow to stand for 30 min and filter.
Appearance Reference solution Dissolve 2.5 mg of emetine
Light grey or yellowish-brown powder. hydrochloride CRS and 3 mg of cephaeline hydrochloride CRS in
IDENTIFICATION methanol R and dilute to 20 mL with the same solvent.
A. Microscopic examination (2.8.23). The powder is light Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silua gel
grey or yellowish-brown. Examine under a microscope using F254plate R (2-10 µm)].
2023 Ipecacuanha Preparations IV-303
Mobile phase concentrated ammonia R, methanol R, ethyl 100 °C for 5 min. Dissolve the residue in 5 mL of previously
acetate R, toluene R (2:15:18:65 V/V/V/V). neutralised ethanol (90 per cent V/V) R, warming on a water-
Application 10 µL [or 5 µL] as bands of 10 mm [or 8 mm]. bath. Add 15.0 mL of 0.1 M hydrochloric acid and titrate the
excess acid with 0.1 M sodium hydroxide using 0.5 mL of
methyl red mixed solution R as indicator.
Drying In air.
1 mL of 0.1 M hydrochloric acid is equivalent to 24.03 mg of
Detection A Examine in ultraviolet light at 365 nm. total alkaloids, expressed as emetine.
chromatograms obtained with the reference solution and the STORAGE
test solution. Furthermore, other faint fluorescent zones may In an airtight container.
solution.
Top of the plate

lpecacuanha Liquid Extract
- - --
DEFINITION
Emeline: a blue fluorescent zone A blue fluorescent zone (emetine)
Ipecacuanha Liquid Extract is prepared from Ipecacuanha by
Cephaeline: a blue fluorescent zone A blue or faint blue fluorescent zone a method stated under the general monograph for Extracts.
(cephaeline) It contains not less than 1.90% and not more than 2.10% of
-- -- total alkaloids, calculated as emetine, C 29 H 40 N 2 0 4 •
EXTEMPORANEOUS PREPARATION
Detection B Treat with a 5 g/L solution of iodine R in ethanol Prepare by extracting Ipecacuanha with Ethanol
(96 per cent) R, heat at 60 °C for 10 min and examine in (80 per cent) according to the following formula and
ultraviolet light at 365 nm. directions.
- the blue fluorescent zone due to cephaeline and the Ipecacuanha in fine powder 1000 g
Ethanol (80 per cent) A sufficient quantity
yellow fluorescent zone due to emetine are clearly
separated.
Exhaust the Ipecacuanha by percolation, Appendix XI F, with
Results B See below the sequence of zones present in the Ethanol (80 per cent), reserving the first 750 mL of the
chromatograms obtained with the reference solution and the percolate. Remove the ethanol from the remainder of the
test solution. Furthermore, other faint fluorescent zones may percolate by evaporation under reduced pressure at a
be present in the chromatogram obtained with the test temperature not exceeding 60° and dissolve the residual
solution. extract in the reserved portion. Determine the proportion of
alkaloids in the liquid thus obtained by the Assay described
Top of the plate below. To the remainder of the liquid add sufficient Ethanol
(80%) to produce an Ipecacuanha Liquid Extract containing
-- -- 2 % w/v of total alkaloids calculated as emetine. Allow to
Emeline: a yellow fluorescent zone A yellow fluorescent zone (emetine) stand for not less than 24 hours; filter.
Cephaeline: a blue fluorescent zone A blue fluorescent zone (cephaeline) The extract complies with the requirements stated under Extracts
and with the following requirements.
- - - -
TESTS
Reference solution Test solution Ethanol content
TESTS Relative density
Loss on drying (2.2.32) 0.910 to 0.960, Appendix VG.
Maximum 5.0 per cent, determined on 1.000 g of the herbal Dry residue
drug by drying in an oven at 105 °C. The requirement for Dry residue does not apply to
Total ash (2.4.16) Ipecacuanha Liquid Extract.
Maximum 5.0 per cent. ASSAY
Ash insoluble in hydrochloric acid (2.8.1) To 5 mL in a separating funnel add 20 mL of water, 5 mL of
Maximum 3.0 per cent. 1M suljuric acid and 10 mL of chloroform and shake well.
ASSAY Transfer the chloroform extract to a second separating funnel
containing a mixture of 4 mL of ethanol (96%) and 20 mL of
Place 7.5 g of the herbal drug in a dry flask, add 100 mL of
0.05M sulfuric acid, shake, allow to separate and discard the
ether Rand shake for 5 min. Add 5 mL of dilute ammonia Rl,
chloroform layer. Continue the extraction of the liquid in the
shake for 1 h, add 5 mL of water Rand shake vigorously.
first separating funnel with two further 10 mL quantities of
Decant the ether layer into a flask through a plug of cotton.
chloroform, transferring the chloroform solution each time to
Wash the residue in the flask with 2 quantities, each of
the second separating funnel and washing as before. Transfer
25 mL, of ether R, decanting each portion through the same
the acidic liquid from the second separating funnel to the
plug of cotton. Combine the ether solutions and eliminate
first separating funnel, make distinctly alkaline with
the ether by distillation. Dissolve the residue in 2 mL of
5M ammonia and shake with successive quantities of
ethanol (90 per cent V/V) R, evaporate to dryness and heat at
IV-304 Ipecacuanha Preparations 2023
chloroform until complete extraction of the alkaloids is effected, Top of the plate
Appendix XI G, washing each chloroform solution with the
same 10 mL of water contained in a third separating funnel. -- --
Remove the chloroform, add to the residue 2 mL of ethanol Emetine: a blue fluorescent zone A blue fluorescent zone (emetine)
(96%), evaporate to dryness and dry for 5 minutes at 80° in
Cephaeline: a blue fluorescent zone A blue or faint blue fluorescent zone
a current of air. Dissolve the residue in 2 mL of ethanol (cephaeline)
(96%), previously neutralised to methyl red solution, add
10 mL of 0.05M sulfuric acid VS and titrate with 0.lM sodium -- --
hydroxide VS using methyl red mixed solution as indicator. Reference solution Test solution
Each mL of 0.05M sulfuric acid VS is equivalent to 24.03 mg
of total alkaloids, calculated as emetine, C 29H 40N 2 O4.
Detection B Treat with a 5 g/L solution of iodine R in ethanol
(96 per cent) R, heat at 60 °C for 10 min and examine in
Standardised lpecacuanha Liquid - the blue fluorescent zone due to cephaeline and the
Extract yellow fluorescent zone due to emetine are clearly
separated.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION test solution. Furthermore, other faint fluorescent zones may
Standardised liquid extract produced from Ipecacuanha root be present in the chromatogram obtained with .the test
(0094). solution.
Content
1.80 per cent to 2.20 per cent of total alkaloids, expressed as Top of the plate
emetine (C 29H 40N 2 0 4 ; Mr 480.6). -- --

PRODUCTION Emeline: a yellow fluorescent zone A yellow fluorescent zone (emetine)
procedure using ethanol (60-80 per cent V/V). Cephaeline: a blue fluorescent zone A blue fluorescent zone (cephaeline)
CHARACTERS -- --
Appearance Reference solution Test solution
Dark brown liquid.
IDENTIFICATION
TESTS
Ethanol (2.9.10)
Test solution Dilute 5.0 mL of the extract to be examined to 9 5 per cent to 105 per cent of the quantity stated on the
50 mL with ethanol (70 per cent V/V) R. To 2.0 mL of this label.
solution add 2 mL of water R and O.1 mL of concentrated
ammonia R. Add 10 mL of ether R and shake. Separate the ASSAY
upper layer, dry it over about 2 g of anhydrous sodium Dilute 1.00 g of the extract to be examined to 10 mL with
sulfate R and filter. ethanol (70 per cent V/V) R and transfer to a chromatography
column about 0.2 m long and about 15 mm in internal
Reference solution Dissolve 2.5 mg of emetine
diameter, containing 8 g of basic aluminium oxide R. After
hydrochloride CRS and 3 mg of cephaeline hydrochloride CRS in
infiltration into the aluminium oxide layer, rinse the internal
methanol R and dilute to 10 mL with the same solvent.
wall of the column with 3 quantities, each of 2 mL, of
Plate TLC silica gel F 254 plate R (5-40 µm) [or TLC silica ethanol (70 per cent V/V) R. Elute in portions, with 40 mL of
gel F 254 plate R (2-10 µm)]. ethanol (70 per cent V/V) R. Avoid disturbance or drying of
Mobile phase concentrated ammonia R, methanol R, ethyl the surface of the aluminium oxide layer. Collect the whole
acetate R, toluene R (2:15:18:65 VIVIVIV). of the eluate. Evaporate the eluate on a water-bath to about
Application 10 µL [or 5 µL] as bands of 10 mm [or 8 mm]. 10 mL. Allow to cool. Add 10.0 mL of 0.02 M hydrochloric
Development Over a path of 10 cm [or 6 cm]. acid and 20 mL of carbon dioxide-free water R. Titrate the
excess acid with 0.02 M sodium hydroxide using 0.15 mL of
Drying In air.
methyl red mixed solution R as indicator.
Perform a blank assay replacing the extract to be examined
Results A See below the sequence of zones present in the with 10.0 mL of ethanol of the strength stated on the label.
1 mL of 0. 02 M hydrochloric acid is equivalent to 4.807 mg of
total alkaloids, expressed as emetine.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
solution.
2023 lpecacuanha Preparations IV-305

Standardised lpecacuanha solution.
Tincture
(Ph. Bur. monograph 1530) Top of the plate
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ -- --
DEFINITION Emeline: a yellow fluorescent zone A yellow fluorescent zone (emetine)

Tincture produced from Ipecacuanha root (0094). Cephaeline: a blue fluorescent zone A blue fluorescent zone (cephaeline)
Content
- - --
0.18 per cent mlm to 0.22 per cent m/m of total alkaloids,
expressed as emetine (C 29 H 40N 2 O 4 ; Mr 480.6). Reference solution Test solution
PRODUCTION
The tincture is produced from the herbal drug by a suitable TESTS
procedure using ethanol (70 per cent V/V). Ethanol (2.9.10)
CHARACTERS 95 per cent to 1OS per cent of the quantity stated on the
Appearance label.
Yellowish-brown liquid. ASSAY
IDENTIFICATION Transfer 10.00 g of the tincture to be examined to a
Thin-layer chromatography (2.2.27). chromatography column about 0.2 m long and about 15 mm
in internal diameter, containing 8 g of basic aluminium
Test solution To 2.0 mL of the tincture to be examined add
oxide R. After infiltration into the aluminium oxide layer,
2 mL of water Rand 0.1 mL of concentrated ammonia R.
rinse the internal wall of the column with 3 quantities, each
Add 10 mL of ether Rand shake. Separate the ether layer,
of 2 mL, of ethanol (70 per cent V/V) R. Elute in portions,
dry it over about 2 g of anhydrous sodium sulfate R and filter.
with 40 mL of ethanol (70 per cent V/V) R. Avoid disturbance
Reference solution Dissolve 2.5 mg of emetine or drying of the surface of the aluminium oxide layer. Collect
hydrochloride CRS and 3 mg of cephaeline hydrochloride CRS in the whole of the eluate. Evaporate the eluate on a water-bath
methanol R and dilute to 10 mL with the same solvent. to about 10 mL. Allow to cool. Add 10.0 mL of
Plate TLC silica gel F254 plate R (S-40 µm) [or TLC sili,ca gel 0.02 M hydrochloric acid and 20 mL of carbon dioxide-free
F254 plate R (2-10 µm)]. water R. Titrate the excess acid with 0. 02 M sodium hydroxide
Mobile phase concentrated ammonia R, methanol R, ethyl using 0.15 mL of methyl red mixed solution Ras indicator.
acetate R, toluene R (2:15:18:65 V/V/V/V). Perform a blank assay replacing the tincture to be examined
Application 10 µL [or S µL] as bands of 10 mm [or 8 mm]. with 10.0 mL of ethanol of the strength stated on the label.
Development Over a path of 10 cm [or 6 cm]. 1 mL of 0.02 M hydrochl.oric acid is equivalent to 4.807 mg of
Drying In air. total alkaloids, expressed as emetine.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
be present in the chromatogram obtained with the test Paediatric lpecacuanha Emetic Mixture
solution. Paediatric lpecacuanha Emetic; Paediatric lpecacuanha
Emetic Oral Solution
Top of the plate DEFINITION
-- --
Paediatric lpecacuanha Emetic Mixture is an oral solution.
Emetine: a blue fluorescent zone A blue fluorescent zone (emetine) Ipecacuanha Liquid Extract 70mL
Hydrochloric Acid 2.5 mL
Cephaeline: a blue fluorescent zone A blue or faint blue fluorescent zone
Glycerol 100 mL
(cephaeline)
Syrup Sufficient to produce 1000 mL
-- --

The mixture complies with the requirements stated under Oral
Liquids and with the following requirements.
Content of total alkaloids
Detection B Treat with a S g/L solution of iodine R in ethanol
0.12 to 0.16% w/v, calculated as emetine, C 29 H 40N 2 O 4 •
(96 per cent) R, heat at 60 °C for 10 min and examine in
ultraviolet light at 365 nm. IDENTIFICATION
System suitability Reference solution: Carry out the method for thin-layer chromatography,
- the blue fluorescent zone due to cephaeline and the Appendix III A, using the following solutions.
yellow fluorescent zone due to emetine are clearly (1) Mix S mL with 10 mL of lm sulfuric acid, shake with two
separated. 10 mL quantities of chloroform and discard the chloroform.
Results B See below the sequence of zones present in the Add sufficient Sm ammonia to make the aqueous solution
chromatograms obtained with the reference solution and the distinctly alkaline to litmus paper, extract with four 10 mL
test solution. Furthermore, other faint fluorescent zones may quantities of chloroform, evaporate the combined extracts to
dryness, cool the residue and dissolve it in 0.5 mL of ethanol
(96%).
IV-306 Isatis Root 2023
(2) 0.1 % w/v of cephaeline hydrochloride EPCRS in ethanol bark, darkest near the cambium, sometimes appearing as a
(96%). thin dark line, and a yellow or brown wood.
(3) 0.1 % w/v of emetine hydrochloride EPCRS in ethanol Fragmented drug It occurs as transverse or oblique slices,
(96%). rounded or elliptical, or as short, thin, cylindrical pieces,
CHROMATOGRAPHIC CONDITIONS 0.3-1.3 cm in diameter. It is externally greyish-yellow or
brownish-yellow, wrinkled longitudinally, and transverse
(a) Use as the coating silica gel G.
lenticels, rootlets and rootlet scars are sometimes visible.
(b) Use the mobile phase as described below. The transversely cut surface shows a yellowish-white, brown
(c) Apply 2 µL of each solution. or dark brown bark, darkest near the cambium, sometimes
(d) Develop the plate to 15 cm. appearing as a thin dark line, and a yellow or brown wood.
(e) After removal of the plate, dry it at 105° to 110° for B. Microscopic examination (2.8.23). The powder is whitish-
30 minutes, allow to cool and spray with dilute potassium yellow, yellow or light brown. Examine under a microscope
iodobismuthate solutwn. using chloral hydrate solutwn R. The powder shows the
following diagnostic characters (Figure 2566.-1): fragments of
MOBILE PHASE
cork consisting of thin-walled cells (surface view [A],
10 volumes of diethylamine and 90 volumes of chloroform. transverse section [BJ); fragments of xylem [D] consisting of
CONFIRMATION reticulate, pitted or, more rarely, spiral vessels [Da] included
The principal spots in the chromatogram obtained with in thin-walled parenchyma cells [Db]; groups of lignified
solution (1) correspond in colour and position to the spots in xylem fibres, occasionally accompanying vessels, with sparse
the chromatograms obtained with solutions (2) and (3). irregularly-positioned pits may be present; thin-walled
Disregard any secondary spots. parenchyma cells (longitudinal section [E], transverse section
[F]); groups of greenish-yellow sclereids with thick, pined
ASSAY walls embedded in parenchyma tissue may be present.
To 25 mL in a separating funnel add 20 mL of water and Examine under a microscope using a 50 per cent V/V
5 mL of lM sulfuric acid, shake with three 10 mL quantities solution of glycerol R. The powder shows abundant, single or
of chloroform and wash each chloroform extract with a compound (mostly 2, or less frequently, 3 or 4) starch
mixture of 20 mL of 0.05M suljuric acid and 4 mL of ethanol granules, 1.5-18 µm in diameter, free [C] or included in
(96%) contained in a second separating funnel. Transfer the parenchyma [G], with a punctiform, slit-shaped or V-shaped
acid-ethanol mixture from the second separating funnel to hilum.
the first, make the combined liquids distinctly alkaline to
litmus paper with 5M ammonia and extract with successive
quantities of chloroform until complete extractwn of the
alkaloids is effected, Appendix XI G. Wash each chloroform
extract with the same 10 mL of water, combine the
chloroform extracts, evaporate the chloroform, add 2 mL of
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 solutwn, add 10 mL of 0.01M sulfuric acid VS
and titrate the excess of acid with 0. 02M sodium hydroxide VS
using methyl red solution as indicator. Each mL of 0.01M
sulfuric acid VS is equivalent to 4.806 mg of C 29 H 40N 2 0 4 •
lsatis Root
~~---------------------
DEFINITION
Whole or fragmented, dried root of Isatis tinctoria L.
(I. indigotica Fortune ex Lindl.) collected in autumn.
Content
Minimum 1.0 per cent of arginine (C 6H 14N 4 0 2 ; Mr 174.2) Figure 2566.-1. - Illustratwnfor identificatwn test B of powdered
(dried drug). herbal drug of isatis root
IDENTIFICATION
A. W'hole drug. It is cylindrical, slightly tortuous, 8-22 cm
long, 1.5 cm in diameter, externally greyish-yellow or Test solutwn 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
brownish-yellow, wrinkled longitudinally and lenticellate
sonicate for 10 min. Centrifuge and use the supernatant.
transversally, with rootlets or rootlet scars. The root crown is
slightly expanded, exhibiting dark green or dark brown Reference solutwn Dissolve 4 mg of arginine R and 4 mg of
petiole bases arranged in whorls, and dense tubercles. cysteine hydrochloride R in 1 mL of ethanol
The texture is compact and easily broken. The transversely (70 per cent V/V) R.
cut surface shows a yellowish-white, brown or dark brown Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel
F254 plate R (2-10 µm)].
2023 Ispaghula Husk IV-307
Mobile phase anhydrous formic acid R, water R, acetonitrile R Detection Evaporative light-scattering detector; the following
(2:8:30 V/V/V). settings have been found to be suitable; if the detector has
Application 4 µL as bands of 10 mm [or 8 mm]. different setting parameters, adjust the detector settings so as
to comply with the system suitability criterion for the signal-
Development Over a path of 8.5 cm [or 6 cm].
to-noise ratio:
Drying In air. - carrier gas: nitrogen R;
Detection Expose to concentrated ammonia R vapour for - pressure: 330 kPa;
5 min, treat with ninhydrin solution R4, then heat at 120 °C - evaporator temperature: 80 °C.
for 3 min. Injection 10 µL.
Results See below the sequence of zones present in 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
-- - -
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.
Calculate the percentage content of arginine using the
Arginine: a brown zone A brown zone (arginine)
A faint brown zone
10'1
mx 10
A logarithm of the concentration of arginine in the test solution,

TESTS determined from the calibration curve;
Loss on drying (2.2.32) m mass of the herbal drug to be examined used to prepare the test
Maximum 9.0 per cent, determined on 1.000 g of the solution, in grams.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
105 °C for 2 h.
Total ash (2.4.16)
lspaghula Husk
Liquid chromatography (2.2.29). Preparations
Test solution To 0.100 g of the powdered herbal drug (355) lspaghula Husk Oral Powder
(2. 9.12) add 20 mL of ethanol (70 per cent V!V) R, sonicate lspaghula Husk Granules
for 20 min, filter, and evaporate the filtrate to dryness.
lspaghula Husk Effervescent Granules
Dissolve the residue in ethanol (70 per cent V/V) R and dilute
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
to 10.0 mL with the same solvent.
Reference solution (a) Dissolve 25.0 mg of arginine CRS in DEFINITION
ethanol (70 per cent V/V) R and dilute to 50.0 mL with the Episperm and collapsed adjacent layers removed from the
same solvent. seeds of Plantago ovata Forssk. (P. ispaghula Roxb.).
Reference solution (b) Dissolve 3.0 mg of cysteine IDENTIFICATION
hydrochloride R in 6.0 mL of reference solution (a) and dilute A. The husk consists of pinkish-beige fragments or flakes up
to 10.0 mL with ethanol (70 per cent V/V) R. to about 2 mm long and 1 mm wide, some showing a light
Reference solutions (c), (d), (e), (j), (g), (h) Dilute reference brown spot corresponding to the location of the embryo
solution (a) to obtain 6 reference solutions of arginine, the before it was removed from the seed.
concentrations of which span the expected value in the test B. Microscopic examination (2.8.23). The powder is pale
solution. yellow. Examine under a microscope using lactic reagent R.
Column: The powder shows the following diagnostic characters:
- size: l = 0.15 m, 0 = 4.6 mm; mainly fragments of the episperm with polygonal cells filled
- stationary phase: base-deactivated end-capped octadecylsilyl with mucilage; fragments of the inner layers of the testa with
silica gel for chromatography R (3 µm); brownish thin-walled cells often associated with the outer
- temperature: 30 °C. layers of the endosperm. Examine under a microscope using
Mobile phase trifiuoroacetic acid R, water for chromatography R a 50 per cent V/V solution of glycerol R. The powder shows
(0.2:99.8 V/V). occasional starch granules, single or in groups of 2-4,
Flow rate 0.2 mL/min. measuring 3-25 µm in diameter.
IV-308 Ispaghula Preparations 2023
C. Thin-layer chromatography (2.2.27). TESTS

Test solution To 10 mg of the powdered herbal drug (355) Swelling index
(2. 9.12) in a thick-walled centrifuge tube, add 2 mL of a Not less than 40, Appendix XI C. Use a quantity of the
230 g/L solution of trijluoroacetic acid R and shake vigorously. granules containing 1.0 g of Ispaghula Husk and a 100-mL
Stopper the test tube and heat at 120 °C for 1 h. Centrifuge ground-glass-stoppered cylinder graduated in 1 mL divisions.
the hydrolysate, transfer the clear supernatant into a 50 mL Loss on drying
flask, add 10 mL of water R and evaporate to dryness under When dried at 100° to 105°, loses not more than 12.0% of
reduced pressure. Take up the residue in 10 mL of water R its weight. Use 1 g.
and evaporate again to dryness under reduced pressure. Take
Ash
up the residue with 2 mL of methanol R.
Reference solution (a) Dissolve 10 mg of arabinose Rina
small quantity of water R and dilute to 10 mL with STORAGE
methanol R. lspaghula Husk Granules should be protected from moisture.
Reference solution (b) Dissolve 10 mg of xylose R in a small
quantity of water R and dilute to 10 mL with methanol R.
Reference solution (c) Dissolve 10 mg of galactose R in a
small quantity of water R and dilute to 10 mL with
lspaghula Husk Effervescent Granules
methanol R. DEFINITION
Plate TLC silica gel plate R. lspaghula Husk Effervescent Granules contain lspaghula
Mobile phase water R, acetonitrile R (15:85 V/V). Husk in a suitable, effervescent basis.
Application 10 µL, as bands. The granules comply with the requirements stated under Granules
Detection Spray with aminohippuric acid reagent R and heat TESTS
at 120 °C for 5 min; examine in daylight. Disintegration
Carry out the test stated under Effervescent Granules with
the following modifications. Stir the contents of the beaker
shows 2 orange-pink zones (arabinose and xylose) and a
occasionally to disperse the mucilage formed; evolution of gas
yellow zone (galactose) similar in position and colour to the
is complete after 5 minutes.
zones in the chromatograms obtained with the reference
solutions. Swelling index
Not less than 40, Appendix XI C. Use a quantity of the
TESTS powdered granules containing 1.0 g of Ispaghula Husk and a
Foreign matter (2.8.2) 100 mL ground-glass-stoppered cylinder graduated in 1 mL
Carry out the determination using 5.0 g. divisions.
105 °C for 2 h. lspaghula Husk Oral Powder
Total ash (2.4.16) DEFINITION
lspaghula Husk Oral Powder contains lspaghula Husk with
Swelling index (2.8.4) or without suitable excipients.
Minimum 40, determined on 0.1 g of the powdered herbal The powder complies with the requirements stated under Oral
drug (355) (2.9.12). Powders and with the following requirements.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
IDENTIFICATION
A. Carry out the method for thin-layer chromatography,
(1) Add 2 mL of a 23% w/v solution of trijluoroacetic acid to
lspaghula Husk Granules a quantity of the powder containing 10 mg of lspaghula
DEFINITION Husk in a thick-walled centrifuge tube, shake vigorously,
Ispaghula Husk Granules contain Ispaghula Husk with or close the tube and heat at 120° for 1 hour. Centrifuge the
without suitable excipients. hydrolysate, transfer the clear supernatant liquid into a
The granules comply with the requirements stated under Granules 50 mL flask, add 10 mL of water and evaporate the solution
and with the following requirements. to dryness under reduced pressure. Take up the residue in
10 mL of water, again evaporate to dryness under reduced
IDENTIFICATION
pressure and take up the residue in 2 mL of methanol.
A. Powder the granules and examine under a microscope
using lactic reagent. Fragments of the episperm with polygonal (2) Dissolve 10 mg of arabinose in a small quantity of water
cells filled with mucilage and fragments of the inner layers of and dilute to 10 mL with methanol.
the testa with brownish thin-walled cells often associated with (3) Dissolve 10 mg of xylose in a small quantity of water and
the outer layers of the endosperm are seen. dilute to 10 mL with methanol.
B. When mounted in ruthenium red solution, the particles of (4) Dissolve 10 mg of galactose in a small quantity of water
the powder are stained red. and dilute to 10 mL with methanol.
(a) Use as the coating silica gel.
2023 Ivy Leaf IV-309
(b) Use the mobile phase as described below. 120 °C for 1 h. Centrifuge the hydrolysate, transfer the clear
(c) Apply 10 µL of each solution. supernatant into a 50 mL flask, add 10 mL of water R and
(d) Develop the plate to 15 cm. evaporate the solution to dryness under reduced pressure.
Take up the residue in 10 mL of water R and evaporate again
(e) After removal of the plate, dry in air, spray with to dryness under reduced pressure. Take up the residue in
aminohippuric add reagent, heat at 120° for 5 minutes and 2 mL of methanol R.
Reference solution (a) Dissolve 10 mg of arabinose R in a
MOBILE PHASE small quantity of water R and dilute to 10 mL with
15 volumes of water and 85 volumes of acetonitrile. methanol R.
CONFIRMATION Reference solution (b) Dissolve 10 mg of xylose R in a small
The chromatogram obtained with solution (1) shows two quantity of water R and dilute to 10 mL with methanol R.
orange-pink zones (arabinose and xylose) and a yellow zone Reference solution (c) Dissolve 10 mg of galactose R in a
(galactose) similar in position and colour to the zones in the small quantity of water R and dilute to 10 mL with
chromatograms obtained with solutions (2), (3) and (4). methanol R.
B. When mounted in ruthenium red solution, the particles of Plate TLC silica gel plate R
the powder are stained red. Mobile phase water R, acetonitrile R (15:85 V/Jl).
TESTS Application 10 µL, as bands.
Swelling index Development Over a path of 15 cm.
Not less than 40, Appendix XI C. Use a quantity of the oral Detection Spray with aminohippuric add reagent R and heat
powder containing 1.0 g oflspaghula Husk and a 100 mL at 120 °C for 5 min. Examine in daylight.
ground-glass-stoppered cylinder graduated in 1 mL divisions.
Ash chromatograms obtained with the reference and the test
Not more than 4.0%, Appendix XI J, Method II. Use a solutions.
quantity of the powder containing 1 g of lspaghula Husk.
STORAGE Top of the plate
lspaghula Husk Oral Powder should be protected from
Xylose: an orange-pink zone An orange-pink zone (xylose)
moisture.
Arabinose: an orange-pink zone An orange-pink zone (arabinose)
Galactose: a yellow zone A yellow zone (galactose)
lspaghula Seed Reference solution Test solution

TESTS
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Foreign matter (2.8.2)
DEFINITION Carry out the determination using 10.0 g.
Dried ripe seeds of Plantago ovata Forssk. (P. ispaghula Swelling index (2.8.4)
Roxb.). Minimum 9.
IDENTIFICATION Loss on drying (2.2.32)
A. lspaghula seed is pinkish-beige, smooth, boat-shaped and Maximum 10.0 per cent, determined on 1.000 g of the
curved. It is 1.5 mm to 3.5 mm long, 1.5 mm to 2 mm wide powdered herbal drug (355) (2. 9.12) by drying in an oven at
and l mm to 1.5 mm thick. The concave surface shows in 105 °C for 2 h.
the centre a light coloured spot corresponding to the hilum. Total ash (2.4.16)
The convex surface shows a light brown spot corresponding Maximum 4.0 per cent.
to the location of the embryo and takes up about one quarter - - - - - - - - - - - - - - - - - - - - - Ph Eur
of the length of the seed.
brown. Examine under a microscope using lactic reagent R.
The powder shows mainly fragments of the episperm with Ivy Leaf
polygonal cells filled with mucilage; fragments of the inner
layers of the testa with brownish thin-walled cells often (Ph. Bur. monograph 2148)
associated with the outer layers of the endosperm; fragments
of the endosperm with cells with thick cellulose walls
containing aleurone grains and oil droplets; a few fragments DEFINITION
of embryo with thin-walled cells. Examine under a Whole or cut, dried leaves of Hedera helix L., collected in
microscope using a 50 per cent V/V solution of glycerol R. spring and summer.
The powder shows starch granules, single or in groups of Content
2 to 4 and measuring 3 µm to 25 µm in diameter. Minimum 3.0 per cent of hederacoside C (C 59H 96 O 26;
C. Thin-layer chromatography (2.2.27). Mr 1221) (dried drug).
Test solution To 50 mg of the powdered herbal drug (355) IDENTIFICATION
(2. 9.12) in a thick-walled centrifuge tube add 2 mL of a
A. Whole leaves are coriaceous, 4-10 cm in length and width,
230 g/L solution of trifiuvroacetic acid R, and shake
cordate at the base. The lamina is palmately 3-5 lobed, the
vigorously. Stopper the test tube and heat the mixture at
lobes more or less triangular with entire margins. The upper
IV-310 Ivy Leaf 2023
surface is dark green with a paler, radiate venation, the lower Reference solution Dissolve 1.0 mg of hederacoside C R and
surface more greyish-green and the venation is distinctly 1.0 mg of a-hederin R in 1.0 mL of methanol R.
raised. The petioles are long, cylindrical, about 2 mm in Plate TLC silica gel plate R.
diameter and grooved longitudinally. Scattered white hairs Mobile phase anhydrous formic acid R, acetone R, methanol R,
occur on the petioles and on the surfaces of younger leaves, ethyl acetate R (4:20:20:30 VIVIVIV).
the older leaves are glabrous. Occasional entire, ovate-
rhombic to lanceolate leaves 3-8 cm long from the flowering Application 20 µL 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 At 100-105 °C.
Examine under a microscope using chloral hydrate solutwn R. Detection Treat with alcoholic solution of sulfuric acid R, heat
The powder shows the following diagnostic characters at 110 °C for 10 min and examine in daylight.
(Figure 2148.-1): fragments of the upper epidermis (surface Results See below the sequence of zones present in the
view [F]), showing cells with thickened, rather sinuous, finely chromatograms obtained with the reference solution and the
pitted anticlinal walls [Fa] usually accompanied by test solution. Furthermore, other zones may be present in the
underlying palisade parenchyma [Fb] including some cells chromatogram obtained with the test solution.
containing cluster crystals of calcium oxalate [Fe]; fragments
of the lower epidermis (surface view [El), showing cells with Top of the plate
sinuous, irregularly thickened and pitted walls [Ea], stomata
that are mostly anomocytic [Eb] but occasionally anisocytic A green zone
(2.8.3), surrounded by cells including some that show faint
-- - -
cuticular striations; the lower epidermis is accompanied by
underlying spongy parenchyma [Ee] including some cells ix-Hederin: a purple zone A very faint purple zone (ix-hederin)
containing cluster crystals of calcium oxalate [Ed]; scattered A broad yellow zone
stellate covering trichomes may be present, composed of 2-3 purple or green zones
4-8 branches joined at the base on a multicellular, biseriate
-- - -
stalk (surface view [BJ, side view [Al); cluster crystals of
calcium oxalate, about 40 µm in diameter, scattered [C] or Hederacoside C: a purple zone A purple zone (hederacoside C)
occurring throughout the parenchyma [Ed, Fe]; groups of Reference solution Test solution
lignified fibro-vascular tissue from the veins [D].
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 R, methanol R (20:80 VIV).
Test solution To 1. 00 g of the powdered herbal drug (35 5)
(2. 9.12) in a 250 mL round-bottomed flask add 50 mL of
the 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 Rand sonicate for 10 min. Filter through
Figure 2148.-1. - Illustratwnfor identification test B of powdered a membrane filter (nominal pore size 0.45 µm).
herbal drug of ivy leaf Column:

=
- size: l 0.125 m, 0 =
4 mm;
Test solution Extract 0.50 g of the powdered herbal drug chromatography R (5 µm).
(355) (2.9.12) under a reflux condenser in a water-bath at
60 °C with 5 mL of methanol R for 30 min. Cool and filter.
2023 Java Tea IV-311
Mobile phase: Occasionally, inflorescences in clusters of bluish-white or

- mobile phase A: mix 14 volumes of acetonitrile R with violet flowers, not yet opened, are found.
88 volumes of water R and adjust to pH 2.0 with B. Microscopic examination (2.8.23). The powder is green or
phosphoric acid R; dark green. Examine under a microscope using chloral hydrate
- mobile phase B: phosphoric acid R, acetonitrile R solutwn R. The powder shows the following diagnostic
(0.2:99.8 V/V); characters (Figure 1229.-1): articulated uniseriate covering
trichomes up to 450 µm long, consisting of 3-8 cells with
Time Mobile phase A Mobile phase B thick pined walls, usually broken [C, E] but sometimes
attached to epidermis (surface view [He], side view ill);
0-5 100 0 unicellular or bicellular conical covering trichomes [Fa],
5-6 100 ➔ 94 0 ➔ 6 mainly present on the margins of the lamina [F]; secretory
6 - 40 94---+ 60 6---+ 40 trichomes with unicellular stalks and tetracellular heads,
40 - 41 60 ➔ 0 40 ➔ 100 isolated (surface view [G]) or attached to epidermis (surface
41 - 55 0 100 view [Db], side view [Aa]); secretory trichomes with
unicellular stalks and unicellular (surface view [Hb], side
Flow rate 1.5 mUmin. view [Ab]) or bicellular heads (surface view [Bal); fragments
Detection Spectrophotometer at 205 nm. of upper epidermis (surface view [B]) with cells with sinuous
outlines, and with underlying palisade parenchyma [Bb];
Injection 20 µL.
fragments of lower epidermis [D, HJ with diacytic stomata
System suitability Reference solution: [Da, Ha] (2.8.3); fragments of lamina (transverse section
- retention time: hederacoside C = about 20 min; [Al) showing usually 2 layers of palisade parenchyma [Ac].
if necessary, adjust the time intervals of the gradient.
Calculate the percentage content of hederacoside C with
reference to the dried drug using the following expression:
area of the peak due to hederacoside C in the chromatogram

solution, in grams;
mass of ivy leaf dry extract HRS used to prepare the reference
solution, in grams;
p percentage content of hederacoside C in ivy leaf dry
extract HRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Java Tea
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION Ha
Whole or fragmented, dried leaf and top of stem of
Orthosiphon aristatus (Blume) Miq. var. aristatus (syn. 50 µm
1------1
Orthosiphon stamineus Benth.). J
Content
Minimum 0.3 per cent of rosmarinic acid (C 18H 16 0s; Mr
360.3) (dried drug).
Figure 1229.-1. - Illustration for identificatwn test B of powdered
IDENTIFICATION herbal drug of Java tea
A. The leaf is friable, whole or fragmented, the entire leaf
measuring on average 7.5 cm in length and 2.5 cm in width. C. Thin-layer chromatography (2.2.27).
The petiole is short. The lamina is oval or lanceolate, the Test solution Shake 1 g of the powdered herbal drug (355)
apex acuminate and the base cuneate. The abaxial surface of (2. 9.12) with 10 mL of methanol R in a water-bath at 60 °C
the leaves is light greyish-green and the adaxial surface is for 5 min and filter the cooled solution.
green or dark green. The venation is pinnate with few
Reference solutwn Dissolve 1 mg of sinensetin R and 1 mg of
secondary veins. Examined under a lens ( x 10), the
scopoletin R in methanol R and dilute to 20 mL with the same
secondary veins, after running parallel to the midrib, diverge
solvent.
at an acute angle. The margin is irregularly and roughly
dentate, sometimes crenate, and the abaxial surface is slightly Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
curved. The petioles are thin, quadrangular, 4-8 mm long plate R (2-10 µm)].
and, like the primary venation, usually violet-coloured.
IV-312 Juniper 2023
Mobile phase methanol R, ethyl acetate R, toluene R Mobile phase:

(5:40:55 V/V/V). - mobile phase A: phosphoric acid R, acetonitrile R, water R
Application 10 µL [or 2 µL] as bands of 15 mm [or 8 mm]. (1:19:80 VIVIV);
Development Over a path of 10 cm [or 6 cm]. - mobile phase B: phosphoric acid R, methanol R, acetonitrile R
(1:40:59 VIVIV);
Drying In air.
Detection Examine in ultraviolet light at 365 nm. Time Mobile phase A Mobile phase B
System suitability Reference solution: (min) (per cent VIII) (per cent VIII)
- the zones due to sinensetin and scopoletin are clearly 0-2 100 0
separated. 2 - 20 100---> 55 0---> 45
Results See below the sequence of zones present in the 20 - 25 55 ---> 0 45---> 100

test solution. Furthermore, red fluorescent zones are present Flow rate 1.2 mUmin.
in the lower third and near the solvent front of the Detection Spectrophotometer at 330 nm.
chromatogram obtained with the test solution. Injection 20 µL.
Relative retention With reference to rosmarinic acid
Top of the plate
(retention time = about 11 min): ferulic acid= about 0.8.
- resolution: minimum 4.0 between the peaks due to ferulic
-- --
acid and rosmarinic acid.
Calculate the percentage content of rosmarinic acid using the
Scopoletin: a blue fluorescent zone following expression:
Sinensetin: a blue fluorescent zone A prominent blue fluorescent zone A1 x m2 x p x 0.4
(sinensetin)
A2xm1
-- --
AI area of the peak due to rosmarinic acid in the chromatogram
I or 2 bluish fluorescent zones obtained with the test solution;
A2 area of the peak due to rosmarinic acid in the chromatogram
Reference solution Test solution obtained with reference solution (a);
TESTS mass of the herbal drug to be examined used to prepare the test
Foreign matter (2.8.2) solution, in grams;
rn 2 mass of rosmarinic acid CRS used to prepare reference
Maximum 5 per cent of stems with a diameter greater than solution (a), in grams;
1 mm and maximum 2 per cent of other foreign matter. p percentage content of rosmarinic acid in rosmarinic acid CRS.
Maximum 11.0 per cent, determined on 1.000 g of the - - - - - - - - - - - - - - - - - - - - - - Ph Eur
105 °C for 2 h.
Total ash (2.4.16)
Maximum 12.5 per cent. Juniper
Liquid chromatography (2.2.29). Carry out the assay protected
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
from light.
Test solution Disperse 0.500 g of the powdered herbal drug DEFINITION
(355) (2.9.12) in 90 mL of ethanol (50 per cent V/v'.) R. Boil Dried ripe cone berry of Juniperns communis L.
in a water-bath under a reflux condenser for 30 min, cool, Content
and filter into a 100 mL volumetric flask. Rinse the flask and Minimum 10 mUkg of essential oil (anhydrous drug).
the filter with 10 mL of ethanol (50 per cent Vlv'.) R and
dilute to 100.0 mL with the same solvent. Filter through a CHARACTERS
membrane filter (nominal pore size 0.45 µm). Strongly aromatic odour reminiscent of terpinen-4-ol,
especially if crushed.
Reference solution (a) Dissolve 10.0 mg of rosmarinic
acid CRS in ethanol (50 per cent Vlv'.) R and dilute to IDENTIFICATION
50.0 mL with the same solvent (solution A). Dilute 4.0 mL A. The berry-shaped cone is globular, up to 10 mm in
of solution A to 20.0 mL with ethanol (50 per cent V/V) R. diameter, and violet-brown or blackish-brown, frequently
Reference solution (b) Dissolve 5.0 mg offerulic acid R in with a bluish bloom. It consists of 3 fleshy scales. The apex
ethanol (50 per cent V/v'.) R and dilute to 50.0 mL with the has a 3-rayed closed cleft and 3 not-very-clearly defined
same solvent. To 5.0 mL of the solution add 5.0 mL of projections. A remnant of peduncle is frequently attached at
solution A. the base. The fleshy part is crumbly and brownish.
It contains 3 or, more rarely, 2 small, elongated, extremely
Column:
hard seeds that have 3 sharp edges and are slightly rounded
- size: l = 0.25 m, 0 = 4.6 mm;
at the back, acuminate at the apex. The seeds are fused with
the fleshy part of the cone berry in the lower part on the
outside of their bases. Very large, oval oil glands containing
sticky resin lie at the outer surface of the seeds.
2023 Juniper Oil IV-313
B. Microscopic examination (2.8.23). The powder is brown. Drying In air.

Examine under a microscope using chloral hydrate solution R. Detection Treat with anisaldehyde solution R, heat at
The powder shows the following diagnostic characters 100-105 °C for 5-10 min and examine in daylight.
(Figure 1532.-1): fragments of epidermis of the cone berry Results The chromatogram obtained with the reference
wall (surface view [C], transverse section [D]) consisting of solution shows a red zone (guaiazulene) in the upper half and
cells with thick, pitted, colourless walls and brown granular a brownish-violet or greyish-violet zone (cineole) in the lower
contents [Ca, Da], occasionally with anomocytic stomata half; the chromatogram obtained with the test solution shows
(2.8.3) [Cb]; fragments of the 3-rayed apical cleft of the cone
a strong violet zone (mono- and sesquiterpenes) similar in
berry with epidermal cells interlocked by papillous position to the zone due to guaiazulene in the chromatogram
outgrowths (transverse section [G]); fragments of the obtained with the reference solution, a reddish-violet zone a
hypodermis with thickened collenchymatous cells ITT; little above the zone due to cineole in the chromatogram
fragments of the mesocarp consisting of large, thin-walled obtained with the reference solution, a greyish-violet zone
parenchymatous cells, usually rounded, with large (terpinen-4-ol) a little below the zone due to cineole in the
intercellular spaces and irregular, large, usually scarcely chromatogram obtained with the reference solution, and just
pitted, yellow idioblasts [F]; fragments of secretory canals below that a blue zone; a faint violet zone may be present in
(transverse section [B]) with schizogenous oil cells; fragments a similar position to the zone due to cineole; further zones
of the testa with thick-walled, pitted, colourless sclereids [E] are present.
containing 1 or more prism crystals of calcium oxalate [Ea];
fragments of the seed (surface view [A], transverse TESTS
section [H]) with a finely pitted testa [Aa, Ha] and thin- Foreign matter (2.8.2)
walled endosperm cells containing droplets of fatty oil and Maximum 5 per cent of unripe or discoloured cone berries
aleurone grains [Ab, Hb]. and maximum 2 per cent of other foreign matter.
Water (2.2.13)
Maximum 120 mUkg, determined on 20.0 g of the crushed
drug.
Total ash (2.4.16)
ASSAY
Use 20.0 g of the herbal drug reduced to a coarse powder
using a suitable mill immediately before the assay, a 500 mL
liquid and 0.5 mL of xylene R in the graduated tube. Distil at
a rate of 3-4 mUmin for 90 min.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Juniper Oil
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINffiON
Essential oil obtained by steam distillation from the ripe,
non-fermented berry cones of Juniperus communis L.
A suitable antioxidant may be added.
CHARACTERS
Appearance
Mobile, colourless or yellowish liquid.
Characteristic odour.
Figure 1532.-1. - Illustration for identification test B of powdered IDENTIFICATION
herbal drng of juniper
C. Thin-layer chromatography (2.2.27). Second identification: A.
Test solution Dilute the oil-xylene mixture obtained in the A. Thin-layer chromatography (2.2.27).
assay to 5.0 mL with hexane R. Test solution Dissolve 0.2 mL of the substance to be
Reference solution Dissolve 4.0 mg of guaiazulene Rand examined in 5 mL of heptane R.
50 µL of cineole R in 10 mL of hexane R. Reference solution Dissolve 20 mg of a-terpineol R and 20 µL
Plate TLC silica gel plate R. of terpinen-4-ol R in 25 mL of heptane R.
Mobile phase ethyl acetate R, toluene R (5:95 V/V). Plate TLC silica gel plate R.
Application 20 µL of the test solution and 10 µL of the Mobile phase ethyl acetate R, toluene R (5:95 V/V).
reference solution, as bands. Application 20 µL, as bands.
IV-314 Kelp 2023
Development Over a path of 12 cm. Temperature:

Drying In air.
Time Temperature
Detection Treat with anisaldehyde solution R and heat at (min) CC)
100-105 °C until the zones appear; examine immediately in
Column 0- I 60
daylight.
I - 58 60---+230
Results See below the sequence of zones present in the Injection port 250
chromatograms obtained with the reference solution and the Detector 250
test solution.
Top of the plate
Injection 0.5 µL.
An intense brownish-violet zone Elution order Order indicated in the composition of the
A brown zone reference solution. Record the retention times of these
substances.
A violet-pink zone
Terpinen-4-ol: a brownish-violet A brownish-violet zone (terpinen-4- - resolution: minimum 1.5 between the peaks due to
zone ol) sabinene and ~-pinene.
A violet zone
ct-Terpineol: a violet or brownish- A violet or brownish-violet zone
violet zone (ct-terpineol)
chromatogram obtained with the reference solution, locate
Reference solution Test solution chromatogram obtained with the test solution.
Determine the percentage content of the components.
B. Examine the chromatograms obtained in the test for Disregard the peak due to trimethylpentane and peaks
chromatographic profile. comprising less than 0.01 per cent of the total surface area.
Results The characteristic peaks in the chromatogram The percentages are within the following ranges:
obtained with the test solution are similar in retention time to - a-pinene: 20 per cent to 50 per cent;
those in the chromatogram obtained with the reference - sabinene: maximum 20 per cent;
solution. - /3-pinene: 1.0 per cent to 12 per cent;
- /3-myrcene: 1.0 per cent to 35 per cent;
TESTS - a-phellandrene: maximum 1.0 per cent;
Relative density (2.2.5) - limonene: 2.0 per cent to 12 per cent;
0.857 to 0.876. - terpinen-4-ol: 0.5 per cent to 10 per cent;
Refractive index (2.2.6) - bornyl acetate: maximum 2.0 per cent;
1.471 to 1.483. - /3-caryophyllene: maximum 7 .0 per cent.
Optical rotation (2. 2. 7) STORAGE
-15° to -0.5°. At a temperature not exceeding 25 °C.
Peroxide value (2.5.5)
Maximum 20.
Fatty oils and resinified essential oils (2.8. 7)
It complies with the test for fatty oils and resinified essential
oils. Kelp
Gas chromatography (2.2.28): use the normalisation Bladderwrack Fucus
procedure. (Ph. Bur. monograph 1426)
Test solution Dissolve 60 mg of the substance to be
examined in trimethylpentane Rand dilute to 5.0 mL with the DEFINITION
same solvent. Fragmented dried thallus of Fucus vesiculosus L.
Reference solution Mix 25 µL each of a-pinene R, sabinene R, or F. serratus L. or Ascophyllum nodosum Le Jolis.
/3-pinene R, /3-myrcene R, a-phellandrene R, limonene R, Content
terpinen-4-ol R, bornyl acetate R and /3-caryophyllene R and Minimum 0.03 per cent and maximum 0.2 per cent of total
dilute to 25.0 mL with trimethylpentane R. iodine (Ar 126.9) (dried drug).
Column:
- material: fused silica; CHARACTERS
- size: l = 30 m (a film thickness of 1 µm may be used) to Salty and mucilaginous taste.
60 m (a film thickness of 0.2 µm may be used), Unpleasant marine odour.
0 = 0.25-0.53 mm; IDENTIFICATION
- stationary phase: phenyl(S)methyl(95)polysiloxane R. A. The drug consists of fragments with a corneous
Carrier gas helium for chromatography R. consistency, blackish-brown to greenish-brown, sometimes
Flow rate 2.0 mLJmin. covered with whitish efflorescence. The thallus consists of a
Split ratio 1:50. ribbon-like blade, branching dichotomously with prominent
central ribs (pseudoveins). F. vesiculosus typically shows a
foliose blade with smooth edges and bears occasional ovoid,
single or paired, air vesicles. The ends of certain branches are
2023 Knotgrass IV-315
of ovoid shape and a little widened. They bear numerous 1 mL of 0.01 M sodium thiosulfate is equivalent to 0.2115 mg
reproductive organs (conceptacles). F. serratus has a foliose of iodine.
blade with a serrate margin and no vesicles, the branches LABELLING
bearing conceptacles are less swollen. The thallus of
The label states the species of kelp present.
A. nodosum is irregularly branched, without pseudo-midrib.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
It shows single ovoid air vesicles; the falciform conceptacles
are located at the end of small branches.
greenish-brown. Examine under a microscope using chloral
hydrate solution R. The powder shows fragments of surface Knotgrass
tissue with regular isodiametric cells with brown contents,
and fragments of deep tissue with colourless, elongated cells (Ph. Bur. monograph 1885)
arranged in long filaments with large mucilaginous spaces ~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
between them. Thick-walled cells in files and in closely
DEFINITION
packed groups, from the pseudovein, are sometimes visible.
Whole or fragmented, dried flowering aerial parts of
C. To 1 g of the powdered herbal drug (355) (2.9.12) add Polygonum aviculare L. s.l.
20 mL of a 2 per cent V/V solution of hydrochloric acid R.
Shake vigorously and filter. Wash the residue with 10 mL of Content
water Rand filter. To the residue add 10 mL of a 200 g/L Minimum 0.30 per cent of flavonoids, expressed as
solution of sodium carbonate R. Shake and centrifuge. Collect hyperoside (C 21 H 20 O 12; Mr 464.4) (dried drug).
the supernatant. Adjust to pH 1.5 using sulfuric acid R. IDENTIFICATION
A white, flocculent precipitate is slowly formed. A. The stem is 0.5-2 mm thick, branched, with nodes,
TESTS cylindrical or slightly angular, and longitudinally striated.
Arsenic (2.4.27) It bears sessile or shortly petiolate, glabrous, entire leaves,
Maximum 90 ppm. which differ widely in shape and size. The sheath-like stipules
(ochrea) are lacerate and silvery. The small, axillary flowers
Cadmium (2.4.27) have 5 greenish-white perianth segments, the tips of which
Maximum 4 ppm. are often red. The dry, indehiscent fruits are 2-4 mm, brown
Lead (2.4.27) or black, triangular, usually punctate or striate.
Maximum 5 ppm. B. Microscopic examination (2.8.23). The powder is
Mercury (2.4.27') greenish-brown. Examine under a microscope using chloral
Maximum 0.1 ppm. hydrate solution R. The powder shows the following diagnostic
Swelling index (2.8.4) characters (Figure 1885.-1): fragments of lower [A] and
Minimum 6. upper [D] leaf epidermises with a striated cuticle and
anisocytic stomata (2.8.3) [Aa, Da]; polygonal cells of the
Loss on drying (2.2.32) upper epidermis [D] with slightly thickened beaded walls,
Maximum 15.0 per cent, determined on 1.000 g by drying in often associated with palisade parenchyma [Db]; cells of the
an oven at 105 °C, for 2 h. lower epidermis [A], with thin, sinuous walls; fragments of
Total ash (2.4.16) the margin of the lamina of the leaf with irregular cells [J];
Maximum 24 per cent. fragments of parenchyma [G] with numerous cells containing
Ash insoluble in hydrochloric acid (2.8.1) cluster crystals of calcium oxalate, some of which are very
Maximum 3.0 per cent. large [Ga], often associated with vessels [Gb]; groups of
fibres [B, C] with thick walls [Ba, Cb] from the hypodermis
ASSAY of the stem associated either with the epidennis [Ca] or with
Total iodine parenchyma consisting of cells containing cluster crystals of
To 1.000 g of the powdered herbal drug, in a tall silica calcium oxalate [Bb]; fragments of the ochrea [El with
crucible, add 5 mL of water R and 5 g of potassium elongated, thin-walled cells [Ea], along which run very
hydroxide R. Stir with a magnesium rod. Heat on a water elongated fibres [Eb]; globular pollen grains with a smooth
bath. Add 1 g of potassium carbonate R. Mix, add the tip of exine and 3 germinal pores [H]; occasional brown fragments
the magnesium rod with the residues of the drug and dry, of the exocarp composed of cells with thick, sinuous
first on a water-bath then over an open flame. Incinerate walls [F].
raising the temperature progressively to not more than
600 °C. Allow to cool. Add 20 mL of water R and heat
gently to boiling, stirring with a glass rod. Filter the hot Test solution To 1.0 g of the powdered herbal drug (355)
mixture through an unpleated filter, into a conical flask. (2.9.12) add 10 mL of methanol R. Heat the mixture in a
Rinse the residue with 4 quantities, each of 20 mL, of hot water-bath under a reflux condenser for 10 min. Cool and
water R. Rinse the filter and the crucible with 50 mL of hot filter.
water R. Combine the solutions. Allow to cool. Neutralise Reference solution Dissolve 1 mg of cajfeic acid R, 1 mg of
with dilute sulfuric acid R in the presence of methyl orange chlorogenic acid R and 2.5 mg of hyperoside R in 10 mL of
solution R. Add 3 mL of dilute sulfuric acid R and 1 mL of methanol R.
bromine water R. The solution is yellow. After 5 min add Plate TLC silica gel plate R.
0.6 mL of a 50 g/L solution of phenol R. The solution is Mobile phase anhydrous Jonnie acid R, glacial acetic acid R,
clear. Acidify with 5 mL of phosphoric acid Rand add 0.2 g of water R, ethyl acetate R (7 :7: 14:72 VIVIVIV).
potassium iodide R. Allow to stand for 5 min protected from
light. Add 1 mL of starch solution Rand titrate with 0.01 M
sodium thiosulfate. Development Over a path of 10 cm.
IV-316 Kudzuvine Root 2023
TESTS
Maximum 2 per cent of roots and maximum 2 per cent of
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Stock solution In a 100 mL round-bottomed flask, place
0.800 g of the powdered herbal drug (355) (2.9.12), and add
of acetone Rand 2 mL of hydrochloric acid Rl. Boil the
10 min. Allow to cool, filter each extract through the plug of
absorbent cotton into the flask. Filter the combined acetone
extracts through a filter paper into a volumetric flask and
dilute to 100.0 mL with acetone R, rinsing the flask and the
filter paper. Introduce 20.0 mL of the 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 and wash with 2 quantities,
each of 50 mL, of water R. Dry the extracts over 10 g of
Figure 188 5. -1. - Illustratum for identification test B of powdered
anhydrous sodium sulfate R, filter into a 50 mL volumetric
herbal drug of knotgrass
flask and dilute to volume with ethyl acetate R.
Drying At 100-105 °C. Test solution To 10.0 mL of the stock solution add 1 mL of
Detection treat with a 10 g/L solution of diphenylboric acid aluminium chloride reagent Rand dilute to 25.0 mL with a
aminoethyl ester R in methanol R; subsequently treat with a 5 per cent V/V solution of glacial acetic acid R in methanol R.
50 g/L solution of macrogol 400 R in methanol R. Allow to dry Compensation liquid Dilute 10.0 mL of the stock solution to
in air for about 30 min. Examine in ultraviolet light at 25.0 mL with a 5 per cent V/V solution of glacial acetic
365 nm. acid R in methanol R.
Results See below the sequence of fluorescent zones present Measure the absorbance (2. 2. 25) of the test solution after
in the chromatograms obtained with the reference solution 30 min by comparison with the compensation liquid at
and the test solution. Furthermore, other fluorescent zones 425 nm. Calculate the percentage content of flavonoids,
are present in the chromatogram obtained with the test calculated as hyperoside, using the following expression:
solution. Ax 1.25
m
Top of the plate
i.e. taking the specific absorbance ofhyperoside to be 500.
Caffeic acid: a light blue fluorescent I or 2 blue fluorescent zones (caffeic
zone acid)
-- --
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
I or 2 yellowish-green fluorescent
zones
Hyperoside: a yellowish-brown
Kudzuvine Root
fluorescent zone
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
fluorescent zone (chlorogenic acid) DEFINITION
Fragmented, dried root of Pueraria montana (Lour.) Merr.
- - - -
var. lobata (Willd.) Maesen & S.M.Almeida ex Sanjappa &
A yellowish-brown fluorescent zone Predeep (syn. Pueraria lobata (Willd.) Ohwi).
Content
Minimum 6.5 per cent of total isoflavonoids, expressed as
puerarin (C 21 H 20 O 9 ; Mr 416.4) (dried drug), of which
minimum 45 per cent consists of puerarin.
2023 Kudzuvine Root IV-31 7
IDENTIFICATION Detection Examine in ultraviolet light at 254 nm.

A. Small, square pieces or thick, rectangular slices, 5-35 cm Results See below the sequence of zones present in the
long and 0.5-1 cm thick. The outer bark is pale brown, with chromatograms obtained with the reference solution and the
longitudinal wrinkles and rough; the section is yellowish- test solution. Furthermore, other zones may be present in the
white and shows indistinct striations. The texture is strongly chromatogram obtained with the test solution.
fibrous.
B. Microscopic examination (2.8.23). The powder is pale Top of the plate
A weak quenching zone
characters (Figure 2434.-1): thick-walled lignified fibres, -- --
which occur in bundles, surrounded by a calcium oxalate
A quenching zone
prism sheath [E]; cork fragments with polygonal cells
(surface view [DJ, transverse section [BJ); fragments of xylem Daidzin: a quenching zone A quenching zone
[CJ consisting of relatively large vessels with hexagonal or
Puerarin: a quenching zone A quenching zone
elliptical bordered-pits, arranged very densely [Ca] and xylem
parenchyma cells with slightly thickened and pitted walls -- - -
[Cb]; rare sclereids, subrounded or elliptical, about 50 µm in

At least 5 quenching zones
diameter [F]; fragments of parenchyma [A]. Examine under
a microscope using a 50 per cent V/V solution of glycerol R. Reference solution Test solution
The powder shows numerous starch granules [G], simple or
2-20 compound; the individual starch granules, 15-30 µm in
diameter, are spheroidal, semi-rounded or polygonal, with a TESTS
punctiform, slit-shaped or stellate hilum. Foreign matter (2.8.2)
Maximum 5 per cent.
105 °C.
Total ash (2.4.16)
ASSAY
(355) (2. 9.12) into a 250 mL conical flask, add 50.0 mL of
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 V/V) R, mix well
and filter.
Reference solution Introduce an amount of kudzuvine root dry
extract HRS corresponding to 3.0 mg of puerarin into a
250 mL conical flask, add 50.0 mL of ethanol
(30 per cent V/V) 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 V/V) R, mix well
and filter.
Column 2 columns coupled in series:
- size: l = 0.10 m, 0 = 4.6 mm;
Figure 2434.-1. - Illustration for identification test B of powdered - stationary phase: end-capped monolithic octadecylsilyl silica gel
herbal drug of kudzuvine root for chromatography R.
C. Thin-layer chromatography (2.2.27). Mobile phase:
- mobile phase A: glacial acetic acid R, water for
Test solution Sonicate 0.5 g of the powdered herbal drug
(355) (2. 9.12) with 5 mL of methanol R, then centrifuge; use
- mobz1e phase B: acetonitrile R;
the supernatant.
Reference solution Dissolve 5 mg of puerarin R and 5 mg of Time Mobile phase A Mobile phase B
daidzin R in 5 mL of methanol R. (min) (per cent VIJJ) (per cent V/JJ)
Plate TLC silica gel F254 plate R (2-10 µm). 0 - 16.5 90----> 71 10 ----> 29
Mobile phase water R, methylene chloride R, methanol R, ethyl

acetate R (10:20:22:40 VIVIVIV); use the lower layer. Flow rate 3.0 mUmin.
Application 7 µL as bands of 8 mm. Detection Spectrophotometer at 260 nm.
Development Over a path of 6 cm. Injection l O µL.
Drying In air.
IV-318 Thomson Kudzuvine Root 2023
Identification of peaks Use the chromatogram supplied with IDENTIFICATION

kudzuvine root dry extract HRS and the chromatogram A. Cylindrical, subfusiform or semi-cylindrical, 12-15 cm
obtained with the reference solution to identify the peaks due long and 4-8 cm in diameter, sometimes in longitudinally or
to the isoflavonoids (3-hydroxypuerarin, puerarin, obliquely cut thick slices, varying in size. Externally
3-methoxypuerarin, 6-O"-o-xylosylpuerarin and daidzin). yellowish-white or pale brown. The root is heavy, texture
Relative retention With reference to puerarin (retention hard and starchy. A transverse section shows pale brown
time= about 3.4 min): 3-hydroxypuerarin = about 0.7; concentric rings formed by fibres. A longitudinal section
3-methoxypuerarin = about 1.09; 6-O"-o- shows several longitudinal striations formed by fibres.
xylosylpuerarin = about 1.15; daidzin = about 1.4. B. Microscopic examination (2.8.23). The powder is
System suitability Reference solution: yellowish-white. Examine under a microscope using chloral
- peak-to-valley ratio: minimum 10, where Hp = height hydrate solution R. The powder shows the following diagnostic
above the baseline of the peak due to 3-methoxypuerarin characters (Figure 2483.-1): thick-walled lignified fibres,
and Hv = height above the baseline of the lowest point of which occur in bundles, surrounded by a calcium oxalate
the curve separating this peak from the peak due to prism sheath [A]; rare sclereids, subrounded or elliptical,
puerarin. about 50 µm in diameter [E]; fragments of xylem [F]
Calculate the percentage content of puerarin using the consisting of relatively large vessels with hexagonal or
following expression: elliptical bordered-pits, arranged very densely [Fa] and xylem
parenchyma cells with slightly thickened and pitted walls
[Fb], fragments ofparenchyma [C]; isolated prism crystals
[BJ. Examine under a microscope using a 50 per cent V/V
solution of glycerol R. The powder shows numerous starch
A1 area of the peak due to puerarin in the chromatogram obtained granules [DJ, simple or 2-20 compound; the individual
with the test solution; granules, 15-30 µm in diameter, are spheroidal, semi-
A2 area of the peak due to puerarin in the chromatogram obtained
rounded or polygonal with a punctiform, slit-shaped or
m1 mass of the herbal drug to be examined used to prepare the test stellate hilum.
solution, in grams;
m2 mass of kudzuvine root dry extract HRS used to prepare the
p percentage content of puerarin in kudzuvine root dry
extract HRS.
Calculate the percentage content of total isoflavonoids

(3-hydroxypuerarin, puerarin, 3-methoxypuerarin, 6-0" -D-
xylosylpuerarin and daidzin) using the following expression:
A1 sum of the areas of the peaks due to the isoflavonoids

(3-hydroxypuerarin, puerarin, 3-methoxypuerarin, 6-0 11 -D-
xylosylpuerarin and daidzin) in the chromatogram obtained with
the test solution;
A2 area of the peak due to puerarin in the chromatogram obtained
solution, in grams;
m2 mass of kudzuvine root dry extract HRS used to prepare the
p percentage content of puerarin in kudzuvine root dry
extract HRS. Fb
25 µm
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Figure 2483.-1 - Illustration for identification test B of powdered

herbal drug of Thomson kudzuvine root
Thomson Kudzuvine Root C. Thin-layer chromatography (2.2.27).

Test solution Sonicate 0.5 g of the powdered herbal drug
(Ph. Bur. monograph 2483) (355) (2.9.12) with 5 mL of methanol R, then centrifuge; use
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ the supernatant.
DEFINITION Reference solution Dissolve 5 mg of daidzin R and 5 mg of
Whole or fragmented, dried root of Pueraria montana (Lour.) puerarin R in 5 mL of methanol R.
Merr. var. thomsonii (Benth.) M.R.Almeida (syn. Pueraria Plate TLC silica gel F 254 plate R (2-10 µm).
thomsonii (Benth.)), with the outer bark removed. Mobile phase water R, methylene chloride R, methanol R, ethyl
Content acetate R (10:20:22:40 V/VIVIV); use the lower layer.
Minimum 0.4 per cent of total isoflavonoids, expressed as Application7 µL as bands of 8 mm.
puerarin (C 21 H 20 O9 ; Mr 416.4) (dried drug), of which Development Over a path of 6 cm.
minimum 55 per cent consists of puerarin. Drying In air.
2023 Lavender Flower IV-31 9
Results See below the sequence of zones present in the to the isoflavonoids (puerarin, 3-methoxypuerarin, 6-0 11 -D-
chromatograms obtained with the reference solution and the xylosylpuerarin and daidzin).
test solution. Furthermore, other zones may be present in the Relative retention With reference to puerarin (retention
chromatogram obtained with the test solution. time= about 3.4 min): 6-O 11 -D-xylosylpuerarin = about 1.15;
daidzin = about 1.4.
Top of the plate System suitability Reference solution:
A weak quenching zone - peak-to-valley ratio: minimum 10, where Hp = height
above the baseline of the peak due to 3-methoxypuerarin
-- --- and Hv = height above the baseline of the lowest point of
Daidzin: a quenching zone A weak quenching zone the curve separating this peak from the peak due to
puerarin.
Puerarin: a quenching zone A weak quenching zone
Calculate the percentage content of puerarin using the
--- --- following expression:
Several quenching zones
area of the peak due to puerarin in the chromatogram obtained

TESTS with the test solution;
area of the peak due to puerarin in the chromatogram obtained
Foreign matter (2.8.2) with the reference solution;
Maximum 5 per cent. mass of the herbal drug to be examined used to prepare the test
mass of kudzuvine root dry extract HRS used to prepare the
Maximum 10.0 per cent, determined on 1.000 g of the reference solution, in grams;
powdered herbal drug (355) (2.9.12) by drying in an oven at p percentage content of puerarin in kudzuvine root dry
105 °C. extract HRS.
Total ash (2.4.16)

Calculate the percentage content of total isoflavonoids
(puerarin, 6-0" -D-xylosylpuerarin and daidzin) using the
Ash insoluble in hydrochloric acid (2.8.1) following expression:
ASSAY
Test solution Introduce 1.00 g of the powdered herbal drug sum of the areas of the peaks due to the isoflavonoids (puerarin,
(355) (2.9.12) into a 250 mL conical flask, add 50.0 mL of 6-0" -D-xylosylpuerarin and daidzin) in the chromatogram
ethanol (30 per cent Viii] R and weigh. Heat under a reflux obtained with the test solution;
condenser for 30 min. Allow to cool and weigh again. Adjust area of the peak due to puerarin in the chromatogram obtained
to the initial mass with ethanol (30 per cent V/V) R, mix well mass of the herbal drug to be examined used to prepare the test
and filter. solution, in grams;
Reference solution Introduce an amount of kudzuvine root dry mass of kudzuviue root dry extraa HRS used to prepare the
extract HRS corresponding to 3.0 mg of puerarin into a percentage content of puerarin in kudzuvine root dry
p
250 mL conical flask, add 50.0 mL of ethanol extract HRS.
(30 per cent Viii] R and weigh. Heat under a reflux
condenser for 30 min. Allow to cool and weigh again. Adjust PhEur
to the initial mass with ethanol (30 per cent Viii] R, mix well
and filter.
Column 2 columns coupled in series:
- size: l = 0.10 m, 0 = 4.6 mm; Lavender Flower
- stationary phase: end-capped monolithic octadecylsil,yl silica gel
for chromatography R. (Ph. Eur. monograph 1534)
Mobile phase:
- mobil,e phase A: glacial acetic acid R, water for
chromatography R (0.1:99.9 VIV); DEFINITION
- mobil,e phase B: acetonitril,e R; Dried flower of Lavandula angustifolia Mill. (L. officinalis
Chaix).
Time Mobile phase A Mobile phase B Content
(min) (per cent VIV) (per cent VIJJ) Minimum 13 mUkg of essential oil (anhydrous drug).
0 - 16.5 90 ➔ 71 10 ➔ 29
IDENTIFICATION
First identification: A, B, D.
Second identification: A, B, C.
A. The flower has a short peduncle and consists of a bluish-
Injection l O µL. grey tubular calyx divided distally into 4 very short teeth and
Identification of peaks Use the chromatogram supplied with a small rounded lobe, a blue bilabial corolla with the upper
kudzuvine root dry extract HRS and the chromatogram lip bifid and the lower lip trilobate and 4 didynamous
obtained with the reference solution to identify the peaks due stamens with ovoid anthers.
IV-320 Lavender Flower 2023
B. Microscopic examination (2.8.23). The powder is bluish- Top of the plate

grey. Examine under a microscope using chloral, hydrate
characters (Figure 1534.-1): covering trichomes bifurcating at A violet zone
one or more levels [C, LJ; secretory trichomes with short
stalks and 8-celled heads of the Lamiaceae type (side -- --
view [H], surface view [MJ); glandular trichomes with Linalyl acetate: a violet zone A violet zone
unicellular [OJ or multicellular [KJ stalks and unicellular
A pink zone
heads; glandular trichomes with long uneven stalks and
unicellular heads, separated from the stalk by an intermediary - -
--
cell with a smooth cuticle, certain trichomes show a crown of
small spheroid protuberances just below the insertion point
of the intermediary cell on the stalk [GJ; fragments of 1,8-Cineole: a violet or brown zone
papillose epidermis from the inner surface of the petals
(surface view W, side view [P]); fragments of calyx epidermis
with sinuous-walled cells and containing prismatic crystals of
calcium oxalate [Q]; spherical pollen grains which have a Linalol: a violet zone A violet zone (linalol)
diameter of about 45 µm and an exine with 6 slit-like
germinal pores and 6 ribbon-like groins radiating from the
poles [A, D, E, F]; rare fragments of leaf epidermis with Reference solution Test solution
stomata, mostly of the diacytic type (2.8.3) [BJ; fragments of
vascular tissue with spiral vessels included in parenchyma
with some cells containing small cluster crystals of calcium D. Examine the chromatograms obtained in the test for other
oxalate [N]. species and varieties of lavender.
Results The 5 principal peaks in the chromatogram obtained
corresponding peaks in the chromatogram obtained with the
reference solution; the 2 main peaks are due to linalol and
linalyl acetate.
A TESTS
CA Maximum 3 per cent of stems and maximum 2 per cent of
\GJo other foreign matter.
°'V (-.:<
E , ,-
<~
F
Lavandin flower (Lavandula x intermed:ia Emeric ex
Loisel)
(2. 9.12) add 5 mL of toluene R, sonicate for 5 min and filter.
Reference solution Dilute 10 µL of cineole R, 5 µL of lina/,ol R
and 5 µL of linalyl acetate R to 1 mL with toluene R.
N Drying In air.
Detection Treat with anisauiehyde solution R and heat at
100 °C for 5 min; examine immediately in daylight.
shows no zone between the zones due to 1,8-cineole and
linalol in the chromatogram obtained with the reference
t---1
25 µm solution.
Other species and varieties of lavender
Figure 1534.-1. - Illustration for identification test B of powdered Gas chromatography (2.2.28): use the normalisation
herbal drug of lavender flower procedure.
Test solution Dilute 0.2 mL of the essential oil-xylene
C. Examine the chromatograms obtained in the test for mixture obtained in the assay to 5 mL with heptane R, add
lavandin flower. 1 g of anhydrous sodium sulfate R, shake and use the
Results See below the sequence of zones present in the supernatant.
chromatograms obtained with the reference solution and the Reference solution Dissolve 0.05 g of camphor Rand 0.2 g of
test solution. Furthermore, other faint zones may be present a-terpineol R in heptane R, add 0.1 g of limonene R, 0.2 g of
in the chromatogram obtained with the test solution. cineole R, 0.4 g of linalol Rand 0.6 g of linalyl acetate R, then
dilute to 100 mL with heptane R.
2023 Lavender Oil IV-321
- material: fused silica; First identification: B.
- size: l = 60 m, 0 = 0.25 mm; Second identification: A.
0.25 µm).
Test solution Dilute 20 µL of the essential oil to be
examined to 1 mL with toluene R.
Flow rate 1.5 milmin. Reference solution Dilute 10 µL of cineole R, 5 µL of linalol R
Split ratio l: 100. and 5 µL of linalyl acetate R to 1 mL with toluene R.
Temperature: Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel
F2 54 plate R (2-10 µm)].
Time Temperature
(min) CC)
Column 0 - 15 70
15 - 70 70---+ 180 Development Over a path of 10 cm [or 6 cm].
Injection port 220 Drying In air.
Detector 220 Detection Treat with anisaldehyde solution R and heat at
100 °C for 5 min; examine immediately in daylight.
Detection Flame ionisation. Results See below the sequence of zones present in the
Injection The same volume of each solution. chromatograms obtained with the reference solution and the
Elution order Limonene, 1,8-cineole, camphor, linalol, linalyl test solution. Furthermore, other faint zones may be present
acetate, ix-terpineol. in the chromatogram obtained with the test solution.
- resolution: minimum 1.5 between the peaks due to Top of the plate
limonene and 1,8-cineole; A violet 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 zone An intense violet zone (linalyl
chromatogram obtained with the reference solution, locate acetate)
the 6 components of the reference solution in the A pink zone
chromatogram obtained with the test solution. Disregard the
peaks due to heptane and xylene.
Limit: -- - -
- camphor. maximum 1 per cent. 1,8-Cineole: a violet or brown zone Possibly a weak violet-brown zone
Water (2.2.13) (1,8-cineole)
Maximum 100 milkg, determined on 20.0 g.
Total ash (2.4.16) Linalol: a violet zone An intense violet zone (linalol)
A greyish or brownish zone
ASSAY
Use 20.0 g of the herbal drug, a 1000 mL round-bottomed Reference solution Test solution
flask, 500 mL of water Ras the distillation liquid and 0.5 mL
of xylene R in the graduated tube. Distil at a rate of
2-3 mLJmin for 2 h. B. The essential oil to be examined complies with the limits
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
of the test for chromatographic profile.
TESTS
0.878 to 0.892.
Lavender Oil Refractive index (2.2.6)
1.455 to 1.466.
(Ph. Eur. monograph 1338) Optical rotation (2.2. 7)
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ -12.5° to - 6.0°.
DEFINITION Acid value (2.5.1)
Essential oil obtained by steam distillation of the flowering Maximum 1.0, determined on 5.00 g of the essential oil to
tops of Lavandula angustifolia Mill. (Lavandula officinalis be examined dissolved in 50 mL of the prescribed mixture of
Chaix). solvents.
CHARACTERS Chromatographic profile

Appearance
procedure.
Colourless or pale yellow, clear liquid.
Odour
examined to 10.0 mL with heptane R.
Complex, reminiscent of linalyl acetate.
Reference solution ( a) Dilute 200 µL of lavender oil for peak
identification HRS to 10.0 mL with heptane R.
IV-322 Lavender Oil 2023
Reference solution (b) Dilute 5 µL of limonene R to 50.0 mL Gamer gas helium for chromatography R.
with heptane R. Dilute 0.5 mL of this solution to 5.0 mL Flow rate 1.3 mUmin.
with heptane R. Split ratio 1:30.
Column: Temperature:
- size: l = 60 m, 0 = 0.25 mm; Time Temperature
- stationary phase: macrogol 20 000 R (film thickness (min) CC)
0.25 µm). Column 0 - 65 50 ➔ 180
Gamer gas helium for chromatography R. Injection port 230
Flow rate 1.5 mUmin. Detector 230
Split ratio 1:50.
Temperature: Detection Flame ionisation.
Injection 1 µL.
Time Temperature Elution order (R)-linalol, (S)-linalol, bomeol, (R)-linalyl
(min) CC) acetate, (S)-linalyl acetate; depending on the operating
Column 0 - 15 70 conditions and the state of the column, bomeol may elute
15 - 70 70 ➔ 180 before or after (S)-linalol.
Injection port 220
Detector 220
- resolution: minimum 5.5 berween the peaks due to
(R)-linalol and (S)-linalol; minimum 2.9 between the
Detection Flame ionisation. peaks due to (S)-linalol and borneol; minimum 2.0
Injection 1 µL. berween the peaks due to (R)-linalyl acetate and
Elution order Limonene, 1,8-cineole, 3-octanone, camphor, (S)-linalyl acetate.
linalol, linalyl acetate, terpinen-4-ol, lavandulyl acetate, Calculate the percentage content of the specified
lavandulol, IX-terpineol. (S)-enantiomers using the following expression:
Identification of peaks V se the chromatogram supplied with
lavender oil for peak identification HRS and the chromatogram A AsA x 100
obtained with reference solution (a) to identify the peaks due s+ R
to limonene, 1,8-cineole, 3-octanone, camphor, linalol, linalyl

As area of the peak due to the corresponding (S)-enantiomer;
acetate, terpinen-4-ol, lavandulyl acetate, lavandulol and AR area of the peak due to the corresponding (R)-enantiomer.
IX-terpineol.
System suitability Reference solution (a): Limits:
- resolution: minimum 1.4 berween the peaks due to - (S)-linalol: maximum 12 per cent;
terpinen-4-ol and lavandulyl acetate. - (S)-linalyl acetate: maximum 1 per cent.
Determine the percentage content of each of the following STORAGE
components. The percentages are within the following At a temperature not exceeding 25 °C.
ranges:
- limonene: maximum 1.0 per cent; ---------------------~~
- 1,8-cineole: maximum 2.5 per cent;
- 3-octanone: 0.1 per cent to 5.0 per cent;
- camphor. maximum 1.2 per cent;
- linalol: 20.0 per cent to 45.0 per cent; Spike Lavender Oil
- linalyl acetate: 25.0 per cent to 47.0 per cent;
- terpinen-4-ol: 0.1 per cent to 8.0 per cent; (Ph. Bur. monograph 2419)
- lavandulyl acetate: minimum 0.2 per cent; PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
- lavandulol: minimum 0.1 per cent;
DEFINITION
- rx-terpineol: maximum 2.0 per cent;
Essential oil obtained by steam distillation of the flowering
- reporting threshold: the area of the principal peak in the
tops of Lavandula latifolia Medik.
(0.05 per cent). CHARACTERS
Chiral purity Appearance
Gas chromatography (2.2.28). Clear, mobile, light yellow or greenish-yellow liquid.
Test solution Dilute 0.02 g of the essential oil to be Odour: reminiscent of cineole and camphor.
examined to 10 mL with pentane R. IDENTIFICATION
Reference solution Dissolve 5 mg of borneol R in pentane R, First identification B.
add 10 µL of linalol R (mixture of (R)-linalol and (S)-linalol) Second identification A.
and 10 µL of linalyl acetate R (mixture of (R)-linalyl acetate A. Thin-layer chromatography (2.2.27).
and (S)-linalyl acetate) and dilute to 10 mL with pentane R.
Column: examined to 1 mL with toluene R.
- size: l = 25 m, 0 = 0.25 mm; Reference solution Dilute 10 µL of cineole R, 5 µL of linalol R
- stationary phase: modified {3-cyclodextrin for chiral and 5 µL of linalyl acetate R to 1 mL with toluene R.
chromatography R (film thickness 0.25 µm). Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel
2023 Lemon Balm IV-323
Mobile phase ethyl acetate R, toluene R (5:95 VIV). Carrier gas helium for chromatography R.
Application 6 µL [or 2 µL] as bands of 10 mm [or 8 mm]. Flow rate 1.5 mL/min.
Development Over a path of 10 cm [or 6 cm]. Split ratio 1:50.
Drying In air. Temperature:
100 °C for 5 min; examine immediately in daylight. Time Temperature
(min) CC)
Column 0 - 15 70
chromatograms obtained with the reference solution and the 15 - 70 70 - 180
test solution. Furthermore, other faint zones may be present Injection port 220
in the chromatogram obtained with the test solution. Detector 220
Top of the plate

A violet zone Injection 1 µL.
-- --
Elution order Limonene, 1,8-cineole, camphor, linalol, linalyl
acetate, ix-terpineol, trans-ix-bisabolene.
Linalyl acetate: a violet zone A faint violet or brown zone may be
present (linalyl acetate) Identification of peaks Use the chromatogram supplied with
spike lavender oil CRS and the chromatogram obtained with
A pink zone
reference solution (a) to identify the peaks due to limonene,
- - - - 1,8-cineole, camphor, linalol, linalyl acetate, ix-terpineol and
trans-ix-bisabolene.
I ,8-Cineole: a violet or brown zone A violet or brown zone (1,8-cineole) - the chromatogram obtained is similar to the
chromatogram supplied with spike lavender oil CRS;
Linalol: a violet zone An intense violet zone (linalol)
limonene and 1,8-cineole.
A greyish or brownisb zone Determine the percentage content of each of the following
ranges:
Reference solution Test solution - limonene: 0.5 per cent to 3.0 per cent;
- 1,8-cineole: 16.0 per cent to 39.0 per cent;
B. The essential oil to be examined complies with the limits - camphor. 8.0 per cent to 16.0 per cent;
of the test for chromatographic profile. - linalol: 34.0 per cent to 50.0 per cent;
- linalyl acetate: maximum 1.6 per cent;
TESTS - rx-terpineol: 0.2 per cent to 2.0 per cent;
Relative density (2.2.5) - trans-rx-bisabolene: 0.4 per cent to 2.5 per cent;
0.894 to 0.907. - reporting threshold: the area of the principal peak in the
Refractive index (2.2.6) chromatogram obtained with reference solution (b)
1.461 to 1.468. (0.05 per cent).
Optical rotation (2. 2. 7) STORAGE
-7° to + 2°. At a temperature not exceeding 25 °C.
Acid value (2.5.J) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Maximum 1.5, determined on 5.00 g of the essential oil to
be examined.
Solubility in alcohol (2.8.10)
1.0 mL of the essential oil to be examined is soluble, Lemon Balm
sometimes with opalescence, in 3.0 mL of ethanol
(70 per cent Vlv'.) R. (Melissa Leaf, Ph. Bur. monograph 1447)
Chromatographic profile Preparation
Gas chromatography (2.2.28): use the normalisation Lemon Balm Dry Extract
procedure. PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried leaf of Melissa officinalis L.
Reference solution (a) Dilute 200 µL of spike lavender oil CRS
to 10.0 mL with heptane R. Content
Minimum 1.0 per cent ofrosmarinic acid (C 18H 160 8;
Reference solution (b) Dilute 5 µL of limonene R to 50.0 mL Mr 360.3) (dried drug).
with heptane R. Dilute 0.5 mL of this solution to 5.0 mL
with heptane R. CHARACTERS
Column: Odour reminiscent of lemon.
- material: fused silica; IDENTIFICATION
- size: l = 60 m, 0 = 0.25 mm; A. The leaves have a petiole of varying length; the lamina is
- stationary phase: macrogol 20 000 R (film thickness broadly ovate, up to about 8 cm long and 5 cm wide, acute
0.25 µm). at the apex and rounded to cordate at the base; the margins
IV-324 Lemon Balm 2023
are crenate to dentate. The upper surface is intense green, Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
the lower surface is paler green and shows a conspicuous plate R (2-10 µm)].
midrib and a raised, reticulate venation; scattered hairs occur Mobile phase ethyl acetate R, hexane R (10:90 V/V).
on the upper surface and along the veins on the lower Application 20 µL [or 4 µL] as bands.
surface, which is also finely punctuate.
Development In an unsaturated tank over a path of 15 cm
[or6cm).
greenish. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic Drying In air.
characters (Figure 144 7. -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. 8.3) [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 (surface
view [Ga]); secretory trichomes with unicellular to tricellular -- --
stalks and unicellular or, more rarely, bicellular heads Citronella!: a grey or greyish-violet A grey or greyish-violet zone
(surface view [Ba], transverse section [F]). zone at the border between the (citronella[) at the border between
upper and middle thirds the upper and middle thirds
-- --
Citral: 2 greyish-violet or bluish- 2 greyish-violet or bluish-violet zones

violet zones at the border between (citral) at the border between the
the middle and lower thirds middle and lower 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 V/V) 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) Rand dilute to 100.0 mL with the
Figure 144 7. -1.- Illustration for identification test B of powdered same solvent. Filter through a 0.45 µm filter.
herbal drug of melissa leaf
C. Thin-layer chromatography (2.2.27). acid CRS in ethanol (50 per cent V/V) R and dilute to
100.0 mL with the same solvent. Dilute 20.0 mL of this
Test solution Place 2.0 g of the powdered herbal drug (355)
solution to 100.0 mL with ethanol (50 per cent V/V) R.
(2.9.12) in a 250 mL round-bottomed flask and add 100 mL
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 Column:
the graduated tube of the apparatus with the aid of a small =
- size: l 0.25 m, 0 =
4.6 mm;
portion of xylene R, and dilute to 1.0 mL with the same - stationary phase: octadecylsilyl silica gel for chromatography R
solvent. (5 µm).
Reference solution Dissolve 1.0 µL of citronella! R and Mobile phase:
10.0 µL of citral R (composed ofneral and geranial) in - mobile phase A: phosphoric acid R, acetonitrile R, water R
25 mL of xylene R. (1:19:80 VIVIV);
2023 Lemon Balm Preparations IV-325
- mobile phase B: phosphoric acid R, methanol R, acetonitrile R Mobile phase anhydrous formic acid R, water R, ethyl acetate R
(1:40:59 VIVIV); (6:6:90 VIVIV).
Application 10 µL [or 2 µL] as bands of 15 mm [or 8 mm] .
(min) (per cent VIV) (per cent VIP)
0 - 20 100 ➔ 55 0 ➔ 45
Drying In air.
20 - 25 55 ➔ 0 45 ➔ 100 Detection Heat at 100 °C for 5 min, spray the plate whilst
25 - 30 0 ➔ 100 100 ➔ 0 still hot with a 5 g/L solution of diphenylboric acid aminoethyl
ester R in ethyl acetate R, and examine in ultraviolet light at
Flow rate 1.2 mIJmin. 365 nm.
Detection Spectrophotometer at 330 nm. Results See below the sequence of fluorescent zones present
Injection 20 µL. and the test solution. Furthermore, other weaker fluorescent
Relative retention With reference to rosmarinic acid zones may be present in the chromatogram obtained with the
(retention time = about 11 min): ferulic acid = about 0.8. test solution.
- resolution: minimum 4.0 between the peaks due to ferulic Top of the plate
following expression: Rosmarinic acid: a light blue An intense light blue fluorescent
fluorescent zone zone (rosmarinic acid)
A1Xmzxpxo.2 A blue fluorescent zone
A 2 xm1
- - --
obtained with reference solution (a); -- --
m1 mass of the herbal drug to be examined used to prepare the test Hyperoside: an orange or greenish-
solution, in grams; yellow fluorescent zone
m2 mass of rosmarinic acid CRS used to prepare reference
solution (a), in grams; A light blue fluorescent zone
p percentage content of rosmarinic acid in rosman'nic acid CRS.
Rutoside: an orange or greenish-
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur yellow fluorescent zone
TESTS
Lemon Balm Dry Extract Loss on drying (2.8.17)
(Melissa Leaf Dry Extract, Ph. Bur. monograph 2524) Maximum 6.0 per cent.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ ASSAY
DEFINITION
Test solution Use brown glass flasks. To 0.200 g of the
Dry extract produced from Melissa leaf (1447).
extract to be examined add 50 mL of ethanol
Content (50 per cent VIV,) R. Sonicate for 10 min and dilute to
Minimum 2.0 per cent of rosmarinic acid (C 18 H 16 0s; 100.0 mL with ethanol (50 per cent VIV,) R. Filter through a
Mr 360.3) (dried extract). membrane filter (nominal pore size 0.45 µm).
PRODUCTION Reference solution (a) Dissolve 20.0 mg of rosmarinic
The extract is produced from the herbal drug by a suitable acid CRS in ethanol (50 per cent Vlv,) R and dilute to
procedure using either hot water (not less than 70 °C) or a 100.0 mL with the same solvent. Dilute 20.0 mL ofthis
hydroalcoholic solvent that is at most equivalent in strength solution to 100.0 mL with ethanol (50 per cent VIV,) R.
to ethanol (70 per cent V/V). Reference solution (b) Dissolve 5 mg of ferulic acid R in
CHARACTERS reference solution (a) and dilute to 50 mL with reference
Appearance solution (a).
Brown or greenish-brown, amorphous powder. Column:
- size: l = 0.25 m, 0 = 4.6 mm;
IDENTIFICATION
- stationary phase: octadecylsi{yl silica gel for chromatography R
Thin-layer chromatography (2.2.27). (5 µm).
Test solution To 0.2 g of the extract to be examined add Mobile phase:
5 mL of methanol R. Sonicate for 5 min and filter. - mobile phase A: phosphoric acid R, acetonitrile R, water R
Reference solution Dissolve 1.0 mg of hyperoside R, 1.0 mg of (1: 19:80 VIVIV);
rutoside trihydrate Rand 5.0 mg of rosmarinic acid R in 10 mL - mobile phase B: phosphoric acid R, methanol R, acetonitrile R
of methanol R. (1:40:59 VIVIV);
IV-326 Lemon Peel 2023
Time Mobile phase A Mobile phase B (c) Apply 20 µL of each solution as bands.
0 - 20 100 ➔ 55 0 ➔ 45
20 - 25 55 ➔ 0 45 ➔ 100
(e) After removal of the plate, dry the plate at 105° and spray
the warm plate with a 1% w/v solution of diphenylboric acid
aminoethyl ester in methanol and then with a 5% w/v solution
Flow rate 1.2 mUmin. of polyethylene glycol 400 in methanol. Allow the plate to stand
Detection Spectrophotometer at 330 run. for 30 minutes and examine under ultraviolet light (365 nm).
Injection 20 µL. MOBILE PHASE
Relative retention With reference to rosmarinic acid A mixture of 10 volumes of anhydrous formic acid, I 0 volumes
=
(retention time about 11 min): ferulic acid about 0.8. = of water, 30 volumes of butan-2-one and 50 volumes of ethyl
System suitability Reference solution (b): acetate.
- resolution: minimum 4.0 between the peaks due to ferulic CONFIRMATION
The chromatogram obtained with solution (1):
following expression: exhibits a yellowish-brown fluorescent zone corresponding in
colour and position to the zone for rutin in the
A1 xmzxpx0.2 chromatogram obtained with solution (2);
A2xm1 above it an intense red fluorescent zone and further above a
area of the peak due to rosmarinic acid in the chromatogram very weak greenish fluorescent zone corresponding in colour
obtained with the test solution; and position to the zone for naringin in the chromatogram
area of the peak due to rosmarinic acid in the chromatogram obtained with solution (2);
other coloured zones are present in the chromatogram.
solution, in grams; TESTS
mass of rosmarinic acid CRS used to prepare reference
solution (a), in grams; Volatile oil
p percentage content of rosmarinic acid in rosman·nic acid CRS. Not less than 2.0% v/w (1. 7% w/w), Appendix XI E, using
300 mL of water as the distillation liquid and no xylene in
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. Bur. monograph 0620)
the ripe, or nearly ripe, fruit of Citrus limon (L.) Burm. f.
Preparation
CHARACTERISTICS Terpeneless Lemon Oil
It has the macroscopical and microscopical characters
described under 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 limon (L.) Burm.
marked thickening of the epicarp around the calyx. The outer
f.
surface is yellow and somewhat rough from the presence of
numerous minute pits, each corresponding to an oil gland; CHARACTERS
the inner surface with only a small remnant of white, spongy Appearance
pericarp. Fracture, short. Clear, mobile, pale yellow or greenish-yellow liquid. It may
B. Prepare thin cross sections from material softened by become cloudy at low temperatures.
soaking in water and examine under a microscope using Characteristic odour.
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
small strands of vascular tissue; prismatic crystals of calcium Second identification: A.
oxalate occur throughout the parenchyma and are A. Thin-layer chromatography (2.2.27).
particularly abundant in the layers adjacent to the epidermis. Test solution Mix 1 mL of the substance to be examined in
C. Carry out the method for thin-layer chromatography, 1 mL of toluene R.
Appendix III A, using the following solutions. Reference solution Dissolve 10 mg of citropten Rand 50 µL of
( 1) Add 1 g of freshly cut peel to 10 mL of methanol and citral R in toluene R and dilute to 10 mL with the same
heat in a water-bath at 65° for 5 minutes, shaking frequently. solvent.
Allow to cool and filter. Plate TLC silica gel GF254 plate R.
(2) 0.01 % w/v each of caffeic acid and naringi,n and Mobile phase ethyl acetate R, toluene R (15:85 VIV).
0.025% w/v of rutin in methanol. Application 10 µL, as bands.
CHROMATOGRAPHIC CONDITIONS DevelO[>ment Over a path of 15 cm.
(a) Using a TLC silica gel plate. Drying In air.
2023 Lemon Oil IV-327
Detection A Examine in ultraviolet light at 254 nm. abscissae. Draw as a baseline the tangent between A and B
Results A See below the sequence of the zones present in (Figure 0620.-1). The absorption maximum C is situated at
the chromatograms obtained with the reference solution and 315 ± 3 nm. From C draw a line perpendicular to the axis
the test solution. of abscissae and intersecting AB at D. Deduct the
absorbance corresponding to point D from that
corresponding to point C. The value C - Dis 0.20 to 0.96
Top of the plate
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) 1.00r---,-,-----,------,---,-----,

A dark blue zone (5-geranyloxy-7- C.
methoxycoumarin)
Citropten: a light blue fluorescent A light blue fluorescent zone
zone (citropten)
A quenching zone (psoralen

derivative)
A quenching zone (biakangelicin)

Q)
0
C
Detection B Examine in ultraviolet light at 365 nm. CU
_o
Results B See below the sequence of the zones present in 0
(/)
the chromatograms obtained with the reference solution and _o
the test solution. <C
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 fluorescent A bright violet-blue fluorescent zone
zone (citropten)
260 280 300 320 340 360 380 400
A yellow fluorescent zone (psoralen Wavelength (nm)
derivative)
An orange zone (biakangelicin)
Figure 0620.-1. - Typical spectrum of lemon oil for the test for
Reference solution Test solution absorbance
Fatty oils and resinified essential oils (2. 8. 7)

B. Examine the chromatograms obtained in the test for It complies with the test for fatty oils and resinified essential
chromatographic profile. oils.
obtained with the test solution are similar in retention rime to
procedure.
solution.
Test solutum The substance to be examined.
TESTS Reference solution Dissolve 20 µL of fJ-pinene R, 10 µL of
Relative density (2.2.5) sabinene R, 100 µL of limonene R, 10 µL of y-terpinene R,
0.850 to 0.858. 5 µL of /3-caryophyllene R, 20 µL of citral R, 5 µL of
Refractive index (2.2.6) rx-terpineol R, 5 µL of neryl acetate R and 5 µL of geranyl
1.473 to 1.476. acetate R in 1 mL of acetone R.
Optical rotation (2. 2. 7) Column:
+ 5.7° to+ 7.0° (measured in a 0.1 dm tube). - material: fused silica;
Absorbance (2.2.25) - size: l = 30 m (a film thickness of 1 µm may be used) to
Dissolve 0.250 g of the substance to be examined in ethanol 60 m (a film thickness of 0.2 µm may be used),
(96 per cent) R, mix and dilute to 100.0 mL with the same 0 = 0.25-0.53 mm;
solvent. Measure the absorbance over the range 260 nm to - stationary phase: macrogol 20 000 R.
400 nm. If a manual instrument is used, measure the Carrier gas helium for chromatography R.
absorbance at 5 nm intervals from 260 nm to about 12 nm Flow rate 1.0 mUmin.
before the expected absorption maximum, then at 3 nm Split ratio 1: 100.
intervals for 3 readings and at 1 nm intervals to about 5 nm
beyond the maximum and finally at 10 nm intervals to
400 nm. Plot a curve representing the absorption spectrum
with the absorbances as ordinates and the wavelengths as
IV-328 Lemon Oil 2023
Temperature: TESTS
Optical rotation
Time Temperature -5° to +2°, Appendix VF.
(min) (°C)
Refractive index
Column 0-6 45
6 - 21 45--> 90
1.475 to 1.485, Appendix VE.
21 - 39 90 --> 180 Solubility in ethanol
39 - 55 180 Soluble, at 20°, in 1 volume of ethanol (80%),
Injection port 220 Appendix X M.
Detector 220
Weight per mL
0.880 to 0.895 g, Appendix V G.
ASSAY
Injection 0.5 µL of the reference solution and 0.2 µL of the
test solution. Carry out the method for determination of aldehydes,
Appendix X K, using 1 g, omitting the toluene and using a
Elution order Order indicated in the composition of the volume, not less than 7 mL, of alcoholic hydroxylamine solutwn
reference solution. Record the retention times of these that exceeds by 1 to 2 mL the volume of 0.5M potassium
substances. hydroxide in ethanol (60%) VS required. Each mL of
System suitability Reference solution: 0.5M potassium hydroxide in ethanol (60%) VS is equivalent to
- resolution: minimum 1.5 between the peaks due to 76.73 mg ofC10H16O.
~-pinene and sabinene; minimum 1.5 between the peaks
due to geranial and geranyl acetate. STORAGE
Terpeneless Lemon Oil should be kept in a well-filled
container and protected from light.
Determine the percentage content of these components. Lemon Spirit
The percentages are within the following ranges:
DEFINffiON
- {3-pinene: 7 .0 per cent to 17 .0 per cent;
- sabinene: 1.0 per cent to 3.0 per cent;
T erpeneless Lemon Oil lOOmL
- limonene: 56.0 per cent to 78.0 per cent; Ethanol (96 per cent) Sufficient to produce 1000 mL
- y-terpinene: 6.0 per cent to 12.0 per cent;
- {3-caryophyllene: maximum 0.5 per cent; The spirit complies with the requirements stated under Spirits and
- neral: 0.3 per cent to 1.5 per cent; with the following requirements.
- rx-terpineol: maximum 0.6 per cent;
Content of aldehydes
- neryl acetate: 0.2 per cent to 0.9 per cent;
3.45 to 4.60% w/v, calculated as citral, C10H16O.
- geranial: 0.5 per cent to 2.3 per cent;
- geranyl acetate: 0.1 per cent to 0.8 per cent. TESTS
Residue on evaporation (2.8.9) Ethanol content
1.8 per cent to 3.6 per cent after heating on a water-bath 84 to 88% v/v, Appendix VIII F.
for 4 h. Weight per mL
STORAGE 0.814 to 0.823 g, Appendix VG.
At a temperature not exceeding 25 °C. ASSAY
LABELLING Carry out the method for the determination of aldehydes,
Appendix X K, using 10 mL, omitting the toluene and using
The label states, where applicable, that the contents are
a volume, not less than 7 mL, of alcoholic hydroxylamine
Italian-type lemon oil.
solution that exceeds by 1 to 2 mL the volume of
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
0.5M potassium hydroxide in ethanol (60%) VS required.
Each mL of 0.5M potassium hydroxide in ethanol (60%) VS is
equivalent to 76.73 mg of C10H16O.
Terpeneless Lemon Oil

Preparations
Lemon Spirit
Lemon Syrup
Compound Orange Spirit DEFINITION
DEFINITION Lemon Spirit 5mL

Terpeneless Lemon Oil may be prepared by concentrating Citric Acid Monohydrate 25 g
Lemon Oil under reduced pressure until most of the terpenes Invert Syrup IOOmL
Syrup Sufficient to produce 1000 mL
have been removed or by solvent partition. It contains not
less than 40% w/w of aldehydes calculated as citral,
C10H16O.
CHARACTERISTICS Dissolve the Citric Acid Monohydrate in some of the Syrup,
A clear, colourless or pale yellow liquid, visibly free from add the Invert Syrup, the Lemon Spirit and sufficient Syrup
water; odour and taste, those of lemon. to produce 1000 mL and mix.
2023 Lemon Verbena Leaf IV-329
CHARACTERISTICS
Lemon Syrup has a weight per mL of about 1.33 g.
The syrup complies with the requirements stated under Oral
Content of citric acid monohydrate, C6 H8 0 7 ,H20
2.2 to 2.6% w/v.
ASSAY
Mix 8 g with 100 mL of water and titrate with 0.1 M sodium
hydroxide VS using phenolphthalein solution Rl as indicator.
Each mL of O. IM sodium hydroxide VS is equivalent to
7.005 mg of C 6 H 8 O 7,H2O. Determine the weight per mL,
Appendix V G, and calculate the content of C 6 H 8 O1,H2O,
weight in volume.
Lemon Verbena Leaf

DEFINITION
Whole or fragmented, dried leaves of Aloysia citrodllra Palau
(syn. Aloysia triphylla (L'Her.) Kuntze; Verbena triphylla
L'Her.; Lippia citriodllra Kunth).
Content
- acteoside (C 29 H 36 O 15; Mr 625): minimum 2.5 per cent,
expressed as ferulic acid (dried drug);
- essential oil: minimum 3.0 mL/kg for the whole drug and
minimum 2.0 mL/kg for the fragmented drug (dried Figure 1834.-1. - Illustration for identification test B of powdered
drug). herbal drug of lemon verbena leaf
CHARACTERS
After grinding, it has a characteristic odour reminiscent of Verbena officinalis.
lemon.
IDENTIFICATION chromatograms obtained with the reference solution and the
A. The leaves are simple with short petioles. They are test solution. Furthermore, other faint zones may be present
narrow, lanceolate, and about 4 times longer than they are in the chromatogram obtained with the test solution. Zones
wide. The entire, slightly undulating margins are curled may be present in the chromatogram obtained with the test
towards the upper surface. The upper surface is dark green solution below the zone due to rutoside in the chromatogram
and rough to the touch; the lower surface is paler green and obtained with the reference solution.
shows a prominent midrib with secondary veins running to
the margins. Top of the plate
green. Examine under a microscope using chloral hydrate ~- --
solution R. The powder shows the following diagnostic An intense greyish-green zone
characters (Figure 1834.-1 ): fragments of the upper
Arbutin: a blue or brown zone
epidermis of the lamina (surface view [A, B, H]), composed
of polygonal cells with numerous short, unicellular, thick-
walled cystolithic trichomes, each arising from a rosette of
Rutoside: a dark brownish-yellow A blue or violet zone
cells at the base and containing calcium concretions [B], zone
glandular trichomes with a unicellular stalk and a unicellular,
globular head of variable size (surface view [Ha], transverse -- --
section [D, F]); these fragments are usually accompanied by Reference solution Test solution
palisade parenchyma [Aa, Hb]; fragments of the lower
epidermis of the lamina (surface view [El) covered by a
striated cuticle and composed of cells more irregular and TESTS
somewhat sinuous in outline, with abundant anomocytic Verbena officinalis
stomata (2.8.3) [Ea] and numerous glandular trichomes in Thin-layer chromatography (2.2.27).
surface view [Eb] and/or their scars [Ee]; fragments of the Test solution To 0.50 g of the powdered herbal drug (710)
lamina (transverse section [G]) with 2 layers of palisade (2. 9.12) add 5 mL of methanol R. Heat in a water-bath at
parenchyma [Ga] and spongy parenchyma [Gb]; lignified 60 °C for 10 min. Cool and filter.
tissue from the veins [C].
Reference solution Dissolve 10 mg of arbutin Rand 10 mg of
rutoside trihydrate R in methanol R and dilute to 10 mL with
the same solvent.
IV-330 Ligusticum Root and Rhizome 2023
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel A1 x m2 xpx 0.5 x 3.1
plate R (2-10 µm)]. A2 xm1
Mobile phase anhydrous formic acid R, glacial acetic acid R, area of the peak due to acteoside in the chromatogram obtained
water R, ethyl acetate R (11:11:27:100 VIVIVIV). with the test solution;
area of the peak due to ferulic acid in the chromatogram
Development Over a path of about 12 cm [or 6 cm]. mass of the herbal drug in the test solution, in grams;
Drying In air. mass of fernlic acid CRS in the reference solution, in grams;
percentage content of ferulic acid in ferulic acid CRS;
Detection Spray with anisaldehyde solution R and dry at correlation factor between acteoside and ferulic acid.
100-105 °C for about 10 min; examine in daylight.
Results The chromatogram obtained with the test solution Essential oil (2. 8.12)
shows no brownish-grey zone at a position between that of Introduce 25.0 g of the freshly crushed herbal drug into a
arbutin and rutoside in the chromatogram obtained with the 1000 mL flask and add 500 mL of a 10 g/L solution of
reference solution. sodium chloride R as the distillation liquid. Use 0.50 mL of
Loss on drying (2.2.32) xylene R in the graduated tube. Distil at a rate of
Maximum 10.0 per cent, determined on 1.000 g of the 3.0-3.5 mLJmin for 3 h.
powdered herbal drug (710) (2. 9.12) by drying in an oven at _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
105 °C for 2 h.
Total ash (2.4.16)
Ligusticum Root and Rhizome
Maximum 3.5 per cent. (Ph. Eur. monograph 2431)
ASSAY PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Acteoside
Test solution To 1.00 g of the powdered herbal drug (710) Whole or fragmented, dried rhizome and root of Ligusticum
(2.9.12) add 50.0 mL of the reference solution and stir for sinense Oliv. or Ligusticum jeholense (Nakai & Kitag.) Nakai &
2 h with a magnetic stirrer. Centrifuge for 15 min and pass Kitag.
the supernatant through a membrane filter (nominal pore Content
size 0.45 µm). Minimum 5.0 mUkg of essential oil (dried drug).
Reference solution Dissolve 10.0 mg offerulic acid CRS in IDENTIFICATION
ethanol (60 per cent V/V) Rand dilute to 100.0 mL with the A. The rhizome is short, up to 3 cm in diameter, brown or
same solvent. yellowish-brown, simple or somewhat twisted. The roots are
Precolumn: usually up to 1.5 mm thick, show no ramification, and are
=
- size: l 0.01 m, 0 = 4.0 mm; the same colour as the rhizome; the fracture is usually
- stationary phase: octadecylsilyl silica gel for chromatography R fibrous; the remaining stem bases are cylindrical, 5-6 mm
(5 µm). thick and their longitudinal fracture shows a yellowish-white
Column: medulla. The fragmented rhizome occurs as more or less
= =
- size: l 0.25 m, 0 4.0 mm; thick slices.
- stationary phase: octadecylsilyl silica gel for chromatography R The rhizome of Ligusticum sinense shows an irregular brown
(5 µm); or blackish-brown outer surface; the yellowish-white or pale
- temperature: 20 °C. yellowish-brown transverse section is fibrous, porous and
Mobile phase: cracked.
- mobile phase A: 0.3 per cent V/V solution of phosphoric The rhizome of Ligusticum jeholense shows root scars and the
acid R; spiny remains of roots on its outer surface; a transverse
- mobile phase B: acetonitrile R; section shows radial striations and cracks in the xylem.
Time Mobile phase A Mobile phase B brownish-beige. Examine under a microscope using chloral
(min) (per cent V/Jl) (per cent V/Jl)
0 - 20 93 ➔ 83 7 ➔ 17 characters (Figure 2431.-1): cork fragments made up of cells
20 - 30 83 17 with brown contents which are polygonal in surface view [C];
30 - 35 83 ➔ 75 17-, 25 numerous fragments of parenchyma consisting of rounded or
35 - 40 75-, 20 25-, 80 ovoid cells, with thin or slightly thickened walls [H];
40 - 45 20-, 93 80 ➔ 7 fragments of brownish-yellow secretory canals up to 170 µm
wide (transverse section [A], longitudinal section [El);
Flow rate 1.0 mIJmin. fragments of reticulate vessels about 80 µm in diameter,
Detection Spectrophotometer at 330 nm. isolated or in groups of 2 or 3 [BJ; groups of smaller
Injection 20 µL. reticulate vessels surrounded by either xylem parenchyma [G]
System suitability Test solution:
or by lignified fibres with thick, moderately pitted walls m;
fibres are isolated [L] or in groups of 2 or 3, colourless, often
- resolution: minimum 3.5 between the peaks due to ferulic whole with pitted walls, sometimes reaching 700 µm in
acid and acteoside. length and about 20 µm wide; round or rectangular sclereids,
Calculate the percentage content of acteoside, expressed as isolated [F] or grouped in clusters [DJ, with thickened and
ferulic acid, using the following expression: channelled walls (some of the larger sclereids may be up to
2023 Ligusticum Root and Rhizome IV-331
100 µm long). Examine under a microscope using a be present in the chromatogram obtained with the test
50 per cent V/V solution of glycerol R. The powder shows solution.
many isolated, small (5-10 µm), round or ovoid starch
granules [Kl . Top of the plate
A quenching zone (L. jeholense)
(Z)-Ligustilide: a blue fluorescent A blue fluorescent zone ((Z)-

zone ligustilide)
--- ---
2 quenching zones
Osthole: a quenching zone
Imperatorin: a quenching zone
--- ---

test solution. Furthermore, other faint zones of various
colours may be present in the chromatogram obtained with
the test solution.
Top of the plate
2 prominent reddish zones
A brown zone (L. jeholense)
(Z)-Ligustilide: a grey zone
A violet zone
--- ---
Imperatorin: a reddish-grey zone A broad reddish zone

herbal drug of ligusticum root and rhizome A violet zone
C. Thin-layer chromatography (2.2.27). ---

---
Examine the chromatograms obtained in the test for officinal Several faint violet zones
species of Angelica and Levisticum.
test solution. Furthermore, other faint fluorescent zones may TESTS
be present in the chromatogram obtained with the test Officinal species of Angelica and Levisticum
solution. Thin-layer chromatography (2.2.27).
Test solution To 1 g of the freshly powdered herbal drug
Top of the plate (355) (2.9.12) add 4 mL of methanol Rand sonicate for
(Z)-Ligustilide: a bluish-white A bluish-white fluorescent zone ((Z)-
5 min. Centrifuge and use the supernatant.
fluorescent zone ligustilide) Reference solution Dissolve 1 mg of imperatonn R, l mg of
(Z)-ligustilide R and 1 mg of osthole R in 1 mL of methanol R.
A faint bluish-white fluorescent zone
--- ---
Mobile phase glacial acetic acid R, ethyl acetate R, toluene R
Osthole: a blue fluorescent zone (1:10:90 VIVIV).
lmperatorin: a whitish fluorescent A faint blue fluorescent zone Application 4 µL as bands of 8 mm.
zone Development Over a path of 6 cm.
-- ---
Drying In air.
A faint bluish-white fluorescent zone
Reference solution Test solution shows no prominent blue fluorescent zone directly below or
above the zone due to imperatorin in the chromatogram
Results B See below the sequence of zones present in the obtained with the reference solution.
chromatograms obtained with the reference solution and the Detection B Examine in ultraviolet light at 254 nm.
IV-332 Lime Flower 2023
Results B The chromatogram obtained with the test solution sinuous anticlinal walls [HJ and an abaxial epidermis [CJ
shows no zone corresponding in position to, or directly with cells with wavy-sinuous walls [Ca] and anomocytic
below, the zone due to imperatorin in the chromatogram stomata (2.8.3) [Cb], covered by a finely striated cuticle;
obtained with the reference solution. fragments of the sepals [A] with epidermal cells with rigid
Detection C Treat with a 10 per cent VIV solution of sulfuric anticlinal walls [Aa] with rounded to oval covering trichome
acid R in methanol R; heat at 100 °C for 5 min and examine scars with thick, channelled and coarsely pined walls [Ac],
in daylight. thick-walled covering trichomes, unicellular, isolated [Ab] or
stellate with up to 5 cells and measuring up to 350 µm [K);
Results C The chromatogram obtained with the test solution
fragments of the mesophyll of the sepals or of the lamina of
shows no distinct greenish zone below the zone due to
the petals (transverse section [J]), composed of
imperatorin in the chromatogram obtained with the reference
parenchymatous cells containing cluster crystals of calcium
solution.
oxalate Ua] and large mucilaginous cells Ubl; fragments of
Foreign matter (2.8.2) the adaxial epidermis of the petals [BJ consisting of straight-
Maximum 3 per cent, determined on 50 g. walled cells [Ba] and elongated multicellular glandular
Loss on drying (2.2.32) trichomes [Bb); pollen grains with 3 germinal pores and a
Maximum 12.0 per cent, determined on 1.000 g of the finely granulated exine, about 30-40 µm in diameter [G];
powdered herbal drug (355) (2. 9.12) by drying in an oven at twisted, unicellular trichomes from the ovary may be present,
105 °C. isolated [D, E, F] or stellate with 2-4 branches.
Total ash (2.4.16)
ASSAY
Use 25.0 g of the powdered herbal drug (500) (2.9.12)
immediately before the assay using a blade grinder
refrigerated at 5 °C, a 1000 mL round-bottomed flask,
500 mL of water R as the distillation liquid, 10 drops of
liquid paraffin R, a few grains of pumice and 0.50 mL of
o-xylene R in the graduated tube. Distil at a rate of
2-3 mUmin for 3 h.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Lime Flower
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole, dried inflorescence of Tilia cordata Mill., of Tilia
platyphyllos Scop., of Tilia x europaea L. or a mixture of
these.
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 Figure 0957.-1. - Illustration for identification test B of powdered
which is joined for up to about half of its length with the herbal drug of lime flower
peduncle. The inflorescence usually consists of 2-7 flowers,
occasionally up to 16. The sepals are detached easily from C. High-performance thin-layer chromatography (2.8.25).
the perianth; they are up to 6 mm long, their abaxial surface Test solution To 0.5 g of the powdered herbal drug (355)
is usually glabrous, their adaxial surface and their borders are (2.9.12) add 5.0 mL of methanol R. Sonicate for 15 min,
strongly pubescent. The 5 spatulate, thin petals are yellowish- filter or centrifuge and use the filtrate or supernatant.
white, up to 8 mm long. They show fine venation and their
borders only are sometimes covered with isolated trichomes.
5.0 mg of rutoside trihydrate R in methanol Rand dilute to
The numerous stamens are free and usually constitute
5 groups. The superior ovary has a pistil with a somewhat
5-lobate stigma. Reference solution (b) Dilute 2.5 mL of reference solution (a)
yellowish-green. Examine under a microscope using chloral Reference solution (c) Dissolve 5 mg of hyperoside R and
hydrate solution R. The powder shows the following diagnostic 2 mg of chlorogenic acid R in methanol R and dilute to 10 mL
characters (Figure 0957.-1): fragments of the bract showing with the same solvent.
an adaxial epidermis with cells with straight or slightly
2023 Linseed IV-3 3 3
lntensiry markers Hyperoside for the yellow or orange zones Loss on drying (2.2.32)
in the middle third of the chromatogram and rutoside for the Maximum 12.0 per cent, determined on 1.000 g of the
yellow or orange zones at the top of the lower third of the powdered herbal drug (355) (2. 9.12) by drying in an oven at
chromatogram. 105 °C for 2 h.
Plate TLC silica gel F 254 plate R (2-10 µm). Total ash (2.4.16)
Mobile phase anhydrous formic acid R, water R, methyl ethyl Maximum 8.0 per cent.
ketone R, ethyl acetate R (10:10:30:50 VIV!VIV). _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur

Linseed
400 R in methanol R or, alternatively, dip the warm plate in a When Powdered Linseed is prescribed or demanded, material
5 g/L solution of diphenylboric acid aminoethyl ester R in ethyl complying with the appropriate requirements below shall be
acetate R, then in a 50 g/L solution of macrogol 400 R in dispensed or supplied.
methylene chloride R; allow to dry in air for about 1 min and PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried, ripe seeds of Linum usitatissimum L.
zones, which may be touching; the lower zone IDENTIFICATION
(chlorogenic acid) shows a light blue fluorescence and A. The seed has a flattened, elongated ovoid shape. The testa
the upper zone (hyperoside) shows a yellow or orange is dark reddish-brown or yellow, smooth and shiny.
fluorescence. The seeds are 4-6 mm long, 2-3 mm wide and 1.5-2 mm
Results See below the sequence of fluorescent zones present thick; one end is rounded and the other end forms an
in the chromatograms obtained with reference solution (a) oblique point near which the hilum appears as a slight
and the test solution. Furthermore, other faint blue, yellow or depression. When viewed with a lens, the surface of the seed-
orange, brownish and greenish fluorescent zones may be coat is seen to be minutely pitted. Inside the testa a narrow,
present in the chromatogram obtained with the test solution, whitish endosperm and an embryo composed of 2 large,
especially in the upper and lower thirds. The zones below flattened, yellowish and oily cotyledons are present; the
hyperoside show a relatively high variation in intensity and radicle points towards the hilum.
colour. B. Microscopic examination (2.8.23). The powder is greasy
to the touch. Examine under a microscope using chloral
Top of the plate hydrate solution R. The powder shows the following diagnostic
characters (Figure 0095.-1): fragments of the outer testa [A,
A blue zone, faint to equivalent BJ with cells that are polygonal (surface view [Aa]) or narrow
(transverse section [Bal) and filled with mucilage [Bb];
-- --
fragments of the collenchymatously thickened sub-epidermal
2 yellow or orange zones, faint to layer (transverse section [Be], surface view [Ab]) with
equivalent
rounded cells with triangular intercellular spaces often
A yellow or orange zone, equivalent attached to the sclerenchymatous layer composed of
Hyperoside: a yellow or orange zone elongated cells, with thickened and pitted walls [Ca], some
with strongly thickened and pitted walls (G]; fragments
A brownish zone, very faint to faint (surface view [CJ) consisting of the hyaline layer with thin-
walled cells [Cb] often remaining attached to the layer of
elongated sclereids and crossing them at approximately right
A yellow, orange or greenish zone,
angles [Ca]; fragments of the inner testa (surface view [DJ),
faint
composed of moderately thickened polygonal cells filled with
- - - -
brown-orange pigment; small polyhedral masses of pigment
Rutoside: a yellow or orange zone A yellow, orange or bluish zone, faint [HJ; numerous fragments of parenchyma from the testae,
to equivalent with large, slightly and regularly thickened cells (surface view
Reference solution (a)
U, L]); parenchyma of the endosperm [K] and cotyledons
Test solution
[E] containing aleurone grains and oil droplets; very
numerous isolated oil droplets [F] .
TESTS TESTS
Foreign matter (2.8.2) Foreign matter (2.8.2)
Maximum 2 per cent, determined on 30 g. Maximum 10 per cent of seeds with a dull coat and
Tilia americana L. or Tilia tomentosa Moench maximum 1.5 per cent of other foreign matter.
There are no inflorescences with a bract bearing on the Swelling index (2.8.4)
abaxial surface, stellate, 5- to 8-rayed covering trichomes and Minimum 4.
flowers having an apparent double corolla by transformation
Cadmium (2.4.27)
of 5 stamens into petal-like staminoids, the pistil is neither
Maximum 0.5 ppm.
lobular nor indented.
IV-334 Liquorice 2023
Bb 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.
yellow or faintly greyish. Examine under a microscope using
. . chloral hydrate solution R. The powder shows the following
C diagnostic characters: fragments of yellow thick-walled fibres,
. E
700-1200 µm long and 10-20 µm wide with a punctiform
.. o.,,l@H lumen, often accompanied by crystal sheaths containing
~ :1~ ~oil,
prisms of calcium oxalate 10-35 µm long and 2-5 µm wide.
The walls of the vessels are yellow, 5-10 µm 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.
The powder shows the following diagnostic characters:
J simple, round or oval starch granules, 2-20 µm in diameter.
(2.9.12) in a 50 mL round-bottomed flask add 16.0 mL of
water R and 4.0 mL of hydrochloric acid Rl and heat on a
water-bath under a reflux condenser for 30 min. Cool and
filter. Dry the filter and the round-bottomed flask at 105 °C
for 60 min. Place the filter in the round-bottomed flask, add
herbal drng of linseed
20.0 mL of ether R and heat in a water-bath at 40 °C under
Loss on drying (2.2.32) a reflux condenser for 5 min. Cool and filter. Evaporate the
Maximum 8.0 per cent, determined on 1.000 g of the filtrate to dryness. Dissolve the residue in 5.0 mL of ether R.
powdered herbal drug (355) (2. 9.12) by drying in an oven at Reference solution Dissolve 5.0 mg of glycyrrhetic acid Rand
105 °C for 2 h. 5.0 mg of thymol R in 5.0 mL of ether R.
Total ash (2.4.16) Plate TLC silica gel F254 plate R.
Maximum 5.0 per cent. Mobile phase concentrated ammonia R, water R, ethanol
(96 per cent) R, ethyl acetate R (1:9:25:65 VIVIVIV).
Application l O µL.
Liquorice Detection A Examine in ultraviolet light at 254 nm.
(Liquorice Root, Ph. Bur. monograph 0277) Results A The chromatograms obtained with the test
solution 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 R, 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
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 (isoliquiridigenine)
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.
chromatogram obtained with the reference solution. Further
Content zones may be present.
Minimum 4.0 per cent of 18~-glycyrrhizic acid (C4 2H62016;
Mr 823) (dried drug). TESTS
IDENTIFICATION Maximum 10.0 per cent, determined on 1.000 g of the
A. The root has few branches. Its bark is brown or brownish- powdered herbal drug (355) (2.9.12) by drying in an oven at
grey with longitudinal striations and bears traces of lateral 105 °C for 2 h.
roots. The cylindrical stolons are 1-2 cm in diameter; their
2023 Liquorice Preparations IV-335
Total ash (2.4.16)

Maximum 10.0 per cent for the unpeeled drug and
Liquorice Dry Extract for
maximum 6.0 per cent for the peeled drug. Flavouring Purposes
Ash insoluble in hydrochloric acid (2.8.1) (Ph. Bur. monograph 2378)
Maximum 2.0 per cent for the unpeeled drug and maximum
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
0.5 per cent for the peeled drug.
Ochratoxin A (2.8.22) DEFINITION
Maximum 20 µg per kilogram of herbal drug. Dry extract produced from Liquorice root (0277).
ASSAY Content
Liquid chromatography (2.2.29). 5.0 per cent to 7.0 per cent of 18~-glycyrrhizic acid
(C42H62O16; M, 823) (dried extract).
(180) (2.9.12) in a 150 mL ground-glass conical flask. PRODUCTION
Add 100.0 mL of an 8 g/L solution of ammonia Rand treat The extract is produced from the cut herbal drug by a
in an ultrasonic bath for 30 min. Centrifuge a part of the suitable procedure using water.
solution and dilute 1.0 mL of the supernatant layer to CHARACTERS
5.0 mL with an 8 g/L solution of ammonia R. Filter through Appearance
a membrane filter (nominal pore size 0.45 µm); use the Yellowish-brown or brown powder.
filtrate as the test solution.
Very sweet taste.
Solution A Dissolve 0.130 g of monoammonium
glycyrrhizate CRS in an 8 g/L solution of ammonia R and IDENTIFICATION
dilute to 100.0 mL with the same solvent. Thin-layer chromatography (2.2.27).
Reference solution (a) Dilute 5.0 mL of solution A to Solvent mixture ethyl acetate R, methanol R (50:50 V/V).
100.0 mL with an 8 g/L solution of ammonia R. Test solution To 0.30 g of the extract to be examined add
Reference solution (b) Dilute 10.0 mL of solution A to 30 mL of hydrochloric acid Rl and boil on a water-bath under
100.0 mL with an 8 g/L solution of ammonia R. a reflux condenser for 60 min. After cooling, extract the
Reference solution (c) Dilute 15.0 mL of solution A to mixture with 2 quantities, each of 20 mL, of ethyl acetate R.
100.0 mL with an 8 g/L solution of ammonia R. Combine the organic layers and filter through a filter covered
with anhydrous sodium sulfate R. Evaporate the filtrate to
Column:
dryness in vacuo and dissolve the residue in 2.0 mL of the
- size: l = 0.10 m, 0 = 4 mm;
solvent mixture.
- stationary phase: octadecylsilyl siHca gel for chromatography R
(5 µm). Reference solution Dissolve 5.0 mg of glycyrrhetic acid Rand
5.0 mg of thymol R in 5.0 mL of the solvent mixture.
Mobile phase glacial acetic acid R, acetonitrile R, water R
(6:30:64 VIVIV). Plate TLC silica gel F 254 plate R (5-40 µm) [or TLC silica
gel F2s4plate R (2-10 µm)].
Mobile phase concentrated ammonia R, water R, ethanol
(96 per cent) R, ethyl acetate R (1:9:25:65 VIVIVIV).
Injection I O µL.
Establish a calibration curve with the mass of
Develvpment Over a path of 15 cm [or 7 cm].
monoammonium glycyrrhizate in the reference solutions, in
grams, as the abscissa and the corresponding peak areas as Drying In air for 5 min.
the ordinate. Detection Spray with anisaldehyde solution R and heat at
Using the retention times and the peak areas determined 100-105 °C for 5-10 min; examine in daylight.
from the chromatograms obtained with the reference Results See below the sequence of zones present in the
solutions, locate and integrate the peak due to 18~- chromatograms obtained with the reference solution and the
glycyrrhizic acid in the chromatogram obtained with the test test solution. Furthermore, other faint zones may be present
solution. in the chromatogram obtained with the test solution.
Calculate the percentage content of 18~-glycyrrhizic acid
using the following expression: Top of the plate
5 823
Ax-xBx-
m 840 Thymol: a red zone
A mass equivalent of monoammonium glycyrrhizate in the test A yellow zone

solution, determined from the calibration curve, in grams;
B declared percentage content of monoarnmonium -- - -
glycyrrhizate CRS;
- - --
solution, in grams; Glycyrrhetic acid: a violet zone A violet zone (glycyrrhetic acid)
823 molecular mass of l 8!3-glycyrrhizic acid;
840 molecular mass of monoammonium glycyrrhizate (without any
water of crystallisation).
LABELLING
The label states whether the drug is peeled or unpeeled.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
IV-336 Liquorice Liquid Extract 2023
TESTS
Liquorice Liquid Extract
Maximum 7 .0 per cent. DEFINITION
Ochratoxin A (2.8.22) Liquorice Liquid Extract is produced from Liquorice.
Maximum 80 µg per kilogram of extract. It contains not less than 1.4% w/v of 18~-glycyrrhizic acid,
C42H62016•
The maximum content applies to the pure undiluted extract.
Where excipients are added to reduce the strength of the CHARACTERISTICS
extract, the maximum content should be reduced A dark brown, clear liquid containing no more than a trace
proportionally. of deposit.
ASSAY PRODUCTION
Liquid chromatography (2.2.29). The liquid extract is produced from Liquorice by extraction
Solvent mixture water R, methanol R (20:80 V/V). with water, precipitation of unwanted constituents, followed
by concentration of the extraction liquors and the addition of
Test solution Place 0.200 g of the extract to be examined in
sufficient ethanol (96%) and water to produce the required
a 150 mL ground-glass conical flask. Add 100.0 mL of the
volume. The liquid extract must stand for a minimum of 4 to
solvent mixture and sonicate for 2 min. Filter through a
8 weeks prior to decantation of the clear supernatant liquid.
The extract complies with the requirements stated under Extracts
Reference solution Dissolve 50.0 mg of monoammonium
glycyrrhizate CRS in the solvent mixture and dilute to
50.0 mL with the solvent mixture. Dilute 1.0 mL of this IDENTIFICATION
solution to 10.0 mL with the solvent mixture. Carry out the method for high performance thin-layer
Column: chromatography, Appendix XI W, using the following
- size: l = 0.10 m, 0 = 4.0 mm; solutions.
- stationary phase: octadecylsilyl silica gel for chromatography R ( 1) Dilute 1 volume of the preparation being examined to
(5 µm). 10 volumes with ethanol (70%).
Mobile phase glacial acetic acid R, acetonitrile R, water R (2) 0.05% w/v of hyperoside and 0.05% w/v of rutin in
(6:30:64 VIVIV). methanol.
Flow rate 1.5 mUmin. (3) Dilute 1 volume of solution (2) to 4 volumes of methanol.
Detection Spectrophotometer at 254 nm. (4) 0.05% w/v of hyperoside and 0.05% w/v of chlorogenic acid
Injection 10 µL. in methanol.
Run time 3 times the retention time of 18~-glycyrrhizic acid. (5) 1.4% w/v of 18{3-glycyrrhizic acid in methanol.
Retention time 18~-glycyrrhizic acid = about 9 min. CHROMATOGRAPHIC CONDITIONS
Identification of peaks Use the chromatogram supplied with (a) Use as the coating silica gel F 254 (Merck silica gel 60 F 254
monoammonium glycyrrhizate CRS and the chromatogram plates are suitable).
obtained with the reference solution to identify the peaks due (b) Use the mobile phase as described below.
to 18~-glycyrrhizic acid and 181:l-glycyrrhizic acid. (c) Apply 2 µL of each solution as 8 mm bands.
System suitability Reference solution: (d) Develop the plate to 7 cm.
- the chromatogram obtained with the reference solution is
(e) Remove the plate and allow to dry in air.
similar to the chromatogram supplied with
monoammonium g(ycyrrhizate CRS; (f) Dip the plate in methanolic sulfuric acid (10%), heat at
- resolution: minimum 2.0 between the peaks due to 18~- 100° for 10 minutes and examine under white light.
glycyrrhizic acid and 181:l-glycyrrhizic acid. MOBILE PHASE
Calculate the percentage content of l Sj)-glycyrrhizic acid, 1 volume of glacial acetic acid, 1 volume of anhydrous formic
using the following expression: acid, 2 volumes of water and 15 volumes of ethyl acetate.
SYSTEM SUITABILITY
A 1 xm 2 xpx0.979
A2 x m1 x 5 The test is not valid unless the chromatogram obtained with
solution (4) shows two clearly separated bands.
A, area of the peak due to I sp-g!ycyrrhizic acid in the CONFIRMATION
area of the peak due to 1SP-glycyrrhizic acid in the When examined in daylight, the chromatogram obtained with
chromatogram obtained with the reference solution; solution (1) shows a yellow band at the top of the plate.
m, mass of the extract to be examined used to prepare the test There is a faint yellow band above a faint to equivalent
solution, in grams;
mass of monoammonium glycyrrhizate CRS used to prepare
yellow band in the middle third of the plate. There are two
the reference solution, in grams; yellow bands above the brown band corresponding to 18~-
p percentage content of 1sp-g!ycyrrhizic acid in glycyrrhizic acid and a broad brown band in the bottom third
monoammonium glycyrrhizate CRS; of the plate. Other faint bands may be present.
0.979 peak correlation factor between 1sp-g!ycyrrhizic acid and
monoammonium glycyrrhizate.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
2023 Liquorice Liquid Extract IV-337
TOD of the olate A Percentage equivalent of monoammonium g]ycyrrhizate in

solution (I) determined from the calibration curve in % w/v;
A yellow band p declared percentage content of manoammonium
g/ycyrrhizate EPCRS;
823 molecular weight of 18~-g]ycyrrhizic acid;
840 molecular weight of monoammonium glycyrrhizate (without
A faint yellow band
A faint to equivalent any water of crystallisation).
yellow band A yellow band A yellow band
(hyperoside) (hyperoside)
A pink band
A yellow band (chlorogenic acid)
A yellow band
A brown band A yellow band A brown band Liquorice Root for use in TCM
(glycyrrhizic acid) (Rutin) (glycyrrhizic acid)
A broad brown band DEFINITION
Liquorice Root for use in TCM is the dried unpeeled root
Solution (1) Solution (2) Solution (4) Solution (5) and rhizome of Glycyrrhiza uralensis Fisch., and/or Glycyrrhiza
and 131
infiata Bat. and/or Glycyrrhiza glabra L.
It is collected in spring and autumn, separated from the
TESTS rootlets and dried in the sun.
Ethanol It contains not less than 2.0% of glycyrrhizic acid
16 to 20 % v/v, Appendix VIII F, Method III. (C42H62O16), calculated with reference to the dried material.
Methanol IDENTIFICATION
Not more than 0.05% v/v, Appendix VIII G, Method B. A. The root has few branches. Its bark is brownish-grey or
Ochratoxin A brown with longitudinal striations and bears traces of lateral
Not more than 20 ppb, Appendix XI S2. roots. The cylindrical stolons are 1-2 cm in diameter; their
external appearance is similar to that of the root but there are
ASSAY occasional small buds. The fracture of the root and the
Carry out the method for liquid chromatography, stolon is granular and fibrous. The cork layer is thin; the
Appendix III D, using an aqueous solvent mixture containing secondary phloem region is thick and light yellow with radial
8 g/L solution of ammonia. striations. The yellow xylem cylinder is compact, with a
(1) Dilute 2 mL of the preparation being examined to radiate structure. The stolon has a central pith, which is
100 mL and centrifuge. Dilute 10 mL of the supernatant absent from the root.
liquid to 25 mL. B. Reduce to a powder. The powder is yellowish-brown.
(2) 0.0065% w/v of monoammonium glycyrrhizate EPCRS. Examine under a microscope using chloral hydrate solution.
(3) 0.013% w/v of monoammonium glycyrrhizate EPCRS. The powder shows abundant fibres occurring in groups, each
group surrounded by a calcium oxalate prism crystal sheath,
(4) 0.020% w/v of monoammonium glycyrrhizate EPCRS.
the individual fibres with very thick, partially lignified walls
CHROMATOGRAPHIC CONDITIONS and few small pits; lignified bordered-pitted vessels, singly or
(a) Use a stainless steel column (10 cm x 4 mm) packed in small groups and sometimes accompanied by lignified
with octadecylsilyl silica gel for chromatography (5 µm) (Hypersil parenchymatous cells with moderately thickened and pitted
BDS Cl8 is suitable). walls, some of the individual vessels are very large, with pit
(b) Use isocratic elution and the mobile phase described apertures much elongated and the borders difficult to
below. discern; prism crystals of calcium oxalate up to about 35 µm
(c) Use a flow rate of 1.5 mL per minute. long occur scattered and in parenchymatous tissue; fragments
of cork composed of thin-walled, slightly lignified polygonal
(d) Use a detection wavelength of 254 nm.
cells. Examine under a microscope using 50% w/w glycerol
(e) Inject 10 µL of each solution. in water. The powder shows abundant starch granules,
MOBILE PHASE mostly simple, spherical to ovoid, 3 µm to 20 µm in
6 volumes of glacial acetic acid, 30 volumes of acetonitrile and diameter.
64 volumes of water. C. Carry out the method for thin-layer chromatography,
(1) Add 16 mL of water and 4 mL of hydrochloric acid Rl to
Establish a calibration curve with the content of
0.5 g of the powdered drug, heat on a water-bath under
monoammonium glycyrrhizate in solutions (2) to (4), in
reflux for 30 minutes, cool and filter. Dry the filter paper and
% w/v, as the abscissa and the corresponding peak areas as
residue in a flask at 105° for 60 minutes, add 20 mL of
the ordinate.
ether, heat on a water-bath at 40°C under reflux for
Using the retention times and the peak areas determined 5 minutes, cool and filter. Evaporate the filtrate to dryness
from the chromatograms obtained with the solutions (2) to and dissolve the residue in 5 mL of ether.
(4), locate and integrate the peak due to 18~-glycyrrhizic acid
(2) 0.1 % w/v each of glycyrrhetic acid and thymol in ether.
in the chromatogram obtained with solution (1).
Calculate the percentage content of 18~-glycyrrhizic acid CHROMATOGRAPHIC CONDITIONS
using the following expression: (a) Use as the coating substance silica gel F254 (Merck
10 x 20 cm plates are suitable).
Axl.25xPx823 (b) Use the mobile phase as described below.
840 (c) Apply 10 µL of each solution as a band.
Where: (e) Remove the plate and allow it to dry in air for 5 minutes.
Examine under ultraviolet light (254 nm). Spray the plate with
IV-338 Liquorice Liquid Extract 2023
anisaldehyde solutwn and heat at 100° to 105° for 10 minutes DETERMINATION OF CONTENT
and examine in daylight. Inject solution (4). Adjust the sensitivity of the system so that
MOBILE PHASE the height of the peaks is at least 50% of the full scale of the
1 volume of concentrated ammonia, 9 volumes of water, recorder. Inject solutions (2), (3) and (4) and determine the
25 volumes of ethanol (96%) and 65 volumes of ethyl acetate. peak areas. Prepare a calibration curve with the concentration
of the solutions (g per 100 mL) as the abscissa and the
SYSTEM SUITABILITY
corresponding areas as the ordinate. Inject solution (1).
Under ultra-violet light, the chromatogram obtained with Using the retention time and the peak area from the
solution (2) shows in the lower half a quenching zone due to chromatograms obtained with solutions (2), (3) and (4),
glycyrrhetic acid. When sprayed, the chromatogram obtained locate and integrate the peak due to glycyrrhizic acid in the
with solution (2) shows in the lower half the violet zone of chromatogram obtained with solution (1).
glycyrrhetic acid and in the upper third the red zone of
Calculate the percentage content of glycyrrhizic acid from the
thymol.
CONFIRMATION
The chromatogram obtained with solution (1) shows in the 5 822
Ax-xBx-
lower half a violet zone corresponding to the zone of m 840
glycyrrhetic acid in the chromatogram obtained with solution
(2) and a yellow zone (isoliquiridigenine) in the upper third
under the zone of thymol in the chromatogram obtained with A concentration of rnonoarnrnonium glycyrrhizate in solution (I)
determined from the calibration curve, in g per 100 rnL,
solution (2). Further zones may be present. B declared percentage content of monoammonium glycyrrhizate EPCRS,
TESTS m weight of the substance being examined in grams,
822 molecular weight of glycyrrhizic acid,
Total Ash 840 molecular weight of monoammonium glycyrrhizate (without any
Not more than 7.0%, Appendix XI J, Method II. water of crystallisation).
Acid-insoluble ash
Not more than 2.0%, Appendix XI K, Method II. STORAGE
Loss on drying Liquorice Root for use in TCM should be protected from
When dried for 2 hours at 100° to 105°, loses not more than moisture.
12.0%. Use 1 g.
Ochratoxin A
Not more than 20 ppb, Appendix XI S2.
ASSAY Processed Liquorice Root for use in
Carry out the method for liquid chromatography, TCM
Appendix III D, using the following solutions.
DEFINITION
(1) Mix 1 g of the powdered drug with 100 mL of 0.8% w/v
Processed Liquorice Root for use in TCM is the processed
of ammonia, place in an ultrasonic bath for 30 minutes,
Liquorice Root for use in TCM.
centrifuge, dilute 1 mL of supernatant solution to 5 mL with
0.8% w/v of ammonia and filter through a 0.45-µm filter. It contains not less than 2.0% of glycyrrhizic acid
(C 42 H 62 0 16) calculated with reference to the dried material.
(2) 0.0065% w/v of monoammonium glycyrrhizate EPCRS in
0.8% w/v of ammonia. PRODUCTION
(3) 0.013% w/v of monoammonium glycyrrhizate EPCRS in Processed Liquorice Root for use in TCM is cleaned,
0.8% w/v of ammonia. softened thoroughly, sliced transversely or longitudinally to
form uniform pieces and dried.
0.8% w/v of ammonia. IDENTIFICATION
CHROMATOGRAPHIC CONDITIONS A. The transversely-cut pieces are 0.5 to 2.5 cm in diameter,
irregularly circular to ovoid, up to about 3 mm thick.
(a) Use a stainless steel column (12.5 cm x 4 mm) packed
The outer surface is dark reddish-brown and longitudinally
with octadecylsilyl silica gel for chromatography (5 µm) (Hypersil
wrinkled. The transverse surface is cream to yellow and
ODS SS is suitable).
shows a thin layer of cork and a pale brown cambium line
(b) Use isocratic elution and the mobile phase described separating the radiate phloem region from the distinctly
below. radiate xylem. In pieces cut from the rhizome there is a
(c) Use a flow rate of 1.5 mL per minute. central, whitish pith; in those cut from the roots the radiate
(d) Use ambient column temperature. xylem continues to the centre.
(e) Use a detection wavelength of 254 nm. The longitudinally-cut pieces have been sliced obliquely and
(f) Inject 10 µL of each solution. usually include a small portion of the outer surface at the
tapering ends; they are about 6 to 8 cm long, 1 to 1.5 cm
MOBILE PHASE
wide and 3 mm thick. The outer surface is reddish-brown
6 volumes of glacial acetic acid, 30 volumes of acetonitrile and and longitudinally wrinkled with scattered transverse ridges;
64 volumes of water. the smooth cut surface is cream to yellow, faintly fibrous and
SYSTEM SUITABILITY shows a distinct cambium; on some of the pieces the vessel
The assay is not valid unless (a) the column efficiency, cavities are visible in the central region.
determined on the peak due to glycyrrhizic acid in the B. Reduce to a powder. The powder is yellowish-brown.
chromatogram obtained with solution (2), is at least 30,000 Examine under a microscope using chloral hydrate solution.
theoretical plates per metre and (b) the symmetry factor of the The powder shows abundant fibres occurring in groups, each
peak is not more than 1.3. group surrounded by a calcium oxalate prism crystal sheath,
2023 Liquorice Liquid Extract IV-339
the individual fibres have very thick, partially lignified walls ASSAY
and few small pits; lignified bordered-pitted vessels, singly or Carry out the method for liquid chromatography,
in small groups and sometimes accompanied by lignified Appendix III D, using the following solutions.
parenchymatous cells with moderately thickened and pitted (1) Mix 1 g of the powdered drug with 100 mL of0.8% w/v
walls, some of the individual vessels are very large, with pit of ammonia, place in an ultrasound bath for 30 minutes,
apertures much elongated and the borders difficult to centrifuge, dilute 1 mL of the supernatant solution to 5 mL
discern; prism crystals of calcium oxalate up to about 35 µm with 0.8% w/v of ammonia and filter through a 0.45-µm
long occur scattered and in parenchymatous tissue; fragments filter.
of cork composed of thin-walled, slightly lignified polygonal
cells. Examine under a microscope using 50% w/w glycerol
0.8% w/v of ammonia.
in water. The powder shows abundant starch granules,
mostly simple, spherical to ovoid, 3 µm to 20 µm in (3) 0.013% w/v of monoammonium glycyrrhizate EPCRS in
diameter. 0.8% w/v of ammonia.
C. Carry out the method for thin-layer chromatography, (4) 0.0195% w/v of monoammonium glycyrrhizate EPCRS in
Appendix III A, using the following solutions. 0.8% w/v of ammonia.
(1) Add 16 mL of water and 4 mL of hydrochloric acid (25%) CHROMATOGRAPHIC CONDITIONS
to 0.5 g of the powdered drug, heat on a water-bath under (a) Use a stainless steel column (12.5 cm x 4 mm) packed
reflux for 30 minutes, cool and filter. Dry the filter paper and with octadecylsilyl silica gel for chromatography (5 µm) (Hypersil
residue in a flask at 105° for 60 minutes, add 20 mL of ether, ODS SS is suitable).
heat on a water-bath at 40° under reflux for 5 minutes, cool (b) Use isocratic elution and the mobile phase described
and filter. Evaporate the filtrate to dryness and dissolve the below.
residue in 5 mL of ether. (c) Use a flow rate of 1.5 mL per minute.
(2) 0.1 % w/v each of glycyrrhetic acid and thymol in ether.
(d) Use ambient column temperature.
CHROMATOGRAPHIC CONDITIONS (e) Use a detection wavelength of 254 nm.
(a) Use as the coating substance silica gel F254 (Merck (f) Injection 10 µL of each solution.
10 x 20 cm plates are suitable).
MOBILE PHASE
6 volumes of glacial acetic acid, 30 volumes of acetonitrile and
(c) Apply 10 µL of each solution as a band.
64 volumes of water.
SYSTEM SUITABILITY
(e) Remove the plate and allow it to dry in air for 5 minutes.
The assay is not valid unless (a) the column efficiency,
determined on the peak due to glycyrrhizic acid in the
anisaldehyde solution and heat at 100° to 105° for 10 minutes
chromatogram obtained with solution (2), is at least 30,000
and examine in daylight.
theoretical plates per metre and (b) the symmetry factor of the
MOBILE PHASE peak is not more than 1.3.
1 volume of concentrated ammonia, 9 volumes of water, DETERMINATION OF CONTENT
25 volumes of ethanol (96%) and 65 volumes of ethyl acetate.
Inject solution (4). Adjust the sensitivity of the system so that
SYSTEM SUITABILITY the height of the peaks is at least 50% of the full scale of the
Under ultra-violet light, the chromatogram obtained with recorder. Inject solutions (2), (3) and (4) and determine the
solution (2) shows in the lower half a quenching zone due to peak areas. Prepare a calibration curve with the concentration
glycyrrhetic acid. When sprayed, the chromatogram obtained of the solutions (g per 100 mL) as the abscissa and the
with solution (2) shows in the lower half the violet zone of corresponding areas as the ordinate. Inject solution (1).
glycyrrhetic acid and in the upper third the red zone of Using the retention time and the peak area from the
thymol. chromatograms obtained with solutions (2), (3) and (4),
CONFIRMATION locate and integrate the peak due to glycyrrhizic acid in the
chromatogram obtained with solution (1).
The chromatogram obtained with solution (1) shows in the
lower half of violet zone corresponding to the zone of Calculate the percentage content of glycyrrhizic acid from the
glycyrrhetic acid in the chromatogram obtained with solution following expression:
(2) and a yellow zone (isoliquiridigenine) in the upper third 5 822
under the zone of thymol in the chromatogram obtained with Ax-xBx-
solution (2). Further zones may be present. m 840
TESTS
A concentration of monoammonium glycyrrhizate in the solution (I)
Total Ash
determined from the calibration curve, in g per l 00 mL,
Not more than 5.0%, Appendix XI J, Method II. B declared percentage content of monoammonium g/ycyrrhizate EPCRS,
Acid-insoluble ash m weight of the substance being examined in grams,
822 molecular weight of glycyrrhizic acid,
Not more than 1.0%, Appendix XI K, Method II. 840 molecular weight of the monoammonium glycyrrhizate (without any
Loss on drying water of crystallisation).
When dried for 2 hours at 100° to 105°, loses not more than
10.0% of its weight. Use 1 g. STORAGE
Ochratoxin A Processed Liquorice Root for use in TCM should be
Not more than 20 ppb, Appendix XI S2. protected from moisture.
IV-340 Long Pepper 2023
Long Pepper \
~i
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
1-7
DEFINITION
Dried, ripe or nearly ripe fruiting spikes of Pi.per longum L. //!(I///!(:~'
or Pi.per retrofractum Vahl (syn. P. chaba Hunter and
P. officinarum (Miq.) C. DC.) or a mixture of both species. Ac
Content
- essential oil: minimum 6.0 mUkg (dried drug);
- piperine (C 17 H 19N03 ; Mr 285.3): minimum 3.0 per cent
(dried drug).
IDENTIFICATION
A. P. longum. The fruiting spikes are cylindrical or irregularly
cylindrical, 1-2.5 cm long (rarely longer than 2.5 cm),
3-5 mm in diameter, blackish-brown or almost black.
The spikes are quite compact, tough, composed of small
fruits firmly fixed on the receptacle in regular or oblique ""' Hb
rows. The berries are spherical, about 1 mm in diameter.
The bracts are black, small, punctiform, confined to
depressions between adjacent berries. The remains of the
peduncle may be present at the base of the cylinder. Spikes H . '•,. Ha
can be easily broken; the fracture is irregular and granular.
' ·:-,-::::~
P. retrofractum The fruiting spikes are similar to those of 25µm
1-----i
P. longum but clearly more robust, straight and cylindrical,
2.5-4 cm long (rarely smaller than 2.5 cm), 5-8 mm in
diameter, brown or reddish-brown. The berries are also
firmly fixed on the receptacle but, in contrast to those of
P. longum, arranged more obviously in spiral rows. Figure 2453.-1. - Illustration for identification test B of powdered
The bracts are more prominent than those of P. longum. herbal drug of long pepper
B. Microscopic examination (2.8.23). The powder is grey or
light brown. Examine under a microscope using chloral Reference solution Dissolve 10 mg of borneol R and 15 mg of
hydrate solution R. The powder shows the following diagnostic piperine R in 10 mL of methanol R.
characters (Figure 2453.-1): fragments of the endocarp Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel
(surface view [A]), consisting of sclereids, more or less F254plate R (2-10 µm)].
elongated, about 75 µm long, pitted, with irregularly Mobile phase ethyl acetate R, cyclohexane R (30:50 VIV).
thickened walls and wide channels [Aa], associated with the
reddish-brown pigmented layer of the testa with indistinct
cells [Ab] and also associated with the inner testa with Development Over a path of 15 cm [or 6 cm].
rectangular, brown, thin-walled cells [Ac]; fragments of the Drying In air.
endocarp (transverse section [D]), showing sclereids with Detection A Examine in ultraviolet light at 254 nm.
irregularly thickened inner walls on the 3 lower sides [Da], Results A See below the sequence of zones present in the
usually associated with the testa (namely the pigmented layer chromatograms obtained with the reference solution and the
with indistinct cells [Db] and the layer of the inner testa test solution. Furthermore, other faint quenching zones may
[De]); fragments of the parenchyma of the mesocarp [B, C] be present in the chromatogram obtained with the test
containing more or less polygonal sclereids, isolated [Ba] or solution.
in groups [Bb], and oil cells about 50 µm in diameter [Ca];
numerous ovoid or polygonal cells of the parenchyma of the
Top of the plate
seed [Dd]; fragments of the epicarp [F] with thin-walled
cells; rare fragments from the centre of the spike [E], -- --
consisting of parenchymatous cells [Eb] and elongated
2 strong quenching zones
sclereids up to 400 µm long [Ea]; a few fragments of vascular
tissue [HJ with spiral or striated vessels [Ha] and fibres [Hb]. A quenching zone
solution of glycerol R. Starch is visible as rounded, compound -- --
granules, each about 20 µm in diameter [Ga], made up of A quenching zone
tiny individual granules, ovoid or polyhedral by compression, Piperine: a quenching zone A strong quenching zone (piperine)
free [Gb] or included in the parenchymatous cells of the
seed [G].
(2. 9.12) add 5 mL of methanol R. Sonicate for 10 min,
2023 Loosestrife IV-341
Detection B Treat with anisaldehyde solution R and heat at Mobile phase:

100 °C for 5 min; examine in daylight. - mobile phase A: water R;
Results B See below the sequence of zones present in the - mobile phase B: acetonitrile R;
test solution. Furthermore, other zones may be present in the Time Mobile phase A Mobile phase B
(min) (per cent V/JI) (per cent Vil')
0-5 so so
5 - 20 so-, 5 so -, 95
Top of the plate
20 - 22 5 -, 0 95 -, JOO
A purple-grey zone

-- --
A purple zone
Injection 10 µL.
Borneo!: a yellowish-brown zone A violet zone
A purple-grey zone long pepper for system suitability HRS and the chromatogram
obtained with reference solution (b) to identify the peak due
-- --
to piperine and peak 2.
Piperine: a green or brownish zone A green or brownish zone (piperine) Retention time Piperine = about 10 min.
- peak-to-valley ratio: minimum 4, where Hp = height above
the baseline of peak 2 and Hv = height above the baseline
Reference solution Test solution of the lowest point of the curve separating the peak due to
piperine from peak 2.
Calculate the percentage content of piperine using the
TESTS
Maximum 3 per cent.
A1 area of the peak due to piperine in the chromatogram obtained
freshly powdered herbal drug (355) (2.9.12) by drying in an with the test solution;
oven at 105 °C for 2 h. A2 area of the peak due to piperine in the chromatogram obtained
Total ash (2.4.16) m1 mass of the herbal drug to be examined used to prepare the test
Maximum 5.0 per cent. solution, in grams;
m2 mass of piperine CRS used to prepare reference solution (a), in
ASSAY grams;
Essential oil (2.8.12) p percentage content of piperine in piperine CRS.
Use 25.0 g of the freshly powdered herbal drug (1400)
(2.9.12), a 1000 mL round-bottomed flask, 400 mL of _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
water R as the distillation liquid and 0.5 mL of xylene R in
the graduated tube. Distil at a rate of 2-3 mUmin for 3 h.
Piperine
Liquid chromatography (2.2.29). Carry out the assay protected Loosestrife
from light.
Test solution Disperse 0.250 g of the powdered herbal drug (Ph. Bur. monograph 1537)
(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) R, combine the filtrate and washings DEFINITION
and dilute to 50.0 mL with the same solvent. Filter through Dried flowering tops, whole or cut, of Lythrum salicaria L.
a membrane filter (nominal pore size 0.45 µm). Content
Reference solution (a) Dissolve 15.0 mg of piperine CRS in Minimum 5 .0 per cent of tannins, expressed as pyrogallol
ethanol (96 per cent) Rand dilute to 100.0 mL with the same (C 6 H60 3 ; Mr 126.1) (dried drug).
solvent. IDENTIFICATION
Reference solution (b) Disperse 0.250 g of long pepper for A. The stems are rigid, 4-angled, branching at the top,
system suitability HRS (355) (2.9.12) in 40 mL of ethanol brownish-green, longitudinally wrinkled and pubescent.
(96 per cent) R. Sonicate for 20 min and filter. Rinse the flask The leaves are opposite, decussate, rarely verticillate in threes
and the filter with 5 mL of ethanol (96 per cent) R, combine and sometimes alternate at the inflorescence which forms a
the filtrate and washings and dilute to 50.0 mL with the long terminal spike. The leaves are sessile, lanceolate and
same solvent. Filter through a membrane filter (nominal pore cordate at the base, 5-15 cm long and 1-2.5 cm wide,
size 0.45 µm). pubescent on the lower surface; the subsidiary veins form
Column: arcs that anastomose near the leaf margin. The flowers have
- size: l = 0.15 m, 0 = 4.6 mm; a pubescent, tubular, persistent gamosepalous calyx, 4-8 mm
- stationary phase: end-capped octadecylsilyl silica gel for long, consisting of 6 sepals bearing 6 small, triangular teeth
chromatography R (5 µm). 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
IV-342 Lovage Root 2023
narrowing at the base. The androecium consists of 2 verticils Reference solution Dissolve 0.5 mg of chlorogenic acid R, 1 mg
of 6 stamens ( 1 verticil with short, barely emerging stamens, of hyperoside R, 1 mg of rutoside trihydrate R and 1 mg of
the other with long stamens extending well out of the vitexin R in 10 mL of methanol R.
corolla). The fruit, if formed, is a small capsule included in Plate TLC silica gel plate R.
the persistent calyx.
Mobile phase anhydrous acetic acid R, anhydrous formic acid R,
B. Microscopic examination (2.8.23). The powder is water R, ethyl acetate R (7.5:7.5: 18:67 V/VIVIV).
greenish-yellow. Examine under a microscope using chloral
characters (Figure 1537.-1): unicellular [Ea] or Development Over a path of 15 cm.
bicellular [Aa], uniseriate, thick-walled, finely pitted covering Drying At 100-105 °C.
trichomes from the epidermis of the leaf [A] and stem [E]; Detection treat the still-warm plate with a 10 g/L solution of
numerous uniseriate, unicellular [Ga] or bicellular [Gb], diphenylboric acid aminoethyl ester R in methanol R.
thin-walled, finely pitted, annularly striated covering Subsequently treat with a 50 g/L solution of macrogol 400 R
trichomes from the calyx (side view [G]); transparent violet- in methanol R. Allow to dry in air for 30 min and examine in
pink fragments from the petals [F] consisting of epidermal ultraviolet light at 365 nm.
cells with sinuous walls and a grainy cuticle [Fa], covering Results The chromatogram obtained with the reference
fine spiral vessels [Fb]; fragments of parenchyma from the solution shows in the lower third a yellowish-brown
leaf [D] with numerous cells containing cluster crystals of fluorescent zone due to rutoside and in the middle third a
calcium oxalate [Da], associated with spiral vessels [Db]; light blue fluorescent zone due to chlorogenic acid, above it a
pollen grains with 3 pores and a thin and slightly granular yellowish-brown fluorescent zone due to hyperoside and a
exine [C]; fragments of the upper epidermis of the leaf [A] green fluorescent zone due to vitexin. The chromatogram
with large polygonal cells and sinuous walls, covered by a obtained with the test solution shows a bright green
finely striated cuticle [Ab]; fragments of the lower epidermis fluorescent zone slightly above the zone due to rutoside in
of the leaf [BJ with smaller polygonal cells [Ba] and the chromatogram obtained with the reference solution, a
anomocytic stomata [Bb] (2.8.3); fragments of the stem [E) yellow fluorescent zone similar in position to the zone due to
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)
---------------------~&
Lovage Root
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or cut, dried rhizome and root of Levisticum officinale
E W.D.J.Koch.
Eb ~~.
,_____. Essential oil content:
25µm Ga - for the whole drug, minimum 4.0 mIJkg (dried drug);
- for the cut drug, minimum 3.0 mIJkg (dried drug).
Figure 1537.-1. - Illustration for identification test B of powdered IDENTIFICATION
herbal drug of loosestrife A. The rhizome and the large roots are often split
longitudinally. The rhizome is short, up to 5 cm in diameter,
C. Thin-layer chromatography (2.2.27). light greyish-brown or yellowish-brown, simple or with
Test solution To 1.0 g of the powdered herbal drug (355) several protuberances. The roots, showing little ramification,
(2.9.12) add 10 mL of methanol Rand heat in a water-bath are the same colour as the rhizome; they are usually up to
at 65 °C for 5 min with frequent shaking. Cool and filter. 1.5 cm thick and up to about 25 cm long; the fracture is
Dilute the filtrate to 10 mL with methanol R.
2023 Lavage Root IV-343
usually smooth and shows a very wide yellowish-white bark Top of the plate
and a narrow brownish-yellow wood.
(Z)-Ligustilide; a bluish-white A bluish-white fluorescent zone
fluorescent zone
--- ---
Osthole: a blue fluorescent zone
Imperatorin: a whitish fluorescent A weak whitish fluorescent zone

zone
--- - -
A weak whitish fluorescent zone

0 1e®
@ 0 I'.)
Oo<ifo
@ 0 0 Results B See below the sequence of zones present in the
(90
test solution. Furthermore, other weak quenching zones may
solution.
Top of the plate
(Z)-Ligustilide; a bluish fluorescent A bluish fluorescent zone

zone
--- --
Osthole; a quenching zone A weak quenching zone
lmperatorin: a quenching zone
--- ---
Figure 1233.-1. - Illustration for identification test B of powdered Results C See below the sequence of zones present in the
herbal drug of lmJage root chromatograms obtained with the reference solution and the
B. Microscopic examination (2.8.23). The powder is in the chromatogram obtained with the test solution.
Top of the plate
characters (Figure 1233.-1): cork cells, polygonal or rounded
(surface view [C]), with brown contents; abundant 2 prominent reddish zones
parenchyma, consisting of mostly thin-walled and rounded
(Z)-Ligusrilide: a grey zone A grey zone
cells (transverse section [El) but some with thicker walls,
some elongated with walls showing fine criss-cross striations --- ---
(longitudinal section [H]); groups of small, reticulately
thickened vessels embedded in small-celled, unlignified
parenchyma [G]; isolated fragments of reticulate vessels [F] lmperatorin: a grey zone
up to 125 µm in diameter; fragments of secretary canals
--- ---
(transverse section [A], longitudinal section [BJ) up to
180 µm wide. Examine under a microscope using a 2 purple zones
50 per cent V/V solution of glycerol R. The powder shows A distinct brown zone
starch granules, simple, rounded or ovoid, up to about
12 µm in size, and numerous larger, compound granules, Reference solution Test solution
many with several components [DJ.
C. Examine the chromatograms obtained in the test for TESTS
species of Angelica and Ligusticum described in the European
Species of Angelica and Ligusticum described in the
Pharmacopoeia. European Pharmacopoeia
Results A See below the sequence of zones present in the Thin-layer chromatography (2.2.27).
chromatograms obtained with the reference solution and the Test solution To 1 g of the freshly powdered herbal drug
test solution. Furthermore, other weak fluorescent zones may
(355) (2.9.12) add 4 mL of heptane Rand sonicate for 5 min.
Centrifuge the mixture and use the supernatant.
solution.
Reference solution Dissolve 1 mg of imperatorin R, l mg of
(Z)-ligustilide Rand 1 mg of osthole R in methanol Rand
IV-344 Lycopus Lucidus Herb 2023
Mobile phase glacial acetic acid R, ethyl acetate R, toluene R reddish nodes covered in white hairs. The leaves are
(1: 10:90 VIVIV). opposite, with short petioles or almost sessile; when whole,
Application 4 µL, as bands of 8 mm. the lamina, usually crumpled, is lanceolate or oblong,
Development Over a path of 6 cm. 5-10 cm long, with dentate margins, an acute apex and a
slightly decurrent base, and is pubescent on both surfaces;
Drying In air. the upper surface is dark green or blackish-green while the
Detection A Examine in ultraviolet light at 365 nm. lower surface is greyish-green, and densely dotted with
Results A The chromatogram obtained with the test solution glandular trichomes; the inflorescence is grouped in axillary
shows no blue fluorescent zone just below or above the zone whorls and consists only of persistent calyces and leaf-like
due to imperatorin in the chromatogram obtained with the bracts, as the corollas have usually fallen off. The bracts are
reference solution. lanceolate with hairs on the margins. The calyx is hairy, and
Detection B Examine in ultraviolet light at 254 nm. 2-lipped with 5 teeth. The texture is fragile.
Results B The chromatogram obtained with the test solution Fragmented drug It occurs in various sizes 1-3 cm long.
shows no zone at or just below the zone due to imperatorin The fragments of the stems are flattened, angular,
in the chromatogram obtained with the reference solution. longitudinally striated and have a hollowed whitish pith;
some fragments have nodes covered in white hairs; the
Detection C Treat the plate with a solution of 20 mL of
fragments of the leaves often show the dentate margin of the
sulfuric acid R in 180 mL of ice-cooled methanol R; heat at
lamina, and prominent veins on the lower surface; some
100-105 °C for 5 min and examine in daylight.
fragments have inflorescences in axillary whorls, reduced to
Results C The chromatogram obtained with the test solution brown 2-lipped calyces.
shows no purple zone between the 2 reddish zones at the top
of the chromatogram and the zone due to (Z)-ligustilide in
green or brownish-green. Examine under a microscope using
the chromatogram obtained with the reference solution; the
chromatogram obtained with the test solution shows no
purple zone between the zones due to (Z)-ligustilide and
upper epidermis of the leaves (surface view [BJ) consisting of
osthole in the chromatogram obtained with the reference
cells usually with rigid walls [Ba] bearing covering trichomes
solution.
and glandular trichomes; the covering trichomes have spiny
Foreign matter (2.8.2) walls, some are unicellular, short and conical [Bb], others are
Maximum 3 per cent, determined on 50 g. uni- [C, E] or multicellular (2-5 cells) [G], occasionally
Loss on drying remaining only as circular, thickened scars on the
(2.2.32): maximum 12.0 per cent, determined on 1.000 g of epidermis [F]; the glandular trichomes, rarer, have a
the powdered herbal drug (355) (2.9.12) by drying in an unicellular stalk and an ovoid head, either uni- or
oven at 105 °C for 2 h. bicellular [Be], sometimes associated with loosely arranged
Total ash (2.4.16) palisade parenchyma [Bd]; fragments of the lower epidermis
Maximum 8.0 per cent. of the lamina (surface view [Al) covered by a striated cuticle
composed of cells with sinuous walls [Aa], diacytic
Ash insoluble in hydrochloric acid (2.8.1) stomata [Ab], uni- or multicellular covering trichomes similar
Maximum 2.0 per cent. to those of the upper epidermis, mainly visible on the veins,
ASSAY and numerous glandular trichomes, sometimes with a
Essential oil (2.8.12) unicellular stalk and a unicellular head ([Ac], isolated side
Use 40.0 g of the herbal drug reduced to a powder (500) view [DJ), or a unicellular stalk and a bicellular head [Ad],
(2. 9.12) immediately before the determination, a 2 L flask, or a unicellular stalk and an octocellular head of laminaceous
10 drops of liquid paraffin R, 500 mL of water R as the type [Ae] about 60-80 µm in diameter; bundles of long
distillation liquid and 0.50 mL of xylene R in the graduated fibres [H, K] from the stems, with thick walls [Ha, Ka],
tube. Distil at a rate of 2-3 mUmin for 4 h. sometimes associated with bordered-pitted vessels [Hb] or
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur parenchymatous cells of the pith [Kb]; frequent sclereids m,
usually isolated Ua, Jb] or in small groups Uc], ovoid to
rectangular, with varying thickened and channelled walls,
from the pith near the nodes.
Lycopus Lucidus Herb C. Thin-layer chromatography (2.2.27).
(Ph. Bur. monograph 2723) (2. 9.12) add 5 mL of ethyl acetate R. Sonicate for 10 min.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Centrifuge and use the supernatant.
Reference solution Dissolve 1.0 mg of luteolin Rand 1.0 mg
DEFINITION of ursolic acid R in 2.0 mL of ethyl acetate R.
Whole or fragmented, dried aerial parts of Lycopus lucidus
var. hirtus (Regel) Makino & Nemoto.
Mobile phase anhydrous formic acid R, methylene chloride R,
Content ethyl acetate R, cyclohexane R (1:5:8:20 V/V/V/V).
Minimum 0.15 per cent ofrosmarinic acid (C 18 H 16 0 8 ;
Mr 360.3) (dried drug). Application 4 µL as bands of 8 mm.
IDENTIFICATION
Drying In air.
A. Wnole drug. The stems are square in cross-section with a
hollowed pith and few branches, 50-100 cm long and Detection Treat with 2.5 M alcoholic sulfuric acid Rand
2-6 mm in diameter; the greenish-yellow or reddish outer heat at 110 °C for 3 min. Examine in daylight.
surface shows, on each side, shallow longitudinal furrows and
2023 Lycopus Lucidus Herb IV-345

(2. 9.12) add 25.0 mL of a 70 per cent V/V solution of
methanol R and weigh. Boil on a water-bath under a reflux
condenser for 30 min. Allow to cool and weigh again.
Compensate for the loss of solvent with a 70 per cent V/V
solution of methanol R and mix. Filter the solution and dilute
2.0 mL of the filtrate to 10.0 mL with a 70 per cent V/V
solution of methanol R. Filter this solution through a
acid CRS in 20.0 mL of a 70 per cent V/V solution of
methanol R. Dilute 1.0 mL of this solution to 25.0 mL with a
70 per cent V/V solution of methanol R.
Reference solution (b) Dissolve 5 mg offernlic acid R in
25 mL of a 70 per cent V/V solution of methanol R. Dilute
1 mL of this solution to 10.0 mL with reference solution (a).
Column:
- size: l =0.25 m, 0 =4.0 mm;
Mobile phase:
- mobile phase A: phosphoric acid R, acetonitrile R, water for
chromatography R (1:19:80 VIVIV);
- mobile phase B: phosphoric acid R, methanol R, acetonitrile R
(1:40:59 VIVIV);

0 - 20 100 ➔ 70 0 ➔ 30
Figure 2723.-1. - Illustration for identification test B of powdered 20 - 25 70 ➔ 0 30 ➔ 100
herbal drng of lycopus lucidus herb 25 - 30 0 ➔ 100 100 ➔ 0
Results See below the sequence of zones present in the Flow rate 1.0 mUmin.
Top of the plate reference solution (a) to identify the peak due to rosmarinic
acid.
Retention time Ferulic acid = about 10 min; rosmarinic
A pale violet zone acid = about 14 min.
--- ---
- resolution: minimum 5.0 between the peaks due to ferulic
A reddish-violet zone acid and rosmarinic acid.
Ursolic acid: a violet zone A violet zone (ursolic acid) Calculate the percentage content of rosmarinic acid using the
--- ---
2 dark violet zones AI area of the peak due to rosmarinic acid in the chromatogram
Luteolin: a yellow zone A2 area of the peak due to rosmarinic acid in the chromatogram
solution, in grams;
"12 mass of rosmarinic acid CRS used to prepare reference
TESTS solution (a), in grams;
p percentage content of rosmarinic acid in rosmarinic acid CRS.
- - - - - - - - - - - - - - - - - - - - - - - Ph Eur
powdered herbal drug (35 5) (2. 9.12) by drying in an oven at
105 °C for 2 h.
Total ash (2.4.16')
ASSAY
IV-346 Magnolia Biondii Flower 2023
Magnolia Biondii Flower

PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole, dried flower bud of Magnolia biondii Pamp. (syn.
Yulania biondii (Pamp.) D.L.Fu).
Content
- essential ou: minimum 14.0 ml.Jkg (anhydrous drug);
- magnolin (C23H 28O 7 ; Mr 416.5): minimum 3.0 per cent
(anhydrous drug).
IDENTIFICATION
A. The flower bud, whose texture is light and fragile, is ovoid
to elongated-ovoid, whitish-grey, yellowish-grey or greenish-
grey, silky, pubescent, with a pointed apex; 1.2-3.0 cm long
and 8-16 mm in diameter. The ligneous, short peduncle,
covered in whitish lenticels, is usually present at the base of
the flower bud. The flower bud is surrounded by 2-3 whorls
of bracts: each bract has an outer surface densely covered in
silky hairs, and a glabrous, brownish inner surface.
The perianth consists of 9 light yellow or brownish tepals F
arranged in 3 whorls; the outer whorl consists of 3 sepaloid •.
tepals, linear, whose length is about a quarter of that of the ~ ·. .. .
.
~ . .
.
·,_
-
other tepals; the petaloid tepals are arranged in 2 inner . '
whorls, each consisting of 3 tepals. The stamens and carpels ~

are numerous, arranged in a spiral.
B. Microscopic examination (2.8.23). The unsieved powder
is greenish to brownish-grey, dark-brown or slightly greenish-
yellow. Examine under a microscope using chloral hydrate Figure 27 42.-1. - Illustration for identification test B of powdered
solution R. The powder shows the following diagnostic herbal drug of Magnolia biondii flower bud
characters (Figure 2742.-1): isolated and fragmented,
multicellular (2-6 cells when whole) covering trichomes [B, Top of the plate
C, DJ, whose cells have thick walls, often with spiral
striations [Ba]; their bases are enlarged and consist of cells
with very thick walls like sclereids, with granular orange-
-- --
brown contents [Bb]; numerous sclereids, more or less
branched, isolated [F] or in groups [E], with very thick, Fargesin: a dark blue fluorescent A dark blue fluorescent zone
zone (fargesin)
striated, channelled walls, up to 60 µm in diameter;
fragments of perianth segments (surface view [A]; sectional -- --
view [G]) covered by a cuticle [Ga] and composed of
Magnolin: a quenching zone A quenching zone (magnolin)
polyhedral epidermal cells [Aa, Gb], paracytic stomata
(2.8.3) [Ab] and parenchyma consisting of rounded cells
[Ad, Ge] and including rounded oil cells (up to 100 µm in
diameter) with orange-yellow contents [Ac, Gd].
C. Thin-layer chromatography (2.2.27) as described in the
test for Magnolia officinalis Rehder & E.H.Wilson with the Detection B Treat with vanulin reagent R, heat at
following modifications. 100-105 °C for 5-7 min and examine in daylight.
Detection A Examine in ultraviolet light at 254 nm. Results B See below the sequence of zones present in the
Results A See below the sequence of zones present in the chromatograms obtained with the reference solution and the
chromatograms obtained with the reference solution and the test solution. Furthermore, other faint zones of various
test solution. Furthermore, other faint zones may be present colours may be present in the chromatogram obtained with
in the chromatogram obtained with the test solution. the test solution.
2023 Magnolia Biondii Flower IV-347
Top of the plate methanol R the first time and 20 mL of methanol R the
second time. Combine the supernatants, cool, and dilute to
A faint violet zone
100.0 mL with methanol R. Dilute 1.0 mL of the solution to
-- -- 2.0 mL with a mixture of 10 volumes of acetonitrile Rand
90 volumes of water for chromatography R. Filter through a
F argesin: a brown zone A brown zone (fargesin)
A dark blue zone Reference solution (a) Dissolve 5.0 mg of magnolin CRS in
- - - -
Dilute 1.0 mL of the solution to 5.0 mL with a mixture of
A faint pinkish-violet zone 10 volumes of acetonitrile R and 90 volumes of water for
Magnolin: a pinkish-violet zone A pinkish-violet zone (magnolin) chromatography R.
Reference solution (b) To 0.250 g of magnolia biondiifiower
for system suitability HRS add 35 mL of methanol R. Sonicate
Reference solution Test solution for 1 h, changing the water of the ultrasonic bath after
30 min of sonication to prevent heating. Centrifuge for
15 min. Transfer the supernatant to a 100 mL volumetric
TESTS flask. Repeat the extraction twice, adding to the residue
Magnolia officinalis Rehder & E.H. Wilson 35 mL of methanol R the first time and 20 mL of methanol R
Thin-layer chromatography (2.2.27). the second time. Combine the supernatants, cool, and dilute
Test solution Reduce the herbal drug to a powder using a to 100.0 mL with methanol R. Dilute 1.0 mL of the solution
blade grinder to prevent heating. To 0.5 g of the powdered to 2.0 mL with a mixture of 10 volumes of acetonitrile R and
herbal drug add 3.0 mL of methanol R. Sonicate for 15 min 90 volumes of water for chromatography R. Filter through a
and centrifuge for 15 min. Transfer the supernatant to a membrane filter (nominal pore size 0.45 µm).
5 mL flask. Add 2 mL of methanol R to the residue, sonicate Column:
for 15 min and centrifuge. Transfer the supernatant into the - size: l = 0.15 m, 0 = 4.6 mm;
same 5 mL flask. Dilute the combined supernatants to - stationary phase: end-capped octadecylsilyl silica gel for
5.0 mL with methanol R. Filter through a membrane filter chromatography R (5 µm);
(nominal pore size 0.45 µm) if necessary. - temperature: 30 °C.
Reference solution Dissolve 2.0 mg offargesin Rand 2.0 mg Mobile phase:
of magnolin R in methanol Rand dilute to 5.0 mL with the - mobile phase A: anhydrous acetic acid R, acetonitrile R, water
same solvent. for chromatography R (0.1:14:85.9 VIVIV);
Plate TLC silica gel F254 plate R (2-10 µm). - mobile phase B: anhydrous acetic acid R, water for
Mobile phase methanol R, ethyl acetate R, toluene R chromatography R, acetonitrile R (0.1 :4. 9:95 VIVIV);
(1:5:30 VIV!V).
(min) (per cent V/JJ) (per cent VIV)
Development Over a path of 7 cm. 0 - 12 68 32
Drying In air. 12 - 20 68 ➔ 0 32 ➔ 100
Detection Treat with vamllin reagent R, heat at 100-105 °C 20 - 28 0 100
for 5-7 min and examine in daylight.
Results The chromatogram obtained with the test solution Flow rate 1.0 mL/min.
shows no pinkish zone (due to magnolol) just below the zone Detection Spectrophotometer at 278 nm.
due to fargesin in the chromatogram obtained with the
Injection 20 µL.
reference solution.
Water (2.2.13) magnolia biondii flower for system suitability HRS and the
Maximum 100 mL/kg, determined on 25.0 g of the finely cut chromatogram obtained with reference solution (b) to
herbal drug. identify the peak due to eudesmin; use the chromatogram
Total ash (2.4.16) obtained with reference solution (a) to identify the peak due
Maximum 4.0 per cent. to magnolin.
ASSAY Retention time Eudesmin = about 12 min; magnolin = about
Essential oil (2. 8. 12) 13 min.
Use 15.0 g of freshly, finely cut herbal drug, a 1 L round- System suitability Reference solution (b):
bottomed flask, 300 mL of water R as the distillation liquid - resolution: minimum 1.8 between the peaks due to
and 0.50 mL of xylene R in the graduated tube. Distil at a eudesmin and magnolin.
rate of 2-3 mL/min for 3 h. If necessary, dilute the test solution with a mixture of
Magnolin 10 volumes of acetonitrile R and 90 volumes of water for
Liquid chromatography (2.2.29). chromatography R to obtain a peak due to magnolin with a
Test solution Reduce the herbal drug to a powder using a height that is similar to or smaller than the height of the
blade grinder to prevent heating. To 0.250 g of the powdered corresponding peak in the chromatogram obtained with
herbal drug add 35 mL of methanol R. Sonicate for 1 h, reference solution (a). Take into account the additional
changing the water of the ultrasonic bath after 30 min of dilution factor d.
sonication to prevent heating. Centrifuge for 15 min. Calculate the percentage content of magnolin using the
Transfer the supernatant to a 100 mL volumetric flask. following expression:
Repeat the extraction twice, adding to the residue 35 mL of
IV-348 Magnolia Officinalis Bark 2023
A 1 x m2 xp x 4 x d
A2 xm1
A1 area of the peak due to magnolin in the chromatogram obtained
A2 = area of the peak due to magnolin in the chromatogram obtained
solution, in grams;
m2 = mass of magnolin CRS used to prepare reference solution (a), in
grams;
p percentage content of magnolin in magnolin CRS;
d additional dilution factor.
Magnolia Officinalis Bark

Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried bark from the stem and branch of Magnolia officinalis
Rehder et E.H. Wilson.
Content
Minimum 2.0 per cent for the sum of magnolol (C 18H 18 O 2 ;
M, 266.3) and honokiol (C 18H 180 2 ; Mr 266.3) (dried drug).
IDENTIFICATION
A. Fragments of stem and branch bark, quilled singly or
double quilled, about 30 cm long and 2-7 mm thick.
Figure 2567.-1. - Illustrati.on for identification test B of powdered
The outer surface is brownish-grey, rough, sometimes scaly,
herbal drug of Magnolia officinalis bark
easily exfoliated, with distinct lenticels and longitudinal
striations. The inner surface is reddish-brown or dark brown, Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel
smooth, with numerous fine, longitudinal striations. F254plat.e R (2-10 µm)].
The texture is hard and difficult to break. The fracture is
Mobile phase methanol R, ethyl acetate R, toluene R
granular, brownish-grey in the outer layers and reddish-
(4:8:120 VIVIV).
brown or dark brown in the inner layers.
yellowish-brown. Examine under a microscope using chloral Development Over a path of 15 cm [or 7 cm].
hydrat.e solution R. The powder shows the following diagnostic Drying In air.
characters (Figure 2567.-1): numerous sclereids [DJ of Det.ection A Examine in ultraviolet light at 254 nm.
various shapes and sizes, free or in groups, often branched, Results A See below the sequence of zones present in the
up to 100 µm long, with very thick, striated walls and chromatograms obtained with the reference solution and the
conspicuous pit canals; oval [F] or rounded [G] oil cells, up test solution. Furthermore, other faint zones may be present
to 100 µm in diameter, with orange-yellow contents; narrow, in the chromatogram obtained with the test solution.
thick-walled fibres, often in bundles [C, HJ, accompanied by
fusiform medullary rays (tangential section [Ca]) or
Top of the plate
consisting of files of rectangular cells (longitudinal section
[Ha]); brown cork fragments with regularly and finely -- --
thickened walls [BJ; fragments of phloem parenchyma [A]
Eugenol: a faint quenching zone
consisting of cells with irregular walls [Aa], accompanied by
fusiform medullary rays (tangential section [Ab]). Examine
under a microscope using a 50 per cent V/V solution of Magnolol: a dark blue fluorescent A dark blue fluorescent zone
glycerol R. The powder shows rounded, ovoid or polyhedral zone (magnolol)
starch granules, about 2-12 µm in diameter, simple or 2-6
compound, free [J] or included in parenchyma cells [E]. Honokiol: a quenching zone A quenching zone (honokiol)
C. Thin-layer chromatography (2.2.27). -- --

Test solution Reduce to a powder (355) (2.9.12), avoiding Reference solution Test solution
heating. To 0.5 g of the powdered herbal drug add 5 mL of
methanol R, sonicate for 5 min, centrifuge, and use the
supernatant. Filter through a membrane filter (nominal pore Detection B Treat with vanillin reagent R, heat at
size 0.45 µm) if necessary. 100-105 °C for 5-10 min and examine in daylight.
Reference soluti.on Dissolve 1 mg of honoki.ol R, 1 mg of Results B See below the sequence of zones present in the
magnolol R and 2 mg of eugenol R in 1 mL of methanol R. chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones of various
2023 Magnolia Officinalis Flower IV-349
colours may be present in the chromatogram obtained with System suitability Reference solution (b):
the test solution. - resolution: minimum 1.8 between the peaks due to
honokiol monoacetate isomers 1 and 2.
Top of the plate Calculate the sum of the percentage contents of honokiol and
magnolol using the following expression:
- - -- Ai x m2 xpi x 5 + A3 x m3 xp2 x 5
Eugenol: a brown zone
A2 x m1 A4 xm 1
A, area of the peak due to honokiol in the chromatogram obtained
Magnolol: a pinkish-violet zone A pinkish-violet zone (magnolol) A2 area of the peak due to honokiol in the chromatogram obtained
Honokiol: a dark violet zone A dark violet zone (honokiol) A3 area of the peak due to magnolol in the chromatogram obtained
A bluish-violet zone A4 area of the peak due to magnolol in the chromatogram obtained
-- -- m, mass of the herbal drug to be examined used to prepare the test
solution, in grams;
m2 mass of honokiol CRS used to prepare reference solution (a), in
grams;
mass of magnolol CRS used to prepare reference solution (a), in
TESTS grams;
Loss on drying (2.2.32) p, percentage content of honokiol in honokiol CRS;
Maximum 11.0 per cent, determined on 1.000 g of the Pz percentage content of magnolol in magnolol CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEw
105 °C for 2 h.
Total ash (2.4.16)
Ash insoluble in hydrochloric acid (2. 8.1) Magnolia Officinalis Flower
Liquid chromatography (2.2.29). ffi& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __

DEFINITION
(2.9.12) add 80 mL of methanol Rand heat in a water-bath
Steamed and dried, unopened flower of Magnolia officinalis
under a reflux condenser for 30 min. Cool, then dilute to
Rehder et E.H. Wilson.
(nominal pore size 0.45 µm). Content
Reference solution (a) Dissolve 4.0 mg of honokiol CRS and Minimum 0.20 per cent of the sum ofmagnolol (C 18H 18O 2;
4.0 mg of magnolol CRS in methanol Rand dilute to 20.0 mL Mr 266.3) and honokiol (C1sH1sO2; Mr 266.3) (dried drug).
with the same solvent. IDENTIFICATION
Reference solution (b) Dissolve 2.0 mg of honokiol CRS in A. The greyish-yellow pedicel is short (0.5-2 cm) and densely
2.0 mL of acetonitrile R. To 1.0 mL of the solution add tomentose. The brown or reddish-brown flower bud is
15 µL of acetic anhydride R and mix. Heat at 50 °C for elongated, conical, 4-7 cm long and 1.5-2.5 cm in diameter
60 min. Cool. Add successively, mixing after each addition, at the base; it usually consists of 12 perianth segments in
16 µL of concentrated ammonia R, 1.0 mL of acetonitrile R and several whorls. The stamens are numerous with a fine, short
2.0 mL of water R. Filter through a membrane filter filament and a linear, yellowish-brown anther. The carpels
(nominal pore size 0.45 µm). are free and numerous, spirally arranged on a conical
Column: receptacle.
- size: l = 0.25 m, 0 = 4.6 mm; B. Microscopic examination (2.8.23). The powder is reddish-
- stationary phase: end-capped octadecylsilyl silica gel for brown. Examine under a microscope using chloral hydrate
chromatography Rl (5 µm); solution R. The powder shows the following diagnostic
- temperature: 30 °C. characters: fragments of the perianth segments with
Mobile phase 0.5 per cent V/V solution of acetic acid R, polyhedral or elliptical epidermal cells, with irregularly
acetonitrile for chromatography R (40:60 V/V). thickened walls and anomocytic stomata (4-6 subsidiary cells)
(2.8.3), accompanied by parenchyma that includes oval or
rounded oil cells about 50 µm in diameter with orange-
Detection Spectrophotometer at 290 nm. yellow contents; certain fragments contain epidermal cells
Injection 10 µL. with rounded papillae; numerous, branched sclereids, with
Run time Twice the retention time of honokiol for the test channelled walls and a large lumen, about 15 µm in
solution and reference solution (a); 3 times the retention time diameter; numerous elliptical pollen grains about 50 µm long
of honokiol for reference solution (b). and 40 µm wide, with a smooth exine.
Relative retention With reference to honokiol (retention C. Examine the chromatograms obtained in the test for other
time =about 8 min): magnolol =about 1.4; honokiol Magnolia species.
monoacetate isomer 1 = about 1.5; honokiol monoacetate Detection A Examine in ultraviolet light at 254 nm.
isomer 2 = about 1.6; honokiol diacetate = about 2.6. Results A See below the sequence of zones present in the
IV-350 Magnolia Officinalis Flower 2023
test solution. Furthermore, other faint zones may be present Results The chromatogram obtained with the test solution
in the chromatogram obtained with the test solution. shows no blue fluorescent zone in the lower part of the plate
and no green fluorescent zone in the upper part, nor any
Top of the plate other fluorescent zone.
- - - -
Eugenol: a faint quenching zone powdered herbal drug (710) (2. 9.12) by drying in an oven at
105 °C.
Total ash (2.4.16)
Magnolol: a dark blue fluorescent A dark blue fluorescent zone
zone (magnolol)
Honokiol: a quenching zone A quenching zone (honokiol)

ASSAY
-- -- Test solutwn Reduce the herbal drug to a powder (710)
Reference solution Test solution (2. 9.12) using a blade grinder equipped with a double-walled
grinding chamber cooled to a temperature of about 10 °C.
To 0.500 g of the powdered herbal drug add 10 mL of
Detection B Treat with vanillin reagent R, heat at methanol R. Sonicate for 1 h at a power of 80 W and a
100-105 °C for 5-10 min and examine in daylight. frequency of 37 kHz (sonication time may be adapted
Results B See below the sequence of zones present in the according to the power and frequency used). Change the
chromatograms obtained with the reference solution and the water of the ultrasonic bath after 30 min of sonication to
test solution. Furthermore, other faint zones of various prevent heating. Centrifuge at 1500-2000 g for 15 min.
colours may be present in the chromatogram obtained with Transfer the supernatant to a 20.0 mL flask. Add 9.5 mL of
the test solution. methanol R to the residue. Repeat the sonication for 1 h.
Change the water of the ultrasonic bath after 30 min of
Top of the plate sonication to prevent heating. Centrifuge. Transfer the
supernatant to the same 20.0 mL flask. Cool, then dilute to
- - - - (nominal pore size 0.45 µm).
Eugenol: a brown zone Reference solutwn (a) Dissolve 5.0 mg of honokwl CRS in
Magnolol: a pinkish-violet zone A pinkish-violet zone (magnolol) Reference solutwn (b) Dissolve 6.0 mg of magnolol CRS in
Honokiol: a dark violet zone A dark violet zone (honokiol)
Reference solutwn (c) Dissolve 2.0 mg of honokwl R in
A bluish-violet zone 2.0 mL of acetonitrile R. Add 30 µL of acetic anhydride R and
mix. Heat at 50 °C for 60 min. Cool. Add successively,
-- --
mixing after each addition, 32 µL of concentrated ammonia R,
Reference solution Test solution 2.0 mL of acetonitrile Rand 4.0 mL of water R. Filter
TESTS Column:
Other Magnolia species - size: l =0.15 m, 0 =4.6 mm;
Thin-layer chromatography (2.2.27). - statwnary phase: end-capped ethylene-bridged polar-embedded
octadecylsifyl silica gel for chromatography (hybrid material) R
Test solutwn Reduce the herbal drug to a powder (710)
(3.5 µm);
(2.9.12), avoiding heating. To 0.5 g of the powdered herbal
- temperature: 25 ± 2 °C.
drug add 2.5 mL of methanol R. Sonicate for 15 min at a
power of 80 Wand a frequency of 37 kHz (sonication time Mobile phase:
may be adapted according to the power and frequency used), - mobile phase A: anhydrous formic acid R, water R
then centrifuge at 1500-2000 g for 10 min and transfer the (0.1:99.9 V/V);
supernatant to a 5 mL flask. Add 2 mL of methanol R to the - mobile phase B: acetonitrile for chromatography R;
residue, sonicate for 15 min and centrifuge. Transfer the
supernatant into the same 5 mL flask. Dilute to 5 mL with Time Mobile phase A Mobile phase B
size 0.45 µm) if necessary. 0 - 20 47 53
20 - 22 47 ➔ 5 53---> 95
Reference solutwn Dissolve 1 mg of honokwl R, 1 mg of
22 - 27 5 95
magnolol R and 2 mg of eugenol R in 4 mL of methanol R.
Mobile phase methanol R, ethyl acetate R, toluene R
(1:5:30 V/V/V).
Injectwn 20 µL.
Relative retention With reference to honokiol (retention
Drying In air.
= =
time about 10 min): magnolol about 1.3; honokiol
monoacetate isomer 1 = about 1.4; honokiol monoacetate
Detection Examine in ultraviolet light at 365 nm. = =
isomer 2 about 1.5; honokiol diacetate about 1.9.
2023 Mallow Flower IV-351
System suitability Reference solution (c): (surface view [F], transverse section [G]); fragments of the
- resolution: minimum 2.0 between the peaks due to mesophyll of the calyx and the epicalyx whose cells contain
honokiol monoacetate isomers 1 and 2. small cluster crystals of calcium oxalate [K]; veins of the
If necessary, dilute the test solution to obtain peaks of sepals [P] with vessels [Pa] accompanied by cells with cluster
honokiol and magnolol that are similar in height to the crystals of calcium oxalate [Pb]; fragments of petal epidermis,
corresponding peaks in reference solutions (a) and (b). with elongated cells and sinuous margins, narrow in the wild
Calculate the sum of the percentage contents of honokiol and plant [A], shorter and broader in the cultivated varieties [BJ,
magnolol using the following expression: bearing sessile glandular trichomes with multicellular club-
shaped heads [Ba, C, E]; fragments of petal mesophyll [HJ
A 1 x m 2 x0.16xp, xd A3 xm3 XP2 xd consisting of large mucilage cells [He], sometimes cells with
- - - A2
- -xm,
----+-- ----
A4 xm,
small cluster crystals of calcium oxalate (Hb] and spiral
vessels [Ha]; spherical pollen grains, about 150 µm in
AI area of the peak due to honokiol in the chromatogram obtained diameter, with a roughly spiny exine [M].
area of the peak due to honokiol in the chromatogram obtained
area of the peak due to magnolol in the chromatogram obtained
area of the peak due to magnolol in the chromatogram obtained
with reference solution (b);
solution, in grams;
m2 mass of honokiol CRS used to prepare reference solution (a), in
grams;
mass of magnolol CRS used to prepare reference solution (b), in
grams;
P1 percentage content of honokiol in honokwl CRS;
Pz percentage content of magnolol in magnolo/ CRS;
d dilution factor of the test solution.
----------------------~& Ha /
Mallow Flower
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
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 Figure 1541. -1. - Illustratwn for identificatwn test B of powdered
pubescent triangular lobes, gamosepalous at the base; herbal drug of mallow flower
a corolla 3-4 times longer than the calyx with 5 wedge-
shaped, notched petals fused to the stamina! tube at their C. Thin-layer chromatography (2.2.27).
base; numerous stamens, the filaments of which fuse into a Test solution To 1 g of the powdered herbal drug (355)
stamina! tube covered by small star-shaped trichomes and (2. 9.12) add 10 mL of ethanol (60 per cent V/V) R. Stir for
occasional simple trichomes visible using a lens; numerous 15 min and filter.
wrinkled carpels, glabrous or sometimes pubescent, enclosed
Reference solutwn 0.5 g/L solution of quinaldine red R in
in the stamina! tube and arranged into a circle around a
ethanol (96 per cent) R.
central style ending with numerous filiform stigmas.
In cultivated varieties, the epicalyx is 3-7 partite, the calyx Plate TLC silica gel plate R.
5-8 partite and the corolla 5-10 partite. Mobile phase glacial acetic acid R, water R, butanol R
B. Microscopic examination (2.8.23). The powder is bluish- (15:30:60 V/V/V).
grey. Examine under a microscope using chloral hydrate Applicatwn 10 µL of the test solution and 5 µL of the
solution R. The powder shows the following diagnostic reference solution, as bands.
characters (Figure 1541.-1): unicellular, thick-walled, Development Over a path of 10 cm.
flexuous covering trichomes, from the calyx and the epicalyx, Drying In air.
up to 2 mm in length, whole [L] or, most often, fragmented
[Q]; fragments of the epidermis of the sepals (surface view
[D, TI) with anomocytic stomata (2.8.3) [De]; club-shaped Results The chromatogram obtained with the reference
glandular trichomes with multicellular heads [Db] and short solution shows an orange-red zone in the upper part of the
unicellular covering trichomes, somewhat curved, either middle third ; the chromatogram obtained with the test
isolated [J] or in star-shaped groups of 2-6 [Da]; fragments solution shows, below the zone in the chromatogram
of covering trichomes [N]; isolated glandular trichomes obtained with the reference solution, 2 violet zones in the
IV-352 Mallow Leaf 2023
middle third, with the principal zone (6 11 -malonyl malvin)

situated just below the other violet zone (malvin).
TESTS
powdered herbal drug by drying in an oven at 105 °C.
Total ash (2.4.16)
Swelling index (2.8.4)
Minimum 15, determined on 0.2 g of the powdered herbal
drug (710) (2.9.12) moistened with 0.5 mL of anhydrous
ethanol R.
---------------------~&
Mallow Leaf
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or fragmented, dried leaf of Malva sylvestris L., Malva
neglecta 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 Figure 2391.-1. - Illustration for identification test B of powdered
brownish-green. The abaxial surface of the lamina bears herbal drug of malww leaf
more hairs and shows a more prominent venation than the
adaxial surface. The major veins on the upper surface of the C. Thin-layer chromatography (2.2.27).
leaves and the petioles may be violet. The petioles are as long Test solution To 2.0 g of the powdered herbal drug (710)
as the leaves, up to 2 mm wide, rounded and somewhat (2. 9.12) add 20 mL of an 80 per cent V/V solution of
flattened, longitudinally slightly grooved, green or brownish- tetrahydrofuran R; extract for 10 min using sonication and
green or violet. The fragmented drug consists of occasionally filter.
agglomerated, crumpled pieces of leaves showing prominent Reference solution Dissolve 3 mg of hyperoside R and 3 mg of
veins. rutoside trihydrate R in 20 mL of methanol R.
B. Microscopic examination (2.8.23). The powder is green or Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
yellowish-green. Examine under a microscope using chloral plate R (2-10 µm)].
hydrate solution R. The powder shows the following diagnostic Mobile phase anhydrous formic acid R, anhydrous acetic acid R,
characters (Figure 2391.-1): fragments of the lamina water R, ethyl formate R, 3-pentanone R
(transverse section [F]), consisting of the lower epidermis (4:11:14:20:50 VIVIVIV/V).
(surface view [C]) and the upper epidermis (surface view Application 10 µL [or 4 µL] as bands of 10 mm [or 8 mm].
[DJ, transverse section [Fb]), with cells that show straight, or
more or less sinuous anticlinal walls; stomata mostly Devewpment Over a path of 10-12 cm [or 6 cm].
anisocytic (2.8.3) on both surfaces [Ca, Da]; long covering Drying In air.
trichomes with thickened walls and tapering to a point at the Detection Heat at 100 °C for 10 min; spray or dip the warm
apex, usually unicellular, whole [A, Fa] or fragmented [Db], plate in a 10 g/L solution of diphenylboric acid aminoethyl
but in M. Sylvestris they may be stellate with 2-8 components ester R in methanol R; remove the solvent with cold air; spray
[HJ, each strongly pitted at the base; club-shaped glandular or dip the plate in a 50 g/L solution of macrogol 400 R in
trichomes composed of 2-6 cells [E] occur in both species; methanol R, dry in air and examine after 15 min in ultraviolet
fragments of the mesophyll consisting of palisade parenchyma light at 365 run.
(surface view [De], transverse section [Fe]) and spongy Results See below the sequence of fluorescent zones present
mesophyll cells containing mucilage, cells containing cluster in the chromatograms obtained with the reference solution
crystals of calcium oxalate, often associated with vessels [BJ; and the test solution. Furthermore, other faint fluorescent
occasional spherical pollen grains, 110-170 µm in diameter, zones may be present in the chromatogram obtained with the
with a spiny exine [G]. test solution.
2023 Mandarin Epicarp and Mesocarp IV-353
Top of the plate yellowish-brown or orange, reddish-brown or more rarely,

blackish- or greenish-brown; often finely wrinkled and slightly
-- -- shiny. Scattered, concave or raised oil glands are dense on
Hyperoside: a yellow fluorescent the outer surface; when visible in transverse section oil glands
zone appear as irregularly shaped cavities beneath the epicarp.
The inner surface of the pericarp appears whitish or whitish-
yellowish, rough and often fibrous in texture, bearing
-- -- yellowish-white or yellowish-brown raised, often branched
Rutoside: a yellow fluorescent zone vascular bundles. The texture is mostly hard, stiff and fragile,
easily broken.
A light blue fluorescent zone yellowish or orange-brown, more rarely reddish-brown.
An orange fluorescent zone (Figure 2430.-1): fragments of epicarp (surface view [HJ)
consisting of small polygonal cells, sub-square or rectangular,
with slightly thickened anticlinal walls [Ha] and occasional
rounded stomata [Hb) (18-27 µm in diameter) encircled by a
TESTS combination of small polygonal plus narrow, elongate
Foreign matter (2.8.2) subsidiary cells; epicarp usually accompanied by sub-
Maximum 5 per cent of foreign organs, maximum 5 per cent epicarpal layers of thin-walled cells containing prisms of
of leaves with blisters of spores of Puccinia malvacearum and calcium oxalate [He]; prisms commonly biconical, also
maximum 2 per cent of foreign elements. rhomboidal or polyhedral prisms present, 5-30 µm long and
3-18 µm in diameter; fragments of pericarp (transverse
Foreign organs can be flowers, fruits and parts of the stem.
section [El) showing the epicarp covered by a thick
The blisters of spores on the leaves are mostly 1 mm wide,
cuticle [Ea] and sub-epicarpal layers composed of cells, some
and red or brown. Examine under a microscope using chloral
of which contain prisms of calcium oxalate [Eb ); sometimes
hydrate solution R. The spores of Puccinia malvacearum are
more than 1 prism crystal is found in the same cell [Aa, Hd];
oblong or oval with brownish walls and a small appendage.
fragments of schizolysigenous oil glands [CJ; numerous
Loss on drying (2.2.32) groups of mesocarp cells (transverse section [A], longitudinal
Maximum 12.0 per cent, determined on 1.000 g of the section [D]) with irregularly thickened, weakly lignified cell
powdered herbal drug (710) (2. 9.12) by drying in an oven at walls; free prism crystals of calcium oxalate or single crystals
105 °C for 2 h. within isolated cells [BJ; yellow hesperidin crystals sometimes
Total ash (2.4.16) abundant in cells; small droplets of orange-yellow essential oil
Maximum 17.0 per cent. occasionally present; numerous small vascular bundles [F]
Ash insoluble in hydrochloric acid (2.8.1) with narrow reticulate or spiral xylem [Fa] (4-23 µm in
Maximum 3.0 per cent. diameter), and occasional pitted tracheids; vasculature,
sometimes accompanied by crystal sheaths [Fb]; occasional
Swelling index (2.8.4) isolated, short vessels [G]. Examine under a microscope
Minimum 7, determined on 1.0 g of the powdered herbal
using a 20 g/L solution of potassium hydroxide R.
drug (710) (2.9.12).
The mounting medium becomes yellow because of the
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
presence of hesperidin.
(2. 9.12) add 10 mL of methanol R and heat in a water-bath
Mandarin Epicarp and Mesocarp at 65 °C for 5 min, shaking frequently. Allow to cool and
filter.
Reference solution Dissolve 1 mg of caffeic acid R and 2 mg
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
of hesperidin R in 5 mL of methanol R.
DEFINITION Plate TLC silica gel plate R (2-10 µm).
Dried epicarp and mesocarp of the ripe fruit of Citrus Mobile phase water R, anhydrous formic acid R, ethyl acetate R
reticulata Blanco or its cultivars, partly freed from the white (10:15:75 V/V/V).
spongy tissue of the mesocarp. Application 5 µL of the test solution and 10 µL of the
Content reference solution as bands of 8 mm.
Minimum 3.5 per cent of hesperidin (C 28 H 34 O 15; Mr 611) Development Over a path of 6 cm.
(dried drug).
Drying In air, then heat at 110-120 °C for 5 min.
IDENTIFICATION Detection Treat with a 10 g/L solution of aluminium
A. The pericarp consists of variously sized irregular pieces, or chloride R in ethanol (96 per cent) Rand heat at 110-120 °C
it is cut into strips, which may be folded or form compressed for 5 min; treat the warm plate with a 10 g/L solution of
loops. The larger pieces are mostly lobed, the lobes attached diphenylboric acid aminoethyl ester R in methanol R, and then
at the base of the pericarp where the remains of a fruit stalk with a 50 g/L solution of macrogol 400 R in methanol R. After
or stalk scar may be present. Isolated lobes of pericarp may 60 min examine the chromatograms in ultraviolet light at
also be present. Irregular pieces 0.5-7.6 cm long, and 365 nm.
0.4-4.6 cm wide; strips up to 7 .6 cm long and up to 2 cm
wide; both forms 1-4.5 mm thick. The outer surface is
IV-354 Mandarin Oil 2023
ASSAY
(355) (2.9.12) in a 100 mL round-bottomed flask.
Add 50.0 mL of methanol R, stir for 2 hand filter through a
Reference solution (a) Dissolve 10.0 mg of hesperidin CRS in
Reference solution (b) Dissolve 5.0 mg of naringin R in
reference solution (a) and dilute to 25.0 mL with reference
solution (a).
Column:
- size: l = 0.25 m, 0 = 4.6 mm;
Mobile phase acetonitrile R, glacial acetic acid R, methanol R,
water R (2.7:3.7:22:71.6 VIVIVIV).
G Flow rate 1 mUmin.
~ 25 µm
,___._. Detection Spectrophotometer at 283 nm.
~
Ir!jection 10 µL.
Run time Twice the retention time of hesperidin.
herbal drug of mandarin epicarp and mesocarp
=
Retention time Naringin about 15 min; hesperidin about =
20 min.
Results See below the sequence of fluorescent zones present System suitability Reference solution (b):
in the chromatograms obtained with the reference solution - resolution: minimum 2.0 between the peaks due to
and the test solution. Furthermore, other faint fluorescent naringin and hesperidin.
zones may be present in the chromatogram obtained with the Calculate the percentage content of hesperidin using the
test solution. following expression:
Top of the plate
Caffeic acid: a light blue fluorescent

zone A1 area of the peak due to hesperidin in the chromatogram
A blue fluorescent zone A2 area of the peak due to hesperidin in the chromatogram
-- -- m1 = mass of the herbal drug to be examined used to prepare the test
solution, in grams;
m2 mass of hesperidin CRS used to prepare reference solution (a), in
Hesperidin: a greenish-brown A greenish-brown fluorescent zone grams;
fluorescent zone (hesperidin) p = percentage content of hesperidin in hesperidin CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
A yellowish-green fluorescent zone
-- - -
A greenish fluorescent zone Mandarin Oil

~~----------------------
TESTS
DEFINITION
Bitter-orange epicarp and mesocarp
Essential oil obtained without heating, by suitable mechanical
Examine the chromatograms obtained in the assay.
treatment, from the peel of the fresh fruit of Citrus reticulata
Results The chromatogram obtained with the test solution Blanco.
shows no peak at the retention time of naringin with an area
of more than 1 per cent of the area of the peak due to CHARACTERS
hesperidin. Appearance
Greenish, yellow or reddish orange liquid showing blue
fluorescence.
powdered herbal drug (355) (2.9.12) by drying in an oven at Characteristic odour.
105 °C for 2 h. IDENTIFICATION
Total ash (2.4.16) First identification: B.
Maximum 7 .0 per cent. Second identification: A.
2023 Mandarin Oil IV-35 5
Test solutwn Dilute 0.1 mL of the substance to be examined Reference solutwn (a) Dilute 5 µL of a-pinene R, 5 µL of
to 1 mL with toluene R. sabinene R, 5 µL of /J-pinene R, 5 µL of fJ-myrcene R, 5 µL of
Reference solutwn Dissolve 2 µL of methyl p-cymene R, 70 µL of limonene R, 20 µL of y-terpinene Rand
N-methylanthranilate R, 4 mg of guaiazulene Rand 10 mg of 5 µL of methyl N-methylanthranilate R to 5.0 mL with
a-terpineol R in 10 mL of toluene R. heptane R.
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel Reference solutwn (b) Dissolve 5 µL of limonene R in 50 mL
plate R (2-10 µm)]. of heptane R. Dilute 0.5 mL of the solution to 5.0 mL with
Mobile phase ethyl acetate R, toluene R (15:85 VIV). heptane R.
Application 10 µL [or 2 µL] as bands. Column:
Development Over a path of 15 cm [or 6 cm] . = =
- size: l 60 m, 0 0.25 mm;
Drying In air. - stationary phase: phenyl(S)methyl(95)polysiloxane R (film
Detection A Examine in ultraviolet light at 365 nm. thickness 0.25 µm).
Results A The intense blue fluorescent zone in the Carrier gas helium for chromatography R.
chromatogram obtained with the test solution is similar in Flow rate 1.4 mlJmin.
position and fluorescence to the zone due to methyl N- Split ratw 1:70.
methylanthranilate in the chromatogram obtained with the
Temperature:
reference solution. Furthermore, other fluorescent zones may
Time Temperature
solution. (min) CC)
Detection B Spray with a 200 g/L solution of Column 0 - 90 50 - 230
phosphomolybdic acid R in ethanol (96 per cent) R and heat at Injection port 250
100 °C for 10 min; examine in daylight. Detector 250
the chromatograms obtained with the reference solution and Detection Flame ionisation.
the test solution. Furthermore, other zones may be present in
Injection 1 µL.
reference solution (a); record the retention times of these
Top of the plate
substances.
A blue zone System suitability Reference solution (a):
Guaiazulene: a blue zone A blue zone - resolution: minimum 1.5 between the peaks due to
sabinene and P-pinene; minimum 1.5 between the peaks
A blue zone due to p-cymene and limonene.
-- -·- Identification of components Using the retention times
determined from the chromatogram obtained with reference
A blue zone
solution (a), locate the components of reference solution (a)
-- -- in the chromatogram obtained with the test solution.
oc-Terpineol: a blue zone A blue zone (oc-terpineol)
Disregard the peak due to heptane.
Reference solution Test solution components. The limits are within the following ranges:
- a-pinene: 1.6 per cent to 3.0 per cent;
B. Examine the chromatograms obtained in the test for - sabinene: maximum 0.3 per cent;
chromatographic profile. - /J-pinene: 1.2 per cent to 2.0 per cent;
Results The characteristic peaks in the chromatogram - /J-myrcene: 1.5 per cent to 2.0 per cent;
obtained with the test solution are similar in retention time to - p-cymene: maximum 1.0 per cent;
those in the chromatogram obtained with the reference - limonene: 65.0 per cent to 75.0 per cent;
solution. - y-terpinene: 16.0 per cent to 22.0 per cent;
- methyl N-methylanthranilate: 0.30 per cent to
TESTS 0.60 per cent;
Relative density (2.2.5) - disregard limit: area of the principal peak in the
0.848 to 0.855. chromatogram obtained with reference solution (b).
Refractive index (2.2.6) Residue on evaporation (2.8.9)
1.474 to 1.478. 1.6 per cent to 4.0 per cent, determined after heating on a
Optical rotation (2. 2. 7) water-bath for 4 h.
+ 6.4° to+ 7.5° (measured in a 0.1 dm tube). STORAGE
Fatty oils and resinified essential oils (2. 8. 7) At a temperature not exceeding 25 °C.
It complies with the test. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
procedure.
Test solutwn Dilute 0.20 g of the substance to be examined
to 10.0 mL with heptane R.
IV-356 Marshmallow Leaf 2023
Marshmallow Leaf
DEFINITION
Whole or cut, dried leaf of Althaea officinalis L.
IDENTIFICATION
A. The leaves have long petioles and are about 7-10 cm long;
the lamina is cordate or ovate with 3-5 shallow lobes and
crenate or dentate margins; the venation is palmate.
The petioles and both surfaces of the lamina are greyish-
green and densely pubescent. Rarely, fragments of the
inflorescence or immature fruits may be present.
characters (Figure 1856.-1): numerous long, rigid, unicellular
covering trichomes with thick walls, pointed at the apex,
often fragmented [C], angular and pitted at the base where
they are sometimes still united to form stellate structures with
up to 8 components (surface view [BJ, transverse
section [El); few secretary trichomes, isolated, with
unicellular stalks and globular, multicellular heads [F];
fragments of the lower [A] and upper [D] leaf epidermises in
surface view with anomocytic [Aa] or paracytic [Da] stomata
(2.8.3), glandular trichomes [Ab] and basal cells of covering
trichomes [Ac], often accompanied by palisade
parenchyrna [Db]; cluster crystals of calcium oxalate,
isolated [HJ or included in the parenchyrna of the Figure 1856.-1. - Illustration for identification test B of powdered
mesophyll [Ge, Kb]; fragments of veins [G] with small, herbal drug of marshmallow leaf
spiral [Gb] or annular [Ga] vessels, often accompanied by
sheaths containing cluster crystals of calcium oxalate [Ge];
fragments of the lamina, in transverse section [Kl, showing Top of the plate
the epidermises bearing broken covering trichomes [Ka], a A blue fluorescent zone
symmetrical, heterogeneous mesophyll with some cells
containing cluster crystals of calcium oxalate [Kb]; occasional A yellow fluorescent zone
pollen grains, spherical, with a roughly spiny exine, about Quercitrin: an orange zone
150 µm in diameter m. Examine under a microscope using
ruthenium red solution R. The powder shows groups of -- --
parenchyrna containing mucilage, which stains orange-red. An orange fluorescent zone
(2. 9.12) add 10 rnL of methanol R. Heat in a water-bath -- --
under a reflux condenser for 5 min. Allow to cool and filter. Chlorogenic acid: a blue fluorescent
Distil the filtrate under reduced pressure until the zone
total volume is about 2 mL.
Reference solution Dissolve 2.5 mg of chlorogenic acid Rand
2.5 mg of quercitrin R in 10 mL of methanol R. An orange fluorescent zone
Plate TLC silica gel plate R. An intense yellow fluorescent zone

Mobile phase anhydrous formic acid R, glacial acetic acid R, Reference solution Test solution
water R, ethyl acetate R (11:11:27:100 VIVIVIV).
TESTS
Drying At 100-105 °C.
Maximum 4 per cent of leaves infected by Puccinia
Detection Spray with a 10 g/L solution of diphenylboric acid malvacearum, showing red spots, and maximum 2 per cent of
aminoethyl ester R in methanol R, then with a 50 g/L solution other foreign matter.
of macrogol 400 R in methanol R; allow to dry in air for
30 min and examine in ultraviolet light at 365 nm.
Results See below the sequence of zones present in the powdered herbal drug (355) (2. 9.12) by drying in an oven at
chromatograms obtained with the reference solution and the 105 °C for 2 h.
present in the chromatogram obtained with the test solution. Total ash (2.4.16)
2023 Mastic IV-357
Ash insoluble in hydrochloric acid (2.8.1) The powder shows the following diagnostic characters
Maximum 2.0 per cent. (Figure 1126.-1): fragments of colourless, mainly unlignified,
Swelling index (2.8.4) thick-walled fibres [C, D, M) with split or pointed ends [DJ,
Minimum 12, determined on 0.2 g of the powdered herbal sometimes accompanied by parenchymatous cells of the
drug (355) (2.9.12). medullary rays [M], or grouped [CJ; fragments of vessels,
bordered-pitted or with reticulate or scalariform thickenings
- - - - - - - - - - - - - - - - - - - - - PhEur
[G, HJ; cluster crystals of calcium oxalate about 20-35 µm,
mostly 25-30 µm in size, isolated [Kl or included in
parenchymatous cells [BJ; fragments of parenchyma [E] with
cells containing mucilage [Ea, F]; fragments of cork with
Marshmallow Root thin-walled, tabular cells (surface view [A], transverse section
[L)) (unpeeled root). Examine under a microscope using
(Ph. Bur. monograph 1126) ruthenium red solution R. The powder shows groups of
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ parenchyma containing mucilage, which stains orange-red.
Examine under a microscope using water R. The powder
DEFINITION
shows numerous starch granules ITT, about 3-25 µm in size,
Peeled or unpeeled, whole or cut, dried root of Althaea occasionally with a longitudinal hilum. The starch granules
officinalis L. are mostly simple ITa], a few being 2-4 compound ITb].
A. The unpeeled, non-fragmented drug consists of Foreign matter (2.8.2)
cylindrical, slightly twisted roots, up to 2 cm thick, with deep Maximum 2 per cent of brown deteriorated drug.
longitudinal furrows. The outer surface is greyish-brown and
bears numerous rootlet scars. The fracture is fibrous Loss on drying (2.2.32)
externally, rugged and granular internally. The section shows Maximum 12.0 per cent, determined on 1.000 g of the
a more or less thick, whitish bark with brownish periderm, powdered herbal drug (710) (2. 9.12) by drying in an oven at
separated by the well-marked, brownish cambium from a 105 °C for 2 h.
white xylem. The stratified structure of the bark and the Total ash (2.4.16)
radiate structure of xylem become more distinct when Maximum 6.0 per cent for the peeled root and maximum
moistened. 8.0 per cent for the unpeeled root.
The peeled drug has a greyish-white, finely fibrous outer Swelling index (2.8.4)
surface. Cork and external cortical parenchyma are absent. Minimum 10, determined on the powdered herbal drug
(710) (2.9.12).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Mastic
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried resinous exudate obtained from stems and branches of
Pistacia lentiscus L.
Content
Minimum 10 mUkg of essential oil (anhydrous drug).
IDENTIFICATION
A. Small light yellow to greenish-yellow, non-uniform,
spherical or pyriform, clear or opaque, hard glassy fragments.
Test solution Dissolve 1 g of the substance to be examined
in 10 mL of methylene chloride Rand filter after 1-2 min.
Reference solution Dissolve 25 mg of eugenol Rand 25 mg of
borneol R in 3 mL of methylene chloride R.
Mobile phase light petroleum R, toluene R (5:95 V/V).
Application l µL as bands.
Figure 1126.-1. - Illustration for identification test B of p(Yl()dered Drying In air.
herbal drug of marshmallow root Detection Spray with vanillin reagent R and heat at
100-105 °C for 5 min.
brown (unpeeled root) or whitish (peeled root). Examine
IV-358 Mate Leaf 2023
test solution. Furthermore, other zones of various colours

solution.
Top of the plate
A violet zone
-- --
A pale violet zone
A very pale violet zone
-- --
Eugena!: a brown zone A blue zone
Borneo!: a greenish-blue zone A bluish-violet zone
A dark violet zone
TESTS
Acid value (2.5.1)
Water (2.2.13)
Maximum 10 mUkg, determined on 25.0 g of the drug
reduced to a powder (1400) (2. 9. 12).
Total ash (2.4. 16)
Maximum 0.5 per cent. Figure 2678.-1. - Illustration for identification test B of powdered
herbal drug of mate leaf
ASSAY
Essential oil (2.8.12) B. Microscopic examination (2.8.23). The powder is brown.
Use a 500 mL round-bottomed flask and 200 mL of water R Examine under a microscope using chloral hydrate solution R.
as the distillation liquid. Reduce the drug to a powder (1400) The powder shows the following diagnostic characters
(2.9.12) and immediately use 20.0 g for the determination. (Figure 2678.-1): fragments of the upper epidermis of the
Introduce 0.50 mL of xylene R in the graduated tube. Distil lamina (surface view [F], transverse section [BJ), covered
at a rate of 2-3 mUmin for 2 h. with an irregularly striated cuticle [Fa, Ba] and consisting of
STORAGE cells with slightly and regularly thickened walls [Fa, Bb], with
Do not powder. some cells containing a prism of calcium oxalate [Fb, Be],
and accompanied by underlying palisade parenchyma [Fe,
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Bd]; fragments of the upper epidermis of the lamina from the
area near a vein (surface view [K]), consisting of nearly
rectangular cells in a regular arrangement; fragments of the
lower epidermis of the lamina (surface view [A], transverse
Mate Leaf section [L]), covered by a striated cuticle [Aa, La] consisting
of polygonal cells [Aa] and anomocytic stomata (2.8.3) [Lb,
(Ph. Bur. monograph 2678) Ab]; fragments of spongy parenchyma (surface view [DJ),
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ sometimes accompanying the lower epidermis (transverse
section [Le]); cluster crystals of calcium oxalate of various
DEFINITION
Leaf of flex paraguariensis A.St.-Hil., rapidly desiccated by
sizes that may exceed 30 µm in diameter, isolated m or
included in parenchyma cells [G]; groups of pericyclic
heating and cut. fibres [C]; rectangular, thick-walled and channelled cells
Content from the endodermis [E], often accompanying pericyclic
Minimum 1.0 per cent of caffeine (C 8 H 10N 4 0 2 ; Mr 194.2) fibres [Ea]; vascular bundles [HJ consisting of spiral vessels
(dried drug). with a small diameter [Ha], accompanied by fibres [Hb] and
IDENTIFICATION sometimes by parenchyma, with some cells containing a
cluster crystal of calcium oxalate [He].
A. The yellowish-green or brownish-green leaf is tough and
brittle with a short petiole. It is 8-12 cm long and 5-6 cm C. Thin-layer chromatography (2.2.27).
wide. The lamina is elliptic oval and glabrous, with dentate Test solution To 1.0 g of the powdered herbal drug (355)
margins. The venation is pinnate and prominent on the lower (2. 9.12) add 10 mL of methanol R and sonicate for 10 min.
surface. The cut herbal drug occurs as glabrous, yellowish- Centrifuge or filter.
green, irregular, angular pieces ranging in length from Reference solution Dissolve 7 mg of caffeine R, I mg of
2-4 mm. chlorogenic acid R and 1 mg of rutoside trihydrate R in 5 mL of
methanol R.
2023 Mate Leaf IV-359
Mobile phase toluene R, water R, anhydrous formic acid R, TESTS

ethyl formate R (3:6:8:60 VIVIVIV). Loss on drying (2.2.32)
Application 4 µL as bands of 8 mm. Maximum 8.0 per cent, determined on 1.000 g of the
Development Over a path of 6 cm. powdered herbal drug (355) (2. 9.12) by drying in an oven at
105 °C.
Drying In a current of air at room temperature.
Total ash (2. 4. 16)
chromatograms obtained with the reference solution and the ASSAY
test solution. Furthermore, other faint quenching zones may Liquid chromatography (2.2.29).
be present in the chromatogram obtained with the test Solution A Dissolve 30.0 mg of caffeine CRS in the mobile
solution. phase and dilute to 100.0 mL with the mobile phase.
Top of the plate (355) (2.9.12) into a 100 mL round-bottomed flask and add
40 mL of methanol R. Heat in a water-bath at 70 °C under a
reflux condenser for 30 min. Allow to cool and filter through
-- --- a plug of absorbent cotton into a 100 mL volumetric flask.
A quenching zone
Transfer the absorbent cotton with the drug residue into the
round-bottomed flask. Add 40 mL of methanol R, proceed as
Caffeine: a quenching zone A quenching zone (caffeine) before and filter into the same volumetric flask. Rinse the
round-bottomed flask and the filter with methanol R, add the
rinsings to the volumetric flask and dilute to 100.0 mL with
A quenching zone methanol R. Shake to obtain a homogeneous solution.
Transfer 10.0 mL of this solution into a volumetric flask, add
30 mL of water R and dilute to 50.0 mL with the mobile
Chlorogenic acid: a quenching zone A quenching zone (chlorogenic acid) phase. Filter through a membrane filter (nominal pore size
0.45 µm).
--- ---
Rutoside: a quenching zone A quenching zone (rutoside)
50.0 mL with the mobile phase.
Reference solution Test solution Reference solution (b) Dissolve 15. 0 mg of (-)-epicatechin R
in the mobile phase and dilute to 25.0 mL with the mobile
Detection B Heat at 100-105 °C for 5 min. Treat the warm phase. To 10.0 mL of the solution add 5.0 mL of solution A
plate with a 10 g/L solution of diphenylboric acid aminoethyl and dilute to 50.0 mL with the mobile phase.
ester R in methanol R, then with a 50 g/L solution of macrogol Column:
400 R in methanol R. Allow the plate to dry in air. Examine - size: l = 0.25 m, 0 = 4.6 mm;
in ultraviolet light at 366 nm. - stationary phase: end-capped octadecylsilyl silica gel for
Results B See below the sequence of zones present in the chromatography R (5 µm).
chromatograms obtained with the reference solution and the Mobile phase methanol R, water for chromatography R
test solution. 1 or 2 faint blue fluorescent zones may be (25:75 VIV).
present in the upper third of the chromatogram obtained Flow rate I mL/min.
with the test solution. Furthermore, other faint fluorescent Detection Spectrophotometer at 272 nm.
Injection I O µL.
test solution.
Run time 3 times the retention time of caffeine.
Top of the plate Retention time Caffeine = about 11 min.
- resolution: minimum 2.0 between the peaks due to caffeine
1 or 2 red fluorescent zones and (-)-epicatechin.
--- --
An intense blue fluorescent zone

AI area of the peak due to caffeine in the chromatogram obtained
Chlorogenic acid: a blue fluorescent An intense blue fluorescent zone
zone (chlorogenic acid)
solution, in grams;
--- --
m2 mass of caffeine CRS used to prepare solution A, in grams;
Rutoside: an orange fluorescent zone An orange fluorescent zone p percentage content of caffeine in caffeine CRS.
(rutoside)
- - - - - - - - - - - - - - - - - - - - - - - Ph Eur
IV-360 Matricaria Flowers 2023
Matricaria Flowers
(Matricaria Flower, Ph. Bur. monograph 0404)
Preparation
Matricaria Liquid Extract
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried capitula of Matricaria recutita L. (Chamomilla
recutita (L.) Rauschert).
Content
- blue essential oil: minimum 4 mIJkg (dried drug);
- total apigenin 7-glucoside (C 21 H 20 O 10; Mr 432.4):
minimum 0.25 per cent (dried drug).
IDENTIFICATION
A. Capitula, when spread out, consisting of an involucre
made up of many bracts arranged in 1-3 rows; an elongated-
conical receptacle, occasionally hemispherical (young
capitula); 12-20 marginal ligulate florets with a white ligule;
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 stamens
and a gynoecium similar to that of the ligulate florets.
hydrate solution R. The powder shows the following diagnostic Figure 0404. -1. - Illustration for identification test B of powdered
characters (Figure 0404.-1): fragments of the outer epidermis herbal drng of matricaria flower
of the bracts of the involucre (surface view [Kl), consisting
of, on the margin, thin-walled cells covered by a finely Reference solution Dissolve 2 µL of chamazulene R, 5 µL of
(-)-rx-bisabolol R and 10 mg of bomyl acetate R in 5 mL of
striated cuticle [Ka], anomocytic stomata (2.8.3) [Kb] and
occasional biseriate glandular trichomes [Kc], and a central toluene R.
region 01 composed of elongated sclereids with moderately Plate TLC silica gel plate R.
thickened and channelled walls Ua]; fragments of the inner Mobile phase ethyl acetate R, toluene R (5:95 V/V).
epidermis of the corolla of the ligulate florets consisting of Application 10 µL as bands.
thin-walled, polygonal cells, slightly papillose [BJ; fragments
of the outer epidermis of the ligulate florets consisting of
sinuous cells, covered by a striated cuticle [DJ, often Drying In air.
accompanied by underlying narrow vessels [Da]; fragments Detection Spray with anisaldehyde solution R and heat at
from the apex of the lobes of the corolla of the tubular florets 100-105 °C for 5-10 min; examine immediately in daylight.
[A] with elongated cells on the margin [Aa] and slightly Results See below the sequence of zones present in the
papillose cells fAb]; biseriate glandular trichomes with a short chromatograms obtained with the reference solution and the
stalk (1 or 2 tiers of 2 cells) and a head of 2-3 tiers of 2 cells, test solution. Furthermore, other zones are present in the
on the epidermises of the corollas of both types of floret and chromatogram obtained with the test solution.
of the bracts of the involucre and on the ovary (surface view
[Eb, G, Kc], side view [Hal); fragments of the base of the Top of the plate
flower where the ovary is located, with a ring of thick-walled
sclerous cells [CJ; fragments of the epidermis surrounding I or 2 blue or bluish-violet zones
the ovary (surface view [El) consisting of thin, longitudinally Chamazulene: a red or reddish-violet A red or reddish-violet zone
elongated cells [Ea], with numerous glandular trichomes zone (chamazulene)
[Eb], alternating with large elongated cells filled with
mucilage in transversally folded layers [Ee]; fragments of the -- --
epidermis surrounding the ovary (side view [HJ) bearing Bornyl acetate: a yellowish-brown
glandular trichomes [Ha] and underlying parenchymatous zone
cells containing small cluster crystals of calcium oxalate [Hb]; A brown zone (en-yne-dicycloether)
groups of cells at the apex of the stigmas forming elongated
papillae [L]; pollen grains spherical or triangular, about -- --
25 µm in diameter, with 3 pores and a spiny exine [F]. (-)-a-Bisabolol: a reddish-violet or A reddish-violet or bluish-violet zone
C. Thin-layer chromatography (2.2.27). bluish-violet zone ((-)-a-bisabolol)
Test solution Dilute 50 µL of essential oil obtained in the Reference solution Test solution
assay of essential oil in 1 mL of xylene R.
2023 Matricaria Oil IV-361
TESTS Time Mobile phase A Mobile phase B

Broken drug
Maximum 25 per cent, determined on 20.0 g, passes through 0-9 75 25
a sieve (710) (2.9.12). 9 - I9 75 ➔ 25 25 ➔ 75
19 - 24 25 75
powdered herbal drug (355) (2. 9.12) by drying in an oven at Fww rate 1 mUmin.
105 °C for 2 h. Detection Spectrophotometer at 340 nm.
Total ash (2.4.16) Injection 20 µL.
Maximum 13.0 per cent. System suitability Reference solution (b):
ASSAY
apigenin 7-glucoside and 5, 7-dihydroxy-4-
methylcoumarin.
Use 30 g of whole herbal drug, a 1000 mL flask, 500 mL of
water Ras distillation liquid and 0.50 mL of 1,2,4- Calculate the percentage content of total apigenin 7-glucoside
trimethylbenzene R in the graduated tube. Distil at a rate of using the following expression:
3-4 mUmin for 4 h. Towards the end of this period, stop the
A 1 x m2 xp x 0.625
flow of water to the condenser assembly but continue
distilling until the blue, steam-volatile components have A2 xm1
reached the lower end of the condenser. Immediately re-start
AI area of the peak due to apigenin 7-glucoside in the
the flow of water to the condenser assembly to avoid chromatogram obtained with the test solution;
warming the separation space. Stop the distillation after a A2 area of the peak due to apigenin 7-glucoside in the
further 10 min. chromatogram obtained with reference solution (a);
Total apigenin 7-glucoside solution, in grams;
Liquid chromatography (2.2.29). m2 mass of apigenin 7-glucoside CRS used to prepare reference
Solvent mixture Mobile phase B, mobile phase A p percentage content of apigenin 7 -glucoside in apigenin
(25:75 V/V). 7-glucoside CRS.
Test solution Reduce 40 g of the herbal drug to a
powder (500) (2.9.12). Place 2.00 g of the powdered herbal _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
drug in a 500 mL round-bottomed flask. Add 200 mL of

ethanol (96 per cent) R. Heat the mixture under a reflux
condenser on a water-bath for 15 min. Cool and filter. Rinse
the filter and the residue with a few millilitres of ethanol Matricaria Oil
(96 per cent) R. To the filtrate add 10 mL of freshly prepared
dilute sodium hydroxide solution R and heat the mixture under (Ph. Bur. monograph 1836)
a reflux condenser on a water-bath for about 1 h. Cool. PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Dilute to 250.0 mL with ethanol (96 per cent) R. To 50.0 mL
of the solution add 0.5 g of citric acid monohydrate R. Shake DEFINITION
for 5 min and filter. Dilute 5.0 mL of this solution to Blue essential oil obtained by steam distillation from the fresh
10.0 mL with the solvent mixture. or dried flower-heads or flowering tops of Matricaria
Reference solution (a) Dissolve 10.0 mg of apigenin recutita L. (Chamomilla recutita L. Rauschert). There are
7-glucoside CRS in 100.0 mL of methanol R. Dilute 25.0 mL 2 types of matricaria oil which are characterised as rich in
of this solution to 200.0 mL with the solvent mixture. bisabolol oxides, or rich in (-)-rt-bisabolol.
Reference solution (b) Dissolve 10.0 mg of 5,7-dihydroxy-4- CHARACTERS
methylcoumarin R in 100. 0 mL of methanol R. Dilute 2 5. 0 mL Appearance
of this solution to 100. 0 mL with the solvent mixture. Clear, intensely blue, viscous liquid.
To 4.0 mL of this solution add 4.0 mL of reference Intense characteristic odour.
solution (a) and dilute to 10.0 mL with the solvent mixture.
IDENTIFICATION
Precolumn:
=
- size: l 8 mm, 0 =4.6 mm;
- stationary phase: octadecylsilyl silica gel for chromatography R Second identification: A.
(5 µm). A. Thin-layer chromatography (2.2.27).
Column: Test solution Dissolve 20 µL of the substance to be
=
- size: l 0.25 m, 0 =
4.6 mm; examined in 1.0 mL of toluene R.
- stationary phase: end-capped octadecylsilyl silica gel for Reference solution Dissolve 2 mg of guaiazulene R, 5 µL of
chromatography R (5 µm). (-)-a-bisabowl R and I O mg of horny! acetare R in 5.0 mL of
Mobile phase: toluene R.
- mobile phase A: 0.5 per cent V/V solution of phosphoric Plate TLC silica gel plate R.
acid R; Mobile phase ethyl acetate R, toluene R (5:95 V/V).
- mobile phase B: phosphoric acid R, acetonitrile R
Application I O µL, as bands.
(0.5:99.5 V/V);
Development Over a path of I O cm.
IV-362 Matricaria Oil 2023
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 Split ratio 1: 100.
in the chromatograms obtained with the reference solution Temperature:
and the test solution.
Time Temperature
Top of the plate (min) (°C)
Guaiazulene: a blue zone A blue zone (chamazulene) Column 0 - 40 70---> 230

40 - 50 230
-- -- Injection port 250
Detector 250
-- --

Injection 1.0 µL.
Detection B Spray with anisaldehyde solution R and heat at
100-105 °C for 5-10 min. Examine immediately in daylight.
Results B See below for the sequence of the zones present in substances.
Relative retention With reference to chamazulene (retention
the test solution. Furthermore, yellowish-brown to greenish-
time = about 34.4 min): ~-farnesene = about 0.5; bisabolol
yellow zones (lower third), violet zones (lower third) and
oxide B = about 0.8; bisabolone = about 0.87; (-)-cr-
further weak zones may be present in the chromatogram
bisabolol = about 0.9; bisabolol oxide A= about 1.02.
chamazulene and guaiazulene.
1 or 2 blue to bluish-violet zones
Guaiazulene: a red to reddish-violet A red to reddish-violet zone chromatogram obtained with the reference solution, locate
zone (chamazulene) (-)-cr-bisabolol and chamazulene in the chromatogram
obtained with the test solution; locate bisabolol oxides
-- --
(bisabolol oxide B, bisabolone and bisabolol oxide A) using
Bomyl acetate: a yellowish-brown to A brown zone (en-yne-dicycloether)
Figures 1836.-1 and 1836.-2 (disregard the peak due to
greyish-green zone
cyclohexane). The chromatogram obtained with the test
-- -- solution does not show a peak with the retention time of
guaiazulene.
(-)-ct-Bisabolol: a reddish-violet to A reddish-violet to bluish-violet zone
bluish-violet zone ((-)-ct-bisabolol) Determine the percentage content of the components.
The limits are within the following ranges.
A brownish zone
Reference solution Test solution Matricaria oil rich in Matricaria oil rich
bisabolol oxides in (-)-ct-bisabolol
(per cent) (per cent)
B. Examine the chromatograms obtained in the test for Bisabolol oxides 29 - 81
chromatographic profile. (-)-();-Bisabolol 10 - 65
Results The characteristic peaks due to (-)-<"t-bisabolol and Chamazulene 2 1.0 2 1.0
chamazulene in the chromatogram obtained with the test Total of bisabolol 2 20
solution are similar in retention time to those in the oxides and (-)-ct-
Bisabolol
TESTS
Chromatographic profile STORAGE
Gas chromatography (2.2.28): use the normalisation At a temperature not exceeding 25 °C.
procedure. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Test solution Dissolve 20 µL of the essential oil to be

examined in cydohexane Rand dilute to 5.0 mL with the
same solvent.
Reference solution Dissolve 20 µL of (-)-rx-bisabolol R, 5 mg
of chamazulene R and 6 mg of guaiazulene R in cyclohexane R
Column:
- size: l = 30 m (a film thickness of 1 µm may be used) to
60 m (a film thickness of 0.2 µm may be used),
0 = 0.25-0.53 mm, when using a column longer than
30 m, an adjustment of the temperature programme may
be necessary,
2023 Matricaria Oil IV-363
3
2
4 5
0 5 10 15 20 25 30 35 40 45 min
I. P-farnesene 3. bisabolone 5. chamazulene

2. bisabolol oxide B 4. (-)-a-bisabolol 6. bisabolol oxide A
Figure 1836.-1. - Chromatogram of matricaria oil rich in bisabolol oxides

1 4
-
5
2
-
6
-
.I .I I ~I .1
3
I
I I
I
0 5 10 15 20 25 30 35 40 45 min
1. B-farnesene 3. bisabolone 5. chamazulene

2. bisabolol oxide B 4. (-)-c,-bisabolol 6. bisabolol oxide A
Figure 1836.-2. - Chromatogram of matricaria oil rich in (-)-rx-bisabolol

IV-364 Matricaria Preparations 2023
reddish-violet zone (chamazulene) corresponding to

Matricaria Liquid Extract guaiazulene in the chromatogram obtained with the reference
(Ph. Bur. monograph 1544) solution and immediately above it 1 or 2 blue or bluish-violet
zones; further weak zones may be present in the
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION B. Thin-layer chromatography (2.2.27).
Liquid extract produced from Matricaria flower (0404). Test solution The extract to be examined.
Content Reference solution Dissolve 1.0 mg of chlorogenic acid R,
Minimum 0.30 per cent of blue residual oil. 2.5 mg of hyperoside R and 2.5 mg of rutoside trihydrate R in
PRODUCTION 10 mL of methanol R.
The extract is produced from the herbal drug by a suitable Plate TLC silica gel plate R.
procedure for liquid extracts using a mixture of 2.5 volumes Mobile phase anhydrous formic acid R, glacial acetic acid R,
of a 10 per cent mlm solution of ammonia (NH 3 ), water R, ethyl acetate R (7.5:7.5:18:67 V/V/V/V).
47.5 volumes of water and 50 volumes of ethanol Application 10 µL as bands.
(96 per cent). Development Over a path of 15 cm.
CHARACTERS Drying At 100-105 °C.
Appearance Detection Spray the warm plate with a 10 g/L solution of
Brownish, clear liquid. diphenylboric acid aminoethyl ester R in methanol R;
Intense characteristic odour and characteristic bitter taste. subsequently spray with a 50 g/L solution of macrogol 400 R
Solubility in methanol R; allow to dry in air for about 30 min and
Miscible with water and with ethanol (96 per cent) with examine in ultraviolet light at 365 nm.
development of turbidity, soluble in ethanol Results The chromatogram obtained with the reference
(50 per cent V/V). solution shows in the middle part a light blue fluorescent
IDENTIFICATION zone (chlorogenic acid), below it a yellowish-brown
fluorescent zone (rutoside) and above it a yellowish-brown
fluorescent zone (hyperoside). The chromatogram obtained
Test solution Place 10 mL of the extract to be examined in a with the test solution shows a yellowish-brown fluorescent
separating funnel and shake with 2 quantities, each of zone corresponding to the zone of rutoside in the
10 mL, of pentane R. Combine the pentane layers, dry over chromatogram obtained with the reference solution, a light
2 g of anhydrous sodium sulfate R and filter. Evaporate the blue fluorescent zone corresponding to the zone of
filtrate to dryness on a water-bath and dissolve the residue in chlorogenic acid in the chromatogram obtained with the
0.5 mL of toluene R. reference solution, a yellowish-brown fluorescent zone similar
Reference solution Dissolve 4 mg of guaiazulene R, 20 mg of in position to the zone of hyperoside in the chromatogram
(-)-a-bisabolol Rand 20 mg of bornyl acetate R in 10 mL of obtained with the reference solution; it also shows above the
toluene R. yellowish-brown fluorescent zone a green fluorescent zone,
Plate TLC silica gel F 254 plate R. then several bluish or greenish fluorescent zones and near the
Mobile phase ethyl acetate R, toluene R (5:95 V/V). solvent front a yellowish fluorescent zone.
Application 10 µL as bands. TESTS
Development Over a path of 10 cm. Ethanol (2.9.10)
Drying In air. 38 per cent V/V to 53 per cent V/V.
Detection A Examine in ultraviolet light at 254 nm. Dry residue (2. 8.16)
shows several quenching zones, of which 2 main zones are in ASSAY
the middle third (en-yne-dicycloether). Place 20.0 gin a 1000 mL round-bottomed flask, add
Detection B Examine in ultraviolet light at 365 nm. 300 mL of water R and distil until 200 mL has been
collected in a flask. Transfer the distillate into a separating
funnel. Dissolve 65 g of sodium chloride R in the distillate and
shows in the middle part an intense blue fluorescent zone
(herniarin). shake with 3 quantities, each of 30 mL, of pentane R
previously used to rinse the reflux condenser and the flask.
Detection C Spray with anisaldehyde solution R and examine Combine the pentane layers, dry over 2 g of anhydrous sodium
in daylight while heating at 100-105 °C for 5-10 min. sulfate R and filter into a tared 100 mL round-bottomed flask
Results C The chromatogram obtained with the reference which has been dried in a desiccator for 3 h. Rinse the
solution shows in the lower third a reddish-violet or bluish- anhydrous sodium sulfate and the filter with 2 quantities,
violet zone ( (-)-o:-bisabolol), in the middle third a yellowish- each of 20 mL, of pentane R. Evaporate the pentane in a
brown or greyish-green zone (bornyl acetate) and in the water-bath at 45 °C. The residue of pentane is eliminated in
upper third a red or reddish-violet zone (guaiazulene). a current of air for 3 min. Dry the flask in a desiccator for
The chromatogram obtained with the test solution shows in 3 hand weigh. The residual oil is blue (chamazulene).
the lower third yellowish-brown or greenish-yellow and violet _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
zones and a reddish-violet or bluish-violet zone due to (-)-o:-
bisabolol in the chromatogram obtained with the 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
2023 Meadowsweet IV-365

Meadowsweet Test solution Xylene solution obtained in the assay.
(Ph. Bur. monograph 1868) Reference solution Dissolve 0.1 mL of methyl salicylate Rand
0.1 mL of salicylaldehyde R in xylene R and dilute to 5 mL
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION Plate TLC silica gel plate R.
Whole or cut, dried flowering tops of Filipendula ulmaria (L.) Mobile phase hexane R, toluene R (50:50 V/V).
Maxim. (syn. Spiraea ulmaria L.). Application 10 µL as bands.
Content Development Over a path of 10 cm.
Minimum 1 mUkg of essential oil (dried drug). Drying In air.
CHARACTERS Detection treat with 3 mL of ferric chloride solution R3 and
Aromatic odour of methyl salicylate, after crushing. examine in daylight.
IDENTIFICATION
A. The stem, up to 5 mm in diameter, is greenish-brown,
stiff, angular, hollow except at the apex, and has regular,
straight, longitudinal furrows. The petiolate leaf, compound
imparipinnate, has 2 reddish-brown angular stipules.
It consists of 3-9 pairs of leaflets, unevenly dentate, some of
which are small and fan-shaped. The leaflets are dark green
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
prominent and brown on the lower surface.
The inflorescence is complex and composed of very
numerous flowers arranged in irregular cymose panicles.
The flowers are creamish-white and about 3-6 mm in
diameter; the calyx consists of 5 dark green, reflexed and
hairy sepals fused at the base to a concave receptacle; the
5 free petals, which are readily detached, are pale yellow,
obovate and distinctly narrowed at the base; the stamens are
numerous with rounded anthers and they extend beyond the
petals; the gynoecium consists of about 4-6 carpels, each with
a short style and a globular stigma; the carpels become
twisted together spirally to form yellowish-brown fruits with a
helicoidal twist. Unopened flower buds are frequently
present. If the fruit is present, it has a helicoidal twist and
contains brownish seeds.
B. Microscopic examination (2.8.23). The powder is green or
yellowish-green. Examine under a microscope using chloral
characters (Figure 1868. -1): fragments of the epidermises of
the leaves and sepals [C, E, F] with sinuous or wavy cells [Ca,
Ea, Fa], short, thick-walled, conical covering trichomes Figure 1868.-1. - Illustration for identification test B of powdered
thickened at the base (surface view [Eb], side view m), herbal drug of meadowsweet
unicellular covering trichomes, thin-walled, very long and
flexuous, with pointed ends (surface view [Fe], side view [Al) Results See below the sequence of zones present in the
or their scars (flexuous trichome [Fd], conical trichome [Fe)) chromatograms obtained with the reference solution and the
and occasional clavate glandular trichomes with a 1- to test solution. Furthermore, other zones are present in the
3-celled ([Ed] and [G), respectively), uniseriate stalk, a chromatogram obtained with the test solution.
multicellular head and dense brown contents; fragments of the
upper epidermis often accompanied by palisade Top of the plate
parenchyma [Cb] including some hypertrophied cells
containing a cluster crystal of calcium oxalate [Cc]; fragments -- --
of the lower epidermis with anomocytic stomata (2.8.3) [Ee, Methyl salicylate: a violet-brown A violet-brown zone (methyl
Fb], sometimes accompanied by spongy parenchyma [Ff] with zone salicylate)
some cells containing cluster crystals of calcium oxalate [Fg];
Salicylaldehyde: a violet-brown zone A violet-brown zone (salicylaldehyde)
fragments of the petals [HJ with thin-walled epidermal cells,
some showing rounded papillae [Ha]; numerous spherical -- --
pollen grains with 3 pores and a faintly pitted exine [Bb];
fragments of the anther [B, DJ whose fibrous layer shows
specific thickenings (surface view [D), side view [Ba));
fragments of the ovary [K] with an epidermis bearing TESTS
stomata [Ka] and with parenchyma containing prism crystals Foreign matter (2.8.2)
of calcium oxalate [Kb]; fragments of vascular tissue [L] with Maximum 5.0 per cent of stems with a diameter greater than
annular, spiral or pitted vessels from the leaves and stems. 5 mm and maximum 2.0 per cent of other foreign matter.
IV-366 Melilot 2023
Loss on drying (2.2.32) C. Thin-layer chromatography (2.2.27).

Maximum 12.0 per cent, determined on 1.000 g of the Test solutwn To 0.3 g of the powdered herbal drug (355)
powdered herbal drug (355) (2.9.12) by drying in an oven at (2. 9.12) add 3 mL of methanol R. Heat on a water-bath at
105 °C for 2 h. 100 °C for 1 min and filter.
Total ash (2.4.16) Reference solutwn Dissolve 50 mg of coumarin CRS and
Maximum 7.0 per cent. 20 mg of o-coumaric acid R in 50 mL of methanol R.
ASSAY Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
Essential oil (2.8.12) plate R (2-10 µm)].
Use 50.0 g of the cut herbal drug, a 1000 mL flask, 300 mL Mobile phase dilute acetic acid R, ether R, toluene R
of dilute hydrochloric acid R as the distillation liquid, and (10:50:50 VIVIV); use the upper layer.
0.5 mL of xylene R in the graduated tube. Distil at a rate of Applicatwn 25 µL [or 3 µL] as bands of 10 mm [or 8 mm].
2-3 mUmin for 2 h. Development Over a path of 12 cm [or 6 cm].
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Drying In air.
Melilot
(Ph. Bur. monograph 2120) e
I
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ i>
),
DEFINITION I, A
Whole or cut, dried aerial parts of Melilotus officinalis (L.) '/,
Lam. A
i
Content
Minimum 0.3 per cent of coumarin (C 9H 6 O 2 ; Mr 146.1)
(dried drug). 0
IDENTIFICATION
A. The stem is green, cylindrical, glabrous and finely ridged.
The leaves are alternate, petiolate and trifoliate with
2 lanceolate stipules; the leaflets are up to about 3 cm long
and 20 mm wide, elongated or ovate with a finely dentate
margin, acute at the apex and base; the upper surface is dark
green and glabrous, the lower surface paler green with short,
fine hairs, especially at the base. The inflorescence is
racemose with numerous pale yellow flowers, about 7 mm
long, each having a hairy calyx with 5 deeply-divided, Ja
unequal teeth, and a papilionate corolla. The fruit is an
indehiscent pod, often persistent within the calyx, yellowish-
brown, short and tapering at the apex; the surface is glabrous
and transversely wrinkled.
B. Microscopic examination (2.8.23). The powder is M
hydrate solutwn R. The powder shows the following diagnostic Figure 2120.-1. - Illustration for identificatwn test B of powdered
characters (Figure 2120. -1): fragments of the leaf lamina herbal drug of melilot
(surface view [DJ) showing unevenly thickened, slightly
sinuous epidermal cells; numerous stomata [Db], mostly Detection Spray with 2 M alcoholic potassium hydroxide R and
anomocytic (2.8.3) with 3-6 subsidiary cells [Da] and examine in ultraviolet light at 365 nm.
frequently, underlying palisade parenchyma [De]; uniseriate Results See below the sequence of zones present in the
covering trichomes with 2 short, smooth-walled basal cells chromatograms obtained with the reference solution and the
and a long terminal cell, bent at right angles, with a thick test solution. Furthermore, other faint zones of various
wall and a warty cuticle [A, BJ; occasional glandular colours may be present in the chromatogram obtained with
trichomes with a short, 2- or 3- celled stalk and ovoid, the test solution.
biseriate head with 4 indistinct cells [HJ; fragments of the
petals composed of cells with wavy walls [M]; fragments of Top of the plate
vascular tissue from the stem [F, G], including large
Coumarin: a greenish-yellow A greenish-yellow fluorescent zone
vessels [G], sometimes associated with unlignified septate fluorescent zone (coumarin)
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 A blue fluorescent zone
crystals of calcium oxalate [Ja]; fragments of the stem
epidermis with elongated, straight-walled cells and o-Coumaric acid: a greenish-yellow A greenish-yellow fluorescent zone
fluorescent zone (o-coumaric acid) may be present
anomocytic (2. 8.3) stomata [L]; fragments of the fibrous
layer of the anthers (surface view [E], transverse section [Kl); - - --
spherical or ovoid pollen grains about 25 µm long with
3 germinal pores and a smooth exine [C].
2023 Milk-thistle Fruit IV-367
TESTS Content of total balsamic acids

Foreign matter (2.8.2) Not less than 2.8% w/v, calculated as cinnamic acid,
Maximum 2 per cent of stems with a diameter greater than C9Hs02.
3 mm and maximum 2 per cent of other foreign matter. Total solids
Loss on drying (2.2.32) 9.0 to 12.0% w/v when determined by drying at 105° for
Maximum 12.0 per cent, determined on 1.000 g of the 4 hours, Appendix XI A. Use 2 mL.
powdered herbal drug (355) (2. 9.12) by drying in an oven at ASSAY
105 °C for 2 h. Boil 10 mL with 25 mL of 0.5M ethanolic potassium hydroxide
Total ash (2.4.16') under a reflux condenser for 1 hour. Evaporate the ethanol,
Maximum 10.0 per cent. disperse the residue in 50 mL of hot water, cool, add 80 mL
ASSAY of water and 1.5 g of magnesium sulfate dissolved in 50 mL of
Liquid chromatography (2.2.29). water. Mix thoroughly and allow to stand for 10 minutes.
Filter, wash the residue on the filter with 20 mL of water,
Test solutwn Completely reduce about 50 g of the herbal
acidify the combined filtrate and washings with hydrochloric
drug to a powder (500) (2.9.12). To 5.00 g of the powdered
acid and extract with four 40 mL quantities of ether. Discard
herbal drug add 90 mL of methanol R and boil under a reflux
the aqueous solution, combine the ether extracts and extract
condenser for 30 min. Allow to cool. Filter under vacuum
with successive quantities of 20, 20, 10, 10 and 10 mL of
through a fibre-glass filter. Take up the residue and the
sodium hydrogen carbonate solution, washing each aqueous
fragmented filter with 90 mL of metharwl R. Treat in the
extract with the same 20 mL of ether. Discard the ether
same manner as before. Combine the filtrates and dilute to
layers, carefully acidify the combined aqueous extracts with
hydrochloric acid and extract with successive quantities of 30,
Reference solutwn Dissolve 25.0 mg of coumarin CRS in 20, 20 and 10 mL of chloroform, filtering each extract through
methanol Rand dilute to 250.0 mL with the same solvent. anhydrous sodium sulfate supported on absorbent cotton. Distil
Column: the chloroform from the combined filtrates until 10 mL
- size: l = 0.25 m, 0 = 4 mm; remains and remove the remainder in a current of air.
- stationary phase: end-capped octadecylsilyl silica gel for Dissolve the residue, with the aid of gentle heat, in 10 mL of
chromatography R (5 µm). ethanol (96%), previously neutralised to phenol red solution,
Mobile phase acetonitrile R, 5 g/L solution of phosphoric cool and titrate with 0.1M sodium hydroxide VS using phenol
acid R (22:78 V/V). red solutwn as indicator. Each mL of 0.1M sodium hydroxide
Flow rate l .7 mL/min. VS is equivalent to 14.82 mg of total balsamic acids,
Detection Spectrophotometer at 275 nm. calculated as cinnamic acid, C 9 H 8 0 2.
Injection 20 µL.
System suitability:
- retention time: coumarin = about 7 .8 min. Milk-thistle Fruit
Calculate the percentage content of coumarin using the
following expression: (Ph. Eur. monograph 1860)
Preparation
Refined and Standardised Milk Thistle Dry Extract
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
area of the peak due to coumarin in the chromatogram obtained
with the test solution; DEFINITION
area of the peak due to coumarin in the chromatogram obtained
Mature fruit, devoid of the pappus, of Silybum marianum L.
mass of the herbal drug to be examined used to prepare the test Gaertn.
solution, in grams;
Content
mass of coumarin CRS used to prepare the reference solution, in
grams; Minimum 1.5 per cent of silymarin, expressed as silibinin
p percentage content of coumarin in couman·n CRS. (C2sH22010; Mr 482.4) (dried drug).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur CHARACTERS
No rancid odour.
IDENTIFICATION
Menthol and Benzoin Inhalation A. The achene is strongly compressed, elongate-obovate,
Menthol and Benzoin Inhalation Vapour about 6-8 mm long, 3 mm broad and 1.5 mm thick; the
outer surface is smooth and shiny, with variable longitudinal
DEFINITION dark brown streaks against a grey or pale brown background
Menthol and Benzoin Inhalation is an inhalatwn vapour, giving an overall pale greyish or brown colour; the fruit is
solution. tapering at the base and crowned at the apex with a
Racementhol or Levomenthol 20 g glistening, pale yellow extension forming a collar about 1 mm
Benzoin Inhalation Sufficient to produce 1000 mL high surrounding the remains of the style. Cut transversely,
Extemporaneous preparation the fruit shows a narrow, brown outer area and 2 large,
The following directions apply. dense, white, oily cotyledons.
Dissolve the Racementhol or the Levomenthol in a portion of B. Microscopic examination (2.8.23). The powder is
the Benzoin Inhalation, add sufficient Benzoin Inhalation to brownish-yellow with darker specks. Examine under a
produce 1000 mL and mix. microscope using chloral hydrate solutwn R. The powder
The inhalatwn complies with the requirements stated under shows the following diagnostic characters: fragments of the
Preparations for Inhalation and with the following requirements. epicarp composed of colourless cells, polygonal in surface
IV-368 Milk-thistle Fruit 2023
view, the lumen appearing fairly large or as a small slit, Add 100 mL of light petroleum R and heat in a water-bath for
depending on the orientation; groups of parenchymatous cells 8 h. Allow the defatted drug to dry at room temperature.
from the pigment layer, some of them containing colouring In a continuous-extraction apparatus, extract the latter with
matter which appears bright red; very abundant groups of 100 mL of methanol R in a water-bath for 5 h. Evaporate the
large sclereids from the testa with bright yellow pitted walls methanolic extract in vacuo to a volume of about 30 mL.
and a narrow lumen; occasionally fragments of small-celled Filter into a 50 mL volumetric flask, rinsing the extraction
parenchyma with pitted and beaded walls; abundant thin- flask and the filter, and diluting to 50.0 mL with methanol R.
walled parenchymatous cells from the cotyledons containing Dilute 5.0 mL of this solution to 50.0 mL with methanol R.
oil globules and scattered cluster crystals of calcium oxalate; Reference solutwn Dissolve a quantity of milk thistle dry
a few larger, prismatic crystals of calcium oxalate. extract HRS corresponding to 10.0 mg of silibinin in
C. Thin-layer chromatography (2.2.27). methanol Rand dilute to 100.0 mL with the same solvent.
Test solutwn To 1.0 g of the powdered herbal drug (500) Column:
(2.9.12) add 10 mL of methanol R. Heat under reflux in a - size: l = 0.125 m, 0 = 4 mm;
water-bath at 70 °C for 5 min. Cool and filter. Evaporate the - statwnary phase: end-capped octadecylsilyl silica gel for
filtrate to dryness and dissolve the residue in 1.0 mL of chromatography R (5 µm).
methanol R. Mobile phase:
Reference solutwn Dissolve 2 mg of silibinin R and 5 mg of - mobile phase A: phosphoric acid R; methanol R, water for
taxifolin R in methanol R and dilute to 10 mL with the same chromatography R (0.5:35:65 V/V/V);
solvent. - mobile phase B: phosphoric acid R; methanol R, water for
Plate TLC silica gel plate R. chromatography R (0.5:50:50 V/V/V);
Mobile phase anhydrous formic acid R, acetone R, methylene
chloride R (8.5:16.5:75 VIVIV). (min) (per cent V/JJ) (per cent V/JJ)
Applicatwn 30 µL of the test solution and 10 µL of the 0 - 28 100 ➔ 0 0 ➔ 100
reference solution, as bands. 28 - 35 0 100
Development Over a path of 10 cm. 35 - 36 0 ➔ 100 100 ➔ 0
Drying At 100-105 °C.
Detection Treat the still-warm plate with a 10 g/L solution
of diphenylboric acid aminoethyl ester R in methanol R and Flow rate 0.8 mIJmin.
subsequently treat with a 50 g/L solution of macrogol 400 R Detection Spectrophotometer at 288 nm.
in methanol R. Allow to dry for 30 min and examine in Injection 10 µL.
ultraviolet light at 365 nm. Identification of peaks Use the chromatogram supplied with
Results See below the sequence of zones present in the milk thistle dry extract HRS and the chromatogram obtained
chromatograms obtained with the reference solution and the with the reference solution to identify the peaks due to
test solution. Furthermore, other orange and yellowish-green silicristin, silidianin, silibinin A, silibinin B, isosilibinin A and
fluorescent zones are present between the zones due to isosilibinin B. A small shoulder or peak due to
silibinin and taxifolin in the chromatogram obtained with the dihydrosilibinin B may appear in the tail of the peak due to
test solution. silibinin B and is included in its area. In the chromatogram
obtained with the test solution the peak due to silidianin may
Top of the plate vary in size, be absent or be present as the principal peak.
Silibinin: a yellowish-green A yellowish-green fluorescent zone Retention time Silibinin B = about 30 min; if necessary,
fluorescent zone (silibinin) adjust the time periods of the gradient.
-- --
Taxifolin: an orange fluorescent An orange fluorescent zone (taxifolin)
silibinin A and silibinin B;
zone
A yellowish-green fluorescent zone chromatogram supplied with milk thistle dry extract HRS.
(silicrisrin)
Calculate the percentage content of total silymarin, expressed
-- -- as silibinin, using the following expression:
A light blue fluorescent zone (line of
application) (A 1 +A 2 +A3 +A4 +As +A6) x m1 xpx 5
Reference solution Test solution (A1 +As) x m2

area of the peak due to silicristin in the chromatogram obtained
TESTS with the test solution;
area of the peak due to silidianin in the chromatogram obtained
Loss on drying (2.2.32) with the test solution;
Maximum 8.0 per cent, determined on 1.000 g of the A, area of the peak due to silibinin A in the chromatogram
powdered herbal drug (500) (2. 9.12) by drying in an oven at obtained with the test solution;
105 °C for 2 h. area of the peak due to silibinin B and dihydrosilibinin B in the
Total ash (2.4.16) A, area of the peak due to isosilibinin A in the chromatogram
Maximum 8.0 per cent. obtained with the test solution;
area of the peak due to isosilibinin B in the chromatogram
Liquid chromatography (2.2.29). area of the peak due to silibinin A in the chromatogram
Test solutwn Place 5 .00 g of the powdered herbal drug area of the peak due to silibinin B and dihydrosilibinin B in the
(500) (2. 9.12) in a continuous-extraction apparatus. chromatogram obtained with the reference solution;
2023 Milk Thistle Preparations IV-369
mass of milk thfr!le dry extract HRS used to prepare the reference Results See below the sequence of zones present in the
solution, in grams;
solution, in grams; test solution. Furthermore, other yellowish-green fluorescent
p combined percentage content of silibinin A and silibinin B in zones may be present between the zones due to silibinin and
milk thfrtle dry extract HRS. taxifolin in the chromatogram obtained with the test solution.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Top of the plate
Silibinin: a yellowish-green A yellowish-green fluorescent zone

Refined and Standardised Milk fluorescent zone (silibinin)
Thistle Dry Extract - -
(Ph. Bur. monograph 2071) Taxifolin: an orange fluorescent An orange fluorescent zone (taxifolin)
zone
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION (silicristin)
Dry extract, refined and standardised, produced from Milk - -
thistle fruit (1860).
A fluorescent zone (line of
Content application)
90 per cent to 110 per cent of the nominal content of
silymarin, expressed as silibinin (C 25 H 22 O 10; Mr 482.4), Reference solution Test solution
stated on the label. The nominal content of silymarin is
within the range 30 per cent mlm to 65 per cent mlm TESTS
(anhydrous extract). Water (2.5.12)
The content of silymarin corresponds to: Maximum 4.0 per cent, determined on 0.500 g.
- sum of the contents of silicristin and silidianin (both ASSAY
C2sH22010; Mr 482.4): 20 per cent to 45 per cent, Liquid chromatography (2.2.29).
calculated with reference to total silymarin;
Test solutwn Dissolve 60.0 mg of the extract to be examined
- sum of the contents of silibinin A and silibinin B (both
in methanol R and dilute to 100.0 mL with the same solvent.
C 25 H22010; Mr 482.4): 40 per cent to 65 per cent,
calculated with reference to total silymarin; Reference solutwn Dissolve a quantity of milk thistle dry
- sum of the contents of isosilibinin A and isosilibinin B (both extract HRS corresponding to 10.0 mg of silibinin in
C 25 H 22 0 10; Mr 482.4): 10 per cent to 20 per cent, methanol Rand dilute to 100.0 mL with the same solvent.
calculated with reference to total silymarin. Column:
PRODUCTION - size: l = 0.125 m, 0 = 4 mm;
The extract is produced from the herbal drug by an
appropriate procedure, using one or more of the following chromatography R (5 µm).
solvents: Mobile phase:
- ethyl acetate; - mobile phase A: phosphoric acid R, methanol R, water for
- acetone or mixture of acetone and water; chromatography R (0.5:35:65 VIVIV);
- ethanol or mixture of ethanol and water; - mobile phase B: phosphoric acid R, methanol R, water for
- methanol or mixture of methanol and water. chromatography R (0.5:50:50 VIVIV);
CHARACTERS Time Mobile phase A Mobile phase B
Appearance (min) (per cent V/J!) (per cent V/J!)
Yellowish-brown, amorphous powder. 0 - 28 100 ➔ 0 0---, 100
28 - 35 0 100
IDENTIFICATION
35 - 36 0---, 100 100---, 0
Test solution Dissolve 0.250 g of the extract to be examined
in methanol R and dilute to 5 mL with the same solvent. Fww rate 0.8 mUmin.
Reference solutwn Dissolve 2 mg of silibinin R and 5 mg of Detection Spectrophotometer at 288 nm.
taxifolin R in methanol R and dilute to 10 mL with the same Injection l O µL.
solvent. Identification of peaks Use the chromatogram supplied with
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel milk thistle dry extract HRS and the chromatogram obtained
plate R (2-10 µm)]. with the reference solution to identify the peaks due to
Mobile phase anhydrous formic acid R, acetone R, methylene silicristin, silidianin, silibinin A, silibinin B, isosilibinin A and
chloride R (8.5:16.5:75 V/V/V). isosilibinin B. A small shoulder or peak due to
Applicatwn 10 µL [or 8 µL] of the test solution and 10 µL dihydrosilibinin B may appear in the tail of the peak due to
[or 2 µL] of the reference solution, as bands. silibinin B and is included in its area. In the chromatogram
Development Over a path of 10 cm [or 6 cm). obtained with the test solution the peak due to silidianin may
Drying At 100-105 °C. vary in size or be absent.
Detection Treat the still-warm plate with a 10 g/L solution Retention time Silibinin B = about 30 min; if necessary,
of diphenylboric acid aminoethyl ester R in methanol R and adjust the time periods of the gradient.
subsequently treat with a 50 g/L solution of macrogol 400 R System suitability Reference solution:
in methanol R; allow to dry for 30 min and examine in - resolution: minimum 1.8 between the peaks due to
ultraviolet light at 365 nm. silibinin A and silibinin B;
IV-370 Mint Oil 2023
- the chromatogram is similar to the chromatogram Second identification: A.

supplied with milk thistle dry extract HRS. A. Thin-layer chromatography (2.2.27).
Calculate the percentage content of total silymarin, expressed Test solution Dissolve O.1 mL of the substance to be
as silibinin, using the following expression: examined in 1.0 mL of toluene R.
(A1 +A2 +A3 +A4 +As +A6) xm1 xp Reference solution Dissolve 4 µL of carvone R, 4 µL of
(A1 +As) xm2 pulegone R, l O µL of menthyl acetate R, 20 µL of cineole R and
50 mg of menthol R in 5 mL of toluene R.
Calculate the percentage content of the sum of silicristin and Plate TLC silica gel F 254 plate R.
silidianin, with reference to total silymarin, using the Mobile phase ethyl acetate R, toluene R (5:95 V/V).
(A1 +A2) x 100 Development Over a path of 15 cm.
Drying In air.
Calculate the percentage content of the sum of silibinin A Detection A Examine in ultraviolet light at 254 nm.
and silibinin B, with reference to total silymarin, using the Results A See below the sequence of the zones present in
following expression: the chromatograms obtained with the reference solution and
the test solution. Furthermore, a quenching zone may be
present in the upper third of the chromatogram obtained
with the test solution.
Calculate the percentage content of the sum of isosilibinin A
and isosilibinin B, with reference to total silymarin, using the Top of the plate
following expression: Carvone and pulegone: a quenching A quenching zone
zone
A quenching zone
area of the peak due to silicristin in the chromatogram obtained Reference solution Test solution
area of the peak due to silidianin in the chromatogram obtained
with the test solution; Detection B Spray with anisaldehyde solution R and heat at
area of the peak due to silibinin A in the chromatogram
100-105 °C for 5-10 min. Examine immediately in daylight.
area of the peak due to silibinin B and dihydrosilibinin B in the Results B See below the sequence of the zones present in
chromatogram obtained with the test solution; the chromatograms obtained with the reference solution and
area of the peak due to isosilibinin A in the chromatogram
the test solution. Furthermore, the zone due to cineole in the
area of the peak due to isosilibinin B in the chromatogram reference solution is absent in the chromatogram obtained
obtained with the test solution; with the test solution. No yellowish-brown zone below the
A, area of the peak due to silibinin A in the chromatogram intense reddish-violet zone is present in the chromatogram
obtained with the reference solution; obtained with the test solution.
area of the peak due to silibinin B and dihydtosilibinin B in the
mass of milk thistle dry extract HRS used to prepare the reference Top of the plate
solution, in grams;
m2 mass of the extract to be examined used to prepare the test An intense reddish-violet zone (near
solution, in grams; the solvent front)
p combined percentage content of silibinin A and silibinin B in
Menthyl acetate: a bluish-violet zone A bluish-violet zone (menthyl
milk thistle dry extract HRS.
acetate)
A strongly greenish zone
A greenish zone
Dementholised Mint Oil Carvone and pulegone: a reddish

zone
A reddish zone
(Partly Dementholised Mint Ou, Ph. Eur. monograph Cineole: a violet zone
1838)
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 Reference solution Test solution
canadensis L. (syn. M. arvensis L. var. glabrata (Benth) Fem.,
M. arvensis var. piperascens Malinv. ex Holmes), followed by
partial separation of menthol by crystallisation. B. Examine the chromatograms obtained in the test for
CHARACTERS
Appearance
obtained with the test solution are approximately similar in
Colourless, pale yellow or greenish-yellow liquid.
retention time to those in the chromatogram obtained with
Characteristic odour. the reference solution. Carvone may be absent from the
IDENTIFICATION chromatogram obtained with the test solution.
2023 Marinda Root IV-3 71
TESTS - pulegone: maximum 2.5 per cent,

Relative density (2.2.5) - carvone: maximum 2.0 per cent.
0.888 to 0.910. The ratio of cineole content to limonene content is less
Refractive index (2.2.6) than 1.
1.456 to 1.470. STORAGE
Optical rotation (2.2. 7) At a temperature not exceeding 25 °C.
-16.0° to -34.0°. - - - - - - - - - - - - - - - - - - - - - - PhEur
Acid value (2.5.J)
Maximum 1.0, determined on 5.00 g of the substance to be
examined dissolved in 50 mL of the prescribed mixture of
solvents. Morinda Root
Gas chromatography (2.2.28): use the normalisation (Ph. Bur. monograph 2977)
procedure. ~~----------------------
DEFINITION
examined in hexane Rand dilute to 10.0 mL with the same
Dried, whole root of Gynochthodes officinalis (F.C.How)
solvent.
Razafim. & B.Bremer (syn. Marinda officinalis F.C.How),
Reference solution Dissolve 10 mg of limonene R, 20 mg of gently beaten to flatten it, with secondary roots removed.
cineole R, 40 mg of menthone R, 10 mg of (+)-isomenthone R,
40 mg of menthyl acetate R, 20 mg of isopulegol R, 60 mg of Content
Minimum 1.3 per cent for the sum of monotropein and
menthol R, 20 mg of pulegone R and 10 mg of carvone R in
deacetylasperulosidic acid, expressed as monotropein
hexane Rand dilute to 10.0 mL with the same solvent.
(C16H220J1; Mr 390.3) (dried drug).
Column:
- material: fused silica, IDENTIFICATION
- size: l = 30 m (a film thickness of 1 µm may be used) to A. The root is slightly curved, varying in length and 0.4-2 cm
60 m (a film thickness of 0.2 µm may be used), in diameter. The outer surface is dull and yellowish-grey to
0 = 0.25-0.53 mm, brownish-grey. When flattened, the roots are 0.2-0.5 cm
- stationary phase: macrogol 20 000 R. thick and their external surface is longitudinally wrinkled and
Gamer gas helium for chromatography R. transversely cracked; pieces of bark may become detached
from the central xylem (wood), resulting in the characteristic
segmented appearance of the roots, with broad bark
Split ratio l: 100. fragments alternating with sections of narrow wood; wood
Temperature: 0.1-0.6 cm in diameter. The texture is tough. The fractured
surface shows a thin yellowish-grey cork layer and a thick,
Time Temperature horny reddish-violet to dark brown (to blackish-violet in
(min) ("C) older roots) bark. The central xylem is hard and often
Column 0 - 10 60 protrudes from the fractured surface; it is yellowish-white to
10 - 70 60 --+ 180 yellowish-brown (or darker brown in older roots); raphides
70 - 75 180 are often visible on its outer surface.
Injection pon 200 B. Microscopic examination (2.8.23). The powder is variable
Detector 220 in colour, pale or dark reddish-violet to violet-brown.
Detection Flame ionisation. The powder shows the following diagnostic characters
Injection 1.0 µL. (Figure 2977.-1): numerous sclereids, isolated [G] or in
Elution order Order indicated in the composition of the groups [C], variable in shape (e.g. rounded, elliptical,
reference solution. Record the retention times of these subrectangular) and up to 95 µm in diameter, some sclereids
substances. with very thick walls, others with thin walls; walls are often
greenish-yellow or golden brownish-yellow, with distinct pit
canals and sparse pits, some sclereids showing distinctly
striated walls [Be]; needle-shaped crystals of calcium oxalate
limonene and cineole.
up to 180 µm long, occasionally longer (up to 280 µm),
Using the retention times determined from the isolated [E] or included in parenchymatous cells [F]; the
chromatogram obtained with the reference solution, locate needle-shaped crystals are often broken; fragments of cork
the components of the reference solution in the with superimposed, polyhedral cells (surface view [Al);
chromatogram obtained with the test solution. fragments of dermal tissue (transverse section [BJ), consisting
Determine the percentage content of these components. of cork [Ba] and phelloderm [Bb] containing sclereids [Be];
The percentages are within the following ranges: numerous fragments of thin-walled parenchyma cells,
- limonene: 1.5 per cent to 7.0 per cent, occasionally violet or violet-brown in colour; fragments of
- cineole: maximum 1.5 per cent, xylem vessels [D, HJ bordered-pitted [Da, Ha], up to
- menthone: 17.0 per cent to 35.0 per cent, 105 µm in diameter, mostly associated with yellowish fibre-
- isomenthone: 5.0 per cent to 13.0 per cent, tracheids [Db, Hb, He] which show sparse bordered pits
- menthyl acetate: 1.5 per cent to 7.0 per cent, whose central openings may be slit-shaped, V-shaped or
- isopulegol: 1.0 per cent to 3.0 per cent, cross-shaped; rare free xylem vessels; a few fragments of
- menthol: 30.0 per cent to 50.0 per cent, medullary rays consisting of rectangular cells with slightly
thickened and pitted walls [Hd] .
IV-372 Morinda Root 2023
Top of the plate
A brown zone, faint
-- --
Glucose: a brown zone A brown zone, equivalent (glucose)
. ,
A brown zone, faint
,\.-•• .. •••y A
A brown zone, very faint
.. . .; : .: . ,'. ~,
-- --
..-f~- -
Nystose: a brown zone A brown zone, faint (nystose)
A series of 5 brown zones, equivalent
TESTS
105 °C for 3 h.
Total ash (2.4.16)
ASSAY
herbal drng of morinda root Liquid chromatography (2.2.29).
C. High-performance thin-layer chromatography (2.8.25). (2. 9.12) add 25.0 mL of a 30 per cent V/V solution of
Test solution To 0.1 g of the powdered herbal drug (710) methanol R. Weigh and sonicate for 10 min. Cool and weigh
(2. 9.12) add 5.0 mL of methanol R. Sonicate for 15 min. again. Compensate the loss of solvent with a 30 per cent V/V
Centrifuge and use the supernatant. solution of methanol R. Shake thoroughly. Filter through a
Reference solution (a) Dissolve 5.0 mg of nystose Rand membrane filter (nominal pore size 0.45 µm).
10.0 mg of glucose R in 3.0 mL of water Rand dilute to Reference solution (a) Dissolve 2.5 mg of monotropein CRS in
5.0 mL with methanol R. a 30 per cent V/V solution of methanol R and dilute to
Reference solution (b) Dilute 2.5 mL of reference solution (a) 25.0 mL with the same solution.
to 10.0 mL with methanol R. Reference solution (b) To 0.5 g of morinda root for system
Reference solution (c) Dissolve 2.5 mg of sucrose Rand suitability HRS add 25 mL of a 30 per cent V/V solution of
10 mg of glucose R in 3 mL of water R and dilute to 5 mL methanol R. Sonicate for 10 min and shake thoroughly. Filter
with methanol R. through a membrane filter (nominal pore size 0.45 µm).
Intensity marker Glucose. Column;
- size: l = 0.25 m, 0 = 4.6 mm;
- stationary phase: silica gel for chromatography, alkyl-bonded
Mobile phase glacial acetic acid R, anhydrous formic acid R, for use with highly aqueous mobile phases R (5 µm).
water R, ethyl acetate R (20:25:30:60 VIVIV!V).
Mobile phase phosphoric acid R, methanol Rl, water for
Application 3 µL as bands of 8 mm. chromatography R (0.01:49.99:950 VIVIV).
Development 70 mm from the lower edge of the plate. Flow rate 1.0 mUmin.
Drying In a current of cold air for 5 min. Detection Spectrophotometer at 237 nm.
Detection Treat with a 100 g/L solution of suljuric acid R in Injection 10 µL.
ethanol (96 per cent) Rand heat at 120 °C for 3 min; examine
Run time Twice the retention time of monotropein.
in daylight.
morinda root for system suitability HRS and the chromatogram
to deacetylasperulosidic acid; use the chromatogram obtained
(sucrose) is brown and the upper zone (glucose) is
with reference solution (a) to identify the peak due to
intense brown.
monotropein.
Relative retention With reference to monotropein (retention
time = about 7 min): deacetylasperulosidic acid = about 1.2.
with the test solution, other intense brown zones may be
present.
2023 Motherwort IV-373
System suitability Reference solution (b): with a short unicellular stalk and a globular head composed
- resolution: minimum 1.5 between the peaks due to of 8-16 cells [Cc], glandular trichomes with a uni- or
monotropein and deacetylasperulosidic acid. bicellular stalk and a bi- or tetracellular head [Cd] and
Calculate the percentage content of the sum of monotropein sometimes covering trichomes; fragments of the lamina, in
and deacetylasperulosidic acid, expressed as monotropein, transverse section [DJ, composed of epidermises bearing
using the following expression: glandular trichomes with a globular head consisting of
8-16 cells [Da) or a bi- or tetracellular head [Db], a
A 1 x m2 x p A 3 x m2 x p x 1.16 I-layered palisade mesophyll extending almost halfway across
----+------- the section [De], and a loosely arranged spongy
A2 x m1 A2 x m1
parenchyma [Dd]; fragments of the calyx [G] with an
A1 area of the peak due to monotropein in the chromatogram epidermis consisting of polygonal cells bearing uni- or
obtained with the test solution; bicellular conical covering trichomes, with spiny walls [Ga],
A2 area of the peak due to monotropein in the chromatogram
often associated with fusiform mesophyll cells with thick
A3 area of the peak due to deacetylasperulosidic acid in the walls and containing small prism crystals of calcium oxalate
chromatogram obtained with the test solution; (Gb]; isolated glandular trichomes [HJ, either with a
m1 mass of the herbal drug to be examined used to prepare the test multicellular stalk and a unicellular head from the
solution, in grams;
mass of monotropein CRS used to prepare reference solution (a),
anthers [Ha] or a uni- or multicellular stalk and bi- to
in grams; tetracellular head [Hb); spherical pollen grains, about
p percentage content of monotropein in monotropein CRS; 25-30 µm in diameter, with 3 pores and 3 furrows and a
1.16 peak correlation factor between monotropein and smooth exine [E]; thick-walled, lignified fibres [F]; fragments
deacetylasperulosidic acid.
from the stem with spirally and annularly thickened
vessels [K]; occasional fragments of pericarp DJ consisting of
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
lobed cells with thick, pitted walls, each containing a single
prism crystal of calcium oxalate Ua).
Motherwort
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or cut, dried flowering aerial parts of Leonurus
cardiaca L.
Content
IDENTIFICATION
A. The stem pieces are hairy, longitudinally striated,
quadrangular, hollow, and up to about 10 mm wide; they
bear opposite and decussate, petiolate leaves and, in the axils
of the upper leaves, about 6-12 small flowers, arranged in
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
slightly trifid, lanceolate with a serrate margin and cuneate at
the base. The upper surface of the leaves is green with
scattered hairs, the lower surface is paler green, densely
pubescent and shows a prominent palmate and reticulate
venation. The flowers have a funnel-shaped calyx, 3 mm to
5 mm long with 5 stiff, recurved teeth; the corolla is
2-lipped, the upper lip pink and pubescent on the outer
surface, the lower lip white with purplish spots; stamens 4,
densely pubescent.
B. Microscopic examination (2.8.23). The powder is green.
Examine under a microscope using chloral, hydrate solution R. Figure 1833.-1. - Illustration for identification test B of powdered
The powder shows the following diagnostic characters herbal drug of motherwort
(Figure 1833.-1): numerous covering trichomes [A],
whole [Aa] or fragmented [Ab), uniseriate, with warty walls, C. Thin-layer chromatography (2.2.27).
composed of 2-8 cells with slight swellings at the junctions, Test solution To 0.5 g of the powdered herbal drug (355)
up to 1500 µm long; fragments of the upper epidermis (2. 9.12) add 5 mL of methanol R. Heat on a water-bath at
(surface view [BJ) with cells with straight or sinuous 65 °C for 5 min with shaking. Cool and filter.
anticlinal walls [Ba], often accompanied by palisade
Reference solution Dissolve 5 mg of naphthol yellow S R and
parenchyma [Bb ); fragments of the lower epidermis (surface
2.0 mg of catalpol R in 5.0 mL of methanol R.
view [CJ) with cells with sinuous anticlinal walls [Ca],
diacytic stomata (2.8.3) [Cb], bearing glandular trichomes Plate TLC silica gel plate R.
IV-374 Moutan Bark 2023
Mobile phase glacial acetic acid R, water R, ethyl acetate R Compensation liquid Dilute 10.0 mL of the stock solution to
(20:20:60 VIVIV). 25.0 mL with a 5 per cent V/V solution of glacial acetic
Application 20 µL as bands. acid R in methanol R.
Devek>pment Over a path of 10 cm. Measure the absorbance (2.2.25) of the test solution after
Drying In air. 30 min, by comparison with the compensation liquid at
Detection Treat with dimethylaminobenzaldehyde solution R2,
calculated as hyperoside, using the following expression:
using about 5 mL for a plate 200 mm square; heat at
100-105 °C for 10 min until the spots appear; examine in Ax 1.25
daylight.
m
chromatograms obtained with the reference solution and the i.e. taking the specific absorbance of hyperoside to be 500.
test solution. Furthermore, other weak greyish-blue zones
may be present in the chromatogram obtained with the test A absorbance at 425 run;
solution. m == mass of the substance to be examined, in grams.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Top of the plate
A wide white zone
A greyish-blue zone (iridoid)

Moutan Bark
--- --
Naphthol yellow S: an intense yellow 1 or 2 greyish-blue zones (iridoid)
zone PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Catalpa!: a greyish-blue zone DEFINITION

Dried, whole or fragmented, scraped or unscraped root bark
-- --
of Paeonia x suffruticosa Andrews, collected in autumn.
Content
- paeonol (C 9H 10O 3; Mr 166.2): minimum 2.2 per cent
TESTS (dried drug);
Foreign matter (2.8.2) - paeonifiorin (C 23H2sOll; M, 480.5): minimum
Maximum 2 per cent of brown or yellow leaves and 1.1 per cent (dried drug).
maximum 2 per cent of other foreign matter. IDENTIFICATION
Loss on drying (2.2.32) A. The whole bark is quilled or crescent-shaped, about
Maximum 12.0 per cent, determined on 1.000 g of the 2-4 mm thick, 0.6-1.4 cm in diameter and up to 20 cm long.
powdered herbal drug (355) (2. 9.12) by drying in an oven at The fragmented bark occurs as cleft rings, about 2-4 mm
105 °C for 2 h. thick and 0.5-1 cm in diameter. The greyish-brown or
Total ash (2.4.16) yellowish-brown outer surface of the unscraped bark shows
Maximum 12.0 per cent. long transverse lenticels and rootlet scars; where the dermal
tissue is absent, the exposed surface is pink. The outer
ASSAY surface of the scraped bark is pinkish to greyish-white;
Stock solution In a 100 mL round-bottomed flask place transverse lenticels and rootlet scars are present; sometimes
1.00 g of the powdered herbal drug (355) (2.9.12), add remains of the outer bark are visible. The pale greyish-yellow
1 mL of a 5 g/L solution of hexamethylenetetramine R, 20 mL or pale brown inner surface shows fine longitudinal striations.
of acetone R and 2 mL of hydrochloric acid Rl. Boil the The texture is hard and fragile, easily broken. The fracture is
mixture under a reflux condenser for 30 min. Filter the greyish-white to pinkish or light brown and finely granular.
brown to pinkish. Examine under a microscope using chloral
characters (Figure 2474.-1): numerous fragments of
10 min. Allow to cool and filter each extract through the
parenchyma consisting of cells of various shapes and sizes:
plug of absorbent cotton into the flask. After cooling, filter
some rounded with slightly and regularly thickened and
the combined acetone extracts through a paper filter into a
pitted walls [DJ, some with irregularly thickened beaded
volumetric flask and dilute to 100.0 mL with acewne R by
walls [J] and some [HJ containing one [Ha] or more [Hb]
rinsing the flask and the paper filter. Introduce 20.0 mL of
cluster crystals of calcium oxalate up to 45 µm in diameter;
the solution into a separating funnel, add 20 mL of water R
files of cells containing cluster crystals of calcium oxalate may
and shake the mixture with 1 quantity of 15 mL and then
be present [A]; very numerous free cluster crystals of calcium
3 quantities, each of 10 mL, of ethyl acetate R. Combine the
oxalate [CJ; more or less abundant fragments of cork with
ethyl acetate extracts in a separating funnel, wash with
superimposed polyhedral cells (surface view [BJ, longitudinal
2 quantities, each of 50 mL, of water R, filter the extracts
section [El). Examine under a microscope using a
over 1O g of anhydrous sodium sulfate R into a volumetric flask
and dilute to 50.0 mL with ethyl acetate R.
very numerous rounded or polygonal starch granules about
Test solution To 10.0 mL of the stock solution add 1 mL of 3-15 µm in diameter, simple or 2-6 compound, free [G] or
aluminium chloride reagent Rand dilute to 25.0 mL with a included in parenchyma cells [F]; the punctiform, cleft, V-,
5 per cent V/V solution of glacial acetic acid R in methanol R. Y-, or X-shaped hilum is clearly visible.
2023 Moutan Bark IV-375
Top of the plate
Paeonol: a quenching zone A quenching zone (paeonol)
--- ---
A quenching zone
A quenching zone
--- ---
Paeoniflorin: a quenching zone A quenching zone (paeoniflorin)
A quenching zone
Detection B Treat with a 10 per cent V/V solution of sulfuric

Figure 2474.-1. - Illustration for identification test B of powdered acid R in ethanol (96 per cent) R and heat at 100 °C for
herbal drug of mcrutan bark 5 min; examine in daylight.
C. Thin-layer chromatography (2.2.27). chromatograms obtained with the reference solution and 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 metharwl R and heat in a water-bath at chromatogram obtained with the test solution.
60 °C for 1 min. Centrifuge and use the supernatant.
Reference solution Dissolve 1 mg of paeonijlorin R and 1 mg Top of the plate
of paeonol R in 1 mL of metharwl R.
Mobile phase anhydrous formic acid R, ethyl acetate R, Paeonol: a yellow zone A yellow zone (paeonol)
methanol R, methylene chloride R (3:5:5:35 VIVIVIV).
2 violet zones
Drying In air.
Detection A Examine in ultraviolet light at 254 nm. --- --
Results A See below the sequence of zones present in the A sharp, prominent violet zone
in the chromatogram obtained with the test solution. 2 violet zones
A diffuse yellowish-brown zone
--- ---
Paeoniflorin: a brown zone A brown zone (paeoniflorin)
A brown zone

IV-376 Mullein Flower 2023
TESTS area of the peak due to paeoniflorin in the chromatogram

Foreign matter (2.8.2) area of the peak due to paeoniflorin in the chromatogram
Maximum 1.0 per cent. obtained with reference solution (d);
Loss on drying (2.2.32) mass of the herbal drug to be examined used to prepare the test
solution, in grams;
Maximum 11.0 per cent, determined on 1.000 g of the mass of paeoniflorin CRS used to prepare reference solution (a),
powdered herbal drug (355) (2. 9.12) by drying in an oven at in grams;
105 °C for 2 h. percentage content of paeoniflorin in paeoniflorin CRS.
Total ash (2.4. 16)

Maximum 5.0 per cent. Calculate the percentage content of paeonol using the
Maximum 1.0 per cent. A3 x m3 xp 2 x 0.3
ASSAY A 4 xm1
liquid chromatography (2.2.29). area of the peak due to paeonol in the chromatogram obtained
Test solution To 0.200 g of the powdered herbal drug (355) with the test solution;
(2. 9.12) add 3.0 mL of methanol Rl and sonicate for 30 min. area of the peak due to paeonol in the chromatogram obtained
with reference solution (b );
Dilute to 10.0 mL with a 6.8 g/L solution of potassium mass of the herbal drug to be examined used to prepare the test
dihydrogen phosphate R and filter through a membrane filter solution} in grams;
(nominal pore size 0.45 µm). mass of paeonol CRS used to prepare reference solution (b), in
grams;
Reference solution (a) Dissolve 5.0 mg of paeonifiorin CRS in
percentage content of paeonol in paeonol CRS.
methanol Rl and dilute to 5.0 mL with the same solvent.
Reference solution (b) Dissolve 7.0 mg of paeonol CRS in _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
methmwl Rl and dilute to 10.0 mL with the same solvent.
Dilute 3.0 mL of the solution to 10.0 mL with a 6.8 g/L
solution of potassium dihydrogen phosphate R.
Reference solution (c) Dissolve 2.5 mg of 4'- Mullein Flower
hydroxyacetophenone R in 2.5 mL of reference solution (a),
add 12.5 mL of methanol Rl and dilute to 50.0 mL with a (Ph. Bur. monograph 1853)
6.8 g/L solution of potassium dihydrogen phosphate R. Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
to 7.5 mL with methanol Rl. Dilute this solution to 25.0 mL DEFINITION
with a 6.8 g/L solution of potassium dihydrogen phosphate R. Dried flower, reduced to the corolla and the androecium, of
Column: Verbascum thapsus L., V. densifiorum Berto!. (syn.
- size: l = 0.15 m, 0 = 4.6 mm; V. thapsiforme Schrad.) or V. phlomoides L., or their hybrids,
- stationary phase: end-capped octadecylsilyl silica gel for or any mixture amongst the three species and their hybrids.
chromatography R (5 µm). IDENTIFICATION
Mobile phase: A. The corolla, 12-50 mm in diameter, is funnel-shaped and
- mobile phase A: 6.8 g/L solution of potassium dihydrogen pale to bright yellow, or sometimes orange. It is deeply
phosphate R; divided into 5 lobes, 2 of which are smaller than the others,
- mobile phase B: methanol Rl; with rounded apices. The outer surface has a dense covering
of branched and stellate trichomes; the inner surface is
Time Mobile phase A Mobile phase B glabrous, with a network of fine yellow to light brown veins.
(min) (per cent V/JI) (per cent V/JI) The 5 stamens are adnate to the base of the corolla, and
0 - 15 70 30 alternate with the lobes; 2 stamens are long, with glabrous,
15 - 18 70 - 40 30 - 60 greenish to yellow filaments, the other 3 are shorter, with
18 - 25 40 60 densely lanate yellow to orange filaments. The capitate
trichomes on the filaments are white to yellow. The anthers
Flow rate l .O mUmin. are attached transversely. V. thapsus can be distinguished
from the other two species by its smaller funnel-shaped
flowers (up to 25 mm in diameter) and narrower corolla
Injection l 0 µL of the test solution and reference lobes.
solutions (b), (c) and (d).
B. Microscopic examination (2.8.23). The powder is yellow
Retention time Paeoniflorin = about 9.5 min; or yellowish-brown. Examine under a microscope using
=
4'-hydroxyacetophenone about 10.5 min; paeonol about = chloral hydrate solution R. The powder shows the following
23 min. diagnostic characters (Figure 1853.-1): many covering
System suitability Reference solution (c): trichomes from the corolla, whole or fragmented,
- resolution: minimum 1.5 between the peaks due to pluricellular, of the candelabra type, with a central uniseriate
paeoniflorin and 4 '-hydroxyacetophenone. axis from which whorls of branch cells arise at the position of
Calculate the percentage content of paeoniflorin using the the cross walls and at the apex (side view [A, BJ, surface
following expression: view [F]); the covering trichomes from the stamen filaments
[G] are unicellular, long, thin-walled and tubular, have a
distinctly granular or striated surface with a sharp tip [Ga] or
sometimes with a club-shaped tip [Gb, Ge]; numerous ovoid,
tricolpate pollen grains with a finely granular exine [D];
fragments of the endothecium of the anther with thickened
2023 Mullein Flower IV-377
walls giving a characteristic star-shaped appearance [CJ; System suitability Reference solution (c):
yellow fragments of the petals (surface view [El) with - the chromatogram shows in the lower third 2 distinct
polygonal and isodiametric epidermal cells [Ea]; fragments of zones, which may be touching; the lower zone
the underlying mesophyll consisting of irregular (acteoside) shows a light blue fluorescence and the
parenchymatous cells [Eb] sometimes accompanied by spiral upper zone (isoquercitrin) shows an orange
vessels [Ee]. fluorescence.
obtained with the test solution, a reddish fluorescent zone
and 1 or 2 very faint to equivalent yellow fluorescent zones
may be present in the upper third.
In the case of V. thapsus, one of the 2 blue fluorescent zones
in the middle third and the greenish fluorescent zone below
the zone due to acteoside may be absent. An orange
fluorescent zone at the level of isoquercitrin (reference
solution (c)) and another orange fluorescent zone below the
zone due to acteoside may be present.
Top of the plate
Luteolin: a yellow zone 1-2 yellow zones, very faint to

equivalent (may be absent)
-- --
2 blue zones, faint to intense
-- --
Acteoside: a blue zone A blue zone, faint to intense
A greenish zone, faint to equivalent

(may be absent)
Figure 1853.-1. ~ Illustration for identification test B of powdered
herbal drng of mullein flower
(2. 9.12) add 10.0 mL of ethanol (96 per cent) R. Sonicate for D. Boil 1.0 g of the powdered herbal drug (355) (2.9.12)
10 min, centrifuge and use the supernatant. 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
Reference solution (a) Dissolve 1.0 mg of luteolin Rand
develops and, after a few minutes, cloudiness appears and
2.0 mg of acteoside R in methanol Rand dilute to 10.0 mL
then a blackish precipitate (iridoids).
to 10.0 mL with methanol R. Foreign matter (2.8.2)
Reference solution (c) Dissolve 1 mg of isoquercitrin R and Maximum 5 per cent of brown petals and maximum
2 mg of acteoside R in methanol R and dilute to 10 mL with 2 per cent of fragments of the calyx and other foreign matter.
the same solvent. Carry out the determination using 20 g.
Intensity marker Acteoside. Swelling index (2.8.4)
Plate TLC silica gel F 254 plate R (2-10 µm). Minimum 9, determined on the powdered herbal drug (710)
Mobile phase formic acid R, water R, ethyl acetate R (2. 9.12), moistened with 2 mL of ethanol (96 per cent) R.
(1:1:15 VIV!V). Loss on drying (2.2.32)
Application 6 µL, as bands of 8 mm. Maximum 12.0 per cent, determined on 1.000 g of the
Development 70 mm from the lower edge of the plate. powdered herbal drug (710) (2. 9.12) by drying in an oven at
Drying In a current of air at room temperature for 5 min. 105 °C for 2 h.
Detection Heat at 100-105 °C for 3 min; spray the warm Total ash (2.4.16)
plate with a 10 g/L solution of diphenylboric acid aminoethyl Maximum 6.0 per cent.
ester R in methanol R, then with a 50 g/L solution of macrogol Ash insoluble in hydrochloric acid (2.8.1)
400 R in methanol R or, alternatively, dip the warm plate in a Maximum 2.0 per cent.
acetate R, then in a 50 g/L solution of macrogol 400 R in STORAGE
methylene chloride R; allow to dry in air for about 1 min and In an airtight container.
IV-378 Myrrh 2023
Reference solution Dissolve 10 mg of thymol R and 40 µL of

Myrrh anethole R in 10 mL of ethanol (96 per cent) R.
(Ph. Bur. monograph 1349) Plate TLC silica gel F254 plate R (2-10 µm).
Preparation Mobile phase glacial acetic acid R, di-isopropyl ether R,
Myrrh Tincture cyclohexane R (10:40:60 V/V/V).
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Application 5 µL as bands of 8 mm.
DEFINITION
Drying In air.
Gum-resin, hardened in air, obtained by incision or produced
by spontaneous exudation from the stem and branches of Detection A Examine in ultraviolet light at 366 nm.
Commiphora myrrha (Nees) Engl. (syn. Commiphora molmol Results A The chromatogram obtained with the test solution
(Engl.) Engl. ex Tschirch) and/or other species of shows no blue or violet fluorescent zones in the lower third
Commiphora. of the chromatogram.
CHARACTERS Detection B Treat with anisaldehyde solutwn R, heat at
Bitter taste. 100 °C for 2 min and examine in daylight.
Matter insoluble in ethanol
IDENTIFICATION Maximum 75 per cent.
A. The light or dark orange-brown, irregular or roundish
Place 1.00 g of the powdered herbal drug (250) (2.9.12) in a
grains or pieces of different size show components of various
flask. Add 30 mL of ethanol (96 per cent) R and shake
colours. Their surface is mostly covered with grey or
vigorously for 10 min. Filter the supernatant through a tared
yellowish-brown dust.
sintered-glass filter (16) (2.1.2), avoiding the transfer of
B. Microscopic examination (2.8.23). The powder is sediment from the flask. Repeat the extraction with
brownish-yellow or reddish-brown. Examine under a 2 quantities, each of 20 mL, of ethanol (96 per cent) R.
microscope, using chloral hydrate solutwn R. The powder Quantitatively transfer the sediment to the filter by rinsing
shows the following diagnostic characters: a few tissue the flask with ethanol (96 per cent) R. Dry the filter and the
fragments from the original plants including: reddish-brown residue in an oven at 100-105 °C and weigh.
cork fragments; single or grouped polyhedral or elongated
stone cells with partly strongly thickened, pitted and lignified
walls with a brownish content; fragments of thin-walled
parenchyma and sclerenchymatous fibres; irregular prismatic
105 °C for 2 h.
or polyhedral crystals of calcium oxalate, about 10-25 µm in
size. Total ash (2.4.16)
C. Examine the chromatograms obtained in the test for _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Commiphora mukul.
test solution. Furthermore, other faint zones may be present Myrrh Tincture
Top of the plate Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
A reddish-violet zone DEFINITION

Anethole: a violet zone Tincture produced from Myrrh (1349).
-- --
PRODUCTION
A red zone
of ethanol (90 per cent V/V) by a suitable procedure.
Thymol: an orange zone CHARACTERS
Appearance
Clear, yellowish-brown or orange-brown liquid.
IDENTIFICATION
A violet zone
A brown zone Test solution Dilute 5 mL of the tincture to be examined to
10 mL with ethanol (96 per cent) R.
-- --
Reference solutwn Dissolve 10 mg of thymol R and 40 µL of
anethole R in 10 mL of ethanol (96 per cent) R.
Reference solution Test solution Plate TLC silica gel F254 plate R (2-10 µm).
TESTS cyclohexane R (10:40:60 VIVIV).
Commiphora mukul Application 5 µL as bands of 8 mm.
Thin-layer chromatography (2.2.27). Development Over a path of 6 cm.
Test solution To 0.2 g of the powdered herbal drug (355) Drying In air.
(2.9.12) add 4.0 mL of ethanol (96 per cent) R, sonicate for Detectwn Treat with anisaldehyde solution R, heat at 100 °C
10 min and filter. for 2 min and examine in daylight.
2023 Cineole Type Niaouli Oil IV-379
Results See below the sequence of zones present in the Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
chromatograms obtained with the reference solution and the plate R (2-10 µm)].
test solution. Furthermore, other faint zones may be present Mobile phase ethyl acetate R, toluene R (5:95 V/V).
in the chromatogram obtained with the test solution. Application 10 µL [or 2 µL] as bands of 10 mm [or 8 mm].
Top of the plate
Drying In air.
Anethole: a violet zone 100-105 °C for 3 min; examine in daylight.
-- --
A red zone test solution. Furthermore, other faint zones may be present
Thymol: an orange zone
Top of the plate
-- --
A violet zone
A faint grey zone
A brown zone
-- -- A purple zone
1,8-Cineole: a violet-brown zone An intense violet-brown zone (1,8-

Reference solution cineole)
Test solution
TESTS rrans-Nerolidol: a dark violet zone
Ethanol (2. 9.10) -- - -
82 per cent V/V to 88 per cent V/V.

An intense violet-brown zone
Methanol and 2-propanol (2. 9.11)
Maximum 0.05 per cent V/V of methanol and maximum A violet-brown zone
0.05 per cent V/V of 2-propanol. Reference solution Test solution
STORAGE chromatographic profile.
Plastic containers are not recommended. Results The characteristic peaks in the chromatogram
- - - - - - - - - - - - - - - - - - - - - - PhEur obtained with the test solution are similar in retention time to
those in the chromatogram obtained with reference
solution (a).
TESTS
Cineole Type Niaouli Oil Relative density (2.2.5)
0.904 to 0.925.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
1.463 to 1.472.
DEFINITION Optical rotation (2.2. 7)
Essential oil obtained by steam distillation from young leafy -4° to+ 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 µL of methyleugenol R and 5 µL
Aromatic odour of cineole. of isomethyleugenol R in heptane Rand dilute to 50.0 mL with
the same solvent. Dilute 0.5 mL of the solution to 5.0 mL
IDENTIFICATION with heptane R.
First identification B.
Second identification A. reference solution; record the retention times of
A. Thin-layer chromatography (2.2.21). methyleugenol and isomethyleugenol.
Test solution Dissolve 100 µL of the essential oil to be Identification of peaks Using the retention times determined
examined in toluene Rand 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 µL of trans-nerolidol R and chromatogram obtained with the test solution.
SO µL of cineole R in toluene Rand dilute to 5.0 mL with the Limits:
same solvent. - methyleugenol: maximum 0.05 per cent;
- isomethyleugenol: maximum 0.05 per cent.
IV-380 Neroli Oil 2023
Neroli Oil
procedure. (Ph. Bur. monograph 1175)
Test solution Dilute 0.2 mL of the essential oil to be PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Reference solution (a) Dilute 10 µL of rx-pinene R, 5 µL of DEFINITION
{3-pinene R, 10 µL of limonene R, 50 µL of cineole R, 5 µL of Neroli oil is obtained by steam distillation from the fresh
p-cymene R, 5 µL of benzaldehyde R, 5 mg of rx-terpineol R and flowers of Citrus aurantium L. subsp. aurantium L.
5 µL of trans-nerolidol R in heptane R and dilute to 10 mL (C. aurantium L. subsp. amara Engl.).
with the same solvent. CHARACTERS
Reference solution (b) Dissolve 5 µL of limonene R in Appearance
heptane R and dilute to 50.0 mL with the same solvent. Clear, pale-yellow or dark-yellow liquid.
Dilute 0.5 mL of the solution to 5.0 mL with heptane R. Characteristic odour.
- material: fused silica; First identification: B.
- size: l = 60 m, 0 = 0.25 mm;
A. Examine the chromatograms obtained in the test for
0.25 µrn).
bergapten.
Flow rate 1.3 mLJmin. chromatograms obtained with the reference solution and the
Split ratio 1:50. test solution. Furthermore other zones may be present in the
Temperature: chromatograms obtained with the test solution.
Time Temperature Top of the plate

(min) (°C)
--- - -
Column 0-5 65
5 - 65 65-, 185 Methyl anthranilate: a blue A faint blue fluorescent zone (methyl
fluorescent zone anthranilate)
65 - 80 185 ➔ 230
Injection port 230
Detector 250
Bergapten: a greenish-yellow
fluorescent zone
--- ---
Injection 1 µL.
substances. Detection B Spray with anisaldehyde solution R; heat at
100-105 °C for 10 min; examine the chromatograms in
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
chromatogram obtained with the test solution; the peak due chromatograms obtained with the reference solution and the
to viridiflorol elutes with a relative retention of about 1.02 test solution. In the chromatogram obtained with the test
with reference to trans-nerolidol. solution the zone due to linalol is more intense than the zone
due to linalyl acetate.
- resolution: minimum 1.5 between the peaks due to Top of the plate
limonene and 1,8-cineole.
A brown fluorescent zone
Determine the percentage content of each of the following
components. The limits are within the following ranges: Llnalyl acetate: a brownish-red An intense brownish-red fluorescent
- rx-pinene: 5.0 per cent to 15.0 per cent; fluorescent zone zone (linalyl acetate)
- {3-pinene: 1.0 per cent to 4.0 per cent;
--- --
- limonene: 5.0 per cent to 10.0 per cent;
- 1,8-cineole: 45.0 per cent to 65.0 per cent; Methyl anthranilate: a blue A faint blue fluorescent zone (methyl
fluorescent zone anthranilate)
- p-cymene: 0.05 per cent to 4.0 per cent;
- benzaldehyde: 0.05 per cent to 0.5 per cent; A faint brownish-red fluorescent
- rx-terpineol: 3.0 per cent to 8.0 per cent; zone
- trans-nerolicwl: 0.05 per cent to 1.5 per cent; Linalol: a brownish-red fluorescent A brownish-red fluorescent zone
- viridifiorol: 2.5 per cent to 9.0 per cent; zone (linalol)
- disregard limit: the area of the principal peak in the Bergapten: a greenish-yellow
chromatogram obtained with reference fluorescent zone
solution (b) (0.05 per cent).
--- ---
STORAGE
Several blue and brownish-red
fluorescent zones
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
2023 Neroli Oil IV-381
B. Examine the chromatograms obtained in the test for Temperature:

Results The principal peaks in the chromatogram obtained Time Temperature
(min) (°C)
principal peaks in the chromatogram obtained with the Column 0-4 75
4 - 42.8 75 --> 230
reference solution. 42.8 - 63 230
TESTS Injection port 270
Relative density (2.2.5) Detector 270
0.863 to 0.880.
Refractive index (2.2.6) Detectwn Flame ionisation.
1.464 to 1.474. Injection 0.2 µL.
Optical rotation (2.2. 7) Elution order Order indicated in the composition of
+ 1.5° to+ ll.5°. reference solution (a). Record the retention times of these
Acid value (2.5.1) substances.
Maximum 2.0. System suitability Reference solution (a):
Bergapten - resolution: minimum 1.5 between the peaks due to
Thin-layer chromatography (2.2.27). ~-pinene and sabinene.
Test solution Dissolve 0.1 g of the substance to be examined
in ethanol (96 per cent) Rand dilute to 5.0 mL with the same chromatogram obtained with reference solution (a), locate
solvent. the components of reference solution (a) in the
Reference solution Dissolve 2 µL of methyl anthranilate R,
10 µL of linalyl acetate R, 20 µL of linalol R and 5 mg of Limits:
bergapten R in ethanol (96 per cent) Rand dilute to 10.0 mL - {3-pinene: 7.0 per cent to 17.0 per cent,
with the same solvent. - limonene: 9.0 per cent to 18.0 per cent,
- linalol: 28.0 per cent to 44.0 per cent,
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel - linalyl acetate: 2.0 per cent to 15.0 per cent,
plate R (2-10 µm)]. - rJ,-terpineol: 2.0 per cent to 5.5 per cent,
Mobile phase ethyl acetate R, toluene R (15:85 V/V). - neryl acetate: maximum 2.5 per cent,
Application 10 µL [or 2 µL] as bands. - geranyl acetate: 1.0 per cent to 5.0 per cent,
Development Over a path of 15 cm [or 8 cm]. - trans-nerolidol: 1.0 per cent to 5.0 per cent,
- methyl anthranilate: 0.1 per cent to 1.0 per cent,
Drying In air.
- (E,E)-farnesol: 0.8 per cent to 4.0 per cent,
Detection Examine in ultraviolet light at 365 nm. - disregard limit: area of the peak in the chromatogram
Results The chromatogram obtained with the test solution obtained with reference solution (b).
does not show a zone corresponding to the zone due to
Chiral purity
bergapten in the chromatogram obtained with the reference
Gas chromatography (2.2.28).
solution.
Test solution Dissolve 20 mg of the substance to be
Chromatographic profile examined in pentane R and dilute to 10. 0 mL with the same
solvent.
procedure.
Reference solution To 10 µL of linalol R add 10 µL of linalyl
Test solution The substance to be examined. acetate R. Dilute to 10.0 mL with pentane R.
Reference solution (a) Dissolve 20 µL of /3-pinene R, 5 mg of Column:
sabinene R, 40 µL of limonene R, 40 µL of linalol R, 20 µL of - material: fused silica,
linalyl acetate R, 5 mg of rJ,-terpineol R, 5 µL of neryl acetate R, - size: l = 25 m, 0 = 0.25 mm,
5 µL of geranyl acetate R, 5 µL of trans-nerolidol R, 5 µL of - stationary phase: modified {3-cyclodextrin for chiral
methyl anthranilate Rand 5 µL (E,E)-farnesol R in 2 mL of chromatography R (film thickness 0.25 µm).
heptane R.
Reference solution (b) Dissolve 5 µL of methyl anthranilate R
in heptane Rand dilute to 10 mL with the same solvent.
Split ratw 1:30.
Column:
- material: fused silica, Temperature:
- size: l = 60 m, 0 = 0.25 mm,
- stationary phase: macrogol 20 000 R (film thickness Time Temperature
(min) CC)
0.25 µm).
Column 0 - 65 50--> 180
Injection port 230
Flow rate 1.5 mUmin. Detector 230
Split ratio 1:100.
Injection 1 µL.
- resolution: minimum 5.5 between the peaks due to (R)(-)-
linalol (1 st peak) and (S)(+)-linalol (2nd peak);
IV-382 Nettle Leaf 2023
minimum 2.7 between the peaks due to (R)(-)-linalyl [He], transverse section [Del) containing small cluster
acetate (3 rd peak) and (S)(+)-linalyl acetate 4th peak). crystals of calcium oxalate (surface view [Hd], transverse
Calculate the percentage content of the specified section [Dt]); occasional small groups of vessels,
(S)-enantiomers from the following expression: accompanied by parenchyma containing cluster crystals of
calcium oxalate m.
A1
- A xlOO
A 1+ 2
A1 area of the corresponding (S)-enantiomer,

A2 area of the corresponding (R)-enantiomer.
Limits:
- (S)(+)-linalol: maximum 30 per cent,
- (S)(+)-linalyl acetate: maximum 5 per cent.
STORAGE
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Nettle Leaf
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or cut dried leaves of Urtica dioica L., Urtica urens L.,
or a mixture of the 2 species.
Content
Minimum 0.3 per cent for the sum of caffeoylmalic acid and
chlorogenic acid, expressed as chlorogenic acid (C 16 H 18 0 9 ;
IDENTIFICATION
A. The leaves are dark green, dark greyish-green or
brownish-green on the upper surface, paler on the lower
surface; scattered stinging hairs occur on both surfaces, also
small covering trichomes that are more numerous along the Figure 1897. -1. - Illustration for identification test B of powdered
margins and on the veins on the lower surface. The lamina is herbal drug of nettle leaf
strongly shrunken, ovate or oblong, up to 100 mm long and C. Thin-layer chromatography (2.2.27).
50 mm wide, with a coarsely serrate margin and a cordate or
rounded base. The venation is reticulate and distinctly
(2. 9.12) add 10 mL of methanol R. Boil under a reflux
prominent on the lower surface. The petiole is green or
condenser for 15 min. Cool and filter. Evaporate to dryness
brownish-green, rounded or flattened, about 1 mm wide,
in vacua at 40 °C. Dissolve the residue in 2 mL of
longitudinally furrowed and twisted; it bears stinging hairs
and covering trichomes. methanol R.
Reference solution Dissolve 1 mg of scopoletin R and 2 mg of
B. Microscopic examination (2.8.23). The powder is green or
greyish-green. Examine under a microscope using en/oral cnlorogenic acid R in 20 mL of methanol R.
hydrate solution R. The powder shows the following diagnostic Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
characters (Figure 1897.-1): fragments of unicellular stinging plate R (2-10 µm)].
hairs [A, B, C], up to 2 mm long, composed of an elongated Mobile phase anhydrous formic acid R, methanol R, water R,
tapering cell with a slightly swollen stinging tip that readily ethyl acetate R (2.5:4:4:50 V/V/V/V).
breaks off, arising from a raised, multicellular base [Ca]; Application 10 µL [or 4 µL] as bands of 10 mm [or 8 mm].
small glandular trichomes [F] (35-65 µm), with a uni- or Development Over a path of 8 cm [or 6 cm].
bicellular stalk and a bi- or quadricellular head, isolated [Fa],
or on fragments of the epidermis [Fb]; fragments of the Drying In air.
upper epidermis of the leaves (surface view [G], transverse Detection Heat at 100 °C for 5 min; spray the still-warm
section [D]) showing slightly sinuous cells [Da, Ge], plate with a 10 g/L solution of diphenylboric acid aminoethyl
unicellular, straight or slightly curved covering trichomes, ester R in methanol R; examine in ultraviolet light at 365 run.
enlarged at the base, up to 700 µm long [De, Ga] and Results See below the sequence of zones present in the
abundant large cystoliths [Db, Ea, Gb], empty or containing chromatograms obtained with the reference solution and the
dense, granular masses of calcium carbonate; palisade test solution. Furthermore, other faint blue or yellow
parenchyma (surface view [El) with rounded cells [Eb] fluorescent zones may be present in the lower half of the
surrounding cystoliths [Ea] (transverse section [Dd]); chromatogram obtained with the test solution.
fragments of lower epidermis of leaves showing sinuous or
wavy-walled cells [H], anomocytic [Ha] or anisocytic stomata
[Hb] (2.8.3) accompanied by spongy mesophyll (surface view
2023 Nettle Root IV-383
Top of the plate Injection 20 µL.

2 red zones
Relative retention With reference to chlorogenic acid
(retention time = about 13 min): caffeoylmalic
Scopoletin: an intense blue A blue fluorescent zone (scopoletin) acid = about 2.2.
fluorescent zone
Calculate the percentage content of caffeoylmalic acid and
A blue fluorescent zone chlorogenic acid, expressed as chlorogenic acid, using the
-- --
-- --
Chlorogenic acid: a blue fluorescent A blue fluorescent zone (chlorogenic AI sum of the areas of the peaks due to caffeoylmalic acid and
zone acid) chlorogenic acid in the chromatogram obtained with the test
solution;
A brownish-yellow zone A2 area of the peak due to chlorogenic acid in the chromatogram
solution, in grams;
m2 mass of chlnrogenic acid CRS used to prepare the reference
TESTS solution, in grams;
p percentage content of chlorogenic acid in ch/nrogenic acid CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
other foreign matter (including inflorescences).
105 °C for 2 h.
Nettle Root
Total ash (2.4.16) (Ph. Eur. monograph 2538)
Maximum 20.0 per cent. Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Dried, whole or fragmented underground parts of Urtica
ASSAY dwica L. or Urtica urens L., their hybrids or their mixtures.
IDENTIFICATION
A. Irregular cylindrical rhizome, greyish-brown, sporadically
(2.9.12) add 25.0 mL of a 40 per cent V/V solution of
purple or greenish, 3-10 mm in diameter, the internodes are
methanol R. Extract for 30 min in an ultrasonic bath at 40 °C
up to 2-3 cm long, with deep longitudinal furrows,
and filter.
alternating with short, slightly swollen nodes; the nodes bear
Reference solutwn Dissolve 10.0 mg of chlorogenic acid CRS numerous roots that are externally greyish-brown, 0.5-2 mm
in 100.0 mL of a 40 per cent V/V solution of methanol R. in diameter and up to about 10 cm long. The transverse
Dilute 5.0 mL of this solution to 25.0 mL with a section shows a thin, dark bark, the inner part is creamish-
40 per cent VIV solution of methanol R. white with a hollow centre. The fracture of the rhizome is
Precolumn: fibrous and uneven, especially in the inner part.
- size: l = 4 mm, 0 = 4 mm; B. Microscopic examination (2.8.23). The powder is pale
- stationary phase: end-capped octadecylsilyl silica gel for yellowish-brown. Examine under a microscope using chloral
chromatography R (5 µm). hydrate solutwn R. The powder shows the following diagnostic
Column: characters: fragments of brownish cork with thin-walled cells;
=
- size: l 0.125 m, 0 4 mm; = fragments of vessels with bordered pits, 50-150 µm in
- stationary phase: end-capped octadecylsilyl silica gel for diameter; fragments of fibres with thick lignified walls,
chromatography R (5 µm); occurring singly or in groups; abundant fragments of
- temperature: 25 °C. parenchyma with thin-walled cells, some containing large
Mobile phase: cluster crystals of calcium oxalate; rare, scattered, simple
- mobile phase A: mix 15 volumes of methanol R and crystals of calcium oxalate; fragments of the medullary rays
85 volumes of water Rand adjust to pH 2.0 with dilute with moderately thick-walled, pined cells.
phosphoric acid R; C. Thin-layer chromatography (2.2.27).
- mobile phase B: methanol R; Test solutwn To 2 g of the powdered herbal drug (355)
(2. 9.12) add 10 mL of methanol R. Sonicate for 10 min and
filter. Evaporate the filtrate to dryness and dissolve the
(min) (per cent VIJI) (per cent VIJI)
residue in 2 mL of methanol R.
0- I 100 0
JOO--, 85 0--, 15
Reference solutwn Dissolve 1 mg of scopoletin R in 10 mL of
I - 25
25 - 35 85 15
methanol R (solution A). Dissolve 10 mg of arbutin Rand
35 - 36 85 __, 0 15--, 100
20 mg of /3-sitosterol R in methanol R, add 1 mL of solution A
and dilute to 10 mL with methanol R.
Flow rate 1 mUmin.
F2s4 plate R (2-10 µm)).
IV-384 Notoginseng Root 2023
Mobile phase anhydrous formic acid R, methanol R, water R, Extractable matter

ethyl acetate R (2.5:4:4:50 VIVIVIV). Minimum 7.0 per cent.
Applicatwn 20 µL [or 10 µL] as bands of 10 mm [or To 2.000 g of the powdered herbal drug (250) (2.9.12) add a
8 mm]. mixture of 6 g of water R and 14 g of ethanol (96 per cent) R
Development Over a path of 8 cm [or 6 cm]. and extract for 2 h, shaking frequently. Filter, evaporate
5. 000 g of the filtrate to dryness on a water-bath and dry in
an oven at 105 °C for 2 h. The residue weighs a minimum of
Detection A Examine in ultraviolet light at 365 nm. 35 mg.
Results A See below the sequence of zones present in the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Top of the plate

Notoginseng Root
A blue or greenish-blue fluorescent (Ph. Bur. monograph 2383)
zone Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Scopoletin: a blue fluorescent zone A blue fluorescent zone (scopoletin) Whole or fragmented taproot, without secondary roots, of
-- --
Panax notoginseng (Burkill) F.H.Chen [Panax pseudoginseng
var. notoginseng (Burki!!) G.Hoo & C.L.Tseng] treated with
- - - -
steam and dried.
Content
Minimum 3.8 per cent for the sum of ginsenosides Rgl
(C42H12O14,2H2O; Mr 837) and Rbl (Cs4H92O23,3H2O;
Detection B Treat with anisaldehyde solution R and heat at
100-105 °C for 5-10 min; examine in daylight. IDENTIFICATION
A. The primary root is conical, subconical or cylindrical, up
to 6 cm long and 4 cm in diameter. The outer surface,
showing shallow transverse striations and secondary root
scars, is brownish-grey or yellowish-grey. The aerial stem scar
is surrounded by warty protuberances at the crown.
The texture of the root is compact. The fracture is smooth,
Top of the plate shiny, brownish-grey and shows a yellowish-grey ring
A purple zone (cambial zone) and many radial striations.
yellowish-grey. Examine under a microscope using chloral
~-sitosterol: a purple zone A purple zone (~-sitosterol) hydrate solutwn R. The powder shows the following diagnostic
characters (Figure 2383.-1): abundant fragments of
- - - -
parenchymatous cells with rounded or ovoid cells [D];
A faint purple zone fragments of secretory canals (transverse section [G],
longitudinal section [El) with thin-walled cells [Ea, Ga] and
-- --
containing a granular yellowish-brown resin diffused in the
Arbutin: a greenish-brown zone cells [Eb, Gb]; rare vessels, reticulate [BJ or pitted, ranging
from 20-50 µm in diameter; rare fragments of cork (surface
view [A], transverse section [F]). Examine under a
Reference solution Test solution microscope using a 50 per cent V/V solution of glycerol R.
The starch granules, sometimes deformed, single, with a
diameter of 2-20 µm, or in groups of 2-9, are very
TESTS abundant [CJ.
Maximum 5 per cent of stem bases and maximum 2 per cent
Panax ginseng or Panax quinquefolium.
105 °C for 2 h.
Lead (2.4.27)
Maximum 7.0 ppm.
Total ash (2.4.16)
2023 Notoginseng Root IV-385
Mobile phase ethyl acetate R, water R, butanol R

(25:50:100 VIVIV); allow to stand for 10 min and use the
upper layer.
Application 20 µL, as bands of 15 mm [or 4 µL of the test
solution and 2 µL of the reference solution, as bands of
8 mm].
Development In an unsaturated tank, over a path of 10 cm
[or 5 cm].
solution, the absence of a violet zone immediately above the
zone due to arbutin in the chromatogram obtained with the
reference solution indicates the presence of Panax ginseng-,
in the chromatogram obtained with the test solution, the
presence of a brown zone immediately below the violet zone
due to ginsenosides Rgl + Rg2 indicates adulteration with
Panax quinquefolium.
105 °C for 2 h.
Total ash (2.4.16')
>-------< Ga Gb Ash insoluble in hydrochloric acid (2.8.1)
Figure 2383.-1. - Illustration for identification test B of powdered ASSAY

herbal drng of notogimeng root Liquid chromatography (2.2.29).
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
Top of the plate 50 per cent V/V solution of methanol R in a 250 mL round-
A violet zone (at the solvent front)
bottomed flask. After adding a few grains of pumice, boil on
a water-bath under a reflux condenser for 1 h. After cooling,
A violet zone centrifuge and collect the supernatant. Treat the residue as
Arbutin: a brown zone
described above. Mix the collected liquids and evaporate to
dryness under reduced pressure at a temperature not
-- -- exceeding 60 °C. Take up the residue with 10.0 mL of a
A violet zone (ginsenosides Rgl buffer solution, adjusted to pH 4.5, containing 3.5 g of
+ Rg2) sodium dihydrogen phosphate R and 7 .2 g of potassium
2 violet zones dihydrogen phosphate R in 1000 mL of water R (solution A).
Wash a cartridge containing about 0.36 g of octadecylsilyl
2 faint violet zones silica gel for chromatography R with 5 mL of methanol R
-- --
followed by 20 mL of water for chromatography R. Apply
5.0 mL of solution A to the cartridge. Elute with 20 mL of
Aescin: a grey zone A violet zone water for chromatography R, followed by 15 mL of a
Several violet and greenish zones 30 per cent V/V solution of methanol R. Discard the eluates
after confirming that no ginsenosides are present, otherwise
Reference solution Test solution repeat the assay with another type of cartridge. Elute the
cartridge with 20 mL of methanol R and evaporate the eluate
TESTS to dryness. Take up the residue with 5.0 mL of methanol R.
Panax ginseng or Panax quinquefolium Reference solution Dissolve 3.0 mg of ginsenoside Rbl CRS,
Thin-layer chromatography (2.2.27). 3.0 mg of ginsenoside Rf Rand 3.0 mg of ginsenoside Rgl CRS
Test solution To 1.0 g of the powdered herbal drug (355) in methanol Rand dilute to 5.0 mL with the same solvent.
(2. 9.12) add 10 mL of a 70 per cent V/V solution of Column:
methanol R and boil under a reflux condenser for 15 min. - size: l =0.10 m, 0 =4.6 mm;
Filter after cooling and dilute to 10.0 mL with methanol R. - stationary phase: aminopropylsilyl silica gel for
Reference solution Dissolve 5.0 mg of aescin R and 5.0 mg of
arbutin R in 1 mL of methanol R. Mobile phase:
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel - mobile phase A: acetonitrile Rl;
plate R (2-10 µm)]. - mobile phase B: water for chromatography R;
IV-386 Notopterygium Rhizome and Root 2023
Time Mobile phase A Mobile phase B xylem surrounds the pith; the pith (absent in roots) is
(min) (per cent V/JJ) (per cent V/JJ) whitish-yellow or yellow with numerous reddish-brown oil
0 - 14 90 10 cavities dotted in the loosely arranged tissue. Oil cavities are
14 - 18 90 ➔ 80 10 ➔ 20 also abundant in the cortex and phloem.
18 - 55 80 20
The fragmented drug occurs as transverse or longitudinal
slices of rhizome or as irregular pieces of rhizome and roots
Flow rate 2 mIJmin. (rhizome fragments, 0.5-2 cm in diameter; root fragments,
Detection Spectrophotometer at 203 nm. 0.2-1.6 cm in diameter). The external surface is brown to
Infection 20 µL. blackish-brown. Rhizome fragments bear distinct dotted or
tuberculate adventitious root scars. Root fragments are short,
with longitudinal striations. The cut surface (transverse
slices) of the rhizome shows a distinctive yellowish-brown to
ginsenosides Rgl and Rf.
brown cortex and phloem region and a light yellow ring of
Calculate the sum of the percentage contents of xylem surrounding a yellowish-white or brown pith (pith
ginsenosides Rbl and Rgl using the following expression: absent in roots); radially arranged clefts are common; the
cortex, phloem and pith are dotted with reddish-brown oil
A1xm2x2xp1 A2xm3x2x~
------+----,------
m1XA3 m1xA4
cavities. The pith tissue is loosely arranged, showing clearly
visible lacunae in longitudinal slices. The texture is light and
A1 area of the peak due to ginsenoside Rb I in the chromatogram
fragile, easily broken; the fracture is uneven.
obtained with the test solution; B. Microscopic examination (2.8.23). The powder is brown
A2 area of the peak due to ginsenoside Rgl in the chromatogram or dark golden-brown. Examine under a microscope using
A3 area of the peak due to ginsenoside Rb! in the chromatogram
obtained with the reference solution; diagnostic characters (Figure 2662.-1): numerous fragments
A4 area of the peak due to ginsenoside Rgl in the chromatogram of secretary canals (transverse section [CJ, longitudinal
obtained with the reference solution; section [E]) usually containing orange-brown secretions, in
droplets [Ca, Ea]; yellowish-brown fragments of cork (surface
solution, in grams;
m2 mass of ginsenoside Rbl CRS used to prepare the reference view [A], transverse section [F]) consisting of thin-walled
solution, in grams; cells; fragments of cortical parenchyma with relatively thick-
m3 mass of ginsenoside Rgl CRS used to prepare the reference walled, elongated (transverse section [B]) or rounded
solution, in grams; (tangential section [G]) cells; fragments of xylem parenchyma
p, percentage content of ginsenoside Rb! in ginsenoside Rbl CRS;
p2 percentage content of ginsenoside Rgl in ginsenoside Rg1 CRS. common; numerous, mainly reticulate vessels, isolated or
embedded in xylem parenchyma with thin-walled cells [D];
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur droplets of orange-brown oil common [Ca, Eb].
Notopterygium Rhizome and Root

Ph Eur - - - - - - - - - - - - - - - - - - - - - ~
DEFINITION
Dried, whole or fragmented rhizome and root of Hansenia
weberbaueriana (Fedde ex H.Wolff) Pimenov & Kljuykov
(syn. Notopterygi,um incisum KC.Ting ex H.T.Chang).
Content
- essential oil: minimum 14 mIJkg (anhydrous drug);
- minimum 0.4 per cent and maximum 0.8 per cent for the
sum of isoimperatorin (C 16H 140 4 ; Mr 270.3) and
notopterol (C 21 H 220 5 ; Mr 354.4) (anhydrous drug).
IDENfIFICATION
A. The whole rhizome is cylindrical or conical and somewhat
curved, about 3-13 cm long and 0.5-2.5 cm in diameter; it is
occasionally branched. Externally brown to blackish-brown
with raised annulations; the internodes are either very short
(silkworm-type, dense annulations) or elongated (bamboo-
type, sparse annulations).
Each node bears numerous dotted or tuberculate adventitious
root scars and more rarely the remains of thin, brown to
blackish-brown, longitudinally striated roots; fragmented
brown scales are sometimes present. The root crown may
show the remains of non-annulated stems.
The texture is light and fragile, easily broken. The fracture is herbal drug of notopterygi,um rhizome and root
uneven, with numerous radial clefts; the cortex and phloem
are yellowish-brown to brown; a ring of light yellow or yellow C. Thin-layer chromatography (2.8.25).
2023 Notopterygium Rhizome and Root IV-387
Test solution To 1.0 g of the powdered drug add 5.0 mL of Top of the plate
methanol R. Shake for 10 min, centrifuge and use the
supernatant as the test solution.
Reference solution (a) Dissolve 2.0 mg offerulic acid Rand --- ---
2.0 mg of isoimperatorin R in 4.0 mL of methanol R. A green zone, very faint
Isoimperatorin: a green A green zone
zone (isoimperatorin)
Reference solution (c) Dissolve 2 mg of imperatorin R and
Osthole: a blue zone A blue zone
1 mg of osthole R in 2 mL of methanol R.
Intensity marker Isoimperatorin. lmperatorin: a green One or two blue zones
zone
Mobile phase acetic acid R, ethyl acetate R, toluene R --- --- ---
(1:10:90 VIVIV). A blue zone
A blue zone, intense
Ferulic acid: a blue A blue zone
Drying In air.
zone
A green zone
chromatograms obtained with reference solutions (a) and (b) Two blue zones
Reference solution (c) Reference solutions (a) Test solution
obtained with the test solution, other faint zones may be and (b)
present.
Top of the plate

Detection C Spray or dip the plate in a 10 per cent solution
of suljuric acid R in methanol Rand heat at 100 °C for
10 min. Examine in daylight.
--- ---
chromatograms obtained with reference solutions (a) and (b)
Isoimperatorin: a A quenching zone
quenching zone (isoimperatorin)
obtained with the test solution, other faint zones may be
Osthole: a blue zone present.
Imperatorin: a A quenching zone
quenching zone Top of the plate
A pink zone
--- --- ---
A quenching zone, faint --- ---

to equivalent (may be
absent)
A quenching zone, faint Isoimperatorin: a green A green zone

to equivalent zone (isoimperatorin)
Ferulic acid: a A quenching zone,
Osthole: a pink zone
quenching zone intense
lmperatorin: a yellow
zone
and (b)
--- --- ---
Detection B Examine in ultraviolet light at 366 nm. Two overlapping pink

System suitability Reference solution (c): or violet zones
- the chromatogram shows in the middle third 2 distinct A broad pink zone,
zones, which may be touching; the lower zone intense
(isoimperatorin) shows a green colour and the upper A brown zone, intense
zone (osthole) shows a blue colour. Ferulic acid: a pink
Results B See below the sequence of fluorescent zones zone
present in the chromatograms obtained with reference
A violet zone
solutions (a) and (b) and the test solution. Furthermore, in
the chromatogram obtained with the test solution, other faint
fluorescent zones may be present.
and (b)
Detection D Examine the chromatogram obtained after

derivatisation under ultraviolet light at 366 nm.
IV-388 Nutmeg 2023
Results D See below the sequence of fluorescent zones methanol R. Mix. Centrifuge for 5 min. Filter through a
present in the chromatograms obtained with reference membrane filter (nominal pore size 0.45 µm).
solutions (a) and (b) and the test solution. Furthermore, in Column:
the chromatogram obtained with the test solution, other faint - size: l = 0.25 m, 0 = 4.6 mm;
fluorescent zones may be present. - statwnary phase: end-capped octadecylsilyl silica gel for
chromatography Rl (5 µm);
Top of the plate - temperature: 25 °C.
Mobile phase:
A pink zone - mobile phase B: acetonitrile R;
--- ---
A blue zone, faint (may (min) (per cent V/Jl) (per cent V/Jl)
be absent)
0 ➔ 30 50 ➔ 38 50 ➔ 62
Isoimperatorin: a blue
zone
Osthole: a blue zone A blue zone
A blue or green zone, Injection 10 µL.
very faint to faint
reference solution (a) to identify the peak due to
- - --- ---
isoimperatorin, use the chromatogram obtained with
reference solution (b) and the chromatogram supplied with
A blue or green zone
notopterygium rhizome and root for system suitability HRS to
identify the peaks due to notopterol and peak 2.
Ferulic acid: a green A blue or dark zone
Relative retention With reference to isoimperatorin (retention
zone time= about 18.4 min): notopterol = about 0.66; peak
2 = about 0.70.
Two blue or green - resolution: minimum 1.5 between the peak due to
zones notopterol and peak 2.
Calculate the percentage content of the sum isoimperatorin
Reference solution (c) Reference solutions (a) Test solution and notopterol using the following expression:
and (b)
A3 x m2 x 0.4 x 1.5
A 1 x m2 xp x 0.4
----:------+-------=-----
A2 xm1 A2 xm1
TESTS
area of the peak due to isoimperatorin in the chromatogram
Water (2.2.13) obtained with the test solution;
Maximum 110 mUkg, determined on 20.0 g of the area of the peak due to isoimperatorin in the chromatogram
powdered herbal drug (355) (2.9.12). obtained with reference solution (a);
area of the peak due to notopterol in the chromatogram
Total ash (2.4. 16) obtained with the test solution;
Maximum 7 .0 per cent. mass of the herbal drug to be examined used to prepare the test
solution, in grams;
Ash insoluble in hydrochloric acid (2.8.1) mass of isoimperatorin used to prepare reference solution (a), in
Maximum 1.5 per cent. grams;
p percentage content of isoimperatorin in isoimperatorin CRS;
ASSAY 1.5 peak correlation factor between notopterol and isoimperatorin.
Use 50.0 g of the powdered herbal drug (500) (2.9.12), a _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
2 L round-bottomed flask and 1000 mL of water R as the
distillation liquid. Distil at a rate of 2-3 mUmin for 3 h.
Isoimperatorin and notopterol
Liquid chromatography (2.2.29). Nutmeg
Test solutwn Place 0 .200 g of the powdered herbal drug DEFINITION
(355) (2.9.12) in a 50 mL centrifuge tube, add 20.0 mL of Nutmeg is the endosperm of dried seeds of Myristica fragrans
methanol R and weigh. Sonicate for 30 min and weigh again. Houtt. It contains not less than 5.0% v/w of essential oils,
Compensate for the loss of solvent with methanol R. Mix. calculated with reference to the dried drug.
Centrifuge for 5 min. Filter through a membrane filter
(nominal pore size 0.45 µm). IDENTIFICATION
A. Seed ellipsoid, 20 to 30 mm long and up to 20 mm
Reference solution (a) Dissolve 5.0 mg of isoimperatorin CRS
broad; externally greyish-brown and reticulately furrowed,
in methanol Rand dilute to 50.0 mL with the same solvent.
sometimes marked with small irregular darker brown patches
Reference solution (b) Place 0.2 g of notopterygium rhizome or points; one end is a small lighter-coloured area with
and root for system suitabiliry HRS in a 50 mL centrifuge tube, brown lines radiating from the hilum, and surrounded by a
add 20 mL of methanol R and weigh. Sonicate for 30 min raised ring, from which an ill-defined groove, an imprint or
and weigh again. Compensate for the loss of solvent with the raphe, runs to the chalaza at the opposite end, where
there is a small, dark, depression.
2023 Nutmeg Oil IV-389
B. Reduce to a powder, Appendix XVII A. The powder is Table 2: Visualisation under white light
dark brown. Examine under a microscope using chloral
hydrate solution. The powder contains numerous fragments of Too of the olate
reddish-brown parenchyma of the perisperm, those from the

outer layers composed of polygonal or rounded with slightly A faint grey-brown
thickened walls and occasional small intercellular spaces; band
some containing prism crystals; parenchyma of the inner A brownish band Myristicine (A
A purple band brownish band)
layers composed of smaller cells with darker reddish-brown
contents and large, rounded oil cells which occur singly or in A purple band lsoeugenol acetate
groups and are frequently broken; small groups of lignified (A purple band)
vessels; thin-walled parenchyma of the endosperm composed lsoeugenol (A purple
of closely-packed polygonal cells filled with starch granules; band)
when fully cleared of starch these cells can be seen to contain
elongated prism crystals. Examine under glycerol: abundant, An orange-brown
small starch granules, usually simple and spherical, but band
occasionally compound 2-8, with a stellate or slit-shaped A faint purple Terpinen-4-ol (A
hilum. band purple band)
C. Carry out the method for high performance thin-layer A purple band
chromatography, Appendix XI W, using the following An intense violet
solutions. band
(1) To 0.5 g of powdered herbal drug (355) add 5 mL of
Solution (1) Solution (2) and Solution (4)
methanol. Mix with the aid of ultrasound, centrifuge, and use (3)
the supernatant liquid.
(2) 0.09% w/v of myristicine and 0.1 % w/v of terpinen-4-ol in CONFIRMATION
methanol.
The chromatogram obtained with solution (1) shows the
(3) Dilute 1 volume of solution (2) to 4 volumes with toluene.
zones in Table 1 when examined under ultraviolet light and
(4) 0.025% v/v of isoeugenol and 0.05% v/v of isoeugenyl the fluorescent zones in Table 2 when examined under white
acetate in toluene. light. Other bands may be present.
CHROMATOGRAPHIC CONDITIONS TESTS
(a) Use as the coating high performance silica gel F254 (Merck Foreign matter
silica gel 60 F 254 HPTLC plates are suitable). Not more than 2.0%, Appendix XI D.
(b) Use the mobile phase as described below. Water
(c) Apply 4 µL of each solution as 8 mm bands. Not more than 10.0% v/w, Appendix IX C, method II.
(d) Develop the plate to 7 cm. Use 8 g of powdered drug.
(e) After the removal of the plate, dry in a current of cold Total Ash
air, and examine under ultraviolet light (254 nm). To a Not more than 3.0%, Appendix XI J, Method II. Use 1 g of
mixture of 1 volume of sulfuric acid, 2 volumes of acetic acid, powdered drug.
and 17 volumes of methanol, add 0.1 volumes of anisaldehyde. ASSAY
Dip the plate in this solution, and heat the plate to 100° for Carry out the method for Essential Oils in Herbal Drugs,
3 minutes, and examine under white light. Appendix XI E, using 18 g of freshly prepared powdered
MOBILE PHASE drug (425) with 250 mL of water as the distillation liquid.
Distil at a rate of 2 to 3 mL per minute for 2 hours using
5 volumes of ethyl acetate and 95 volumes of toluene.
0.50 mL of toluene in the graduated tube. Measure the
SYSTEM SUITABILITY quantity of essential oil distilled.
The test is not valid unless the chromatogram obtained with ANNEX
solutions (2) and (3) shows a zone corresponding to This section is non-mandatory.
myristicin.
Table 1: Visualisation at 254 nm A DNA reference sequence for the identity of Nutmeg is
published in Supplementary Chapter VII D.
TOD of the Dlate
Nutmeg Oil
A quenching zone Myristicine (A
quenching zone)
A quenching zone
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
A quenching zone lsoeugenol acetate
(A quenching zone) DEFINITION
lsoeugenol (A Essential oil obtained by steam distillation of the dried and
quenching zone) crushed kernels of Myristica fragrans Houtt.
A quenching zone CHARACTERS
Appearance
Solution (1) Solution (2) and Solution (4) Colourless or pale yellow liquid.
(3)
Spicy odour.
IV-390 Oak Bark 2023
IDENTIFICATION Detection Flame ionisation.

First identificatwn: B. Injection 0.2 µL.
Second identificatwn: A. Elution order Order indicated in the composition of the
A. Thin-layer chromatography (2.2.27). reference solution; record the retention times of these
Test solutwn Dissolve 1 mL of the substance to be examined substances.
in toluene R and dilute to 10 mL with the same solvent. System suitability Reference solution:
Reference solutwn Dissolve 20 µL of myristicine R in 10 mL - resolution: minimum 1.5 between the peaks due to
of toluene R. ~-pinene and sabinene.
Plate TLC silica gel plate R. Identification of components Using the retention times
determined from the chromatogram obtained with the
reference solution, locate the components of the reference
Application 10 µL as bands. solution in the chromatogram obtained with the test solution.
Development Over a path of 15 cm. Determine the percentage content of each of these
Drying In air. components. The percentages are within the following
Detection Spray with vanillin reagent R, heat at 100-105 °C ranges:
for 10 min and examine in daylight. - a-pinene: 15 per cent to 28 per cent;
Results The chromatogram obtained with the reference - f]-pinene: 13 per cent to 18 per cent;
solution shows in the upper third a pink or reddish-brown - sabinene: 14 per cent to 29 per cent;
zone (myristicine); the chromatogram obtained with the test - car-3-ene: 0.5 per cent to 2.0 per cent;
solution shows a series of zones of which 1 is similar in - limonene: 2.0 per cent to 7.0 per cent;
position and colour to the zone in the chromatogram - y-terpinene: 2.0 per cent to 6.0 per cent;
obtained with the reference solution; above this zone a - terpinen-4-ol: 2.0 per cent to 6.0 per cent;
brownish zone (safrole) and a violet zone (hydrocarbons) are - safrole: maximum 2.5 per cent;
present; below the myristicine zone, 5 blue zones of variable - myristicine: 5.0 per cent to 12.0 per cent.
intensity are present. STORAGE
B. Examine the chromatograms obtained in the test for Protected from heat.
chromatographic profile. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Results The principal peaks in the chromatogram obtained
with the test solution are similar in retention time to those in
TESTS Oak Bark
0.885 to 0.905. (Ph. Bur. monograph 1887)
Refractive index (2.2.6) PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
1.475 to 1.485.
DEFINITION
Optical rotation (2.2. 7) Cut and dried bark from the fresh young branches of Quercus
+ 8° to+ 18°. robur L., Q. petraea (Matt.) Liebl. and Q. pubescens Willd.
Chromatographic profile Content
Gas chromatography (2.2.28): use the normalisation Minimum 3.0 per cent of tannins, expressed as pyrogallol
procedure. (C6H603; Mr 126.1) (dried drug).
IDENTIFICATION
Reference solutwn Dissolve 15 µL of a-pinene R, 15 µL of
A. The bark occurs in channelled or quilled pieces, not more
/3-pinene R, 15 µL of sabinene R, 5 µL of car-3-ene R, 5 µL of
than 3 mm thick. The outer surface is light grey or greenish-
limonene R, 5 µL of ·y-terpinene R, 5 µL of terpinen-4-ol R,
grey, rather smooth, with occasional lenticels. The inner
5 µL of safrole R and 10 µL of myristicine R in 1 mL of
surface is dull brown or reddish-brown and has slightly raised
hexane R.
longitudinal striations about 0.5-1 mm wide. The fracture is
Column: splintery and fibrous.
- size: l =25-60 m, 0 =about 0.3 mm; brown or reddish-brown and fibrous. Examine under a
- statwnary phase: macrogol 20 000 R.
microscope using chloral hydrate solutwn R. The powder
Carrier gas helium for chromatography R. shows the following diagnostic characters: groups of thick-
Flow rate 1.5 mUmin. walled fibres surrounded by a moderately thickened
Split ratw 1:100. parenchymatous sheath containing prism crystals of calcium
Temperature: oxalate; fragments of cork composed of thin-walled tabular
cells filled with brownish or reddish contents; abundant
Time Temperature sclereids, isolated and in groups, some large with thick,
(min) CC) stratified walls and branching pits, others smaller and
Column 0 - 10 50 thinner-walled with simple pits, often with dense brown
10 - 75 50-+ 180 contents; fragments of parenchyma containing cluster crystals
75 - 130 180 of calcium oxalate; occasional fragments of sieve tissue, thin-
Injection port 200 - 220 walled, some showing sieve areas on the oblique end-walls.
Detector 240 - 250
2023 Olive Leaf IV-391
C. To 1 g of the powdered herbal drug (710) (2.9.12) 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
and filter. To 1 mL of this solution add 2 mL of a 10 g/L
solution of vanillin R in hydrochloric acid R. A red colour
develops.
TESTS
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Tannins (2.8.14)
---------------------~~
Olive Leaf
Preparation
Olive Leaf Dry Extract
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried leaf of Olea europaea L.
Content Figure 1878.-1. - Illustration for identification test B of powdered
Minimum 5.0 per cent of oleuropein (C 25 H 32 O 13 ; Mr 540.5)
herbal drug of olive leaf
(dried drug).
A. The leaf is simple, thick and coriaceous, lanceolate to Test solution To 1.0 g of the powdered herbal drug (35 5)
obovate, 30-50 mm long and 10-15 mm wide, with a (2. 9.12) add 10 mL of methanol R. Boil under a reflux
mucronate apex and tapering at the base to a short petiole; condenser for 15 min. Cool and filter.
the margins are entire and reflexed abaxially. The upper Reference solution Dissolve 10 mg of oleuropein R and 1 mg
surface is greyish-green, smooth and shiny, the lower surface of rutoside trihydrate R in 1 mL of methanol R.
paler and pubescent, particularly along the midrib and main Plate TLC silica gel plate R.
lateral veins.
B. Microscopic examination (2.8.23). The powder is (1.5:15:85 VIVIV).
characters (Figure 1878.-1.): numerous broken sclereids, very Development Over a path of 10 cm.
thick walled and mostly fibre-like with blunt or, occasionally, Drying In air.
forked ends, isolated [G, HJ or associated with the Detection Spray with vanillin reagent R and heat at
parenchyma of the mesophyll [D, L]; numerous, very large, 100-105 °C for 5 min; examine in daylight.
peltate covering trichomes (surface view [A], side view [BJ), Results See below the sequence of zones present in the
with a central unicellular stalk from which radiate some chromatograms obtained with the reference solution and the
10-30 thin-walled cells that become free from the adjoining test solution. Furthermore, other faint zones may be present
cells at the margin of the shield, giving an uneven, jagged in the chromatogram obtained with the test solution.
appearance; fragments of the upper epidermis (surface view
[Kl), with small, thick-walled polygonal cells, accompanied Top of the plate
by underlying palisade parenchyma [Ka] and fragments of
A dark violet-blue zone (solvent
sclereids [Kb]; fragments of the lower epidermis [J], with
front)
small rigid-walled cells, anomocytic stomata (2.8.3) Ua] and A dark violet-blue zone
scars of peltate covering trichomes Ubl; fragments of the
-- --
lamina (transverse section [F]) showing a thick cuticle [Fa],
palisade parenchyma composed of 3 layers of cells [Fb] and Oleuropein: a brownish-green zone A brownish-green zone (oleuropein)
small-celled spongy parenchyma [Fe]; a few isolated
-- --
fragments of palisade parenchyma [C] or of spongy
parenchyma [E]. Rutoside: a brownish-yellow zone

IV-392 Olive Leaf Preparations 2023
TESTS Olive Leaf Dry Extract

Maximum 10.0 per cent, determined on 1.000 g of the (Ph. Bur. monograph 2313)
105 °C for 2 h.
Total ash (2.4.16) DEFINITION
Maximum 9.0 per cent. Dry extract produced from Olive leaf (1878).
ASSAY Content
Minimum 16.0 per cent of oleuropein (C 25H 32 O 13;
Mr 540.5) (dried extract).
Test solution In a flask, place 1.000 g of the powdered
herbal drug (355) (2.9.12) and add 50 mL of methanol R. PRODUCTION
Heat in a water-bath at 60 °C for 30 min with shaking. The extract is produced from the herbal drug by a suitable
Allow to cool and filter into a 100 mL volumetric flask. procedure using ethanol (65-96 per cent V/V).
Rinse the flask and the filter with methanol R and dilute to CHARACTERS
100.0 mL with the same solvent. Dilute 2.5 mL of the Appearance
solution to 25.0 mL with water R. Greenish-brown or brown, amorphous powder.
Reference solution (a) Dissolve 10.0 mg of oleuropein CRS in
IDENTIFICATION
10.0 mL of methanol Rand dilute to 25.0 mL with water R
(solution A). Dilute 1.0 mL of solution A to 10.0 mL with
water R. Test solution To 0.25 g of the extract to be examined add
10 mL of methanol R. Sonicate for 15 min and filter.
Reference solution (b) Dissolve 4 mg of rutoside trihydrate R
in 10 mL of solution A. Reference solution Dissolve 5 mg of oleuropein R and 1 mg of
rutoside trihydrate R in 1 mL of methanol R.
Column:
- size: l = 0.15 m, 0 = 4.6 mm; Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
- stationary phase: end-capped octadecylsilyl silica gel for plate R (2-10 µm)].
chromatography R (5 µm); Mobile phase water R, anhydrous formic acid R, ethyl acetate R
- temperature: 25 °C. (7:13:80 V/V/V).
Mobile phase trifluoroacetic acid R, methanol Rl, water for Application 10 µL [or 2 µL] as bands of 10 mm [or 8 mm].
chromawgraphy R (0.1:40:60 V/V/V). Development Over a path of 10 cm [or 6 cm].
Flow rate 1 mIJmin. Drying In air.
Detection Spectrophotometer at 233 nm. Detection Spray with anisaldehyde solution R and heat at
Injection 20 µL. 100-105 °C for 5 min; examine in daylight.
Run time Twice the retention time of oleuropein. Results See below the sequence of zones present in the
Relative retention With reference to oleuropein (retention chromatograms obtained with the reference solution and the
time= about 11 min): rutoside = about 0.7. test solution. Furthermore, other faint zones may be present
rutoside and oleuropein. Top of the plate
Calculate the percentage content of oleuropein using the A dark violet-blue zone
-- --
Oleuropein: a brownish-green zone A brownish-green zone (oleuropein)
-- --
area of the peak due to oleuropein in the chromatogram
obtained with the test solution; Rutoside: a yellow zone
area of the peak due to oleuropein in the chromatogram
obtained with reference solution (a); Reference solution Test solution
solution, in grams;
mass of oleuropein CRS used to prepare reference solution (a), in TESTS
grams;
p percentage content of oleuropein in oleuropein CRS.
- - - - - - - - - - - - - - - - - - - - - PhEur ASSAY
Liquid chromatography (2.2.29). Prepare the solutions
immediately before use.
50 mL of methanol R. Sonicate for 15 min and filter into a
100 mL volumetric flask. Rinse the flask and the filter with
2 mL of methanol Rand dilute to 100.0 mL with water R.
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.
Reference solution (b) Dissolve 4 mg of rutoside trihydrate R
in 10 mL of reference solution (a).
2023 Opium IV-393
Column: A. Microscopic examination (2.8.23). The powder (500)

- size: l = 0.15 m, 0 = 4.6 mm; (2. 9.12) is light brown. Examine under a microscope using
- stationary phase: end-capped octadecylsilyl silica gel for chloral hydrate solution R. Before heating, the powder shows
chromatography R (5 µm); the following diagnostic characters (Figure 0777.-1): granules
- temperature: 25 °C. of latex agglomerated in irregular masses [A] and light brown
Mobile phase trifiuoroacetic acid R, methanol R, water R elongated filaments. After heating, some fragments of
(1:400:600 V/V/V). vessels U, K] and rather elongated, refringent crystals [F] are
also visible, as well as a smaller number of round pollen
Flow rate 1 mIJmin.
grains with 3 pores and a very finely pitted exine [E] and
fragments of elongated fibres [DJ. Fragments of epicarp
Injection 20 µL. (surface view [B, C, G], transverse section [HJ), consisting of
Run time Twice the retention time of oleuropein. polygonal cells with thick walls defining a stellate lumen, and
Relative retention With reference to oleuropein (retention sometimes anomocytic stomata (2.8.3) [Ba] may also be
time= about 11 min): rutoside = about 0.7. present. Some elements of various origin introduced during
handling of the latex may also be present in small quantities
(fragments of covering trichomes and starch granules).
rutoside and oleuropein.
Calculate the percentage content of oleuropein using the
AI area of the peak due to oleuropein in the chromatogram

A2 area of the peak due to oleuropein in the chromatogram
solution} in grams;
m2 mass of okuropein CRS used to prepare reference solution (a), in
grams;
p percentage content of oleuropein in okuropein CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Opium
(Raw Opium, Ph. Bur. monograph 0777)
Figure 0777 .-1. - Illustration for identification test A of powdered
Preparations raw opium
Opium Tincture
Prepared Opium B. Thin-layer chromatography (2.2.27).
Standardised Opium Dry Extract Test solution Triturate 0.10 g of the powdered substance to
be 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
Raw opium is intended only as starting material for the rinsings to the same 25 mL conical flask. Heat in a water-
manufacture of galenical preparations. It is not dispensed as such. bath at 50-60 °C, while stirring, for 30 min. Cool, filter,
DEFINITION wash the filter with ethanol (70 per cent VIV,) R and dilute the
combined filtrates to 10 mL with the same solvent.
Air-dried latex obtained by incision from the unripe capsules
of Papaver somniferum L. Reference solutwn Dissolve 5 mg of morphine hydrochloride R
in a solution prepared as follows and dilute to 5 mL with the
Content
same solution: dissolve 2 mg of papaverine hydrochloride R,
- morphine (C 17H 19N03 ; 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 (C 18H 21 N0 3 ; Mr 299.4): minimum 2.0 per cent
25 mL with the same solvent.
(dried drug).
CHARACTERS plate R (2-10 µm)].
Appearance Mobile phase concentrated ammonia R, ethanol (96 per cent) R,
Blackish-brown masses of various sizes, which tend to be soft acetone R, toluene R (2:6:40:40 V/VIVIV); use a freshly
and shiny and, after drying, become hard and brittle.
prepared mixture.
IDENTIFICATION Applicatwn 20 µL [or 6 µL] as bands of 10 mm [or 8 mm].
Strip off any covering, cut the substance to be examined into thin Development Over a path of 15 cm [or 8 cm].
slices, dry at about 60 °C for 48 h, if necessary, and reduce to a
powder (500) (2.9.12).
IV-394 Opium 2023
Detection Allow to cool and treat with potassium Column:

iodobismuthate solution R2 and then with a 4 g/L solution of =
- size: l 0.25 m, 0 4.0 mm;=
sulfuric acid R; examine in daylight. - stationary phase: end-capped octylsilyl silica gel for
Results See below the sequence of zones present in the chromatography R (5 µm).
chromatograms obtained with the reference solution and the Mobile phase Dissolve 1.0 g of sodium heptanesulfonate
test solution. A dark red zone (thebaine) situated between monohydrate R in 420 mL of water R, adjust to pH 3.2 with a
the zone due to codeine and the zone due to papaverine may 4.9 g/L solution of phosphoric acid Rand add 180 mL of
be present in the chromatogram obtained with the test acetonitrile R.
solution. Furthermore, other faint zones may be present in Flow rate 1.5 mIJmin.
Top of the plate
solutions (a) and (c).
Noscapine: an orange-red or red An orange-red or red zone Run time Twice the retention time of thebaine.
zone (noscapine)
-- -- - resolution: minimum 2.5 between the peaks due to
morphine and codeine.
Papaverine: an orange-red or red An orange-red or red zone
zone (papaverine) Calculate the percentage content of the relevant alkaloid
--- --
Codeine: an orange-red or red zone An orange-red or red zone (codeine) Ai xm 2 xFxp

A2xm1
Morphine: an orange-red or red zone An orange-red or red zone
(morphine)
A, area of the peak due to the relevant alkaloid in the
Reference solution Test solution chromatogram obtained with the test solution;
A2 area of the peak due to the relevant alkaloid in the
chromatogram obtained with reference solution (a) for thebaine
or reference solution (c) for morphine and codeine;
C. To 1.0 g of the powdered substance (500) (2.9.12) add
m1 mass of the substance to be examined used to prepare the test
5 mL of water R, shake for 5 min and filter. To the filtrate solution, in grams;
add 0.25 mL of ferric chloride solution R2. A red colour m2 mass of the relevant alkaloid used to prepare reference
develops which does not disappear upon the addition of solution (a) for thebaine, reference solution (b) for morphine or
0.5 mL of dilute hydrochloric acid R. reference solution (c) for codeine, in grams;
F 6.250 for the determination of thebaine;
TESTS p percentage content of the relevant alkaloid in the
corresponding CRS.
Thehaine
Limit:
Test solution Suspend 1.000 g of the substance to be - thebaine: maximum 3.0 per cent (dried drug).
examined, cut into thin slices, in 50 mL of ethanol
(50 per cent V/V) R, mix using sonication for 1 h, allow to Loss on drying (2.2.32)
cool and dilute to 100.0 mL with the same solvent. Allow to Maximum 15.0 per cent, determined on 1.000 g of the
stand. To 10.0 mL of the supernatant add 5 mL of substance to be examined, cut into thin slices, by drying in
ammonium chloride buffer solution pH 9.5 R, dilute to 25.0 mL an oven at 105 °C for 4 h.
with water R and mix. Transfer 20.0 mL of this solution to a Total ash (2.4.16)
chromatography column about 0.15 m long and about Maximum 6.0 per cent.
30 mm in internal diameter containing 15 g of kieselguhr for ASSAY
chromatography R. Allow to stand for 15 min. Elute with
2 quantities, each of 40 mL, of a mixture of 15 volumes of
thebaine with the following modifications.
2-propanol R and 85 volumes of methylene chloride R.
Evaporate the combined eluates to dryness in vacua at 40 °C. Injection Test solution and reference solution (c).
Transfer the residue to a volumetric flask using the mobile System suitability Reference solution (c):
phase and dilute to 25.0 mL with the mobile phase. - repeatability: maximum relative standard deviation of
Reference solution (a). Dissolve 5.0 mg of thebaine CRS in 1.0 per cent for the area of the peak due to morphine
the mobile phase and dilute to 50.0 mL with the mobile after 6 injections.
phase. Calculate the percentage content of morphine and the
Reference solution (b) Dissolve 12.0 mg of morphine percentage content of codeine using the expression given in
hydrochloride trihydrate CRS in the mobile phase and dilute to the test for thebaine, with F = 10.417 for morphine and
15.0 mL with the mobile phase. F = 3.125 for codeine.
Reference solution (c) Dissolve 10.0 mg of codeine CRS in the To obtain the p value to be used for the calculation of the
mobile phase and dilute to 50.0 mL with the mobile phase. morphine content, multiply the percentage content of
To 10.0 mL of the solution add 10.0 mL ofreference morphine hydrochloride in morphine hydrochloride
solution (b). trihydrate CRS by 0.887.
Precolumn:
- size: l = 4 mm, 0 = 4.0 mm;
(5 µm).
2023 Opium IV-395
25 mL conical flask. Heat in a water-bath at 50-60 °C, while

Prepared Opium stirring, for 30 min. Cool, filter, wash the filter with ethanol
(Ph. Bur. monograph 1840) (70 per cent V/V) R and dilute the combined filtrates to
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Reference solutwn Dissolve 5 mg of morphine hydrochloride R
DEFINITION in a solution prepared as follows and dilute to 5 mL with the
Raw opium (0777) powdered (180) (2.9.12) and dried at a same solution: dissolve 2 mg of papaverine hydrochloride R,
temperature not exceeding 70 °C, with a morphine content 12 mg of codeine phosphate Rand 12 mg of noscapine
adjusted, if necessary, by adding a suitable excipient or raw hydrochloride R in ethanol (70 per cent V/V) R and dilute to
opium powder with a lower alkaloidal content. 25 mL with the same solvent.
Content Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
- morphine (C 17H 19 NO 3 ; Mr 285.3): 9.5 per cent to plate R (2-10 µm)].
10.5 per cent (dried preparation); Mobile phase concentrated ammonia R, ethanol (96 per cent) R,
- codeine (C 18H 21 NO 3; 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 Applicatwn 20 µL [or 6 µL] 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
A. Microscopic examination (2.8.23). The powder (500) iodobismuthate solutwn R2 and then with a 4 g/L solution of
(2. 9.12) is light brown. Examine under a microscope using sulfuric acid R; examine in daylight.
chloral hydrate solutwn 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
U, 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 [DJ. Fragments of epicarp (surface view
[B, C, G], transverse section [HJ), consisting of polygonal Top of the plate
cells with thick walls defining a stellate lumen, and
sometimes anomocytic stomata (2.8.3) [Ba] may also be Noscapine: an orange-red or red An orange-red or red zone
zone (noscapine)
present. Some elements of various origin introduced during
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 zone An orange-red or red zone (codeine)

(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.
G TESTS
Thebaine
I ~ffln!)
K
25µm
1---1
Figure 1840.-1. - Illustratwnfor identificatwn test A of prepared

opium
Test solutwn 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 solutwn
pH 9.5 R, dilute to 25.0 mL with water Rand mix. Transfer
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
Test solutwn Triturate 0.10 g of the preparation to be containing 15 g of kieselguhr for chromatography R. Allow to
examined with 5 mL of ethanol (70 per cent V/V) R. Transfer stand for 15 min. Elute with 2 quantities, each of 40 mL, of
to a 25 mL conical flask. Rinse with 3 mL of ethanol a mixture of 15 volumes of 2-propanol Rand 85 volumes of
(70 per cent VIV) R and transfer the rinsings to the same methylene chloride R. Evaporate the combined eluates to
IV-396 Opium Preparations 2023
dryness in vacua at 40 °C. Transfer the residue to a System suitability Reference solution (c):
volumetric flask using the mobile phase and dilute to - repeatability: maximum relative standard deviation of
25.0 mL with the mobile phase. 1.0 per cent for the area of the peak due to morphine
Reference solution (a) Dissolve 5.0 mg of thebaine CRS in the after 6 injections.
mobile phase and dilute to 50.0 mL with the mobile phase. Calculate the percentage content of morphine and the
Reference solution (b) Dissolve 12.0 mg of morphine percentage content of codeine using the expression given in
hydrochloride trihydrate CRS in the mobile phase and dilute to the test for thebaine, with F = 10.417 for morphine and
15.0 mL with the mobile phase. F = 3.125 for codeine.
Reference solution (c) Dissolve 10.0 mg of codeine CRS in the To obtain the p value to be used for the calculation of the
To 10.0 mL of the solution add 10.0 mL of reference morphine hydrochloride in morphine hydrochloride
solution (b). trihydrate CRS by 0.887.
Precolumn: - - - - - - - - - - - - - - - - - - - - - - Ph Eur
- size: l = 4 mm, 0 = 4.0 mm;
(5 µm).
Column: Standardised Opium Dry Extract
= =
- size: l 0.25 m, 0 4.0 mm;
- stationary phase: end-capped octylsilyl silica gel for (Ph. Bur. monograph 1839)
chromatography R (5 µm). Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
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 dry extract produced from Raw opium (0777).
4.9 g/L solution of phosphoric acid Rand add 180 mL of
acetonitrile R. Content
Flow rate 1.5 mIJmin. - morphine (C 17H 19N0 3 ; Mr 285.3): 19.0 per cent to
21.0 per cent (dried extract);
Detection Spectrophotometer at 280 nm. - codeine (C 18H 21 N03 ; Mr 299.4): minimum 2.0 per cent
Injection 20 µL of the test solution and reference (dried extract).
solutions (a) and (c). Content adjusted if necessary by adding a suitable excipient
Run time Twice the retention time of thebaine. (e.g. lactose, dextrin).
System suitability Reference solution (c): PRODUCTION
The extract is produced from the drug by a suitable
procedure using water.
Calculate the percentage content of the relevant alkaloid
using the following expression: CHARACTERS
Appearance
Brown, amorphous powder.
IDENTIFICATION
AI area of the peak due to the relevant alkaloid in the
chromatogram obtained with the test solution; Test solution Triturate 50 mg of the extract to be examined
A2 area of the peak due to the relevant alkaloid in the with 5 mL of ethanol (70 per cent V/V) R. Transfer to a
chromatogram obtained with reference solution (a) for thebaine 25 mL conical flask. Rinse with 3 mL of ethanol
m1 mass of the preparation to be examined used to prepare the test
(70 per cent VIV) R and transfer the rinsings to the same
solution, in grams; 25 mL conical flask. Heat in a water-bath at 50-60 °C, while
m2 mass of the relevant alkaloid used to prepare reference stirring, for 30 min. Cool, filter, wash the filter with ethanol
solution (a) for thebaine, reference solution (b) for morphlne or (70 per cent V/V) R and dilute the combined filtrates to
reference solution (c) for codeine, in grams;
F 6.250 for the determination of thebaine;
p percentage content of the relevant alkaloid in the Reference solutwn Dissolve 5 mg of morphine hydrochloride R
corresponding CRS. in a solution prepared as follows and dilute to 5 mL with the
same solution: dissolve 2 mg of papaverine hydrochloride R,
Limit: 12 mg of codeine phosphate R and 12 mg of noscapine
- thebaine: maximum 3.0 per cent (dried preparation). hydrochloride R in ethanol (70 per cent V/V) R and dilute to
Loss on drying (2.2.32) 25 mL with the same solvent.
Maximum 8.0 per cent, determined on 1.000 g of the Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
preparation to be examined by drying in an oven at 105 °C plate R (2-10 µm)].
for 4 h. Mobile phase concentrated ammonia R, ethanol (96 per cent) R,
Total ash (2.4.16) acetone R, toluene R (2:6:40:40 V/V/V/V); use a freshly
Maximum 6.0 per cent. prepared mixture.
ASSAY Application 20 µL [or 6 µL] as bands of 10 mm [or 8 mm].
Liquid chromatography (2.2.29) as described in the test for Development Over a path of 15 cm [or 8 cm].
thebaine with the following modifications. Drying At 100-105 °C for 15 min.
Injection Test solution and reference solution (c).
2023 Opium Preparations IV-397
Detection Allow to cool and treat with potassium - stationary phase: end-capped octylsilyl silica gel for
iodobismuthate solutwn R2 and then with a 4 g/L solution of chromatography R (5 µm).
sulfuric acid R; examine in daylight. Mobile phase Dissolve 1.0 g of sodium heptanesulfonate
Results See below the sequence of zones present in the monohydrate R in 420 mL of water for chromatography R,
chromatograms obtained with the reference solution and the adjust to pH 3.2 with a 4. 9 g/L solution of phosphoric acid R
test solution. A dark red zone (thebaine) situated between and add 180 mL of acetonitrile R.
the zone due to codeine and the zone due to papaverine may Flow rate 1.5 mUmin.
be present in the chromatogram obtained with the test Detection Spectrophotometer at 280 nm.
solution. Furthermore, other faint zones may be present in
the chromatogram obtained with the test solution. Injection 20 µL of the test solution and reference
Top of the plate
Run time Twice the retention time of thebaine.
Noscapine: an orange-red or red An orange-red or red zone
zone (noscapine)
-- -- Calculate the percentage content of the relevant alkaloid
Papaverine: an orange-red or red An orange-red or red zone using the following expression:
zone (papaverine)
A1 xm 2 xFxp
- - -- A 2 xm1
Codeine: an orange-red or red zone An orange-red or red zone (codeine) AI area of the peak due to the relevant alkaloid in the
Morphine: an orange-red or red zone An orange-red or red zone A2 area of the peak due to the relevant alkaloid in the
(morphine) chromatogram obtained with reference solution (a) for thebaine
solution, in grams;
mass of the relevant alkaloid used to prepare reference
B. To 0.5 g of the extract to be examined add 5 mL of solution (a) for thebaine, reference solution (b) for morphine or
water R, shake for 5 min and filter. To the filtrate add reference solution (c) for codeine, in grams;
0.25 mL of ferric chloride solutwn R2. A red colour develops F 6.250 for the determination of thebaine;
which does not disappear upon the addition of 0.5 mL of p percentage content of the relevant alkaloid in the
corresponding CRS.
dilute hydrochloric acid R.
TESTS Limit:
Thebaine - thebaine: maximum 6.0 per cent (dried extract).
Liquid chromatography (2.2.29). Loss on drying (2.2.32)
Test solutwn Suspend 0.500 g of the extract to be examined Maximum 5.0 per cent, determined on 1.000 g of the extract
in 50 mL of ethanol (50 per cent V/V) R, mix using sonication to be examined by drying in an oven at 105 °C for 4 h.
for 1 h, allow to cool and dilute to 100.0 mL with the same
ASSAY
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
25.0 mL with water R and mix. Transfer 20.0 mL of this thebaine with the following modifications.
solution to a chromatography column about 0.15 m long and Injection Test solution and reference solution (c).
about 30 mm in internal diameter containing 15 g of System suitability Reference solution (c):
kieselguhr for chromatography R. Allow to stand for 15 min. - repeatability: maximum relative standard deviation of
Elute with 2 quantities, each of 40 mL, of a mixture of 1.0 per cent for the area of the peak due to morphine
15 volumes of 2-propanol R and 85 volumes of methylene after 6 injections.
chloride R. Evaporate the combined eluates to dryness in Calculate the percentage content of morphine and the
vacuo at 40 °C. Transfer the residue to a volumetric flask percentage content of codeine using the expression given in
using the mobile phase and dilute to 25.0 mL with the the test for thebaine, with F = 10.417 for morphine and
mobile phase. F = 3.125 for codeine.
Reference solutwn (a) Dissolve 5.0 mg of thebaine CRS in the To obtain the p value to be used for the calculation of the
Reference solution (b) Dissolve 12.0 mg of morphine morphine hydrochloride in morphine hydrochloride
hydrochloride trihydrate CRS in the mobile phase and dilute to trihydrate CRS by 0.887.
15.0 mL with the mobile phase. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Reference solutwn (c) Dissolve 10.0 mg of codeine CRS in the
To 10.0 mL of the solution add 10.0 mL of reference
solution (b).
Precolumn:
- size: l = 4 mm, 0 = 4.0 mm;
- stationary phase: octylsilyl silica gel for chromawgraphy R
(5 µm).
Column:
- size: l = 0.25 m, 0 = 4.0 mm;
IV-398 Opium Preparations 2023
TESTS
Standardised Opium Tincture Ethanol (2. 9.10)
(Ph. Bur. monograph 1841) 31 per cent V/V to 34 per cent V/V.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Thebaine
DEFINITION Test solution Dilute 2.000 g of the tincture to be examined
Standardised tincture produced from Raw opi,um (0777). to 25.0 mL with ethanol (50 per cent V/V) R. To 10.0 mL of
Content the solution add 5 mL of ammonium chloride buffer solution
- morphine (C 17H 19N03 ; Mr 285.3): 0.95 per cent to pH 9.5 R, dilute to 25.0 mL with water Rand mix. Transfer
1.05 per cent; 20.0 mL of this solution to a chromatography column about
- codeine (C 18H 21N0 3; Mr 299.4): minimum 0.1 per cent. 0.15 m long and about 30 mm in internal diameter
containing 15 g of kieselguhr for chromatography R. Allow to
PRODUCTION
stand for 15 min. Elute with 2 quantities, each of 40 mL, of
The tincture is produced from the drug by an appropriate
a mixture of 15 volumes of 2-propanol R and 85 volumes of
procedure using equal volumes of ethanol (70 per cent V/V)
methylene chloride R. Evaporate the combinated eluates to
and water.
dryness in vacua at 40 °C. Transfer the residue to a
CHARACTERS volumetric flask using the mobile phase and dilute to
Appearance 25.0 mL with the mobile phase.
Reddish-brown liquid. Reference solution (a) Dissolve 5.0 mg of thebaine CRS in the
IDENTIFICATION mobile phase and dilute to 50.0 mL with the mobile phase.
Thin-layer chromatography (2.2.27). Reference solution (b) Dissolve 12.0 mg of morphine
Test solution Dilute 1.0 mL of the tincture to be examined hydrochloride trihydrate CRS in the mobile phase and dilute to
to 10 mL with ethanol (70 per cent V/V) R. 15.0 mL with the mobile phase.
Reference solution Dissolve 5 mg of morphine hydrochloride R Reference solution (c) Dissolve 10.0 mg of codeine CRS in the
in a solution prepared as follows and dilute to 5 mL with the mobile phase and dilute to 50.0 mL with the mobile phase.
same solution: dissolve 2 mg of papaverine hydrochloride R, To 10.0 mL of the solution add 10.0 mL ofreference
12 mg of codeine phosphate Rand 12 mg of noscapi,ne solution (b).
hydrochloride R in ethanol (70 per cent V/V) R and dilute to Precolumn:
25 mL with the same solvent. - size: l = 4 mm, 0 = 4.0 mm;
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel - stationary phase: octylsilyl silica gel for chromatography R
plate R (2-10 µm)]. (5 µm).
Mobile phase concentrated ammonia R, ethanol (96 per cent) R, Column:
acetone R, toluene R (2:6:40:40 VIVIVIV); use a freshly = =
- size: l 0.25 m, 0 4.0 mm;
prepared mixture. - stationary phase: end-capped octylsilyl silica gel for
Application 20 µL [or 6 µL] as bands of 10 mm [or 8 mm]. chromatography R (5 µm).
Mobile phase Dissolve 1.0 g of sodium heptanesulfonate
monohydrate R in 420 mL of water R, adjust to pH 3.2 with a
Drying At 100-105 °C for 15 min. 4.9 g/L solution of phosphoric acid Rand add 180 mL of
Detection Allow to cool and treat with potassium acetonitrile R.
iodobismuthate solution R2 and then with a 4 g/L solution of Flow rate 1.5 mL/min.
suljuric acid R; examine in daylight.
test solution. A dark red zone (thebaine) situated between
the zone due to codeine and the zone due to papaverine may Run time Twice the retention time of thebaine.
be present in the chromatogram obtained with the test System suitability Reference solution (c):
solution. Furthermore, other faint zones may be present in - resolution: minimum 2.5 between the peaks due to
the chromatogram obtained with the test solution. morphine and codeine.
Calculate the percentage content of the relevant alkaloid
Top of the plate using the following expression:
Noscapine: an orange-red or red An orange-red or red zone
zone (noscapine)
- - - -
A1 area of the peak due to the relevant alkaloid in the
Papaverine: an orange-red or red An orange-red or red zone chromatogram obtained with the test solution;
zone (papaverine) A2 area of the peak due to the relevant alkaloid in the
chromatogram obtained with reference solution (a) for thebaine
-- -- or reference solution (c) for morphine and codeine;
m1 mass of the tincture to be examined used to prepare the test
Codeine: an orange-red or red zone An orange-red or red zone (codeine) solution, in grams;
m2 mass of the relevant alkaloid used to prepare reference
solution (a) for thebaine, reference solution (b) for morphine or
(morphine)
reference solution (c) for codeine, in grams;
Reference solution Test solution F 1.563 for the determination of thebaine;
p percentage content of the relevant alkaloid in the
corresponding CRS.
2023 Opium Preparations IV-399
Limit: calcium hydroxide solution. To the combined filtrate and

- thebaine: maximum 0.3 per cent. washings add 0.1 g of ammonium sulfate, extract with two
Dry residue (2.8.16) 10 mL quantities of ethanol-free chloroform, wash the
Minimum 4.0 per cent, determined on 3.00 g. combined extracts with 10 mL of water and discard the
chloroform solution. To the combined alkaline liquid and
ASSAY aqueous washings add 10 mL of lM hydrochloric acid, heat on
Liquid chromatography (2.2.29) as described in the test for a water bath to remove any chloroform, cool and dilute to
thebaine with the following modifications. 100 mL with water. To 10 mL of this solution add 10 mL of
Injection Test solution and reference solution (c). O.lM hydrochloric acid and 8 mL of a freshly prepared
System suitability Reference solution (c): 1.0% w/v solution of sodium nitrite, allow to stand for
- repeatability: maximum relative standard deviation of 15 minutes, add 12 mL of SM ammonia, dilute to 50 mL
1.0 per cent for the area of the peak due to morphine with water and measure the absorbance of a 4-cm layer of the
after 6 injections. resulting solution at the maximum at 442 nm,
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 ofC 17 H 19N0 3 taking 124 as the value of
A(l %, 1 cm) at the maximum at 442 nm.
To obtain the p value to be used for the calculation of the
morphine content, multiply the percentage content of
morphine hydrochloride in morphine hydrochloride
trihydrate CRS by 0.887. Concentrated Camphorated Opium
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Tincture
DEFINITION
Opium Tincture Opium Tincture 400 mL

Benzoic Acid 40 g
DEFINITION
Racemic Camphor 24 g
Anise Oil or Star Anise Oil 24mL
Opium, sliced 200 g Ethanol (96 per cent) 400 mL
Ethanol (90 per cent) A sufficient quantity Water Sufficient to produce 1000 mL
Purified Water A sufficient quantity
Extemporaneous preparation The following directions apply.
The following directions apply. Dissolve the Benzoic Acid, the Racemic Camphor and the
Pour 500 mL of boiling Purified Water on to the Opium and Anise Oil or Star Anise Oil in the Ethanol (96 per cent), add
allow to stand for 6 hours; add 500 mL of Ethanol the Opium Tincture and sufficient Water to produce
(90 per cent), mix thoroughly and allow to stand in a 1000 mL, mix and filter if necessary.
covered vessel for 24 hours; strain, press the mare, mix the The tincture complies with the requirements for Tinctures stated
liquids and allow to stand for not less than 24 hours; filter. under Extracts and with the following requirements.
Determine the concentration of morphine, calculated as Content of anhydrous morphine, C 17H 1gN'0 3
anhydrous morphine, in the tincture so prepared by the 0.36 to 0.44% w/v.
Assay. To the remainder of the liquid add sufficient of a
mixture of equal volumes of Ethanol (90 per cent) and TESTS
Purified Water to produce an Opium Tincture containing Ethanol content
1% w/v of anhydrous morphine. 54 to 59% v/v, Appendix VIII F, Method III.
The tincture complies with the requirements for Tinctures stated Relative density
under Extracts and with the following requirements. 0.912 to 0.930, Appendix VG.
Content of anhydrous morphine, C 17H1gN'0 3 ASSAY
0.925 to 1.075% w/v. Dilute 10 mL to 100 mL with ethanol (50%) and carry out
the Assay described under Camphorated Opium Tincture
TESTS
using 10 mL of the diluted solution.
Ethanol content
Relative density
0.898 to 0.969, Appendix VG. Camphorated Opium Tincture
ASSAY DEFINITION
Dilute 5 mL to 100 mL with ethanol (45%). To 10 mL of
Opium Tincture 50mL
the resulting solution add 5 mL of water and 1 mL of
Benzoic Acid 5g
5M ammonia and extract with 30 mL of a mixture of equal Racemic Camphor 3g
volumes of ethanol (96%) and chloroform and then with two Anise Oil or Star Anise Oil 3mL
22.5 mL quantities of a mixture of 2 volumes of chloroform Ethanol (60 per cent) Sufficient to produce 1000 mL
and 1 volume of ethanol (96%), washing each extract with the
same 20 mL of a mixture of equal volumes of ethanol (96%) Extemporaneous preparation
and water. Evaporate the combined extracts just to dryness, The following directions apply.
extract the residue with two 5 mL quantities of calcium Dissolve the Benzoic Acid, the Racemic Camphor and the
hydroxide solution, filter and wash the filter with 10 mL of Anise Oil or Star Anise Oil in 900 mL of Ethanol
IV-400 Bitter-Orange Flower 2023
(60 per cent), add the Opium Tincture and sufficient groups of 4 or 5; the ovary is superior, brownish-black and
Ethanol (60 per cent) to produce 1000 mL and mix. Filter, if spherical, consists of 8-10 multi-ovular loculi and is
necessary. surrounded at the base by an annular granular hypogynous
The tincture complies with the requirements for Tinctures stated disc; the thick, cylindrical style ends in a capitate stigma.
under Extracts and with the following requirements. B. Microscopic examination (2.8.23). The powder is
Content of anhydrous morphine, C 17H 1~O 3 brownish-yellow. Examine under a microscope using chloral
0.045 to 0.055% w/v. hydrate solution R. The powder shows the following diagnostic
characters (Figure 1810.-1): very numerous spherical pollen
TESTS grains, with a finely pitted exine and 3-5 germinal pores [H,
Ethanol content K]; fragments of the epidermis of the sepals (surface view
56 to 60% v/v, Appendix VIII F, Method III. [D], transverse section [A, C]) accompanied by underlying
Relative density mesophyll [B], some cells of which contain prisms of calcium
0.90 to 0.92, Appendix VG. oxalate [Aa, Ba, Db], unicellular covering trichomes [Ca]
and numerous anomocytic stomata (2.8.3) [Da]; fragments of
ASSAY
the epidermis of the petals (surface view [F, G, TI), with a
To 10 mL add 5 mL of water and 1 mL of 5M ammonia and
distinctly striated cuticle; fragments of large schizolysigenous
extract with 30 mL of a mixture of equal volumes of ethanol
oil glands in transverse section [E], which measure up to
(96%) and chloroform and then with two 22.5 mL quantities
100 µm in diameter. Examine under a microscope using a
of a mixture of 2 volumes of chlorofonn and 1 volume of
20 g/L solution of potassium hydroxide R. The mounting
ethanol (96%), washing each extract with the same 20 mL of
medium becomes yellow because of the presence of
a mixture of equal volumes of ethanol (96%) and water.
hesperidin in the drug.
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
combined 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 0.1 M 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 C 17H 19N03 taking
124 as the value of A(l %, 1 cm) at the maximum at
442 nm.
Bitter-Orange Flower
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. - Illustratwn for identification test B of powdered
naringin (C 27 H 32 0 14; Mr 580.5) (dried drug). herbal drug of bitter-orange flower
IDENTIFICATION C. Examine the chromatograms obtained in the test for

A. The flower buds are white or yellowish-white and may sweet-orange flower.
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
attached to the yellowish-green peduncle, which is about
5-10 mm long. The flower buds contain at least 20 stamens
with yellow anthers and with filaments fused at the base into
2023 Orange Oil IV-401
Top of the plate After 10 min, measure the absorbance (2.2.25) of the test
solution at 530 nm.
A weak yellow fluorescent zone
A weak yellow fluorescent zone expressed as naringin, using the following expression:
Hesperidin: a greenish-yellow A greenish-yellow fluorescent zone
fluorescent zone (hesperidin) Ax 9.62
m
Naringin: a yellow fluorescent zone A yellow fluorescent zone (naringin)
A red fluorescent zone i.e. taking the specific absorbance of the reaction product of
(neoeriocitrin) naringin to be 52.
A yellow fluorescent zone (diosmin
and neodiosmin) A absorbance at 530 nm;
- - - - - - - - - - - - - - - - - - - - - - PhEur
TESTS
Sweet-orange flower
Orange Oil
(2. 9.12) add 5 mL of methanol R. Heat with stirring at 40 °C DEFINITION
for 10 min. Filter. Orange Oil is obtained by mechanical means from the fresh
Reference solution Dissolve 3.0 mg of naringin R and 3.0 mg peel of the sweet orange, Citrus sinensis (L.) Osbeck.
of hesperidin R in 10 mL of methanol R. CHARACTERISTICS
Plate TLC silica gel plate R. A yellow to yellowish brown liquid, visibly free from water;
Mobile phase water R, anhydrous formic acid R, ethyl acetate R odour, that of orange.
(10:15:75 V/V/V). TESTS
Application 10 µL as bands. Optical rotation
Development Over a path of 10 cm. +94° to +99°, Appendix VF. On distillation, the first 10%
Drying In air; heat in an oven at 110-120 °C for 5 min. of the distillate has an optical rotation the same as, or only
slightly lower than, the original oil.
Detection Spray the hot plate with a 10 g/L solution of
diphenylboric acid aminoethyl ester R in methanol R and then Refractive index
with a 50 g/L solution of macrogol 400 R in methanol R; after 1.472 to 1.476, Appendix VE.
at least 1 h, examine in ultraviolet light at 365 nm. Residue on evaporation
Results The chromatogram obtained with the test solution 1.0 to 5.0% when determined by the method for residue on
shows a yellow zone similar in position to the zone of evaporation of volatile oils, Appendix X M. Use 2 g and heat
naringin in the chromatogram obtained with the reference for 4 hours.
solution, and immediately below it a red zone (neoeriocitrin). Solubility in ethanol
Loss on drying (2.2.32) Soluble at 20°, in 7 parts of ethanol (90%), Appendix X M.
Maximum 11.0 per cent, determined on 1.000 g of the A bright solution is rarely obtained due to the presence of
powdered herbal drug (355) (2.9.12) by drying in an oven at waxy non-volatile substances.
105 °C. Weight per mL
Total ash (2.4.16) 0.842 to 0.848 g, Appendix V G.
Maximum 10.0 per cent. Content of aldehydes
ASSAY Not less than 1.0% w/w, calculated as decanal, C 10H 20 O.
Stock solution To O.175 g of the powdered herbal drug Carry out the method for the determination of aldehydes,
(355) (2.9.12) add 95 mL of ethanol (50 per cent Vlv'.) R. Appendix X K, using 10 g, omitting the toluene and using a
Heat on a water-bath under a reflux condenser for 30 min. volume, not less than 7 mL, of alcoholic hydroxylamine solution
Allow to cool and filter through a sintered-glass filter (2. 1. 2). that exceeds by 1 to 2 mL the volume of 0.5M potassium
hydroxide in ethanol (60%) VS required. Each mL of
Rinse the filter with 5 mL of ethanol (50 per cent Vlv'.) R.
Combine the filtrate and the rinsings in a volumetric flask 0.5M potassium hydroxide in ethanol (60%) VS is equivalent to
and dilute to 100.0 mL with ethanol (50 per cent V/V) R. 78.76 mg ofC 10H 20 O.
Test solution Into a test tube (10 mm x 180 mm) introduce STORAGE
0.150 g of powdered magnesium R (250) (2.9.12), a magnetic Orange Oil should be kept in a well-filled container and
stirring bar 25 mm long and 2.00 mL of the stock solution. protected from light.
Maintain the test tube upright, centrifuge at 125 g and
carefully add dropwise, especially at the beginning, 2.0 mL of
hydrochloric acid R, and then 6.0 mL of ethanol
(50 per cent Vlv'.) R. Stopper the tube and mix by inverting.
Compensation solution Into a 2nd test tube, introduce
2.00 mL of the stock solution and carefully add dropwise,
especially at the beginning, 2.0 mL of hydrochloric acid R and
then 6.0 mL of ethanol (50 per cent Vlv'.) R.
IV-402 Orange Oil 2023

Sweet Orange Oil 1.470 to 1.476.
(Ph. Bur. monograph 1811) Optical rotation (2.2. 7)
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ + 9.4° to+ 9.9° (measured in a 0.1 dm tube).
Peroxide value (2.5.5, Method B)
DEFINITION Maximum 20.
Essential oil obtained without heating, by suitable mechanical
treatment from the fresh peel of the fruit of Fatty oils and resinified essential oils (2.8.7)
Citrus x sinensis (L.) Osbeck. A suitable antioxidant may be
added. Bergapten
CHARACTERS
Appearance Test solution Dilute 0.2 mL of the substance to be examined
Clear, pale yellow or orange, mobile liquid, which may in I mL of ethanol (96 per cent) R.
become cloudy when chilled. Reference solution Dissolve 2 mg of bergapten R, 10 µL of
linalol R and 20 µL of linalyl acetate R in 10 mL of ethanol
IDENTIFICATION (96 per cent) R.
bergapten.
Drying In air.
chromatograms obtained with the reference solution and the Detection A Examine in ultraviolet light at 365 nm.
test solution. Results A The chromatogram obtained with the test solution
shows no greenish-yellow fluorescent zone corresponding to
Top of the plate that of bergapten 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.
-- -- Chromatographic profile
Many blue fluorescent zones
procedure.
Reference solution Test solution Test solution Dilute 200 µL of the substance to be examined
to 10.0 mL with heptane R.
Results B See below the sequence of zones present in the Reference solution (a) Dilute 5 µL of rx-pinene R, 5 µL of
chromatograms obtained with the reference solution and the sabinene R, 5 µL of {3-pinene R, 5 µL of /3-myrcene R, 5 µL of
test solution. octanal R, 70 µL of limonene R, 5 µL of linalol R, 5 µL of
decanal R, 10 µL of citral R (composed of neral and geranial)
Top of the plate and 5 µL of valencene R to 5.0 mL with heptane R.
Reference solution (b) Dilute 5 µL of limonene R to 50.0 mL
with heptane R. Dilute 0.1 mL of the solution to 5.0 mL with
Linalyl acetate: a brownish-orange A faint brownish-orange fluorescent heptane R.
fluorescent zone zone (linalyl acetate)
Column:
-- -- - material: fused silica;
- size: l = 60 m, 0 = 0.25 mm;
Many orange fluorescent zones
- stationary phase: phenyl(5)methyl(95)polysiwxane R (film
Linalol: a brownish-orange A brownish-orange fluorescent zone thickness 0.25 µm).
fluorescent zone (linalol) Carrier gas helium for chromatography R.
Bergapten: a faint greenish-yellow Fww rate 1.4 mUmin.
fluorescent zone
Split ratw 1:70.
Many brownish-orange fluorescent Temperature:
zones
-- -- Time Temperature
(min) (°C)
Many blue fluorescent zones
Co!urno 0 - 90 50 ➔ 230
Reference solution Test solution Injection port 250
Detector 250
B. It complies with the limits of the test for chromatographic
profile. Detection Flame ionisation.
TESTS Injection 1 µL.
0.842 to 0.850.
2023 Bitter-Orange Peel IV-403
Elution order Order indicated in the composition of 0.5M potassium hydroxide in ethanol (60%) VS is equivalent to
reference solution (a); record the retention times of these 78. 76 mg of C1oH20O,
substances. STORAGE
Identification of peaks Using the retention times determined Terpeneless Orange Oil should be kept in a well-filled
from the chromatogram obtained with reference solution (a), container and protected from light.
locate the components of reference solution (a) in the
- resolution: minimum 1.5 between the peaks due to Compound Orange Spirit
sabinene and P-pinene.
DEFINITION
Terpeneless Orange Oil 2.5 mL
ranges: Terpeneless Lemon Oil 1.3 mL
- a-pinene: 0.4 per cent to 0.6 per cent; Anise Oil or Star Anise Oil 4.25 mL
- sabinene: 0.2 per cent to 1.1 per cent; Coriander Oil 6.25 mL
- [3--pinene: 0.02 per cent to 0.3 per cent; Ethanol (90 per cent) Sufficient to produce 1000 mL
- [3-myrcene: 1.5 per cent to 2.5 per cent;
- octanal: 0.1 per cent to 0.4 per cent; The spirit complies with the requirements stated under Spirits and
- limonene: 92.0 per cent to 97.0 per cent; with the following requirements.
- linalol: 0.2 per cent to 0.7 per cent; TESTS
- decanal: 0.1 per cent to 0.4 per cent; Ethanol content
- neral: 0.02 per cent to 0.10 per cent; 86 to 90% v/v, Appendix VIII F.
- geranial: 0.03 per cent to 0.20 per cent; Weight per mL
- valencene: 0.02 per cent to 0.5 per cent. 0.828 to 0.841 g, Appendix VG.
Reporting threshold 0.01 per cent (reference solution (b)).
Residue on evaporation
1.0 per cent to 5.0 per cent.
Evaporate 5.0 g to dryness on a water-bath and dry at Dried Bitter-Orange Peel
100-105 °C for 4 h.
(Bitter-Orange Epicarp and Mesocarp, Ph. Bur.
STORAGE monograph 1603)
Preparations
Orange Peel Infusion
Orange Tincture
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Terpeneless Orange Oil DEFINlTION

Preparation Dried epicarp and mesocarp of the ripe fruit of Citrus
Compound Orange Spirit aurantium L. ssp. aurantium (C. aurantium L. ssp. amara
Engl.) partly freed from the white spongy tissue of the
DEFINITION mesocarp and endocarp.
Terpeneless Orange Oil may be prepared by concentrating
Content
orange oil under reduced pressure until most of the terpenes
Minimum 20 mL/kg of essential oil (anhydrous drug).
have been removed or by solvent partition.
IDENTIFICATION
CHARACTERISTICS
A. The herbal drug consists of elliptical or irregular pieces
A clear, yellow or orange-yellow liquid, visibly free from
5-8 cm long, 3-5 cm broad and about 3 mm thick.
water.
The outer surface is yellowish or reddish-brown and
TESTS distinctly punctate, the inner surface is yellowish or
Optical rotation brownish-white.
Not more than +60°, Appendix VF. B. Microscopic examination (2.8.23). The powder is light
Refractive index brown. Examine under a microscope using chloral hydrate
1.461 to 1.473, Appendix VE. solution R. The powder shows the following diagnostic
Solubility in ethanol characters (Figure 1603.-1): fragments of epicarp (surface
Soluble, at 20°, in 1 part of ethanol (90%), Appendix X M. view [BJ) composed of small polygonal cells with slightly
thickened anticlinal walls, filled with chromatophores and
Weight per mL some anomocytic stomata (2.8.3); fragments of epicarp
0.855 to 0.880 g, Appendix V G. showing the thickened cuticle (side view [N]); fragments of
Content of aldehydes pericarp (transverse section [Al) showing the cuticularised
Not less than 18% w/w, calculated as decanal, C 10 H 20 O. epicarp [Aa], the sub-epicarpal layers with cells with
Carry out the method for the determination of aldehydes, collenchymatous thickening [Ab], some of which contain a
Appendix X K, using 1.5 g, omitting the toluene and using a prism of calcium oxalate [Ac], and fragments of
volume, not less than 7 mL, of alcoholic hydroxylamine solution schizolysigenous oil glands [Ad]; fragments of the
that exceeds by 1 to 2 mL the volume of 0.5M potassium collenchymatous sub-epicarpal layers (surface view [G]);
hydroxide in ethanol (60%) VS required. Each mL of groups of parenchyma cells [L], some of which contain a
IV-404 Orange Peel Preparations 2023
prism of calcium oxalate [CJ; numerous fragments of Top of the plate

mesocarp [D, E, F, H, K, M}; free prisms of calcium
oxalate m. Examine under a microscope using a 20 g/L A light blue fluorescent zone
solution of potassium hydroxuie R. The mounting medium A light blue fluorescent zone
becomes yellow due to the presence of hesperidin in the
Caffeic acid: a light blue fluorescent
herbal drug. zone
Naringin: a dark green fluorescent A dark green fluorescent zone

zone (naringin)
A red fluorescent zone (neoeriocitrin)
TESTS
Water (2.2.13)
Maximum 10.0 per cent, determined by distillation on 20.0 g
of the powdered herbal drug (355) (2.9.12).
Total ash (2.4.16)
Extractable matter
10.0 mL of a mixture of 30 volumes of water Rand
70 volumes of ethanol (96 per cent) Rand extract for 2 h,
shaking frequently. Filter, evaporate 2.000 mL of the filtrate
to dryness on a water-bath and dry in an oven at 100-105 °C
for 3 h. Allow to cool in a desiccator over diphosphorus
pentoxide R and weigh. The residue weighs a minimum of
0.100 g.
ASSAY
25µm
Use a 500 mL round-bottomed flask, 200 mL of water R as
the distillation liquid and 0.50 mL of xylene R in the
Figure 1603.-1. - Illustration for identification test B of powdered graduated tube. Reduce the drug to a powder (710) (2. 9.12)
herbal drug of bitter-orange epicarp and mesocarp and immediately use 15.0 g for the determination. Distil at a
C. Thin-layer chromatography (2.2.27). rate of 2-3 mUmin for 90 min.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
(2. 9.12) add 10 mL of methanol R and heat in a water-bath
at 65 °C for 5 min with shaking. Allow to cool and filter.
Reference solution Dissolve 1.0 mg of naringin R and 1.0 mg
of cajfeic acid R in 1 mL of methanol R.
Orange Peel Infusion
DEFINITION
Mobile phase water R, anhydrous formic acid R, ethyl acetate R Concentrated Orange Peel Infusion !O0mL
(10:15:75 V/V/V). Water Sufficient to produce
Application 20 µL as bands. !O00mL

The infusion complies with the requirements stated under Infusions.
Drying In air, then in an oven at 110-120 °C for 5 min.
Detection Spray the warm plate with a 10 g/L solution of CONCENTRATED ORANGE PEEL
diphenylboric acui aminoethyl ester R in methanol R and then INFUSION
with a 50 g/L solution of macrogol 400 R in methanol R. After
at least 1 h, examine in ultraviolet light at 365 nm. DEFINITION
Dried Biner-Orange Peel, cut small 500 g
chromatograms obtained with the reference solution and the Ethanol (25 per cent) 1350 mL
test solution. Furthermore, other fluorescent zones are
present in the chromatogram obtained with the test solution. Extemporaneous preparation
Macerate the Dried Bitter-Orange Peel in a covered vessel for
48 hours with 1000 mL of the Ethanol (25 per cent) and
2023 Oregano IV-405
press out the liquid. To the pressed mare add 350 mL of the Top of the plate
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 and filter. A light blue fluorescent zone
TESTS Caffeic acid: a light blue fluorescent

Ethanol content zone
18 to 23% v/v, Appendix VIII F. A light blue fluorescent zone
Total solids
10 to 15% w/v, Appendix XIA. Use 1 mL.
Weight per mL Naringin: a dark green fluorescent A dark green fluorescent zone
zone (naringin)
(neoeriocitrin)
Orange Tincture Reference solution Test solution
(Bitter-Orange Epi,carp and Mesocarp Tincture,

Ph. Bur. monograph 1604) TESTS
Ethanol content (2. 9.10)
63 per cent to 67 per cent V/V.
DEFINITION
Methanol and 2-propanol (2. 9.11)
Tincture produced from Bitter--orange epicarp and
Maximum 0.05 per cent V/V of methanol and maximum
mesocarp (1603).
0.05 per cent V/V of 2-propanol.
PRODUCTION Dry residue
The tincture is produced from 1 part of the freshly powdered Minimum 6.0 per cent mlm, determined on 2.00 g of
herbal drug (2000) (2. 9.12) and 5 parts of alcohol tincture to be examined.
(70 per cent V/V) by an appropriate procedure.
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
CHARACTERS
Liquid with a bitter taste.
IDENTIFICATION
Examine by thin-layer chromatography (2.2.27). Orange Syrup
Test solution The tincture to be examined. DEFINITION
Reference solution Dissolve 1.0 mg of naringin Rand 1.0 mg
of caffeic acid R in 1 mL of methanol R. Orange Tincture 60mL
Syrup Sufficient to produce I 000 mL
Mobile phase water R, anhydrous formic acid R, ethyl acetate R The syrup complies with the requirements stated under Oral
(10:15:75 VIVIV). Liquids and with the following requirement.
Application 20 µL, as bands. TESTS
Development Over a path of 10 cm. Weight per mL
Drying In air, and heat in an oven at 110-120 °C for 5 min. 1.29 to 1.31 g, Appendix V G.
Detection Spray the warm plate 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. After
1 h, examine in ultraviolet light at 365 nm. Oregano
chromatograms obtained with the reference and test (Ph. Bur. monograph 1880)
solutions. Furthermore, other zones are present in the
DEFINITION
Dried leaves and flowers separated from the stems of
Origanum onites L. or Origanum vulgare L. subsp. hirtum
(Link) Ietsw., or a mixture of both species.
Content
- essential oil: minimum 25 mIJkg (anhydrous drug);
- sum of the contents of carvacrol and thymol (both C 10H14 0;
Mr 150.2): minimum 60 per cent in the essential oil.
IDENTIFICATION
A. 0. onites. The leaf is yellowish-green, usually 4-22 mm
long and 3-14 mm wide. It has a long or short petiole or is
sessile. The lamina is ovate, elliptic or ovate-lanceolate.
Margins are entire or serrate, the apex is acute or obtuse.
The veins are yellowish and conspicuous on the adaxial
IV-406 Oregano 2023
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
flowers, or inconspicuous. The bracts are imbricate and
green like the leaves. The drug contains yellowish or
yellowish-brown stem parts.
0. vulgare (subsp. hirtum). The leafis green and usually
3-28 mm long and 2.5-19 mm wide. It is petiolate or sessile.
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-
yellow and imbricate. The calyx is corolla-like and
inconspicuous. The corolla is white, on top of inflorescences,
slightly conspicuous or inconspicuous.
(710) (2.9.12). The powder is green (0. vulgare) or
yellowish-green (0. onites). Examine under a microscope
using chloral hydrate solutwn R (Figure 1880.-1).
0. onites Powder shows fragments of leaf epidermis [A, D,
G] composed of cells with sinuous walls, diacytic stomata
(2.8.3) [Ga], covering trichomes and glandular trichomes;
there are 2 types of glandular trichomes: some of lamiaceous
type with 8-16 cells (surface view [Da]) and a very common ,,t
t
type with a unicellular head and uni- [Ge], bi- [H] or
tricellular stalk; the covering trichomes have smooth, thick :' .:·,
walls; some are multicellular [B, Gb], often broken [Aa], and .•~11 '.
contain prisms of calcium oxalate, while others, which are
rare, are unicellular and conical [CJ; scars from covering and .-··-· 25µ
glandular trichomes are visible on the epidermises [Gd, Ge];
pollen grains, with smooth exine, are frequent [E, F].
Figure 1880. -1. - Illustratwn for identificatwn test B of powdered
0. vulgare Subsp. hirtum powder shows fragments of the herbal drug of oregano
upper epidermis with cells with sinuous, beaded walls,
accompanied by palisade parenchyma m; fragments of the
lower epidermis [N] composed of cells with finely and Top of the plate
irregularly thickened walls, diacytic stomata (2.8.3) [Na],
covering trichomes and glandular trichomes; there are 2 types A bluish-purple zone
of glandular trichomes: some of lamiaceous type with -- --

12 cells, in surface view [Nb), and a rare type with a
A pale green zone
unicellular head [Ne] and bi- or tricellular stalk; the covering
trichomes have thick, warty walls and contain fine needles of Thymol: a pink zone A pink zone (thymol)
calcium oxalate; some are conical, multicellular and serrate
Carvacrol: a pale violet zone A pale violet zone (carvacrol)
[L, M], while others, which are rare, are unicellular [K];
there are occasional pollen grains, with smooth exine [E, F] . -- --
C. Thin-layer chromatography (2.2.27). A pale purple zone
A grey zone
(2. 9.12) add 5 mL of methylene chlaride R and shake for
3 min, then filter through about 2 g of anhydrous sodium A pale green zone
sulfate R.
A bluish-purple zone
Reference solutwn Dissolve 1 mg of thymol R and 10 µL of
carvacrol R in 10 mL of methylene chloride R. An intense brown zone
Plate TLC silica gel plate R. Reference solution Test solution

Mobile phase methylene chloride R.
Applicatwn 20 µL as bands.
TESTS
Development Over a path of 15 cm. Water (2.2.13)
Drying In air. Maximum 120 mIJkg, determined on 20.0 g of the
Detection Spray with anisaldehyde solution R using 10 mL for powdered herbal drug (355) (2.9.12).
a plate 200 mm square and heat at 100-105 °C for 10 min. Total ash (2.4.16)
Results See below the sequence of zones present in the Maximum 15.0 per cent.
chromatograms obtained with the reference solution and the Ash insoluble in hydrochloric acid (2.8.1)
test solution. Furthermore, other zones are present in the Maximum 4.0 per cent.
lower third and upper part of the chromatogram obtained
with the test solution.
2023 Orientvine Stem IV-407
ASSAY IDENTIFICATION
Essential oil (2.8. 12) A. Whole drug. Long cylindrical stem, somewhat curved,
Use 30.0 g of the drug to be examined, a 1000 mL round- 60 cm long or more, 0.5-2 cm in diameter. The outer bark is
bottomed flask and 400 mL of water R as the distillation greenish-brown or brown, sometimes greyish-brown, with
liquid. Distil at a rate of 2-3 mUmin for 2 h without xylene R relatively wide longitudinal striations and prominent
in the graduated tube. verrucose lenticels; the nodes are slightly swollen and
Carvacrol and thymol branched. The texture is light, hard and difficult to break;
Gas chromatography (2.2.28): use the normalisation the fracture is uneven, greyish-yellow or pale greyish-brown;
procedure. the bark is thin (about 1/10 of the diameter); the medullary
rays are very conspicuous; the pith is yellowish-white or pale
Test solution Filter the essential oil obtained in the assay of
yellowish-brown.
essential oil over a small amount of anhydrous sodium sulfate R
and dilute to 5.0 mL with heptane R by rinsing the apparatus Fragmented drug Fragments of stems, in discs, about 1.5 cm
and the anhydrous sodium sulfate. in diameter and 0.3 cm thick, with greenish-brown, brown or
greyish-brown outer surface; a transverse section shows a
Reference solution Dissolve 0.20 g of thymol R and 50 mg of
narrow, pale yellow cortical zone, it is mainly occupied by
carvacrol R in heptane R and dilute to 5.0 mL with the same
the vascular system (about 3/4 of the section) consisting of
solvent.
very numerous vascular bundles (about 15-20) in a circle
Column: around the yellowish-white or pale yellowish-brown, small,
- material: fused silica; circular pith; each bundle is delimited on the outside by a
= =
- size: l 60 m, 0 0.25 mm; narrow and continuous, wavy, light brown zone and is
- stationary phase: macrogol 20 000 R (film thickness separated from the next bundle by a narrow, light brown
0.25 µm). medullary ray; the xylem vessels with a relatively wide
Gamer gas nitrogen for chromatography R or helium for interior lumen are clearly visible.
chromatography R. B. Microscopic examination (2.8.23). The powder is
Flow rate 1.5 mLJmin. yellowish-brown or greyish-brown. Examine under a
Split ratio 1:100. microscope using chloral hydrate solution R. The powder
Temperature: shows the following diagnostic characters: rare fragments of
epidermis with polyhedral cells, in surface view, covered with
Temperature a thick, pale yellow cuticle W; sclereids, isolated or in
(°C) groups, of various sizes and shapes (cubical [HJ,
Column 0 - 45 40 ➔ 250 fusiform [F], elliptical or irregular [B, El), with more or less
Injection port 190 thick walls, pitted and distinctly channelled, free or included
Detector 210 in fragments of parenchyma; pale yellow or yellow fibres,
30-70 µm in diameter with thick, channelled walls and a very
narrow lumen [G]; fragments of parenchyma with thin-
walled cells containing fine, needle-shaped crystals of calcium
Injection 0.2 µL. oxalate (C]; fragments of xylem consisting of reticulate or
Elution order Order indicated in the composition of the bordered-pitted vessels [L), up to 200 µm in diameter,
reference solution; record the retention times of these accompanied by cells of xylem parenchyma [A, D] with
substances. slightly and regularly thickened and pitted walls. Examine
System suitability Reference solution: under a microscope using a 50 per cent V/V solution of
- resolution: minimum 1.5 between the peaks due to thymol glycerol R. The powder shows spherical starch granules, about
and carvacrol. 10 µm in diameter, simple or 2- to 3-compound, free or
Using the retention times determined from the included in parenchymatous cells [K].
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.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur Reference solution Dissolve 5 mg of sinomenine R and 5 mg of
papavenne hydrochloride R in 5 mL of ethanol (96 per cent) R.
Mobile phase concentrated ammonia R, water R, toluene R,
Orientvine Stem methanol R, ethyl acetate R (2:10:20:30:40 VIVIVIVIV).
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Drying In air.
DEFINITION Detection A Examine in ultraviolet light at 254 nm.
Dried, whole or fragmented stem of Sinomenium acutum Results A See below the sequence of zones present in the
(Thunb.) Rehder & 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 be present in the chromatogram obtained with the test
Minimum 0.5 per cent of sinomenine (C 19H 23N0 4 ; solution.
IV-408 Orientvine Stem 2023
/ ·- Top of the plate

~~~~~-;.,,,;J'. • •••
A . _. , • • Papaverine: an orange zone An orange zone
-- --
3 light orange zones

.. .
Sinomenine: an orange zone An orange zone (sinomenine)
C C
D '~ -- --
2 orange zones
A light orange zone
1
TESTS
·y Maximum 10.0 per cent, determined on 1.000 g of the
105 °C for 3 h.
Total ash (2.4.16)
if Maximum 6.0 per cent.
ll Aristolochic acids (2.8.21, Method A)

ASSAY
(355) (2. 9.12) in 20.0 mL of ethanol (70 per cent V/V) R in a
conical flask and weigh. Sonicate for 20 min. Cool and weigh
Figure 2450.-1. - Illustration for identification test B of powdered again. Compensate the loss of solvent with ethanol
herbal drug of orientvine stem (70 per cent V/V) R and stopper the flask. Shake thoroughly
and filter through a membrane filter (nominal pore size
0.45 µm).
Top of the plate
Reference solution (a) Dissolve 3.0 mg of sinomenine CRS in
Papaverine: a quenching zone methanol Rand dilute to 10.0 mL with the same solvent.
A quenching zone Reference solution (b) Disperse 0.250 g of orientvine
stem HRS in 10.0 mL of ethanol (70 per cent VIV) Rina
-- -- conical flask and weigh. Sonicate for 20 min. Cool and weigh
again. Compensate the loss of solvent with ethanol
(70 per cent V/V) R and stopper the flask. Shake thoroughly
Sinomenine: a quenching zone A quenching zone (sinomenine) and filter through a membrane filter (nominal pore size
-- --
0.45 µm).
Column:
- size: l =0.15 m, 0 =4.6 mm;
A dark blue fluorescent zone - stationary phase: end-capped octadecylsifyl silica gel for
Mobile phase Adjust a 1.8 g/L solution of disodium hydrogen
phosphate dodecahydrate R to pH 8.0 with a 0.8 g/L solution
Detection B Treat with a 10 g/L solution of sodium nitrite R of sodium dihydrogen phosphate R, then adjust to pH 9.0 with
in potassium iodobismuthate solution RS and allow to dry in air. a 10 g/L solution of triethylamine R. Mix 60 volumes of this
Examine in daylight. solution with 40 volumes of acetonitrile R.
Results B See below the sequence of zones present in the Flow rate 1.0 mUmin.
Retention time Sinomenine = about 3 min.
- resolution: minimum 1.5 between peak 1 and the peak due
to sinomenine; identify peak 1 using the chromatogram
supplied with orientvine stem HRS.
Calculate the percentage content of sinomenine using the
2023 Pansy IV-409
Test solutwn Heat in a water-bath at 65 °C for 5 min, with

frequent stirring, 2.0 g of the powdered herbal drug (355)
(2. 9.12) in 10 mL of alcohol (70 per cent V/V) R. Cool and
area of the peak due to sinomenine in the chromatogram
obtained with the test solution; filter.
area of the peak due to sinomenine in the chromatogram Reference solutwn Dissolve 2.5 mg of rutoside trihydrate R,
2.5 mg of hyperoside R and 1.0 mg of caffeu: acid R in
solution, in grams; methanol R and dilute to 10 mL with the same solvent.
mass of sinomenine CRS used to prepare reference solution (a), Plate TLC silica gel plate R.
in grams;
p assigned percentage content of sinomenine in sinomenine CRS. Mobile phase anhydrous formic acid R, acetic acid R, water R,
ethyl acetate R (11:11:27:100 Jl/VIVIV).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur Application 10 µL, as bands.
Drying At 100-105 °c.
Detection Spray with a solution containing 10 g/L of
Wild Pansy diphenylboru: acid aminoethyl ester R and 50 g/L of macrogol
400 R in methanol R. Allow the plate to dry in air for 30 min.
(Wz"ld Pansy (Flowering Aerial Parts), Ph. Bur.
Examine in ultraviolet light at 365 nm.
monograph 1855)
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION test solution. Furthermore, other zones may be present in the
Dried flowering aerial parts of Viola arvensis Murray and/or chromatogram obtained with the test solution.
Viola tricolor L.
Top of the plate
Content
Minimum 1.5 per cent of flavonoids, expressed as violanthin Caffeic acid: a greenish-blue to light
(C21H30O14; Mr 578.5) (dried drug). blue fluorescent zone
IDENTIFICATION A blue fluorescent zone

A. The stem is angular and hollow. The leaves are oval,
petiolate, with a cordate base or elongated and obtuse, with
lyrate stipules, divided in the middle. The flowers, with a -- --
long peduncle, are zygomorphic, with 5 oval, lanceolate
sepals, an appendage pointed outwards and 5 petals of which
the lower one bears a spur; in Viola arvensis, the petals are Hyperoside: a yellowish-brown
fluorescent zone
shorter than the calyx, the lower petal is cream coloured,
with black lines, the 4 upper petals may be cream coloured A yellowish-green fluorescent zone
or violet blue; in Viola tricolor, the petals are longer than the
calyx and violet coloured, more or less tinged with yellow.
The androecium consisting of 5 stamens bears at the apex a --
membranous connective appendage with 2 spurs. --
Rutoside: a yellowish-brown An intense yellowish-brown
The trilocular ovary shows a short style and globular fluorescent zone fluorescent zone (rutoside)
stigmata. The fruit are navicular capsules, three-lobed,
yellowish brown, 5 mm to 10 mm long. The pale yellow,
pyriform seeds are about 1 mm long, bearing a caruncle. A yellowish-green fluorescent zone
greenish. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic A yellowish-green fluorescent zone
characters: fragments of the epidermis of the leaves in surface
view with wavy-walled cells and anomocytic stomata (2.8.3);
conical unicellular covering trichomes, widened at the base Reference solution Test solution
and sharply pointed at the apex, with a striated cuticle;
glandular trichomes with a multicellular head, and a short,
multicellular stalk in the indentations of the leaf margins; TESTS
cluster crystals of calcium oxalate, sometimes included in Foreign matter (2.8.2)
parenchyma; fragments of the corolla with wavy-walled Maximum 3 per cent.
epidermal cells, those from the mid-region papillose and with Swelling index (2.8.4)
some extended to form flask or bottle-shaped projections, Minimum 9, determined on the powdered herbal drug (355)
those from the base of the petals with covering trichomes up (2. 9.12).
to about 300 µm long with characteristic hump-like swellings Loss on drying (2.2.32)
along their length; spherical or polyhedral pollen grains, Maximum 12.0 per cent, determined on 1.000 g of the
60 µm to 80 µm in diameter, with finely pitted exines and 5 powdered herbal drug (355) (2.9.12) by drying in an oven at
pores ( Viola arvensis) or 4 pores ( Viola tricolor); occasional 105 °C for 2 h.
fragments of spiral and reticulate vessels and groups of fibres
from the stem. Total ash (2.4.16)
IV-410 Passionflower Herb 2023
ASSAY chemotype or a mixture of the two. It may also contain

Stock solution In a 200 mL flask, introduce 0.300 g of the flowers and/or fruits.
powdered herbal drug (250) (2. 9.12) and 40 mL of alcohol Content
(60 per cent V/V) R. Heat in a water-bath at 60 °C for Minimum 1.0 per cent of total flavonoids, expressed as
10 min, shaking frequently. Allow to cool and filter through a isovitexin (C 21 H 20 0 10; M, 432.4) (dried drug).
plug of absorbent conon into a 100 mL volumetric flask.
Transfer the absorbent cotton with the drug residue back IDENTIFICATION
into the 200 mL flask, add 40 mL of alcohol A. The green or greenish-grey or brownish stem is ligneous,
(60 per cent V/V) R and heat again in a water-bath at 60 °C hollow, longitudinally striated, glabrous or very slightly
for 10 min, shaking frequently. Allow to cool and filter into pubescent, with a diameter that is generally less than 8 mm.
the same 100 mL volumetric flask as used previously. Rinse The green or greenish-brown leaves are alternate, finely
the 200 mL flask with a further quantity of alcohol dentate and pubescent, deeply divided into 3 acute lobes of
(60 per cent VIV) R, filter and transfer to the same 100 mL which the central lobe is the largest. The midrib is much
volumetric flask. Dilute to volume with alcohol more prominent on the lower surface. The petiole is
(60 per cent V/V) R and filter. pubescent and bears 2 dark nectaries near the lamina.
The tendrils are very numerous and grow from the axils of
Test solution Introduce 5.0 mL of the stock solution into a
the leaves; they are fine, smooth, round and terminated in
round-bottomed flask and evaporate to dryness under
cylindrical spirals. The radiate flowers, if present, have
reduced pressure. Take up the residue with 8 mL of a
3 small bracts and a corolla consisting of 5 white, elongated
mixture of 10 volumes of methanol R and 100 volumes of
petals with several rows of filiform, petaloid appendices.
glacial acetic acid R and transfer into a 25 mL volumetric
If present, the greenish or brownish fruit is flattened and
flask. Rinse the round-bottomed flask with 3 mL of a
oval; it contains several flattened, brownish-yellow, pitted
seeds.
glacial acetic acid R and transfer into the same 25 mL
volumetric flask as used previously. Add 10.0 mL of a B. Microscopic examination (2.8.23). The powder is light
solution containing 25.0 g/L of boric acid R and 20.0 g/L of green. Examine under a microscope using chloral hydrate
oxalic acid R in anhydrous formic acid R and dilute to 25.0 mL solution R. The powder shows the following diagnostic
with anhydrous acetic acid R. characters (Figure 1459.-1): fragments of the upper
epidermis of the leaf (surface view [A]) consisting of cells
Compensation liquid Introduce 5 .0 mL of the stock solution
with sinuous, almost angular walls, accompanied by
into a round-bottomed flask and evaporate to dryness under
underlying palisade parenchyma [Aa]; fragments of the lower
reduced pressure. Take up the residue with 8 mL of a
epidermis of the leaf (surface view [B]) consisting of cells
mixture of 10 volumes of methanol Rand 100 volumes of
with sinuous walls and anomocytic [Ba] or anisocytic [Bb]
glacial acetic acid R and transfer into a 25 mL volumetric
stomata (2.8.3); fragments of the veins of the leaf [C, F] with
flask. Rinse the round-bottomed flask with 3 mL of a
annular or spiral vessels [Ca, Fb], and sheaths containing
cluster crystals of calcium oxalate [Cb, Fa]; fragments of the
glacial acetic acid R and transfer into the same 25 mL
stem (longitudinal section [D]) with numerous fibres [Db]
volumetric flask as used previously. Add 10.0 mL of
accompanied by pitted or reticulate vessels [Da] and
anhydrous formic acid R and dilute to 25.0 mL with anhydrous
medullary rays [De]; fragments of the central stem
acetic acid R.
parenchyma [K] with cells with slightly thickened and pitted
Measure the absorbance (2.2.25) of the test solution at
405 nm after 30 min.
walls; uniseriate trichomes m, with 1 to 3 thin-walled cells,
straight or slightly curved, ending in a point. In addition, if
Calculate the percentage content of total flavonoids, flowers are present, the powder shows fragments of the
expressed as violanthin from the expression: epidermis of the petals and appendages with papillose
cells [G] and pollen grains with a reticulate exine [E];
Ax 1.25 if mature fruits are present, the powder may also show
m scattered brown tannin cells and fragments of brownish-
yellow testa comprising cells with regularly thickened and
taking the specific absorbance ofviolanthin to be 400. strongly channelled walls [H].
A measured absorbance at 405 nm,
m mass of the herbal drug to be examined, in grams. Test solution To 0.5 g of the powdered herbal drug (355)
(2. 9.12) add 5.0 mL of methanol R. Sonicate for 15 min,
then filter or centrifuge; use the filtrate or the supernatant.
Reference solution (a) Dissolve 1.5 mg of homoorientin Rand
1.5 mg of isovitexin R in methanol Rand dilute to 10.0 mL
Passionflower Herb Reference solution (b) Dilute 2.5 mL of reference solution (a)
Passiflora
Reference solution (c) Dissolve 1.5 mg of isovitexin R and
(Passion Flower, Ph. Bur. monograph 1459) 1.5 mg of orientin R in methanol R and dilute to 10 mL with
Preparation the same solvent.
Passionflower Herb Dry Extract Intensity markers Homoorientin for the yellow fluorescent
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ zones and isovitexin for the green or greenish-blue
fluorescent zones.
DEFINITION
Fragmented or cut, dried aerial parts of Passijfora Plate TLC silica gel F254 plate R (2-10 µm).
incarnata L. of the swertisin chemotype or the isovitexin
2023 Passionflower Herb IV-411
Top of the plate
2 red zones, intense 2 red zones, intense
-- -- --
A green or greenish- A green or greenish-

blue zone, faint to blue zone, faint to
equivalent equivalent
A yellow zone, faint to A yellow zone, faint to

equivalent equivalent
Isovitexin: a green or A green or greenish-

greenish-blue zone blue zone, faint to
equivalent (isovitexin)
A bluish-green zone
(swertisin)
Homoorientin: a yellow A yellow zone, faint to A yellow zone, faint to
zone equivalent equivalent
(homoorientin) (homoorientin)
-- -- --

blue zone, very faint to blue zone, very faint to
faint faint
Reference Test solution Test solution

solution (a) (isovitexin-type) (swertisin-type)
TESTS
Figure 1459.-1. - Illustration for identification test B of powdered Total ash (2.4.16)
herbal drug of passionflower herb Maximum 13.0 per cent.
ketone R, ethyl acetate R (10:10:30:50 V/V/V/V).
Application 4 µL as bands of 8 mm. 105 °C for 2 h.
ASSAY
Drying In a current of air at room temperature for 5 min. Liquid chromatography (2.2.29).
Detection Heat at 100-105 °C for 5 min; spray the warm Solvent mixture water R, methanol R (20:80 V/V).
(355) (2.9.12) into a screw-cap bottle and add 25.0 mL of
the solvent mixture. Sonicate for 40 min. Shake the tube
every 10 min and at the end of the process. Subsequently
centrifuge for 5 min at about 9000-10 000 g and transfer the
methylene chloride R; allow to dry in air for about 1 min.
supernatant to a vial.
Reference solution (a) Dissolve 5.0 mg of isovitexin CRS in
the solvent mixture and dilute to 50.0 mL with the solvent
mixture. Sonicate for about 10 min.
zones which may be touching. The lower zone
(isovitexin) shows a green or greenish-blue Reference solution (b) Dissolve 0.5 mg of homoorientin Rand
fluorescence and the upper zone (orientin) shows a 0.5 mg of orientin R in the solvent mixture and dilute to
light yellow fluorescence. 10.0 mL with the solvent mixture.
Results See below the sequence of fluorescent zones present Reference solution (c) Dilute 1.0 mL of reference solution (a)
in the chromatograms obtained with reference solution (a) to 50.0 mL with the solvent mixture.
and the test solution. Furthermore, in the chromatogram Column:
obtained with the test solution, the faint to very faint green - size: l = 0.10 m, 0 = 2.1 mm;
or greenish-blue fluorescent zone in the lower third of the - stationary phase: end-capped octadecylsilyl silica gel for
chromatogram may overlap with a faint yellow fluorescent chromatography R (1.8 µm);
zone just below it, and other faint to very faint, yellow, - temperature: 50 °C.
greenish-yellow or brownish-yellow and green or greenish- Mobile phase:
blue fluorescent zones may be present, especially in the lower - mobile phase A: anhydrous formic acid R, warer for
third of the chromatogram. chromatography R (1: 1000 V/V);
- mobile phase C: methanol R;
IV-412 Passionflower Preparations 2023
Time Mobile phase A Mobile phase B Mobile phase C Reference solution (c) Dissolve 1.5 mg of isovitexin R and
(rnin) (per cent Vil') (per cent Vil') (per cent Vil')
1.5 mg of orientin R in methanol Rand dilute to 10 mL with
0 - 0.5 95 0 the same solvent.
0.5 - 26.5 95---> 84 0 ---> II
Intensity markers Homoorientin for the yellow fluorescent
zones and isovitexin for the green or greenish-blue
Flow rate 0.7 mLJmin. fluorescent zones.
Detection Spectrophotometer at 338 nm. Plate TLC silica gel F254 plate R (2-10 µm).
Injection 2 µL. Mobile phase anhydrous formic acid R, water R, methyl ethyl
Identification of peaks Use the chromatogram obtained with ketone R, ethyl acetate R (10:10:30:50 VIVIVIV).
reference solution (a) to identify the peak due to isovitexin; Application 4 µL as bands of 8 mm.
identify the peaks due to flavonoids with relative retentions Development 70 mm from the lower edge of the plate.
between 0.64 and 1.12 with reference to isovitexin.
- resolution: minimum 3.0 between the peaks due to orientin Detection Heat at 100-105 °C for 5 min; spray the warm
and homoorientin.
Reporting threshold 0.006 per cent (reference solution (c)). 400 R in methanol R or, alternatively, dip the warm plate in a
Calculate the percentage content of the sum of flavonoids, 5 g/L solution of diphenylboric acid aminoethyl ester R in ethyl
expressed as isovitexin, using the following expression: acetate R, then in a 50 g/L solution of macrogol 400 R in
methylene chloride R; allow to dry in air for about 1 min.
A1 sum of the peak areas due to flavonoids which elute with
relative retentions between 0.64 and 1.12 with reference to zones which may be touching. The lower zone (isovitexin)
isovitexin in the chromatogram obtained with the test solution; shows a green or greenish-blue fluorescence and the upper
A2 area of the peak due to isovitexin in the chromatogram obtained zone (orientin) shows a light yellow fluorescence.
m1 mass of the herbal drug to be examined used to prepare the test Results See below the sequence of fluorescent zones present
solution, in grams; in the chromatograms obtained with reference solution (a)
m2 mass of isovitexin CRS used to prepare reference solution (a), in and the test solution. Furthermore, in the chromatogram
grams;
p percentage content of isovitexin in zsovitexin CRS.
obtained with the test solution, the very faint to faint green
or greenish-blue fluorescent zone in the lower third of the
chromatogram may overlap with a faint yellow fluorescent
zone below it, and other very faint to faint, yellow, greenish-
yellow or brownish-yellow and green or greenish-blue
fluorescent zones may be present, especially in the lower
third of the chromatogram.
Passionflower Herb Dry Extract
Passion Flower Dry Extract Top of the plate
(Ph. Bur. monograph 1882) --- --- ---

blue zone, faint to blue zone, faint to
DEFINITION equivalent equivalent
Dry extract produced from Passionflower herb (1459).
A yellow zone, faint to A yellow zone, very
Content equivalent faint to faint
Minimum 1.5 per cent of total flavonoids, expressed as
isovitexin (C 21 H 20 0 10 ; M, 432.4) (anhydrous extract). Isovitexin: a green or A green or greenish-
greenish-blue zone blue zone, faint to
PRODUCTION equivalent (isovitexin)
A bluish-green zone
procedure using ethanol (40-90 per cent VIV), methanol (swertisin)
(60 per cent V/V) or acetone (40 per cent V/V).
Homoorientin: a yellow A yellow zone, faint to A yellow zone, faint to
CHARACTERS zone equivalent equivalent
(homoorientin) (homoorientin)
Appearance
Greenish-brown amorphous powder. --- --- ---
IDENTIFICATION A green or greenish- A green or greenish-
High-performance thin-layer chromatography (2. 8. 25). blue zone, very faint to blue zone, very faint to
faint faint
5.0 mL of methanol R. Sonicate for 15 min, then filter or
centrifuge; use the filtrate or supernatant.
Reference Test solution Test solution
Reference solution (a) Dissolve 1.5 mg of homoorientin Rand solution (a) (isovitexin-type) (swertisin-type)
1.5 mg of isovitexin R in methanol Rand dilute to 10.0 mL
2023 Peach Seed IV-413
TESTS
Water
Peach Seed
(2.5.12): maximum 5.0 per cent, determined on 0.500 g. (Ph. Bur. monograph 2975)
ASSAY Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried, ripe seed, freed from the shell, of Prunus persica (L.)
Test solution Introduce 0.450 g of the extract to be Batsch or Prunus davidiana (Carriere) Franch.
examined into a screw-cap bottle and add 25.0 mL of the
solvent mixture. Sonicate for 40 min. Shake the tube every Content
10 min and at the end of the process. Subsequently Minimum 2.0 per cent of amygdalin (C 20 H 27 NO 11 ;
centrifuge for 5 min at about 9000-10 000 g and transfer the Mr 457.4) (dried drug).
supernatant to a vial. IDENTIFICATION
Reference solution (a) Dissolve 5.0 mg of isovitexin CRS in A. Elongated ovoid to sub-ovoid seed; strongly laterally
the solvent mixture and dilute to 50.0 mL with the solvent compressed, 1.1-1.8 cm long, 0.8-1.2 cm wide and
mixture. Sonicate for about 10 min. 0.2-0.5 cm thick (Prunus persica) or sightly laterally
Reference solution (b) Dissolve 0.5 mg of homoorientin Rand compressed, 0.9-1.3 cm long, 0.7-0.9 cm wide and
0.5 mg of orientin R in the solvent mixture and dilute to 0.4-0.7 cm thick (Prunus davidiana). The outer surface,
10.0 mL with the solvent mixture. consisting of the yellowish-brown to reddish-brown testa, is
covered by fine granular protuberances; one end is acute to
Reference solution (c) Dilute 1.0 mL of reference solution (a)
acuminate, the middle part is convex, the other end is
to 50.0 mL with the solvent mixture.
obtuse-rounded and slightly oblique. The short and linear
Column: hilum is clearly visible at the acute end; the chalaza is brown
- size: l = 0.10 m, 0 = 2.1 mm; to blackish-brown and clearly visible at the rounded end.
- stationary phase: end-capped octadecylsilyl silica gel for Numerous vascular bundles form prominent longitudinal
chromatography R (1.8 µm); lines running along the surface from the chalaza to the hilum.
- temperature: 50 °C. A transverse section of the seed shows a thin testa and 2
Mobile phase: yellowish-white, smooth and oily cotyledons.
- mobile phase A: anhydrous formic acid R, water for B. Microscopic examination (2.8.23). The powder is
chromatography R (0.1:100 VIV); yellowish-white with reddish-brown flecks. Examine under a
- mobz7e phase B: acetonitrile R; microscope using chloral hydrate solution R. The powder
- mobile phase C: methanol R; shows the following diagnostic characters (Figure 2975.-1):
fragments of the testa (surface view [A, BJ) consisting of the
Time Mobile phase A Mobile phase B Mobile phase C outer testa with sclereids, isolated or in groups of 2 or 3 [Aa,
(min) (per cent VIJI) (per cent VIJI) (per cent VIJI)
Ba] connected to thin-walled cells [Ab, Bb], several layers of
0-0.S 95 0 s parenchyma with ovoid cells [Ac, Be], and the protein layer
0.5-26.5 95 ➔ 84 0 ➔ 11 5
with polyhedral cells with slightly thickened walls [Ad, Bd];
isolated yellow sclereids, up to 270 µm long and 190 µm
Flow rate 0.7 mIJmin. wide at the base, varying in shape; in surface view [HJ the
Detection Spectrophotometer at 338 nm. sclereids are rounded to ovoid, with thick walls, wide
Injection 2 µL. channels and large pits (Prunus persica) [Ba] or fine pits
(Prunus davidiana) [Aa] in the bottom of the cell walls,
whereas in side view [C, F] the sclereids are elliptical, with
reference solution (a) to identify the peak due to isovitexin;
an oval or rounded outer surface with very thick,
identify the peaks due to flavonoids with relative retentions
longitudinally striated, unchannelled walls [Ca, Fa] and a
between 0. 64 and 1. 12 with reference to isovitexin.
more or less flat, thinner and channelled inner surface [Cb,
System suitability Reference solution (b): Fb]; numerous fragments of cotyledons with thin-walled
- resolution: minimum 3.0 between the peaks due to orientin polyhedral cells containing oil droplets and small crystals of
and homoorientin. calcium oxalate, especially in the outermost layers (transverse
Reporting threshold 0.01 per cent (reference solution (c)). section [D, L]); fragments of the epidermis of the cotyledons
Calculate the percentage content of total flavonoids, (surface view [El); numerous fragments of spiral or annular
expressed as isovitexin, using the following expression: vessels [G, TI; numerous isolated oil droplets [K].
(2. 9.12) add 5.0 mL of methanol R and sonicate for 20 min.
AI sum of the peak areas due to flavonoids with relative retentions
between 0.64 and 1.12 with reference to isovitexin in the Reference solution (a) Dissolve 10.0 mg of amygdalin Rand
chromatogram obtained with the test solution; 5.0 mg of sucrose R in methanol Rand dilute to 5.0 mL with
A2 area of the peak due to isovitexin in the chromatogram obtained
the same solvent.
m1 mass of the herbal drug to be examined used to prepare the test Reference solution (b) Dilute 2.5 mL of reference solution (a)
solution, in grams; to 10.0 mL with methanol R.
m2 mass of isavitexin CRS used to prepare reference solution (a), in
grams; Reference solution (c) Dissolve 5 mg of glucose R and 5 mg of
p percentage content of isovitexin in isovitexin CRS. sucrose R in methanol R and dilute to 5 mL with the same
solvent.
- - - - - - - - - - - - - - - - - - - - - - Ph Eur Intensity marker Amygdalin.
IV-414 Peach Seed 2023
Top of the plate
A blue fluorescent zone, intense
A blue fluorescent zone, intense
-- --
Amygdalin: a brown zone A brown zone, very faint to faint

(amygdalin)
-- --
L
A brown zone, faint
Sucrose: a brown zone A brown zone (sucrose)
TESTS
Maximum 9.0.
Figure 2 97 5. -1. - Illustration for identificatwn test B of powdered
herbal drug of peach seed. Primus davidiana: [A, D, E, F, H, J, Introduce 35.0 g of the powdered herbal drug (1400)
K]. Prunus persica: [B, C, G, K, L] (2. 9.12) into a 250 mL round-bonomed flask. Add 150 mL
of heptane R and boil under a reflux condenser for 30 min.
Plate TLC silica gel F 254 plate R (2-10 µm). Allow to cool, filter through a paper filter and evaporate the
Mobile phase water R, methylene chloride R, methanol R, ethyl filtrate to dryness to obtain the oil. Use the oil to determine
acetate R (10:15:22:40 VIVIVIV). the peroxide value.
Applicatwn 5 µL as bands of 8 mm. Foreign matter (2.8.2)
Drying In air for 5 min. Loss on drying (2.2.32)
Detection Treat with a 10 per cent VIV solution of sulfuric
acid R in ethanol (96 per cent) R and heat at 105 °C for
105 °C for 5 h.
3 min; examine in ultraviolet light at 365 nm.
Total ash (2.4.16)
- the chromatogram shows in the lower third 2 distinct
zones, which may be touching; the lower zone ASSAY
(sucrose) and the upper zone (glucose) are brown. Liquid chromatography (2.2.29).
Results See below the sequence of zones present in the Test solutwn To 0.20 g of the powdered herbal drug (710)
chromatograms obtained with reference solution (a) and the (2.9.12) add 50.0 mL of anhydrous methanol R. Weigh and
test solution. Furthermore, in the chromatogram obtained sonicate for 45 min. Allow to cool and weigh again.
with the test solution, other faint blue fluorescent zones or Compensate for the loss of solvent with anhydrous
faint brown zones may be present. methanol R. Filter. Transfer about 5 mL of the filtrate to a
test tube containing 0.5 g of end-capped octadecylsilyl silica gel
for chromatography R. Shake, and allow to stand for 15 min.
Filter through a membrane filter (nominal pore size
0.45 µm).
Reference solutwn (a) Dissolve 5.0 mg of amygdalin CRS in
Reference solution (b) Dissolve 1 mg of primeverin R in
reference solution (a) and dilute to 20 mL with the same
solution.
Column:
= =
- size: l 0.25 m, 0 4.6 mm;
2023 Pelargonium Root IV-415
Mobile phase: C. Thin-layer chromatography (2.2.27).

- mobile phase A: water for chromatography R; Test solution To 0.5 g of the powdered herbal drug (355)
- mobile phase B: acetonitrile Rl; (2. 9.12) add 10 mL of methanol R, shake for 15 min and
filter.
Reference solution Dissolve 1 mg of scopoletin R and 2 mg of
esculin R in 20 mL of methanol R.
0-3 95 5
3 - 13,5 95 ➔ 48 5 ➔ 52
13,5 - 15 48 ➔ 5 52 ➔ 95
F254 plate R (2-10 µm)].
15 - 20 5 95 Mobile phase water R, methanol R, ethyl acetate R
(10:14:76 V/V/V).
Flow rate 1.2 mUmin. Application 10 µL [or 5 µL] as bands.
Detection Spectrophotometer at 210 nm. Development Over a path of 10 cm [or 6 cm].
Injection 10 µL. Drying In air.
Retention time Amygdalin = about 10 ruin; Detection Spray with alcoholic potassium hydroxide solution R.
primeverin = about 11.6 min. Examine in ultraviolet light at 365 nm.
System suitability Reference solution (b): Results See below the sequence of zones present in the
- resolution: minimum 5.0 between the peaks due to chromatograms obtained with the reference solution and the
amygdalin and primeverin. test solution. Furthermore, other blue fluorescent zones may
Calculate the percentage content of amygdalin using the be present in the chromatogram obtained with the test
following expression: solution.
Top of the plate
AI area of the peak due to amygdalin in the chromatogram Scopoletin: a very bright blue A weak blue fluorescent zone
obtained with the test solution; fluorescent zone (scopoletin)
obtained with reference solution (a); -- --
solution, in grams; One or two bright blue fluorescent
m2 mass of amygdalin CRS used to prepare reference solution (a), zones
in grams; Esculin: a very bright blue
p percentage content of amygdalin in amygdalin CRS. fluorescent zone

- - - - - - - - - - - - - - - - - - - - - - Ph Eur
A weak blue fluorescent zone
-- --
Pelargonium Root A blue fluorescent zone

PhEur - - - - - - - - - - - - - - - - - - - - - ~
TESTS
DEFINITION
Dried, usually fragmented, underground organs of Maximum 12.0 per cent, determined on 1.000 g of the
Pelargonium sidoides DC and/or Pelargonium reniforme Curt. powdered herbal drug (355) (2.9.12) by drying in an oven at
Content 105 °C.
Minimum 2.0 per cent of tannins, expressed as pyrogallol Total ash (2.4.16)
(C6H6O3; Mr 126.1) (dried drug). Maximum 12.0 per cent.
IDENflFICATION Ash insoluble in hydrochloric acid (2.8.1)
A. The root is covered with dark, partly reddish-brown, Maximum 3.0 per cent.
longitudinally fissured bark. The transverse section shows,
underneath the cork layer, yellow or white wood, which ASSAY
clearly shows partly brownish medullary rays. Tannins (2.8.14)
brownish-red. Examine under a microscope using chloral - - - - - - - - - - - - - - - - - - - - - ~ Ph Eur
characters: multilayer cork cells consisting of almost uniform,
rectangular cells; fragments of parenchyma underneath the
cork containing sclereids with a wide lumen; numerous
calcium oxalate cluster crystals. Examine under a microscope
using a 50 per cent V!V solution of glycerol R. The powder
shows simple starch granules without striations or cracks.
IV-416 Peony Root 2023
Red Peony Root

Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried, whole or fragmented root of Paeonia lactifiora Pall.
or Paeonia veitchii Lynch or a mixture of the two, with
rhizome and rootlets removed.
Content
Minimum 1.8 per cent of paeoniflorin (C 23H2 8011;
IDENTIFICATION
A. Whole drug. The whole root is cylindrical, slightly curved.
Truncated at both ends, 4-40 cm long, 0.5-3 cm in diameter,
externally brown or reddish-brown, rough, longitudinally
furrowed and wrinkled, showing rootlet scars and transversely
prominent lenticels. The outer bark is sometimes easily
exfoliated. The texture is hard and fragile, easily broken.
Fragmented drug The fragmented root usually occurs as
longitudinal slices, 2-8 cm long, about 1-5 mm thick and F
0.5-3 cm in diameter, or as transversal slices, about 2-4 mm
thick and 0.5-3 cm in diameter. The edges are brown, the
0 000
cut surface is chalk-white or pinkish, the bark is narrow and
the wood shows distinct radial striations, sometimes with
("\o 0
clefts. 0 'vO 25 µm
B. Microscopic examination (2.8.23). The powder is greyish- >--<
white or pale brown. Examine under a microscope using
diagnostic characters (Figure 2425.-1): reticulate or bordered Figure 2425.-1. - Illustration for identification test B of powdered
pitted vessels [G] up to 65 µm in diameter; cluster crystals of herbal drug of peony root, red
calcium oxalate, 10-35 µm in diameter, isolated [C] or
included in parenchymatous cells [B], sometimes several in Top of the plate
the same cell [Ba], sometimes arranged in rows [Ga]; long,
fusiform, ligneous fibres, with thickened and slightly lignified Paeonol: a quenching zone
walls with large oblique or cross pits [E]; brown cork
fragments (surface view [A], transverse section [D]).
Examine under a microscope using a 50 per cent V/V A diffuse quenching zone
solution of glycerol R. The powder shows simple, spheroidal
starch granules up to 50 µm in diameter [F]. -- --
A diffuse quenching zone

(2. 9.12) add 5 mL of methanol R and heat in a water-bath at
Reference solution Dissolve 1 mg of paeonifiorin R and 1 mg
of paeonol R in 1 mL of methanol R. A quenching zone
-- --
Paeoniflorin: a quenching zone A quenching zone (paeoniflorin)
methanol R, methylene chloride R (3:5:5:35 V!VIVIV).
Development Over a path of 6 cm. Reference solution Test solution
Drying In air.
Detection A Examine in ultraviolet light at 254 nm. Detection B Treat with a 10 per cent V/V solution of su[furic
Results A See below the sequence of zones present in the acid R in ethanol (96 per cent) R, heat at 100 °C for 5 min;
chromatograms obtained with the reference solution and the examine in daylight.
test solution. Furthermore, other faint zones may be present Results B See below the sequence of zones present in the
in the chromatogram obtained with the test solution. chromatograms obtained with the reference solution and the
2023 Peony Root IV-41 7
Top of the plate System suitability Reference solution (b):

Paeonol: a yellow zone
paeoniflorin and 4 '-hydroxyacetophenone.
Calculate the percentage content of paeoniflorin using the
2 violet zones
-- --
2 violet zones
A1 area of the peak due to paeoniflorin in the chromatogram

2 violet zones A2 area of the peak due to paeoniflorin in the chromatogram
obtained with reference solution (c);
solution, in grams;
A diffuse yellowish-brown zone
m2 mass of paeonifiorin CRS used to prepare reference solution (a),
in grams;
-- --
p percentage content of paeoniflorin in paeoniflorin CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
A brown zone

White Peony Root
TESTS (Ph. Bur. monograph 2424)
Loss on drying (2.2.32) PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
powdered herbal drug (35 5) (2. 9.12) by drying in an oven at DEFINITION
105 °C for 2 h. Whole or fragmented, peeled root, of Paeonia lactifiora Pall.
with rhizome and rootlets removed, treated with boiling
Total ash (2.4.16)
water and dried.
Ash insoluble in hydrochloric acid (2.8.1) Content
Minimum 1.6 per cent of paeoniflorin (C 23H 28 0ll;
ASSAY
IDENTIFICATION
A. Whole drug. The whole root is cylindrical, straight or
Test solution To 30.0 mg of the powdered herbal drug (355) slightly curved, truncated at both ends, 5-18 cm long,
(2.9.12) add 3.0 mL of methanol Rand sonicate for 30 min. 1-2.5 cm in diameter, externally whitish or pale reddish-
Dilute to 10.0 mL with a 6.8 g/L solution of potassium brown, glossy or with longitudinal wrinkles and rootlet scars.
dihydrogen phosphate R. Filter through a membrane filter Blackish-brown zones due to lesions, especially those around
(nominal pore size 0.45 µm). the rootlet scars, and remains of brown cork are occasionally
Reference solution (a) Dissolve 5.0 mg of paeoniflorin CRS in present. The texture is compact, relatively even and not
methanol Rand dilute to 25.0 mL with the same solvent. easily broken.
Reference solution (b) Dissolve 1.0 mg of 4 1- Fragmented drug The fragmented root usually occurs as
hydroxyacetophenone R in 6.0 mL of reference solution (a). longitudinal slices, 2-8 cm long, about 1-5 mm thick and
Dilute 3.0 mL of this solution to 10.0 mL with a 6.8 g/L 1-2.5 cm in diameter, or as transversal slices, about 2-4 mm
solution of potassium dihydrogen phosphate R. thick and 1-2.5 cm in diameter. The edges are whitish or
Reference solution (c) Dilute 2.5 mL of reference solution (a) pale reddish-brown and the cut surface is whitish, often with
to 10.0 mL with a 6.8 g/L solution of potassium dihydrogen a pinkish tone. The cambium ring and radial striations are
phosphate R. distinct.
Column: B. Microscopic examination (2.8.23). The powder is
- size: l = 0.15 m, 0 = 4.6 mm; yellowish-white. Examine under a microscope using chloral
- stationary phase: end-capped octadecylsilyl silica gel for hydrate solution R. The powder shows the following diagnostic
chromatography R (5 µm). characters (Figure 2424.-1): bordered-pitted or reticulate
Mobile phase methanol R, 6.8 g/L solution of potassium vessels [A, E], 20-65 µm in diameter; cluster crystals of
dihydrogen phosphate R (30:70 V/V). calcium oxalate, 10-35 µm in diameter, isolated [C, D] or
included in parenchymatous cells [B, F], sometimes several
in the same cell [Fa], often arranged in rows [Ba]; ligneous
Detection Spectrophotometer at 230 nm. fibres with thickened walls with large pits [Ea]. Examine
Injection 10 µL of the test solution and reference under a microscope using a 50 per cent V/V solution of
solutions (b) and (c). glycerol R. The powder shows very numerous masses of
Run time 18 min. gelatinised starch, either isolated [H] or associated with the
Retention time Paeoniflorin = about 8 min; remains of cells [G].
4'-hydroxyacetophenone = about 9 min.
IV-418 Peony Root 2023
Detection B Treat with a 10 per cent V/V solution of suljuric

acid R in ethanol (96 per cent) R, heat at 100 °C for 5 min;
Top of the plate
Paeonol: a yellow zone
2 violet zones
-- ---
A sharp violet zone
A violet zone
A violet zone
-- --
A brown zone
herbal drug of peony root, white Reference solution Test solution

TESTS
(2.9.12) add 5 mL of methanol Rand heat in a water-bath at
Reference solution Dissolve 1 mg of paeoniflorin R and 1 mg 105 °C for 2 h.
of paeonol R in 1 mL of methanol R.
Total ash (2.4.16)
methanol R, methylene chloride R (3:5:5:35 VIVIV/V).
ASSAY
Drying In air.
Test solution To 30.0 mg of the powdered herbal drug (355)
Detection A Examine in ultraviolet light at 254 nm. (2. 9.12) add 3.0 mL of methanol R and sonicate for 30 min.
Results A See below the sequence of zones present in the Dilute to 10.0 mL with a 6.8 g/L solution of potassium
chromatograms obtained with the reference solution and the dihydrogen phosphate R. Filter through a membrane filter
test solution. Furthermore, other faint zones may be present (nominal pore size 0.45 µm).
in the chromatogram obtained with the test solution. Reference solution (a) Dissolve 5.0 mg of paeoniflorin CRS in
Top of the plate
Reference solution (b) Dissolve 1.0 mg of 4' -
Paeonol: a quenching zone hydroxyacetophenone R in 6.0 mL of reference solution (a).
Dilute 3.0 mL of this solution to 10.0 mL with a 6.8 g/L
solution of potassium dihydrogen phosphate R.
-- -- Reference solution (c) Dilute 2.5 mL of reference solution (a)
to 10.0 mL with a 6.8 g/L solution of potassium dihydrogen
-- -- phosphate R.
Paeoniflorin: a quenching zone A faint quenching zone
(paeoniflorin)
Column:
=
- size: l 0.15 m, 0 4.6 mm; =
Reference solution Test solution chromatography R (5 µm).
2023 Pepper IV-419
Mobile phase methanol R, 6.8 g/L solution of potassium

dihydrogen phosphate R (30:70 V!V).
solutions (b) and (c).
Run time 18 min.
Retention time Paeoniflorin = about 8 min;
4'-hydroxyacetophenone = about 9 min.
paeoniflorin and 4 '-hydroxyacetophenone. Fa
Calculate the percentage content of paeoniflorin using the
•• Hb
e--
A.i•··
. -• .,o ..
solution, in grams;
m2 mass of paeonifiorin CRS used to prepare reference solution (a), ~
in grams;
p percentage content of paeoniflorin in paeonifiorin CRS.
herbal drug of pepper
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
W'hite pepper The powder is light grey. Examine under a

Pepper fragments of the endocarp in surface view, consisting of more
or less polygonal sclereids about 20-30 µm in diameter,
(Ph. Bur. monograph 2477) which have irregularly thickened walls [Ac, C, Fa] and which
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ may or may not be associated with the testa [A, F],
consisting of a layer of indistinct, reddish-brown pigmented
DEFINITION
cells constituting the 'pigmented layer' [Ab, Fb] and a layer
Dried, ripe or nearly ripe fruit of Piper nigrum L. with an of very thin-walled polygonal cells constituting the 'hyaline
unbroken pericarp (black pepper) or with the outer layers of layer' [Aa]; fragments of the endocarp, in transverse
the pericarp removed (white pepper). section [G], showing sclereids with thickened inner walls on
Content the 3 lower sides [Ga], usually associated with the testa
- essential oil: minimum 25 mlJkg (anhydrous drug); (pigmented layer [Gb] and hyaline layer [Gel); fragments of
- piperine (C 17H 19N0 3 ; Mr 285.3): minimum 3.0 per cent the parenchyma of the mesocarp [DJ containing large oil
(anhydrous drug). cells 50-75 µm in diameter [Da]; numerous thin-walled,
IDENTIFICATION ovoid or polygonal cells of the parenchyma of the seed [E];
A. W'hite pepper. Spheroid berries, 3-5 mm in diameter, rare, elongated sclereids, with thickened walls, from the fruit
slightly flattened at one pole and with a small protuberance peduncle [BJ; a few fragments of vascular tissue with narrow
at the other, with smooth, externally matt, brownish-grey, spiral vessels ill. Examine under a microscope using a
greyish-white or pale yellowish-white surface, with numerous 50 per cent V/V solution of glycerol R. Rounded, compound
pale, linear striations between apex and base. starch granules [H], about 30 µm in diameter, made up of
tiny individual granules, ovoid or polyhedral by compression,
Black pepper Spheroid berries, 3-6 mm in diameter,
free [Hb] or included in the parenchymatous cells of the
externally blackish-brown, with raised reticular wrinkles,
seed [Ha].
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 Black pepper The powder is grey. Examine under a
stripped, the endocarp is greyish-white or pale yellow. microscope using chloral hydrate solution R. In addition to the
The fracture is greyish-white, starchy, possessing a small diagnostic characters described for white pepper, the
space at the centre. powdered black pepper shows the following diagnostic
characters (Figure 2477.-1): fragments of the epicarp [Kl
B. Microscopic examination (2.8.23).
with extremely thin-walled, brownish-red pigmented,
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
with strongly thickened walls [Kb].
IV-420 Pepper 2023
Test solutwn To 0.5 g of the powdered herbal drug (355) Water (2.2.13)
(2. 9.12) add 5 mL of methanol R. Sonicate for 10 min, Maximum 120 mlJkg, determined on 20.0 g of the freshly
centrifuge and use the supernatant. powdered herbal drug (1400) (2.9.12) reduced using a knife
Reference solutwn Dissolve 10 mg of borneol R and 15 mg of mill.
piperine R in 10 mL of methanol R. Total ash (2.4.16)
Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel Maximum 6.0 per cent.
F2 54 plate R (2-10 µm)]. ASSAY
Mobile phase ethyl acetate R, cyclohexane R (30:50 V/V). Essential oil (2.8.12)
Application 10 µL [or 5 µL] as bands of 10 mm [or 8 mm]. Use 10.0 g of the freshly powdered herbal drug (1400)
Development Over a path of 15 cm [or 6 cm]. (2.9.12), a 1000 mL round-bottomed flask, 400 mL of
water Ras the distillation liquid and 0.5 mL of xylene R in
Drying In air.
the graduated tube. Distil at a rate of 2-3 mlJmin for 3 h.
Piperine
Results A See below the sequence of zones present in the Liquid chromatography (2.2.29). Carry out the assay protected
from light.
be present in the chromatogram obtained with the test Test solutwn Disperse 0.250 g of the powdered herbal drug
solution. (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) R, combine the filtrate and washings
Top of the plate
and dilute to 50.0 mL with the same solvent. Filter through
-- -- a membrane filter (nominal pore size 0.45 µm).
3 quenching zones
Reference solutwn (a) Dissolve 15.0 mg of piperine CRS in
ethanol (96 per cent) Rand dilute to 100.0 mL with the same
-- -- solvent.
A quenching zone Reference solutwn (b) Disperse 0.250 g of long pepper for
system suitability HRS (355) (2.9.12) in 40 mL of ethanol
Piperine: a quenching zon_e A strong quenching zone (piperine)
(96 per cent) R. Sonicate for 20 min and filter. Rinse the flask
and the filter with 5 mL of ethanol (96 per cent) R, combine
the filtrate and washings and dilute to 50.0 mL with the
same solvent. Filter through a membrane filter (nominal pore
Reference solution Test solution size 0.45 µm).
Column:
Detection B Treat with anisaldehyde solutwn R and heat at - size: l = 0.15 m, 0 = 4.6 mm;
100 °C for 5 min; examine in daylight. - statwnary phase: end-capped octadecylsilyl silica gel for
chromatograms obtained with the reference solution and the Mobile phase:
test solution. Furthermore, other zones may be present in the - mobile phase A: water R;
chromatogram obtained with the test solution. - mobile phase B: acetonitrile R;

Top of the plate
(rnin) (per cent V/Jl) (per cent VIV)
A strong purple zone 0-5 50 50
5 - 20 50--. 5 50--. 95
A purple zone
20 - 22 5--. 0 95 --. 100
-- --
Borneo!: a yellowish-brown zone Flow rate 1.0 mlJmin.
A purple-grey zone
Injection 10 µL.
-- -- Retention time Piperine = about 10 min.
A violet-grey zone Identificatwn of peaks Use the chromatogram supplied with
A grey zone long pepper for system suitability HRS and the chromatogram
Piperine: a green or brownish zone A green or brownish zone (piperine) to piperine and peak 2.
- peak-to-valley ratw: minimum 4, where Hp = height above
A grey zone
the baseline of peak 2 and Hv = height above the baseline
Reference solution Test solution of the lowest point of the curve separating the peak due to
piperine from peak 2.
Calculate the percentage content of piperine using the
TESTS
Maximum 3 per cent.
2023 Peppermint Leaf IV-421
area of the peak due to piperine in the chromatogram obtained

area of the peak due to piperine in the chromatogram obtained
solution, in grams;
mass of piperine CRS used to prepare reference solution (a), in
grams;
p percentage content of piperine in piperine CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Peppermint Leaf
Preparation
Peppermint Leaf Dry Extract
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or cut, dried leaf of Mentha x piperita L.
Essential oil content:
- for the whole drug, minimum 12 mL'kg (anhydrous
drug);
- for the cut drug, minimum 9 mL'kg (anhydrous drug).
CHARACTERS
Penetrating odour reminiscent of menthol.
Peppermint leaf is green or brownish-green, with brownish-
violet veins in some varieties. The petioles are green or
brownish-violet.
IDENTIFICATION herbal drng of peppermint leaf
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. C. Thin-layer chromatography (2.2.27).
The lamina is oval or lanceolate, the apex acuminate, the Test solution To 0.5 g of the powdered herbal drug (355)
margin sharply dentate and the base asymmetrical. Venation (2. 9.12) add 2.5 mL of methanol R and 2.5 mL of water R,
is pinnate, prominent on the lower surface, with lateral veins sonicate for 5 min and filter.
leaving the midrib at about 45°. The lower surface is slightly Reference solutwn Dissolve 2 mg of luteolin-7-glucoside R,
pubescent and secretory trichomes are visible under a lens
2 mg of rntoside trihydrate R and 5 mg of rosmarinic acid R in
(6 x ) as bright yellowish points. The petiole is grooved, methanol R and dilute to 10 mL with the same solvent.
usually up to 1 mm in diameter and 0.5-1 cm long.
brownish-green. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic ethyl acetate R (7:7:18:68 VIVIVIV).
characters (Figure 0406.-1): fragments of epidermises bearing Applicatwn 4 µL as bands of 8 mm.
covering and glandular trichomes; adaxial epidermis (surface Development Over a path of 6 cm.
view [B, H]) having cells with sinuous-wavy walls [Ha] and Drying In air.
cuticle striated over the veins [BJ associated with palisade Detection Heat at 100-105 °C for 5 min; spray the warm
parenchyma [Hb]; abaxial epidermis [C] with diacytic
stomata (2.8.3) [Ca); covering trichomes are usually
fragmented, elongated, uniseriate with 3-8 cells with striated
cuticle [A, E]; glandular trichomes of 2 types: a) unicellular
stalk with small, rounded unicellular head 15-25 µm in
diameter (surface view [Ba, Cb], transverse
section [DJ), b) unicellular stalk with enlarged oval head
55-70 µm in diameter composed of 8 radiating cells (surface
view [Bb], transverse section [Ga]); fragments from near the Results See below the sequence of zones present in the
leaf margin [F] with isodiametric cells whose anticlinal walls chromatograms obtained with the reference solution and the
are more-or-less straight and beaded [Fa] and short, conical, test solution. Furthermore, other faint fluorescent zones may
unicellular or bicellular covering trichomes [Fb ); dorsiventral be present in the chromatogram obtained with the test
mesophyll fragments (transverse section [G]), with a single solution.
palisade layer [Ge] and 4-6 layers of spongy parenchyma
[Gb]. Yellowish crystals of menthol under the cuticle of
secretory cells may be present.
IV-422 Peppermint Oil 2023
Top of the plate IDENTIFICATION

First identificatwn: B.
Rosmarinic acid: a light blue A light blue fluorescent zone
fluorescent zone (rosmarinic acid) Second identification: A.
--- ---
Test solutwn Mix 0.1 g of the substance to be examined
with toluene R and dilute to 10 mL with the same solvent.
Luteolin-7-glucoside: an orange Reference solutwn Dissolve 50 mg of menthol R, 20 µL of
fluorescent zone cineole R, l O mg of thymol R and 10 µL of menthyl acetate R
An orange-yellow fluorescent zone in toluene R and dilute to 10 mL with the same solvent.
F2 54 plate R (2-10 µm)].
Rutoside: an orange fluorescent zone An orange-yellow fluorescent zone Mobile phase ethyl acetate R, toluene R (5:95 VIV).
--- --- Application l O µL [or 1 µL] of the reference solution and
20 µL [or 2 µL] of the test solution, as bands of 10 mm [or
An orange-yellow fluorescent zone
8 mm].
Drying In air.

TESTS test solution.
Maximum 5 per cent of stems, whose diameter is not greater Top of the plate
than 1.5 mm; maximum 2 per cent of foreign elements; not
more than 8 per cent of the leaves show brown stains due to --- ---
Puccinia menthae. Thymol: a quenching zone
Carry out the determination using 10 g of the drug.
Water (2.2.13)
Maximum 110 mUkg, determined on 20.0 g. Quenching zones may be present
(carvone, pulegone)
Total ash (2.4.16)
Maximum 15.0 per cent. --- ---
Ash insoluble in hydrochloric acid (2.8.1) Reference solution Test solution

ASSAY Detection B Treat with anisaldehyde solutwn R and heat at
Essential oil (2. 8.12) 100-105 °C for 5-10 min; examine immediately in daylight.
Use 20.0 g of the crushed herbal drug, a 500 mL flask, Results B See below the sequence of zones present in the
200 mL of water R as the distillation liquid and 0.50 mL of chromatograms obtained with the reference solution and the
xylene R in the graduated tube. Distil at a rate of 3-4 mUmin test solution. Furthermore, other less intensely coloured
for 2 h. zones may be present in the chromatogram obtained with the
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur test solution.
Top of the plate
An intense violet-red zone (near the

Peppermint Oil solvent front) (hydrocarbons)
A brownish-yellow zone
(menthofuran)
--- ---
Preparation
Peppermint Oil Gastro-resistant Capsules Menthyl acetate: a violet-blue zone A violet-blue zone (menthyl acetate)
A greenish-blue zone (menthone)
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Thymol: a pink zone
DEFINITION
Light pink or greyish-blue or greyish-
Essential oil obtained by steam distillation from the fresh
green zones may be present
aerial parts of Mentha x piperita L. (carvone, pulegone, isomenthone)
CHARACTERS 1,8-Cineole: a violet-blue or brown A faint violet-blue or brown zone
Appearance Colourless, pale yellow or pale greenish-yellow zone (1,8-cineole)
liquid.
--- ---
Characteristic odour and taste followed by a sensation of
Menthol: an intense blue or violet An intense blue or violet zone
cold. zone (menthol)
Soluhility Miscible with ethanol (96 per cent) and with
methylene chloride.
2023 Peppermint Oil Preparations IV-423
B. Examine the chromatograms obtained in the test for Elution order Order indicated in the composition of
chromatographic profile. reference solution (a); record the retention times of these
Results The characteristic peaks due to limonene, substances.
1,8-cineole, menthone, menthofuran, isomenthone, menthyl Identification of peaks Using the retention times determined
acetate and menthol in the chromatogram obtained with the from the chromatogram obtained with reference solution (a),
test solution are similar in retention time to those in the locate the components of reference solution (a) in the
chromatogram obtained with reference solution (a). chromatogram obtained with the test solution.
Isopulegol, pulegone and carvone may be present in the System suitability Reference solution (a):
chromatogram obtained with the test solution. - resolution: minimum 1.5 between the peaks due to
TESTS limonene and 1,8-cineole; minimum 1.5 between the
Relative density (2.2.5) peaks due to piperitone and carvone.
0.900 to 0.916. Determine the percentage content of each of the following
Refractive index (2.2.6) components. The limits are within the following ranges:
1.457 to 1.467. - limonene: 1.0 per cent to 3.5 per cent;
- 1,8-cineok: 3.5 per cent to 8.0 per cent;
Optical rotation (2.2. 7) - menthone: 14.0 per cent to 32.0 per cent;
-30° to -10°. - menthofuran: 1.0 per cent to 8.0 per cent;
Acid value (2.5. J) - isomenthone: 1.5 per cent to 10.0 per cent;
Maximum 1.4, determined on 5.0 g diluted in 50 mL of the - menthyl acetate: 2.8 per cent to 10.0 per cent;
prescribed mixture of solvents. - isopukgol: maximum 0.2 per cent;
Fatty oils and resinified essential oils (2.8.7) - menthol: 30.0 per cent to 55.0 per cent;
It complies with the test for fatty oils and resinified essential - pulegone: maximum 3.0 per cent;
oils. - carvone: maximum 1.0 per cent;
Mint oil
(0.05 per cent).
The ratio of 1,8-cineole content to limonene content is a
minimum of 2.
does not show a peak with the retention time of isopulegol
that has an area of more than 0.2 per cent of the total area. STORAGE
Chromatographic profile At a temperature not exceeding 25 °C.
Gas chromatography (2.2.28): use the normalisation _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
procedure.
Test soluti.on Mix 0.20 mL of the substance to be examined
with heptane Rand dilute to 10.0 mL with the same solvent.
Reference soluti.on (a) Dissolve 10 µL of limonene R, 20 µL Peppermint Oil Gastro-resistant
of cineok R, 40 µL of menthone R, 10 µL of menthofuran R, Capsules
10 µL of (+)-isomenthone R, 40 µL of menthyl acetate R,
20 µL of isopukgol R, 60 mg of menthol R, 20 µL of Gastro-resistant Peppermint Oil Capsules
pulegone R, 10 µL of piperitone R and 10 µL of carvone R in DEFINITION
heptane R and dilute to 10.0 mL with the same solvent. Peppermint Oil Gastro-resistant Capsules contain
Reference soluti.on (b) Dissolve 5 µL of isopukgol R in Peppermint Oil. They are covered with a gastro-resistant
heptane R and dilute to 10.0 mL with the same solvent. coating.
Dilute 0.1 mL of the solution to 5.0 mL with heptane R. The capsuks comply with the requirements stated under Capsuks
Column: and with the following requirements.
- material: fused silica; IDENTIFICATION
- size: l = 60 m, 0 = 0.25 mm;
A. Carry out the method for thin-layer chromatography,
0.25 µm).
(1) Dissolve a quantity of the contents of the capsules
containing 0.1 g of Peppermint Oil in sufficient toluene to
Flow rate 1.5 mUmin. produce 10 mL.
Split rati.o 1:50. (2) Dissolve 10 mg of thymol, 10 µL of menthyl acetate, 20 µL
Temperature: of cineok and 50 mg of menthol in toluene and dilute to
Time Temperature
(min) CC)
Column 0 - 10 60
(a) Use a TLC silica gel GF 254 plate.
10 - 70 60 ➔ 180 (b) Use the mobile phase as described below.
70 - 75 180 (c) Apply 20 µL of solution (I) and 10 µL of solution (2) as
Injection port 200 bands.
Detector 220 (d) After removal of the plate, dry in air and examine under
ultraviolet light (254 nm), spray with anisaldehyde solution and
Detection Flame ionisation. examine in daylight for 5 to 10 minutes while heating at 100°
Injection 1 µL. to 105°.
IV-424 Peppermint Leaf Preparations 2023
MOBILE PHASE Time Temperature Comments

5 volumes of ethyl acetate and 95 volumes of toluene. (minutes)
CONFIRMATION 0----> 10 60° isothermal
When examined under ultraviolet light (254 nm) the
chromatogram obtained with solution (1) may show 10----> 70 60° ---->180° linear gradient
quenching zones (carvone, pulegone) situated just below the
70----> 75 180° isothermal
level of the zone (thymol) in the chromatogram obtained
with solution (2).
When examined in daylight the chromatogram obtained with SYSTEM SUITABILITY
solution (2) shows, in order of increasing Rf value, an intense
blue to violet zone (menthol) in the lower third; a violet-blue The test is not valid unless the number of theoretical plates
to brown zone (cineole); a pink zone (thymol); and a violet- calculated from the limonene peak at 110° is at least 30,000
blue zone (menthyl acetate). In the chromatogram obtained and the resolution factor between the peaks corresponding to
with solution (1) there is a zone due to menthol (the most limonene and cineole is at least 1.5 in the chromatogram
intense) and a faint zone due to cineole; at Rf values between
those of the cineole and thymol zones in the chromatogram DETERMINATION OF CONTENT
obtained with solution (2), there may be light pink or Using the retention times determined from the
greyish-blue or greenish-grey zones (carvone, pulegone, chromatogram obtained with solution (2), locate the
isomenthone); in the middle of the chromatogram, there is a components of solution (2) on the chromatogram obtained
violet-blue zone (menthyl acetate) and just below it a with solution (1) (disregard the peak due to hexane).
greenish-blue zone (menthone); an intense violet-red zone Determine the percentage content of the components by the
(hydrocarbons) appears near the solvent front and below it normalisation procedure.
there may be a brownish-yellow zone (menthofuran); other
less intensely coloured zones may also appear.
B. Examine the chromatograms obtained in the test for Limonene 1.0 to 3.5%,
Chromatographic profile. The retention time of the principal Cineole 3.5 to 8.0%,
peaks in the chromatogram obtained with solution (1) is Menthone 14.0 to 32.0%,
similar to that of the principal peaks in the chromatogram Menthofuran 1.0 to 8.0%,
Isomenthone 1.5 to 10.0%,
obtained with solution (2). Carvone and pulegone may be Menthyl acetate 2.8 to 10.0%,
absent from the chromatogram obtained with solution (1). Isopulegol Maximum 0.2%
Menthol 30.0 to 55.0%
TESTS Pulegone Not more than 3.0%,
Disintegration test Carvone Not more than 1.0%.
Complies with the requirements stated under Gastro-resistant
Capsules. The ratio of cineole content to limonene content is greater
Refractive index than 2.
1.457 to 1.467, determined on the contents of the capsules, STORAGE
Appendix VG. Peppermint Oil Gastro-resistant Capsules should be
Relative density protected from light.
0.900 to 0.916, determined on the contents of the capsules,
Appendix V E.
Composition of peppermint oil
Carry out the method for gas chromatography, Peppermint Leaf Dry Extract
Appendix III B, using the following freshly prepared
solutions. (Ph. Bur. monograph 2382)
(1) Use the contents of the capsules. PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
(2) Dissolve 0.1 g of limonene, 0.2 g of dneole, 0.4 g of DEFINITION

menthone, 0.1 g of menthofuran, 0.1 g of isomenthone, 0.4 g of Dry extract produced from Peppennint leaf (0406).
menthyl acetate, 0.6 g of menthol, 0.2 g of pulegone and 0.1 g
Content
of carvone in 1 mL of hexane.
Minimum 0.5 per cent ofrosmarinic acid (C 18H160s;
CHROMATOGRAPHIC CONDITIONS Mr 360.3) (dried extract).
(a) Use a fused-silica capillary column (60 m x 0.25 mm) PRODUCTION
coated with macrogol 20,000 as the bonded phase.
(b) Use helium as the carrier gas at 1.5 mL per minute. procedure using ethanol (30-50 per cent V/V) or water at
(c) Use the gradient conditions described below. minimum 60 °C.
(d) Use an inlet temperature of 220°. CHARACTERS
(e) Use a flame ionisation detector at a temperature of 220°. Appearance
(f) Inject 0.2 µL of each solution. Brown, amorphous powder.
(g) Use a split ratio of 1: 100. IDENTIFICATION
When the chromatograms are recorded in the prescribed Thin-layer chromatography (2.2.27).
conditions, the components elute in the order indicated in Test solution To 0.2 g of the extract to be examined add
the composition of solution (2). Record the retention times 2.5 mL of methanol Rand 2.5 mL of water R, sonicate for
of these substances. 5 min and filter.
2023 Peru Balsam IV-425
Reference solution Dissolve 2 mg of luteolin-7-glucoside R, Mobile phase:

2 mg of rutoside trihydrate R and 5 mg of rosmarinic acid R in - mobile phase A: phosphoric acid R, acetonitrile R, water for
methanol R and dilute to 10 mL with the same solvent. chromatography R (1:19:80 VIVIV);
Plate TLC silica gel F254 plate R (2-10 µm). - mobile phase B: phosphoric acid R, methanol R, acetonitrile R
(1 :40:59 VIVIV);
Application 4 µL as bands of 8 mm. (min) (per cent VIV) (per cent VIV)
Development Over a path of 6 cm. 0 - 20 100 ➔ 55 0 ➔ 45
Drying In air. 20 - 25 55 ➔ 0 45 ➔ JOO
25 - 30 0 ➔ 100 JOO ➔ 0
ester R in methanol R, then with a 50 g/L solution of macrogol Flow rate 1.2 mL/min.
400 R in methanol R or, alternatively, dip the warm plate in a Detection Spectrophotometer at 330 nm.
Injection 20 µL.
methylene chloride R; allow to dry in air for about 1 min and Identification of peaks Use the chromatogram obtained with
reference solution (a) to identify the peak due to rosmarinic
acid.
chromatograms obtained with the reference solution and the Relative retention With reference to rosmarinic acid
test solution. Furthermore, other faint fluorescent zones may =
(retention time about 11 min): ferulic acid about 0.8. =
be present in the chromatogram obtained with the test System suitability Reference solution (b):
solution. - resolution: minimum 4.0 between the peaks due to ferulic
Top of the plate Calculate the percentage content of rosmarinic acid using the
Rosmarinic acid: a light blue A light blue fluorescent zone
fluorescent zone (rosmarinic acid) A 1 x m2 xpx 0.2
-- --- A2 xm1
Luteolin-7-glucoside: an orange A faint orange fluorescent zone A2 area of the peak due to rosmarinic acid in the chromatogram
fluorescent zone obtained with reference solution (a);
solution, in grams;
m2 mass of rosmarinic acid CRS used to prepare reference
Rutoside: an orange fluorescent zone An orange-yellow fluorescent zone p percentage content of rosmarinic acid in rosmarinic acid CRS.
--- -- _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Reference solution Test solution Peru Balsam

ASSAY
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Test solution Use brown glass flasks. To 0.400 g of the DEFINITION
extract to be examined add 15 mL of ethanol Balsam obtained from the scorched and wounded trunk of
(50 per cent VIV) R, sonicate for 10 min and filter into a Myroxylon balsamum (L.) Harms var. pereirae (Royle) Harms.
20 mL volumetric flask. Rinse the flask and the filter with Content
ethanol (50 per cent Viii'.) R and dilute to 20.0 mL with the 45.0 per cent m/m to 70.0 per cent mlm of esters, mainly
same solvent. benzyl benzoate and benzyl cinnamate.
CHARACTERS
acid CRS in ethanol (50 per cent Viii'.) R and dilute to
Appearance
Dark brown, viscous liquid which is transparent and
Reference solution (b) Dissolve 5 mg offerulic acid R in yellowish-brown when viewed in a thin layer; it is not sticky,
reference solution (a) and dilute to 50 mL with the same non-drying and does not form threads.
solution.
Solubility
Column: Practically insoluble in water, freely soluble in anhydrous
- size: l = 0.25 m, 0 = 4.6 mm; ethanol, not miscible with fatty oils, except for castor oil.
chromatography R (5 µm). IDENTIFICATION
A. Dissolve 0.20 g in 10 mL of ethanol (96 per cent) R.
Add 0.2 mL of ferric chloride solution Rl. A green or
yellowish-green colour develops.
IV-426 Phellodendron Amurense Bark 2023
B. Thin-layer chromatography (2.2.27). 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 Protected from light.
benzyl cinnamate R and 80 µL of benzyl benzoate R in 5 mL _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
of ethyl acetate R.
Plate TLC silica gel GF254 plate R.
Mobile phase glacial acetic acid R, ethyl acetate R, hexane R
(0.5:10:90 VIVIV). Phellodendron Amurense Bark
Application l O µL, as bands of 20 mm by 3 mm. DEFINITION
Development Twice over a path of 10 cm. Phellodendron Amurense Bark is the dried bark of
Drying In air. Phellodendron amurense Ruprecht.
Detection A Examine in ultraviolet light at 254 nm and It is collected between spring and summer and the rough
mark the quenching zones. part of the bark is removed and sundried.
Results A The chromatogram obtained with the reference It contains not less than 0.4% w/w ofberberine
solution shows in the upper third 2 quenching zones, the (C 20H 18NO 4 ) and not less than 0.2% w/w palmatine
higher one due to benzyl benzoate and the lower one due to (C 21 H 22NO 4 ) calculated with reference to the dried material.
benzyl cinnamate. The chromatogram obtained with the test
IDENTIFICATION
solution shows 2 quenching zones at the same levels and of
A. The bark pieces are usually rectangular or shallowly
approximately the same size.
quilled, of variable length and width; between 2 and 6 mm
Detection B Spray with a freshly prepared 200 g/L solution thick. Prepared, cut slices are transversely sliced into
of phosphomolybdu acid R in ethanol (96 per cent) R, using rectangular pieces and occasionally show the remains of the
10 mL for a plate 200 mm square and examine in daylight corky outer bark. The outer surface is relatively smooth with
while heating at 100-105 °C for 5-10 min. yellow, yellowish-green or yellowish-brown inner bark
Results B The zones due to benzyl benzoate and benzyl (secondary phloem) interrupted by irregularly shaped remains
cinnamate are blue against a yellow background. of brown or greyish-brown outer bark (cork or rhytidoma),
The chromatogram obtained with the reference solution the latter fissured or sometimes raised, angular and somewhat
shows at about the middle a violet-grey zone (thymol). In the spongy. Lenticels are occasionally visible in the outer bark.
chromatogram obtained with the test solution, a blue zone The inner surface is smooth and yellow or yellowish-brown
(nerolidol) is seen just below the level of the zone due to throughout with fine longitudinal striations. The texture is
thymol in the chromatogram obtained with the reference light and hard. The fracture is fibrous, bright yellow or
solution. Just below the zone due to nerolidol, no blue zone yellowish-green.
is seen corresponding to a quenching zone seen when B. Reduce to a powder, Appendix XVII A. The powder is
examined in ultraviolet light at 254 nm (colophony). In the yellow or yellowish-brown. Examine under a microscope
upper and lower part of the chromatogram obtained with the using chloral hydrate solution. The powder shows the following
test solution, other faint blue zones may be seen. diagnostic characters: abundant fragments of fibres, yellow or
TESTS pale yellow, mainly in bundles or sheets, with thickened walls
Relative density (2.2.5) and a long, narrow lumen, surrounded by a crystal sheath of
1.14 to 1.17. parenchyma cells containing prisms of calcium oxalate.
Scattered calcium oxalate prism crystals, up to about 24 µm,
Saponification value (2.5.6)
are also seen in the powder. The fibre sheets are interspersed
230 to 255, determined on the residue obtained in the assay.
with medullary rays composed of polygonal cells two to three
Artificial balsams cells wide. Numerous large stone cells, bright yellow, sub-
Shake 0.20 g with 6 mL of light petroleum Rl. The light orbicular, fusiform or irregular in shape, some branched or
petroleum solution is clear and colourless and the whole of with a pointed apex, with thickened, distinctly striated walls,
the insoluble parts of the balsam stick to the wall of the test- are present, commonly arranged in groups. Groups of
tube. tabular, colourless, cork cells in layers are present.
Fatty oils C. Carry out the method for thin-layer chromatography,
Shake 1 g with 3 mL of a 1000 g/L solution of chloral Appendix III A, using the following solutions.
hydrate R. The resulting solution is as clear as the 1000 g/L (1) To 0.25 g of the finely powdered drug,
solution of chloral hydrate R. Appendix XVII A, add 4 mL of a mixture of 20 mL of water
Turpentine and 80 mL of methanol. Mix with the aid of ultrasound for
Evaporate to dryness 4 mL of the solution obtained in the 10 minutes and filter. Extract the remaining residue in the
test for artificial balsams. The residue has no odour of filter with two 2-mL quantities of methanol. Combine the
turpentine. solutions and dilute to 20 mL with methanol.
ASSAY (2) 0.04% w/v each of palmatine chloride BPCRS and berberine
To 2.50 gin a separating funnel add 7.5 mL of dilute sodium chloride BPCRS in methanol.
hydroxide solution R and 40 mL of peroxide-free ether R and CHROMATOGRAPHIC CONDITIONS
shake vigorously for 10 min. Separate the lower layer and (a) Use as the coating octadecylsilyl silua gel for HPTLC
shake it with 3 quantities, each of 15 mL, of peroxide-free (Merck silica gel HPTLC plates are suitable)
ether R. Combine the ether layers, dry over 10 g of anhydrous
sodium sulfate Rand filter. Wash the sodium sulfate with
2 quantities, each of 10 mL, of peroxide-free ether R. Combine (c) Apply 20 µL of each solution, as 8 mm bands.
2023 Phellodendron Chinense Bark IV-427
(d) Develop the plate to 6 cm from the point of application. (f) Inject 50 µl of each solution.
(e) After removal of the plate, dry in air for 5 minutes, dip MOBILE PHASE
the plate in ethanolic sulfuric acid (20%) and then heat at 105° 27 volumes of acetonitn'le and 73 volumes of a 1.36% w/v
until coloured bands appear. Examine under ultraviolet light solution of potassium dihydrogen orthophosphate in water.
(366 nm).
SYSTEM SUITABILITY
MOBILE PHASE
10 volumes of anhydrous formic acid, 10 volumes of water and with solution (2), the resolution between the peaks due to
80 volumes of ethyl acetate. palmatine (retention time approximately 8 minutes) and
SYSTEM SUITABILITY berberine (retention time approximately 9 minutes) is
The test is not valid unless the chromatogram obtained with at least 2.0.
solution (2) shows two clearly separated bands. DETERMINATION OF CONTENT
RESULTS Using the retention times and the peak areas from the
See the sequence of zones present in the chromatograms chromatograms obtained with solution (2), locate and
obtained with the solution (1) and solution (2). Other faint integrate the peaks due to berberine and palmatine in the
zones may be present. chromatogram obtained with solution (1). Calculate the
content of berberine and of palmatine in the sample using
Top of the plate the declared content of berberine in berberine chloride BPCRS
and palmatine in palmatine chloride BPCRS and the following
expression:
A1 xm2 xp xl2.5
A 2 xm 1
A green band (berberine) A green band (berberine)
An orange band A1 Area of the peak due to berberine/palmatine in the

chromatogram obtained with solution (!).
A green band (palmatine) A green band (palmatine) A2 Area of the peak due to berberine/palmatine in the
An orange band
m1 Weight of the drug being examined in g.
A blue band m2 Weight of berberine chwridelpalmatine chloride in the reference
solution in g.
A blue band p Percentage content of berberine/palmatine in berberine
chwridelpalmatine chloride.
Solur:iim (1) Solution (2) STORAGE

Phellodendron Amurense Bark should be protected from
moisture.
TESTS
ANNEX
Loss on drying
When dried at 100° to 105° for 2 hours, loses not more than This section is non-mandatory.
10.0% of its weight, Appendix IX D. Use 1 g. DNA reference sequence
Total ash A DNA reference sequence for the identity of Phellodendron
Amurense Bark is published in Supplementary Chapter VII D.
ASSAY
Carry out the method for liquid chromatography,
Appendix III D, using the following solutions. Phellodendron Chinense Bark
( 1) To 0 .1 g of powdered sample, add 80 mL of a mixture of
DEFINITION
equal volumes of acetonitrile and 0.1 % v/v solution of
orthophosphoric acid. Mix with the aid of ultrasound for Phellodendron Chinense Bark is the dried bark of
40 minutes and allow to cool. Dilute to 100 mL with mobile Phellodendron chinense Schneid.
phase and filter (Whatman GF/C is suitable). It is collected between spring and summer and the rough
(2) 0.01 % w/v of palmatine chloride BPCRS and 0.01 % w/v outer bark is removed and discarded. The remainder is
berberine chloride BPCRS in mobile phase. sundried.
It contains not less than 3.0% w/w of berberine
(C 20 H1 8 NO4 ), calculated with reference to the dried
(a) Use a stainless steel column (15 cm x 4.6 mm) packed material.
with end-capped octadecylsilyl silica gel for chromatography
(5 µm) (Phenomenex Luna ClS is suitable). IDENTIFICATION
A. The bark pieces are usually rectangular or shallowly
quilled of widely varying length and width; up to 6 mm
below.
thick. Prepared, cut, slices are sliced transversely into
(c) Use a flow rate of 1.2 mL per minute. rectangular pieces. The outer surface is greyish or yellowish
(d) Use an ambient column temperature. (depending on the amount of thin outer cork bark removed),
(e) Use a detection wavelength of 235 nm. with irregularly scattered raised transverse lenticels. The inner
surface is smooth and yellow to yellowish-brown throughout
IV-428 Phellodendron Chinense Bark 2023
with fine longitudinal striations. The texture is light and Top of the plate
hard. The fracture is fibrous, yellow or yellowish green; older
barks are thicker and include a hard sponge-like zone on the
innermost side.
yellow or yellowish-brown. Examine under a microscope
using chloral hydrate solution. The powder shows the following A green band (berberine) A green band (berberine)
diagnostic characters: abundant fragments of short fibres, A yellow band
yellow or pale yellow, mainly in bundles or sheets, with
thickened walls and a long, narrow lumen, surrounded by a A green band (palmatine)
crystal sheath of parenchyma cells containing prisms of
calcium oxalate. Scattered calcium oxalate prism crystals, up
to about 40 µm, are also seen in the powder. The fibre sheets
are interspersed with conspicuous medullary rays composed
of polygonal cells two to three cells wide. Numerous large
stone cells, bright yellow, sub-orbicular, fusiform or irregular Solution (1) Solution (2)
in shape, some branched or with a pointed apex, with
thickened, distinctly striated walls, are present, commonly TESTS
arranged in groups.
Phellodendron amurense
C. Carry out the method for thin-layer chromatography, In identification test C, the chromatogram obtained with
Appendix III A, using the following solutions. solution (1) shows no orange band between the bands for
(1) To 0.25 g of the finely powdered drug, berberine and palmatine and no orange or blue bands below
Appendix XVII A, add 4 mL of a mixture of 20 mL of water the band for palmatine given in the chromatogram obtained
and 80 mL of methanol. Mix with the aid of ultrasound for with solution (2).
10 minutes and filter. Extract the remaining residue in the Loss on drying
filter with two 2-mL quantities of methanol. Combine the When dried at 100° to 105° for 2 hours, loses not more than
solutions and dilute to 20 mL with methanol. 10.0% of its weight, Appendix IX D. Use 1 g.
(2) 0.04% w/v each of palmatine chloride BPCRS and berben·ne Total ash
chloride BPCRS in methanol. Not more than 8.0%, Appendix XI J, Method II.
ASSAY
(a) Use as the coating octadecylsilyl silica gel for HPTLC Carry out the method for liquid chromatography,
(Merck silica gel HPTLC plates are suitable). Appendix III D, using the following solutions.
(b) Use the mobile phase as described below. (1) To 0.1 g of powdered sample, add 80 mL of a mixture of
(c) Apply 20 µL of each solution, as 8 mm bands. equal volumes of acetonitrile and 0.1 % v/v solution of
(d) Develop the plate to 6 cm from the point of application. orthophosphoric acid. Mix with the aid of ultrasound for
(e) After removal of the plate, dry in air for 5 minutes, dip 40 minutes and allow to cool. Dilute to 100 mL with mobile
the plate in ethanolic sulfuric acid (20%) and then heat at 105° phase and filter (Whatman GF/C is suitable).
until coloured bands appear. Examine under ultraviolet light (2) 0.01% w/v ofpalmatine chloride BPCRS and 0.01% w/v
(366 nm). berberine chloride BPCRS in mobile phase.
MOBILE PHASE CHROMATOGRAPHIC CONDITIONS
10 volumes of anhydrous formic acid, 10 volumes of water and (a) Use a stainless steel column (15 cm x 4.6 mm) packed
80 volumes of ethyl acetate. with end-capped octadecylsilyl silica gel for chromatography
(5 µm) (Phenomenex Luna C18 is suitable).
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with
solution (2) shows two clearly separated bands. below.
RESULTS
See the diagram for the sequence of zones present in the (d) Use an ambient column temperature.
chromatograms obtained with solution (1) and solution (2). (e) Use a detection wavelength of 235 nm.
Furthermore, in the chromatogram obtained with solution (f) Inject 50 µL of each solution.
(1) a faint or more intense green band corresponding to MOBILE PHASE
palmatine may be present. Other faint zones may be present.
27 volumes of acetonitrile and 73 volumes of a 1.36% w/v
solution of potassium dihydrogen orthophosphate in water.
SYSTEM SUITABILITY
with solution (2), the resolution between the peaks due to
palmatine (retention time of approximately 8 minutes) and
berberine (retention time of approximately 9 minutes) is
at least 2.0.
Using the retention time and the peak area from the
chromatograms obtained with solution (2), locate and
2023 Phyllanthus Emblica Pericarp IV-429
integrate the peak due to berberine in the chromatogram CHROMA TO GRAPHIC CONDITIONS
obtained with solution (1). Calculate the content of berberine (a) Use as the coating silica gel 60 F254 or high-performance
in the sample using the declared content of berberine in silica gel 60 F254 (Merck silica gel 60 F 254 HPTLC plates are
berberine chloride BPCRS and the following expression: suitable).
A1 xm2 xp xl2.5 (c) Apply 10 µL [or 2 µL] of each solution, as bands.
A 2 xm 1 (d) Develop the plate to 15 cm [or 7 cm].
(e) After removal of the plate, dry in air, spray with a 5% w/v
solution of iron(III) chloride in ethanol and examine in
Area of the peak due to berberine in the chromatogram daylight.
obtained with solution (!). MOBILE PHASE
Area of the peak due to berberine in the chromatogram
obtained with solution (2). 1.5 volumes of toluene, 3 volumes of acetic acid, 4 volumes of
m1 Weight of the drug being examined in g. formic acid and 30 volumes of ethyl acetate.
m, Weight of berberine chloride in the reference solution in g.
p Percentage content of berberine in berbenne chloride. SYSTEM SUIT ABILITY
STORAGE solution (2) shows two clearly separated bands.
Phellodendron Chinense Bark should be protected from CONFIRMATION
moisture. The chromatogram obtained with solution (1) shows a dark
ANNEX blue band with an Rf value of 0.6 corresponding in colour
This section is non-mandatory. and position to the band obtained with ellagic acid in
solution (2) and a dark blue band with an Rf value of
approximately 0.9 corresponding in position to the dark blue
A DNA reference sequence for the identity of Phellodendron
band obtained with gallic acid in solution (2). Two or three
Chinense Bark is published in Supplementary Chapter VII D.
separated blue or faint blue bands with Rf values of between
0.2 and 0.3 are also present. Other bands may be present in
solution (1).
Phyllanthus Emblica Pericarp Top of the plate
DEFINITION
Dark blue band Gallic acid: a dark blue band
Phyllanthus Emblica Pericarp is the pericarp of dried mature
fruits of Phyllanthus emblica L. (syn. Emblica officinalis
Gaertn.)
It contains not less than 6.0% tannins, expressed as Dark blue band Ellagic acid: a dark blue band
pyrogallol (C 6 H 6 0 3 • Mr 126.1), calculated with reference to
the dried drug.
IDENTIFICATION
A. Irregular pieces of the pericarp showing a dark brownish Blue/faint blue band
to black outer surface, much wrinkled and grooved with Blue/faint blue band
occasional greyish-white patches; the underlying brown
mesocarp is about 2 to 3 mm wide surrounding a thin, paler Solution (1) Solution (2)
brown endocarp. Infrequently whole seeds, or portions of the
creamish white testa may be present, attached to the
endocarp; whole seeds are subspherical, about 1 cm in TESTS
diameter and the testa is marked with 6 equidistant Ethanol-soluble extractive
longitudinal ridges. Occasional whole fruits may also be Not less than 15.0%, Appendix XI Bl
present; they are spherical to ovoid or irregular, about 2 cm
Foreign matter
in diameter and show a depression at one end.
Not more than 5% of foreign matter including seed material,
B. The powder is yellow-green or pale brown. It shows small Appendix XI D.
polygonal cells from the exocarp covered with a thick cuticle,
numerous mesocarp, some filled with amorphous grey
Not less than 50%, Appendix XI B2.
birefractive masses, some with mucilage, and some with
calcium oxalate crystals in the form of needles, large prism or Ash
microsphenoids; thick-walled, pitted sclereids from the Not more than 7.0%, Appendix XI J, method II.
mesocarp found singly and usually surrounded by Loss on drying
parenchyma, and fragments of the endocarp consisting of a Not more than 10.0%, Appendix IX D. Use 1 g.
thick layer of pitted fibres and sclereids.
ASSAY
C. Carry out the method for thin-layer chromatography, Carry out the determination of tannins in herbal drugs,
Appendix III A, using the following solutions. Appendix XI M. Use 1.0 g of powdered drug.
(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
methanol.
IV-430 Dwarf Pine Oil 2023
Optical rotation (2.2.7)

Dwarf Pine Oil -15° to -6°.
(Ph. Bur. monograph 2377) Acid value (2.5.J)
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Maximum 1.0.
DEFINITION Maximum 20.
Essential oil obtained by steam distillation of the fresh leaves
and twigs of Pinus mugo Turra. A suitable antioxidant may be
Fatty oils and resinified oils (2. 8. 7)
added.
CHARACTERS
Appearance procedure.
Test solution Dilute 200 µL of the substance to be examined
IDENTIFICATION to 10.0 mL with heptane R.
First identification: B. Reference solution (a) Dilute 30 µL of rJ.-pinene R, 5 mg of
Second identification: A. camphene R, 10 µL of {J-pinene R, 20 µL of car-3-ene R, 5 µL
A. Thin-layer chromatography (2.2.27). of fJ-myrcene R, 10 µL of limonene R, 5 µL of p-cymene R,
Test solution Dilute 1 mL of the substance to be examined 10 µL of terpinolene R, 5 µL of bornyl acetate R and 5 µL of
to 10 mL with toluene R. {J-caryophyllene R in heptane R and dilute to 5 mL with the
same solvent.
Reference solution Dissolve 10 mg of borneol R and 10 µL of
bornyl acetate R in toluene R and dilute to 10 mL with the Reference solution (b) Dissolve 5 mg of camphene R in
same solvent. heptane Rand dilute to 50.0 mL with the same solvent.
Dilute 1.0 mL ofthis solution to 10.0 mL with heptane R.
plate R (2-10 µm)]. Column:
- size: l = 60 m, 0 = 0.25 mm;
Application 10 µL [or 2 µL], as bands. - stationary phase: macrogol 20 000 R (film thickness
Development Over a path of 15 cm [or 6 cm]. 0.25 µm).
Drying In air. Carrier gas helium for chromatography R.
Detection Spray with anisaldehyde solution R and heat at Flow rate 1.5 mLJmin.
100-105 °C for 5-10 min; examine in daylight. Split ratio 1:50.
Results 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
41 - 50 220
A pink zone
Injection pon 220
- - -- Detector 250
Bomyl acetate: a brown or greyish- A brown or greyish-brown zone

brown zone (bornyl acetate) Detection Flame ionisation.
A pink zone Injection 1 µL.
- - --
Borneo!: a brown or greyish-brown A cluster of violet zones substances.
zone
car-3-ene and ~-myrcene.
Identification of components Using the retention times
determined from the chromatogram obtained with reference
B. Examine the chromatograms obtained in the test for solution (a), locate the components of reference solution (a)
chromatographic profile. in the chromatogram obtained with the test solution; the
Results The characteristic peaks in the chromatogram peak due to ~-phellandrene is eluted after the peak due to
obtained with the test solution are similar in retention time to limonene with a relative retention of about 1.03 with
those in the chromatogram obtained with reference reference to limonene.
solution (a). Determine the percentage content of each of these
TESTS components. The limits are within the following ranges:
Relative density (2.2.5) - rJ.-pinene: 10.0 per cent to 30.0 per cent;
0.857 to 0.870. - camphene: maximum 2.0 per cent;
- {J-pinene: 3.0 per cent to 14.0 per cent;
- car-3-ene: 10.0 per cent to 40.0 per cent;
1.474 to 1.480. - {J-myrcene: 3.0 per cent to 12.0 per cent;
2023 Pine Silvestris Oil IV-431
- limonene: 5.0 per cent to 14.0 per cent; Top of the plate
- /J-phellandrene: 9.0 per cent to 19.0 per cent;
A pink zone (hydrocarbons)
- p-cymene: maximum 2.5 per cent;
- terpinolene: maximum 8.0 per cent; -- --
- bornyl acetate: 0.5 per cent to 5.0 per cent;
Bomyl acetate: a brown or grey- A brown or grey-brown zone (bornyl
- /3-caryophyllene: 0.5 per cent to 5.0 per cent; bro\\-11 zone acetate)
- reporting threshold: 0.05 per cent (principal peak in the
chromatogram obtained with reference solution (b)). A pink zone
STORAGE -- --
In an inert container and at a temperature not exceeding Borneo!: a brown or grey-brown A cluster of violet zones
25 °C. zone
Pine Silvestris Oil B. Examine the chromatograms obtained in the test for
(Ph. Bur. monograph 1842) Results The characteristic peaks in the chromatogram
DEFINITION
solution (a).
Essential oil obtained by steam distillation of the fresh leaves
and branches of Pinus sylvestris L. A suitable antioxidant may TESTS
be added. Relative density (2.2.5)
0.855 to 0.875.
CHARACTERS
Appearance Refractive index (2.2.6)
Clear, colourless or pale yellow liquid. 1.465 to 1.480.
Characteristic odour. Optical rotation (2. 2. 7)
-9° to -30°.
IDENTIFICATION
First identification: B. Acid value (2.5.J)
Maximum 1.0.
Maximum 20.
Test solutwn Dilute 1 mL of the substance to be examined
to 10 mL with toluene R. Fatty oils and resinified oils (2.8.7)
Reference solutwn Dissolve 10 mg of borneol Rand 10 µL of
horny! acetate R in toluene R and dilute to 10 mL with the Chromatographic profile
same solvent. Gas chromatography (2.2.28): use the normalisation
procedure.
plate R (2-10 µm)). Test solutwn The substance to be examined.
Mobile phase ethyl acetate R, toluene R (5:95 V!V). Reference solutwn (a) Dissolve 30 µL of rx-pinene R, 10 mg
of camphene R, 20 µL of /3-pinene R, 10 µL of car-3-ene R,
10 µL of /3-myrcene R, 20 µL of limonene R, 10 µL of
Development Over a path of 15 cm [or 6 cm). p-cymene R, 10 µL of terpinolene R, 10 µL of horny! acetate R
Drying In air. and 10 µL of /3-caryophyllene R in 1 mL of heptane R.
Detection Treat with anisaldehyde solution R, heat at Reference solutwn (b) Dissolve 10 mg of camphene R in
100-105 °C for 5-10 min and examine in daylight. heptane R and dilute to 2 mL with the same solvent. Dilute
Results See below the sequence of the zones present in the 0 .1 mL of the solution to 1 mL with heptane R.
chromatograms obtained with the reference solution and the Column:
test solution. Furthermore other faint zones may be present - material: fused silica,
in the chromatogram obtained with the test solution. - size: l = 60 m, 0 = 0.22 mm,
- stationary phase: macrogol 20 000 R (0.2 µm).
Split ratw 1:100.
IV-432 Plantain 2023
Temperature: length and running almost parallel. Hairs may be almost

absent, sparsely scattered or sometimes abundant, especially
Time Temperature on the lower surface and over the veins. The scape is
(min) (°C) brownish-green, longer than the leaves, 3-4 mm in diameter
Column 0 - 10 65 and is deeply grooved longitudinally, with 5-7 conspicuous
JO - 41 65 - 220 ribs. The surface is usually covered with fine hairs.
41 - 50 220 B. Microscopic examination (2.8.23). The powder is
Injection port 220 yellowish-green. Examine under a microscope using chloral,
Detector 250 hydrate solution R. The powder shows the following diagnostic
characters (Figure 1884.-1): fragments of epidermis,
Detection Flame ionisation. composed of cells with irregularly sinuous anticlinal walls, the
Injection 0.2 µL. fragments of the upper epidermis of the lamina (surface
Elution order Order indicated in the preparation of reference view [HJ, transverse section [DJ) are accompanied by
solution (a). Record the retention times of these substances. palisade parenchyma [Da, Ha], and those of the lower
epidermis (surface view [G]) show stomata (2.8.3) mostly of
System suitability Reference solution (a): the diacytic type [Ga] and sometimes of the anomocytic
- resolution: minimum 1.5 between the peaks due to type [Gb]; the multicellular, uniseriate, conical covering
car-3-ene and ~-myrcene. trichomes are highly characteristic, whole [C] or mostly
Identification of components Using the retention times fragmented [A], with a basal cell larger than the other
determined from the chromatogram obtained with reference epidermal cells followed by a short cell supporting 2 or more
solution (a), locate the components of reference solution (a) elongated cells with the lumen narrow and variable, occluded
in the chromatogram obtained with the test solution. at intervals corresponding to slight swellings in the trichome
The peak due to ~-phellandrene is eluted after the peak due and giving a jointed appearance, the terminal cell has an
to limonene with a relative retention of about 1.03 with acute apex and a filiform lumen; the glandular trichomes
reference to limonene. have a unicellular, cylindrical stalk and a multicellular,
Determine the percentage content of these components. elongated, conical head consisting of several rows of small
The limits are within the following ranges: cells and a single terminal cell [B, Ge]; dense groups of
- a-pinene: 32.0 per cent to 60.0 per cent, lignified fibro-vascular tissue with narrow, spirally and
- camphene: 0.5 per cent to 2.0 per cent, annularly thickened vessels and slender, moderately thickened
- /3-pinene: 5.0 per cent to 22.0 per cent, fibres [F]; fragments of the scape [E] with cells with
- car-3-ene: 6.0 per cent to 18.0 per cent, thickened walls and a coarsely ridged cuticle, stomata [Ee],
- /3-myrcene: 1.5 per cent to 10.0 per cent, multicellular, uniseriate covering trichomes [Eb] and
- limonene: 7.0 per cent to 12.0 per cent, glandular trichomes [Ea] of the type previously described.
- /3-phellandrene: maximum 2.5 per cent,
- p-cymene: maximum 2.0 per cent,
- terpinolene: maximum 4.0 per cent,
- bornyl acetate: 1.0 per cent to 4.0 per cent,
- /3-caryophyllene: 1.0 per cent to 6.0 per cent,
chromatogram obtained with reference solution (b).
STORAGE
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Plantain
(Ribwort Plantain, Ph. Eur. monograph 1884)
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or fragmented, dried leaf and scape of Plantago
lanceolata L. s. l.
Content
Minimum 1.5 per cent of total ortho-dihydroxycinnamic acid
derivatives expressed as acteoside (C 29 H 360 15 ; 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 Figure 1884.-1. - Illustration for identification test B of powdered
undulate. It has 3, 5 or 7 primary veins, nearly equal in herbal drug of ribwort plantain
2023 Platycodon Root IV-433
C. Examine the chromatograms obtained in the test for 0.5 M hydrochloric acid, 2 mL of a solution prepared by
Digitalis lanata leaves. dissolving 10 g of sodium nitrite R and 10 g of sodium
Results A See below the sequence of zones present in the molybdate R in 100 mL of water R, and 2 mL of dilute sodium
chromatogram obtained with the reference solution and the hydroxide solution R. Dilute to 10.0 mL with water R.
test solution. Furthermore, other zones may be present in the Immediately measure the absorbance (2.2.25) of the test
chromatogram obtained with the test solution. solution at 525 nm using as compensation liquid a solution
prepared as follows: to a 10 mL volumetric flask add 1.0 mL
Top of the plate of the stock solution, 2 mL of 0.5 M hydrochloric acid and
2 mL of dilute sodium hydroxide solution R, and dilute to
-- -- 10.0 mL with water R.
Acteoside: a yellow zone A yellow zone (acteoside) Calculate the percentage content of total
ortho-dihydroxycinnamic acid derivatives, expressed as
acteoside, using the following expression:
-- --
Ax 1000
Aucubin: a blue zone A blue zone (aucubin)
185 X m
i.e. taking the specific absorbance to be 185 for acteoside at
525 nm.
TESTS
Digitalis lanata leaves A absorbance of the test solution at 525 nm;
Thin-layer chromatography (2.2.27). m mass of the substance to be examined, in grams.

- - - - - - - - - - - - - - - - - - - - - ~ Ph Eur
Test solution Use a freshly prepared solution. To I g of the
powdered herbal drug (355) (2. 9.12) in a 25 mL flask, add
10 mL of the solvent mixture and shake for 30 min. Filter,
rinse the flask and the filter with 2 quantities, each of 5 mL,
of the solvent mixture. Dilute to 25 mL with the solvent
Platycodon Root
mixture. (Ph. Bur. monograph 2660)
Reference solution Dissolve I mg of acteoside R and 1 mg of
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
aucubin R in 1 mL of the solvent mixture.
Plate TLC silica gel F 254 plate R. DEFINITION
Mobile phase anhydrous formic acid R, glacial acetic acid R, Dried whole or fragmented root of Plat:ycodiJn grandifiorus
water R, ethyl acetate R (11: 11:27:100 VIVIVIV). Oacq.) A.DC., peeled or unpeeled, with rootlets removed,
collected in spring or autumn.
Application 10 µLas bands.
Development Over a path of 8 cm; heat immediately after Content
development at about 120 °C for 5-10 min. Minimum 0.3 per cent of total saponins, expressed as
platycodin D (C 57H 92 0 28; Mr 1225) (dried drug).
Detection A Examine in daylight.
IDENTIFICATION
A. Whole drug. The whole root is cylindrical or slightly
fusiform, gradually tapering downwards, sometimes
shows no bright blue fluorescent zone just below the reddish-
branched, slightly twisted, about 5-20 cm long and 0.3-2 cm
brown fluorescent zone corresponding to aucubin in the
in diameter. It shows deep, longitudinally twisted furrows
and rootlet scars or transverse lenticel-like scars; the margins
Foreign matter (2.8.2) of the rootlet scars may appear shiny and slightly translucent;
Maximum 5 per cent of leaves of different colour and the upper part of the root also shows transverse striations.
maximum 2 per cent of other foreign matter. The outer surface of the peeled root is white or brownish-
Loss on drying (2.2.32) yellow, whilst the outer surface of the unpeeled root is
Maximum 10.0 per cent, determined on 1.000 g of the brownish-yellow or brownish-grey. Sometimes the upper part
powdered herbal drug (355) (2. 9.12) by drying in an oven at shows a relatively short or inconspicuous rhizome, marked by
105 °C for 2 h. several crescent-shaped or irregularly round stem scars.
Total ash (2.4.16) The texture is fragile and the fracture uneven.
Maximum 14.0 per cent. Fragmented drug The fragmented root occurs as thin discs,
about 1.5-4 mm thick, sometimes oblique or irregular; most
ASSAY come from peeled roots, but sometimes showing patches of
Stock solution In a flask, place 1.000 g of the powdered bark still adhering. The outer surface may sometimes also be
herbal drug (355) (2.9.12) and add 90 mL of ethanol cracked. A transverse section shows a distinctly crenate
(50 per cent VIV) R. Boil in a water-bath under a reflux outline, a yellow or pale brown cortex, a distinct, more or
condenser for 30 min. Allow to cool and filter into a 100 mL less dark brown cambium ring and a well-developed yellow
volumetric flask. Rinse the flask and the filter with 10 mL of or pale brown xylem showing radial striations and occasional
ethanol (50 per cent V/V) R. Combine the filtrate and the clefts.
rinsings and dilute to 100.0 mL with ethanol
(50 per cent VIV) R.
yellowish-white or light brown. Examine under a microscope
Test solution To a 10 mL volumetric flask add, mixing after using chloral hydrate solution R. The powder shows the
each addition, 1.0 mL of the stock solution, 2 mL of following diagnostic characters (Figure 2660.-1): reticulate or
IV-434 Platycodon Root 2023
pitted xylem vessels (longitudinal section [B]) up to 56 µm in Mobile phase water R, methanol R, glacial acetic acid R,
diameter, free or in groups of 2, 3 or more, accompanied by methylene chloride R (2:3:8:15 V/V/V/V).
thin-walled parenchyma cells [Ba]; the sieve plates of the Applicatwn 10 µL as bands of 8 mm.
vessels have a thickened outline giving the appearance of a Development Over a path of 6 cm.
hole; numerous fragments of parenchyma consisting of ovoid
Drying In air.
cells [A] with slightly thickened and lignified walls; fragments
of phloem containing articulated laticiferous tubes, with thick Detection Treat with anisaldehyde solution R and heat at
non-lignified walls and granular yellowish-grey or brown 100 °C for 3 min; examine in daylight.
contents (longitudinal section [F], transverse section [D]); Results See below the sequence of zones present in the
fragments of orange cork, consisting of several layers of chromatograms obtained with the reference solution and the
superimposed cells (surface view [C], transverse section [E]), test solution. Furthermore, other faint zones may be present
frequent in unpeeled roots and relatively rare in peeled roots; in the chromatogram obtained with the test solution.
small calcium oxalate prism crystals may be present in the
parenchymatous cells. Examine under a microscope using Top of the plate
glycerol R. The powder shows numerous pieces of inulin,
angular, irregular [HJ or crystallised in a fan shape, free or
included in parenchyma cells [G]. -- --
Xylose: a yellowish-brown zone A yellowish-brown zone
-- --
Glucose: a yellowish-brown zone 2 yellowish-brown zones
TESTS
Codonopsis pilosula (Franch.) Nannf
Examine the herbal drug as described in identification test B.
The presence of sclereids, isolated or in groups, and starch
granules indicates adulteration by Codonopsis pilosula
(Franch.) Nannf.
105 °C for 2 h.
Total ash (2.4.16)
Ash insoluble in hydrochloric acid (2. 8. 1)
ASSAY
Solvent mixture water R, anhydrous ethanol R (30:70 V/V).
(2.9.12) add 50.0 mL of the solvent mixture and sonicate for
45 min. Allow to cool and filter. Rinse the filter with 10 mL
Figure 2 660. -1. - Illustration for identificatwn test B of powdered
of solvent mixture. Combine the filtrate and washings, and
herbal drug of platycodon root
evaporate to dryness under reduced pressure. Take up the
C. Thin-layer chromatography (2.2.27). residue with the solvent mixture, transfer to a volumetric
flask and dilute to 10.0 mL with the solvent mixture. Shake
well and filter through a membrane filter (nominal pore size
(2.9.12) add 5.0 mL of ethanol (70 per cent V/V) R. Sonicate
0.45 µm).
for 10 min and centrifuge or filter. Evaporate the supernatant
or filtrate to dryness under reduced pressure. Dissolve the Reference solutwn (a) Dissolve 8.0 mg of platycodin D CRS
residue in 1.0 mL of water R. Prepare a ready-to-use sample in the solvent mixture and dilute to 10.0 mL with the solvent
preparation cartridge containing 50 mg of octadecylsilyl silica mixture.
gel (55 µm), using 3 mL of methanol R followed by 3 mL of Reference solutwns (b), (c), (d), (e), (j), (g) Dilute reference
water R. Apply 1.0 mL of the solution to be analysed to the solution (a) to obtain 6 reference solutions of platycodin D,
top of the cartridge. Elute the cartridge with 3 mL of water R the concentrations of which span the expected value in the
and collect the eluate. Evaporate the eluate to dryness under test solution.
reduced pressure. Dissolve the residue in 1.0 mL of Reference solutwn (h) Dissolve 5.0 mg of platycodon root dry
methanol R. extract for system suitability HRS in 1. 0 mL of methanol R.
Reference solution Dissolve 1.0 mg of glucose R and 1.0 mg of Column:
xylose R in 2.0 mL of methanol R. - size: l =0.15 m, 0 =4.6 mm;
2023 Podophyllum Resin IV-435
- stationary phase: end-capped solid core octadecylsilyl silica gel

for chromatography R (2. 7 µm);
Podophyllum Resin
- temperature: 30 °C. Podophyllin
Mobile phase: Action and use
- mobile phase A: water for chromatography R; Used in treatment of warts.
- mobile phase B: acetonitrile Rl; Preparation
Compound Podophyllin Paint
(min) (per cent VIV) (per cent VIV) DEFINITION
0 - 10 85 --> 75 15 --, 25 Podophyllum Resin is the resin obtained from rhizomes and
10 - 30 75 25 roots of Podophyllum hexandrum Royle (P. emodi Wall.).
It contains not less than 50.0% of total aryltetralin lignans,
Flow rate I .O mUmin. calculated as podophyllotoxin.
Detection Evaporative light-scattering detector; the following CHARACTERISTICS
settings have been found to be suitable; if the detector has An amorphous powder varying in colour from light brown to
different setting parameters, adjust the detector settings so as greenish yellow, or brownish grey masses; odour,
to comply with the system suitability criteria: characteristic; caustic.
- carrier gas: nitrogen R; Partly soluble in hot water, from which it is precipitated on
- pressure: 350 kPa; cooling, in chlorofcmn, in ether and in 5M ammonia.
- evaporator temperature: 45 °C.
Injection 10 µL. IDENTIFICATION
Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions in methanol.
plarycodon root dry extract for system suitability HRS and the
chromatogram obtained with reference solution (h) to (I) I% w/v of the substance being examined.
identify the peaks due to saponins I, 2 (platycodin D), 3, 4 (2) 0.5% w/v of podophyllotoxin.
and 5; use the chromatogram obtained with reference (3) 0.1 % w/v of phenazone.
solution (a) to identify the peak due to platycodin D. CHROMATOGRAPHIC CONDITIONS
Relative retention With reference to platycodin D (a) Use as the coating silica gel GF254•
(retention time = about 21 min): saponin I = about 0.98;
saponin 3 = about 1.03; saponin 4 = about 1.06;
saponin 5 = about 1.08. Additional peaks may be present. (c) Apply as bands 10 µL of each solution.
System suitability: (d) Develop the plate to 10 cm.
- resolution: minimum 1.5 between the peaks due to (e) After removal of the plate, allow it to dry in air and
saponin I and platycodin D in the chromatogram examine under ultraviolet light (254 nm).Spray the plate with
obtained with reference solution (h); methanolic sulfuric acid (50%) and heat at 130° for
- signal-to-noise ratw: minimum 50 for the principal peak in 10 minutes.
the chromatogram obtained with reference solution (a). MOBILE PHASE
Establish a calibration curve with the common logarithm of I volume of methanol and 25 volumes of chloroform.
the concentration (in milligrams per millilitre) of reference
solutions (a), (b), (c), (d), (e), (t) and (g) (corrected by the CONFIRMATION
assigned percentage content of plarycodin D CRS) as the When viewed under ultravwlet light (254 nm), the
abscissa and the common logarithm of the corresponding chromatogram obtained with solution (1) exhibits quenching
peak area as the ordinate. zones corresponding in position to the principal quenching
Calculate the percentage content of saponins I, 2, 3, 4 and 5 zones in the chromatograms obtained with solutions (2) and
using the following expression: (3). Other quenching zones may be present.
When viewed after spraying, the chromatogram obtained
with solution (I) exhibits a purplish zone (podophyllotoxin)
m corresponding in position and colour to the principal zone in
solution (2) and a purplish zone
A common logarithm of the concentration of the peak due to each (4 1-demethylpodophyllotoxin) corresponding in position to
saponin in the chromatogram obtained with the test solution,
the quenching zone found in solution (3). Other coloured
determined from the calibration curve;
m mass of the herbal drug to be examined used to prepare the test zones may be present.
solution, in grams.
TESTS
Matter insoluble in ethanol (96%)
Calculate the percentage content of total saponins, expressed
Shake I g, finely powdered, with 20 mL of ethanol (96%) for
as platycodin D, by taking the sum of the percentage
5 minutes, filter through a sintered-glass crucible (ISO 4 793,
contents of saponins I, 2, 3, 4 and 5.
porosity grade 2, is suitable), wash the filter with ethanol
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
(96%) and dry at 105°. The residue weighs not more than
25 mg.
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
IV-436 Podophyllin Paint 2023
filtering and washing being not more than 10 minutes. (d) Develop the plate to 10 cm.
Dry the filter and residue to constant weight at 105°. (e) After removal of the plate, dry in air and examine under
The residue weighs not less than 0.18 g and not more than ultraviolet light (254 nm) to locate the quenching zone due to
0.30 g. phenazone in solution (3). Spray the plate with methanolic
Loss on drying sulfuric acid (50%) and heat at 130° for 10 minutes.
When dried to constant weight at 105°, loses not more than MOBILE PHASE
5.0% of its weight. Use 1 g.
1 volume of methanol and 25 volumes of chloroform.
Sulfated ash
CONFIRMATION
Not more than 1.0%, Appendix IX A.
The chromatogram obtained with solution (1) exhibits a
ASSAY purplish zone (podophyllotoxin) corresponding in position
Dissolve 0.5 gin sufficient ethanol (96%) to produce and colour to the principal zone in solution (2) and a
100 mL. To 10 mL of this solution in a separating funnel purplish zone (4 '-demethylpodophyllotoxin) corresponding in
add 190 mL of water and extract with six 30-mL quantities position to the quenching zone found in solution (3). Other
of dichloromethane. Combine the dichloromethane layers, coloured zones are present in the chromatogram obtained
extract with 10 mL of 0.2M sodium hydroxide followed by five with solution (1).
10-mL quantities of water and wash each of the six aqueous
layers separately with the same 20-mL quantity of TESTS
dichloromethane. Combine the dichloromethane solutions, Weight per mL
filter through absorbent cotton and evaporate the filtrate to 0.925 to 0.975 g, Appendix VG.
dryness. Dissolve the residue in sufficient ethanol (96%) to Total solids
produce 100 mL, dilute 10 mL of this solution to 50 mL 27.0 to 33.0% w/v when determined by evaporating 1 mL to
with ethanol (96%) and measure the absorbance of the dryness on a water bath and drying the residue at 105° for
resulting solution at the maximum at 292 nm, 4 hours.
Appendix II 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 Polygonum Cuspidatum Rhizome
Podophyllum Resin should be protected from light. and Root
On exposure to light, or to temperatures above 25°, it
becomes darker in colour. (Ph. Bur. monograph 2724)
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
LABELLING
The label states the botanical source. DEFINITION
Dried, fragmented rhizome and root, with rootlets removed,
of Reynoutria japonica Houtt. (syn. Polygonum cuspidatum
Sieb. et Zucc.) collected in spring or autumn.
Compound Podophyllin Paint Content
Compound Podophyllin Cutaneous Solution - emodin (C 15 H 10 0 5 ; Mr 270.2): minimum 1.0 per cent
DEFINITION (dried drug),
- polydatin (C 20 H 220 8; Mr 390.4): minimum 1.5 per cent
Compound Podophyllin Paint is a cutaneous solution.
(dried drug).
Podophyllum Resin 150 g IDENTIFICATION
Compound Benzoin Tincture Sufficient to produce 1000 mL A. Irregular thick slices or sections, about 2-9 cm long and
5-27 mm in diameter or sometimes cylindrical pieces.
In making the Compound Benzoin Tincture used to prepare The outer surface is yellowish-brown, with longitudinal
Compound Podophyllin Paint, the Ethanol (90 per cent) striations and rootlet scars. In transverse section, the brown
may be replaced by Industrial Methylated Spirit 1 diluted so or reddish-brown bark is relatively thin, separating easily
as to be of equivalent ethanolic strength. from the wood. The wood is thick, yellowish-brown, with
The paint complies with the requirements stated under Li,quids for numerous radial striations. The pith of the rhizome is large,
Cutaneous Application and with the following requirements. hollowed and transversally septate.
IDENTIFICATION B. Microscopic examination (2.8.23). The powder is
Carry out the method for thin-layer chromatography, yellowish-brown. Examine under a microscope using chloral
Appendix III A, using the following solutions in methanol. hydrate solution R. The powder shows the following diagnostic
(1) 7% v/v of the paint. characters (Figure 2724.-1): very numerous cluster crystals of
calcium oxalate, up to 100 µm in diameter, usually free [H],
(2) 0.5% w/v of podophyUotoxin.
or sometimes inside parenchyma cells [Ca]; brownish-yellow
(3) 0.1 % w/v of phenazone. fragments of cork (surface view [A]), with wavy striations in
CHROMATOGRAPHIC CONDITIONS the cells; sclereids, elongated and ramified at one end, with
(a) Use as the coating silica gel GF2s4, clearly channelled walls, isolated or in small groups [G];
bundles of phloem fibres [El, thick-walled and channelled;
fragments of the medullary rays [BJ, with rectangular or sub-
(c) Apply 10 µL of each solution. rectangular cells with slightly thickened, beaded and pitted
walls; fragments of large vessels, usually with bordered
1 The law and the sratuwry regulations governing the use of Industrial
pits [D, F]; fragments ofparenchyma with large ovoid cells
Methylated Spirit must be observed.
2023 Polygonum Cuspidatum Rhizome and Root IV-43 7
having slightly thickened and pitted walls [C]. Examine Top of the plate
under a microscope using a 50 per cent V/V solution of
2 yellow fluorescent zones
glycerol R. The powder shows numerous starch granules,
rounded or elliptical, on average 10 µm in diameter and with
a punctiform or slightly eccentric slit-shaped hilum, simple or
2-10 compound m. Resveratrol: a blue fluorescent zone A blue fluorescent zone (resveratrol)
-- --
A broad orange fluorescent zone
-- --
Polydatin: a blue fluorescent zone A blue fluorescent zone (polydatin)
TESTS
105 °C for 4 h.
Total ash (2.4.16)
ASSAY
Emodin
(2.9.12) in a round-bottomed flask add 150.0 mL of a
14 per cent V/V solution of sulfuric acid Rand 150.0 mL of
methylene chloride R. Heat under a reflux condenser in a
Figure 2724.-1. - Illustration for identificatwn test B of powdered water-bath at 60 °C for 2 h. Allow to cool and transfer the
herbal drug of Polygonum cuspidatum rhizome and root contents to a separating funnel. Separate the organic layer
and shake the acid layer with 3 quantities, each of 15 mL, of
C. Thin-layer chromatography (2.2.27). methylene chloride R. Combine the organic extracts and
Test solutwn To 0.5 g of the powdered herbal drug (355) evaporate to dryness. Dissolve the residue in methanol R and
(2. 9.12) add 5 mL of methanol R and sonicate for 10 min. dilute to 100.0 mL with the same solvent. Filter the solution
Centrifuge or filter, and use the supernatant or the filtrate as through a membrane filter (nominal pore size 0.45 µm).
the test solution. Reference solutwn (a) Dissolve 5.0 mg of emodin CRS in
Reference solutwn Dissolve 1 mg of polydatin R and 1 mg of methanol Rand dilute to 50.0 mL with the same solvent.
resveratrol R in 1 mL of methanol R. Dilute 5.0 mL of the solution to 20.0 mL with methanol R.
Plate TLC silica gel plate R (2-10 µm). Reference solutwn (b) Dissolve 2.5 mg of rhein R in
Mobile phase methanol R, methylene chloride R (20:80 V/V). methanol R and dilute to 25.0 mL with the same solvent.
Applicatwn 8 µL as bands of 8 mm. Dilute 1.0 mL of the solution to 10.0 mL with reference
solution (a).
Column:
Drying In air.
- size: l =0.25 m, 0 =4.0 mm;
Detection Examine in ultraviolet light at 365 nm. - stationary phase: octadecylsilyl silica gel for chromatography R
Results See below the sequence of zones present in the (5 µm);
chromatograms obtained with the reference solution and the - temperature: 30 °C.
test solution. Furthermore, other faint zones may be present Mobile phase:
in the chromatogram obtained with the test solution. - mobile phase A: phosphoric acid R, water for
0 - 11 15 85
11 - 11.1 15 ➔ 5 85 ➔ 95
II.I -22 5 95
IV-438 Polygonum Orientale Fruit 2023
Flow rate 1.0 mL'min. Calculate the percentage content of polydatin using the
Detection Spectrophotometer at 254 nm. following expression:
Injection 20 µL. A 1 xm 2 xpx 1.67
Retention time Rhein = about 4 min; A2xm1
emodin = about 6 min.
area of the peak due to polydatin in the chromatogram obtained
System suitability Reference solution (b): with the test solution;
- resolution: minimum 5 between the peaks due to rhein and area of the peak due to polydatin in the chromatogram obtained
emodin. with reference solution (a);
Calculate the percentage content of emodin using the solution, in grams;
following expression: mass of po/ydatin CRS used to prepare reference solution (a), in
grams;
p percentage content of polydatin in polydatin CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
area of the peak due to emodin in the chromatogram obtained
area of the peak due to emodin in the chromatogram obtained
solution, in grams;
Polygonum Orientale Fruit
mass of emodin CRS used to prepare reference solution (a), in
grams; (Ph. Bur. monograph 2726)
p percentage content of emodin in emodin CRS. Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Polydatin DEFINITION
Liquid chromatography (2.2.29). Carry out the assay protected Dried ripe fruit of Persicaria orientalis (L.) Spach (syn.
from bright light. Polygonum orientate L.).
Test solution To 0.200 g of the powdered herbal drug (355) Content
(2.9.12) in a round-bottomed flask add 50.0 mL of ethanol Minimum 0.15 per cent oftaxifolin (C 15H 12 0 7 ; Mr 304.3)
(50 per cent V/V) R and weigh. Heat under a reflux (dried drug).
condenser in a water-bath at 95 °C for 30 min. Allow to IDENTIFICATION
cool, weigh and adjust to the original mass with ethanol A. The fruit is roundish and oblate, the flattened sides
(50 per cent VIV) Rand shake well. Filter and dilute 3.0 mL
slightly concave, about 2-3.5 mm in diameter and about
of the filtrate to 25.0 mL with a 60 per cent VIV solution of 1-1.5 mm thick. The glossy surface is brownish-black or
methanol R. reddish-brown. There is a light brown and slightly protruding
Reference solution (a) Dissolve 5.0 mg of polydatin CRS in pedicel at the base with the remains of the membranous
methanol R and dilute to 50.0 mL with the same solvent. perianth. The texture is hard.
Dilute 10.0 mL of the solution to 50.0 mL with a
60 per cent VIV solution of methanol R.
Reference solution (b) Dissolve 5.0 mg of resveratrol R in a
60 per cent V/V solution of methanol R and dilute to
50.0 mL with the same solution. Dilute 3.0 mL of the
solution to 25.0 mL with reference solution (a).
Column:
=
- size: l 0.25 m, 0 =
4.0 mm;
(5 µm);
Mobile phase:
- mobile phase B: acetonitrile for chromatography R;

{min) (per cent VIP) (per cent VIP)
0 - 12 82 18
12 - 18 82 ➔ 5 18 ➔ 95
18 - 25 5 95
Flow rate l .0 mL'min.

Detection Spectrophotometer at 306 nm. Figure 2726.-1. - Illustration for identification test B of powdered
Injection 20 µL. herbal drug of Polygonum orientate fruit
Retention time Polydatin = about 10 min;
resveratrol = about 17 min. B. Microscopic examination (2.8.23). The powder is greyish-
System suitability Reference solution (b): brown. Examine under a microscope using chloral hydrate
- resolution: minimum 5 between the peaks due to polydatin solution R. The powder shows the following diagnostic
and resveratrol. characters (Figure 2726.-1): fragments of the pericarp
2023 Polygonum Orientale Fruit IV-439
consisting of groups of yellowish-brown or reddish-brown TESTS

palisade cells, 100-190 µm long and 15-30 µm wide, with Loss on drying (2.2.32)
thickened walls [Da], arranged as a single layer (side view Maximum 12.0 per cent, determined on 1.000 g of the
[DJ), which exhibit, on the outer surface, a polygonal or powdered herbal drug (355) (2.9.12) by drying in an oven at
near-circular shape, narrow lumina and pitted walls (surface 105 °C for 2 h.
view [BJ), and on the inner surface, a sinuous, anticlinal
shape and circular lumina (surface view [Cl); fragments of Total ash (2.4.16)
the inner part of the pericarp composed of brown rectangular Maximum 3.0 per cent.
cells [Db]; fragments of the parenchyma of the mesocarp, ASSAY
with colourless thin-walled cells [E], sometimes accompanied Liquid chromatography (2.2.29).
by fine annular or spiral vessels [Ea]; fragments of the testa Test solution 0.500 g of the powdered herbal drug (355)
consisting of a layer of cells with a barely visible zigzag [HJ (2.9.12) in a round-bottomed flask, add 25 mL of a
or polygonal m pattern mostly attached to a layer with a 55 per cent VIV solution of methanol R. Heat under a reflux
sinuous wall structure Ua, F]; fragments of the cotyledons condenser in a water-bath at 90 °C for 1 h. Allow to cool
with thin-walled, small parenchyma cells (transverse section and filter. Rinse the filter with 5.0 mL of hydrochloric acid Rl.
[G]); fragments of the endosperm with thin-walled cells [A]; Combine the filtrate and the rinsings in a volumetric flask
possibly fragments of the perianth with cluster crystals of and dilute to 50.0 mL with a 55 per cent V/V solution of
calcium oxalate up to 50 µm in diameter, small vascular methanol R. Filter through a membrane filter (nominal pore
bundles and a partly papillous epidermis. Examine under a size 0.45 µm).
Reference solution (a) Dissolve 5.0 mg of taxifolin CRS in a
The powder shows numerous simple starch granules 2-9 µm
55 per cent VIV solution of methanol R. Add 2.5 mL of
in diameter, sometimes with a cleft hilum [Kl.
hydrochloric acid Rl and dilute to 25.0 mL with a
C. Thin-layer chromatography (2.2.27). 55 per cent V/V solution of methanol R. To 10.0 mL of this
Test solution To 0.5 g of the powdered herbal drug (355) solution add 4.0 mL of hydrochloric acid Rl and dilute to
(2. 9.12) add 5 mL of methanol R. Sonicate at 60 °C for 50.0 mL with a 55 per cent V/V solution of methanol R.
10 min. Allow to cool and filter; use the filtrate. Reference solution (b) 0.500 g of polygonum orientale fruit for
Reference solution Dissolve 2.0 mg of taxifolin Rand 3.0 mg system suitability HRS in a round-bottomed flask, add
of kaempferol R in 6 mL of methanol R. 25.0 mL of a 55 per cent VIV solution of methanol R. Heat
Plate TLC silica gel plate R (2-10 µm). under a reflux condenser in a water-bath at 90 °C for 1 h.
Mobile phase acetone R, glacial acetic acid R, anhydrous formic Allow to cool and filter. Rinse the filter with 5.0 mL of
acid R, methylene chloride R (4:5:5:50 VIVIVIV). hydrochloric acid Rl. Combine the filtrate and the rinsings in
a volumetric flask and dilute to 50.0 mL with a
55 per cent VIV solution of methanol R. Filter through a
Development Over a path of 6 cm. membrane filter (nominal pore size 0.45 µm).
Drying In air. Column:
Detection Heat at 100 °C for 3 ruin; treat the still-warm - size: l = 0.150 m, 0 = 4.0 mm;
plate with a 5 g/L solution of diphenylboric acid aminoethyl - stationary phase: octadecylsilyl silica gel for chromatography R
ester R in ethyl acetate R, and then with a 50 g/L solution of (5 µm).
macrogol 400 R in methylene chloride R; allow to dry in air for Mobile phase:
60 min. Examine in ultraviolet light at 365 nm. - mobile phase A: phosphon·c acid R, water for
Results See below the sequence of zones present in the chromatography R (0.1:99.9 VIV);
chromatograms obtained with the reference solution and the - mobile phase B: acetonitrile for chromatography R;
in the chromatogram obtained with the test solution. Time Mobile phase A Mobile phase B
Top of the plate 0 - 20 85 15
20 - 21 85-, 0 15-, 100
21 - 30 0 100
-- --
A pale blue zone Flow rate 1.0 mUmin.
Kaempferol: a pale blue zone Detection Spectrophotometer at 290 nm.
Injection 10 µL.
A pale blue zone
3 bluish-green zones polygonum orientale fruit for system suitability HRS and the
chromatogram obtained with reference solution (b) to
- - --
identify the peak due to epitaxifolin.
Taxifolin: an orangish-brown zone An orangish-brown zone (taxifolin)
Retention time Taxifolin = about 14 ruin;
A blue zone epitaxifolin = about 16 min.
An orangish-brown zone
taxifolin and epitaxifolin.
Calculate the percentage content of taxifolin using the
IV-440 Poppy Petals 2023
A1 x m2 x 0.4 xp
Reference solution Dissolve 1 mg of quinaldine red R and
A2 xm1 1 mg of sulfan blue R in 2 mL of methanol R.
area of the peak due to taxifolin in the chromatogram obtained Plate TLC silica gel plate R.
with the test solution; Mobile phase anhydrous formic acid R, water R, butanol R
area of the peak due to taxifolin in the chromatogram obtained
with reference solution (a); (10:12:40 VIVIV).
mass of the herbal drug to be examined used to prepare the test Application 10 µL as bands.
solution, in grams;
mass of taxifolin CRS used to prepare reference solution (a), in
grams; Drying In air.
p percentage content of taxifolin in taxifolin CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur Results See below the sequence of zones present in the
Red Poppy Petals Top of the plate
(Ph. Eur. monograph 1881) 2 yellow zones
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Quinaldine red: an orange-red zone
DEFINITION
-- --
Dried, whole or fragmented petals of Papaver rhoeas L.
A violet principal zone
IDENTIFICATION A violet zone
A. The petal is dark red or dark violet-brown, very thin, A yellow zone
floppy, wrinkled, often crumpled into a ball and velvety to Sulfan blue: a blue zone
the touch. It is broadly ovate with an entire margin, about
6 cm long and 4-6 cm wide, narrowing at the base where -- --
there is a black spot. The vascular bundles radiate from the A compact group of violet zones
base and they anastomose in a continuous arc, all at the
same short distance from the margin.
B. Microscopic examination (2.8.23). Examine under a
microscope using chloral hydrate solution R. The powder has TESTS
an intense reddish-pink colour and shows the following Foreign matter (2.8.2)
diagnostic characters (Figure 1881.-1): fragments of Maximum 2.0 per cent of capsules and maximum
epidermis composed of elongated, sinuous-walled cells [B, D, 1.0 per cent of other foreign matter.
G] with small, rounded, anomocytic stomata (2.8.3) [Ba]; Loss on drying (2.2.32)
numerous vascular bundles with spiral vessels [E) embedded Maximum 12.0 per cent, determined on 1.000 g of the
in the parenchyma; occasional fragments of the fibrous layer powdered herbal drug (355) (2.9.12) by drying in an oven at
of the anthers [F); rounded pollen grains, about 30 µm in 105 °C for 2 h.
diameter, with 3 pores and a finely verrucose exine [A, C,
HJ. Total ash (2.4.16)
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 V/V) R.
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
of hydrochloric acid Rand dilute to 100.0 mL with ethanol
(30 per cent V/V) R. Allow to stand for 10 min.
The absorbance (2.2.25) measured at 523 nm using ethanol
(30 per cent V/V) R as the compensation liquid is not less
than 0.6.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
8 G

herbal drug of red poppy petals

(2. 9.12) add 10 mL of ethanol (60 per cent V/V) R. Stir for
15 min. Filter through a filter paper.
2023 Primula Root IV-441
D. The herbal drug sticks to the pestle when moistened with

Poria water Rand pressed into a mortar.
(Ph. Bur. monograph 2475) E. To a small piece of the herbal drug add 1 drop of
iodinated potassium iodide solution Rl. A deep red colour is
produced.
DEFINITION TESTS
Dried sclerotium without skin of Wolfiporia extensa (Peck) Foreign matter (2.8.2)
Ginns (syn. Poria cocos (Schw.) Wolf; Wolfiporia cocos (F.A. Maximum 0.1 per cent of brown skins and roots of conifer
Wolf) Ryvarden & Gilb.). and maximum 2 per cent of other foreign matter.
IDENTIFICATION Loss on drying (2.2.32)
A. Square, rectangular or polyhedral pieces, or slices, varying Maximum 13.0 per cent, determined on 1.000 g of the
in length and thickness; whitish with a pale brown hue, flat powdered herbal drug (355) (2. 9.12) by drying in an oven at
and smooth, square, rectangular or polyhedral pieces, with 105 °C for 2 h.
no brown skin, difficult to break; slices easily broken, rough
Total ash (2.4.16)
fracture with granular or farinaceous texture.
B. Microscopic examination (2.8.23). The powder is whitish
with a pale brown hue. Examine under a microscope using
diagnostic characters: irregularly shaped and occasionally To 5.00 g of the powdered herbal drug (355) (2.9.12) add
branched colourless particles, which dissolve gradually in 100 mL of boiling water R. Allow to stand for 10 min,
chloral hydrate solution R. Examine under a microscope using shaking occasionally. Allow to cool, dilute to 100.0 mL with
a 50 g/L solution of potassium hydroxide R. The powder water R and filter. Evaporate 25.0 mL of the filtrate to
shows the following diagnostic characters: fragments of dryness on a water-bath. Dry the residue in an oven at
hyphae, colourless, slender, slightly curved, sometimes with 100-105 °C. The residue weighs a minimum of 18.75 mg.
septa, branched, 3-16 µm in diameter. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
(2. 9.12) add a mixture of 2 mL of ethyl acetate R and 3 mL
of methanol R. Sonicate for 10 min, centrifuge and use the Primula Root
supernatant.
Reference solution Dissolve 10 mg of 4-aminobenzoic acid R, (Ph. Bur. monograph 1364)
10 mg of coumarin R and 10 mg of thymol R in 10 mL of Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
methanol R.
DEFINITION
Plate TLC silica gel F 254 plate R (2-10 µm). Whole or cut, dried rhizome and root of Primula veris L.
Mobile phase glacial acetic acid R, 2-propanol R, cyclohexane R or Primula elatior Hill.
(10:10:80 V/V/V).
IDENTIFICATION
Application 5 µL as bands of 8 mm. A. The coarsely torose, greyish-brown rhizome is straight or
Development Over a path of 6 cm. slightly curved, about 1-5 cm long and about 2-4 mm thick.
Drying In air. The rhizome crown often bears the remains of stems and
Detection Examine in ultraviolet light at 254 nm. leaves. Attached to the rhizome are numerous brittle roots,
Results See below the sequence of zones present in the about 1 mm thick and usually 6-8 cm long. The root of
chromatograms obtained with the reference solution and the P. elatior is light brown or reddish-brown, that of P. veris
test solution. Furthermore, other faint quenching zones may light yellow or yellowish-white. The fracture is smooth.
be present in the chromatogram obtained with the test B. Microscopic examination (2.8.23). The powder is greyish-
solution between the zones due to 4-aminobenzoic acid and brown. Examine under a microscope using chloral hydrate
coumarin in the chromatogram obtained with the reference solution R. The powder shows the following diagnostic
solution. characters (Figure 1364. -1): fragments of parenchyma from
the bark of the root or the rhizome and from the medulla of
Top of the plate the rhizome [G, HJ, consisting of rounded or ovoid cells with
irregularly thickened and pitted walls; brownish fragments
from the dermal tissue of the root showing absorbent hairs
-- -- [C]; yellow or brownish fragments of the epidermis of the
Thymol: a quenching zone rhizome covered by a striated cuticle (surface view [Al,
transverse section [F]) accompanied by parenchyma from the
2 quenching zones
bark [Fa]; reticulate vessels [BJ sometimes accompanied by
- - - -
spiral vesselsm; groups of large, strongly pitted, yellowish-
green sclereids from the medullary parenchyma of the
Coumarin: a quenching zone
rhizome [El, which are characteristic of P. elatior. Examine
4-Aminobenzoic acid: a quenching under a microscope using a 50 per cent V/V solution of
zone glycerol R. The powder shows simple or compound starch
granules of various shapes and sizes [D].

IV-442 Psyllium Seed 2023

Results B In the chromatogram obtained with the test
solution no zones of light-blue or greenish fluorescence occur
below the main zone due to aescin in the chromatogram
105 °C for 2 h.
Total ash (2.4.16)
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
,
..
~ ....~
. • V
. Psyllium Seed
.. '
. . -:· 'l
"'· ... J (Ph. Bur. monograph 0858)
:-:·. ~" ~
~
.-· .r~'.:llmmm Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Ripe, whole, dry seeds of Plantago afra L. (Plantago
psyllium L.) or Plantago indica L. (Plantago arenaria Waldstein
and K.itaibel).
25µm
CHARACTERS
Sweet taste.
herbal drug of primula root IDENTIFICATION
P. afra Seeds are light brown to very dark brown but never
C. Thin-layer chromatography (2.2.27) as described in the black, smooth and shiny having an elliptical oblong shape.
test for Vincetoxicum hirundinaria Medik. root with the They are 2-3 mm long and 0.8-1.0 mm wide, one end being
following modifications. wider than the other. Towards the middle of the dorsal
Detection Treat with anisaldehyde solution R, heat at surface there is a fairly marked transverse constriction of light
100-105 °C for 5-10 min and examine in daylight. colour. On the ventral surface, there is a linear lighter-
Results The main zone (aescin) in the chromatogram coloured groove in the middle of which is a clear spot
obtained with the reference solution is bluish-violet and is corresponding to the hilum and bounded by swollen edges.
situated near the boundary between the lower and middle P. indica Seeds are almost identical to the seeds of P. afra,
thirds. The chromatogram obtained with the test solution but a little less shiny; they are 2-3 mm long and have a
shows 1-2 strong dark violet zones a little below the zone due maximum diameter of 1.5 mm.
to aescin in the chromatogram obtained with the reference TESTS
solution; further pale violet, yellowish or brownish-green Swelling index (2.8.4)
zones may be visible. Minimum 10.
TESTS Foreign matter (2.8.2)
Vincetoxicum hirundinaria Medik. root Maximum 1.0 per cent, determined on 10.0 g of the drug,
Thin-layer chromatography (2.2.27). including greenish unripe seeds. Psyllium seed does not
Test solution To 1.0 g of the powdered herbal drug (500) contain seeds having a dark central spot on the groove
(2. 9.12) add 10 mL of ethanol (70 per cent Vlv) R and heat (Plantago lanceolata L. and P. major L.) or seeds with
under a reflux condenser for 15 min. Cool and filter. brownish-grey or pinkish outer coats (P. ovata Forssk.
Reference solution Dissolve 10 mg of aescin R in 1.0 mL of and P. sempervirens Crantz).
ethanol (70 per cent VIV) R. Loss on drying (2.2.32)
Plate TLC silica gel F254 plate R. Maximum 14.0 per cent, determined on 1.000 g of drug by
Mobile phase glacial acetic acid R, water R, butanol R drying in an oven at 105 °C for 2 h.
(10:40:50 VIVIV); use the upper layer. Total ash (2.4.16)
Application 20 µL as bands. Maximum 4.0 per cent.
Development Over a path of 12 cm. STORAGE
Drying In an oven at 100-105 °C. Store protected from moisture.
Results A The chromatograms obtained with the reference
solution and the test solution show a quenching zone (aescin)
near the boundary between the lower and the middle thirds.
Mark this zone.
2023 Purple Coneflower Herb Expressed Juice, Stabilised with Ethanol IV-443
Mobile phase formic acid R, cyclohexane R, ethyl acetate R,

Purple Coneflower Herb toluene R (3:10:20:80 V/V/V/V).
Expressed Juice, Stabilised with Application 25 µL [or 5 µL] as bands of 10 mm [or 8 mm].
Ethanol Development Over a path of about 15 cm [or 6 cm].
Echinacea Purpurea Herb Drying In a stream of cold air for about 10 min.
(Ph. Bur. monograph 2282) Detection Treat with anisaldehyde solution R and heat at
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION chromatograms obtained with the reference solution and the
Expressed juice, stabilised with ethanol (96 per cent), from test solution. Furthermore, other faint zones may be present
the fresh flowering aerial parts of cultivated Echinacea in the chromatogram obtained with the test solution.
purpurea (L.) Moench.
Content Top of the plate
Minimum 0.1 mg/100 mL of N-isobutyldodecatetraenamide A faint bluish-violet or pink zone
(C15H2sNO; Mr 247.4).
A faint violet or pink zone
PRODUCTION
The juice is obtained from the fresh herbal drug. The fresh - - --
herbal drug satisfies the following requirements. A violet or pink zone
Identification
~-sitosterol: a violet or pink zone
The herbaceous perennial plant is usually 60-150 cm, rarely
up to 180 cm, in height. The stem is reddish-green, upright A violet or pink zone
and slightly branched. The leaves are alternate, dark green, Ursolic acid: a violet or pink zone
ovate to ovate-lanceolate, irregularly serrate, rugose on both
surfaces, with prominent light green veins on the lower A faint blue or violet zone
surface; the lamina is thick and shiny. The involucral bracts
-- --
of the large capitulum are arranged in 2-3 rows.
The receptacle is slightly convex and filled with pith. Each of
the outer violet ligulate florets (measuring 4-6 cm) and of the A dark violet-brown zone
inner violet-pink tubular florets is attached to a reddish acute
and coriaceous bract, which overtops the tubular florets. 2 violet-brown zones
The gamosepalous calyx is reduced; the 5 lobes form a very 2-3 dark violet-brown zones
short irregular crown, one of which is up to 1 mm long.
The achenes bearing a pappus, usually with 4 teeth, are
green or light brown. Reference solution Test solution
Maximum 5 per cent. B. Examine the chromatograms obtained in the assay.
The juice is expressed from the fresh herbal drug as soon as Restdts The chromatogram obtained with the test solution
possible and within a time period no more than 24 h after shows 2 peaks due to N-isobutyldodecatetraenamide isomers
harvesting. Harvested herbal drug which has stood for several
1 and 2.
hours is carefully examined and any material which is
discoloured or altered in texture due to TESTS
fermentation/composting is rejected prior to juice expression. Dry residue (2.8.16)
The expressed juice is stabilised with a suitable quantity of Minimum 3.5 per cent m!V.
ethanol (96 per cent). Ethanol (2. 9.10)
CHARACTERS 18. 0 per cent V/V to 30. 0 per cent V/V.
Appearance Relative density (2.2.5)
Turbid, greenish-brown liquid. 0.970 to 1.020.
IDENTIFICATION Microbial contamination (5.1.8, B)
First identification: B. ASSAY
Second identification: A. Liquid chromatography (2.2.29).
A. Thin-layer chromatography (2.2.27). Solvent mixture acetonitrile R, water R (50:50 V/V).
Test solution In a separation funnel, introduce 25 mL of the Test solution Introduce 25. 0 mL of the juice to be examined
juice to be examined. Shake with 3 quantities, each of into a chromatography column (l 0.15 m, 0 = 30 mm), =
20 mL, of methylene chloride R. Combine the 3 methylene containing 15 g of kieselguhr for chromatography R and allow
chloride layers and evaporate to dryness in a pear-shaped to stand for 15 min. Elute with 70 mL of methylene
flask. Dissolve the residue in 1 mL of methanol R. chloride R. Evaporate the eluate to dryness in vacuo in a pear-
Reference solution Dissolve 1 mg of /3-sitosterol R and 1 mg of shaped flask. Dissolve the residue in 1.0 mL of the solvent
ursolic acid R in methanol Rand dilute to 5.0 mL with the mixture.
same solvent. Reference solution (a) Dissolve 10.0 mg of benzanilide CRS
Plate TLC silica gel F 254 plate R (5-40 µm) [or TLC silica gel in methanol R and dilute to 50.0 mL with the same solvent.
F2 54plate R (2-10 µm)]. Dilute 5.0 mL of the solution to 50.0 mL with methanol R.
IV-444 Purple Coneflower Herb Expressed Juice, Stabilised without Ethanol 2023
Reference solution (b) Dissolve 5.0 mg of echinacea dry extract

for system suitability HRS in the solvent mixture and dilute to
Purple Coneflower Herb
2.5 mL with the solvent mixture. Expressed Juice, Stabilised
Column:
- size: l = 0.25 m, 0 = 4.0 mm;
without Ethanol
- stationary phase: end-capped octadecylsilyl silica gel for Echinacea Purpurea Herb
chromatography R (5 µm); (Ph. Bur. monograph 2894)
- temperature: 30 °C. Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Mobz1e phase:
- mobile phase A: water for chromatography R; DEFINITION
- mobile phase B: acetonitrile R; Expressed juice, stabilised without ethanol, produced from
fresh flowering aerial parts of cultivated Echinacea
Time Mobile phase A Mobile phase B purpurea (L.) Moench.
(min) (per cent V/V) (per cent V/V) Content
0 - 30 55 45 - cichoric acid (C 22H 1 s0 12; Mr 474.4): minimum
30 - 40 55-+ 5 45-+ 95 0.25 per cent;
- N-isobutyldodecatetraenamide (C 16H 25 NO; Mr 247.4):
Flow rate 1.5 mUmin. minimum 0.1 mg/100 mL.
Detection Spectrophotometer at 254 nm. PRODUCTION
Injection 25 µL. The juice is obtained from the fresh herbal drug. The fresh
Relative retention With reference to benzanilide herbal drug satisfies the following requirements.
(retention time= about 6.1 min): Identification
N-isobutyldodecatetraenamide isomer 1 = about 4.4; The herbaceous perennial plant is usually 60-150 cm, rarely
N-isobutyldodecatetraenamide isomer 2 = about 4.6. up to 180 cm, in height. The stem is reddish-green, upright
Identification of peaks Use the chromatogram supplied with and slightly branched. The leaves are alternate, dark green,
echinacea dry extract for system suitability HRS and the ovate to ovate-lanceolate, irregularly serrate, rugose on both
chromatogram obtained with reference solution (b) to surfaces, with prominent light green veins on the lower
identify the peaks due to N-isobutyldodecatetraenamide surface; the lamina is thick and shiny. The involucral bracts
isomers 1 and 2. of the large capitulum are arranged in 2-3 rows.
System suitability Reference solution (b): The receptacle is slightly convex and filled with pith. Each of
- peak-to-valley ratio: minimum 2, where Hp = height above the outer violet ligulate florets (4-6 cm) and of the inner
the baseline of the peak due to violet-pink tubular florets is attached to a reddish acute and
N-isobutyldodecatetraenamide isomer 1 and Hv = height coriaceous bract, which overtops the tubular florets.
above the baseline of the lowest point of the curve The gamosepalous calyx is reduced; the 5 lobes form a very
short irregular crown, one of which is up to 1 mm long.
separating this peak from the peak due to
The achenes bearing a pappus, usually with 4 teeth, are
N-isobutyldodecatetraenamide isomer 2.
green or light brown.
Calculate the content of N-isobutyldodecatetraenamide in
milligrams per 100 mL using the following expression: Foreign matter (2.8.2)
Maximum 5 per cent.
A 1 xmx0.61xp The fresh herbal drug is crushed, treated with water vapour
A2 X 125 X 100 and the juice is expressed as soon as possible and within no
more than 24 h after harvesting. Harvested herbal drug
sum of the areas of the peaks due to which has stood for several hours is carefully examined and
N-isobutyldodecatetraenamide isomers I and 2 in the
any material which is discoloured or altered in texture due to
area of the peak due to benzanilide in the chromatogram fermentation/composting is rejected prior to juice expression.
obtained with reference solution (a); The expressed juice is centrifuged and preserved by
m mass of benzanilide CRS used to prepare reference solution (a), pasteurisation.
in milligrams;
0.61 correction factor berween N-isoburyldodecatetraenamide CHARACTERS
isomers I and 2, and benzanilide; Appearance
p percentage content of benzanilide in benzanilide CRS.
Turbid, brown liquid.
LABELLING IDENTIFICATION
The label states that the substance has been stabilised with First identification: C, D.
ethanol (96 per cent). Second identification: A, B.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur A. Thin-layer chromatography (2.2.27).
Test solution The juice to be examined.
Reference solution Dissolve 0.5 mg of caffeic acid R and
0.5 mg of chlorogenic acid R in methanol Rand dilute to
Mobile phase formic acid R, water R, ethyl methyl ketone R,
2023 Purple Coneflower Herb Expressed Juice, Stabilised without Ethanol IV-445
Applicatwn 50 µL [or 10 µL] of the test solution and 10 µL Top of the plate
[or 2 µL] of the reference solution, as bands of 10 mm
A faint bluish-violet or pink zone
[or 8 mm].
Development Over a path of about 15 cm [or 6 cm). A faint violet or pink zone
Drying In a stream of cold air for about 10 min, then heat -- --

at 100 °C for 2 min.
A pink zone
Detectwn Treat the still-warm plate with a 5 g/L solution of
diphenylboric acid aminoethyl ester R in ethyl acetate R; examine P-sitosterol: a violet or pink zone
in ultraviolet light at 365 nm. Ursolic acid: a violet or pink zone
chromatograms obtained with the reference solution and the -- --
test solution. Furthermore, other faint zones may be present A pink zone
in the lower part of the chromatogram obtained with the test
A violet zone
solution.
A violet-blue zone
Top of the plate A pink zone
Caffeic acid: an intense blue A violet zone
fluorescent zone
An intense blue fluorescent zone

--
- -
C. Examine the chromatograms obtained in the assay on
An intense blue fluorescent zone cichoric acid.
Chlorogenic acid: an intense blue Results The chromatogram obtained with the test solution
fluorescent zone shows a peak due to cichoric acid.
D. Examine the chromatograms obtained in the assay of
N-isobutyldodecatetraenamide.
-- Results The chromatogram obtained with the test solution
--
shows 2 peaks due to N-isobutyldodecatetraenamide
isomers 1 and 2.
TESTS
Minimum 3.5 per cent m!V.
Test solutwn In a separation funnel introduce 25 mL of the
juice to be examined. Shake with 3 quantities, each of
1.000 to 1.100.
20 mL, of methylene chloride R. Combine the 3 methylene
chloride layers and evaporate to dryness in a pear-shaped Microbial contamination (5.1.8, B)
flask. Dissolve the residue in 1 mL of methanol R. ASSAY
Reference solutwn Dissolve 1 mg of /3-sitosterol R and 1 mg of Cichoric acid
ursolic acid R in methanol Rand dilute to 5.0 mL with the Liquid chromatography (2.2.29).
same solvent. Solvent mixture water R, acetonitrile R (15:85 VIV).
Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel Test solutwn Dilute 1.000 g of the juice to be examined to
F254 plate R (2-10 µm)). 20.0 mL with the solvent mixture.
Mobile phase Jonnie acid R, cyclohexane R, ethyl acetate R, Reference solutwn (a) Dissolve 10.0 mg of chlorogenic
toluene R (3:10:20:80 VIVIV/V). acid CRS in ethanol (70 per cent VIV) R, sonicate for 15 min
Application 25 µL [or 5 µL) as bands of 10 mm [or 8 mm]. and dilute to 10.0 mL with the same solvent (solution A).
Development Over a path of about 15 cm [or 6 cm). Dilute 4.0 mL of this solution to 100.0 mL with ethanol
Drying In a stream of cold air for about 10 min. (70 per cent V/V) R.
Detection Treat with anisaldehyde solutwn R and heat at Reference solutwn (b) Dissolve 10 mg of caffeic acid R in
100-105 °C for 3 min; examine in daylight. ethanol (70 per cent V/V) R, sonicate for 15 min and dilute to
10 mL with the same solvent. Mix 4.0 mL of this solution
with 4.0 mL of solution A and dilute to 100 mL with ethanol
in the chromatogram obtained with the test solution. Column:
=
- size: l 0.25 m, 0 =
4.6 mm;
(5 µm);
Mobile phase:
- mobile phase A: phosphon·c acid R, water for
chromatography R (1:999 V/V);
IV-446 Pygeum Bark 2023
Time Mobile phase A Mobile phase B Detection Spectrophotometer at 254 nm.

(min) (per cent V/J/) {per cent V/J/)
Injection 25 µL.
0 - 13 90---> 78 10 __, 22
13 - 14 78---, 60 22 ➔ 40
Relative retention With reference to benzanilide
(retention time= about 6.1 min):
14 - 20 60 40
N-isobutyldodecatetraenamide isomer 1 = about 4.4;
N-isobutyldodecatetraenamide isomer 2 = about 4.6.
Flow rate 1.5 mL'min.
Detection Spectrophotometer at 330 nm. echinacea dry extract for system suitability HRS and the
Injection l O µL. chromatogram obtained with reference solution (b) to
Relative retention With reference to chlorogenic acid identify the peaks due to N-isobutyldodecatetraenamide
(retention time= about 7 min): cichoric acid = about 2.3. isomers 1 and 2.
Identification of peaks Use the chromatogram obtained with System suitability Reference solution (b):
reference solution (b) to identify the peaks due to caffeic acid - peak-to-valley ratio: minimum 2, where Hp = height above
and chlorogenic acid. Use the chromatogram supplied in the baseline of the peak due to
Figure 2894.-1 to identify the peak due to cichoric acid. N-isobutyldodecatetraenamide isomer 1 and Hv = height
System suitability Reference solution (b): above the baseline of the lowest point of the curve
- resolution: minimum 5.0 between the peaks due to caffeic separating this peak from the peak due to
acid and chlorogenic acid. N-isobutyldodecatetraenamide isomer 2.
Calculate the percentage content of cichoric acid using the Calculate the content of N-isobutyldodecatetraenamide in
following expression: milligrams per 100 mL using the following expression:
A1 x m2 x 0.695 xp A 1 xmx0.61xp
A2 xm1 x 12.5 A2 x 125 x 100
area of the peak due to cichoric acid in the chromatogram sum of the areas of the peaks due to
obtained with the test solution; N-isobutyldodecatetraenamide isomers I and 2 in the
area of the peak due to chlorogenic acid in the chromatogram chromatogram obtained with the test solution;
obtained with reference solution (a); area of the peak due to benzanilide in the chromatogram
mass of the expressed juice used to prepare the test solution, in obtained with reference solution (a);
milligrams; m mass of benzanilide CRS used to prepare reference solution (a),
mass of ch/orogenic acid CRS used to prepare reference in milligrams;
solution (a), in milligrams; 0.61 correction factor between N-isobutyldodecatetraenamide
0.695 correction factor between chlorogenic acid and cichoric acid; isomers 1 and 2, and benzanilide;
p percentage content of chlorogenic acid in ch/orogenic acid CRS. p percentage content of benzanilide in benzanilide CRS.
N-isobutyldodecatetraenamide
Solvent mixture acetonitrile R, water R (50:50 V/V).
Test solution Introduce 25.0 mL of the juice to be examined
into a chromatography column (l = 0.15 m; 0 = 30 mm)
Pygeum Bark
containing 15 g of kieselguhr for chromatography R and allow
(Pygeum Ajri.canum Bark, Ph. Eur. monograph 1886)
to stand for 15 min. Elute with 70 mL of methylene
chloride R. Evaporate the eluate to dryness in vacuo in a pear- PhEur - - - - - - - - - - - - - - - - - - - - ~ - -
shaped flask. Dissolve the residue in 1.0 mL of the solvent DEFINITION
mixture. Whole or fragmented, dried bark of the stems and branches
Reference solution (a) Dissolve 10.0 mg of benzanilide CRS of Prunus ajri.cana (Hook.f.) Kalkman (syn. Pygeum afri.canum
in methanol R and dilute to 50.0 mL with the same solvent. Hook.f.).
IDENTIFICATION
Reference solution (b) Dissolve 5.0 mg of echinacea dry extract A. The dark brown or reddish-brown bark occurs in curved,
for system suitability HRS in the solvent mixture and dilute to hard, irregular pieces. The outer surface has a wrinkled dark
2.5 mL with the solvent mixture. reddish-brown cork with areas of adhering lichen.
Column: The reddish-brown or dark brown inner surface bears
- size: l = 0.25 m, 0 = 4.0 mm; longitudinal striations. It may also occur in rolled fragments
- stationary phase: end-capped octadecylsilyl silica gel for with a fibrous fracture.
chromatography R (5 µm); B. Microscopic examination (2.8.23). The powder is reddish-
- temperature: 30 °C. brown. Examine under a microscope using chloral hydrate
Mobile phase: solution R. The powder shows the following diagnostic
- mobile phase A: water for chromatography R; characters (Figure 1886.-1): numerous sclereids, varying
- mobile phase B: acetonitrile R; greatly in size, up to more than 500 µm in diameter, with
very thick walls showing concentric striations and a reduced
Time Mobile phase A Mobile phase B lumen, isolated [A] or in groups [BJ sometimes accompanied
(min) (per cent V/J/) (per cent VIV)
by sclereids about 50 µm in diameter [Ba], some with
0 - 30 55 45 granular reddish-brown contents [Bb]; isolated cluster
30 - 40 55 ➔ 5 45---> 95 crystals of calcium oxalate of various sizes [CJ and a few
calcium oxalate prisms [F]; numerous lignified fibres, usually
Flow rate 1.5 mIJmin. broken, thick-walled and channelled with a narrow lumen,
2023 Quillaia IV-44 7
sometimes isolated [L], but usually in groups [G] Top of the plate
accompanied by rectangular cells of the medullary rays [Ga];
A violet zone
fragments of parenchyma with reddish-brown, polygonal or Several weak violet, blue or grey
ovoid cells [DJ, including some with reticulate walls U, M]; zones
fragments of cork (surface view [HJ, side view [El). Examine
- - --
under a microscope using lactic reagent R. The powder shows
a few simple starch granules that stain violet-blue, rounded, P-Sitosterol: a violet zone A violet zone CP-sitosterol)
U rsolic acid: a blue zone A blue zone (ursolic acid)
10-20 µm in diameter, with a punctiform or Y-shaped
Several weak violet, blue or grey
hilum [K]. zones
-- ----
A violet zone CP-sitosterol glucoside)
TESTS
105 °C for 2 h.
Total ash (2.4.16)
Extractable matter
Extract 20.0 g of the powdered herbal drug (250) (2.9.12)
with methylene chloride R for 4 h in a continuous extraction
apparatus (Soxhlet type). Evaporate the solution to dryness
on a water-bath in vacua and then dry the residue at 80 °C
for 2 h. The residue weighs a minimum of 0.10 g.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Quillaia
Preparations
Quillaia Liquid Extract
When Powdered Quillaia is prescribed or demanded, material
herbal drug of pygeum africanum bark
complying with the appropriate requirements below shall be
C. Thin-layer chromatography (2.2.27). dispensed or supplied.
Test solution Extract 15.0 g of the powdered herbal drug DEFINITION
(250) (2. 9.12) with methylene chloride R for 30 min in a Quillaia is the dried inner part of the bark of Quillaja
continuous extraction apparatus (Soxhlet type). Filter. saponaria Molina and of other species of Quillaja.
Evaporate the solvent to dryness under reduced pressure. IDENTIFICATION
Dissolve the residue in 1 mL of methylene chloride R. A. Pieces flat, up to about 1 metre long, 10 to 20 cm broad
Reference solution Dissolve 20 mg of /3-siwsterol R and 20 mg and 3 to 10 mm, usually 6 mm, thick. Outer surface
of ursolic acid R in 10 mL of a mixture of equal volumes of brownish white or pale reddish brown, longitudinally striated
methanol R and methylene chloride R. or coarsely reticulated, with occasional blackish brown
Plate TLC silica gel plate R. patches of adherent outer bark; inner surface yellowish white,
Mobile phase methanol R, methylene chloride R (10:90 V/V). smooth and very hard; fracture splintery and laminated, the
broken surface showing numerous large prisms of calcium
oxalate as glistening points. Smoothed transversely cut
Development Over a path of 15 cm. surface appearing chequered, with delicate radial lines
Drying In air. representing medullary rays and tangential lines formed by
Detection Treat with vanillin reagent R, heat at 100-105 °C alternating tangential bands of fibrous and non-fibrous
for 10 min and allow to cool; examine in daylight. phloem.
Results See below the sequence of zones present in the B. Outer bark, when present, consisting of reddish brown
chromatograms obtained with the reference solution and the cork cells alternating with bands of brown parenchyma
test solution. Furthermore, other zones may be present in the containing numerous groups of phloem fibres and large
chromatogram obtained with the test solution. prisms of calcium oxalate. Inner bark consisting of alternating
bands of tortuous fibres, irregularly enlarged at intervals,
about 500 to 1000 µm long and 20 to 50 µm wide and of
IV-448 Quillaia Bark 2023
sieve tissue mixed with parenchyma. Medullary rays mostly

three to four, but sometimes up to six cells wide, with
occasional pitted, subrectangular sclereids adjacent to the
bundles of phloem fibres. Starch granules 5 to 20 µm,
usually about 10 µm, in diameter and prisms of calcium
oxalate usually 50 to 170 µm long and up to 30 µm wide
present in the parenchymatous cells.
TESTS
Extractive soluble in ethanol (45%)
Not less than 22.0%, Appendix XI Bl.
Acid-insoluble ash
Foreign matter
Complies with the test for foreign matter, Appendix XI D.
Quillaia Bark
DEFINITION
Whole or fragmented, dried bark, with the cork and
underlying parenchyma removed, of Quillaja saponaria
Molina s.l.
Content
quillaia saponin III (C 104H 168 0 55 ; Mr 2298) (dried drug). Figure 1843.-1. - Illustration for identification test B of powdered
IDENTIFICATION herbal drug of quillaia bark
A. Large, fiat pieces of variable length and width, 3-10 mm
Reference solution Dissolve 10 mg of purified quillaia
thick, or smaller, splintered pieces. The outer surface is
saponins R and 2 mg of sucrose R in 1 mL of water R and mix
brownish-white or pale reddish-brown, longitudinally striated
with 1 mL of methanol R.
or coarsely reticulated, with occasional blackish-brown
patches of incompletely removed outer bark. The inner Plate TLC silica gel plate R (2-10 µm).
surface is yellowish-white and smooth. The fracture is Mobile phase anhydrous acetic acid R, ethyl acetate R, water R,
splintery and laminated, the surface often glistening due to propanol R (1.5:30:30:40 V/V/V/V).
the presence of numerous large prisms of calcium oxalate. Application 5 µL as bands of 6 mm.
B. Microscopic examination (2.8.23). The powder is pale Development Over a path of 6 cm.
pinkish-yellow. Examine under a microscope using chloral Drying In hot air.
Detection Treat with a 10 per cent V/V solution of sulfuric
characters (Figure 1843.-1): abundant phloem fibres [E, F],
acid R in methanol R; heat at 120 °C for 5 min and examine
up to 1 mm long, isolated or, more usually, in groups, each
in daylight.
fibre irregular in outline with lignified walls of varying
thickness and an uneven lumen; numerous, multiseriate Results See below the sequence of zones present in the
medullary rays, spindle-shaped (tangential section [Ca, Fb]), chromatograms obtained with the reference solution and the
accompanied by either phloem fibres [Fa] or phloem test solution. Furthermore, other faint zones may be present
parenchyma [CbJ; very numerous prisms of calcium oxalate, in the chromatogram obtained with the test solution.
up to 200 µm long, free, whole or, more usually, fragmented
[AJ or included in phloem parenchyma cells [Cc, CdJ; Top of the plate
occasional sclereids of 2 types: the 1st type is sub-rectangular
--- --
with pitted, slightly thickened walls, isolated [GJ or included
in phloem parenchyma cells [HJ, while the 2nd type has an Quillaia saponins: 3 or more green 3 or more green or brown zones
irregularly shaped outline and very thick walls W, sometimes or bro\Vll. zones (quillaia saponins)
adjacent to the bundles of phloem fibres; occasional dark A blue zone

brown or reddish-brown fragments of cork [DJ. Examine
Sucrose: a brown or blue zone A brown or blue zone (sucrose)
glycerol R. The powder shows numerous, small (5-20 µm), -- --
mainly simple, spherical starch granules, either scattered or as
compacted masses in parenchyma cells [BJ.
C. Thin-layer chromatography (2.2.27). Reference solution Test solution
(2.9.12) add 5 mL of methanol Rand 5 mL of water R.
Sonicate for 10 min and filter.
2023 Quillaia Preparations IV-449
TESTS area of the peak due to glycyrrhizic acid derived from

monoammonium glycyrrhizate in the chromatogram obtained
Loss on drying (2.2.32) with reference solution (a);
Maximum 10.0 per cent, determined on 1.000 g of the mass of the herbal drug to be examined used to prepare the
powdered herbal drug (355) (2. 9.12) by drying in an oven at test solution, in grams;
105 °C for 2 h. mass of quillaia saponin for assay CRS used to prepare
reference solution (a), in grams;
Total ash (2.4.16) p percentage content of monoammonium glycyrrhizate in
Maximum 10.0 per cent. quillaia saponin for assay CRS;
0.6 response factor between monoammonium glycyrrhizate and
monodesmosidic quillaia saponin 3;
Maximum 1.0 per cent. 2298 molecular mass of quillaia saponin III;
ASSAY 957 molecular mass of monodesmosidic quillaia saponin 3.
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
(355) (2.9.12) 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 Quillaia Liquid Extract
absorbent cotton. Add the absorbent cotton to the residue, DEFINITION
add 25 mL of ethanol (96 per cent) R and shake thoroughly. Quillaia Liquid Extract is prepared by extracting Quillaia
Filter. Combine the filtrates and dilute to 50.0 mL with with Ethanol (45 per cent).
water R. Filter through a membrane filter (nominal pore size
0.45 µm).
The following formula and directions apply.
Reference solution (a) Dissolve 12.0 mg of quil/aia saponinfor
assay CRS (containing monoammonium glycyrrhizate) in a Quillaia, in moderately fine powder 1000 g
mixture of equal volumes of ethanol (96 per cent) R and a Ethanol (45 per cent) A sufficient quantity
10 g/L solution of phosphoric acid R, and dilute to 50.0 mL
with the same mixture of solvents. Exhaust the Quillaia in moderately fine powder with Ethanol
Reference solution (b) Introduce 12 mg of purified quil/aia (45 per cent) by percolation, Appendix XI F, and reserve the
saponins HRS into a 50 mL round-bottomed flask, add first 850 mL of percolate. Evaporate the subsequent
20 mL of a 20 g/L solution of potassium hydroxide R and heat percolate to the consistence of a soft extract, dissolve it in the
under a reflux condenser in a water-bath for 2 h. After reserved portion and add sufficient Ethanol (45 per cent) to
cooling, add 2 mL of phosphoric acid R. Add 25 mL of produce 1000 mL. Allow to stand for not less than 24 hours;
ethanol (96 per cent) Rand shake thoroughly. Dilute to filter.
50.0 mL with water R. Filter through a membrane filter The extract compli.es with the requirements stated under Extracts
(nominal pore size 0.45 µm). and with the following requirements.
Column: TESTS
- size: l =0.25 m, 0 =4.6 mm; Ethanol content
(5 µm);
- temperature: 30 ± 2 °C. Dry residue
20 to 30% w/v.
Mobile phase acetonitrile Rl, 1 g/L solution of phosphoric
acid R (35:65 V/V). Relative density
Flow rate 1.0 mUmin. 1.02 to 1.06, Appendix V G.
Injection 50 µL.
Run time 1.2 times the retention time of glycyrrhizic acid.
Quillaia Tincture
purified quil/aia saponins HRS and the chromatogram obtained DEFINITION
with reference solution (b) to identify the peaks due to
monodesmosidic quillaia saponins 1 and 3; a minor peak due Quillaia Liquid Extract 50 mL
Ethanol (45 per cent) Sufficient to produce 1000 mL
to monodesmosidic quillaia saponin 2 may be present
between the peaks due to monodesmosidic quillaia
saponins 1 and 3.
Retention time Monodesmosidic quillaia saponin 1 = about
Mix, allow to stand for not less than 12 hours and filter.
9 min; monodesmosidic quillaia saponin 3 = about 10 min;
glycyrrhizic acid = about 13 min. The tincture compli.es with the requirements for Tinctures stated
Calculate the percentage content of triterpene glycosides,
expressed as quillaia saponin III, using the following TESTS
expression: Ethanol content
A 1 x m2 xpx 2298 x0.6
Dry residue
A 2 x m 1 x 957 1.0 to 1.5% w/v. Use 10 mL.
A1 sum of the areas of the peaks due to monodesmosidic quillaia Relative density
saponins (1, 2 and 3) in the chromatogram obtained with the 0.940 to 0.955, Appendix VG.
test solution;
IV-450 Raspberry Leaf 2023
Raspberry Leaf
(Ph Bur. monograph 2950)
~~---------------------
DEFINITION
Whole or fragmented, dried leaves of Rubus idaeus L.
harvested in spring or early summer.
Content
IDENTIFICATION
A. Whole drug. The leaf is imparipinnate, with 3, 5 or rarely
7 ovate to lanceolate leaflets with sharply serrate or doubly-
serrate margins. The upper surface is dark green to
brownish-green and weakly pubescent; the lower surface is
silvery grey and densely tomentose with a prominent pinnate
venation. The petiole is about 1-2 mm thick, green or
occasionally reddish, slightly channelled on the upper surface,
and sometimes shows small, straight prickles. The stipules
are long and thin. The leaves of the short shoots are often
simple, more or less 3-lobed, or they are trifoliate.
Fragmemed drug It is characterised by clumped fragments of
leaves and petioles.
(71 0) (2. 9.12). The powder is greyish-green and flocculent.
(Figure 2950.-1): very numerous broken unicellular covering
trichomes, either rigid with thickened walls [G], or twisted Figure 2950.-1. - Illustration for identification test B of powdered
with slightly thickened walls [F]; fragments of the upper herbal drug of raspberry leaf
epidermis of the leaflet lamina (surface view [DJ) with
polygonal cells [Da] and rare, rigid, unicellular covering Reference solutwn (b) Dilute 2.5 mL of reference solution (a)
trichomes with thickened walls, measuring from 20 µm to to 10.0 mL with methanol R.
more than 500 µm in length [Db], or their scars, often Reference solutwn (c) Dissolve 3 mg of hyperoside R and
accompanied by palisade parenchyma [De] including some 2.5 mg of chlorogenic acid R in methanol R and dilute to
hypertrophied cells containing a cluster crystal of calcium 10 mL with the same solvent.
oxalate [Dd]; fragments of the lower epidermis of the leaflet Intensity marker Hyperoside.
lamina [A] containing cells with thin, slightly lobed Plate TLC silica gel F254 plate R (2-10 µm).
walls [Aa] and anomocytic stomata (2.8.3) (5-7 subsidiary Mobile phase anhydrous formic acid R, water R, ethyl acetate R
cells) (Ab], with an abundant indumentum composed of (10:10:80 VIVIV).
unicellular, fine, twisted covering trichomes [Ac] up to
500 µm long; annular or spiral vessels from the petioles, Applicatwn 10 µL of the test solution and 4 µL of reference
rachis or principal veins ITT, sometimes accompanied by cells solutions (a), (b) and (c), as bands of 8 mm.
of the pith with slightly thickened, pitted walls Ua]; fragments Development 70 mm from the lower edge of the plate.
of pericyclic fibres [C], frequently with a crystal sheath Drying In a current of air at room temperature for 5 min.
containing small cluster crystals of calcium oxalate [Ca]; Detection Heat at 100-105 °C for 5 min; spray the warm
fragments of the lamina (transverse section [H]) consisting of plate with a 10 g/L solution of diphenylboric acid aminoethyl
the upper epidermis [Ha], 1 or 2 layers of palisade ester R in methanol R, then with a 50 g/L solution of macrogol
parenchyma [Hb], including hypertrophied cells containing a 400 R in methanol R or, alternatively, dip the warm plate in a
cluster crystal of calcium oxalate [He], spongy 5 g/L solution of diphenylboric acid aminoethyl ester R in ethyl
parenchyma [Hd] and the lower epidermis with twisted acetate R, then in a 50 g/L solution of macrogol 400 R in
unicellular covering trichomes [He]; free cluster crystals of methylene chloride R; allow to dry in air for about 1 min and
calcium oxalate [E]; exceptionally, secretory trichomes with a examine in ultraviolet light at 366 nm.
biseriate, multicellular stalk and a globular, multicellular System suitability Reference solution (c):
head, located on the upper epidermis of the leaflet veins [BJ. - the chromatogram shows in the middle third 2 distinct
C. High-performance thin-layer chromatography (2.8.25). zones, which may be touching; the lower zone
Test solution To 0.5 g of the powdered herbal drug (710) (chlorogenic acid) shows a light blue fluorescence and
(2.9.12) add 10.0 mL of methanol R. Sonicate for 15 min, the upper zone (hyperoside) shows a yellow or orange
filter or centrifuge and use the filtrate or supernatant. fluorescence.
Reference solution (a) Dissolve 2.5 mg of hyperoside Rand Results See below the sequence of fluorescent zones present
3.5 mg of rutoside trihydrate R in methanol Rand dilute to in the chromatograms obtained with reference solution (a)
10.0 mL with the same solvent. and the test solution. Furthermore, in the chromatogram
obtained with the test solution, other faint fluorescent zones,
2023 Rehmannia Root IV-451
mainly light blue, greenish and yellow or orange, may be

present, especially in the upper and lower thirds.
Rehmannia Root
Top of the plate
DEFINITION
A red zone Dried root tuber of Rehmannia glutinosa (Gaertn.) DC. (syn.
A red zone, faint
Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C.A.
Mey.) with the crown and rootlets removed.
Content
Minimum 0.2 per cent of catalpol (C 15 H 22 O 10; Mr 362.3)
(dried drug).
-- --
IDENTIFICATION
A. The whole drug usually occurs as irregular or oblong
A light blue zone, faint to equivalent masses, swollen in the centre, slightly tapering at both ends
and bearing lenticular rootlet scars, 6-12 cm long and 3-6 cm
A greenish zone, faint (possibly
overlapping with an orange zone) thick. The fragmented drug occurs as slightly compressed or
twisted slices. The outer surface is blackish-brown or
blackish-grey, and is heavily shrunken with irregular
Hyperoside: a yellow or orange zone A yellow or orange zone, very faint transverse wavy lines. Fracture is difficult, with the cut
to faint (possibly overlapping with a surface being lustrous, blackish-brown or jet black and
blue zone)
viscous.
A yellow or orange zone
B. Microscopic examination (2.8.23). The blackish-brown
powder consists of rather sticky particles. Examine under a
-- -- shows the following diagnostic characters (Figure 2569.-1):
blackish-brown cork fragments consisting of polygonal cells
(surface view [A]) or composed of superimposed layers
(transverse section [E]); fragments ofparenchyma [DJ,
Rutoside: a yellow or orange zone brown, consisting of polygonal or rectangular cells with thin,
A yellow or orange zone, faint to
wavy or wrinkled walls, some containing orange-yellow oil
equivalent droplets [Da]; vessels about 100-200 µm long and 40-60 µm
in diameter, with reticulate or pitted walls [B, C], with the
junction between vessels of similar diameter being clearly
visible.
Reference solution (a) Test solution C. High performance thin-layer chromatography (2.8.25).
(2. 9.12) add 5.0 mL of metha,wl R. Sonicate for 10 min,
TESTS then filter or centrifuge the solution. Use the filtrate or
Rubus fruticosus L supernatant.
Microscopic examination (2.8.23). Examine the powdered
Reference solution (a) Dissolve 10.0 mg of sucrose Rand
herbal drug (710) (2. 9.12) using chloral hydrate solution R.
10.0 mg of raffinose R in the minimum volume of water R
The presence of fasciculate-stellate covering trichomes
and dilute to 2.0 mL with methanol R.
indicates adulteration by Rubus jrnticosus L.
Reference solution (b) Mix 1.0 mL of reference solution (a)
Loss on drying (2.2.32) and 3.0 ml of methanol R.
Reference solution (c) Dissolve 10 mg ofjrnctose Rand
105 °C for 2 h. 10 mg of sucrose R in the minimum amount of water R and
dilute to 2 mL with methanol R.
Total ash (2.4.16)
Intensil;Y marker Sucrose.
ASSAY
Mobile phase glacial acetic acid R, anhydrous formic acid R,
Tannins (2.8.14)
water R, ethyl acetate R (4:5:6:12 VIVIV!V).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Detection Treat with a 10 per cent VIV solution of sulfuru
acid R in ethanol (96 per cent) R, heat at 120 °C for 3 min
and examine in ultraviolet light at 366 nm.
zones, which may be touching; both the lower zone
(sucrose) and the upper zone (fructose) show a brown
colour.
IV-452 Rehmannia Root 2023
TESTS
105 °C for 5 h.
Total ash (2.4.16)
ASSAY
(2.9.12) add 50.0 mL of water R. Weigh and sonicate for
30 min at a temperature below 25 °C. Weigh again and
compensate for the loss of solvent with water R. Shake well,
then centrifuge for 10 min and filter the supernatant through
a membrane filter (nominal pore size 0.45 µm).
Reference solution Dissolve 5.0 mg of catalpa! CRS in water R
Column:
=
- size: l 0.15 m, 0 =
4.6 mm;
Mobile phase:
- mobile phase B: acetonitrile Rl, water for chromatography R
Figure 2569.-1. - Illustration for identification test B of powdered (5:95 V/V);
herbal drug of rehmannia root
Results See below the sequence of zones present in the (min) (per cent V/J/) (per cent V/J/)
chromatograms obtained with reference solution (a) and the 0-3 90 10
test solution. Furthermore, in the chromatogram obtained 3 - 20 90-+ 70 10-+ 30
with the test solution, other faint blue fluorescent zones and
faint brown zones may be present. Flow rate 0.5 mL/min.
Top of the plate
Injection 10 µL.
Retention time Catalpol = about 13 min.
- repeatability: maximum relative standard deviation of
A blue fluorescent zone, faint to
intense
2.0 per cent determined on 6 injections.
Calculate the percentage content of catalpol using the
--- ---
A brown zone, faint
Sucrose: a brown zone A brown zone, faint to equivalent

A1 area of the peak due to catalpol in the chromatogram obtained
(sucrose)
A2 area of the peak due to catalpol in the chromatogram obtained
-- ---
Raffi.nose: a brown zone A brown zone, faint to equivalent m1 mass of the herbal drug to be examined used to prepare the test
(raffinose) solution, in grams;
m2 mass of cata/po/ CRS used to prepare the reference solution, in
grams;
A brown zone, intense p percentage content of catalpol in catalpo/ CRS.
A brown zone, very faint _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur

2023 Restharrow Root IV-453

Restharrow Root Test solution To 1.0 g of the powdered herbal drug (180)
(Ph. Bur. monograph 1879) (2. 9.12) add 15.0 mL of methanol R and boil under a reflux
condenser for 30 min. Cool and filter.
Reference solution Dissolve 10 mg of resorcinol R and 50 mg
DEFINITION of vanillin R in 10 mL of methanol R.
Whole or cut, dried root of Ononis spinosa L. Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica
IDENTIFICATION gel F254 plate R (2-10 µm)].
A. The root is more or less flattened, twisted and branched, Mobile phase ethanol (96 per cent) R, methylene chloride R,
deeply wrinkled, brown and grooved longitudinally. toluene R (10:45:45 VIVIV).
The transversely cut surface shows a thin bark and a xylem Application 20 µL [or 5 µL] as bands of 15 mm [or 8 mm].
cylinder with a conspicuously radiate structure. The fracture Development Over a path of 15 cm [or 6 cm].
of the root is short and fibrous.
Drying In air.
Detection A Examine in ultraviolet light at 254 nm and
brown or brown. Examine under a microscope using chloral
365 nm.
characters (Figure 1879.-1): brown fragments of cork Results A See below the sequence of zones present in the
composed of thin-walled, polygonal cells (surface view [G]); chromatograms obtained with the reference solution and the
groups of thick-walled, narrow fibres, often accompanied by test solution. Furthermore, other fluorescent zones are
a parenchymatous crystal sheath containing prisms of calcium present in the middle third of the chromatogram obtained
oxalate [C]; vascular fragments [D, E] consisting of vessels with the test solution.
with numerous bordered pits, often accompanied by lignified
Top of the plate
fibres with pitted walls [Ea]; thin-walled parenchymatous
cells from the bark, some containing a single prism of Vanillin: a zone visible at 254 nm
calcium oxalate [HJ; ligneous parenchyma cells with slightly
thickened and pitted walls [A, BJ, some of which contain -- --
prisms of calcium oxalate [Aa]; numerous free prisms of Resorcinol: a zone visible at 254 nm An intense blue fluorescent zone
calcium oxalate [F]. Examine under a microscope using a visible at 365 nm
50 per cent V/V solution of glycerol R. The powder shows -- --
very numerous, rounded starch granules, 5-10 µm in
diameter, simple or sometimes 2-4 compound, free mor
inside parenchymatous cells [Kl.

Detection B Treat with anisaldehyde solution R. Heat at
100-105 °C for 5-10 min. Examine in daylight.
test solution.
Top of the plate
Vanillin: a greyish-violet zone
-- --
A violet zone (onocol)
Resorcinol: a red zone
-- --
TESTS
powdered herbal drug (355) (2.9. 12) by drying in an oven at
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
Figure 1879.-1. Illustration for identification test B of powdered
~
minimum of 75 mg.
herbal drug of restharrow root _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
IV-454 Rhatany Root 2023
Rhatany Root
Krameria
Preparation
Rhatany Tincture
When Powdered Rhatany Root is prescribed or demanded,
exception ofidentification test A and the test for Foreign
matter shall be dispensed or supplied.
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried, usually fragmented, underground organs of Krameria
triandra Ruiz et Pav., known as Peruvian rhatany.
Content
Minimum 5 .0 per cent of tannins, expressed as pyrogallol
IDENTIFICATION
A. The taproot is dark reddish-brown and has a thick, knotty
crown. The secondary roots are the same colour and nearly
straight or somewhat tortuous. The bark is rugged or scaly in
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.
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 Figure 0289.-1. - Illustration for identification test B of powdered
numerous fine medullary rays; the central heartwood is often herbal drug of rhatany root
darker.
B. Microscopic examination (2.8.23). The powder is reddish- Detection Treat with a 5 g/L solution of fast blue B salt R,
brown. Examine under a microscope using chloral hydrate allow to dry in air and examine in daylight.
solution R. The powder shows the following diagnostic Results See below the sequence of zones present in the
characters (Figure 0289.-1): cork cells containing dark brown chromatograms obtained with the reference solution and the
phlobaphenes (surface view [A], side view [B]); fragments of test solution. Furthermore, other faint zones may be present
phloem [CJ consisting ofunlignified fibres, usually 12-30 µm in the chromatogram obtained with the test solution.
in diameter with moderately thickened walls [Ca],
parenchyma cells some containing prisms and microcrystals Top of the plate
of calcium oxalate [Cc], and cells of the medullary rays [Cb];
fragments of vessels usually 20-60 µm in diameter with -- --
bordered pits [E]; fragments of tracheids [DJ up to 20 µm Thymol: a brownish-yellow zone A faint violet zone
wide 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
50 per cent V/V solution of glycerol R. The powder shows A bluish-grey zone
rounded, simple or 2- to 4-compound starch granules, an
individual granule measuring up to 30 µm in diameter [HJ
and some granules being found in the cells of the medullary Dichlorophenolindophenol: a
rays and in the parenchyma [G]. greyish-blue zone
C. Thin-layer chromatography (2.2.27). An intense violet zone
Centrifuge or filter. Use the supernatant or filtrate. Reference solution Test solution
Reference solution Dissolve 5 mg of thymol R and 20 mg of
dichlorophenolindophenol, sodium salt R in 20 mL of ethanol TESTS
(60 per cent V/V) R. Foreign matter (2.8.2)
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel Maximum 2 per cent of foreign matter and maximum
plate R (2-10 µm)]. 5 per cent of fragments of crown or root exceeding 25 mm in
Mobile phase methylene chloride R. diameter. Root without bark may be present in very small
Application 10 µL [or 4 µL] as bands of 8 mm [or 8 mm]. quantities.
Development Over a path of 15 cm [or 6 cm]. Loss on drying (2.2.32)
Drying In air.
105 °C.
2023 Rhubarb IV-45 5
Total ash (2.4.16) TESTS

Maximum 5.5 per cent. Ethanol (2. 9.10)
ASSAY 63 per cent V/V to 67 per cent VIV.
Tannins (2.8.14) Methanol and 2-propanol (2. 9.11)
Use 0.750 g of the powdered herbal drug (180) (2.9.12). Maximum 0.05 per cent VIV of methanol and maximum
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur 0.05 per cent V/V of 2-propanol.
ASSAY
Tannins (2.8.14)
Use 2.500 g of the tincture to be examined.
Rhatany Tincture - - - - - - - - - - - - - - - - - - - - - Ph Eur
DEFINITION Rhubarb
Tincture produced from Rhatany root (0289). (Ph. Bur. monograph 0291)
Content
Preparation
Minimum 1.0 per cent m/m of tannins, expressed as
Compound Rhubarb Tincture
pyrogallol (C 6 H 6 0 3; M, 126.1).
When Powdered Rhubarb is prescribed or demanded,
PRODUCTION material complying with the requirements below with the
The tincture is produced from 1 part of the herbal drug and exception of Identification test A and the test for Foreign
5 parts of ethanol (70 per cent VIV) by a suitable procedure. matter shall be dispensed or supplied.
CHARACTERS
Appearance
DEFINITION
Reddish-brown liquid.
Rhubarb consists of the whole or cut, dried underground
IDENTIFICATION parts of Rheum palmatum L. or of Rheum officinale Baillon or
Thin-layer chromatography (2.2.27). of hybrids of these two species or of a mixture.
Test solution The tincture to be examined. The underground parts are often divided; the stem and most
Reference solution Dissolve 5 mg of thymol R and 20 mg of of the bark with the rootlets are removed. It contains not less
dichlorophenolindophenol, sodium salt R in 20 mL of ethanol than 2.2 per cent of hydroxyanthracene derivatives, expressed
(60 per cent VIV) R. as rhein (C 15H 8 06' M, 284.2), calculated with reference to
the dried drug.
plate R (2-10 µm)]. CHARACTERS
Mobile phase methylene chloride R. Characteristic, aromatic odour.
Application 10 µL [or 4 µL] as bands of 8 mm [or 8 mm]. IDENTIFICATION
Development Over a path of 15 cm [or 6 cm]. A. The appearance is variable: disc-shaped pieces up to
Drying In air.
10 cm in diameter and 1 cm to 5 cm in thickness; cylindrical
pieces; oval or planoconvex pieces. The surface has a pinkish
Detection Treat with a 5 g/L solution of fast 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
Results See below the sequence of zones present in the darker lines. This structure causes the marbled appearance of
chromatograms obtained with the reference solution and the the drug. The fracture is granular. The transverse section of
test solution. Furthermore, other faint zones may be present the rhizome shows a narrow outer zone of radiating
in the chromatogram obtained with the test solution. brownish-red lines. These medullary rays are crossed
perpendicularly by a dark cambial ring. Inside this zone is a
Top of the plate ring of small star-spot formations of anomalous vascular
bundles. The root shows a more radiate structure.
-- --
B. Microscopic examination (2.8.23). The powder is orange
Thymol: a brownish-yellow zone A violet zone to brownish-yellow. Examine under a microscope using
-- --
diagnostic characters: large calcium oxalate cluster crystals,
An orange zone which may measure more than 100 µm, and their fragments;
A bluish-grey zone reticulately thickened non-lignified vessels measuring up to
175 µm. Numerous groups of rounded or polygonal, thin-
walled parenchyma cells. Sclereids and fibres are absent.
Dichlorophenolindophenol: a Examine under a microscope using a 50 per cent V/V
greyish-blue zone solution of glycerol R. The powder shows simple, rounded or
compound (2 to 4) starch granules with a star-shaped hilum.
An intense violet zone
C. Examine by thin-layer chromatography (2.2.27), using a
suitable silica gel as the coating substance.
Reference solution Test solution 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
IV-456 Rhubarb Preparations 2023
of hydrochloric acid R and 30 mL of water R. Allow to cool 100 mL round-bottomed flask with a ground-glass neck.
and shake the liquid with 25 mL of ether R. Dry the ether Add 20 mL of ferric chloride solutwn Rl and mix. Heat under
layer over anhydrous sodium sulfate R and filter. Evaporate the a reflux condenser on a water-bath for 20 min, add 1 mL of
ether layer to dryness and dissolve the residue in 0.5 mL of hydrochloric acid R and heat for a further 20 min, shaking
ether R. frequently. Cool, transfer to a separating funnel and shake
Reference solutwn Dissolve 5 mg of emodin R in 5 mL of with three quantities, each of 25 mL, of ether R previously
ether R. used to rinse the flask. Combine the ether extracts and wash
with two quantities, each of 15 mL, of water R. Filter the
Apply separately to the plate as bands 20 µL of each
ether extracts through a plug of absorbent cotton into a
solution. Develop over a path of 10 cm using a mixture of
volumetric flask and dilute to 100.0 mL with ether R.
1 volume of anhydrous formic acid R, 25 volumes of ethyl
Evaporate 10.0 mL carefully to dryness on a water-bath and
acetate R and 75 volumes of light petroleum R. Allow the plate
dissolve the residue in 10.0 mL of a 5 g/L solution of
to dry in air and examine in ultraviolet light at 365 nm.
The chromatogram obtained with the reference solution
magnesium acetate R in methanol R. Measure the absorbance
shows in its central part a zone of orange fluorescence
(2.2.25) at 515 nm, using methanol Ras the compensation
liquid.
(emodin). The chromatogram obtained with the test solution
shows: a zone due to emodin; above the emodin zone, two Calculate the percentage content of rhein from the
zones of similar fluorescence (physcione and chrysophanol, in expression:
order of increasing Rp value); below the emodin zone, also
two zones of similar fluorescence (rhein and aloe-emodin, in Ax 0.64
order of decreasing Rp value). Spray with a 100 g/L solution m
of potassium hydroxide R in methanol R. All the zones become
i.e. taking the specific absorbance of rhein to be 468,
red to violet.
calculated on the basis of the specific absorbance of
D. To about 50 mg of the powdered herbal drug (180) barbaloin.
(2. 9.12) add 25 mL of dilute hydrochloric acid R and heat the
mixture on a water-bath for 15 min. Allow to cool, shake A absorbance at 515 nm,
with 20 mL of ether R and discard the aqueous layer. Shake m mass of the herbal drug used, in grams.
the ether layer with 10 mL of dilute ammonia Rl.
The aqueous layer becomes red to violet. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
TESTS
Rheum rhaponticum
Examine by thin-layer chromatography (2.2.27), using silica
gel G R as the coating substance. Compound Rhubarb Tincture
Test solutwn To O.2 g of the powdered herbal drug (180) DEFINITION
(2. 9.12) add 2 mL of methanol R and boil for 5 min under a
reflux condenser. Allow to cool and filter. Use the filtrate as Rhubarb, in moderately coarse 100 g
the test solution. powder
Cardamom Oil 0.40mL
Reference solutwn Dissolve 10 mg of rhaponticin R in 10 mL Coriander Oil 0.03 mL
of methanol R. Glycerol IO0mL
Ethanol (60 per cent) Sufficient to produce I 000 mL
Apply separately to the plate, as bands not more than 20 mm
by 3 mm, 20 µL of each solution. Develop over a path of
12 cm using a mixture of 20 volumes of methanol Rand Extemporaneous preparation
80 volumes of methylene chloride R. Allow the plate to dry in The following directions apply.
air and spray with phosphomolybdic acid solutwn R. Moisten the Rhubarb with a sufficient quantity of Ethanol
The chromatogram obtained with the test solution does not ( 60 per cent) and prepare 850 mL of tincture by percolation,
show a blue zone near the line of application (rhaponticin) Appendix XI F. Add the Cardamom Oil, the Coriander Oil
corresponding to the zone in the chromatogram obtained and the Glycerol and sufficient Ethanol (60 per cent) to
with the reference solution. produce 1000 mL. Mix and filter, if necessary.
Loss on drying (2.2.32) The tincture complies with the requirements for Tinctures stated
Not more than 12.0 per cent, determined on 1.000 g of the under Extracts and with the following requirements.
powdered herbal drug (180) (2.9.12) by drying in an oven at TESTS
105 °C. Ethanol content
Total ash (2.4.16) 48 to 53% v/v, Appendix VIII F, Method III.
Not more than 12.0 per cent. Glycerol
Ash insoluble in hydrochloric acid (2.8.1) 9.0 to 11.0% v/v when determined by the following method.
Not more than 2.0 per cent. Dilute 20 mL to 100 mL with water, to 10 mL of this
solution add 100 mL of water and 1 g of activated charcoal
ASSAY
and boil under a reflux condenser for 15 minutes. Filter,
wash the filter and charcoal with sufficient water to produce
Introduce 0.100 g of the powdered herbal drug (180) 150 mL, add 0.25 mL of bromocresol purple solutwn and
(2.9.12) into a 100 mL flask. Add 30.0 mL of water R, mix neutralise with 0.lM sodium hydroxide or 0.05M suljuric acid to
and weigh. Heat in a water-bath under a reflux condenser for the blue colour of the indicator. Add 1.4 g of sodium
15 min. Allow to cool, add 50 mg of sodium hydrogen perwdate, allow to stand for 15 minutes, add 3 mL of
carbonate R, weigh and adjust to the original mass with propane-1,2-diol, shake and allow to stand for 5 minutes.
water R. Centrifuge and transfer 10. 0 mL of the liquid to a Add 0.25 mL of bromocresol purple solution and titrate with
2023 Roselle IV-457
O.lM sodium hydroxide VS to the same blue colour. Each mL

of 0. 1M sodium hydroxide VS is equivalent to 9 .210 mg of
glycerol. Calculate the percentage v/v of glycerol, taking
1.260 g as its weight per mL.
Relative density
Roselle
(Ph. Bur. monograph 1623) E~
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
\G
~
DEFINITION H
Whole or cut dried calyces and epicalyces of Hibiscus ;.-r:_ ....
'
sabdarijfa L. collected during fruiting.
Content
Minimum 13.5 per cent of acids, expressed as citric acid
(C5H 8 01; Mr 192.1) (dried drug).
..
CHARACTERS \'
Acidic taste. b
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
herbal drug of roselle
or deep purple, somewhat lighter at the base of the inner
side. Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
B. Microscopic examination (2.8.23). The powder is red or plate R (2-10 µm)].
violet-red. Examine under a microscope using chloral hydrate Mobile phase anhydrous formic acid R, water R, butanol R
solution R. The powder shows the following diagnostic (10:12:40 VIVIV).
characters (Figure 1623.-1): predominantly red
fragments [A, F] consisting of polygonal epidermal cells with
very irregularly thickened walls (surface view [Ac, Fa]), some Development Over a path of 10 cm [or 6 cm].
containing cluster crystals of calcium oxalate [Fb], with Drying In air.
underlying parenchyma consisting of ovoid cells with slightly Detection Examine immediately in daylight.
thickened walls [Aa], some containing cluster crystals of Results See below the sequence of zones present in the
calcium oxalate [Ab] whilst others are filled with mucilage, chromatograms obtained with the reference solution and the
unicellular, long, flexuous, twisted covering trichomes [Ad], test solution. Furthermore, other faint zones may be present
rigid, straight, unicellular covering trichomes, simple or in in the chromatogram obtained with the test solution.
groups of 2-4 [Fd], glandular trichomes with a unicellular
stalk and a globular or oval, multicellular and biseriate
Top of the plate
head [Fe] and stomata usually of the anisocytic type
(2.8.3) [Fe]; numerous fragments of vascular bundles [DJ -- --
with spiral or reticulate vessels [Da], sometimes accompanied
Quinaldinc red: an orange-red zone
by sclerenchymatous fibres with a wide lumen [Db], and
parenchyma [De], of which some cells contain cluster crystals An intense violet zone
of calcium oxalate [Dd], whilst others are mucilage-
Sulfan blue: a blue zone
filled [De]; rare, rectangular, parenchymatous sclereids [HJ;
numerous fragments of rigid [C, G] or flexuous W covering An intense violet-blue zone
trichomes; free cluster crystals of calcium oxalate [BJ and
-- --
glandular trichomes [E]; exceptionally, spherical pollen
grains, about 200 µm in diameter, with a spiny exine. Reference solution Test solution
Test solution To 1 g of the powdered herbal drug (355) TESTS
(2. 9.12) add 10 mL of ethanol (60 per cent VIV) R. Shake for Foreign matter (2.8.2)
15 min and filter. Maximum 2 per cent of fragments of fruits (red funicles and
Reference solution Dissolve 2.5 mg of quinaldine red Rand parts of the 5-caverned capsule with yellowish-grey pericarp,
2.5 mg of sulfan blue R in 10 mL of methanol R. whose thin walls consist of several layers of differently
IV-458 Rosemary Leaf 2023
directed fibres; flattened, reniform seeds with a dotted fragmented [A, C, D]; fragments of the upper epidermis
surface) and maximum 2 per cent of other foreign matter. (surface view [F]) with cells with straight, thickened and
Loss on drying (2.2.32) pitted walls [Fa], and an underlying hypodermis composed of
Maximum 11.0 per cent, determined on 1.000 g of the large, irregular cells with thickened and beaded anticlinal
powdered herbal drug (355) (2.9.12) by drying in an oven at walls [Fb]; fragments of the lamina (transverse section [G]),
105 °C for 2 h. showing the epidermis covered by a very thick cuticle [Ga],
hypodermal cells extending across the mesophyll [Gb] at
Total ash (2.4.16)
intervals, separating 1 or 2 layers of palisade parenchyma into
large, crescent-shaped areas [Ge]; glandular trichomes of 2
Colouring intensity types, the majority with a short, unicellular stalk and a
Reduce 100 g to a powder (1400) (2.9.12) and homogenise. radiate head composed of 8 cells (surface view [E], side view
Further reduce about 10 g of this powder to a finer powder [H]), others, less abundant, with a uni- or bicellular stalk and
(355) (2.9.12). To 1.0 g of this powder in a 100 mL flask a spherical, unicellular head Ua, K].
add 25 mL of boiling water Rand 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 Ras 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.
Filter. To 50.0 mL of the filtrate add 100 mL of carbon
dioxide-free water R. Titrate with 0.1 M sodium hydroxide to
pH 7 .0, determining the end-point potentiometrically
(2.2.20).
1 mL of 0.1 M sodium hydroxide is equivalent to 6.4 mg
of citric acid.
---------------------~~
Rosemary Leaf
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole, dried leaf of Rosmarinus officinalis L.
Content Figure 1560.-1. - Illustration for identification test B of powdered
- minimum 12 mUkg of essential oil (anhydrous drug); herbal drug of rosemary leaf
- minimum 3 per cent of total hydroxycinnamic derivatives,
expressed as rosmarinic acid (C 1sH16Os; Mr 360.3) C. Thin-layer chromatography (2.2.27).
(anhydrous drug). Test solution Dissolve 20 µL of the oil obtained in the assay
CHARACTERS in 1 mL of hexane R.
Strongly aromatic odour. Reference solution Dissolve 5 mg of borneol R, 5 mg of horny!
IDENTIFICATION acetate R and 10 µL of cineole R in 1 mL of hexane R.
A. The leaves are sessile, tough, linear or linear-lanceolate, Plate TLC silica gel plate R.
1-4 cm long and 2-4 mm wide, with recurved edges. Mobile phase ethyl acetate R, toluene R (5:95 V/V).
The upper surface is dark green, glabrous and grainy, the Application 10 µL as bands.
lower surface is greyish-green and densely tomentose with a
prominent midrib.
Drying In air.
green or yellowish-green. Examine under a microscope using Detection Treat with anisaldehyde solution R, heat at
chloral hydrate solution R. The powder shows the following 100-105 °C for 10 min and examine in daylight.
diagnostic characters (Figure 1560.-1): fragments of the Results See below the sequence of zones present in the
lower epidermis (surface view [B, TI) with straight or sinuous- chromatograms obtained with the reference solution and the
walled cells [Ba] and numerous diacytic stomata (2.8.3) [Bb] test solution.
and glandular trichomes Ua] or covering trichomes or their
scars [Be, Bd]; numerous multicellular, mostly branched,
covering trichomes of the lower epidermis, usually
2023 Rosemary Oil IV-459
Top of the plate molybdate R in 100 mL of water R, and then add 2 mL of

dilute sodium hydroxide solution Rand dilute to 10.0 mL with
A red zone
water R; mix.
Bomyl acetate: a yellowish-brown A yellowish-brown zone of low Compensation solution Dilute 1.0 mL of the stock solution to
zone intensity
A coloured zone of low intensity Measure immediately the absorbance (2.2.25) of the test
Cineole: a violet zone A violet zone
solution at 505 nm.
Calculate the percentage content of total hydroxycinnamic
Coloured zones of low intensity derivatives, expressed as rosmarinic acid, using the following
Borneo]: a violet-brown zone A violet-brown zone expression:
A coloured zone of low intensity Ax2.5
Reference solution Test solution m
i.e. taking the specific absorbance of rosmarinic acid to be

D. Thin-layer chromatography (2.2.27). 400.
Test solution Grind 1.0 g of the herbal drug in 10 mL of
methanol Rand filter. A absorbance of the test solution at 505 nm;
Reference solution Dissolve 1.0 mg of cajfeic acid Rand
5.0 mg of rosmarinic acid R in 10 mL of methanol R.
Plate TLC silica gel plate R. Use 25.0 g of the crushed herbal drug, a 1000 mL flask and
Mobile phase anhydrous formic acid R, acetone R, methylene 300 mL of water R as the distillation liquid. Distil at a rate of
chloride R (8.5:25:85 V/V/V). 2-3 mUmin for 3 h.
Application 10 µL of the test solution and 20 µL of the - - - - - - - - - - - - - - - - - - - - - - Ph Eur
Drying In air.
Detection Examine in ultraviolet light at 365 nm. Rosemary Oil
test solution. ~m ______________________
DEFINITION
Top of the plate Essential oil obtained by steam distillation from the flowering
A pink fluorescent zone
aerial parts of Rosmarinus officinalis L.
Caffeic acid: a light blue fluorescent A blue fluorescent zone of low

CHARACTERS
zone intensity Appearance
Clear, mobile, colourless or pale yellow liquid.
Rosmarinic acid: a light blue An intense light blue fluorescent
fluorescent zone zone Characteristic odour.
Reference solution Test solution IDENI1FICATION

TESTS
Test solution Dissolve 0.5 mL of the substance to be
other foreign matter. examined in toluene R and dilute to 10 mL with the same
solvent.
Water (2.2. 13)
Reference solution Dissolve 50 mg of borneol R, 50 mg of
bornyl acetate R and 100 µL of cineole R in toluene R and
Total ash (2.4.16)
ASSAY Application 10 µL, as bands.
Total hyd.roxycinnamic derivatives
Stock solution To 0.200 g of the powdered herbal drug
(355) (2.9.12) add 80 mL of ethanol (50 per cent V/V) R. Boil Drying In air.
in a water-bath under a reflux condenser for 30 min. Allow Detection Spray the plate with vanillin reagent R and heat the
to cool and filter. Rinse the filter with 10 mL of ethanol plate at 100-105 °C for 10 min. Examine immediately in
(50 per cent VIV) R. Combine the filtrate and the rinsings in daylight.
a volumetric flask and dilute to 100.0 mL with ethanol Results See below the sequence of the zones present in the
(50 per cent V/V) R. chromatograms obtained with the reference solution and the
Test solution To 1.0 mL of the stock solution add 2 mL of test solution. Furthermore, several violet-blue to violet-grey
0.5 M hydrochloric acid, 2 mL of a solution prepared by zones of medium intensity (terpene alcohols) are present in
dissolving 10 g of sodium nitrite R and 10 g of sodium
IV-460 Rosemary Oil 2023
the lower third of the chromatogram obtained with the test Temperature:
solution.
Time Temperature
(min) ('C)
Top of the plate
Column 0 - 10 50
An intense violet zone 10 - 85 50 ➔ 200
A violet-grey zone 85 - 110 200
Injection port 200
-- -- Detector 250
Bomyl acetate: a bluish-grey zone of A bluish-grey zone of low intensity
low intensity (bomyl acetate) Detection Flame ionisation.
A violet-pink zone Injection l µL.
-- --
Cineole: an intense blue zone An intense blue zone (cineole) substances.
Borneo!: a violet-blue zone of A violet -blue zone of medium System suitability Reference solution:
medium intensity intensity (bomeol) - resolution: minimum 1.5 between the peaks due to
limonene and cineole and minimum 1.5 between the
peaks due to et-terpineol and borneol.
Results The characteristic peaks in the chromatogram chromatogram obtained with the test solution.
obtained with the test solution are similar in retention time to Determine the percentage content of these components.
solution. For rosemary oil, Spanish type, the percentages are within
the following ranges:
TESTS - rx-pi,nene: 18 per cent to 26 per cent,
Relative density (2.2.5) - camphene: 8.0 per cent to 12.0 per cent,
0.895 to 0.920. - {3-pi,nene: 2.0 per cent to 6.0 per cent,
Refractive index (2. 2. 6) - {3-myrcene: 1.5 per cent to 5.0 per cent,
1.464 to 1.473. - limonene: 2.5 per cent to 5.0 per cent,
- cineole: 16.0 per cent to 25.0 per cent,
-5c to+ 8°. - p-cymene: 1.0 per cent to 2.2 per cent,
- camphor: 13.0 per cent to 21.0 per cent,
Acid value (2.5. J) - bornyl acetate: 0.5 per cent to 2.5 per cent,
Maximum 1.0. - rx-terpineol: 1.0 per cent to 3.5 per cent,
Chromatographic profile - borneol: 2.0 per cent to 4.5 per cent,
Gas chromatography (2.2.28): use the normalisation - verbenone: 0.7 per cent to 2.5 per cent.
procedure. For rosemary oil, Moroccan and Tunisian type, the
Test solutum Dissolve 0.20 mL of the substance to be percentages are within the following ranges:
examined in hexane Rand dilute to 10.0 mL with the same - rx-pi,nene: 9.0 per cent to 14.0 per cent,
solvent. - camphene: 2.5 per cent to 6.0 per cent,
Reference solution Dissolve 20 µL of rx-pi,nene R, l 0 mg of - {3-pi,nene: 4.0 per cent to 9.0 per cent,
camphene R, 20 µL of {3-pi,nene R, 10 µL of {3-myrcene R, - {3-myrcene: 1.0 per cent to 2.0 per cent,
20 µL of limonene R, 50 µL of cineole R, l O µL of p-cymene R, - limonene: 1.5 per cent to 4.0 per cent,
50 mg of camphor R, 30 mg of bornyl acetate R, 10 mg of - cineole: 38.0 per cent to 55.0 per cent,
rx-terpineol R, l O mg of borneol R and 10 µL of verbenone R in - p-cymene: 0.8 per cent to 2.5 per cent,
hexane Rand dilute to 10.0 mL with the same solvent. - camphor: 5.0 per cent to 15.0 per cent,
Column: - bornyl acetate: 0.1 per cent to 1.5 per cent,
- material: fused silica, - rx-terpineol: 1.0 per cent to 2.6 per cent,
- borneol: 1.5 per cent to 5.0 per cent,
- size: l = 30 m (a film thickness of 1 µm may be used) to
60 m (a film thickness of 0.2 µm may be used), - verbenone: maximum 0.4 per cent.
0 = 0.25-0.53 mm, STORAGE
- stationary phase: macrogol 20 000 R. At a temperature not exceeding 25 °C.
Carrier gas helium for chromatography R. LABELLING
Flow rate l mlJmin. The label states that the content is Spanish type or
Split ratio l :50. Moroccan and Tunisian type.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
2023 Round Amomum Fruit IV-461

Round Amomum Fruit glycerol R. The powder shows numerous round cells of the
endosperm, filled with small starch granules aggregated into
masses and free aggregates of starch granules.
A. compactum Microscopic examination (2.8.23).
DEFINITION The powder is yellowish-brown. Examine under a
Dried, whole, peeled or unpeeled ripe fruit of Amomum microscope using chloral hydrate solution R. The powder
krervanh Pierre ex Gagnep. or Amomum compactum Sol. shows the following diagnostic characters: fragments of
ex Maton. epicarp consisting of polyhedral cells, numerous scars of
covering trichomes with thick, channelled walls and rare
Content
stomata, usually paracytic (2.8.3); unicellular covering
- essential oil: minimum 50 mUkg for A. krervanh
(anhydrous drug) and minimum 40 mUkg for trichomes, usually fragmented, with regularly thickened walls,
up to 800 µm in length; fragments of mesocarp composed of
A. compactum (anhydrous drug);
round cells with spaces between them, containing fine
- 1,8-cineole (CrnH 180; Mr 154.3): minimum 65.0 per cent
acicular crystals or prisms of calcium oxalate, and oil cells;
of the essential oil.
groups of ovoid sclereids with thick, channelled walls, about
IDENTIFICATION 50 µm in diameter, from the inner layers of the mesocarp;
A. A. krervanh. The fruit is a trilocular, indehiscent capsule, vascular bundles composed of spiral or reticulate vessels
subspherical, about 1.5-2 cm in diameter. The outer surface accompanied by fibres with thick, channelled walls and
is whitish-yellow or pale brownish-yellow, smooth, and shows sclereids; fragments of the aril consisting of very fine cells,
3 deep longitudinal furrows. The apex bears a prominent some of which contain small crystals; fragments of the pale
stylopodium; the base shows the dentate scar of the stalk. yellow outer testa consisting of elongated cells with indistinct
Both ends and the hollows of the furrows are covered by a walls and rounded ends, accompanied by an underlying layer
pale brown pubescence. The thin, brittle pericarp is easily consisting of rectangular or polyhedral cells with dark orange-
broken, showing 3 locules each containing 4-10 seeds. red contents, perpendicular to the previous layer; fragments
The seeds are hard, irregularly polyhedral, with a slightly of the reddish-brown inner testa composed of thick-walled
raised dorsal surface, 3-4 mm in diameter, brown on the cells, regularly polyhedral in surface view and U-shaped in
surface, finely wrinkled, bearing the remains of the fine, side view; fragments of the endosperm, with polyhedral cells.
membranous aril. Examine under a microscope using a 50 per cent V/V
A. compactum The fruit is a trilocular, indehiscent capsule, solution of glycerol R. The powder shows numerous
subspherical, about 1-2 cm in diameter. The outer surface is fragments of the endosperm with polyhedral cells, filled with
whitish-yellow or pale brownish-yellow, sometimes slightly small starch granules aggregated into masses and free
reddish, and shows about 15 longitudinal furrows, 3 of which aggregates of starch granules.
are deep. The apex bears a prominent stylopodium; the stalk C. Thin-layer chromatography (2.2.27).
is usually fragmented. Both ends and the hollows of the Test solution To 1 g of the powdered herbal drug (355)
furrows are covered by a brownish-yellow pubescence. (2. 9.12) add 5 mL of methylene chloride R. Sonicate for
The thin, brittle pericarp is easily broken, showing 3 locules 10 min. Centrifuge and use the supernatant.
each containing 6-12 seeds. The seeds are hard, irregularly
Reference solution Dissolve 10 µL of bornyl acetate R, 10 µL
polyhedral, 2-3 mm in diameter, blackish-brown on the
of cineole R and 10 mg of borneol R in I mL of toluene R.
surface, wrinkled, bearing the remains of the transparent,
membranous aril. Plate TLC silica gel plate R (2-10 µm).
B. A. krervanh. Microscopic examination (2.8.23). Mobile phase ethyl acetate R, toluene R (7:93 V/V).
The powder is greyish-brown. Examine under a microscope Application5 µL as bands of 8 mm.
using chloral hydrate solution R. The powder shows the Development Over a path of 6 cm.
following diagnostic characters: fragments of epicarp Drying In air.
consisting of polyhedral cells, numerous scars of covering
trichomes with thick, channelled walls and rare stomata,
paracytic or anomocytic (2.8.3); 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 µm 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 µm in diameter, from the inner layers of the mesocarp; Top of the plate
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 outer
testa consisting of elongated cells with distinct, yellow, finely
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, Reference solution Test solution
regularly polyhedral in surface view and U-shaped in side
view; fragments of the endosperm with round cells. Examine
IV-462 Safflower Flower 2023
Detection B Treat with anisaldehyde solution R, heat at Column:

100-105 °C for 3 min and examine in daylight. - material: fused silica;
Results B See below the sequence of zones present in the - size: l = 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 faint zones may be present 0.25 µm).
in the chromatogram obtained with the test solution. Carrier gas helium for chromatography R.
Top of the plate Split ratio 1:50.
A reddish-brown zone Temperature:
-- -- Time Temperature
A bluish-violet zone (min) ('C)
Column 0 - 60 60-, 210
Bomyl acetate: a greyish-brown zone
Injection port 230
Detector 250

Injection 1 µL.
1,8-Cineole: a bluish-violet zone A bluish-violet zone (1,8-cineole) Identification of peaks Use the chromatogram obtained with
A greyish-brown zone reference solution (a) to identify the peaks due to ~-pinene
and 1,8-cineole.
Relative retention With reference to ~-pinene (retention
time = about 11 min): 1,8-cineole = about 1.3.
Borneo]: a greyish-brown zone at the System suitability Reference solution (a):
border between the middle and - resolution: minimum 5 between the peaks due to ~-pinene
lower thirds and 1,8-cineole.
-- --
Calculate the percentage content of 1,8-cineole. Disregard
any peak due to the solvent or with an area less than the area
A bluish-violet zone of the principal peak in the chromatogram obtained with
reference solution (b) (0.05 per cent).
LABELLING
The label states the species present.
I or 2 bluish-violet zones
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
TESTS
Water (2.2.13) Safflower Flower
of the powdered herbal drug (355) (2. 9.12).
PhEw _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Total ash (2.4.16)
ASSAY Dried flower of Carthamus tinctorius L.
Essential oil (2. 8.12) Content
Use 10.0 g of the herbal drug reduced to a powder (1400) Minimum 1.0 per cent of total flavonoids, expressed as
(2. 9.12) immediately before the assay, a 500 mL round- hyperoside (C 21 H200 12; Mr 464.4) (dried drug).
bottomed flask, 200 mL of water R as the distillation liquid IDENTIFICATION
and 0.5 mL of trimethylpentane R in the graduated tube. A. The orange-yellow or reddish-orange, tubular,
Distil at a rate of 3-3.5 mUmin 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 (2.2.28): 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
Test solution Dilute a volume of the essential oil- the hollow cylinder formed by the fused yellow anthers, in
trimethylpentane mixture obtained in the assay of essential oil which the filiform style persists, thickened near the apex.
corresponding to 150 µL of the essential oil in heptane Rand 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 µL of /3-pinene Rand 15 µL solution R. The powder shows the following diagnostic
of cineole R in heptane Rand dilute to 5.0 mL with the same characters (Figure 2386.-1): fragments of the corolla tube [E]
solvent. with epidermis consisting of elongated, thin-walled, finely
Reference solution (b) Dilute 5 µL of cineole R to 100.0 mL striated cells whose margins are lobed [B, Ea, TI, and with
with heptane R. Dilute 1.5 mL of the solution to 10.0 mL parenchyma consisting of small polygonal cells containing
with heptane R. prisms of calcium oxalate [Eb]; outer epidermis bearing
glandular trichomes, usually sheared off, with only the
2023 Safflower Flower IV-463
base [Ja] persisting on the epidermis; these trichomes are Results A See below the sequence of zones present in the
isolated, biseriate with a multicellular stalk and a bicellular chromatograms obtained with the reference solution and the
head [C]; fragments of the lobes of the corolla showing at test solution. Furthermore, other faint zones may be present
their apices a large number of small, rounded, very in the chromatogram obtained with the test solution.
prominent papillae [G]; fragments of parenchyma containing
vascular bundles [Ed] surrounded by secretory canals with Top of the plate
reddish-brown contents [Ee]; fragments of the filaments of
Quercetin: a light yellow zone
the anthers consisting of elongated, thick-walled, pitted
cells [K] and fragments of the characteristic layer of the -- --
anther whose walls show thickenings in bands [H]; fragments
of the stigma, covered with rather long, conical papillae [DJ, -- --
usually accompanied by pollen grains; rounded or elliptical Rutoside: a light yellow zone
pollen grains up to 60 µm in diameter with 3 pores and an
A red zone
echinulate exine [A); isolated prisms of calcium oxalate [F].
tf£S3:3 C
A yellow zone
A yellow zone
Detection B Heat at 100 °C for 3 min; treat the plate whilst

still hot 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; allow to dry in air for about
30 min; examine in ultraviolet light at 365 nm.
Top of the plate
Quercetin: an orange fluorescent

zone
-- --
-- --
Rutoside: a yellow fluorescent zone

herbal drng of saffiower flower A yellow fluorescent zone

Test solution To 1.0 g of the powdered herbal drug (355) A green fluorescent zone
(2. 9.12) add 10 mL of methanol R. Sonicate for 10 min and A brmvn fluorescent zone
centrifuge.
Reference solution Dissolve 1 mg of rntoside trihydrate R and Reference solution Test solution
5 mg of quercetin dihydrate R in 50 mL of methanol R.

Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel TESTS
plate R (2-10 µm)]. Absorbance (2.2.25)
Mobile phase acetic acid R, anhydrous formic acid R, water R, A. Yellow pigment: macerate 0.1 g of the powdered herbal
ethyl acetate R (11:11:27:100 VIVIVIV). drug (355) (2. 9.12) in 150 mL of water R, stir for 1 h, filter
Application 25 µL [or 10 µL] as bands of 15 mm [or through a sintered-glass filter (40) (2.1.2) and dilute to
8 mm]. 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)
Drying In air.
(2. 9.12) add 50 mL of a mixture of 20 volumes of water R
Detection A Examine in daylight. 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
IV-464 Sage Leaf 2023
(40) (2.1.2) and dilute to 100.0 mL, washing the residue B. Microscopic examination (2.8.23). The powder is light
with a mixture of 20 volumes of water R and 80 volumes of grey or brownish-green. Examine under a microscope using
acetone R. The absorbance is not less than 0.40 at 518 nm. chloral hydrate solutwn R. The powder shows the following
Loss on drying (2.2.32) diagnostic characters (Figure 1370.-1): very numerous
Maximum 11.0 per cent, determined on 1.000 g of the articulated and bent covering trichomes with narrow
powdered herbal drug (355) (2.9.12) by drying in an oven at elongated cells and a base cell with very thick walls, whole
105 °c for 2 h. [Be] or fragmented, either isolated [C, G, HJ or on an
epidermis (surface view [Be], transverse section [Ab]);
Total ash (2.4.16)
glandular trichomes of lamiaceous type, with a unicellular
stalk and an 8- to 12-celled head covered by a common
Ash insoluble in hydrochloric acid (2.8.1) cuticle, isolated (side view [DJ) or on an epidermis (surface
Maximum 3.0 per cent. view [Fa]); small glandular trichomes with a unicellular [Aa,
ASSAY Bd] or multicellular [Fb] stalk and a unicellular head, usually
Solutwn A Place 0.250 g of the powdered herbal drug (180) on an epidermis; more rarely, glandular trichomes (surface
(2.9.12) in a 250 mL flask and add 95 mL of methanol R. view [Eb, Ee], side view [Ed]) with a unicellular stalk [Ee]
Heat under a reflux condenser on a water-bath for 30 min. and a bicellular head [Eb, Ed]; fragments of the upper
Allow to cool and filter. Rinse the filter with 5 mL of epidermis (surface view [E], transverse section [A]) with
methanol R. Combine the filtrate and the rinsing solution in a pitted, somewhat polygonal cells [Ea], covering trichomes
volumetric flask and dilute to 100.0 mL with methanol R. and glandular trichomes, sometimes accompanied by 1 or 2
layers of palisade parenchyma [Ac, Ee]; some diacytic
Test solutwn Place 5.0 mL of solution A in a volumetric
stomata (2. 8. 3) may be present; fragments of the lower
flask and dilute to 20.0 mL with a 20 g/L solution of
epidermis [B, F] with sinuous cells [Ba] and numerous
aluminium chloride R in methanol R.
diacytic stomata (2.8.3) [Bb].
Compensatwn solutwn Place 5.0 mL of solution A in a
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
taking the specific absorbance of hyperoside at 420 nm to be

400.
A absorbance of the test solution at 420 nm;

- - - - - - - - - - - - - - - - - - - - - PhEur
Sage Leaf
(Sage Leaf (Salvia officinalis), Ph. Bur. monograph
1370)
Preparation
Sage Tincture
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or cut, dried leaves of Salvia officinalis L.
Essential oil content: Figure 1370.-1. - Illustratwnfor identificatwn test B of powdered
- for the whole drug, minimum 12 mUkg (anhydrous herbal drng of sage leaf
drug);
- for the cut drug, minimum 10 mUkg (anhydrous drug). C. Thin-layer chromatography (2.2.27).
IDENTIFICATION Test solutwn Shake 0.5 g of the freshly powdered herbal
drug (355) (2.9.12) with 5 mL of ethanol R for 5 min.
A. The lamina of whole sage leaf (Salvia officinalis) is about
2-10 cm long and 1-2 cm wide, oblong-ovate, elliptical. Reference solutwn Dissolve 20 µL of thujone R and 25 µL of
The margin is finely crenate to smooth. The apex is rounded cineole R in 20 mL of ethanol R.
or subacute and the base is shrunken at the petiole and Plate TLC silica gel plate R.
rounded or cordate. The upper surface is greenish-grey and Mobile phase ethyl acetate R, toluene R (5:95 V/V).
finely granular; the lower surface is white and pubescent and Application20 µL as bands.
shows a dense network of raised veinlets.
2023 Sage Preparations IV-465
Drying In air. Plate TLC silica gel plate R.

Detection Treat with a 200 g/L solution of phosphomolybdic Mobile phase ethyl acetate R, toluene R (5:95 V/V).
acid R in ethanol Rand heat at 100-105 °C for 10 min; Applicatwn 20 µL, as bands.
examine in daylight. Development Over a path of 15 cm.
Results See below the sequence of zones present in the Drying In air.
test solution. Furthermore, other zones are present in the Detectwn Spray with a 200 g/L solution of phosphomolybdic
chromatogram obtained with the test solution. acid R in ethanol Rand heat at 100-105 °C for 10 min.
Examine in daylight.
Top of the plate
A blue zone (near the solvent front) test solution. Furthermore, other zones are present in the
-- --
o:-Thujone and P-thujone: 2 pinkish- 2 pinkish-violet zones (c,-thujone and

Top of the plate
violet zones !3-thujone)
A blue zone (near the solvent front)
Cineole: a blue zone A blue zone (cineole)
-- --
-- --
o:-Thujone and P-thujone: 2 pinkish- 2 pinkish-violet zones (c,-thujone and
Blue zones
violet zones P-thujone)
Cineole: a blue zone A blue zone (cineole)
-- --
TESTS Blue zones
Water (2.2.13) TESTS
Maximum 100 mLJkg, determined on 20.0 g. Ethanol content (2.9.10)
64 per cent VIV to 69 per cent V/V.
Total ash (2.4.16)
Maximum 10.0 per cent. Methanol and 2-propanol (2. 9.11)
Maximum 0.05 per cent V/V of methanol and maximum
ASSAY
0.05 per cent of 2-propanol.
Use 20.0 g of the herbal drug, cut, if necessary, immediately Dry residue (2.8.16)
before the assay, a 500 mL flask and 250 mL of water R as Minimum 2.0 per cent, determined on 3.00 g.
the distillation liquid. Add 0.50 mL of xylene R in the ASSAY
graduated tube. Distil at a rate of 2-3 mUmin for 2 h. In a 500 mL round-bottomed flask, place 30.0 g of the
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur tincture and add 100 mL of water R. Distil, using a
descending condenser, into a separating funnel which has
been marked beforehand at 50 mL. Stop the distillation
process as soon as the distillate reaches the 50 mL mark.
Sage Tincture Rinse the condenser with 10 mL of pentane R. Dissolve in
the distillate sufficient sodium chloride R to produce a
(Ph. Bur. monograph 1889) saturated solution. Shake with 3 quantities, each of 20 mL,
of pentane R. Dry the combined pentane layers, including the
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
pentane from rinsing the condenser, over anhydrous sodium
DEFINITION sulfate R and filter through a plug of absorbent cotton into a
Tincture produced from Sage leaf (Salvia officinalis) (1370). weighed 100 mL round-bottomed flask. Wash the sodium
sulfate several times with small quantities of pentane R.
Content
Minimum 0.1 per cent mlm of essential oil. Remove the pentane carefully at a temperature not exceeding
40 °C. Dry the residue in a desiccator over diphosphorus
PRODUCTION pentoxide R and hard paraffin at atmospheric pressure and at
The tincture is produced from 1 part of comminuted drug room temperature for 2 h. Weigh the residue (essential oil).
and 10 parts of ethanol (70 per cent V/V) by a suitable _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
procedure.
CHARACTERS
Appearance
Brownish liquid with a characteristic odour.
IDENTIFICATION
Test solution The tincture to be examined.
Reference solution Dissolve 20 µL of thujone R and 25 µL of
cineole R in 20 mL of ethanol R.
IV-466 Sage Leaf 2023
Ad
Three-lobed Sage Leaf
(Ph. Eur. monograph 1561) Ac ·.
C:'Jf:;b
DEFINITION Ca
Whole or cut, dried leaves of Salvia fructicosa Mill. (syn.
Salvia triloba L. fil).
Essential oil content:
- for the whole drug, minimum 18 mUkg (anhydrous
drug); i\~ '.·, \
- for the cut drug, minimum 12 mUkg (anhydrous drug).
\·,
CHARACTERS
Spicy odour when ground, similar to eucalyptus oil.
IDENTIFICATION
A. The lamina of whole three-lobed sage leaf is about
8-50 mm long and about 4-20 mm wide, and oblong-ovate
or lanceolate. The margin is finely crenate and undulate but
indistinct owing to the dense, hairy covering on both
surfaces. The base is obtuse and sometimes bears 1 or 2
more or less developed lobes. The upper surface is grey-
tomentose pubescent, the lower surface is densely white-
tomentose pubescent; the venation is indistinct. The densely
white-tomentose pubescent petiole is about 1 mm in
diameter.
green and tomentose. Examine under a microscope using
diagnostic characters (Figure 1561.-1): very numerous Figure 1561.-1. -Illustration for identification test B of
covering and glandular trichomes, whole and attached to powdered herbal drug of three-lobed sage leaf
fragments of the epidermises [A, D, G, HJ or fragmented
and free [B, C, E, F]; uniseriate covering trichomes, either Plate TLC silica gel plate R.
unicellular [Ab] or multicellular articulated and thick-walled Mobile phase ethyl acetate R, toluene R (5:95 V/V).
[Ad]; those on the upper epidermis are straight [Ga], those Application 20 µL as bands.
on the lower epidermis are tortuous [Da]; glandular
trichomes of 2 types: some with a unicellular [Hd] or
multicellular [Ca, Gb, He] stalk and a unicellular [Cb, Hd] Drying In air.
or bicellular [Cc] head; others of lamiaceous type, with a Detection Treat with a 200 g/L solution of phosphomolybdic
unicellular stalk and a head composed of 8-12 radiating cells acid R in anhydrous ethanol R and heat at 100-105 °C for
with a raised common cuticle [Ae, BJ; the upper epidermis 10 min. Examine in daylight.
(surface view [Al, transverse section [G]) with pitted and Results The chromatogram obtained with the reference
beaded cells [Aa], somewhat polygonal, with a few diacytic solution shows in the middle part a blue zone (cineole) and
stomata (2.8.3), covering trichomes [Ab, Ad, Ga] or their in the upper part a pink-blue zone (thujone).
scars [Ac] and glandular trichomes [Ae, Af, Gb]; the lower The chromatogram obtained with the test solution shows no
epidermis (surface view [H], transverse section [DJ) with zone or a very faint pink-blue zone due to thujone.
sinuous or wavy-walled cells [Ha] and numerous diacytic
stomata (2.8.3) [Hb], glandular trichomes [Db, Hd, He] and
covering trichomes [Da, He], some of which are unicellular
and short, with finely pitted walls [DeJ.
Water (2.2.13)
Maximum 100 mIJkg, determined on 20.0 g.
thujone.
Results The chromatogram obtained with the test solution Total ash (2.4.16)
shows a blue zone due to cineole, equal or greater in size and Maximum 10.0 per cent.
intensity to the zone in the chromatogram obtained with the ASSAY
reference solution. Further zones are present. Essential oil (2.8.12)
TESTS Use 20.0 g of the herbal drug, cut, if necessary, immediately
Thujone before the assay, a 500 mL flask and 250 mL of water R as
Thin-layer chromatography (2.2.27). the distillation liquid. Add 0.50 mL of xylene R in the
graduated tube. Distil at a rate of 2-3 mIJmin for 2 h.
Test solution Shake O.3 g of the freshly powdered herbal
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
drug (355) (2.9.12) with 5.0 mL of anhydrous ethanol R for
5 min.
Reference solution Dilute 20 µL of thujone R and 25 µL of
cineole R in 20 mL of anhydrous ethanol R.
2023 Sage Oil IV-467
Sage Oil Gas chromatography (2.2.28): use the normalisation
(Clary Sage Oil, Ph. Bur. monograph 1850) procedure.
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Test solution The substance to be examined.
Reference solution To 1 g of hexane R, add 5 µL of thujone R,
DEFINITION 5 µL of linalol R, 100 µL of linalyl acetate R, 10 µL of
Essential oil obtained by steam distillation from the fresh or rx-terpineol Rand 25 mg (± 20 per cent) of sclareol R.
dried flowering stems of Salvia sclarea L. Mix thoroughly by stirring.
CHARACTERS Column:
Appearance - material: fused silica,
Colourless or brownish-yellow liquid, usually pale yellow. - size: l = 30 m (a film thickness of 1 µm may be used) to
Characteristic odour. 60 m (a film thickness of 0.2 µm may be used),
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. Split ratio 1:100.
A. Thin-layer chromatography (2.2.27). Temperature:
Test solution Dissolve 1 mL of the substance to be examined
in toluene R and dilute to 10 mL with the same solvent. Time Temperature
Reference solution Dissolve 60 µL of linalol R, 200 µL of (min) CC)
linalyl acetate R and 60 µL of rx-terpineol R in toluene R and Column 0 - 10 60
dilute to 10 mL with the same solvent. 10 - 75 60-+ 190
Plate TLC silica gel plate R. 75 - 120 190

Injection port 220
Detector 240
Injection 0.2 µL.
Drying In air.
Detection Spray with vanillin reagent R and heat at reference solution. Record the retention times of these
100-105 °C for 5-10 min; examine in daylight within 5 min. substances.
Results See below the sequence of the zones present in the System suitability Reference solution:
chromatograms obtained with the reference solution and the - resolution: minimum 1.5 between the peaks due to linalol
test solution. Furthermore, other faint zones are present in and linalyl acetate,
Top of the plate
cr-Terpineol: a dark violet zone A dark violet zone chromatogram obtained with the test solution (disregard any
peak due to hexane). Thujone R is a mixture of a.- and
-- -- ~-thujone. a.-Thujone elutes before ~-thujone under the
Linalyl acetate: a dark violet zone A dark violet zone described conditions.
-- -- components.
Linalol: a dark violet zone A dark violet zone
Also determine the percentage content of germacrene-D.
Reference solution Test solution The germacrene-D peak can be identified in the
chromatogram obtained with the test solution by its relative
retention of 1.23 with reference to linalol under the described
B. Examine the chromatograms obtained in the test for operating conditions.
Results The chromatogram obtained with the test solution - rx- and /3-thujone: maximum 0.2 per cent,
shows 5 peaks similar in position to the 5 peaks in the - linalol: 6.5 per cent to 24 per cent,
chromatogram obtained with the reference solution. The 2 - linalyl acetate: 56 per cent to 78 per cent,
peaks corresponding to a.- and ~-thujone may be absent. - rx-terpineol: maximum 5.0 per cent,
TESTS - germacrene-D: 1.0 per cent to 12 per cent,
Relative density (2.2.5) - sclareol: 0.4 per cent to 2.6 per cent.
0.890 to 0.908. STORAGE
Refractive index (2.2.6) At a temperature not exceeding 25 °C.
1.456 to 1.466. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Optical rotation (2. 2. 7)
-26° to -10°.
Acid value (2.5.1)
Maximum 1.0.
IV-468 Sage Oil 2023
Acid value (2.5.1)

Spanish Sage Oil Maximum 2.0, determined on 5.00 g.
(Ph. Bur. monograph 1849) Solubility in alcohol (2.8.10)
1 volume is soluble in 2 volumes and more of ethanol
Ph Eur - - - - - - - - - - - - - - - - - - - - - -
DEFINITION Chromatographic profile
Essential oil obtained by steam distillation from the aerial Gas chromatography (2.2.28): use the normalisation
parts of Salvia lavandulifolia V ah!, collected at the flowering procedure.
stage.
Test solutwn Dissolve 0.200 g of the essential oil to be
CHARACTERS examined in heptane Rand dilute to 10.0 mL with the same
Appearance solvent.
Clear, colourless or pale yellow, mobile liquid. Reference solutwn (a) Dissolve 0.200 g of Spanish sage oil for
Camphor-like odour. peak identificatwn CRS in heptane Rand dilute to 10.0 mL
IDENTIFICATION
First identificatwn: B. Reference solutwn (b) Dissolve 5 µL of limonene R in
heptane R and dilute to 50.0 mL with the same solvent.
Dilute 0.5 mL of this solution to 5.0 mL with heptane R.
Column:
Test solutwn Dissolve 0.1 mL of the essential oil to be - material: fused silica;
examined in 10 mL of toluene R. - size: l = 60 m, 0 = 0.25 mm;
Reference solutwn Dissolve 20 µL of thujone R and 30 µL of - statwnary phase: macrogol 20 000 R (film thickness
cineole R in 10 mL of toluene R. 0.25 µm).
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel Carrier gas helium for chromatography R.
plate R (2-10 µm)]. Flow rate 1.5 mUmin.
Mobile phase ethyl acetate R, toluene R (5:95 VIV). Split ratw 1:50.
Applicatwn 10 µL [or 3 µL] as bands of 10 mm [or 6 mm]. Temperature:
Drying In air. Time Temperature
(min) ('C)
Detection spray with a freshly prepared 200 g/L solution of
Column 0 - 43 60 ➔ 232
phosphomolybdic acid R in ethanol (96 per cent) R and heat at
Injection port 250
105 °C for 10 min; examine in daylight.
Detector 250
test solution. Furthermore, other faint zones may be present Detection Flame ionisation.
Top of the plate - the chromatogram obtained is similar to the
chromatogram supplied with Spanish sage oil for peak
A blue zone
identification CRS;
-- -- - resolution: minimum 1.5 between the peaks due to
limonene and 1,8-cineole and minimum 1.5 between the
Thujone: 2 pinkish-violet zones
peaks due to o:-terpinyl acetate and bomeol.
Cineole: a blue zone A blue zone (cineole) Use the chromatogram supplied with Spanish sage oil for peak
identification CRS and the chromatogram obtained with
-- --
reference solution (a) to locate the peaks due to o:-pinene,
3 blue zones sabinene, limonene, 1,8-cineole, thujone, camphor, linalol,
Reference solution Test solution linalyl acetate, terpinen-4-ol, sabinyl acetate, o:-terpinyl
acetate and bomeol.
ranges:
Results The characteristic peaks in the chromatogram - rx-pinene: 4.0 per cent to 11.0 per cent;
obtained with the test solution are similar in retention time to - sabinene: 0.1 per cent to 3.5 per cent;
those in the chromatogram obtained with reference - limonene: 2.0 per cent to 6.5 per cent;
solution (a). - 1,8-cineole: 10.0 per cent to 30.5 per cent;
TESTS - thujone: maximum 0.5 per cent;
Relative density (2.2.5) - 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;
- linalyl acetate: maximum 5.0 per cent;
- terpinen-4-ol: maximum 2.0 per cent;
1.465 to 1.473.
- sabinyl acetate: 0.5 per cent to 9.0 per cent;
Optical rotation (2.2. 7) - rx-terpinyl acetate: 0.5 per cent to 9.0 per cent;
+ 7° to+ 17°. - borneol: 1.0 per cent to 7.0 per cent;
2023 Salvia Miltiorrhiza Root and Rhizome IV-469

(0.05 per cent).
STORAGE
At a temperature not exceeding 25 "C.
Salvia Miltiorrhiza Root and

Rhizome
DEFINITION
Dried, whole or fragmented rhizome and root of Salvia
miltiorrhiza Bunge, collected in spring or autumn.
Content
- salvianolic acid B (C36H 30 O 16; Mr 719): minimum
3.0 per cent (dried drug);
- tanshinone !IA (C19H1 8 O 3 ; Mr 294.3): minimum
0.12 per cent (dried drug).
IDENTIFICATION
A. The rhizome is short and thick, sometimes with stem
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
rootlets. The outer surface is reddish-brown or dark reddish-
brown, marked with longitudinal striations. The bark of old
herbal drug of salvia miltiorrhiza root and rhizome
roots comes off usually as purplish-brown scales. The texture
is hard and fragile. The fracture is soft, fissured or slightly Mobile phase methanol R, anhydrous formic acid R, toluene R,
even and dense, with a reddish-brown outer part and a methylene chloride R, ethyl acetate R
greyish-yellow or purplish-brown wood, showing bundles of (5:20:20:30:40 VIVIVIVIV).
yellowish-white vessels, arranged radially.
Cultivars are relatively stout, about 0.5-1.5 cm in diameter.
The outer surface is brownish-red, longitudinally wrinkled.
The bark adheres closely to the wood and is difficult to Drying In air.
remove. The texture is compact; the fracture is relatively Detection A Examine in daylight.
even. Results A See below the sequence of zones present in the
B. Microscopic examination (2.8.23). The powder is chromatograms obtained with the reference solution and the
brownish-red. Examine under a microscope using chloral test solution. Furthermore, other faint zones may be present
hydrate solution R. The powder shows the following diagnostic in the upper third and middle part of the chromatogram
characters (Figure 2663.-1): fragments of cork consisting of obtained with the test solution.
subrectangular or polygonal cells, up to 150 µm in diameter,
containing yellowish-brown pigment (surface view [A), Top of the plate
transverse section [El); fragments of parenchyma
Tanshinone 11.,: a prominent red A prominent red zone
(longitudinal section [Cl) consisting of polygonal or
zone (tanshinone IIA)
elongated cells, often septate [Ca]; fragments of parenchyma
(transverse section [BJ) with rounded cells; xylem fibres An orange zone
usually in bundles [F], long and fusiform, with walls showing
pits shaped as crosses or oblique slits; very numerous -- --
reticulate or pitted vessels, 3-120 µm in diameter, free, in A faint brownish-green zone

bundles (longitudinal section [DJ, transverse section [G)) or Sa!vianolic acid B: a faint grey zone A faint grey zone (salvianolic acid B)
sometimes accompanying the fibres [Fa].
C. Thin-layer chromatography (2.2.27). -- - -

(2. 9.12) add 40 mL of methanol R. Sonicate for 15 min.
Filter. Evaporate the filtrate to 1 mL.
Reference solution Dissolve 2 mg of salvianolic acid B R and
2 mg of tanshinone IIA R in 1 mL of methanol R. Detection B Examine in ultraviolet light at 254 nm.
Plate TLC silica gel F254 plate R (2-10 µm). Results B See below the sequence of zones present in the
IV-470 Salvia Miltiorrhiza Rhizome and Root 2023
in the upper third and middle part of the chromatogram Identification of peaks Use the chromatogram obtained with
obtained with the test solution. reference solution (a) to identify the peak due to
tanshinone IIA and the chromatogram obtained with
Top of the plate reference solution (b) to identify the peak due to salvianolic
acid B.
Tanshinone IIA: a prominent A prominent quenching zone
quenching zone (tan shinone II,0 Relative retention With reference to tanshinone IIA
A quenching zone
=
(retention time about 33 min): rosmarinic acid about =
0.3; salvianolic acid B = about 0.4.
-- -- System suitability:
A quenching zone
rosmarinic acid and salvianolic acid B in the
Salvianolic acid B: a prominent A prominent quenching zone chromatogram obtained with reference solution (c);
quenching zone (salvianolic acid B) - symmetry factor. maximum 2.0 for the peak due to
salvianolic acid B in the chromatogram obtained with
-- --
reference solution (b).
Calculate the percentage content of tanshinone IIA using the
Reference solution Test solution following expression:
TESTS
area of the peak due to tanshinone IIA in the chromatogram
Maximum 10.0 per cent, determined on 1.000 g of the obtained with the test solution;
powdered herbal drug (355) (2. 9.12) by drying in an oven at area of the peak due to tanshinone IIA in the chromatogram
105 °C for 2 h. obtained with reference solution (a);
Total ash (2.4.16) solution, in grams;
Maximum 10.0 per cent. mass of tanshinone IIA CRS used to prepare reference
Ash insoluble in hydrochloric acid (2.8.1) P, percentage content of tanshinone IIA in tanshinone /IA CRS.
ASSAY Calculate the percentage content of salvianolic acid B using
Liquid chromatography (2.2.29). Protect the solutions from the following expression:
light. A3 xm3 xpz x 2
Test solution Disperse 0.30 g of the powdered herbal drug A4 xm1
(355) (2.9.12) in 50.0 mL ofa 70 per cent V/Vsolution of
methanol R. Sonicate for 1 h. Filter through a membrane area of the peak due to salvianolic acid B in the chromatogram
filter (nominal pore size 0.45 µm). area of the peak due to salvianolic acid B in the chromatogram
Reference solution (a) Dissolve 5.0 mg of tanshinone IIA CRS obtained with reference solution (b );
in methanol Rand dilute to 50.0 mL with the same solvent. mass of the herbal drug to be examined used to prepare the test
solution, in grams;
m, mass of salvianolic acid B CRS used to prepare reference
Reference solution (b) Dissolve 5.0 mg of salvianolic solution (b), in grams;
acid B CRS in methanol R and dilute to 25.0 mL with the percentage content of salvianolic acid B in salvianolic acid
BCRS.
same solvent.
Reference solution (c) Dissolve 1 mg of rosmarinic acid R in
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
methanol R, add 5 mL of reference solution (b) and dilute to
Column:
- size: I= 0.25 m, 0 = 4.6 mm;
Processed Salvia Miltiorrhiza Rhizome
chromatography R (5 µm). and Root
Mobile phase: DEFINITION
- mobile phase A: 0.1 per cent V/V solution of anhydrous Processed Salvia Miltiorrhiza Rhizome and Root is Salvia
formic acid R; Miltiorrhiza Rhizome and Root that has been processed.
- mobile phase B: acetonitrile for chromatography R; It contains not less than 0.04% of tanshinone IlA
(C 19H 18 0 3), not less than 0.17% ofrosmarinic acid
Time Mobile phase A Mobile phase B (C 18H 16 0 8 ) and not less than 3.0% of salvianolic acid B
(min) (per cent VIP) (per cent VIP)
(C 36H 36 0 16), calculated with reference to the dried material.
0 - 10 79 ➔ 71 21 ➔ 29
10 - 15 71 ➔ 65 29 ➔ 35 PRODUCTION
15 - 25 65 ➔ 28 35 ➔ 72 It is collected in spring or autumn, separated from soil,
25 - 37 28 ➔ 0 72 ➔ 100 washed clean, softened thoroughly, sliced longitudinally or
transversely and dried. It may be stir baked with wine.
Flow rate 1.0 mL/min. IDENTIFICATION
Detection Spectrophotometer at 280 nm. A. The longitudinally-sliced pieces are up to about 5 cm
Injection 10 µL. long, 1.5 cm wide and 1 to 2 mm thick; those cut from the
thinner roots show the dark brown striated cork covering one
2023 Salvia Miltiorrhiza Rhizome and Root IV-471
longitudinal surface and the yellowish to cream inner tissues Top of the plate
on the other; slices cut from the thicker rhizomes are usually
cut obliquely so that parts of the outer and inner tissues are A fluorescent band Tanshinone IIA: a fluorescent
included on both longitudinal surfaces. The transversely-cut band
slices are irregularly elliptical to nearly circular, 4 to 12 mm
wide and 2 to 3 mm thick; the outer surface is dark brown Several fluorescent bands
and uneven; the smoothed transverse surface shows the outer
layers about I to 2 mm wide separated by a darker line from
the yellowish white, radiate vascular tissue; some pieces show A fluorescent band Rosmarinic acid: a
a small, light brown central pith. fluorescent band
B. Reduce to a powder (355). The powder is reddish-brown. A fluorescent band Salvianolic acid B: a
Examine under a microscope using chloral hydrate solution. fluorescent band
The powder shows a surface view of cork cells almost
rectangular or polygonal, containing yellowish-brown Solution (1) Solution (2)
pigment, 12 to 151 µm in diameter. Parenchymatous cells in
cortex squarish or polygonal, containing reddish-brown
TESTS
pigmental sediments. Xylem fibres usually in bundles, long
Total ash
fusiform, with oblique or criss-cross striations, 11 to 60 µm
Not more than 10%, Appendix XI J.
in diameter, vivid yellow when examined under a polarizing
microscope. Numerous mainly bordered or reticulated Acid-insoluble ash
vessels, 3 to 120 µm in diameter. Not more than 2.0%, Appendix XI K.
C. Carry out the method for thin-layer chromatography, Loss on drying
Appendix III A, using the following solutions. When dried for 2 hours at 105°, loses not more than 12.0%
(1) Place 2 g of the powdered drug (355) in a cellulose of its weight. Use 1 g.
fingerstall in a continuous extraction apparatus (Soxhlet ASSAY
type). Add 75 mL of methanol and heat for 1 hour. For tanshinone IIA
Evaporate the extract to 20 mL, cool and filter if necessary. Carry out the method for liquid chromatography,
(2) 0.1 % w/v each of tanshinone IIA CRS, rosmarinic acid CRS Appendix III D, using the following solutions.
and salvianolic acid B CRS in methanol. (1) Finely powder about 5.0 g of the herbal drug being
CHROMATOGRAPHIC CONDITIONS examined. Transfer 0.5 g of the powder into a 25 mL
(a) Use as the coating silica gel F254 • volumetric flask and add 20 mL of the mobile phase given
below. Shake and mix with the aid of ultrasound for
(b) Use the mobile phase described below.
30 minutes, shaking intermittently. Add the mobile phase to
(c) Apply as bands 8 µL of solution (1) and 5 µL of give a total volume of 25 mL. Filter through a 0.45-µm filter.
solution (2).
(2) 0.002% w/v of tanshinone IIA CRS in the mobile phase.
(e) After removal of the plate, dry in air and examine under
ultraviolet light (366 nm). (a) Use a stainless steel column (25 cm x 4.6 mm) packed
with octadecylsilyl silica gel for chromatography (5 µm)
MOBILE PHASE (Nucleosil ODS is suitable).
10 volumes of water, 13 .5 volumes of methanol and (b) Use isocratic elution and the mobile phase described
100 volumes of ethyl acetate. below.
SYSTEM SUITABILITY (c) Use a flow rate of 1 mL per minute.
The chromatogram obtained with solution (2) shows three (d) Use an ambient column temperature.
clearly separated bands. (e) Use a detection wavelength of 270 nm.
CONFIRMATION (f) Inject 20 µL of each solution.
The blue fluorescent bands with Rf values of approximately MOBILE PHASE
0.7 (tanshinone IIA), 0.2 (rosmarinic acid) and 0.06
0.01M sodium octyl sulfonate in a mixture of 25 volumes of
(salvianolic acid B) in the chromatogram obtained with
water and 75 volumes of methanol adjusted to pH 5.0 with
solution (1) correspond in colour and position to those in the
acetic acid.
chromatogram obtained with solution (2). Other bands may
be present in the chromatogram obtained with solution (1) as SYSTEM SUITABILITY
shown below. The test is not valid, unless in the chromatogram obtained
with solution (2):
- the symmetry factor of the peak due to tanshinone IIA is
less than 1.2;
- the number of theoretical plates is not less than 4500.
Using the retention time and peak area from the
chromatograms obtained with solution (2), locate and
integrate the peak due to tanshinone IIA in the
IV-4 72 Saw Palmetto Extract 2023
Calculate the content of tanshinone IIA in the sample using Area of the peak due to rosmarinic acid in the chromatogram
the declared content of tanshinone IIA (C 19H 1 sO 3 ) in Area of the peak due to rosmarinic acid in the chromatogram
tanshinone /IA CRS and the following expression: obtained with solution (2).
Weight of the drug in mg.
Weight of rosmarinic acid CRS in mg.
A1 m, V, 100 Dilution volume of solution (I) in mL.
-x-x-xpx---
A, V, m, 100-d Dilution volume of solution (2) in mL.
Percentage content of rosmarinic acid in rosmarinic acid CRS.
A1 Area of the peak due to tanshinone !IA in the chromatogram

Salvianolic acid B Using the retention time and peak area
obtained with solution (!).
Area of the peak due to tanshinone IIA in the chromatogram from the chromatogram obtained with solution (3), locate
obtained with solution (2). and integrate the peak due to salvianolic acid B in the
m, Weight of the drug in mg. chromatogram obtained with solution (1).
m2 Weight of tanshinone IIA CRS in mg.
V1 Dilution volume of solution (I) in mL. Calculate the content of salvianolic acid B in the sample
V2 Dilution volume of solution (2) in mL. using the declared content of salvianolic acid B (C 36H 36 0 16)
p Percentage content of tanshinone IIA in tanshinone !IA CRS. in salvianolic acid B CRS and the following expression:
d Percentage loss on drying of the herbal drug being examined.
A, ITu V, 100
For rosmarinic acid and salvianolic acid B -x-x-xpx
Carry out the method for liquid chromatography, A, V2 mi 100-d
Appendix III D, using the following solutions prepared
immediately before use.
(1) Finely powder about 5.0 g of the herbal drug being Area of the peak due to salvianolic acid in the chromatogram
obtained with solution (I).
examined. Transfer 0.5 g of the powder into a 25-mL
Area of the peak due to salvianolic acid in the chromatogram
volumetric flask and add 20 mL of the mobile phase given obtained with solution (3).
below. Shake and mix with ultrasound for 30 minutes, Weight of the drug in mg.
shaking intermittently. Add the mobile phase to give a total Weight of salvianolic acid B CRS in mg.
volume of 25 mL. Filter through a 0.45-µm filter. Dilution volume of solution (1) in mL.
Dilution volume of solution (2) in mL.
(2) 0.003% w/v each of rosmarinic acid CRS andferulic acid in Percentage content of salvianolic acid B in salvianolic acid B CRS.
water. Percentage loss on drying of the herbal drug being examined.
(3) 0.06% w/v of salvianolic acid B CRS in the mobile phase.
STORAGE
Processed Salvia Miltiorrhiza Rhizome and Root should be
(a) Use a stainless steel column (25 cm x 4.6 mm) packed protected from moisture.
with octadecylsilyl silica gel for chromatography (5 µm)
(Nucleosil ODS is suitable).
below. Saw Palmetto Extract
(c) Use a flow rate of 1 mL per minute.
(d) Use an ambient column temperature. (Ph. Bur. monograph 2579)
(e) Use a detection wavelength of 330 nm. PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
(f) Inject 20 µL of each solution. DEFINITION

MOBILE PHASE Extract produced from Saw palmetto fruit (1848).
22 volumes of acetonitrile and 78 volumes of 0.4% v/v of Content
formic acid. - total fatty acids: minimum 80.0 per cent (anhydrous
SYSTEM SUITABILITY extract);
The test is not valid unless, in the chromatogram obtained - !auric acid (C 12H 240 2 ; Mr 200.3): minimum 23.0 per cent
(anhydrous extract);
with solution (2), the resolution factor between the two main
peaks, rosmarinic acid and ferulic acid, is at least 5.0. - total sterols, expressed as /3-sitosterol (C 29 H 500; Mr 414.7):
minimum 0.20 per cent (anhydrous extract);
DETERMINATION OF CONTENT - /3-sitosterol (C 29 H 500; Mr 414.7): minimum 0.10 per cent
Rosmarinic acid Using the retention time and peak area (anhydrous extract).
from the chromatogram obtained with solution (2), locate
PRODUCTION
and integrate the peak due to rosmarinic acid in the
procedure using ethanol (minimum 90 per cent VIV), or
Calculate the content of rosmarinic acid in the sample using supercritical carbon dioxide or a mixture of mainly n-hexane
the declared content of rosmarinic acid (C 18 H 16 0 8 ) in and methylpentanes (bp: 65-70 °C).
rosmarinic acid CRS and the following expression:
CHARACTERS
A1 ITu V1 100 Appearance
-x-x-xpx--- The ethanol extract is a dark greenish-brown, oily liquid; the
A2 V2 mi 100-d supercritical carbon dioxide extract is a yellowish-brown or
orange-brown, oily liquid; the hexane extract is a yellowish-
green or orange-yellow, oily liquid.
Odour: strong but not rancid.
2023 Saw Palmetto Extract IV-473
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,
Test solution Dissolve 0.25 g of the extract to be examined
unless otherwise justified and authorised.
in 20 mL of ethanol (96 per cent) R. ASSAY
Reference solution Dissolve 4 mg of /3-amyri,n Rand 10 mg of Total fatty acids
{3-sitosterol R in 10 mL of ethanol (96 per cent) R. Gas chromatography (2.2.28).
Plate TLC silica gel plate R (2-10 µm). Internal standard solution Dissolve 0.47 g of methyl
Mobile phase anhydrous acetic acid R, ethyl acetate R, margarate R in 20 mL of dimethylformamide R and dilute to
toluene R (1:30:70 V/V/V). 100.0 mL with the same solvent.
Application 2 µL as bands of 8 mm. 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
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 !auric acid CRS
chromatograms obtained with the reference solution and the and 0.870 g of oleic acid CRS in dimethylformamide Rand
test solution. Furthermore, other faint zones may be present, dilute to 10.0 mL with the same solvent. To 1.0 mL of the
especially in the lower third, in the chromatogram obtained solution add 4.0 mL of the internal standard solution and
with the test solution. dilute to 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
Top of the plate trimethylsulfonium hydroxide R in methanol R.
Reference solution (b) Disperse 0.25 g of saw palmetto
A blue zone
extract HRS in 10 mL of dimethylformamide R. Add 4.0 mL
A blue zone of the internal standard solution and dilute to 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
~-Amyrin: a blue zone A strong bluish-violet zone hydroxide R in methanol R.
Column:
~-Sitosterol: a blue zone
- size: l = 25 m, 0 = 0.20 mm;
-- -- - stationary phase: methylpolysiwxane R (film thickness
0.33 µm).
Split ratio 1:40.
B. Examine the chromatograms obtained in the assay of total Temperature:
fatty acids.
Results The peaks due to methyl caproate, methyl caprylate, Time Temperature
methyl caprate, methyl laurate, methyl myristate, methyl (min) CC)
palmitoleate, methyl palmitate, methyl linoleate, methyl Column 0-2 150
linolenate, methyl oleate and methyl stearate in the 2-7 150 ➔ 190
chromatogram obtained with the test solution are similar in 7 - 12 190
retention time to the corresponding peaks in the 12 - 22 190 ➔ 220
chromatogram obtained with reference solution (b); the 22 - 32 220
principal peaks are due to methyl laurate and methyl oleate. Injection port 300
Detector 300
TESTS
Water (2.5.12, Method A)
Maximum 3.0 per cent, determined on 0.5 g. Detection Flame ionisation.
Relative density (2.2.5) Injection 1 µL.
0.850 to 0.950. Identification of peaks Use the chromatogram supplied with
Refractive index (2.2.6) saw palmetto extract HRS and the chromatogram obtained
1.40 to 1.50. with reference solution (b) to identify the peaks due to
methyl caproate, methyl caprylate, methyl caprate, methyl
Acid value (2.5.1) laurate, methyl myristate, methyl palmitoleate, methyl
150.0 to 220.0, determined on 0.500 g. palmitate, methyl linoleate, methyl linolenate, methyl oleate,
Iodine value (2. 5. 4, Method A) methyl stearate and methyl margarate.
30.0 to 60.0. System suitability Reference solution (b):
Peroxide value (2.5.5) - peak-to-valley ratio: minimum 1.2, where Hp = height
Maximum 5.0. above the baseline of the peak due to methyl linolenate
and H,, = height above the baseline of the lowest point of
IV-4 7 4 Saw Palmetto Extract 2023
the curve separating this peak from the peak due to diatomaceous earth R capable of holding 3 mL of aqueous
methyl linoleate. phase. Absorb the solution into the column by applying
Calculate the percentage content of total fatty acids, where vacuum. Maintain the vacuum for at least 20 min, until the
caproic, caprylic, capric, lauric, myristic, palmitoleic, palmitic column returns to room temperature, indicating that the
and stearic acids are expressed as lauric acid (C 12 H 24 O 2 ; methanol is completely evaporated. Rinse the column with
Mr 200.3) and linoleic, linolenic and oleic acids are expressed 90 mL of methylene chloride R and evaporate the eluate to
as oleic acid (C 18H 340 2 ; Mr 282.5), using the following dryness. Dissolve the residue in 1.0 mL of derivatisation
expression: solution (b).
Reference solution (a) To 9.0 mg of /3-sitosterol CRS add
A1 xA4 xm2 xp1 x0.l As xA4 xm3 XP2 x0.l 1.0 mL of the internal standard solution and dilute to
---------+---------
A2XA3Xm1 A6xA3xm1 5.0 mL with methylene chloride R. Evaporate 0.6 mL of this
sum of the areas of the peaks due to methyl caproate, methyl solution to dryness under a stream of nitrogen R. Dissolve the
caprylate, methyl caprate, methyl laurate, methyl myristate, residue in 1.0 mL of derivatisation solution (b).
methyl palmitoleate, methyl palmitate and methyl stearate in the
Reference solution (b) Introduce 1.0 mL of the internal
area of the peak due to methyl laurate in the chromatogram standard solution into a 50 mL round-bottomed flask and
obtained with reference solution (a); evaporate to dryness. Place 3.35 g of saw palmetto
area of the peak due to methyl margarate in the chromatogram extract HRS, accurately weighed, into the round-bottomed
obtained with the test solution; flask and add 20 mL of a solution prepared as follows:
area of the peak due to methyl margarate in the chromatogram
obtained with reference solution (a); dissolve 130 g of potassium hydroxide R in 200 mL of water R
sum of the areas of the peaks due to methyl linoleate, methyl and dilute to 1000 mL with methanol R. Heat under reflux
linolenate and methyl oleate in the chromatogram obtained with for 2 h, transfer quantitatively to a flask and dilute to
the test solution; 25.0 mL with water R. Apply 3.0 mL of this solution to a
area of the peak due to methyl oleate in the chromatogram
cartridge containing diatomaceous earth R capable of holding
mass of the extract to be examined used to prepare the test 3 mL of aqueous phase. Absorb the solution into the column
solution, in grams; by applying vacuum. Maintain the vacuum for at least
mass of /auric acid CRS used to prepare reference solution (a), 20 min, until the column returns to room temperature,
in grams;
mass of oleic acid CRS used to prepare reference solution (a), in
indicating that the methanol is completely evaporated. Rinse
grams; the column with 90 mL of methylene chloride R and evaporate
percentage content of !auric acid in /auric acid CRS; the eluate to dryness. Dissolve the residue in 1.0 mL of
percentage content of oleic acid in oleu: acid CRS. derivatisation solution (b).
Column:
Calculate the percentage content of !auric acid using the
=
- size: l 25 m, 0 = 0.20 mm;
A1xA4xm2xpx0.l - stationary phase: methylpolysiloxane R (film thickness
A2xA3xm1 0.33 µm).
area of the peak due to methyl laurate in the chromatogram
obtained with the test solution; Flow rate 0.5 mUmin.
area of the peak due to methyl laurate in the chromatogram Split ratio 1:40.
area of the peak due to methyl margarate in the chromatogram
obtained with the test solution; Time Temperature
area of the peak due to methyl margarate in the chromatogram (min) ('C)
obtained with reference solution (a); Column 0-3 200
m1 mass of the extract to be examined used to prepare the test 3 - 13 200 ➔ 300
solution, in grams;
m2 mass of /auric acid CRS used to prepare reference solution (a),
13 - 35 300
in grams; Injection port 325
p percentage content of !auric acid in /auric acid CRS. Detector 325
Total sterols Detection Flame ionisation.

Gas chromatography (2.2.28).
Injection l µL.
Derivatisation solution (a) chlorotrimethylsilane R, N,O-bis
( trimethylsilyl) acetamide R, N-trimethylsilylimidazole R
saw palmetto extract HRS and the chromatogram obtained
(2:3:3 VIVIV).
with reference solution (b) to identify the peaks due to the
Derivatisation solution (b) Derivatisation solution (a), N,O- trimethylsilyl derivatives of cholesterol, campesterol,
bis (trimethylsilyl) trifiuoroacetamide R, pyridine R ( 1: 1: 1 V/V/V). stigmasterol, ~-sitosterol and stigmastanol.
Internal standard solution Dissolve 0.25 g of cholesterol R in System suitability Reference solution (b):
25.0 mL of methylene chloride R. - resolution: minimum 1.6 between the peaks due to the
Test solution Introduce 1.0 mL of the internal standard trimethylsilyl derivatives of ~-sitosterol and stigmastanol.
solution into a 50 mL round-bottomed flask and evaporate to Calculate the percentage content of total sterols (campesterol,
dryness. Place 3.35 g of the extract to be examined, stigmasterol, ~-sitosterol and stigmastanol), expressed as
accurately weighed, into the round-bottomed flask and add ~-sitosterol, using the following expression:
20 mL of a solution prepared as follows: dissolve 130 g of
potassium hydroxide R in 200 mL of water R and dilute to A1 xA4 xm2 xp
1000 mL with methanol R. Heat under reflux for 2 h, transfer A2 xA3 x m1
quantitatively to a flask and dilute to 25.0 mL with water R.
Apply 3.0 mL of this solution to a cartridge containing
2023 Saw Palmetto Fruit IV-475
A1 sum of the areas of the peaks due to the trimethylsilyl testa, narrow perisperm and a large area of dense, horny,
derivatives of campesterol, stigmasterol, P-sitosterol and
greyish-white endosperm, with the embryo positioned to one
stigmastanol in the chromatogram obtained with the test
solutioni side.
A2 area of the peak due to the trimethylsilyl derivative of B. Microscopic examination (2.8.23). Reduce a minimum of
!3--sitosterol in the chromatogram obtained with reference
250 g of the herbal drug to a powder (4000) (2. 9.12) and
solution (a);
A, area of the peak due to the trimethylsilyl derivative of homogenise. Further reduce about 50 g of this powder to a
cholesterol in the chromatogram obtained with the test solution; finer powder (1000) (2.9.12). The powder is reddish or
A4 area of the peak due to the trimethylsilyl derivative of blackish-brown and oily. Examine under a microscope using
cholesterol in the chromatogram obtained with reference chloral hydrate solutwn R. The powder shows the following
solution (a);
diagnostic characters (Figure 1848.-1): fragments of epicarp
solution, in grams; [A] consisting of polyhedral cells (10-40 µm), reddish-brown,
m2 mass of fi-sitosrerol CRS used to prepare reference solution (a), pigmented and highly cutinised, in small groups separated by
in grams; thin walls [Aa] and accompanied by considerably larger cells
p percentage content of P-sitosterol in fi-sitosterol CRS.
of the underlying layers [Ab]; groups of parenchyma cells of
the mesocarp [E]; groups of xylem tissue of the mesocarp
Calculate the percentage content of ~-sitosterol using the
showing small, lignified, annular or spirally thickened vessels
[F], sometimes accompanied by parenchyma containing
A 1 xA4xm2xp small sclereids [Fa]; sclereids of the mesocarp (20-200 µm),
A2 xA3 xm1 usually occurring singly W but sometimes in small
groups [G], with walls that are moderately thickened,
A1 area of the peak due to the trimethylsilyl derivative of distinctly striated and finely pitted; fragments of endocarp
P-sitosterol in the chromatogram obtained with the test solution;
A2 area of the peak due to the trimethylsilyl derivative of
containing groups of sclereids about 300 µm long, with
P-sitosterol in the chromatogram obtained with reference strongly thickened walls and numerous pits [BJ; isolated
solution (a); sclereids of the endocarp [K]; fragments of the testa [C]
A3 area of the peak due to the trimethylsilyl derivative of consisting of small, thin-walled cells with brown contents
cholesterol in the chromatogram obtained with the test solution;
[Ca] and underlying sclereids [Cb]; thick-walled endosperm
A4 area of the peak due to the trimethylsilyl derivative of
cholesterol in the chromatogram obtained with reference cells [DJ with large conspicuous pits, and containing
solution (a); aleurone grains and oil; very numerous fragments of the
m1 mass of the extract to be examined used to prepare the test brown cuticle [HJ.
solution, in grams;
m2 mass of /3-sirosrerol CRS used to prepare reference solution (a),
in grams;
p percentage content of P-sitosterol in /3-sitosterol CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Saw Palmetto Fruit

Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried ripe fruit of Serenoa repens (W.Bartram) Small
(syn. Sabal. semdata (Michx.) Schult.f.).
Content
Minimum 11.0 per cent of total fatty acids (dried drug).
CHARACTERS
Odour Strong but not rancid.
IDENTIFICATION
First identification: A, B, D.
Second identificatwn: A, B, C.
A. The fruit is an ovoid or subspherical drupe, with a dark
brown or blackish, roughly wrinkled surface and more or less
coppery sheen, up to 2.5 cm long and 1.5 cm in diameter.
The apex sometimes bears the remains of the style and
tubular calyx, with 3 teeth, and the base bears a small
depression with the scar of the stalk. The epicarp and
underlying mesocarp form a thin fragile layer, which partially
peels off, revealing the thin, hard, pale brown endocarp, Figure 1848.-1. - Illustratwn for identificatwn test B of powdered
which is fibrous and easily separable. The seed is irregularly herbal drug of saw palmetto fruit
spherical or ovoid, up to 12 mm long and 8 mm in diameter,
with a hard, smooth or finely pitted surface which is reddish-
brown with a paler, raised and membranous area over the Test solutwn Reduce about 50 g of the powdered herbal
raphe and micropyle; cut transversely, the seed has a thin drug (4000) (2.9.12) prepared for identification test B to a
IV-476 Saw Palmetto Fruit 2023
finer powder (710) (2.9.12). To 1.5 g of the powdered herbal Test solution Reduce about 50 g of the powdered herbal
drug, add 20 mL of ethanol (96 per cent) R and stir for drug (4000) (2.9.12) prepared for identification test B to a
15 min. Filter. finer powder (200) (2. 9.12). Disperse 4.00 g of the powdered
Reference solution Dissolve 4 mg of P-amyrin Rand 10 mg of herbal drug in 60 mL of dimethylformamide R. Sonicate for
P-sitosterol R in 10 mL of ethanol (96 per cent) R. 15 min and then shake for 30 min. Dilute to 100.0 mL with
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel dimethylformamide R. Allow to stand for a few minutes and
plate R (2-10 µm)]. filter. To 20.0 mL of this solution add 4.0 mL of the internal
standard solution and dilute to 25.0 mL with
Mobile phase acetic acid R, ethyl acetate R, toluene R dimethylformamide R. Mix 0.4 mL of this solution and
(1:30:70 VIVIV). 0.6 mL of an 18.84 g/L solution of trimethylsulfonium
Application 10 µL [or 2 µL] as bands of 10 mm [or 8 mm]. hydroxide R in methanol R.
Development Over a path of 10 cm [or 6 cm]. Reference solution (a) Dissolve 0.699 g of !auric acid CRS
Drying In air. and 0.870 g of oleic acid CRS in dimethylformamide Rand
Detection Treat with anisaldehyde solution R; heat at dilute to 10.0 mL with the same solvent. To 1.0 mL of the
100-105 °C for 5-10 min; examine in daylight. solution add 4.0 mL of the internal standard solution and
Results See below the sequence of zones present in the dilute to 25.0 mL with dimethylformamide R. Mix 0.4 mL of
chromatograms obtained with the reference solution and the this solution and 0.6 mL of an 18.84 g/L solution of
trimethylsulfonium hydroxide R in methanol R.
test solution. Furthermore, other faint zones may be present,
especially in the lower third, in the chromatogram obtained Reference solution (b) Disperse 0.25 g of saw palmetto
with the test solution. extract HRS in 10 mL of dimethylformamide R. Add 4.0 mL
of the internal standard solution and dilute to 25.0 mL with
Top of the plate
dimethylformamide R. Mix 0.4 mL of this solution and
0.6 mL of an 18.84 g/L solution of trimethylsulfonium
A strong blue zone hydroxide R in methanol R.
-- -- Column:
2 faint blue zones - size: l = 25 m, 0 = 0.20 mm;
0-Amyrin: a blue zone - stationary phase: methylpolysiloxane R (film thickness
0.33 µm).
A strong bluish-violet zone
0-Sitosterol: a blue zone A faint blue zone
Split ratio l :40.
- - --
Temperature:
A faint blue zone Time Temperature
(min) CC)
Column 0-2 150
2-7 150 ➔ 190
D. Examine the chromatograms obtained in the assay of total 7 - 12 190
fatty acids. 12 - 22 190 ➔ 220
Results The peaks due to caproic, caprylic, capric, !auric, 22 - 32 220
myristic, palmitoleic, palmitic, linoleic, linolenic, oleic and Injection port 300
stearic acids in the chromatogram obtained with the test Detector 300
solution are similar in retention time to the corresponding
peaks in the chromatogram obtained with reference Detection Flame ionisation.
solution (b); the principal peaks are due to !auric acid and
Injection l µL.
oleic acid.
TESTS saw palmetto extract HRS and the chromatogram obtained
Loss on drying (2.2.32) with reference solution (b) to identify the peaks due to
Maximum 12.0 per cent. caproic, caprylic, capric, !auric, myristic, palmitoleic,
Reduce about 50 g of the powdered herbal drug (4000) palmitic, linoleic, linolenic, oleic and stearic acids and methyl
(2. 9.12) prepared for identification test B to a finer powder margarate.
(710) (2.9.12). Determine the loss on drying on 1.000 g of System suitability Reference solution (b):
the powdered herbal drug by drying in an oven at 105 °C - peak-to-valley ratio: minimum 1.2, where Hp = height
for 2 h. above the baseline of the peak due to linolenic acid and
Total ash (2.4.16) Hv = height above the baseline of the lowest point of the
Maximum 5.0 per cent. curve separating this peak from the peak due to Iinoleic
acid.
ASSAY
Total fatty acids Calculate the percentage content of total fatty acids, where
Gas chromatography (2.2.28). caproic, caprylic, capric, !auric, myristic, palmitoleic, palmitic
and stearic acids are expressed as !auric acid (C 12H 240 2;
Internal standard solution Dissolve 0.4 7 g of methyl
Mr 200.3) and linoleic, linolenic and oleic acids are expressed
margarate R in 20.0 mL of dimethylformamide Rand dilute to
as oleic acid (C 18H 340 2 ; Mr 282.5), using the following
expression:
2023 Schisandra Fruit IV-477
A1 xA4 x m2 xp, x 0.5 +As xA4 x m3 xpz x 0.5 slightly polyhedral starch granules and rare free starch
A2 x A3 x m, A6 x A3 x m, granules, rounded or slightly polyhedral, simple or 2-5
compound [F]; the hilum is often visible.
sum of the areas of the peaks due to caproic, caprylic, capric,
lauric, myrisric, palmitoleic, palmitic and stearic acids in the
A2 area of the peak due to !auric acid in the chromatogram
A3 area of the peak due to methyl margarate in the chromatogram
A4 area of the peak due to methyl margarate in the chromatogram
A5 sum of the areas of the peaks due to linoleic, linolenic and oleic
acids in the chromatogram obtained with the test solution;
A6 area of the peak due to oleic acid in the chromatogram obtained
solution, in grams; A
mass of launc acid CRS used to prepare reference solution (a),
in grams;
m3 mass of o/eic acid CRS used to prepare reference solution (a), in
grams;
P, percentage content of !auric acid in /auric acid CRS;
P2 percentage content of oleic acid in o/eic acid CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Schisandra Fruit
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole, dried or steamed and dried, ripe fruit of Schisandra
chinensis (Turcz.) Baill.
Content
Minimum 0.40 per cent of schisandrin (C 24 H 32 01;
-
25 µm
Mr 432.5) (dried drug). Figure 2428.-1. - Illustration for identification test B of powdered
IDENfIFICATION herbal drug of schisandra fruit
A. The berry is more or less spherical, up to 8 mm in
diameter; red, reddish-brown or blackish outer surface,
Schisandra sphenanthera.
sometimes covered in a whitish frost; strongly shrivelled
pericarp; presence of 1-2 reniform, yellowish-brown, lustrous Results A See below the sequence of quenching zones
seeds, with thin seed-coat. present in the chromatograms obtained with the reference
solution and the test solution. Furthermore, other weak
quenching zones may be present in the chromatogram
reddish-brown. Examine under a microscope using chloral
characters (Figure 2428.-1): reddish-brown fragments of
Top of the plate
pericarp consisting of 1 layer of thin-walled epicarp cells
(surface view [A]), covered by a striated cuticle [Aa] and y-Schisandrin: a quenching zone A quenching zone (y-schisandrin)
accompanied by sparse essential oil cells [Ab], the cuticle is
not striated above the essential oil cells; fragments of -- ---
mesocarp [C] composed of several layers of ovoid, more or A weak quenching zone
less flattened cells; fragments of the testa [B, E] with the
outer layer consisting of polygonal sclereids, about 11-30 µm --- ---
in diameter (surface view [Ba]) or in palisade arrangement Schisandrin: a quenching zone A quenching zone (schisandrin)
(side view [Ea]), with thick, lignified, finely channelled walls
and small lumen with reddish-brown or blackish contents
with the inner layers of the testa consisting of sclereids with
yellowish or slightly reddish walls, isolated or in small groups Results B See below the sequence of zones present in the
or together with the outer sclereids, about 80 µm in chromatograms obtained with the reference solution and the
diameter, with slightly lignified, thickened and markedly test solution. Furthermore, other faint zones may be present
channelled, pitted walls and a large lumen [Bb, Eb]; in the chromatogram obtained with the test solution.
fragments of mostly colourless endosperm [D] consisting of
polyhedral cells containing oil droplets [Da] and aleurone
grains. Examine under a microscope using a 50 per cent VIV
solution of glycerol R. The powder shows parenchymatous
cells of the mesocarp containing numerous small, round or
IV-478 Scrophularia 2023
Top of the plate Time Mobile phase A Mobile phase B

(min) (per cent VIJ') (per cent VIJ')
y-Schisandrin: a brown zone A brown zone (y-schisandrin) 0 - 10 100 0
10 - 16 100 ➔ 58 0 ➔ 42
-- --
16 - 26 58 42
-- -- Flow rate 1 mUmin.

Schisandrin: an intense, brownish- An intense, brownish-green zone Detection Spectrophotometer at 250 nm.
green zone (schisandrin) Injection 10 µL.
Reference solution Test solution Retention time Schisandrin = about 8 min.
System suitability:
- number of theoretical plates: minimum 5000, calculated for
TESTS
the peak due to schisandrin in the chromatogram
Schisandra sphenanthera obtained with the reference solution.
Calculate the percentage content of schisandrin using the
Test solutwn To 2.5 g of the powdered herbal drug (355) following expression:
(2. 9.12) add 10 mL of methanol R. Extract at 25 °C in an
ultrasonic bath for 5 min and centrifuge.
Reference solutwn Dissolve 5 mg of schisandrin R and 5 mg
of y-schisandrin R in 5 mL of methanol R.
area of the peak due to schisandrin in the chromatogram
Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica obtained with the test solution;
gel F2s4 plate R (2-10 µm)]. area of the peak due to schisandrin in the chromatogram
Mobile phase acetic acid R, ethyl acetate R, toluene R
(2:22:46 VIVIV). solution, in grams;
Application 5 µL [or 2 µL] as bands of 10 mm [or 6 mm]. mass of schisandrin CRS used to prepare the reference solution,
in grams;
Development Over a path of 10 cm [or 7 cm]. p percentage content of schisandrin in schisandrin CRS.
Drying In air.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Detection B Spray with a 100 g/L solution of sulfuric acid R
in methanol Rand heat in an oven at 120 °C for 7 min;
Results B The chromatogram obtained with the test solution Scrophularia Root
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. Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Loss on drying (2.2.32) DEFINITION

Maximum 10.0 per cent, determined on 1.000 g of the Whole or fragmented root of Scrophularia ningpoensis Hems!.,
powdered herbal drug (35 5) (2. 9.12) by drying in an oven at collected in winter after the aerial parts have withered, freed
105 °C for 2 h. from the rhizome, buds and rootlets, slowly baked or sun-
Total ash (2.4.16) dried until semi-dried and then piled for 3-6 days until fully
Maximum 6.0 per cent. dry.
ASSAY Content
Liquid chromatography (2.2.29). Minimum 0.45 per cent for the sum ofharpagoside
(C24H30Ou; Mr 494.5) and harpagide (C1sH24O10;
Test solutwn Weigh 1.250 g of the powdered herbal
Mr 364.3), expressed as harpagoside (dried drug).
drug (355) (2.9.12) into a 250 mL conical flask, add 90 mL
of methanol R and sonicate for 30 min. Filter the solution IDENTIFICATION
into a volumetric flask, add 10 mL of methanol R whilst A. Whole drug. The whole root is subcylindrical to slightly
rinsing the filter and dilute to 100.0 mL with the same laterally flattened, sometimes curved, slightly thickened in the
solvent. middle or thick in the upper part and slender in the lower
Reference solutwn Dissolve 5.0 mg of schisandrin CRS in part, 6-28 cm long and 1-4 cm in diameter. The outer
methanol Rand dilute to 100.0 mL with the same solvent. surface is yellowish-grey or greyish-brown, with distinct,
irregular, longitudinal furrows, and transverse lenticels; sparse
Column:
transverse fissures and sunken rootlet scars or protruding
- size: l = 0.25 m, 0 = 4.6 mm;
rootlet remains are sometimes visible. The texture is hard;
the fracture is horny, slightly lustrous and black to brownish-
black.
Fragmented drug The fragmented root occurs as thin,
Mobile phase:
transverse, longitudinal or oblique slices with an irregular
- mobile phase A: water R, methanol R (35:65 V/V);
margin, 0.6-4 cm in diameter; the outer surface is yellowish-
grey or greyish-brown; the cut surface is black to brownish-
black with a thin, lighter bark layer; lighter-coloured wood
may be visible in the centre or as radiating lines in transverse
section; clefts or lacunae are sometimes present between the
2023 Scrophularia IV-479
xylem tissues. The texture is hard; the fracture is horny, Reference solution (c) Dissolve 2 mg of harpagide R and 4 mg
slightly lustrous and black to brownish-black. of catalpol R in methanol R and dilute to 10 mL with the
B. Microscopic examination (2.8.23). The powder is dark same solvent.
brown to greyish-brown. Examine under a microscope using Intensity markers (reference solutions (a) and (b)) Harpagide
chloral hydrate solution R. The powder shows the following and harpagoside.
diagnostic characters (Figure 2973.-1): numerous sclereids, Plate TLC silica gel F254 plate R (2-10 µm).
isolated [B, D, E] or in groups of 2-5 [H], varying greatly in
Mobile phase water R, anhydrous ethanol R, methylene
shape (rectangular, subsquare, rounded, triangular or
chloride R (13:90: 140 VIVIV).
fusiform), 20-130 µm in diameter and 45-250 µm long, walls
4-22 µm thick with concentric striations, distinct pits and Application 6 µL as bands of 8 mm.
branched channels; the relatively large lumen of the sclereids Development 70 mm from lower edge of the plate.
is sometimes filled with reddish-brown contents; numerous Drying In a current of cold air for 5 min.
fragments of parenchyma cells [G] sometimes containing a Detection Treat with vanillin reagent Rand heat at 110 °C
subrounded nucleus-like object; groups of xylem fibres with for 3 min; examine in ultraviolet light at 366 nm.
slightly lignified and obliquely pitted walls m,frequently
associated with vessels; reticulate [F, K] or bordered-pitted
- the chromatogram shows in the lower third an orange
[C] vessels 4-125 µm in diameter; fragments of yellowish-
zone (harpagide) and directly above it a blue zone
brown cork, composed of subrectangular cells with slightly
(catalpol).
thickened walls (surface view [Al).
with the test solution, other faint zones may be present.
Top of the plate
- - - -
C Harpagoside: an orange zone An orange zone, faint to equivalent

(harpagoside)
A green zone, faint to equivalent
-- --
Harpagide: an orange zone An orange zone, faint to equivalent

(harpagide)
TESTS
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
herbal drug of scrophularia root
Solution A 50 per cent V/V solution of methanol R.
C. High-performance thin-layer chromatography (2.8.25). Test solution To 0.500 g of the powdered herbal drug (355)
Store the herbal drug at - 20 °G for at least 24 h before grinding. (2.9.12) add 25.0 mL of solution A. Weigh, macerate for
Test solution To 1.5 g of the powdered herbal drug (355) 10 min at room temperature and sonicate for 30 min. Weigh
(2.9. 12) add 5.0 mL of ethanol (96 per cent) Rand sonicate again and compensate for the loss of solvent with solution A.
for 10 min. Centrifuge and use the supernatant. Mix thoroughly. Filter the supernatant through a membrane
filter (nominal pore size 0.45 µm). Use the filtrate.
Reference solution (a) Dissolve 2.0 mg of harpagide Rand
1.5 mg of harpagoside R in methanol Rand dilute to 10.0 mL Reference solution (a) Dissolve the contents of a vial of
with the same solvent. harpagoside CRS in solution A and dilute to 50.0 mL with
solution A.
to 10.0 mL with methanol R. Reference solution (b) Dissolve 10 mg of trans-cinnamic
acid R in solution A and dilute to 10 mL with solution A.
IV-480 Scutellariae Baicalensis Root 2023
Reference solution (c) Dissolve 1 mg of harpagi,de R in 15 mL IDENTIFICATION

of reference solution (a), add 100 µL of reference A. The whole root is conical, twisted, 8-25 cm long and
solution (b) and dilute to 20 mL with solution A. 1-3 cm in diameter. The outer surface is yellowish-brown or
Column: dark yellow, bearing sparse, warty remnants of rootlets; the
- size: l = 0.25 m, 0 = 4.0 mm; upper part is rough, with longitudinal or irregularly reticulate
- stationary phase: end-capped octadecylsilyl silica gel for wrinkles; the lower part bears longitudinal furrows and fine
chromatography Rl (5 µm); wrinkles. The texture is hard and fragile, easily broken.
- temperature: 25 °C. The fracture is yellow, with some roots showing a yellow
Mobile phase: outer part and a withered, reddish-brown core; the cortex is
- mobile phase A: 0.03 per cent V/V solution of phosphoric narrow. The central part of old roots is dark brown or
acid R; brownish-black, reduced or hollowed. The root of cultivated
- mobile phase B: acetonitrile Rl; plants is tapering, usually branched, with external bark
adhering tightly to the wood. The outer surface is yellowish-
brown, with relatively fine, longitudinal wrinkles.
(min) (per cent VIJ!) (per cent V/J!) The fragmented root occurs as slices, short fragments or
0 95 5 irregular pieces, 0.3-1.9 cm in diameter. The outer surface is
0-8 95 ➔ 90 5 ➔ 10 yellowish-brown or dark yellow, occasionally bearing warty
8 - 23 90 ➔ 50 10 ➔ 50 remnants of rootlets. The bark may be rough or with
23 - 26 50 50 longitudinal furrows. The cut surface has a narrow yellow or
greenish-yellow cortex and lighter yellow wood part; some
Flow rate 1.0 mIJmin. roots with a yellow outer part and a withered, reddish-brown
core; the xylem is radially striated or may appear laminated.
The central part of old roots is dark brown or brownish-
Injection l O µL of the test solution and reference black, reduced or hollowed. The texture is hard and fragile,
solutions (a) and (c). mostly easily broken.
Identification of peaks Use the chromatogram obtained with B. Microscopic examination (2.8.23). The powder is
reference solution (a) to identify the peak due to harpagoside. yellowish or yellowish-brown. Examine under a microscope
Relative retention With reference to harpagoside (retention using chloral hydrate solution R. The powder shows the
time= about 21.6 min): harpagide = about 0.3; cinnamic following diagnostic characters (Figure 2438.-1): phloem
acid = about 1. 1. fibres and sclereids, isolated [F, G] or in groups W,
System suitability: sometimes adhering to parenchymatous cells [B, D, HJ, with
- resolution: minimum 5.0 between the peaks due to thick to very thick, channelled and pitted walls; phloem fibres
harpagoside and cinnamic acid in the chromatogram fusiform, 60-270 µm long and 9-35 µm in diameter [Ba],
obtained with reference solution (c). sclereids ovoid [Ac, Da], rectangular W or subsquare [G],
Calculate the percentage content of the sum of harpagide 26-29 µm in diameter; fragments of yellowish-brown cork,
and harpagoside, expressed as harpagoside, using the with polygonal cells (surface view [C]); fragments of the
following expression: outer bark [A] with several layers of cork [Aa],
phelloderm [Ab] and a few layers of cortical parenchyma
A1 x m1 A3 x m1 x 4.6 consisting of cells with slightly thickened walls [Ad] and
------ +------ sclereids or fibres [Ac]; numerous vessels [L, M],
A2 x m2 x 2000 A 2 x m2 x 2000
reticulate [La] or bordered pitted [E, Lb, Ma], 14-72 µm in
A1 area of the peak due to harpagoside in the chromatogram diameter, accompanied by xylem fibres, frequently broken,
obtained with the test solution; 10-20 µm in diameter, mostly with sparse, oblique pits.
A2 area of the peak due to harpagoside in the chromatogram
obtained with reference solution (a); Examine under a microscope using a 50 per cent V/V
A3 area of the peak due to harpagide in the chromatogram solution of glycerol R. The powder shows very numerous
obtained with the test solution; starch granules [Kl, rounded, mostly 2-10 µm in diameter,
m1 mass of harpagoside contained in I vial of harpagoside CRS, occasionally up to 14 µm in diameter, with a visible hilum,
used to prepare reference solution (a), in milligrams;
simple or 2-3 compound.
solution, in grams. C. Thin-layer chromatography (2.2.27).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Reference solution Dissolve 1 mg of baicalin R and 1 mg of
acteoside R in 10 mL of methanol R.
Scutellariae Baicalensis Root Plate TLC silica gel F254 plate R (2-10 µm).
(Baical Skullcap Root, Ph. Bur. monograph 2438) Mobile phase acetic acid R, anhydrous formic acid R, water R,
ethyl acetate R (1:1:2:15 V/V/V/V).
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Application l O µL as bands.
DEFINITION Development Over a path of 6 cm.
Dried (or steamed or treated with boiling water and dried),
Drying In air.
peeled, whole or fragmented root of Scutellaria baicalensis
Georgi without rootlets. It is collected in spring or autumn.
Content
Not less than 9.0 per cent ofbaicalin (C 21 H 18 0 11 ; Mr 446.4)
(dried drug).
2023 Scutellariae Baicalensis Root IV-481
Total ash (2.4.16')

ASSAY
(2. 9.12) add 40 mL of ethanol (70 per cent V/V) R, heat
under a reflux condenser on a water bath for 3 h, cool and
filter. Transfer the filtrate to a 100 mL volumetric flask.
Wash both the container and the residue several times with a
small volume of ethanol (70 per cent V/V) R and filter the
washings into the same flask. Dilute to 100.0 mL with
ethanol (70 per cent V/V) R. Mix well. Dilute 1.0 mL of the
solution to 10.0 mL with methanol R. Mix well.
Reference solution (a) Dissolve 5.0 mg of baicalin CRS in
Reference solution (b) Dissolve 2 mg of methyl
parahydroxybenzoate R in methanol R, add 20 mL of reference
solution (a) and dilute to 100 mL with methanol R.
Column:
=
- size: l 0.125 m, 0 4 mm; =
(5 µm).
Mobile phase:
- mobile phase A: 0.1 per cent VIV solution of phosphoric
acid R;
herbal drng of baical skullcap root Time Mobile phase A Mobile phase B
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 R,
then 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 Flow rate 1.0 mUmin.
ultraviolet light at 365 nm. Detection Spectrophotometer at 280 nm.
Results See below the sequence of zones present in the Injection 10 µL.
chromatograms obtained with the reference solution and the Retention time Methyl
test solution. Furthermore, other faint blue fluorescent zones parahydroxybenzoate = about 15 min;
may be present in the chromatogram obtained with the test baicalin = about 16 min.
solution. System suitability Reference solution (b):
- resolution: minimum 3.0 between the peaks due to methyl
Top of the plate parahydroxybenzoate and baicalin.
3-4 fluorescent zones Calculate the percentage content of baicalin using the
-- --
2 fluorescent zones
-- --
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), in
grams;
A blue fluorescent zone S1 area of the peak due to baicalin in the chromatogram obtained
Baicalin: a black zone A black zone S2 area of the peak due to baicalin in the chromatogram obtained
p percentage content of baicalin in baicalin CRS.
A weak yellow fluorescent zone

STORAGE
Reference solution Test solution Protected from moisture.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
TESTS
105 °C for 2 h.
IV-482 Selfheal Fruit-Spike 2023
Selfheal Fruit-Spike
(Common Selfheal Fruit-Spike, Ph. Bur. monograph
2439)
~~---------------------~
DEFINITION
Dried fruit-spike of Prunella vulgaris L.
Content
(piD · •,':,
·
Minimum 0 .12 per cent of the sum of oleanolic acid
(C30H4sO3; Mr 456.7) and ursolic acid (C 30H 48 O 3 ;
Mr 456. 7), expressed as ursolic acid, of which not less than
70.0 per cent consists of ursolic acid (dried drug).
IDENTIFICATION
A. Cylindrical, somewhat flattened, 1.5-8 cm long, Po~ . _-,t\\.
D ·,
0.8-1.5 cm in diameter, accompanied by remains of the stem

up to 15 cm long, pale brown or brownish-red. The whole
J ~.
spike is composed of up to 10 or more whorls of persistent 7
calyices and bracts, each whorl with 2 opposite bracts, fan-
shaped, apex acuminate, striations of vein distinct, the outer
surface with white hairs. Each bract is accompanied by
3 flowers, each with a persistent bilabiate calyx but often with
the corolla missing, and by 4 small brown ovoid nutlets, each
white and convex at the acute end. The calyx is closed at the
b
fruiting stage. ' Dd

brown or brown. Examine under a microscope using chloral }__, ~
characters (Figure 2439.-1): very numerous covering
trichomes, multicellular, scattered or on the epidermises, Figure 2439.-1. - Illustration for identification test B of powdered
usually broken [E, Da, De], sometimes exceeding 1 mm long herbal drug of common selfheal fruit-spike
and 125 µm wide at the base, with spiny walls, upper cell
Reference solution Dissolve 1 mg of /3-sitosterol R and 1 mg of
usually short and acuminate, fine needle-shaped crystals may
ursolic acid R in 2 mL of methanol R.
be visible in the cells [Ea, Db]; fragments of the bracts
(surface view [A, C]), with lobed cells on the upper surface Plate TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel
[Aa] and slightly sinuous cells covered by a thin, striated F2 54 plate R (2-10 µm)].
cuticle on the lower surface [Ca], with trichomes, mostly Mobile phase glacial acetic acid R, ethyl acetate R,
unicellular [Cb], sometimes bi- [Cc] or tricellular, conical, cyclohexane R (1:16:40 V/V/V).
acuminate, short and serrate; diacytic stomata (2.8.3) usually Application 10 µL [or 4 µL] as bands of 10 mm [or 8 mm].
accompanied by 2 subsidiary cells very unequal in size [Ab, Development Over a path of 12 cm [or 6 cm].
Cd] and rare glandular trichomes with a unicellular stalk and
Drying In air.
a bicellular head [Ac, G] or a bicellular stalk and a
unicellular head [B]; fragments of the bracts [DJ and/or calyx Detection Treat with a 10 per cent V/V solution of sulfuric
margins with numerous serrate trichomes pointing in the acid R in anhydrous ethanol R and heat at 100 °C for 3 min;
same direction [Dd]; fragments of the calyx (surface view examine in ultraviolet light at 365 nm.
[L]), composed of lobed cells strongly thickened and deeply Results See below the sequence of zones present in the
grooved; fragments of reticulate or bordered-pitted vessels chromatograms obtained with the reference solution and the
from the stems; rare fragments of the nutlets (surface test solution. Furthermore, other faint zones may be present
view [H], transverse section [J]) consisting of the epicarp in the chromatogram obtained with the test solution.
Ua], 3 layers of mesocarp, 2 of which have strongly thickened
Top of the plate
walls Ub, Jc, Ha] and are positioned on either side of the
middle layer whose cells contain microcrystals of calcium A pale violet fluorescent zone
oxalate Ud], and the endocarp Ue, Hb] consisting of cells
with finely striated walls; fragments of the seed (surface view -- --
[K]) consisting of testa cells with finely reticulate walls [Ka], 2 faint yellow fluorescent zones
associated with cells of the endosperm [Kb] with oily
P-sitosterol: a violet fluorescent zone
contents; very numerous oil droplets [Kc]; rare fragments of
the cauline leaves [F] may be present, with an epidermis Ursolic acid: a yellowish-orange A yellowish-orange fluorescent zone
covered with a striated cuticle [Fa] bearing glandular fluorescent zone (ursolic acid)
trichomes of lamiaceous type with 4 secretary cells [Fb] and
diacytic stomata [Fe].
C. Thin-layer chromatography (2.2.27). -- --
Test solution To 0.5 g of the powdered herbal drug (355) 2 faint green fluorescent zones
(2. 9.12) add 5 mL of methanol R, sonicate for 10 min and
centrifuge; use the supernatant.
2023 Senega Root IV-483
TESTS System suitability Reference solution (b):

Foreign matter (2.8.2) - resolution: minimum 1.5 between the peaks due to
Maximum 5 per cent of stems longer than 15 cm and oleanolic acid and ursolic acid.
maximum 2 per cent of other foreign matter. Calculate the percentage contents of ursolic acid and
Loss on drying (2.2.32) oleanolic acid, expressed as ursolic acid, using the following
Maximum 12.0 per cent, determined on 1.000 g of the equations:
105 °C.
Total ash (2. 4. 16)
Maximum 12.0 per cent. A3xm 2 xpx0.l
Ash insoluble in hydrochloric acid (2.8.1) n2 =
n1 percentage content of ursolic acid;
ASSAY n2 percentage content of oleanolic acid;
Liquid chromatography (2.2.29). AI area of the peak due to ursolic acid in the chromatogram
Solvent mixture methanol R, 1, 1-dimethylethyl methyl ether R A2 area of the peak due to ursolic acid in the chromatogram
(20:80 V/V). obtained with reference solution (a);
Test soluti.on Disperse 2.000 g of the powdered herbal drug A3 area of the peak due to oleanolic acid in the chromatogram
(355) (2.9.12) in 20 mL of the solvent mixture, heat under mI mass of the herbal drug to be examined used to prepare the test
reflux at 80 °C for 30 min and filter. Repeat the extraction solution, in grams;
twice. Combine the filtrates and dilute to 100.0 mL with the m2 mass of ursolic acid CRS used to prepare solution A, in grams;
solvent mixture. Evaporate 50.0 mL of this solution to p assigned percentage content of ursolic acid in ursolic acid CRS.
dryness at 40 °C. Dissolve the residue in 1.0 mL of
1,1-dimethylethyl methyl ether R. Rinse the flask 4 times with Calculate the sum of the percentage contents of ursolic acid
1.0 mL of 1,1-dimethylethyl methyl ether R. Pre-condition a and oleanolic acid (n 1 + n2) and the relative content of
3 mL solid phase extraction column, containing 500 mg of ursolic acid using the following expression:
aminopropylsilyl silica gel for chromatography Rl, using 2 mL of n1 X 100
methanol R followed by 2 mL of 1, 1-dimethylethyl methyl
(n1 + n2)
ether R. Subsequently apply the solution and the washings to
the pre-conditioned column. Wash the column with 1.0 mL _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
of 1,1-dimethylethyl methyl ether R followed by 5 quantities,
each of 1.0 mL, of methanol R. Apply 1.0 mL of a
2 per cent V/V solution of anhydrous formic acid R in
methanol R and elute after 5 min. Repeat the elution 3 times
and dilute the eluates to 5.0 mL with a 2 per cent V/V
Senega Root
solution of anhydrous formic acid R in methanol R. Senega
Soluti.on A Dissolve 10.0 mg of ursolic acid CRS in (Ph. Bur. monograph 0202)
When Powdered Senega Root is prescribed or demanded,
Reference soluti.on (a) Dilute 1.0 mL of solution A to material complying with the requirements below with the
10.0 mL with a 2 per cent V/V solution of anhydrous formic exception of Identification test A and the test for Foreign
acid R in methanol R. matter shall be dispensed or supplied.
Reference soluti.on (b) Dissolve 10.0 mg of oleanolic acid R in PhEw _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Mix 1.0 mL of the solution and 1.0 mL of solution A and DEFINITION
dilute to 10.0 mL with a 2 per cent V/V solution of Dried, whole or fragmented root and root crown of Polygala
anhydrous formic acid R in methanol R. senega L. or root of Polygala tenuifolia Willd., with rootlets
Column: removed.
- size: l = 0.15 m, 0 = 4.6 mm; IDENTIFICATION
- stationary phase: octadecylsilyl silica gd for chromatography R A. P. senega. The root crown of the whole drug is greyish-
(5 µm). brown and distinctly wider than the root; it forms an
Mobile phase Mix 25 volumes of a 4.6 g/L solution of irregular head up to 3 cm in diameter consisting of
ammonium dihydrogen phosphate R adjusted to pH 6.0 with numerous, tightly packed remains or scars of stems and
strong sodium hydroxide soluti.on R, 35 volumes of methanol Rl violet-brown buds. The taproot is flexuous and tortuous,
and 40 volumes of acetonitn"le Rl. brown or yellow, usually single but occasionally branched
Flow rate 1.0 mUmin. into 2 or 3 roots, usually 1-8 mm in diameter at the crown,
gradually tapering to the tip; the external surface is
transversely and longitudinally striated and often shows a
Injection 20 µL. more or less distinct, decurrent, elongated spiral ridge.
Run time 1. 1 times the retention time of ursolic acid. The transverse section of the root is more or less triangular.
Elution order Oleanolic acid, ursolic acid. The fracture is short and shows a yellowish cortex of varying
Relative retention With reference to ursolic acid thickness surrounding a paler, central, woody area irregular
(retention time = about 28 min): oleanolic acid = about 0.9. in shape, especially at the ridge. The fragmented drug
consists of pieces of varying shape; fragments of the root
crown comprise the tightly packed remains or scars of stems
and violet-brown buds; fragments of the root have a brown
IV-484 Senega Root 2023
or yellow, transversely and longitudinally striated external C. High-performance thin-layer chromatography (2.8.25).
surface with a paler internal surface. Test solution To 0.5 g of the powdered herbal drug (355)
P. tenuifolia The whole drug consists of the root, light (2. 9.12) add 5.0 mL of ethanol (70 per cent V/V) R. Sonicate
yellowish-brown or brown, straight or curved, 3-15 cm in at 80 °C for 15 min, filter or centrifuge and use the filtrate or
length and 3-8 mm in diameter, showing numerous supernatant.
transverse wrinkles and fissures and, to a lesser extent, Reference solution (a) Dissolve 1 mg of rutoside trihydrate R
longitudinal striations; round rootlet scars are often visible. and 30 mg of puerarin R in methanol Rand dilute to 25.0 mL
The xylem is light yellow and readily detached, with some with the same solvent.
fragments consisting only of rolls of bark with their edges
folded inwards. The fracture shows a yellowish cortex and a
pale yellow, usually circular, central woody area.
The fragmented drug consists of more or less cylindrical Reference solution (c) Dissolve 2 mg of chlorogenic acid R and
slices, solid or hollow in the central part where the xylem has 30 mg of puerarin R in methanol R and dilute to 25 mL with
detached. The external surface is yellowish-grey to brownish- the same solvent.
grey and transversely striated; the transverse section is Intensity marker Puerarin.
yellowish-brown and shows a rounded, pale yellow, central Plate TLC silica gel F254 plate R (2-10 µm).
woody area. Mobile phase anhydrous formic acid R, glacial acetic acid R,
B. Microscopic examination (2.8.23). The powder is light water R, ethyl acetate R (11: 11:26:100 VIVIVIV).
yellow to brown. Examine under a microscope using chloral Application 3 µL as bands of 8 mm.
characters (Figure 0202.-1): fragments of lignified tissue [C,
D, F] made up of numerous pitted tracheids [Ca, Da] and Drying In a current of air at room temperature for 5 min.
slightly larger, reticulate [Cb], pitted [Db] or bordered-pitted Detection Heat at 100-105 °C for 3 min; spray the warm
vessels [Fa]; yellowish parenchyma cells containing oil plate with a 10 g/L solution of diphenylboric acid aminoethyl
droplets [BJ; fragments of cork (surface view [A], transverse ester R in methanol R, then with a 50 g/L solution of macrogol
section [El), sometimes accompanied by phelloderm and 400 R in methanol R or, alternatively, dip the warm plate in a
parenchyma, some cells of which contain oil droplets [Ea]; 5 g/L solution of diphenylboric acid aminoethyl ester R in ethyl
numerous isolated oil droplets U]. P. tenuifolia powder also acetate R, then in a 50 g/L solution of macrogol 400 R in
contains calcium oxalate cluster crystals, isolated [G] or methyl.ene chloride R; allow the plate to dry in air for about
included in parenchymatous cells [HJ, and long, fine, thick- 1 min and examine in ultraviolet light at 366 nm.
walled lignified fibres, most often fragmented, in clusters [M] System suitability Reference solution (c):
or associated with vessels (Fb]. P. senega powder may show - the chromatogram shows in the middle third 2 distinct
occasional fragments of epidermal tissue from bud scales [K, zones; the lower zone (chlorogenic acid) shows a light
L] with stomata [La] and unicellular trichomes [Ka]. blue fluorescence and the upper zone (puerarin) shows
a blue fluorescence.
obtained with the test solution, other faint fluorescent zones
may be present.
Top of the plate
-- -- --
A blue zone
Puerarin: a blue zone A blue zone A blue zone
A blue zone A blue zone
A greyish zone, faint
Rutoside: an orange An orange zone, faint

zone
A greenish-blue zone (at

the border between the
middle and lower third)
-- -- --
A bluish zone
A whitish-blue zone
3 bluish zones
Reference Test solution Test solution (Pofygala

solution (a) (Polygala sem,ga) tenuifolia)
herbal drug of senega root
2023 Senna Preparations IV-485
TESTS
Standardised Senna Fruit Dry
Maximum 12.0 per cent, determined on 1.000 g of the Aqueous Extract
powdered herbal drug (355) (2. 9.12) by drying in an oven at (Ph. Bur. monograph 3084)
105 °C for 2 h.
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Total ash (2.4.16)
Ash insoluble in hydrochloric acid (2.8.1) Standardised dry aqueous extract produced from Senna pods
Maximum 3.0 per cent. (0207).
Extractable matter Content

Minimum 20.0 per cent. 7 .0 per cent to 13.0 per cent of total hydroxyanthracene
glycosides, expressed as sennoside B (C42H 38 O20; Mr 863)
To 1.00 g of the powdered herbal drug (355) (2.9.12) add a
(dried extract). The measured content does not deviate from
mixture of 4.5 g of ethanol (96 per cent) Rand 15.5 g of
the value stated on the label by more than ± 10 per cent.
water Rand allow to macerate for 3 h, shaking frequently.
Filter. Evaporate 5.0 g of the filtrate to dryness on a water- PRODUCTION
bath and dry in an oven at 100-105 °C for 2 h. The residue The extract is produced from the herbal drug by a suitable
weighs a minimum of 50 mg. procedure using water.
Foam index (2.8.24) CHARACTERS
Minimum 3.5, determined on 1.00 g of the powdered herbal Appearance
drug (355) (2.9.12). Brownish or brown powder.
STORAGE IDENTIFICATION
Protected from humidity. High-performance thin-layer chromatography (2. 8. 25).
LABELLING Prepare the test and reference solutions protected from bright light.
The label states the species present. Solvent mixture ethanol (96 per cent) R, water R (50:50 V/V).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur Test solution To 75 mg of the extract to be examined, add
5.0 mL of the solvent mixture. Sonicate for 5 min, then filter
or centrifuge the solution. Use the filtrate or supernatant.
Reference solution (a) Dissolve 3 mg of sennoside AR and
Senna Liquid Extract 3 mg of sennoside B R in a mixture of equal volumes of
ethanol (96 per cent) R and a 1 g/L solution of sodium
DEFINITION hydrogen carbonate Rand dilute to 20.0 mL with the same
mixture of solvents.
Senna Fruit, Alexandrian or Tinnevelly, 1000 g
crushed Reference solution (b) Dilute 2.5 mL of reference solution (a)
Coriander Oil 6mL to 10.0 mL with the solvent mixture.
Ethanol (90 per cent) 250 mL
Reference solution (c) Dissolve 10 mg of senna extract HRS in
Purified Water, freshly boiled and cooled A sufficient quantity
1 mL of the solvent mixture (a slight residue may remain).
Extemporaneous preparation Intensity marker Sennoside A.
The following directions apply. Plate TLC silica gel F 254 plate R (2-10 µm).
Macerate the crushed Senna Fruit in 5 litres of Purified Mobile phase water R, ethyl acetate R, propanol R
Water for 8 hours, decant the clear liquid and strain; repeat (30:40:40 VIVIV).
the process twice using 2 litres of Purified Water for each Application 2 µL as bands of 8 mm.
maceration. Lightly press the mare, strain the expressed
liquid, mix the strained liquid with the previously decanted
liquid and heat the combined liquids at 80° for 3 minutes in Drying In a current of air at room temperature for 5 min.
a covered vessel. Allow to stand for not less than 24 hours; Detection Heat at 110 °C for 10 min; treat the warm plate
filter. with a 50 g/L solution of potassium hydroxide R in the solvent
Evaporate the filtrate to 750 mL under reduced pressure at a mixture and heat at 110 °C for 10 min; examine immediately
temperature not exceeding 60°. Separately, dissolve the in ultraviolet light at 366 nm.
Coriander Oil in the Ethanol (90 per cent), add the solution System suitability Reference solution (c):
to the evaporated filtrate and add sufficient Purified Water to - the chromatogram shows 2 distinct zones, which may be
produce 1000 mL. Allow to stand for not less than 24 hours; touching, near the border between the lower and middle
filter. thirds; the lower zone (sennoside A) shows a light yellow
The extract complies with the requirements stated under Extracts
fluorescence and the upper zone (sennoside D) shows a
faint brownish-yellow fluorescence.
TESTS in the chromatograms obtained with reference solution (a)
Ethanol content and the test solution. Furthermore, in the chromatogram
21 to 24% v/v, Appendix VIII F, Method III. obtained with the test solution, other very faint to faint blue
Dry residue and/or reddish-brown fluorescent zones may be present.
17 to 25% w/v. For sennosides, Rp values vary slightly and the zones may be
Relative density curved depending on the concentration.
1.02 to 1.09, Appendix V G.
IV-486 Senna Preparations 2023
Top of the plate isorhamnetin diglucoside and hydroxyanthracene glycosides

(peaks 2-9); any shoulder appearing on the ascending part of
the peak due to sennoside B (peak 3) is included in its area;
- - - -
peaks 4 and 5 may be co-eluted; peaks 7 and 8 may be
co-eluted.
Relative retention With reference to sennoside B (peak 3,
A light yellow zone, very faint to retention time = about 14.2 min): isorhamnetin
faint (sennoside C) diglucoside = about 0.93; hydroxyanthracene glycoside (peak
2) = about 0.98; hydroxyanthracene glycoside (peak
A brownish-yellow zone, faint
(sennoside D) 5) = about 1.02; hydroxyanthracene glycoside (peak
Sennoside A: a light yellow zone A light yellow zone, equivalent
(sennoside A) 7) = about 1.09; hydroxyanthracene glycoside (peak
9) = about 1.13.
-- -- System suitability Reference solution (a):
isorhamnetin diglucoside and hydroxyanthracene glycoside
Sennoside B: a brownish-yellow zone A brownish-yellow zone, equivalent (peak 2).
(sennoside B)
Calculate the percentage content of total hydroxyanthracene
glycosides (peaks 2-9), expressed as sennoside B, using the
Reference solution (a) Test solution following expression:
TESTS
Maximum 5.0 per cent. A, sum of the areas of the peaks due to hydroxyanthracene
glycosides (peaks 2-9) in the chromatogram obtained with the
ASSAY test solution;
Liquid chromatography (2.2.29). Carry out the assay protected area of the peak due to sennoside B in the chromatogram
from bright light. obtained with reference solution (b);
Solvent mixture 1.0 g,'L solution of sodium hydrogen solution, in grams;
carbonate R, methanol R (30:70 V/V). mass of sennoside B CRS used to prepare reference solution (b),
Test solution Introduce 0.150 g of the extract to be in grams;
p percentage content of sennoside B in sennoside B CRS.
examined into a 250 mL screw-cap bottle and add 100.0 mL
of the solvent mixture. Sonicate for 30 min and shake for
2 h. Filter through a membrane filter (nominal pore size LABELLING
0.45 µm). The label states the content of total hydroxyanthracene
Reference solutwn (a) Dissolve 10 mg of senna extract HRS glycosides.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
in 8 mL of the solvent mixture using sonication for 5 min
and dilute to 10 mL with the solvent mixture (a slight
residue may remain). Filter through a membrane filter
(nominal pore size 0.45 µm). Standardised Senna Fruit Dry
Reference solutwn (b) Dissolve 5.0 mg of sennoside B CRS in
25 mL of methanol R using sonication and dilute to 50.0 mL Hydroalcoholic Extract
with water R. (Ph. Bur. monograph 3127)
Column: PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
- size: l =0.25 m, 0 =4.6 mm;
- statwnary phase: end-capped propoxybenzene silica gel for DEFINITION
chromatography R (4 µm); Standardised dry hydroalcoholic extract produced from Senna
- temperature: 30 °C. pods (0207).
Mobile phase: Content
- mobile phase A: 1.275 per cent V/V solution of formic 14.0 per cent to 22.0 per cent of total hydroxyanthracene
acid R; glycosides, expressed as sennoside B (C 42 H 38O 20; Mr 863)
- mobile phase B: acetonitrile R; (dried extract). The measured content does not deviate from
(min) (per cent V/f') (per cent V/f') PRODUCTION
0-3 87 13 The extract is produced from the herbal drug by a suitable
3 - 40 87--+ 37 13--+ 63 procedure using methanol (40-80 per cent V/V) or ethanol
(40-80 per cent VIV).
Flow rate 1.0 mUmin. CHARACTERS
Detection Spectrophotometer at 270 nm. Appearance
Injection 10 µL. Brownish or brown powder.
Identification of peaks Use the chromatogram supplied with IDENTIFICATION
senna extract HRS and the chromatogram obtained with High-performance thin-layer chromatography (2. 8. 25).
reference solution (a) to identify the peaks due to Prepare the test and reference solutions protected from bright light.
Solvent mixture ethanol (96 per cent) R, water R (50:50 VIV). TESTS
Test solution To 50 mg of the extract to be examined, add Loss on drying (2. 8.1 7)
5 .0 mL of the solvent mixture. Sonicate for 5 min, then filter Maximum 6.0 per cent.
or centrifuge the solution. Use the filtrate or supernatant. ASSAY
Reference solution (a) Dissolve 3 mg of sennoside A Rand Liquid chromatography (2.2.29).
3 mg of sennoside B R in a mixture of equal volumes of Carry out the assay protected from bright light.
ethanol (96 per cent) R and a 1 g/L solution of sodium
Solvent mixture 1.0 g/L solution of sodium hydrogen
hydrogen carbonate Rand dilute to 20.0 mL with the same
carbonate R, methanol R (30:70 V/V).
Test solution Introduce 0.100 g of the extract to be
examined into a 250 mL screw-cap bottle and add 100.0 mL
of the solvent mixture. Sonicate for 30 min and shake for
Reference solution (c) Dissolve 10 mg of senna extract HRS in 2 h. Filter through a membrane filter (nominal pore size
1 mL of the solvent mixture (a slight residue may remain). 0.45 µm).
Intensity marker Sennoside A. Reference solution (a) Dissolve 10 mg of senna extract HRS
Plate TLC silica gel F254 plate R (2-10 µm). in 8 mL of the solvent mixture using sonication for 5 min
Mobile phase water R, ethyl acetate R, propanol R and dilute to 10 mL with the solvent mixture (a slight
(30:40:40 VIVIV). residue may remain). Filter through a membrane filter
Application 2 µL as bands of 8 mm. (nominal pore size 0.45 µm).
Development 70 mm from the lower edge of the plate. Reference solution (b) Dissolve 5.0 mg of sennoside B CRS in
25 mL of methanol R using sonication and dilute to 50.0 mL
with water R.
Column:
with a 50 g/L solution of potassium hydroxide R in the solvent
- size: l = 0.25 m, 0 = 4.6 mm;
mixture and heat at 110 °C for 10 min; examine immediately
- stationary phase: end-capped propoxybenzene silica gel for
in ultraviolet light at 366 nm.
System suitability Reference solution (c): - temperature: 30 °C.
- the chromatogram shows 2 distinct zones, which may be
Mobile phase:
touching, near the border between the lower and middle
- mobile phase A: 1.275 per cent V/V solution of formu:
thirds; the lower zone (sennoside A) shows a light yellow
acid R;
fluorescence and the upper zone (sennoside D) shows a
faint brownish-yellow fluorescence.
Results See below the sequence of fluorescent zones present Time Mobile phase A Mobile phase B
in the chromatograms obtained with reference solution (a) (min) (per cent V/JJ) (per cent V/JJ)
and the test solution. Furthermore, in the chromatogram 0-3 87 13
obtained with the test solution, other very faint to faint blue 3 - 40 87 ➔ 37 13 ➔ 63
fluorescent zones may be present and very faint to equivalent
reddish-brown fluorescent zones may be present.
For sennosides, RF values vary slightly and the zones may be
curved depending on the concentration. Detection Spectrophotometer at 270 nm.
Injection 10 µL.
Top of the plate Identification of peaks Use the chromatogram supplied with
senna extract HRS and the chromatogram obtained with
reference solution (a) to identify the peaks due to
-- -- isorhamnetin diglucoside and hydroxyanthracene glycosides
A light yellow zone, very faint to peaks 4 and 5 may be co-eluted; peaks 7 and 8 may be
faint (sennoside C) co-eluted.
A brownish-yellow zone, faint retention time = about 14.2 min): isorhamnetin
(sennoside D) diglucoside = about 0.93; hydroxyanthracene glycoside (peak
Sennoside A: a light yellow zone A light yellow zone, equivalent 2) = about 0.98; hydroxyanthracene glycoside (peak
(sennoside A) 4) = about 1.01; hydroxyanthracene glycoside (peak
- - -- 7) = about 1.09; hydroxyanthracene glycoside (peak
9) = about 1.13.
Sennoside B: a brownish-yellow zone A brownish-yellow zone, equivalent
(sennoside B) System suitability Reference solution (a):
Reference solution (a) Test solution (peak 2).
Calculate the percentage content of total hydroxyanthracene
glycosides (peaks 2-9), expressed as sennoside B, using the
following expression: (d) Use an ambient column temperature.

(e) Use a detection wavelength of 350 nm.
MOBILE PHASE
sum of the areas of the peaks due to hydroxyanthracene
glycosides (peaks 2-9) in the chromatogram obtained with the 17 volumes of acetonitrile and 83 volumes of a 1% v/v
test solution; solution of glacial acetic acid.
area of the peak due to sennoside B in the chromatogram
obtained with reference solution (b); DETERMINATION OF CONTENT
mass of the extract to be examined used to prepare the test Calculate the total content of sennosides A and B as
solution~ in grams;
mass of sennoside B CRS used to prepare reference solution (b),
sennoside B in the granules using the declared content of
in grams; sennosides in Alexandrian senna fruit powder BPCRS.
p percentage content of sennoside B in sennoside B CRS.
STORAGE
Standardised Senna Granules should be kept in an airtight
LABELLING container.
The label states the content of total hydroxyanthracene
glycosides. LABELLING
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
The quantity of active ingredient is stated in terms of the
equivalent amount of sennoside B.
Standardised Senna Granules Senna Leaflet

DEFINITION
Standardised Senna Granules contain Alexandrian Senna Senna Leaf
Fruit in powder form with suitable excipients. The granules (Ph. Bur. monograph 0206)
contain 0.55% w/w of sennosides, calculated as sennoside B. Preparation
The granules comply with the requirements stated under Granules Standardised Senna Leaf Dry Extract
and with the following requirements. When Powdered Senna Leaf is prescribed or demanded,
Content of sennosides calculated as sennoside B material complying with the requirements below with the
0.467 to 0.633% w/w. exception of Identification test A and the test for Foreign
IDENTIFICATION matter shall be dispensed or supplied.
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
A. To 25 mg, in No. 180 powder, add 50 mL of water and
2 mL of hydrochloric acid, heat in a water bath for DEFINITION
15 minutes, allow to cool and shake with 40 mL of ether. Dried leaflets of Senna alexandrina Mill. (syn. Cassia acutifolia
Dry the ether layer over anhydrous sodium sulfate, filter, Delile and Cassia angustifolia Vah!).
evaporate 5 mL of the filtrate to dryness, cool and add 5 mL
of 6M ammonia to the residue; a yellow or orange colour is Content
produced. Heat on a water bath for 2 minutes; a reddish Minimum 2.0 per cent of total hydroxyanthracene glycosides,
violet colour is produced. expressed as sennoside B (C 42 H 38 0 20 ; Mr 863) (dried drug).
B. In the Assay, the chromatogram obtained with solution IDENTIFICATION
(1) exhibits two peaks corresponding to the peaks due to A. The green to brownish-green leaflets are thin, elongated,
sennoside A and sennoside B in the chromatogram obtained lanceolate, more or less asymmetrical at the base and
with solution (2). mucronate at the apex, 15-50 mm long and 5-20 mm wide.
Loss on drying Both surfaces of the lamina are covered with varying
When dried at 105° for 5 hours, lose not more than 2.0% of numbers of fine, short trichomes and may be marked with
their weight. Use 5 g. transverse or oblique lines. Pinnate venation is visible mainly
on the abaxial surface and usually shows lateral veins
ASSAY anastomosing at the leaf margins.
Carry out the method for liquid chromawgraphy, B. Microscopic examination (2.8.23). The powder is light
Appendix III D, using the following solutions. green or greenish-yellow. Examine under a microscope using
(I) Shake 2 g of the granules with 50 mL of a 0.3% v/v chloral hydrate solution R. The powder shows the following
solution of acetic acid adjusted to pH 5.9 with IM sodium diagnostic characters (Figure 0206.-1): fragments of
hydroxide for 30 minutes, centrifuge, filter through a glass epidermis [A, B, J, K] with polygonal cells [Aa, Ka],
fibre paper (Whatman GF/C is suitable) and use the filtrate. paracytic stomata (2.8.3) [Ab, Ac, Ba, Ja, Kb] and unicellular
(2) Prepare solution (2) in the same manner as solution (1) covering trichomes, conical in shape, with warty walls
but using a quantity of Alexandrian senna fruit powder BPCRS (surface view [Ad], side view [G]), or their scars [Bb, Jb],
containing the equivalent of 11 mg of sennoside B. frequently accompanied by palisade parenchyma [Ae, Jc];
isolated, fragmented covering trichomes [E]; fibres [F]
accompanied by crystal sheaths containing prisms of calcium
(a) Use a stainless steel column (15 cm x 4.6 mm) packed oxalate [Fa]; isolated prisms of calcium oxalate [D]; isolated
with end-capped octadecylsilyl silica gel for chromawgraphy cluster crystals of calcium oxalate [HJ; fragments of median
(5 µm) (Spherisorb ODS 2 is suitable). parenchyma from the lamina [C] with some cells containing
(b) Use isocratic elution and the mobile phase described cluster crystals of calcium oxalate [Ca], often accompanied
below. by palisade parenchyma [Cb] and annular vessels [Cc].
(c) Use a flow rate of 2 mL per minute.
2023 Senna Leaflet IV-489
light yellow fluorescence and the upper zone

(sennoside D) shows a faint brownish-yellow
fluorescence.
obtained with the test solution, other blue and/or reddish
fluorescent zones may be present, mainly above the zone due
to sennoside A. For sennosides, Rp values vary slightly and
the zones may be curved depending on the concentration.
Top of the plate
- - --
A light yellow zone, very faint to

faint (sennoside C)

(sennoside D)
Figure 0206.-1. - Illustration for identification test B of powdered Sennoside A: a light yellow zone A light yellow zone, equivalent
herbal drug of senna leaflet (sennoside A)
- - --
Solvent mixture ethanol (96 per cent) R, water R (50:50 VIV).
(2. 9.12) add 5.0 mL of the solvent mixture. Sonicate for A brownish-yellow zone, equivalent
Sennoside B: a brownish-yellow zone
10 min, then filter or centrifuge the solution. Use the filtrate (sennoside B)
or supernatant.
Reference solution (a) Dissolve 3 mg of sennoside A Rand
3 mg of sennoside B R in a mixture of equal volumes of
ethanol (96 per cent) R and a 1 g/L solution of sodium Reference solution (a) Test solution
hydrogen carbonate R and dilute to 20.0 mL with the same
to 10.0 mL with the solvent mixture. Total anthraquinones (aloe emodin and rhein)
Liquid chromatography (2.2.29). Carry out the test protected
from bright light.
1 mL of the solvent mixture (a slight residue may remain).
Intensity marker Sennoside A.
carbonate R, methanol R (30:70 VIV).
Test solution Introduce 1.00 g of the powdered herbal
Mobile phase water R, ethyl acetate R, propanol R drug (355) (2.9.12) into a 250 mL screw-cap bottle and add
(30:40:40 VIV/V). 100.0 mL of the solvent mixture. Sonicate for 30 min and
Application 1 µL of the test solution and 2 µL of reference shake for 2 h. Filter through a membrane filter (nominal
solutions (a), (b) and (c), as bands of 8 mm. pore size 0.45 µm).
Development 70 mm from the lower edge of the plate. Reference solution (a) Dissolve 10 mg of aloe emodin Rand
Drying In a current of air at room temperature for 5 min. 10.0 mg of rhein CRS in tetrahydrofuran R and dilute to
Detection Heat at 110 °C for 10 min; treat the warm plate 50.0 mL with the same solvent. Dilute 1.0 mL of the
with a 50 g/L solution of potassium hydroxide R in the solvent solution to 20.0 mL with the solvent mixture.
mixture and heat at 110 "C for 10 min; examine immediately Reference solution (b) Dissolve 10 mg of senna extract HRS in
in ultraviolet light at 366 nm. 8 mL of the solvent mixture using sonication for 5 min and
System suitability Reference solution (c): dilute to 10 mL with the solvent mixture (a slight residue
- the chromatogram shows 2 distinct zones, which may may remain). Filter through a membrane filter (nominal pore
be touching, near the border between the lower and size 0.45 µm).
middle thirds; the lower zone (sennoside A) shows a
Reference solution (c) Dissolve 5.0 mg of sennoside B CRS in Calculate the percentage content of total anthraquinones with
25 mL of methanol R using sonication and dilute to 50.0 mL reference to the sum of total hydroxyanthracene glycosides
with water R. (THG, see Assay) and total anthraquinones, using the
Column: following expression:
- size: l = 0.25 m, 0 = 4.6 mm;
- stationary phase: end-capped propoxybenzene silica gel for TA
THG+ TA x lOO
Limit:
Mobile phase: - total anthraquinones (aloe emodin and rhein) expressed as
rhein (C1 sf{8 Or,; Mr 284.2): maximum 7.0 per cent,
formic acid R; calculated with reference to the sum of total
- mobile phase B: acetonitrile R; hydroxyanthracene glycosides and total anthraquinones
(dried drug).
(min) (per cent V/J!) (per cent VIJ!) Foreign matter (2.8.2)
0-3 87 13 Maximum 4 per cent.
3 - 40 87--> 37 13--> 63 Loss on drying (2.2.32)
Flow rate 1.0 mUmin. powdered herbal drug (355) (2.9.12) by drying in an oven at
Detection Spectrophotometer at 270 nm. 105 °C for 2 h.
Injection 10 µL of the test solution and reference Total ash (2.4.16)
solutions (a) and (b). Maximum 12.0 per cent.
Identification of peaks Use the chromatogram supplied with Ash insoluble in hydrochloric acid (2.8.1)
senna extract HRS and the chromatogram obtained with Maximum 2.5 per cent.
reference solution (b) to identify the peaks due to ASSAY
isorhamnetin diglucoside and hydroxyanthracene glycosides Liquid chromatography (2.2.29) as described in the test for
(peaks 2-9); any shoulder appearing on the ascending part of total anthraquinones (aloe emodin and rhein) with the
the peak due to sennoside B (peak 3) is included in its area; following modification.
co-eluted; use the chromatogram obtained with reference
solution (a) to identify the peaks due to aloe emodin and Calculate the percentage content of total hydroxyanthracene
rhein. glycosides (peaks 2-9) (THG), expressed as sennoside B,
retention time = about 14.2 min): isorhamnetin
diglucoside = about 0.93; hydroxyanthracene glycoside
(peak 2) = about 0.98; hydroxyanthracene glycoside
(peak 4) = about 1.01; hydroxyanthracene glycoside A1 sum of the areas of the peaks due to hydroxyanthracene
(peak 5) = about 1.02; hydroxyanthracene glycoside glycosides (peaks 2-9) in the chromatogram obtained with the
(peak 6) = about 1.07; hydroxyanthracene glycoside test solution;
A2 area of the peak due to sennoside B in the chromatogram
(peak 7) = about 1.09; hydroxyanthracene glycoside obtained with reference solution (c);
(peak 8) = about 1.11; hydroxyanthracene glycoside m1 mass of the herbal drug to be examined used to prepare the test
(peak 9) = about 1.13; aloe emodin = about 2.2; solution, in grams;
rhein = about 2.3. m2 mass of sennoside B CRS used to prepare reference solution (c),
in grams;
System suitability Reference solution (b): p percentage content of sennoside B in sennoside B CRS.
isorhamnetin diglucoside and hydroxyanthracene glycoside STORAGE
(peak 2).
Calculate the percentage content of total anthraquinones _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
(aloe emodin and rhein) (TA), expressed as rhein, using the
Standardised Senna Leaflet Dry

sum of the areas of the peaks due to aloe emodin and rhein in
Extract
the chromatogram obtained with the test solution; Standardised Senna Leaf Dry Extract
area of the peak due to rhein in the chromatogram obtained
with reference solution (a); (Ph. Eur. monograph 1261)
mass of the herbal drug to be examined used to prepare the test PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
solution, in grams;
mass of rhein CRS used to prepare reference solution (a), in DEFINITION
grams; Standardised dry extract produced from Senna leaflet (0206).
p percentage content of rhein in rhein CRS.
Content
5 .5 per cent to 12. 0 per cent of total hydroxyanthracene
glycosides, expressed as sennoside B (C 42H 38 0 20; Mr 863)
(dried extract). The measured content does not deviate from Top of the plate
PRODUCTION
The extract is produced from the herbal drug by a suitable - - - -
procedure using methanol (40-80 per cent VIV) or ethanol

(40-80 per cent V/V).
CHARACTERS
A light yellow zone, very faint to
Appearance faint (sennoside C)
Brownish or brown powder.
IDENTIFICATION (sennoside D)
High-performance thin-layer chromatography (2.8.25). Sennoside A: a light yellow zone A light yellow zone, equivalent
Prepare the test and reference solutions protected from bright light. (sennoside A)
Solvent mixture ethanol (96 per cent) R, water R (50:50 V/V). -- --

Test solution To 75 mg of the extract to be examined, add Sennoside B: a brownish-yellow zone A brownish-yellow zone, equivalent
5.0 mL of the solvent mixture. Sonicate for 5 min, then filter (sennoside B)
or centrifuge the solution. Use the filtrate or supernatant.
Reference solution (a) Dissolve 3 mg of sennoside A R and
ethanol (96 per cent) R and a 1 g/L solution of sodium Reference solution (a) Test solution
hydrogen carbonate R and dilute to 20.0 mL with the same
TESTS
Loss on drying (2. 8.17)
1 mL of the solvent mixture (a slight residue may remain). ASSAY
Liquid chromatography (2.2.29). Carry out the assay protected
Intensity marker Sennoside A.
from bright light.
Mobile phase water R, ethyl acetate R, propanol R carbonate R, methanol R (30:70 V/V).
(30:40:40 VIVIV).
Test solution Introduce O.150 g of the extract to be
Application 2 µL as bands of 8 mm. examined into a 250 mL screw-cap bottle and add 100.0 mL
Development 70 mm from the lower edge of the plate. of the solvent mixture. Sonicate for 30 min and shake for
Drying In a current of air at room temperature for 5 min. 2 h. Filter through a membrane filter (nominal pore size
Detection Heat at 110 °C for 10 min; treat the warm plate 0.45 µm).
with a 50 g/L solution of potassium hydroxide R in the solvent Reference solution ( a) Dissolve 10 mg of senna extract HRS
mixture and heat at 110 °C for 10 min; examine immediately in 8 mL of the solvent mixture using sonication for 5 min
in ultraviolet light at 366 nm. and dilute to 10 mL with the solvent mixture (a slight
System suitability Reference solution (c): residue may remain). Filter through a membrane filter
- the chromatogram shows 2 distinct zones, which may be (nominal pore size 0.45 µm).
touching, near the border between the lower and middle Reference solution (b) Dissolve 5.0 mg of sennoside B CRS in
thirds; the lower zone (sennoside A) shows a light yellow 25 mL of methanol R using sonication and dilute to 50.0 mL
fluorescence and the upper zone (sennoside D) shows a with water R.
faint brownish-yellow fluorescence. Column:
Results See below the sequence of fluorescent zones present =
- size: l 0.25 m, 0 4.6 mm; =
in the chromatograms obtained with reference solution (a) - stationary phase: end-capped propoxybenzene silica gd for
and the test solution. Furthermore, in the chromatogram chromatography R (4 µm);
obtained with the test solution, other very faint to equivalent - temperature: 30 °C.
blue and/or reddish-brown fluorescent zones may be present. Mobile phase:
For sennosides, RF values vary slightly and the zones may be - mobi1e phase A: 1.275 per cent V/V solution of formic
curved depending on the concentration. acid R;

(min) (per cent V/J,') (per cent V/Ji)
0-3 87 13
3 - 40 87---. 37 13---. 63

Injection I O µL.
senna extract HRS and the chromatogram obtained with
IV-492 Senna Pods 2023
reference solution (a) to identify the peaks due to Content

isorhamnetin diglucoside and hydroxyanthracene glycosides Minimum 2.0 per cent of total hydroxyanthracene glycosides,
(peaks 2-9); any shoulder appearing on the ascending part of expressed as sennoside B (C 42 H 380 20; Mr 863) (dried drug).
IDENTIFICATION
A. Flattened, more or less reniform pods, yellowish-brown to
co-eluted.
greenish-brown with brown patches at the positions
Relative retention With reference to sennoside B (peak 3, corresponding to the seeds, usually 35-60 mm long and
retention time = about 14.2 min): isorhamnetin 14-20 mm wide. At one end is a stylar point and at the other
diglucoside = about 0.93; hydroxyanthracene glycoside (peak a short stalk. The pods contain 5-8 flattened, obovate seeds,
2) = about 0.98; hydroxyanthracene glycoside (peak green or pale brown, with a more or less prominent network
4) = about 1.01; hydroxyanthracene glycoside (peak of transverse, wavy ridges on the testa.
B. Microscopic examination (2.8.23). The powder is brown.
(Figure 0207.-1): fragments of the epicarp (surface
9) = about 1.13.
view [B, Kl) with polygonal cells, anomocytic [Ba] or
System suitability Reference solution (a): paracytic [Bb, Ka] stomata (2.8.3) and covering
- resolution: minimum 3.0 between the peaks due to trichomes [Kb] or their scars [Be]; isolated conical warty
isorhamnetin diglucoside and hydroxyanthracene glycoside covering trichomes, usually bent [C]; fragments of the
(peak 2). mesocarp (surface view [D]) with fibres [Da] in 2 crossed
Calculate the percentage content of total hydroxyanthracene layers accompanied by a layer of cells containing calcium
glycosides (peaks 2-9), expressed as sennoside B, using the oxalate prisms [Db] and sometimes cells of the underlying
following expression: endocarp [De]; fragments of the outer layers of the testa
(transverse section W) covered by a thick cuticle Ua] with
palisade cells about 50 µm long, with thick walls and a
reduced lumen Ubl, accompanied by the hypodermis with
pillar-like cells Uc]; palisade cells of the testa (surface
A1 sum of the areas of the peaks due to hydroxyanthracene
view [N]) and fragments of the hypodermis of the seed
glycosides (peaks 2-9) in the chromatogram obtained with the
test solution; forming rings (surface view [A]); fragments of cotyledons
A2 area of the peak due to sennoside B in the chromatogram (surface view [F], transverse section [El) consisting of small
obtained with reference solution (b); cells of the epidermis [Ea, Fa] and of the palisade
m1 mass of the extract to be examined used to prepare the test tissue [Eb, Fb]; prisms and cluster crystals of calcium
solution, in grams;
m2 mass of sennoside B CRS used to prepare reference solution (b), oxalate, free [De, Ga] or included in parenchyma [G];
in grams; fragments of vascular bundles [L] with spiral vessels [La] and
p percentage content of sennoside B in sennoside B CRS. fibres with moderately thickened and pitted walls [Lb];
sclereids [M, OJ and fibres [HJ accompanied by crystal
LABELLING sheaths of calcium oxalate [Ha] from fruit stalks.
The label states the content of total hydroxyanthracene C. High-performance thin-layer chromatography (2.8.25).
glycosides. Solvent mixture ethanol (96 per cent) R, water R (50:50 VIV).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
(2. 9.12) add 5.0 mL of the solvent mixture. Sonicate for
10 min, then filter or centrifuge the solution. Use the filtrate
or supernatant.
Senna Pods Reference solutwn ( a) Dissolve 3 mg of sennoside A R and
Alexandrian Senna Fruit ethanol (96 per cent) R and a 1 g/L solution of sodium
(Ph. Bur. monograph 0207) hydrogen carbonate Rand dilute to 20.0 mL with the same
Preparations mixture of solvents.
Senna Liquid Extract Reference solution (b) Dilute 2.5 mL of reference solution (a)
Standardised Senna Granules to 10.0 mL with the solvent mixture.
Senna Tablets Reference solutwn (c) Dissolve 10 mg of senna extract HRS in
When Powdered Alexandrian Senna Fruit is prescribed or 1 mL of the solvent mixture (a slight residue may remain).
demanded, material complying with the requirements below, Intensity marker Sennoside A.
with the exception of Identification test A and the test for Plate TLC silica gel F 254 plate R (2-10 µm).
Foreign matter, shall be dispensed or supplied. Mobile phase water R, ethyl acetate R, propanol R
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ (30:40:40 V/V/V).
DEFINITION Application 1 µL of the test solution and 2 µL of reference
Dried fruit of Senna alexandrina Mill. (syn. Cassia acutifolia solutions (a), (b) and (c), as bands of 8 mm.
Delile and Cassia angustifolia Vah!). Development 70 mm from the lower edge of the plate.
2023 Senna Pods IV-493
Top of the plate
-- --
De
E
• .F
.
--F•
"Ea Fb A light yellow zone, very faint to
faint (sennoside C)
a

J (sennoside D)
fa G
Sennoside A: a light yellow zone A light yellow zone, equivalent to
intense (sennoside A)
-- --
Sennoside B: a brownish-yellow zone A brownish-yellow zone, intense

(sennoside B)
Figure 0207.-1. - Illustrati.onfor identificati.on test B of powdered

herbal drng of senna pods Reference solution (a) Test solution

TESTS
Total anthraquinones (aloe emodin and rhein)
with a 50 g/L solution of potassium hydroxide R in the solvent
Liquid chromatography (2.2.29). Cany out the test protected
mixture and heat at 110 °C for 10 min; examine immediately
from bright light.
in ultraviolet light at 366 nm.
Solvent mixture 1.0 giL solution of sodium hydrogen
carbonate R, methanol R (30:70 V/V).
- the chromatogram shows 2 distinct zones, which may
be touching, near the border between the lower and Test soluti.on Introduce 0.500 g of the powdered herbal drug
middle thirds; the lower zone (sennoside A) shows a (355) (2. 9.12) into a 250 mL screw-cap bottle and add
light yellow fluorescence and the upper zone 100.0 mL of the solvent mixture. Sonicate for 30 min and
(sennoside D) shows a faint brownish-yellow shake for 2 h. Filter through a membrane filter (nominal
fluorescence. pore size 0.45 µm).
Results See below the sequence of fluorescent zones present Reference soluti.on (a) Dissolve 10 mg of aloe emodin Rand
in the chromatograms obtained with reference solution (a) 10.0 mg of rhein CRS in tetrahydrojuran Rand dilute to
and the test solution. Furthermore, in the chromatogram 50.0 mL with the same solvent. Dilute 1.0 mL of the
obtained with the test solution, other blue and/or reddish solution to 20.0 mL with the solvent mixture.
fluorescent zones may be present, mainly above the zone due Reference soluti.on (b) Dissolve 10 mg of senna extract HRS in
to sennoside A. For sennosides, Rp values vary slightly and 8 mL of the solvent mixture using sonication for 5 min and
the zones may be curved depending on the concentration. dilute to 10 mL with the solvent mixture (a slight residue
may remain). Filter through a membrane filter (nominal pore
size 0.45 µm).
Reference soluti.on (c) Dissolve 5.0 mg of sennoside B CRS in
25 mL of methanol R using sonication and dilute to 50.0 mL
with water R.
Column:
- size: l = 0.25 m, 0 = 4.6 mm;
- stationary phase: end-capped propoxybenzene silica gel for
Mobile phase:
formic acid R;
Time Mobile phase A Mobile phase B Loss on drying (2.2.32)

(min) (per cent V/Jl) (per cent VIV) Maximum 12.0 per cent, determined on 1.000 g of the
0-3 87 13 powdered herbal drug (355) (2.9.12) by drying in an oven at
3 - 40 87 ➔ 37 13 ➔ 63 105 °C for 2 h.
Total ash (2.4.16)
Flow rate 1.0 mIJmin. Maximum 9.0 per cent.
Detection Spectrophotometer at 270 run. Ash insoluble in hydrochloric acid (2.8.1)
Injection I O µL of the test solution and reference Maximum 2.0 per cent.
solutions (a) and (b).
ASSAY
Identification of peaks Use the chromatogram supplied with Liquid chromatography (2.2.29) as described in the test for
senna extract HRS and the chromatogram obtained with total anthraquinones (aloe emodin and rhein) with the
reference solution (b) to identify the peaks due to following modification.
isorhamnetin diglucoside and hydroxyanthracene glycosides
the peak due to sennoside B (peak 3) is included in its area; Calculate the percentage content of total hydroxyanthracene
peaks 4 and 5 may be co-eluted; peaks 7 and 8 may be glycosides (peaks 2-9) (THG), expressed as sennoside B,
co-eluted; use the chromatogram obtained with reference using the following expression:
solution (a) to identify the peaks due to aloe emodin and
rhein.
retention time = about 14.2 min): isorhamnetin A, sum of the areas of the peaks due to hydroxyanthracene
diglucoside = about 0.93; hydroxyanthracene glycoside glycosides (peaks 2-9) in the chromatogram obtained with the
(peak 2) = about 0.98; hydroxyanthracene glycoside test solution;
(peak 4) = about 1.01; hydroxyanthracene glycoside area of the peak due to sennoside B in the chromatogram
(peak 5) = about 1.02; hydroxyanthracene glycoside mass of the herbal drug to be examined used to prepare the test
(peak 6) = about 1.07; hydroxyanthracene glycoside solution, in grams_;
(peak 7) = about 1.09; hydroxyanthracene glycoside mass of sennoside B CRS used to prepare reference solution (c),
(peak 8) = about 1.11; hydroxyanthracene glycoside in grams;
p percentage content of sennoside B in sennostde B CRS.
(peak 9) = about 1.13; aloe emodin = about 2.2;
rhein = about 2.3.
STORAGE
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
(peak 2).
Calculate the percentage content of total anthraquinones
(aloe emodin and rhein) (TA), expressed as rhein, using the
following expression: Senna Tablets
DEFINITION
Senna Tablets contain the powdered pericarp of Senna Fruit,
Alexandrian or Tinnevelly.
A, sum of the areas of the peaks due to aloe emodin and rhein in The tablets comply with the requirements stated under Tablets and
the chromatogram obtained with the test solution; with the fallowing requirements.
area of the peak due to rhein in the chromatogram obtained
with reference solution (a); Content of total sennosides, calculated as sennoside B
mass of the herbal drug to be examined used to prepare the test 85.0 to 115.0% of the stated amount.
solution, in grams;
m2 mass of rhein CRS used to prepare reference solution (a), in IDENTIFICATION
grams; The powdered tablets exhibit diagnostic structures of senna
p percentage content of rhein in rhein CRS. pericarp. External epidermis of isodiametric cells with very
thick outer walls. Occasional stomata. Trichomes few,
Calculate the percentage content of total anthraquinones with unicellular, conical and warty. Parenchymatous cells from
reference to the sum of total hydroxyanthracene glycosides inner part of a two-to five-layered zone subjacent to the
(THG, see Assay) and total anthraquinones, using the epidermis, each containing a single prism of calcium oxalate.
following expression: Thick-walled fibres in two to four layers, the fibres of the
outer and inner zones respectively with their long axes at
TA
right angles to each other. Sutural vascular strands sheathed
THG+ TA x lOO
by cells containing prisms of calcium oxalate; elements of
seed tissue may also be present.
Limit.
- total anthraq_uinones (aloe emodin and rhein) expressed as TESTS
rhein (C 15H 8 O 6; M, 284.2): maximum 7.0 per cent, Disintegration
calculated with reference to the sum of total Maximum time, 60 minutes, Appendix XII A.
hydroxyanthracene glycosides and total anthraquinones ASSAY
(dried drug). Weigh and powder 20 tablets. Carry out the following
Foreign matter (2.8.2) procedure protected from light. To a quantity of the powder
Maximum 1 per cent. containing the equivalent of 7 .5 mg of total sennosides add
2023 Serratula Herb IV-495
30 mL of water, weigh, heat under a reflux condenser on a The powder shows the following diagnostic characters
water bath for 15 minutes, allow to cool, weigh and restore (Figure 2754.-1): fragments of the upper epidermis of the
the original weight with water. Centrifuge, transfer 20 mL of leaf (surface view [D]), consisting of polygonal cells with
the supernatant liquid to a separating funnel, add 0.1 mL of slightly sinuous anticlinal walls [Da] and anomocytic stomata
2M hydrochloric acid, shake with two 15 mL quantities of (2.8.3) with 3-5 subsidiary cells [Db], usually accompanied
chloroform, allow to separate and discard the chloroform by palisade parenchyma [De]; fragments of the lower
layers. Add 0.10 g of sodium hydrogen carbonate and shake for epidermis of the leaf (surface view [Cl), composed of
3 minutes; centrifuge and transfer 10 mL of the liquid to a irregularly shaped cells [Ca] and anomocytic stomata with
round-bottomed flask fitted with a ground-glass neck. Add a 3-5 subsidiary cells [Cb] often accompanied by spongy
mixture of 8 mL of iron(m) chloride solution and 12 rnL of parenchyma cells [Cc]; fragments of the margin of the leaf
water and mix. Heat under a reflux condenser on a water lamina showing the remains of the bases of numerous
bath for 20 minutes, add 1 mL of hydrochloric acid and uniseriate covering trichomes [B, HJ composed of 3-5 cells
continue heating for a further 20 minutes, shaking frequently, with thickened walls and a striated cuticle [Ba], occasionally
until the precipitate is dissolved. Allow to cool, transfer the bearing very thin-walled cells [Bb); fragments of the abaxial
mixture to a separating funnel and extract with three 25-mL epidermis of the bracts of the involucre [A, G] with slightly
quantities of ether previously used to rinse the flask. Wash the thickened, elongated cells [Aa] bearing short, unicellular,
combined ether extracts with two 15-mL quantities of water lignified covering trichomes pointing in the same direction,
and add sufficient ether to produce 100 mL. Evaporate fusiform in surface view [Ab] and spiny in transverse
10 mL just to dryness on a water-bath and dissolve the section [Ga], and rare biseriate glandular trichomes [Ac];
residue in 10 mL of lM potassium hydroxide, filtering if fragments of the bristles of the calyx ill; fragments of the
necessary through a sintered-glass filter (ISO 4 793, porosity style [E] composed of fine irregular cells [Ea], 1 or 2
grade 3, is suitable). Measure the absorbance of the resulting secretory canals with orange contents [Eb) lying parallel to
solution without delay at the maximum at 500 nm, the annular vessels [Ee]; pollen grains, 60-70 µm in diameter,
Appendix II B. Calculate the content of total sennosides, as with 3 pores and a thick, spiny exine [F); fragments of the
sennoside B, taking 200 as the value of A(l %, 1 cm) at the ovary with long styloid calcium oxalate crystals and fragments
maximum at 500 nm. of the corolla containing small prismatic crystals of calcium
oxalate may also be present.
LABELLING
The quantity of active ingredient is stated in terms of total
sennosides expressed as the equivalent content of
sennoside B.
Serratula Herb
(Serratula Coronata Herb, Ph. Bur. monograph 2754)
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried, whole or fragmented aerial part of Serratula
coronata L. collected during flowering.
Content
Minimum 5.0 per cent of ~-ecdysterone (C 27 H 44 O 7 ;
IDENfIFICATION
A. The stem is sulcate, erect, glabrous, usually terminally
branched, rarely unbranched. Basal leaves are glabrescent,
varying from pinnately lobed, or sometimes irregularly
pinnatifid, to pinnatisect or pinnate; segments are elliptical to
lanceolate, with hairy, irregularly serrate or dentate margins;
main veins are sunken on the upper surface and prominent
on the lower surface. Cauline leaves are similar to the basal
leaves, but are smaller towards the top of the stem.
The capitula, 15-25 mm in diameter, are campanulate and
arranged as a loose, irregular panicle; they consist of an
involucre, a receptacle with paleae up to 10 mm in length
and numerous florets. Involucral bracts are yellowish-green Figure 2754.-1. - Illustration for identification test B of powdered
tinged with reddish-brown to violet, rigid, acute and herbal drug of Serratula coronata herb
distinctly velutinous. Inner bracts are more elongated,
sometimes hooked at the apex. Florets are pinkish-violet, C. High-performance thin-layer chromatography (2.8.25).
tubular and 20---28 mm in length. Achenes, if present, are Test solution To 0.25 g of the powdered herbal drug (355)
oblanceolate-ellipsoid and glabrous, sometimes still bearing a (2. 9.12) add 5.0 mL of methanol R. Sonicate for 10 min,
pappus of multiseriate, acute, unbranched hairs. centrifuge and use the supernatant.
B. Microscopic examination (2.8.23). The powder is green.
IV-496 Serratula Herb 2023
Reference solution (a) Dissolve 1.5 mg of rutoside trihydrate R TESTS

and 2.5 mg of isoquercitroside R in methanol R and dilute to Loss on drying (2.2.32)
10.0 mL with the same solvent. Maximum 10.0 per cent, determined on 1.000 g of the
Reference solution (b) Dilute 2.5 mL of reference solution (a) powdered herbal drug (355) (2.9.12) by drying in an oven at
to 10.0 mL with methanol R. 105 °C for 2 h.
Reference solution (c) Dissolve 1.5 mg of chlorogenu: acid R Total ash (2.4.16)
and 1.5 mg of hyperoside R in methanol R and dilute to Maximum 12.0 per cent.
ASSAY
Intensity marker Isoquercitroside.
Test solution Place 4.0 g of the powdered herbal drug (355)
Mobile phase anhydrous formic acid R, water R, ethyl acetate R (2. 9.12) in the cartridge of a continuous extraction apparatus
(10:10:80 VIVIV). (Soxhlet type). Place 150 mL of methanol Rand a magnetic
Applu:ation 2 µL as bands of 8 mm. stirrer in a 500 mL round-bottomed flask and heat under
Development 70 mm from the lower edge of the plate. reflux for 8 h. Transfer the resulting extract to a volumetric
Drying In a current of air at room temperature for 5 min. flask and dilute to 200.0 mL with methanol R.
Detection Heat at 100-105 °C for 3 min; spray the warm Reference solution (a) Dissolve 10.0 mg of /3-ecdysterone CRS
plate with a 10 g/L solution of diphenylboru: acid aminoethyl in methanol R using sonication and dilute to 50.0 mL with
ester R in methanol R, then with a 50 g/L solution of macrogol the same solvent.
400 R in methanol R or, alternatively, dip the warm plate in a Reference solution (b) Dissolve 2 mg of makisterone A R in
5 g/L solution of diphenylboru: acid aminoethyl ester R in ethyl reference solution (a) using sonication and dilute to 10 mL
acetate R, then in a 50 g/L solution of macrogol 400 R in with reference solution (a).
methylene chloride R; allow the plate to dry in air for about Column:
1 min and examine in ultraviolet light at 366 nm. =
- size: l 0.25 m, 0 4.0 mm; =
System suitability Reference solution (c): - stationary phase: end-capped octadecylsilyl dial silica gel for
- the chromatogram shows in the middle third 2 distinct chromatography compatible with highly aqueous mobile
zones, which may be touching; the lower zone phases R (5 µm);
(chlorogenic acid) shows a light blue fluorescence and - temperature: 25 °C.
the upper zone (hyperoside) shows a yellow or orange Mobile phase:
fluorescence. - mobile phase A: 0.125 per cent V/V solution of anhydrous
Results See below the sequence of fluorescent zones present formic acid R;
in the chromatograms obtained with reference solution (a) - mobile phase B: acetonitrile for chromatography R;
obtained with the test solution, other faint to very faint (min) (per cent V/V) (per cent V/V)
fluorescent zones may be present. 0-2 85 15
2 - 17 85--+ 72.5 15 --+ 27.5
Top of the plate
17 - 23 72.5 --+ 10 27.5 --+ 90
A red zone, very faint to equivalent
A yellow zone, very faint to intense
Injection 20 µL.
- - -- Identification of peaks Use the chromatogram obtained with
reference solution (a) to identify the peak due to
~-ecdysterone; use the chromatogram obtained with reference
solution (b) to identify the peak due to makisterone A.
2 greenish-blue zones, very faint to Retention time ~-ecdysterone = about 11 min.
equivalent (possibly overlapping with the System suitability Reference solution (b):
yellow or orange zone below)
Isoquercitroside: a yellow or A yellow or orange zone, very faint to ~-ecdysterone and makisterone A.
orange zone faint
Calculate the percentage content of ~-ecdysterone using the
A greenish-blue zone, equivalent following expression:
A bluish zone, very faint to faint
A1 area of the peak due to P-ecdysterone in the chromatogram

- ~ - - A2 area of the peak due to P-ecdysterone in the chromatogram
Rutoside: a yellow or orange A yellow or orange zone, very faint to solution, in grams;
zone faint m2 mass of P-ecdysrerone CRS used to prepare reference
p assigned percentage content of P-ecdysterone in
P-ecdysterone CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
2023 Sophora Flavescens Root IV-497
CONFIRMATION
Sesame Seed The chromatogram obtained with solution (1) shows 4 grey
DEFINITION 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 Loss on drying
from 1.5 to 4 mm long, 1 to 2 mm wide and 0.5 to 1 mm 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 Total Ash
India are predominantly yellow, dark brown or black; and Not more than 9%, Appendix XI J, Method II.
surface texture ranges from smooth to finely reticulate or
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, Sophora Flavescens Root
and a longitudinal line may be visible on one of the flat
(Lightyellow Sophora Root, Ph. Bur. monograph
surfaces, notably in those cultivars with a pale coloured testa.
2440)
Inside the thin testa, a narrow whitish endosperm and an
embryo comprised of a two large, yellowish and oily PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
cotyledons are present. DEFINITION

B. Reduce to a powder. The powder varies in colour from Whole or fragmented, dried root of Sophora flavescens Aiton,
yellow to brown or blackish. Epidermis of the testa, a single with root crown and rootlets removed, collected in spring or
layer of palisade-like cells thin-walled, with yellowish autumn.
coloured contents and an embedded cluster crystal of
Content
calcium oxalate in each cell, except in those of the ridges
Minimum 1.2 per cent for the sum ofmatrine (C 15 H24N2O;
which have no contents; the remainder of the testa is
Mr 248.4) and oxymatrine (C1sH24N2O2; Mr 264.4) (dried
composed of collapsed cells. Endosperm 3 layered, rarely 2
drug).
layered, consisting of polygonal cells containing fixed oils and
small aleurone grains. Epidermis of cotyledons, a single layer IDENTIFICATION
covered with a thin cuticle, with an underlying single layer of A. The root is cylindrical, usually branched in the lower part,
palisade- like cells; endosperm tissue composed of polygonal, 10-30 cm long, 1-6.5 cm in diameter, externally greyish-
parenchyma cells containing fixed oil and aleurone grains. brown or brownish-yellow, exhibiting longitudinal wrinkles
Cluster crystals of calcium oxalate and fragments may be and transverse elongated lenticel-like protuberances.
found scattered in the powder. The outer bark is thin, mostly broken and recurved, easily
C. Carry out the test for the identification of fatty oils by exfoliated, with the exposed surface appearing yellow and
thin-layer chromatography, Appendix X N, using the smooth. The texture is hard and difficult to break.
following solutions. The fracture is fibrous. Slices are 3-6 mm thick; the
transversely cut surface is yellowish-white with radial lines
(1) Add 100 mL of dichloromethane to 10.0 g of the
and cracks, sometimes exhibiting abnormal vascular bundles
powdered herbal drug. Shake for 24 hours on a mechanical
arranged in concentric rings or scattered irregularly.
shaker, filter and evaporate to dryness at 40°. Dissolve 60 mg
of the oily residue in 9 mL of dichloromethane. B. Microscopic examination (2.8.23). The powder is pale
brownish-yellow. Examine under a microscope using chloral
(2) 0.67% w/v of sesame oil in dichloromethane.
CHROMATOGRAPHIC CONDITIONS characters (Figure 2440.-1): very numerous unlignified fibres,
(a) Use as the coating octadecylsilyl silica gel for HPTLC single or in groups [C, G], usually surrounded by crystal
(Merck silica gel 60 RP-18 HPTLC plates are suitable). sheaths of calcium oxalate [Ca]; some fibres accompanied by
(b) Use mobile phase A and mobile B described below. medullary rays with cells having slightly thickened and pitted
(c) Apply lµL of each solution as 6 mm bands. walls [Ga]; xylem vessels, pitted or reticulate [K, L],
50-200 µm in diameter; calcium oxalate prisms, 10-25 µm in
(d) Develop the plate twice to 0.5 cm in mobile phase A, diameter, either isolated [BJ or inside parenchymatous
drying the plate after each application. Develop the plate a cells [Da], sometimes several prisms in the same cell, or
further two times to 8 cm in mobile phase B, drying the plate forming crystal sheaths around the fibres [Ca]; numerous
after each application. fragments of parenchyma with rounded to ovoid, slightly
(e) After removal of the plate, dry in air and spray with a pitted cells [DJ; fragments of cork (surface view [Al)
10% w/v solution of ethanolic phosphomolybdic acid solution, consisting of polygonal cells, finely reticulate inside; rare
heat at 120° for 3 minutes and examine in daylight. sclereids, either in groups [F] or, more often, isolated [E].
MOBILE PHASE Examine under a microscope using a 50 per cent V/V
Mobile phase A ether solution of glycerol R. The powder shows very abundant
Mobile phase B 20 volumes of dichloromethane, 40 volumes
starch granules, either included in parenchyma cells m or
isolated [HJ; some are simple, most are 2-8 compound of
of glacial acetic acid and 50 volumes of acetone.
variable size; the hilum is usually visible.
SYSTEM SUITABILITY
solution (2) shows 4 clearly separated bands.
IV-498 Sophora Flavescens Root 2023
e©s Top of the plate
A faint brown or orange-red zone
A brown or orange-red zone
- -
--
An orange-red zone (brucine)
A brown or orange-red zone

.. (quinine)
E
-- --
F ·. : 't; 2 or 3 prominent brown or orange-

red zones
~-- .· __
TESTS
105 °C.
Total ash (2.4.16)
Figure 2440. -1. - Illustration for identification test B of powdered
ASSAY
herbal drug of lightyellow sophora root
C. Thin-layer chromatography (2.2.27). Test solution To 0.300 g of the powdered herbal drug (355)
Test solution To 1.0 g of the powdered herbal drug (355) (2.9.12) add 0.5 mL of concentrated ammonia Rand 25.0 mL
(2. 9.12) add 20 mL of water R. Heat on a water-bath under of methylene chloride R, weigh and sonicate for 30 min. Allow
a reflux condenser at 90 °C for 30 min. Allow to cool and to cool, weigh and adjust to the original mass with methylene
centrifuge for 5 min. Dilute 5 mL of the supernatant to chloride R, shake well and filter. Transfer 5.0 mL of the
10 mL with methanol R. A white precipitate is formed. filtrate to a chromatographic column about 0.2 m long and
Centrifuge for 5 min and use the clear, slightly yellow 10 mm in internal diameter packed with 5 g of neutral
supernatant. aluminium oxide R (60-210 µm). Elute with 20 mL of a
Reference solution Dissolve 1 mg of brucine R and 1 mg of
methylene chloride R. Evaporate the eluate to dryness. Dissolve
quinine R in 5 mL of ethanol (96 per cent) R.
the residue in ethanol (96 per cent) Rand dilute to 10.0 mL
Plate TLC silica gel F254 plate R (2-10 µm). with the same solvent.
Mobile phase concentrated ammonia R, methanol R, methylene Reference solution Dissolve 5.0 mg of matrine CRS and
chloride R (5:10:85 VIVIV). 5.0 mg of oxymatrine CRS in a mixture of 2 volumes of
Application 5 µL as bands of 8 mm. anhydrous ethanol R and 8 volumes of acetonitrile R, and dilute
Development Over a path of 6 cm. to 5.0 mL with the same mixture of solvents. Dilute 1.0 mL
Drying In air. of the solution to 20.0 mL with a mixture of 2 volumes of
anhydrous ethanol R and 8 volumes of acetonitrile R.
Detection Treat with potassium iodobismuthate solutwn R and
then with dilute hydrogen peroxide solution R until the orange Column:
or brown zones become visible against a yellow background; - size: l = 0.125 m, 0 = 4.0 mm;
examine in daylight. - stationary phase: aminopropylsilyl silica gel for
chromatograms obtained with the reference solution and the Mobile phase 1 per cent V/V solution of phosphoric acid R in
test solution. Furthermore, other faint zones may be present ethanol (96 per cent) R, acetonitrile for chromatography R
in the chromatogram obtained with the test solution. (20:80 VIV).
Injection 20 µL.
Retention time Matrine = about 14 min; oxymatrine = about
24 min.
2023 Sophora Flower IV-499
System suitability Reference solution: cells containing crystalline masses of rutoside [He]; fragments
- resolution: minimum 5.0 between the peaks due to matrine of parenchyma [E] from the sepals containing prisms of
and oxymatrine. calcium oxalate [Ea] and crystalline masses of rutoside [Eb ];
Calculate the percentage content of the sum of matrine and fragments of anthers [N] showing the characteristic fibrous
oxymatrine using the following expression: layer (transverse section [Na], surface view [Kl) and
immature pollen grains [Nb]; free prisms of calcium
A1 x m2 xp1 x 0.50 A3 x m3 xpz x 0.50 oxalate [M]. Examine under a microscope using chloral
-------+------- hydrate solution R, without heating the preparation: brownish-
A2 x m1 A4 x m1
yellow rutoside crystals are visible, free or included in cells,
A1 area of the peak due to matrine in the chromatogram obtained as crystalline masses [Eb, He, TI or in fan-shaped aggregates
of very fine needles [D, Ee, Gb].
A2 area of the peak due to matrine in the chromatogram obtained
area of the peak due to oxymatrine in the chromatogram
area of the peak due to oxymatrine in the chromatogram
solution) in grams;
m2 mass of mam·ne CRS used to prepare the reference solution, in
grams;
m3 mass of oxymatrine CRS used to prepare the reference solution,
in grams;
Pi percentage content of matrine in matrine CRS;
Pz percentage content of oxymarrine in oxymatrine CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Sophora Flower
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried, opened flower of Styphnolobium japonicum (L.) Schott
(syn. Sophora japonica L.).
Content
- minimum 8.0 per cent of total flavonoids, expressed as
rutoside (C27H30O16; Mr 611) (dried drug);
- minimum 6.0 per cent ofrutoside (C 27 H 30O1 6; Mr 611)
(dried drug).
IDENTIFICATION
A. The opened flower is crumpled, rolled, and has a very Figure 2639 .-1. - Illustratwn for identificatwn test B of powdered
thin and short pedicel. The dark green or brown, herbal drug of sophora flower
campanulate calyx is about 3-4 mm long and consists of
5 fused sepals with longitudinal striations at the base, divided C. Thin-layer chromatography (2.2.27).
at the apex into 5 slightly bilabiate lobes. The pale yellow or Test solutwn To 1 g of the powdered herbal drug (355)
light yellowish-brown, papilionaceous type corolla is often (2.9.12) add 5.0 mL of methanol R, sonicate for 10 min and
broken and measures about 10-15 mm; the upper petal is the filter.
largest, subrounded, with a reflexed apex and a bright yellow
Reference solutwn Dissolve 10 mg of hyperoside R and 10 mg
unguis at its internal base. The other 4 petals are oblong.
of rutoside trihydrate R in 10 mL of methanol R.
There are 10 free stamens surrounding a cylindrical and
curved central style. Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
yellowish-green. Examine under a microscope using chloral Mobile phase anhydrous formic acid R, water R, ethyl acetate R
hydrate solutwn R. The powder shows the following diagnostic (10: 10:80 VIV/V).
characters (Figure 2639.-1): roundish [La] or triangular [Lb] Applicatwn 10 µL [or 5 µL] as bands of 10 mm [or 8 mm].
pollen grains [L] with 3 pores and a smooth exine, about Development Over a path of 10 cm [or 6 cm].
18 µm in diameter; isolated covering trichomes [A, B, F, OJ Drying In air.
of varying lengths (60-660 µm), slightly flexed, usually
consisting of 1 or 2 basal cells and a long pointed distal cell,
aminoethyl ester R in methanol Rand then with a 50 g/L
with smooth or slightly warty walls; fragments of sepals [C]
solution of macrogol 400 R in methanol R, allow to dry in air
composed of anomocytic stomata (2.8.3) with 4-8 subsidiary
for about 30 min, and examine in ultraviolet light at 365 nm.
cells [Ca], covering trichomes [Cb] or their scars [Cc];
fragments of petals [G, HJ with cells covered by a finely Results See below the sequence of fluorescent zones present
striated cuticle [Ga, Ha], sometimes accompanied by fine in the chromatograms obtained with the reference solution
annular or spiral vessels [Hb] and parenchyma with some and the test solution. Furthermore, other faint fluorescent
IV-500 Sophora Flower 2023
zones may be present in the chromatogram obtained with the Test solution Place 0.500 g of the powdered herbal drug
test solution. (355) (2.9.12) in a conical flask and add 50.0 mL of
methanol R. Weigh, sonicate for 30 min and allow to cool.
Top of the plate Weigh and compensate for the loss of solvent with
methanol R. Shake vigorously, filter, and dilute 2.0 mL of the
An orange-yellow zone
filtrate to 10.0 mL with methanol R.
-- -- Reference solution (a) Dissolve 10.0 mg of rutoside
trihydrate CRS in 2 mL of methanol Rand dilute to 10.0 mL
A brown zone
with a 50 per cent V/V solution of methanol R. Dilute 2.0 mL
Hyperoside: a yellowish-orange zone of this solution to 10.0 mL with a 50 per cent VIV solution
of methanol R.
-- --
Reference solution (b) Dissolve 10.0 mg of apigenin
2 green zones
7-glucoside R and 10.0 mg of rutoside trihydrate R in 30 mL of
Rutoside: an orange-yellow zone A very intense orange-yellow zone methanol R and dilute to 50.0 mL with a 50 per cent V/V
(rutoside) solution of methanol R.
Reference solution Test solution Column:
- size: l = 0.25 m, 0 = 4.6 mm;
TESTS (5 µm).
Foreign matter (2.8.2) Mobile phase:
Maximum 5 per cent of flower buds and maximum - mobile phase A: 1 per cent V/V solution of glacial acetic
2 per cent of other foreign matter. acid R;
Loss on drying (2.2.32) - mobile phase B: methanol R;
powdered herbal drug (355) (2.9. 12) by drying in an oven at Time Mobile phase A Mobile phase B
105 °C for 2 h. (min) (per cent V/V) (per cent V/V)
0-5 68 32
Total ash (2.4. 16)
5 - 20 68 ➔ 50 32 ➔ 50
20 - 30 50 ➔ 0 50 ➔ 100
ASSAY 30 - 35 0 100
Total flavonoids
Stock solution Place 2.000 g of the powdered herbal Flow rate 1.3 mL/min.
drug (355) (2. 9.12) in the cartridge of a continuous-
extraction apparatus (Soxhlet type). Add 100 mL of Detection Spectrophotometer at 350 nm.
heptane R and heat under a reflux condenser until the Injection 20 µL.
extraction liquid is colourless. Allow to cool and discard the Relative retention With reference to rutoside (retention
heptane. Add 90 mL of methanol R and continue the time= about 17 min): apigenin 7-glucoside = about 1.1.
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
methanolic solution to a 100 mL volumetric flask. Rinse the rutoside and apigenin 7-glucoside.
extraction flask with a few millilitres of methanol R. Combine Calculate the percentage content of rutoside using the
the methanolic solutions and dilute to 100.0 mL with following expression:
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
100.0 mL with a 20 g/L solution of aluminium chloride R in
methanol R. area of the peak due to rutoside in the chromatogram obtained
Compensation solution Dilute 10.0 mL of the stock solution area of the peak due to rutoside in the chromatogram obtained
to 100.0 mL with methanol R. with reference solution (a);
Measure the absorbance (2. 2. 25) of the test solution after mass of the herbal drug to be examined used to prepare the test
solution, in grams;
15 min by comparison with the compensation solution at mass of rutoside tn"hydrate CRS used to prepare reference
425 nm. solution (a), in grams;
Calculate the percentage content of total flavonoids, p assigned percentage content of rutoside in rutoside
trihydrate CRS.
expressed as rutoside, using the following expression:
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Ax 1000
mx37
i.e. taking the specific absorbance of rutoside to be 370.

Rutoside
2023 Sophora Flower-Bud IV-501
Sophora Flower-Bud
(Ph. Bur. mcmograph 2427) ~~/
A
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION D
Whole, dried flower bud of Styphnolobium japonicum (L.)
Schott (syn. Sophora japonica L.).
Content
- minimum 20. 0 per cent of total flavonoids, expressed as
rutoside (C 27 H 30 O 16; M, 611) (dried drug);
- minimum 15.0 per cent ofrutoside (CnH 30 O 16; M, 611)
(dried drug).
IDENTIFICATION
A. The flat flower bud, ovoid or ellipsoid, has a very thin and
short pedicel and is about 7-10 mm long and 3-4 mm thick.
The dark green or brown calyx, forming the lower part of the
bud, is about 3-4 mm long and consists of 5 fused sepals
with longitudinal striations at the base. The pale yellow or
brownish-yellow corolla, unopened, delicate, extends beyond
the calyx and contains 10 free stamens surrounding a central
style.
yellow. Examine under a microscope using chloral hydrate
characters (Figure 2427.-1): roundish [La] or triangular [Lb]
pollen grains [L] with 3 pores and a smooth exine, about
18 µm in diameter; isolated covering trichomes [A, B, F, OJ
of varying lengths (60-660 µm), slightly flexed, usually
Figure 2427 .-1. - Illustratwn for identification test B of powdered
consisting of 1 or 2 basal cells and a long pointed distal cell,
herbal drug of sophora flower-bud
with smooth or slightly warty walls; fragments of sepals [C]
composed of anomocytic stomata (2.8.3) with 4-8 subsidiary Detection Treat with a 10 g/L solution of diphenylboric acid
cells [Ca], covering trichomes [Cb] or their scars [Cc]; aminoethyl ester R in methanol R and then with a 50 g/L
fragments of petals [G, HJ with cells covered by a finely solution of macrogol 400 R in methanol R, allow to dry in air
striated cuticle [Ga, Ha], sometimes accompanied by fine for about 30 min, and examine in ultraviolet light at 365 nm.
annular or spiral vessels [Hb] and parenchyma with some
cells containing crystalline masses of rutoside [He]; fragments
of parenchyma [E] from the sepals containing prisms of
calcium oxalate [Ea] and crystalline masses of rutoside [Eb];
fragments of anthers [N] showing the characteristic fibrous
test solution.
layer (transverse section [Na], surface view [Kl) and
immature pollen grains [Nb]; free prisms of calcium
Top of the plate
oxalate [M]. Examine under a microscope using chloral
hydrate solution R, without heating the preparation: brownish- An orange-yellow zone
yellow rutoside crystals are visible, free or included in cells,
as crystalline masses [Eb, He, TI or in fan-shaped aggregates -- --
of very fine needles [D, Ee, Gb].
A brown zone
Hyperoside: a yello¼i.sh-orange zone
(2.9.12) add 5.0 mL of methanol R, sonicate for 10 min and -- --
filter. 2 green zones
Reference solutwn Dissolve 10 mg of hyperoside R and 10 mg Rutoside: an orange-yellow zone A very intense orange-yellow zone
of rutoside trihydrate R in 10 mL of methanol R. (rutoside)
(10:10:80 V/V/V). TESTS
Applicatwn 10 µL [or 5 µL] as bands of 10 mm [or 8 mm]. Foreign matter (2.8.2)
Development Over a path of 10 cm [or 6 cm]. Maximum 5 per cent of opened flowers and maximum
Drying In air.
powdered herbal drug (35 5) (2. 9.12) by drying in an oven at
105 °C for 2 h.
IV-502 Spearmint Oil 2023
Total ash (2.4.16) Time Mobile phase A Mobile phase B

Maximum 9.0 per cent. (min) (per cent V/V) (per cent VIJ')
0-5 68 32
ASSAY 5 - 20 68 ➔ 50 32 ➔ 50
Total flavonoids 20 - 30 50 ➔ 0 50 ➔ 100
Stock solution Place 1.00 g of the powdered herbal drug 30 - 35 0 100
(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 Flow rate 1.3 mUmin.
colourless. Allow to cool and discard the heptane. Detection Spectrophotometer at 350 nm.
Add 90 mL of methanol R and continue the extraction with Injection 20 µL.
heating under a reflux condenser until the extraction liquid is Relative retention With reference to rutoside (retention
colourless. Allow to cool. Transfer the methanolic solution to time = about 17 min): apigenin 7-glucoside = about 1.1.
a 100 mL volumetric flask. Rinse the extraction flask with a System suitability Reference solution (b):
few millilitres of methanol R. Combine the methanolic - resolution: minimum 1.5 between the peaks due to
solutions and dilute to 100.0 mL with methanol R. Dilute rutoside and apigenin 7-glucoside.
10.0 mL of this solution to 100.0 mL with water Rand shake
vigorously. Calculate the percentage content of rutoside using the
Test solution Dilute 10.0 mL of the stock solution to
100.0 mL with a 20 g/L solution of aluminium chloride R in
methanol R.
Compensation solution Dilute 10.0 mL of the stock solution
to 100.0 mL with methanol R. area of the peak due to rutoside in the chromatogram obtained
Measure the absorbance (2. 2. 25) of the test solution after area of the peak due to rutoside in the chromatogram obtained
15 min by comparison with the compensation solution at with reference solution (a);
425 nm. mass of the herbal drug to be examined used to prepare the test
solution, in grams;
Calculate the percentage content of total flavonoids, mass of rutoside trihydratc CRS used to prepare reference
expressed as rutoside, using the following expression: solution (a), in grams;
p assigned percentage content of rutoside in rutoside
Ax 1000 trihydrate CRS.
mx37
i.e. taking the specific absorbance of rutoside to be 370.

Spearmint Oil
Rutoside DEFINITION
Liquid chromatography (2.2.29). Spearmint Oil is obtained by distillation from fresh flowering
Test solution Place 0.200 g of the powdered herbal drug plants of Mentha spicata L. and Mentha x cardiaca (Gray)
(355) (2.9.12) in a conical flask and add 50.0 mL of Bak.
methanol R. Weigh, sonicate for 30 min and allow to cool. CHARACTERISTICS
Weigh and compensate for the loss of solvent with A clear, colourless, pale yellow or greenish yellow liquid
methanol R. Shake vigorously, filter, and dilute 2.0 mL of the when recently distilled, but becoming darker and viscous on
filtrate to 10.0 mL with methanol R. keeping; visibly free from water; odour, that of spearmint.
Reference solution ( a) Dissolve 10. 0 mg of rutoside
IDENTIFICATION
trihydrate CRS in 2 mL of methanol Rand dilute to 10.0 mL
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 rutoside trihydrate R in 30 mL of
methanol Rand dilute to 50.0 mL with a 50 per cent V/V TESTS
solution of methanol R. Optical rotation
American-type oil, --45° to -60°; Chinese-type oil, -50° to -
Column:
- size: l = 0.25 m, 0 = 4.6 mm; 62°; Appendix V F.
- stationary phase: octadecylsilyl silica gel for chromatography R Refractive index
(5 µm). 1.484 to 1.491, Appendix VE.
Mobile phase: Solubility in ethanol
- mobile phase A: 1 per cent 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 O. 9 34 g; Chinese-type oil,
2023 Squill IV-503
Chromatographic profile IDENTIFICATION

Carry out the method for gas chromatography, A. Transverse slices, about 5 to 8 mm thick, occurring as
Appendix III B, using the following solutions. Solution (1) is straight or curved triangular pieces about 5 to 50 mm long
the substance being examined. For solution (2) mix carefully and 3 to 8 mm wide at mid-point, tapering towards each
0.1 g of limonene, 0.2 g of cineole, 0.4 g of menthone, 0.1 g of end, yellowish white in colour, texture horny, somewhat
(+ )-isomenthone, 0.4 g of menthyl acetate, 0.2 g of pulegone, translucent, breaking with an almost glassy fracture when
0.6 g of menthol and 0.1 g of carvone with 1 g of hexane. quite dry, but readily absorbing moisture when exposed to
The chromatographic procedure may be carried out using the air and becoming tough and flexible; transversely cut
(a) a glass capillary column (25 m to 60 m x about surface showing a single row of prominent, vascular bundles
0.25 mm) coated with polyethylene glycol 20,000 as bonded near the concave edge and numerous smaller bundles
phase (Carbowax 20M is suitable) and (b) helium as the scanered throughout the mesophyll.
carrier gas at a flow rate of 1.5 mL per minute. Maintain the B. Epidermis: cells polygonal and axially elongated, 1 to
temperature of the column at 55° for 6 minutes then increase 2 times longer than wide, cuticle thick, stratified; stomata
it at the rate of 4° per minute to 180°; keep the injection port very rare, anomocytic, Appendix XI H, and nearly circular in
temperature at 220° and the detector at 230°. outline, about 50 to 60 µm in diameter; mesophyll of
Inject 0.1 µL of solution (2). When the chromatograms are colourless, thin-walled parenchyma containing very
recorded in the prescribed conditions, the components elute occasional starch granules, many cells containing bundles of
in the order indicated in the composition of the reference acicular crystals of calcium oxalate embedded in mucilage,
solution. Record the retention times of these substances. crystals up to about I mm long and about 1 to 15 µm wide;
The test is not valid unless the number of theoretical plates other cells containing sinistrin; vascular bundles collateral,
calculated from the limonene peak is at least 30,000 and the scanered throughout the mesophyll; xylem vessels with spiral
resolution factor between the peaks corresponding to limonene and annular wall thickening; trichomes absent.
and cineole is at least 1.5. C. The mucilage contained in the cells of the mesophyll is
Inject O.1 µL of solution ( 1). Using the retention times stained red with alkaline coral/in solution but produces no red
determined from the chromatogram obtained with solution colour with ruthenium red solution and no purple colour with
(2) locate the components of the reference solution on the 0.01M iodine.
chromatogram obtained with solution (1) (disregard the peak TESTS
due to hexane). Determine the percentage content of the Acid-insoluble ash
components by normalisation. The percentages are within the Not more than 1.5%, Appendix XI K, Method I.
following ranges:
Extractive soluble in ethanol (60%)
Limonene 2.0 to 25.0%. Not less than 68.0%, Appendix XI BI. Use material that has
Cineole less than 2.5%. been dried for 2 hours at 105° and powdered.
Menthone less than 2.5%.
Loss on drying
Isomenthone less than 1.0%. When dried at 100° to 105° for 2 hours, loses not more than
Menthyl acetate less than 1.0%. 14. 0% of its weight. Use 1 g.
Pulegone less than 0.5%.
STORAGE
Menthol less than 2.0%.
Squill should be stored in a dry place.
Carvone Not less than 55.0%.
STORAGE
Spearmint Oil should be kept in a well-filled container and
protected from light. Indian Squill
LABELLING Preparations
The label states whether the oil is American-type oil or Squill Oxymel
Chinese-type oil. When Powdered Indian Squill is prescribed or demanded,
material complying with the appropriate requirements below
shall be dispensed or supplied.
DEFINITION
Preparations Indian Squill consists of the bulb of Dn.mia indica (Roxb.) J
Squill Liquid Extract P Jessop, collected soon after the plant has flowered, divested
Squill Oxymel of dry, outer membranous coats and usually cut
longitudinally into slices and dried.
Opiate Squill Linctus
Indian Squill CHARACTERISTICS
Odourless or almost odourless.
Paediatric Opiate Squill Linctus
When Powdered Squill is prescribed or demanded, material Macroscopical
complying with the appropriate requirements below shall be Curved or irregularly shaped strips, about 10 to 50 mm long,
dispensed or supplied. 3 to 10 mm wide and 1 to 3 mm thick, frequently tapering
towards the ends, occasionally grouped three or four together
DEFINITION and anached to a portion of the axis; ridged in the direction
Squill consists of the bulb of Drimia maritima (L.) Steam, of their length and varying in colour from pale yellowish
collected when the foliage and leaves are absent, divested of brown to buff; brinle when dry, but tough and flexible when
its dry, outer, membranous coats, cut into transverse slices exposed to air.
and dried. It is known in commerce as white squill. It may
be powdered.
IV-504 Squill Preparations 2023
Microscopical content of acetic acid, add sufficient Acetic Acid

Epidermis: cells tetrahedral to hexahedral, thin-walled, three (33 per cent) to the remainder of the filtrate to produce a
to five times longer than wide, having a thick, striated cuticle; solution containing about 8.5% w/v of acetic acid and mix.
stomata rare, anomocytic, Appendix XI H, circular in outline, To every three volumes of the resulting solution add seven
40 to 42 µm in diameter; mesophyll of thin-walled polygonal volumes of Purified Honey and mix thoroughly.
cells containing mucilage, some cells also containing bundles Content of acetic acid, C 2 H 4 O 2
of acicular crystals of calcium oxalate, 20 to 900 µm in 2.2 to 2.7% w/v.
length; vascular bundles collateral, scattered throughout the
mesophyll; xylem vessels with spiral and annular wall TESTS
thickening; trichomes and starch absent. Optical rotation
+0.6° to -3.0°, Appendix VF, when measured in a 25% w/v
IDENTIFICATION
solution in water decolourised, if necessary, with activated
The mucilage contained in the cells of the mesophyll is charcoal.
stained red with alkaline corallin solution and reddish purple
with 0.01M iodine. Weight per mL
TESTS
Ash ASSAY
Not more than 6.0%, Appendix XI J. Dilute 20 mL with 20 mL of carbon dioxide-free water and
titrate with lM sodium hydroxide VS using phenolphthalein
STORAGE solutwn Rl as indicator. Each mL of JM sodium hydroxide VS
Indian Squill should be stored in a dry place. is equivalent to 60.05 mg of C 2H 4 0 2 •
Squill Liquid Extract Opiate Squill Linctus

DEFINIDON Compound Squill Linctus; Gee's Linctus; Opiate Squill Oral
Squill Liquid Extract is prepared by extracting Squill with Solution
Ethanol (70 per cent).
DEFINITION
Extemporaneous preparation Opiate Squill Linctus is an opalescent oral solution containing
The following formula and directions apply. 33% v/v each of Squill Oxymel and Camphorated Opium
Tincture in a suitable vehicle with a tolu flavour.
Squill, in coarse powder 1000 g
Ethanol (70 per cent) A sufficient quantity Extemporaneous preparation
The following formula applies.
Exhaust the Squill, in coarse powde,r, with Ethanol
(70 per cent) by percolation, Appendix XI F. Reserve the first Squill Oxymel 300 mL
850 mL of the percolate; evaporate the subsequent percolate Camphorated Opium Tincture 300 mL
to the consistence of a soft extract and dissolve it in the Tolu Syrup 300mL
reserved portion. Add sufficient Ethanol (70 per cent) to
produce 1000 mL and filter. The linctus complies with the requirements stated under Oral
The extract complies with the requirements stated under Extracts Liquids and with the following requirements.
and with the following requirements. Content of anhydrous morphine, C 17H 1~O 3
TESTS 0.013 to 0.020% w/v.
Ethanol content TESTS
34 to 50% v/v, Appendix VIII F, Method III. Ethanol content
Dry residue 18.0 to 22.0% v/v, Appendix VIII F.
40 to 55% w/v. ASSAY
Relative density To 12 g add 5 mL of water and 1 mL of SM ammonia and
1.00 to 1.14, Appendix VG. extract with 30 mL of a mixture of equal volumes of ethanol
(96%) and chloroform and then with two 22.5-mL quantities
of a mixture of 2 volumes of chloroform and 1 volume of
Squill Oxymel ethanol (96%), washing each extract with the same 20 mL of
a mixture of equal volumes of ethanol (96%) and water.
DEFINITION Evaporate the combined extracts, extract the residue with
10 mL of calcium hydroxide solutwn, filter and wash the filter
Squill, bruised or Indian Squill, 50 g with 10 mL of calcium hydroxide solution. To the combined
bruised
Acetic Acid (33 per cent) 90 mL or a sufficient quantity filtrate and washings add 0.1 g of ammonium sulfate, extract
Purified Water, freshly boiled and 250 mL with two 10 mL quantities of ethanol-free chloroform, wash the
cooled combined extracts with 10 mL of water and discard the
Purified Honey A sufficient quantity chloroform solution. To the combined alkaline liquid and
aqueous washings add 10 mL of lM 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
Macerate the Squill or the Indian Squill with the Acetic Acid freshly prepared 1.0% w/v solution of sodium nitrite, allow to
(33 per cent) and the Purified Water for 7 days with stand for 15 minutes, add 12 mL of SM ammonia, dilute to
occasional agitation, strain, press out the liquid, heat the 50 mL with water and measure the absorbance of a 4-cm layer
mixed liquids to boiling, filter whilst hot, cool, determine the of the resulting solution at the maximum at 442 nm,
2023 St. John's Wort IV-505
Appendix II B, using in the reference cell a solution prepared

in the same manner and at the same time but using 8 mL of
St. John's Wort
water in place of the solution of sodium nitrite. Calculate the Hypericum
content of C 17H 19N03 from a calibration curve prepared
using quantities of 2, 4, 6 and 8 mL of a 0.008% w/v
solution of anhydrous morphine in 0.1 M hydrochloric acid, each Preparation
diluted to 20 mL with 0.1M hydrochloric acid and using the St. John's Wort Dry Extract, Quantified
method described above beginning at the words 'add 8 mL Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
... '. Determine the weight per mL of the linctus,
DEFINITION
Appendix VG, and calculate the content of C 17H 19N0 3 ,
weight in volume. 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
Paediatric Opiate Squill Linctus hypericin (C 30H 160s; Mr 504.4) (dried drug).
Opiate Linctus for Infants; Paediatric Opiate Squill Oral IDENTIFICATION
Solution A. The branched and bare stem shows 2 more or less
DEFINITION prominent longitudinal ridges. The leaves are opposite,
Paediatric Opiate Squill Linctus is an oral solutwn containing sessile, exstipulate, oblong-oval and 15-30 mm long; present
6% v/v each of Squill Oxymel and Camphorated Opium on the leaf margins are glands which appear as black dots
Tincture in a suitable vehicle with a tolu flavour. 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 following formula applies.
the apex of the stem. They have 5 green, acute sepals, with
black secretary glands on the margins; 5 orange-yellow
Squill Oxymel 60mL
Camphorated Opium Tincture 60 mL petals, also with black secretary glands on the margins;
Tolu Syrup 60 mL 3 stamina! blades, each divided into many orange-yellow
Glycerol 200 mL stamens and 3 carpels surmounted by red styles.
Syrup Sufficient to produce I 000 mL
The drug may also show the following: immature and ripe
The linctus complies with the requirements stated under Oral fruits and seeds. Immature fruits are green or yellowish, seeds
Liquids and with the foll,owing requirements. are whitish. Occasional ripe fruits may be present; these are
dry trilocular capsules containing numerous seeds, brown,
Content of anhydrous morphine, C 17H 1gN0 3 broad or small-ovate, 5-10 mm long, with broad linear or
0.0024 to 0.0036% w/v. punctiform glands, irregularly striated ducts, conducting
ASSAY secretions. Ripe seeds are 1-1.3 mm long, cylindrical or
To 32 g add 5 mL of water and 1 mL of SM ammonia and trigonous, shortly pointed at both ends, brown or almost
extract with 30 mL of a mixture of equal volumes of ethanol black, minutely pitted longitudinally.
(96%) and chloroform and then with two 22.5-mL quantities B. Microscopic examination (2.8.23). The powder is
of a mixture of 2 volumes of chloroform and 1 volume of greenish-yellow. Examine under a microscope using chloral
ethanol (96%), washing each extract with the same 20 mL of hydrate solutwn R. The powder shows the following diagnostic
a mixture of equal volumes of ethanol (96%) and water. characters (Figure 1438.-1): fragments of the leaf epidermis
Evaporate the combined extracts, extract the residue with [A, BJ or stems [HJ with paracytic [Ab, Ha], anisocytic [Ac,
10 mL of calcium hydroxide solutwn, filter and wash the filter Bb, Hb] or anomocytic [Ae] stomata (2.8.3); fragments of
with 10 mL of calcium hydroxide solution. To the combined the leaf epidermis often accompanied by palisade
filtrate and washings add 0.1 g of ammonium sulfate, extract parenchyma [Ad, Be]; polygonal cells of the upper epidermis
with two 10 mL quantities of ethanol-free chloroform, wash the with thickened and beaded walls [Ba]; more or less sinuous,
combined extracts with 10 mL of water and discard the thin-walled cells of the lower epidermis [Aa]; fragments of
chloroform solution. To the combined alkaline liquid and the leaf and sepal [E] with large, red-pigmented oil glands
aqueous washings add 5 mL of lM hydrochloric acid, heat on [Ea] associated with palisade parenchyma [Eb] and small
a water bath to remove any chloroform, cool and dilute to vessels [Ee]; elongated cells of fragments of the petal
50 mL with water. To 20 mL of this solution add 8 mL of a epidermis with straight or wavy anticlinal wallsm;
freshly prepared 1.0% w/v solution of sodium nitrite, allow to vessels [DJ with reticulate or pitted walls [Da] and groups of
stand for 15 minutes, add 12 mL of SM ammonia, dilute to thick-walled fibres [Db]; fragments of the central parenchyma
50 mL with water and measure the absorbance of a 4-cm layer of the stems [Kl with lignified and pitted rectangular
of the resulting solution at the maximum at 442 nm, cells [Ka] sometimes associated with vessels [Kb]; fragments
Appendix II B, using in the reference cell a solution prepared of the anthers [F] showing the central part consisting of small
in the same manner and at the same time but using 8 mL of cells containing cluster crystals of calcium oxalate [Fb] and
water in place of the solution of sodium nitrite. Calculate the cells from the fibrous layer [Fa]; fragments of the stamina!
content of C 17 H 19N0 3 from a calibration curve prepared filament with elongated, thin-walled cells with a striated
using quantities of 2, 4, 6 and 8 mL of a 0.008% w/v cuticle [CJ; numerous pollen grains with 3 germinal pores
solution of anhydrous morphine in O. lM hydrochloric acid, each and a smooth exine, occurring singly [G] or in dense groups.
diluted to 20 mL with 0.1 M hydrochloric acid and using the
method described above beginning at the words 'add 8 mL
.. .'. Determine the weight per mL of the linctus,
Appendix VG, and calculate the content of C 17 H 19N03 ,
weight in volume.
IV-506 St. John's Wort 2023
fluorescence and the upper zone (hyperoside) shows a

yellow or orange fluorescence.
with the test solution, other faint to very faint fluorescent
zones, which may be blue, red or orange-yellow, may be
present, especially above the red zones due to hypericin and
pseudohypericin; the light blue fluorescent zone due to
chlorogenic acid may be overlapped by the yellow or orange
fluorescent zone due to hyperoside.
Top of the plate
A red fluorescent zone or a faint red

fluorescent zone
-- --
2 red fluorescent zones (hypericin

and pseudohypericin)
2 yellow or orange fluorescent zones
or 2 faint yellow or orange
fluorescent zones
-- --
A yellow or orange fluorescent zone

fluorescent zone
Hyperoside: a yellow or orange A yellow or orange fluorescent zone
fluorescent zone or an intense yellow or orange
Figure 1438.-1. - Illustration for identification test B of powdered fluorescent zone (hyperoside)
herbal drng of St. John's wort A light blue fluorescent zone or a
faint light blue fluorescent zone
C. High-performance thin-layer chromatography (2.8.25). (chlorogenic acid)

then filter or centrifuge the solution and use the filtrate or
supernatant. Rutoside: a yellow or orange A yellow or orange fluorescent zone
fluorescent zone (rutoside)
3.5 mg of rntoside trihydrate R in methanol Rand dilute to
Reference solution (c) Dissolve 2.5 mg of hyperoside R and TESTS
3 mg of chlorogenic acid R in methanol R and dilute to Foreign matter (2.8.2)
10.0 mL with the same solvent. Maximum 3 per cent of stems with a diameter greater than
Intensity marker Hyperoside. 5 mm and maximum 2 per cent of other foreign matter.
Plate TLC silica gel F254 plate R (2-10 µm). Loss on drying (2.2.32)
(6:9:90 V/V/V).
105 °C for 2 h.
Total ash (2.4.16)
Development 70 mm from the lower edge of the plate. Maximum 7.0 per cent.
ASSAY
Detection Heat at 100-105 °C for 5 min. Spray the warm Test solution In a 100 mL round-bottomed flask, introduce
plate with a 10 g/L solution of diphenylboric acid aminoethyl 0.800 g of the powdered herbal drug (500) (2.9.12), 60 mL
ester R in methanol R, then with a 50 g/L solution of macrogol of a mixture of 20 volumes of water R and 80 volumes of
400 R in methanol R or, alternatively, dip the warm plate in a tetrahydrofuran R and a magnetic stirrer. Boil the mixture in a
5 g/L solution of diphenylboric acid aminoethyl ester R in ethyl water-bath at 70 °C under a reflux condenser for 30 min.
acetate R, then in a 50 g/L solution of macrogol 400 R in Centrifuge (2 min at 700 g) and decant the supernatant into
methylene chloride R. Allow the plate to dry in air for about a 250 mL flask. Take up the residue with 60 mL of a
1 min and examine in ultraviolet light at 366 nm. mixture of 20 volumes of water R and 80 volumes of
System suitability Reference solution (c): tetrahydrofuran R. Heat again under a reflux condenser for
- the chromatogram shows 2 distinct zones in the lower 30 min. Centrifuge (2 min at 700 g) and decant the
third which may, however, be partially overlapping. supernatant. Combine the extracts and evaporate to dryness.
The lower zone (chlorogenic acid) shows a light blue
2023 St. John's Wort Preparations IV-507
Take up the residue with 15 mL of methanol R using Application 4 µL as bands of 8 mm.

ultrasound and transfer to a 25 mL measuring flask. Rinse Development 70 mm from the lower edge of the plate.
the 250 mL flask with methanol Rand dilute to 25.0 mL with
the same solvent. Centrifuge again, filter 10 mL through a
syringe filter (0.2 µm). Discard the first 2 millilitres of the Detection Heat at 100-105 °C for 5 min. Spray the warm
filtrate. Introduce 5.0 mL of the filtrate into a measuring plate with a 10 g/L solution of diphenylboric acid aminoethyl
flask and dilute to 25.0 mL with methanol R. ester R in methanol R, then with a 50 g/L solution of macrogol
Compensation lUJuid methanol R.
5 g/L solution of diphenylboric aczd aminoethyl ester R in ethyl
Measure the absorbance (2.2.25) of the test solution at acetate R, then in a 50 g/L solution of macrogol 400 R in
590 nm, by comparison with the compensation liquid. methylene chloride R. Allow the plate to dry in air for about
Calculate the percentage content of total hypericins, 1 min and examine in ultraviolet light at 366 nm.
expressed as hypericin, using the following expression: System suitability Reference solution (c):
- the chromatogram shows 2 distinct zones in the lower
Ax 125
third which may, however, be partially overlapping.
mx870 The lower zone (chlorogenic acid) shows a light blue
i.e. taking the specific absorbance of hypericin to be 870. fluorescence and the upper zone (hyperoside) shows a
yellow or orange fluorescence.
A absorbance at 590 nm; Results See below the sequence of zones present in the
m mass of the herbal drug to be examined, in grams. chromatograms obtained with reference solution (a) and the
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ffi& test solution. Furthermore, in the chromatogram obtained
with the test solution, other faint to very faint fluorescent
zones, which may be blue, red or orange-yellow, may be
present, especially above the red zones due to hypericin and
Quantified St. John's Wort Dry pseudohypericin; the light blue fluorescent zone due to
Extract chlorogenic acid may be overlapped by the yellow or orange
fluorescent zone due to hyperoside.
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Top of the plate

DEFINITION
Quantified dry extract obtained from St. John's wart (1438). fluorescent zone
Content -- --
- total hypericins, expressed as hypericin (C 30H 16 0 8 ;
2 red fluorescent zones (hypericin
Mr 504.4): 0.10 per cent to 0.30 per cent (anhydrous
and pseudohypericin)
extract);
- flavonoids, expressed as rutoside (C27 H 30O 16; Mr 610.5):
or 2 faint yellow or orange
minimum 6.0 per cent (anhydrous extract); fluorescent zones
- hyperjorin (C 35H 52 0 4 ; Mr 536.8): maximum 6.0 per cent
(anhydrous extract) and not more than the content stated
on the label. -- --
PRODUCTION A yellow or orange fluorescent zone
The extract is produced from the herbal drug by a suitable or a faint yellow or orange
fluorescent zone
procedure using ethanol (50-80 per cent V/V) or methanol
(50-80 per cent V/V). Hyperoside: a yellow or orange An intense yellow or orange
fluorescent zone fluorescent zone (hyperoside)
CHARACTERS
Appearance A light blue fluorescent zone or a
faint light blue fluorescent zone
Brownish-grey powder.
(chlorogenic acid)
IDENTIFICATION
High-performance thin-layer chromatography (2.8.25).
5.0 mL of methanol R. Sonicate for 15 min, then filter or Rutoside: a yellow or orange A yellow or orange fluorescent zone
centrifuge the solution and use the filtrate or supernatant. fluorescent zone (rutoside)
3.5 mg of rutoside trihydrate R in methanol Rand dilute to
10.0 mL with the same solvent. Reference solution (a) Test solution

to 10.0 mL with methanol R. TESTS
Reference solution (c) Dissolve 2.5 mg of hyperoside Rand Water (2.5.12)
3 mg of chlorogenic acid R in methanol R and dilute to Maximum 4.0 per cent, determined on 0.500 g.
10.0 mL with the same solvent. ASSAY
Intensity marker Hyperoside. Total hypericins
Plate TLC silica gel F 254 plate R (2-10 µm). Liquid chromatography (2.2.29).
Mobile phase anhydrous formic acid R, water R, ethyl acetate R Test solution Dissolve 70.0 mg of the extract to be examined
(6:9:90 VIVIV). in 25.0 mL of methanol R. Sonicate and centrifuge the
IV-508 St. John's Wort Preparations 2023
solution. Expose the solution to a xenon lamp at about - mobile phase B: phosphoric acid R, acetonitrile R
765 W/m 2 for 8 min. (3:1000 V/V);
Reference solution Dissolve a quantity of St. John's wart dry
Time Mobile phase A Mobile phase B Flow rate
extract HRS corresponding to O.15 mg of hypericin in (min) (per cent V/J/) (per cent V/J/) (mUmin)
25.0 mL of methanol R. Sonicate and centrifuge. Expose the 0- 8 82 18 0.8
solution to a xenon lamp at about 765 W/m2 for 8 min. 8 - 18 82-+ 47 18-+ 53 0.8
Column: 18 - 18.1 47 ➔ 3 53-+ 97 0.8
- size: l = 0.15 m, 0 = 4.6 mm; 18.1 - 19 3 97 0.8 -+ 1.2
- stationary phase: octadecylsilyl silica gel for chromatography R 19 - 31 3 97 1.2
(5 µm);
- temperature: 40 °C. Detection Spectrophotometer at 360 nm, then at 275 nm
Mobile phase Mix 39 volumes of ethyl acetate R, 41 volumes after the elution of biapigenin (about 22 min).
of a 15.6 g/L solution of sodium dihydrogen phosphate R Injection 10 µL.
adjusted to pH 2 with phosphoric acid R and 160 volumes of Identification of peaks Use the chromatogram supplied with
methanol R. St. John's wart dry extract HRS and the chromatogram
Flow rate 1.0 mL'min. obtained with reference solution (b) to identify the peaks due
Detection Spectrophotometer at 590 nm. to rutoside, hyperoside, isoquercitroside, quercitrin,
Injection 20 µL. quercetin, biapigenin, hyperforin and adhyperforin.
Run time 15 min. System suitability Reference solution (b):
chromatogram supplied with St. John's wart dry
St. John's wart dry extract HRS and the chromatogram
extract HRS;
obtained with the reference solution to identify the peaks due
to pseudohypericin and hypericin.
rutoside and hyperoside, and minimum 2.0 between the
System suitability Reference solution: peaks due to hyperforin and adhyperforin.
Calculate the percentage content of hyperforin using the
chromatogram supplied with St. John's wart dry
extract HRS;
- resolution: minimum 2 between the peaks due to A4 x m 4 xp x 2.3
pseudohypericin and hypericin.
As x m3 x 10
Calculate the percentage content of total hypericins,
expressed as hypericin, using the following expression: A4 area of the peak due to hyperforin in the chromatogram
A5 area of the peak due to rutoside in the chromatogram obtained
(A1 +A2) xm2 xp
A3 xm1 m3 mass of the extract to be examined used to prepare the test
solution, in grams;
area of the peak due to pseudohypericin in the chromatogram m4 mass of rutoside trihydrare CRS used to prepare reference
obtained with the test solution; solution (a), in grams;
area of the peak due to hypericin in the chromatogram obtained 2.3 correction factor for hyperforin with respect to rutoside;
with the test solution; p percentage content of rutoside in rutoside trihydrate CRS.
area of the peak due to hypericin in the chromatogram obtained
solution_, in grams; rutoside, using the following expression:
mass of St. John's wort dry extract HRS used to prepare the
reference solution, in grams; m4 xpx (A6 +A1 +As +Ag +A10 +An)
p percentage content of hypericin in St. John's wart dry
m3 xA 5 x 10
extract HRS.
area of the peak due to rutoside in the chromatogram obtained
Hyperforin and flavonoids with reference solution (a);
Liquid chromatography (2.2.29). Carry out the assay protected area of the peak due to rutoside in the chromatogram obtained
from light.
area of the peak due to hyperoside in the chromatogram
Solvent mixture water R, methanol R (20:80 V/V). obtained with the test solution;
As area of the peak due to isoquercitroside in the chromatogram
Test solution Dissolve 75.0 mg of the extract to be examined
in 20.0 mL of the solvent mixture. Sonicate and centrifuge. area of the peak due to quercitrin in the chromatogram
Reference solution (a) Dissolve 20.0 mg of rutoside obtained with the test solution;
trihydrate CRS in 200.0 mL of the solvent mixture. area of the peak due to quercetin in the chromatogram obtained
Reference solution (b) Dissolve 75.0 mg of St. John's wart dry area of the peak due to biapigenin in the chromatogram
extract HRS in 20.0 mL of the solvent mixture. Sonicate and obtained with the test solution;
centrifuge. mass of the eXtract to be examined used to prepare the test
solution, in grams;
Column: mass of rutoside trihydrate CRS used to prepare reference
- size: l =0.15 m, 0 = 4.6 mm; solution (a), in grams;
- stationary phase: octadecylsilyl silica gel for chromatography R p percentage content of rutoside in rutoside trihydrme CRS.
(3 µm).
Mobile phase: LABELLING
- mobile phase A: phosphoric acid R, water R (3: 1000 V/V); The label states the content of hyperforin.
- - - - - - - - - - - - - - - - - - - - - - - Ph Eur
2023 Stephania Tetrandra Root IV-509
Stephania Tetrandra Root Top of the plate
Protopine: an orange zone

(Fourstamen Stephania Root, Ph. Bur. monograph
2478) -- --
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Tetrandrinc: an orange zone An orange zone (tetrandrine)
DEFINITION
Scraped, cut and dried root of Stephania tetrandra S.Moore. An orange zone
Content
-- --
Minimum 1.6 per cent for the sum of tetrandrine and
fangchinoline, expressed as tetrandrine (C 38H 4 2N 206; Reference solution Test solution
A. The root is found as slices or irregularly cylindrical or Aristolochia fangchi
semi-cylindrical pieces, mostly tortuous, about 0.5-1 cm thick Test for aristolochic acids in herbal drugs (2.8.21). The drug
and 1-5 cm in diameter. The greyish-yellow outer surface to be examined complies with method A.
usually shows deep and sinuous transversal striations; the
curved parts are knotty and bumpy. The texture is dense and Maximum 10.0 per cent, determined on 1.000 g of the
compact. The cut surface is greyish-white and shows radial powdered herbal drug (355) (2. 9.12) by drying in an oven at
striations. 105 °C for 2 h.
B. Microscopic examination (2.8.23). The powder is whitish-
Total ash (2.4.16)
grey. Examine under a microscope using chloral hydrate
characters: numerous fragments of parenchyma with cells Ash insoluble in hydrochloric acid (2.8.1)
having slightly thickened and moniliform walls; reticulate or Maximum 1.0 per cent.
pitted xylem vessels accompanied by fibres; fragments of ASSAY
phelloderm containing sclereids; rare cork fragments; rare, Tetrandrine and fangchinoline
fine, rod-shaped calcium oxalate crystals. Examine under a Liquid chromatography (2.2.29).
microscope using a 50 per cent V!V solution of glycerol R.
Test solution In a 50 mL round-bottomed flask, weigh
The powder shows very many round or truncated, simple or
0.500 g of the powdered herbal drug (355) (2.9.12).
2- or 3-compound starch granules, 10-20 µm in diameter,
Add 25 mL of a 2 per cent V/V solution of hydrochloric
with a punctiform hilum.
acid R in methanol R. Weigh. Heat under a reflux condenser
C. Thin-layer chromatography (2.2.27). on a water-bath at 60 °C for 30 min. Cool and weigh. Adjust
Test solution To 0.4 g of the powdered herbal drug (355) to the initial weight using a 2 per cent V/V solution of
(2.9.12) add 10 mL ofa mixture of 1 volume of anhydrous hydrochloric acid R in methanol R. Filter. Dilute 5.0 mL of the
formic acid R, 9 volumes of water R and 40 volumes of filtrate to 10.0 mL with the mobile phase.
methanol R. Sonicate at 25 °C for 10 min and filter. Reference solution Dissolve 10.0 mg of tetrandrine CRS in
Reference solution Dissolve 10 mg of protopine hydrochloride R 5 mL of methanol R and dilute to 10.0 mL with the mobile
and 10 mg of tetrandrine R in methanol R and dilute to phase.
10 mL with the same solvent. Column:
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel - size: l = 0.25 m, 0 = 4.6 mm;
plate R (2-10 µm)]. - stationary phase: octadecylsilyl silica gel for chromatography R
Mobile phase concentrated ammonia R, methanol R, ethyl (5 µm).
acetate R, toluene R (0.3:5:10:10 VIVIVIV). Mobile phase 4.1 g/L solution of sodium laurylsulfonate for
Application 10 µL [or 5 µL] as bands of 10 mm [or 8 mm]. chromatography R in a mixture of 1 volume of glacial acetic
Development Over a path of 10 cm [or 6 cm). acid R, 30 volumes of methanol R, 30 volumes of water R and
40 volumes of acetonitrile R.
Drying In a current of warm air for 5 min.
Flow rate 2.0 mLJmin.
Detection Treat with a 5 g/L solution of iodine R in ethanol
(96 per cent) R until the background becomes yellow; Detection Spectrophotometer at 280 nm.
examine in daylight after the yellow colour has disappeared. Injection 20 µL.
Results See below the sequence of zones present in the Run time 30 min.
chromatograms obtained with the reference solution and the Relative retention With reference to tetrandrine (retention
test solution. Furthermore, other faint zones may be present time= about 18 min): fangchinoline = about 0.7.
in the chromatogram obtained with the test solution. System suitability Test solution:
fangchinoline and tetrandrine.
Calculate the percentage content of tetrandrine and
fangchinoline, expressed as tetrandrine, using the following
expression:
(A1 +A3)xm2xpx5
A2 xm1
IV-510 Sterculia 2023
area of the peak due to tetrandrine in the chromatogram until the gum is completely swollen, and boil gently under a
area of the peak due to tetrandrine in the chromatogram
reflux condenser for 2 hours. Steam distil until 800 mL of
obtained with the reference solution; distillate is obtained and the acid residue measures about
area of the peak due to fangchinoline in the chromatogram 20 mL and titrate the distillate with 0.1M sodium hydroxide
obtained with the test solution; VS using phenolphthalein solution Rl as indicator. Repeat the
operation without the substance being examined.
solution, in grams;
mass of terrandrine CRS used to prepare the reference solution, The difference between the titrations represents the amount
in grams; of alkali required to neutralise the volatile acid. Each mL of
p assigned percentage content of tetrandrine in tetrandrine CRS. 0.1M sodium hydroxide VS is equivalent to 6.005 mg of
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur volatile acid, calculated as C2H4O 2.
Microbial contamination
1.0 g is free from Escherichia coli, Appendix XVI Bl.
Sterculia STORAGE
Sterculia should be stored in a dry place.
Sterculia Gum; Karaya Gum
Preparations
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.
dispensed or supplied. The granules comply with the requirements stated under Granules
DEFINITION and with the following requirements.
Sterculia is the gum obtained from Sterculia urens Roxb. CHARACTERISTICS
and other species of Sterculia. White or buff with a distinct odour of acetic acid;
CHARACTERISTICS transparent, irregular shaped granules of about 1 to 4 mm
which swell when treated with water.
It has the macroscopical and microscopical characters
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
as small, transparent, angular particles of various sizes and
IDENTIFICATION
shapes; the particles lose their sharp edges when water is
A. Irregular or vermiform pieces, about 5 to 20 mm thick; added and each gradually swells until a large, indefinite,
greyish white with a brown or pink tinge; surface striated. almost structureless mass results; when mounted in ruthenium
B. When powdered and mounted in ethanol (96%) it appears red solution the particles are stained red; no blue coloured
as small, transparent, angular particles of various sizes and particles (starch) are visible when mounted in iodine
shapes; the particles lose their sharp edges when water is solution Rl.
added and each gradually swells until a large, indefinite, C. Add 1 g to 80 mL of water and allow to stand for
almost structureless mass results; when mounted in ruthenium 24 hours, shaking occasionally. A tacky and viscous granular
red solution the particles are stained red; no blue coloured mucilage is produced. Retain the mucilage for use in test D.
particles (starch) are visible when mounted in iodine
D. Boil 4 mL of the mucilage obtained in test C with
solution R 1.
0.5 mL of hydrochloric acid, add 1 mL of SM sodium
C. Add 1 g to 80 mL of water and allow to stand for hydroxide, filter, add 3 mL of cupri-tartaric solution Rl to the
24 hours, shaking occasionally. A tacky and viscous granular filtrate and heat. A red precipitate is produced.
mucilage is produced. Retain the mucilage for use in test D.
TESTS
D. Boil 4 mL of the mucilage obtained in test C with
Acid-insoluble ash
0.5 mL of hydrochloric acid, add 1 mL of SM sodium
hydroxide, filter, add 3 mL of cupri-tartaric solution Rl to the
filtrate and heat. A red precipitate is produced. Ash
Not more than 7.0%, Appendix XI J.
E. Warm 0.5 g with 2 mL of SM sodium hydroxide. A brown
colour is produced. Volatile acid
Not less than 13.0%, calculated as acetic acid, C 2H4O2,
TESTS when determined by the following method. To 1 g contained
Acid-insoluble ash in a 700 mL Kjeldahl flask add 100 mL of water and 5 mL
Not more than 1.0%, Appendix XI K. of orthophosphoric acid, allow to stand for several hours, or
Foreign matter until the granules are completely swollen, and boil gently
Complies with the test for forei-gn matter, Appendix XI D. under a reflux condenser for 2 hours. Steam distil until
Ash 800 mL of the distillate is obtained and the acid residue
measures about 20 mL and titrate the distillate with 0.1M
sodium hydroxide VS using phenolphthalein solution Rl as
Volatile acid indicator. Repeat the operation without the substance being
Not less than 14.0%, calculated as acetic acid, C 2H4O2, examined. The difference between the titrations represents
when determined by the following method. To 1 g contained the amount of alkali required to neutralise the volatile acid.
in a 700 mL Kjeldahl flask add 100 mL of water and 5 mL Each mL of 0.1 M sodium hydroxide VS is equivalent to
of orthophosphoric acid, allow to stand for several hours, or 6.005 mg of volatile acid, calculated as C 2H 4 O 2.
2023 Stramonium Leaf IV-511
Loss on drying subspherical pollen grains about 60-80 µm in diameter with

The powdered granules, when dried to constant weight at 3 germinal pores and a nearly smooth exine [G]; fragments
105°, lose not more than 20.0% of their weight. Use 1 g. of the corolla [HJ with wavy-walled cells [Ha] and underlying
Microbial contamination mesophyll [Hb] with some cells containing prisms [He] or
1.0 g is free from Escherichia coli, Appendix XVI Bl. cluster crystals [Hd] of calcium oxalate; seed fragments
containing yellowish-brown, sinuous, thick-walled sclereids of
STORAGE the testa DJ, and occasional prisms and microsphenoidal
Sterculia Granules should be stored in a dry place. crystals of calcium oxalate.
Ab Ca Cb
/-;
Stramonium Leaf Bf--'\ .
Preparation
~1
Prepared Stramonium
When Stramonium Leaf or Powdered Stramonium Leaf is
e ~<fJ
prescribed, Prepared Stramonium shall be dispensed.
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried leaf or dried leaf and flowering, and occasionally fruit-
bearing, tops of Datura stramonium L. and its varieties.
Content
Minimum 0.25 per cent of total alkaloids, expressed as
hyoscyamine (C 17H 23NO 3; M, 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
wrinkled transversely, branched dichasially, with a single
herbal drug of stramonium leaf
flower or an immature fruit in the fork. Flowers, on short
pedicels, have a gamosepalous calyx with 5 lobes and C. Examine the chromatograms obtained in the
trumpet-shaped brownish-white or purplish corolla. The fruit chromatography test.
is a capsule, usually covered with numerous short, stiff
Results The principal zones in the chromatograms obtained
emergences; seeds are brown or black with a minutely pitted
with the test solution are similar in position, colour and size
testa.
to the principal zones in the chromatogram obtained with the
B. Microscopic examination (2.8.23). The powder is greyish- same volume of the reference solution.
green. Examine under a microscope using chloral, hydrate
D. Shake 1 g of the powdered herbal drug (180) (2.9.12)
with 10 mL of dilute suljuric acid Rl for 2 min. Filter and add
characters (Figure 0246.-1): fragments of upper [A] and
to the filtrate 1 mL of concentrated ammonia R and 5 mL of
lower [CJ epidermises of the lamina, in surface view, showing
water R. Shake cautiously with 15 mL of peroxide-free ether R,
cells with slightly wavy anticlinal walls and a smooth cuticle
avoiding the formation of an emulsion. Separate the ether
accompanied by palisade [Aa] and spongy [Ca] parenchyma;
layer and dry over anhydrous sodium sulfate R. Filter and
anisocytic [Ac, Cb] and anomocytic [Ab] stomata (2.8.3),
evaporate the ether in a porcelain dish. Add 0.5 mL of nitric
more frequent on the lower epidermis; fragments of covering
acid R and evaporate to dryness on a water-bath. Add 10 mL
trichomes, conical [El, uniseriate with 3-5 cells with warty
of acetone R and, dropwise, a 30 g/L solution of potassium
walls, some of them collapsed [Ea]; glandular trichomes,
hydroxide R in ethanol (96 per cent) R. A deep violet colour
short and clavate (side view [BJ) with heads formed by 2-7
develops.
cells; dorsiventral mesophyll (transverse section [F]), with a
single layer of palisade cells [Fa] and a spongy TESTS
parenchyma [Fb] containing cluster crystals of calcium Chromatography
oxalate [Fe]; fragments of spongy parenchyma [DJ with some Thin-layer chromatography (2.2.27).
cells containing small cluster crystals of calcium oxalate [Db], Test solution To 1.0 g of the powdered herbal drug (180)
associated with annularly and spirally thickened vessels [Da], (2. 9.12) add 10 mL of dilute suljuric acid Rl, shake for
in surface view. The powdered herbal drug may also show: 15 min and filter. Wash the filter with dilute sulfuric acid Rl
fibres and reticulately thickened vessels from the stems; until 25 mL of filtrate is obtained. To the filtrate add 1 mL
IV-512 Stramonium Preparations 2023
of concentrated ammonia R and shake with 2 quantities, each funnel, rinsing with peroxide-free ether R. Add a quantity of
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 solutwn 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 2nd 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
anhydrous sodium sulfate with 3 quantities, each of 10 mL,
Mobile phase concentrated ammonia R, water R, acetone R of chloroform R. Add the washings to the chloroform extract,
(3:7:90 VIV!V). evaporate to dryness on a water-bath and heat in an oven at
Application 10 µL and 20 µL of each solution, as bands of 100-105 °C for 15 min. Dissolve the residue in a few
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 solutwn 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.
57.88 X (20 - n)
Results A The zones in the chromatograms obtained with
the test solution are similar in position (hyoscyamine in the (100-d) x m
d loss on drying, as a percentage;
chromatograms) and colour to those in the chromatograms n volume of 0. 02 M sodium hydroxide, in millilitres;
obtained with the reference solution. The zones in the m mass of the powdered herbal drug, in grams.
chromatograms obtained with the test solution are at least
equal in size to the corresponding zones in the chromatogram STORAGE
obtained with the same volume of the reference solution. Protected from moisture.
Faint secondary zones may appear, particularly in the middle _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
of the chromatogram obtained with 20 µL of the test solution
or near the point of application in the chromatogram
obtained with 10 µL of the test solution.
coating is transparent; examine after 15 min.
Prepared Stramonium
Results B The zones due to hyoscyamine in the (Ph. Bur. monograph 0247)
chromatograms obtained with the reference solution and the Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
test solution change from brown to reddish-brown but not to
greyish-blue (atropine) and any secondary zones disappear. DEFINITION
Foreign matter (2.8.2) Stramonium leaf powder (180) (2. 9.12) adjusted, if
Maximum 3 per cent of stems with a diameter greater than necessary, by the addition of powdered lactose or
5mm. stramonium leaf of lower content of total alkaloids.
Maximum 20.0 per cent. 0.23 per cent to 0.27 per cent of total alkaloids, expressed as
hyoscyamine (C 17H 23NO 3; Mr 289.4) (dried drug).
Maximum 4.0 per cent. CHARACTERS
ASSAY Appearance
Greyish-green powder.
a) Determine the loss on drying (2.2.32) on 2.000 g of the
powdered herbal drug (180) (2. 9.12) by drying in an oven at Unpleasant odour.
105 °C. IDENTIFICATION
b) Moisten 10.0 g of the powdered herbal drug (180) A. Microscopic examination (2.8.23) of the powder (180)
(2. 9.12) with a mixture of 5 mL of ammonia R, 10 mL of (2. 9.12). Examine under a microscope using chloral hydrate
ethanol (96 per cent) R and 30 mL of peroxide-free ether R and solutwn R. The powder shows the following diagnostic
mix thoroughly. Transfer the mixture to a suitable percolator, characters: fragments of leaf lamina showing epidermal cells
if necessary with the aid of the extracting mixture. Allow to with slightly wavy anticlinal walls and smooth cuticle;
macerate for 4 h and percolate with a mixture of 1 volume of stomata are more frequent on the lower epidermis (anisocytic
chloroform R and 3 volumes of peroxide-free ether R until the and anomocytic) (2.8.3); covering trichomes are conical,
alkaloids are completely extracted. Evaporate to dryness a uniseriate with 3-5 cells and warty walls; glandular trichomes
few millilitres of the liquid flowing from the percolator, are short and clavate with heads formed by 2-7 cells;
dissolve the residue in 0.25 M sulfuric acid and verify the dorsiventral mesophyll, with a single layer of palisade cells
absence of alkaloids using potassium tetrawdomercurate and a spongy parenchyma containing cluster crystals of
solutwn R. Concentrate the percolate to about 50 mL by calcium oxalate; annularly and spirally thickened vessels.
distilling on a water-bath and transfer it to a separating The powdered herbal drug may also show the following
2023 Stramonium Preparations IV-513
diagnostic characters: fibres and reticulately thickened vessels or near the point of application in the chromatogram
from the stems; subspherical pollen grains usually about obtained with 10 µL of the test solution.
60-80 µm in diameter with 3 germinal pores and nearly Detection B Spray with sodium nitrite solution R until the
smooth exine; fragments of the corolla with papillose coating is transparent; examine after 15 min.
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 cent) R, 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
Chromatography test.
Maximum 5.0 per cent, determined on 1.000 g by drying in
Results The principal zones in the chromatogram obtained an oven at 105 °C.
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
same volume of the reference solution.
C. Shake 1 g with 10 mL of dilute sulfuric acid Rl for 2 min. Ash insoluble in hydrochloric acid (2.8.1)
Filter and add to the filtrate 1 mL of concentrated ammonia R Maximum 4.0 per cent.
and 5 mL of water R. Shake cautiously with 15 mL of ASSAY
peroxide-free ether R, avoiding the formation of an emulsion. a) Determine the loss on drying (2.2.32) on 2.000 g by
Separate the ether layer and dry over anhydrous sodium drying in an oven at 105 °C.
sulfate R. Filter and evaporate the ether in a porcelain dish. b) Moisten 10.0 g with a mixture of 5 mL of ammonia R,
Add 0.5 mL of nitric acid Rand evaporate to dryness on a 10 mL of ethanol (96 per cent) R and 30 mL of peroxide-free
water-bath. Add 10 mL of acetone R and, dropwise, a 30 g/L ether R and mix thoroughly. Transfer the mixture to a
solution of potassium hydroxide R in ethanol (96 per cent) R. suitable percolator, if necessary with the aid of the extracting
A deep violet colour develops. mixture. Allow to macerate for 4 h and percolate with a
TESTS mixture of 1 volume of chloroform R and 3 volumes of
Chromatography peroxide-free ether R until the alkaloids are completely
Thin-layer chromatography (2.2.27). extracted. Evaporate to dryness a few millilitres of the liquid
Test solution To 1.0 g of the drug to be examined add flowing from the percolator, dissolve the residue in 0.25 M
10 mL of dilute sulfuric acid Rl, shake for 15 min and filter. sulfuric acid and verify the absence of alkaloids using
Wash the filter with dilute suljuric acid Rl until 25 mL of potassium tetraiodomercurate solution R. Concentrate the
filtrate is obtained. To the filtrate add 1 mL of concentrated percolate to about 50 mL by distilling on a water-bath and
ammonia R and shake with 2 quantities, each of 10 mL, of transfer it to a separating funnel, rinsing with peroxide-free
peroxide-free ether R. If necessary, separate by centrifugation. ether R. Add a quantity of peroxide-free ether R equal to at
Dry the combined ether layers over anhydrous sodium least 2.1 times the volume of the percolate to produce a
sulfate R, filter and evaporate to dryness on a water-bath. liquid of a density well below that of water. Shake the
Dissolve the residue in 0.5 mL of methanol R. solution with no fewer than 3 quantities, each of 20 mL, of
0.25 M sulfuric acid, separate the 2 layers by centrifugation if
necessary and transfer the acid layers to a 2nd separating
funnel. Make the acid layer alkaline with ammonia R and
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
hydrobromide solution and dilute to 10 mL with methanol R.
sulfate R and allow to stand for 30 min with occasional
Plate TLC silica gel G plate R. shaking. Decant the chloroform and wash the sodium sulfate
Mobile phase concentrated ammonia R, water R, acetone R with 3 quantities, each of 10 mL, of chloroform R. Add the
(3:7:90 VIVIV). washings to the chloroform extract, evaporate to dryness on a
Application 10 µL and 20 µL of each solution as bands of water-bath and heat in an oven at 100-105 °C for 15 min.
20 mm by 3 mm, leaving 1 cm between the bands. Dissolve the residue in a few millilitres of chloroform R, add
Development Over a path of 10 cm. 20.0 mL of 0.01 M suljuric acid and remove the chloroform
by evaporation on a water-bath. Titrate the excess of acid
Drying At 100-105 °C for 15 min and allow to cool. with 0.02 M sodium hydroxide using methyl red mixed
Detection A Spray with potassium iodobismuthate solution R2, solution R as indicator.
using about 10 mL for a plate 200 mm square, until the
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 57.88(20 - n)
the test solution are similar in position (hyoscyamine in the (100 - d)m
chromatogram) and colour to those in the chromatograms d loss on drying, as a percentage;
obtained with the reference solution. The zones in the n volume of 0. 02 M sodium hydroxide, in millilitres;
chromatograms obtained with the test solution are at least m mass of the herbal drug, in grams.
equal in size to the corresponding zones in the chromatogram

obtained with the same volume of the reference solution. STORAGE
Faint secondary zones may appear, particularly in the middle In an airtight container.
of the chromatogram obtained with 20 µL of the test solution _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
IV-514 Szechwan Lavage Rhizome 2023
Szechwan Lavage Rhizome

Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or fragmented rhizome, gently baked to dryness, of
Ligusticum sinense Oliv. 'Chuanxiong' (Canioselinum
anthriscoides (H.Boissieu) Pimenov & Kljuykov, syn.
Ligusticum chuanxiong Hort. ex S.H.Qiu, Y.Q.Zeng, KY.Pan,
Y.C.Tang & J.M.Xu), with rootlets removed, collected in
summer when the nodes of the stem become obviously
swollen and purplish.
Content
Minimum 3.5 mlJkg of essential oil.
IDENTIFICATION
A. Whole drug. The whole rhizome is irregularly knotty and
fist-like, 2-7 cm in diameter. Externally, greyish-brown to
yellowish-brown, rough and shrunken, with many nodular
stem bases on the upper part of the rhizome, each with
parallel and raised annulations and concave, subrounded
stem scars. Numerous tuberculous rootlet scars are visible on
the lower part and at the nodes.
Fragmented drug The fragmented rhizome occurs as thick
slices or irregular pieces. The slices have a conspicuously
irregular outline. The cut surface is whitish-yellow to Figure 2634.-1. - Illustration for identification test B of powdered
brownish-yellow. The texture is compact, difficult to break; herbal drug of szechwan lovage rhizome
the fracture shows a yellowish-white or greyish-yellow
medulla, scattered with yellowish-brown oil dots.
The cambium occurs as an undulate ring. Drying In air.
B. Microscopic examination (2.8.23). The powder is pale Detection A Examine in ultraviolet light at 254 nm.
yellow to brown. Examine under a microscope using chloral Results A See below the sequence of zones present in the
hydrate solution R. The powder shows the following diagnostic chromatograms obtained with the reference solution and the
characters (Figure 2634.-1): brownish-yellow cork fragments, test solution. Furthermore, other faint quenching zones may
showing polyhedral cells with slightly sinuous walls (surface be present in the chromatogram obtained with the test
view [Al) or rectangular cells (transverse section [El); very solution.
numerous fragments of parenchyma [B, G] with slightly
thickened cells; cluster crystals of calcium oxalate (10-25 µm Top of the plate
in diameter), isolated [F] or included in the parenchyma cells
[G], more or less numerous depending on the sample;
reticulate or scalariform spiral vessels ( 14-50 µm in -- --
diameter), isolated or included in the parenchyma with thin-
A bluish fluorescent zone
walled cells [D]; secretory canals, usually broken with thin-
walled secretory cells containing droplets of oil, are A weak quenching zone
sometimes visible (longitudinal section [C], transverse section
[H]); very rarely fusiform fibres with thickened walls.
Examine under a microscope using a 50 per cent V/V Osthole: a blue fluorescent zone 2 quenching zones
solution of glycerol R. The powder shows very numerous
starch granules, isolated [Ba] or included in parenchyma cells
[B]; the granules are simple, rounded, ovoid, oblong or Imperatorin: a quenching zone
reniform, 5-16 µm in diameter and up to 22 µm long; the
-- --
punctiform, slit- or V-shaped hilum may be visible.
Occasional 2- to 4-compound starch granules may be
present. 2 quenching zones
(2.9.12) add 5.0 mL of methanol Rand mix for 10 min.
Reference solution Dissolve 1 mg of imperatonn R and 1 mg
of osthole R in 1.0 mL of methanol R.
Detection B Treat with a 10 per cent V/V solution of suljuric
acid R in methanol R, heat at 120 °C for 3 min and examine
Mobile phase glacial acetic acid R, ethyl acetate R, toluene R in daylight.
(1:10:90 V/V/V).
Application 4 µL as bands of 8 mm. chromatograms obtained with the reference solution and the
2023 Tea-tree Oil IV-515
test solution. Furthermore, other faint zones may be present IDENTIFICATION

in the chromatogram obtained with the test solution. First identification: B.
Top of the plate A. Thin-layer chromatography (2.2.27).
Test solution Dissolve O. 1 mL of the substance to be
examined in 5 mL of heptane R.
Reference solution Dissolve 30 µL of cineole R, 60 µL of
A reddish zone terpinen-4-ol R and 10 mg of a-terpineol R in 10 mL of
heptane R.
-- --
Mobile phase ethyl acetate R, heptane R (20:80 VIV).
A weak yellowish zone (Z-ligustilide)
2 pink zones Development Over a path of 10 cm.
Drying In air.
Detection Spray with anisaldehyde solution R. Heat at
100-105 °C for 5-10 min while observing. Examine in
Impcratorin: a yellow zone A brownish zone daylight.
- - - -
A brownish zone
test solution. Furthermore, other zones are present in the
Top of the plate
Cineole: a violet-brown zone A violet-brown zone, less intense

Reference solution Test solution (cineole)
Terpinen-4-ol: a brownish-violet A brownish-violet zone terpinen-4-
zone ol)
TESTS
Loss on drying (2.2.32) a-terpineol: a violet or brownish- A violet or brownish-violet zone
violet zone (cr-terpineol)
105 °C for 2 h.
Total ash (2.4.16')
Maximum 6.0 per cent. Reference solution Test solution

Maximum 2.0 per cent. B. Examine the chromatograms obtained in the test for
ASSAY
Use 25.0 g of the freshly crushed herbal drug, a 500 mL
solution.
liquid and 0.50 mL of trimethylpentane R in the graduated
tube. Distil at a rate of 3-3.5 mUmin for 3 h. TESTS
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur Relative density (2.2.5)
0.885 to 0.906.
1.475 to 1.482.
Tea Tree Oil Optical rotation (2.2. 7)
+ 5° to+ 15°.
Melaleuca Oil
(Ph. Bur. monograph 1837) Gas chromatography (2.2.28): use the normalisation
Ph Eur - - - - - - - - - - - - - - - - - - - - - - procedure.
DEFINITION Test solution Dissolve O.15 mL of the substance to be
Essential oil obtained by steam distillation from the foliage examined in 10 mL of hexane R.
and terminal branchlets of Melaleuca alternifolia (Maiden and Reference solution Dissolve 5 µL of a-pinene R, 5 µL of
Betch) Chee!, M. linarizfolia Smith, M. dissitiflora F. Mueller sabinene R, 15 µL of a-terpinene R, 5 µL of limonene R, 5 µL
and/or other species of Melaleuca. of cineole R, 30 µL of y-terpinene R, 5 µL of p-cymene R, 5 µL
CHARACTERS of terpinolene R, 60 µL of terpinen-4-ol R, 5 µL of
aromadendrene R and 5 mg of a-terpineol R in 10 mL of
Appearance
hexane R.
Clear, mobile, colourless or pale yellow liquid.
Column:
Characteristic odour.
IV-516 Terminalia Arjuna Stem Bark 2023
- size: l = 30 m (a film thickness of 1 µm may be used) to occasional paler brown patches. Fracture short and starchy in
60 m (a film thickness of 0.2 µm may be used), the inner part, the outer part frequently laminated.
0 = 0.25-0.53 mm, B. Reduce to a powder (355). The powder is reddish-brown.
- stationary phase: macrogol 20 000 R. Examine under a microscope using chloral hydrate solution.
Carrier gas helium for chromatography R. The powder shows a variety of parenchymatous cells, some
Flow rate 1.3 mLJmin. thin-walled, square or round and others yellowish, polygonal
and thick-walled. Rectangular or polygonal, pitted, thin-
Split ratio 1:50.
walled, light brown, lightly lignified cells from the cork layer
Temperature: or outer areas of the cortex are present.
Fibres occur singly and in small or large groups, individual
Time Temperature
(min) (°C) cells narrowing to highly pointed ends, and possibly showing
Column 0- I 50
wavy invaginations of the walls where surrounding cells have
I - 37 50----> 230
become detached; degree of lignification varies; walls are
37 - 45 230
yellowish brown, some pitted, others not. Single fibres may
Injection port 240
be complete, but those in groups are usually fragmented,
Detector 240
individual cells being straight or noticeably curved in places.
Rounded cells of the medullary rays, which are one cell wide,
intersperse the fibres.
Small, calcium oxalate cluster crystals occur scattered
Injection 1 µL. throughout as well as being found within parenchymatous
Elution order Order indicated in the composition of the cells, some forming a crystal sheath alongside the fibres.
reference solution. Record the retention times of these Other crystals are very large and less well defined, and
substances. usually free.
System suitability Reference solution: Examine under a microscope using 50% v/v of glycerol.
- resolution: minimum 2.7 between the peaks due to Starch granules are frequent, but not abundant, mainly free,
terpinen-4-ol and aromadendrene. but some in parenchymatous cells. They are small, simple,
Using the retention times determined from the round, oval or irregular in shape, and occasionally in 2 to 3
chromatogram obtained with the reference solution, locate compound granules, without visible hila. More or less
the components of the reference solution in the frequent scattered lumps of brown pigment may be found
chromatogram obtained with the test solution. Disregard the sometimes in parenchymatous cells.
peak due to hexane. C. Carry out the method for thin-layer chromatography,
Determine the percentage content of these components. Appendix III A, using the following solutions.
The percentages are within the following ranges: (1) Shake 1.0 g of the powdered drug with 10 mL of absolute
- rx-pinene: 1.0 per cent to 6.0 per cent, ethanol, centrifuge at 3000 rpm for 5 minutes and filter
- sabinene: maximum 3.5 per cent, (Whatman GF/C is suitable).
- rx-terpinene: 5.0 per cent to 13.0 per cent, (2) 0.01 % w/v each of arjunolu: acid and gallic acid in absolute
- limonene: 0.5 per cent to 4.0 per cent, ethanol.
- cineole: maximum 15.0 per cent,
- y-terpinene: 10.0 per cent to 28.0 per cent, CHROMATOGRAPHIC CONDITIONS
- p-cymene: 0.5 per cent to 12.0 per cent, (a) Use as the coating high performance silica gel (Merck
- terpinolene: 1.5 per cent to 5.0 per cent, silica gel 60 HPTLC plates are suitable).
- terpinen-4-ol: minimum 30.0 per cent, (b) Use the mobile phase described below.
- aromadendrene: maximum 7 .0 per cent, (c) Apply as bands 8 µL of each solution.
- rx-terpineol: 1.5 per cent to 8.0 per cent.
STORAGE (e) Remove the plate and allow it to dry in air for 5 minutes.
At a temperature not exceeding 25 °C. Spray the plate with anisaldehyde solution, heat at 100° to
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur 105° for 5 minutes and examine in daylight.
MOBILE PHASE
15 volumes of ethyl acetate, 15 volumes of formic acid and
Terminalia Arjuna Stem Bark SYSTEM SUITABILITY
DEFINITION The test is not valid unless the chromatogram obtained with
Terminalia Arjuna Stem Bark consists of cut dried bark of solution (2) shows two clearly separated bands.
the stems of Terminalia arjuna W. 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
with an Rf value of approximately 0.30 corresponding in
IDENTIFICATION
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
2023 Terminalia Belerica Fruit IV-51 7
Top of the plate 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
parenchymatous cells.
( 1) Add 10 mL of absolute ethanol to 1. 0 g of the powdered
drug, centrifuge at 3000 rpm for 5 minutes and filter
Dark band (Whatman GF/C is suitable).
(2) 0.01 % w/v each of arjunolic acid, gallic acid and ellagic acid
in absolute ethanol.
Dark band Dark band: arjunolic (a) Use as the coating high performance silica gel F 254
acid (Merck silica gel 60 F 254 HITLC plates are suitable).
Two separated dark Yellow band: gallic (b) Use the mobile phase described below.
bands acid
(c) Apply as bands 8 µL of each solution.
(e) Remove the plate and allow it to dry in air for 5 minutes.
TESTS anisaldehyde solutwn, heat at 100° to 105° for 5 minutes and
Ash examine in daylight.
Not more than 25%, Appendix XI J. MOBILE PHASE
Loss on drying 15 volumes of ethyl acetate, 15 volumes of Jonnie acid and
When dried for 2 hours at 100° to 105°, loses not more than 70 volumes of toluene.
10% of its weight. Use 1 g. SYSTEM SUITABILITY
Water soluble extractive The test is not valid unless the chromatogram obtained with
Not less than 20%, Appendix XI B2. solution (2) shows two clearly separated bands under both
ASSAY ultraviolet light (254 nm) and daylight.
Carry out the determination of tannins in herbal drngs, CONFIRMATION
Appendix XI M. Use 1.0 g of powdered drug. Under ultraviolet light (254 nm) the chromatogram obtained
with solution (1) shows bands with Rf values of
approximately 0.11 and 0.15 corresponding in colour and
Terminalia Belerica Fruit position to the bands obtained with ellagic acid and gallic
acid in solution (2) and light blue bands with Rf values of
DEFINITION approximately 0.04 and 0.36. Other bands may be present.
Terminalia Belerica Fruit consists of pericarp of dried ripe
fruits of Tenninalia belerica Roxb. It contains not less than Top of the plate
10% of tannins, expressed as pyrogallol, calculated with
IDENTIFICATION
A. The dried fruits are spherical to subspherical, about 3 to
5 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.
Light blue band
Examine under a microscope using chlcrral hydrate solution.
The 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 Blue band Blue band: gallic acid
spherical shapes occur in large and small groups; occasional Dark band Dark band: ellagic acid
medium sized reticulate vessels; heavily pitted, lignified Light blue band
parenchymatous cells and others with reticulate thickenings; Solution (1) Solution (2)
many lignified, pitted, thin walled sclereids and groups of
fragmented, thick-walled, pitted, fibres are present. Rarely oil Under daylight after spraying with anisaldehyde solutwn the
globules, starch granules, and calcium oxalate crystals may be chromatogram obtained with solution (1) shows a band with
found in the parenchymatous cells from the embryo. an Rf value of approximately 0.15 corresponding in colour
Examine under a microscope using 50% v/v of glycerol. and position to the band obtained gallic acid in solution (2);
The powder shows minute, either single or 2 to 4 compound a dark band with an Rf value of 0.36; several dark bands in
starch granules, some scattered, but mainly filling the upper part of the plate.
IV-518 Terminalia Chebula Fruit 2023
Top of the plate occasional small, spiral, lignified vessel fragments. Small,
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
Several dark bands 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
slit or stellate hila; simple granules are often not perfectly
spherical. Larger calcium oxalate crystals, with fewer small
ones, are scattered or in parenchymatous cells.
Dark band (1) To 1.0 g of the powdered drug, add 10 mL of absolute
Dark band: arjunolic ethanol, centrifuge at 3000 rpm for 5 minutes and filter
acid (Whatman GF/C is suitable).
Light brown band Light brown band: (2) 0.01 % w/v each of a,junolic acid, gallic acid and ellagic acid
gallic acid in absolute ethanol.
(a) Use as the coating high performance silica gel F 254
Solution (1) Solution (2) (Merck silica gel 60 F 254 HPTLC plates are suitable).
TESTS (c) Apply as bands 8 µL of each solution.
Ash (d) Develop the plate to 8 cm.
Not more than 7%, Appendix XI J. (e) Remove the plate and allow it to dry in air for 5 minutes.
Loss on drying Examine under ultraviolet light (254 nm). Spray the plate with
When dried for 2 hours at 100° to 105°, loses not more than anisalaehyde solution, heat at 100° to 105° for 5 minutes and
5.0% of its weight. Use 1 g. examine in daylight.
Water soluble extractive MOBILE PHASE
Not less than 45%, Appendix XI B2. A mixture of 15 volumes of ethyl acetate, 15 volumes of formic
ASSAY acid and 70 volumes of toluene.
Carry out the determination of tannins in herbal drngs, SYSTEM SUITABILITY
Appendix XI M. Use 1.0 g of powdered drug. The test is not valid unless the chromatogram obtained with
solution (2) shows two clearly separated bands under both
ultraviolet light (254 nm) and daylight.
CONFIRMATION
Terminalia Chebula Fruit When examined under ultraviolet light (254 nm) the
DEFINITION chromatogram obtained with solution (1) shows bands
Terminalia Chebula Fruit consists of pericarp of mature with Rf values of approximately 0.11 and 0.15 corresponding
fruits of Tenninalia chebula Retz. It contains not less than in colour and position to the bands obtained with gallic acid
20% of tannins, expressed as pyrogallol, calculated with and ellagic acid in solution (2).
IDENTIFICATION Top of the plate
A. The dried fruits are sub-globular to ovoid, 3 to 4 cm long
and 1.5 to 2 cm wide, bluntly pointed at the tip and tapering
towards the base. The surface is yellowish to greenish,
sometimes brown, shiny and more or less wrinkled and has
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 Blue band Blue band: gallic acid
occur. A variety of thick-walled, heavily pitted, lignified Dark band Dark band: ellagic acid
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 Solution (1) Solution (2)
sclereids, with broad lumens, are also found. There are very
2023 Thunberg Fritillary Bulb IV-519
When examined under daylight after spraying with Externally whitish, the bulb consists of 2 fleshy scale leaves,
anisaldehyde solutwn the chromatogram obtained with solution slightly reniform, each overlapping the other and surrounding
( 1) shows a band with an Rf value of approximately 0 .15 2 or 3 small central scales and the shrivelled remains of the
corresponding in colour and position to the band obtained stems.
for gallic acid in solution (2) and a dark band with B. Microscopic examination (2.8.23). The powder is whitish
an Rf value of approximately 0.30. There may be some faint to pale yellow. Examine under a microscope using chloral
brown bands with Rf values of approximately 0.40 and 0.70. hydrate solutwn R. The powder shows the following diagnostic
characters (Figure 2588.-1): fragments of epidermis [BJ
Top of the plate consisting of subpolygonal to rectangular cells with pitted,
irregularly thickened and beaded walls [Ba] and rare,
indistinct anomocytic stomata (2.8.3), 40-56 µm in diameter,
surrounded by 4 or 5 subsidiary cells [Bb]; fragments of
parenchyma consisting of subrounded or polygonal cells of
various sizes [A, C, D], some containing one or more small
fusiform, rod-shaped (bacilliform) or prismatic crystals of
calcium oxalate [Ca, Da]; vessels usually with spiral
thickenings 8-40 µm in diameter [E] . Examine under a
microscope using a 50 per cent VIV solution of glycerol R.
The powder shows abundant, simple, ovoid, subtriangular or
elliptical starch granules 6-56 µm in diameter; the short, slit-
shaped, eccentric hilum is sometimes visible in the narrowest
part [F].
Dark band Dark band: arjunolic
acid
Light brown band Lig hi brown band:

gallic acid
TESTS
Ash
Not more than 5%, Appendix XI J.
Loss on drying
When dried for 2 hours at 100° to 105°, loses not more than
10% of its weight. Use 1 g.
Water soluble extractive
Not less than 50%, Appendix XI B2.
ASSAY
Carry out the determination of tannins in herbal drugs,
Appendix XI M. Use 1.0 g of powdered drug.
Thunberg Fritillary Bulb

DEFINITION
Whole or fragmented, dried bulb of Fritillaria thunbergii Miq.,
with the outer scale removed, harvested at the beginning of Figure 2588.-1. - Illustration for identijicatwn test B of powdered
summer. The largest bulbs no longer have the central bud; herbal drug of Thunberg fritil1ary bulb
the smallest bulbs still have the central bud.
IDENTIFICATION peimine and peiminine.
A. The whole bulb is limited to a single scale leaf, fleshy, Results B See below the sequence of fluorescent zones
slightly crescent-shaped, 1-2.1 cm high and 10-45 mm in present in the chromatograms obtained with reference
diameter. The inner and outer surfaces are whitish to pale solutions (c) and (d) (superimposed spots, track 2) and the
yellow. test solution. Furthermore, in the chromatograms obtained
The fragmented bulb occurs in slices, about 2-3.5 mm thick; with the test solution, other faint fluorescent zones may be
the fracture is white or yellowish-white, soft to the touch present.
owing to the presence of starch.
The smallest bulb is always found whole, as small rounded
masses, oblate, 0.9-2.1 cm high and 11-35 mm in diameter.
IV-520 Thunberg Fritillary Bulb 2023
Top of the plate Intensity markers Peimine (reference solutions (c) and (e))
and peiminine (reference solutions (d) and (f)).
Papaverine: a green Mobile phase diethylamine R, acetone R, toluene R
zone
(6:45:45 VIVIV).
Application 5 µL as bands of 8 mm, 8 mm from the lower
edge and at least 15 mm from the left and right edges of the
Yohimbine: a blue Peiminine: a blue zone A blue zone, faint to
zone equivalent (peiminine) plate (see Table 2588.-1).
Development 70 mm from the lower edge of the plate, in an
-- -- -- unsaturated chamber.
A blue zone
Detection A Examine the chromatograms in ultraviolet light
at 366 nm.
A blue zone System suitability The RF values of the quenching zones due
A greenish zone, very faint to yohimbine and papaverine are approximatively 0.61 and
to faint 0.66, respectively.
Peimine: a greenish A greenish zone (peimine) Detection B Treat with a 10 per cent V/V solution of sulfuric
zone acid R in methanol R and dry the plate at room temperature
until the layer is unifonnly white (approximately 1 min).
-- -- --
Heat the plate at 120 °C for 5 min and examine in ultraviolet
A blue zone light at 366 nm within 3 min after derivatisation.
A blue zone, faint to Results C.
equivalent - any zone due to peimine in the chromatogram obtained
with the test solution is at least of the same intensity as
the corresponding zone in the chromatogram obtained
Reference Reference Test solution, with reference solution (g) (minimum 0.06 per cent);
solutions (a) solutions (c), (d) Detection B
and (b) ( applied (applied as - any zone due to peiminine in the chromatogram obtained
as superimposed superimposed bands with the test solution is at least of the same intensity as
bands in track I), in track 2 and (e), (f) the corresponding zone in the chromatogram obtained
Detection A (applied as with reference solution (h) (minimum 0.02 per cent).
superimposed bands
in track 3), Table 2588.-1. - Application scheme
Detection B
Track I 2 3 4 5
TESTS Application 5 5 5 5 5
volume
Peimine and peiminine (µL)
High-performance thin-layer chromatography (2. 8.25).
Solution Reference solution (a) Reference solution (c)
Test solution Introduce 0.500 g of the powdered herbal drug + reference + reference
(355) (2. 9.12) into a centrifuge tube and add 2.5 mL of solution (b) solution (d) _
concentrated ammonia Rl. Stopper the tube and allow to stand Reference Reference solution (g) Test
solution ( e) + reference solution
for 30 min. Add 12.5 mL of methanol Rand shake for
+ reference solution (h)
20 min at 300 r/min. Centrifuge and use the supernatant.
solution (f)
Stock solution A Dissolve 3.0 mg of peimine R in 15.0 mL of
methanol R. Tracks 1-3 and 5 are used for Identification C; tracks 4 and 5 for the test for
peimine and peiminine.
Stock solution B Dissolve 3.0 mg of peiminine R in 15.0 mL
of methanol R.
Reference solution (a) Dissolve 2 mg of papaverine Maximum 12.0 per cent, determined on 5.000 g of the
hydrochloride R in 4 mL of methanol R. powdered herbal drug (355) (2. 9.12) by drying in an oven at
Reference solution (b) Dissolve 1 mg of yohimbine 105 °C for 2 h.
hydrochloride R in 10 mL of methanol R. Total ash (2.4.16)
Reference solution (c) To 1.0 mL of stock solution A, add Maximum 3.0 per cent.
4.0 mL of methanol R. Ash insoluble in hydrochloric acid (2.8.1)
Reference solution ( d) To 1.0 mL of stock solution B, add Maximum 0.4 per cent.
4.0 mL of methanol R. Extractable matter
Reference solution (e) To 1.0 mL of reference solution (c), Minimum 8.0 per cent.
add 3.0 mL of methanol R. To 2.00 g of the powdered herbal drug (710) (2.9.12) add
Reference solution (j) To 1.0 mL of reference solution (d), 100 g of ethanol (50 per cent Vlv,) R. Weigh and allow to
add 3.0 mL of methanol R. macerate for 1 h. Boil under a reflux condenser for 1 h.
Reference solution (g) Dilute 1.0 mL of stock solution A to Allow to cool. Weigh and compensate for the loss of solvent
10.0 mL with methanol R. with ethanol (50 per cent V/v,) R. Shake vigorously. Filter,
Reference solution (h) Dilute 1.0 mL of stock solution B to evaporate 25 g of the filtrate to dryness on a water-bath and
10.0 mL with methanol R. Dilute 6.7 mL of this solution to dry in an oven at 105 °C for 3 h. The residue weighs a
20.0 mL with methanol R. minimum of 40 mg.
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
2023 Thyme IV-521
the abaxial epidermis (transverse section [H, L]) bears

Thyme covering trichomes of different types: unicellular, straight or
(Ph. Bur. monograph 0865) slightly curved [Ha, La]; bicellular or tricellular, articulated
and most often elbow-shaped [Hb, TI (T. vulgaris); bicellular
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
or tricellular, more or less straight [N], or very large,
DEFINITION multicellular [M], at the base of the lamina (T. zygis);
Whole leaves and flowers separated from the previously dried fragments of calyx covered by numerous, uniseriate trichomes
stems of Thymus vulgaris L. or Thymus zygis L. or a mixture with 5-6 cells and a weakly striated cuticle (surface view [E]).
of both species.
~
Content
- essential oil: minimum 12 mLJkg (anhydrous drug);
- sum of the contents of thymol and carvacrol (both C 10H 14 0;
M, 150.2): minimum 40 per cent in the essential oil. V
CHARACTERS
Strong odour reminiscent of thymol.
IDENTIFICATION
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
both surfaces by a grey or greenish-grey indumentum; the
edges are markedly rolled up .10wards 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
lower is longer and has 2 hairy teeth. After flowering, the
calyx tube is closed by a crown of long, stiff hairs. Fb 'I E
1,
The corolla, about twice as long as the calyx, is usually I I
brownish in the dry state and is slightly bilabiate.

I I
The leafof Thymus zygis is usually 1.7-6.5 mm long and I' :
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
shows the following diagnostic characters (Figure 0865.-1
herbal drug of thyme
and Figure 0865.-2): fragments of the outer epidermis of the
corolla (surface view [A, C, F]), consisting of cells with wavy C. Thin-layer chromatography (2.2.27).
and slightly thickened [Fe] or unthickened [Ac] walls, Test solution To 0.5 g of the powdered herbal drug (355)
numerous uniseriate, multicellular, covering trichomes, often (2. 9.12) add 5 mL of methanol R. Sonicate for 10 min.
with 1 cell collapsed [Aa], glandclar trichomes with a Centrifuge or filter; use the supernatant or the filtrate.
unicellular head and a unicellular [Ca, Fb] or
Reference solution Dissolve 1 mg of rutoside trihydrate R and
multicellular [Ab] stalk, diacytic stomata (2.8.3) [Fa] and
glandular trichomes generally with 12 cells [D]; cells of the 1 mg of rosmarinic acid R in 5 mL of methanol R.
epidermis from the base of the corolla, isodiametric with Plate TLC silica gel F 254 plate R (5-40 µm) [or TLC silica gel
slightly thickened walls [C]; pollen grains, relatively rare, F 254 plate R (2-10 µm)].
spherical and smooth, with 6 germinal slit-like pores, Mobile phase anhydrous formic acid R, water R, ethyl acetate R
measuring about 35 µm in diameter [BJ; the powder of T. (1:1:15 VIVIV).
zygis also contains numerous thick bundles of fibres from the Application 20 µL [or 5 µL] as bands of 20 mm [or 8 mm].
main veins and from fragments of stems; the epidermises of
the leaves (surface view [G, K]) have cells with anticlinal
walls that are sinuous and beaded [Ga, Ka], and diacytic Drying In air.
stomata (2.8.3) [Gb]; numerous glandular trichomes made Detection Heat at 100 °C for 3 min, treat the still-hot plate
up of 12 secretory cells, the cuticle of which is generally with a 5 g/L solution of diphenylboric acid aminoethyl ester R in
raised by the secretion to form a globular or ovoid, bladder- ethyl acetate R, then treat with a 50 g/L solution of macrogol
like covering [Kb); glandular trichomes with a unicellular 400 R in methylene chloride R; examine in ultraviolet light at
stalk and a globular or ovoid head [Kc]; in both species, the 365 nm.
adaxial epidermis bears covering trichomes with warty walls
that are shaped as pointed teeth [Ge], and is usually
associated with underlying palisade parenchyma [Gd, Kd];
IV-522 Thyme 2023

solution (a).
TESTS
r
other foreign matter. Stems must not be more than 1 mm in
2,-:;:_:Y diameter and 15 mm in length.
Thymus serpyllum L.
. J
Adulteration with T. serpyllum L. is indicated by the presence
.
of leaves with long trichomes at their base and with weakly
Kd
Kb pubescent other parts.
Water (2.2.13)
Total ash (2.4.16)
La
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 far 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
anhydrous sodium sulfate Rand dilute to 5.0 mL with
Results See below the sequence of zones present in the heptane R by rinsing the apparatus and the anhydrous sodium
chromatograms obtained with the reference solution and the sulfate. Dilute a volume of the filtered solution corresponding
test solution. Furthermore, other faint fluorescent zones may to 100 µL of the essential oil to 5.0 mL with heptane R.
be present in the chromatogram obtained with the test Reference solution (a) Dissolve 0.20 g of thymol Rand 50 mg
solution. of carvacrol R in heptane Rand dilute to 5.0 mL with the
same solvent.
Top of the plate Reference solution (b) Dilute 10 µL of carvacrol R to
2 red fluorescent zones 10.0 mL with heptane R. Dilute 100 µL of the solution to
10.0 mL with heptane R.
Column:
Rosmarinic acid: a blue fluorescent A blue fluorescent zone (rosmarinic - material: fused silica;
zone acid) - size: l = 30-60 m, 0 = 0.25 mm;
- - - -
0.25 µm).
I or 2 blue fluorescent zones
Carrier gas nitrogen for chromatography R or helium for
chromatography R.
Flow rate 1-2 mUmin.
-- --
Split ratio 1: 100.
Temperature:
A green fluorescent zone may be
present Time Temperature
(min) CC)
Column 0 - 45 40--+ 220
Rutoside: an orange-yellow
fluorescent zone Injection pan 190
Detector 210

D. Examine the chromatograms obtained in the assay for Injection 0.2 µL.
thymol and carvacrol.
Results The characteristic peaks in the chromatogram reference solution (a); record the retention times of these
obtained with the test solution are similar in retention time to substances.
2023 Thyme Oil IV-523
System suitability Reference solution (a): Top of the plate

- resolution: minimum 1.5 between the peaks due to thymol
A pink zone
and carvacrol.
Using the retention times determined from the -- --
chromatogram obtained with reference solution (a), locate Thymol: an orange-brown zone An intense orange-brown zone
the components of the reference solution in the (thymol)
chromatogram obtained with the test solution. Carvacrol: an orange-grey zone A faint orange-grey zone (carvacrol)
Determine the percentage content of thymol and carvacrol. may be present
Disregard any peak due to the solvent or with an area less -- --
than the area of the principal peak in the chromatogram
A pink zone
obtained with reference solution (b) (0.05 per cent).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur A violet zone
A brownish-grey zone
Thyme Oil, Thymol Type

(Ph. Bur. monograph 1374) chromatographic profile.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Results The characteristic peaks in the chromatogram
DEFINITION
Essential oil obtained by steam distillation from the fresh
solution (a).
flowering aerial parts of Thymus vulgaris L., T. zygis L. or a
mixture of both species. TESTS
CHARACTERS
0.915 to 0.935.
Appearance
Clear, yellow or very dark reddish-brown, mobile liquid. Refractive index (2.2.6)
1.490 to 1.505.
Odour reminiscent of thymol.
Solubility
Miscible with anhydrous ethanol and with light petroleum.
procedure.
IDENTIFICATION Test solutwn Dissolve 200 µL of the substance to be
First identification: B. examined in heptane Rand dilute to 10.0 mL with the same
Second identificatwn: A. solvent.
A. Thin-layer chromatography (2.2.27). Reference solutwn ( a) Dissolve 5 µL of fJ-myrcene R, 5 µL of
Test solutwn Dissolve 0.2 mL of the substance to be a-terpinene R, 20 µL of p-cymene R, 10 µL of y-terpinene R,
examined in methylene chloride R and dilute to 10 mL with 5 µL of linalol R, 5 µL of terpinen-4-ol R, 40 mg of thymol R
the same solvent. and 5 µL of carvacrol R in 5 mL of heptane R.
Reference solutwn Dissolve 5 mg of thymol R and 10 µL of Reference solutwn (b) Dissolve 10 µL of carvacrol R in
carvacrol R in methylene chloride R and dilute to 10 mL with heptane Rand dilute to 10.0 mL with the same solvent.
the same solvent. Dilute 100 µL of the solution to 10.0 mL with heptane R.
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel Column:
plate R (2-10 µm)]. - material: fused silica;
Mobile phase methylene chloride R. =
- size: l 60 m, 0 =
0.25 mm;
- stationary phase: phenyl(5)methyl(95)polysiloxane R
(film thickness 0.25 µm).
Drying In air.
Detection Treat with anisaldehyde solutwn R and heat at
Split ratw 1:50.
Temperature:
Time Temperature
test solution. Furthermore, other faint zones may be present (rnin) CC)
in the chromatogram obtained with the test solution. Column 0 - 75 65 --> 215
Injection port 230
Detector 250

Injection 1 µL.
substances.
IV-524 Thyme 2023
System suitability Reference solution (a): consisting of cells with sinuous anticlinal walls [Aa, Ba, Fa]
- resolution: minimum 1.5 between the peaks due to thymol and diacytic stomata (2.8.3) [Ab, Bb, Fb]; cells of the adaxial
and carvacrol. leaf epidermis [B] with wavy, irregularly thickened anticlinal
Identification of peaks Using the retention times determined walls [Ba]; numerous covering trichomes on both epidermises
from the chromatogram obtained with reference solution (a), and the leaf margins, with some of the cells containing very
locate the components of reference solution (a) in the small crystals of calcium oxalate [Af, Ca, Fd], the majority
chromatogram obtained with the test solution. The peak due are short, conical, unicellular, with thickened and warty walls
to o:-thujene elutes with a relative retention of about 0.8 with (surface view [Be], side view [Fe]); fewer multicellular
reference to ~-myrcene. The peak due to carvacrol methyl covering trichomes, long, tapering to a point, composed of
ether elutes with a relative retention of about O. 9 with up to 8 cells, slightly swollen at the joints, with finely pitted
reference to thymol. walls, on an epidermis [Ae] or fragmented [C]; abundant
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
- rx-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
- rx-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 [Ee] 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: 37.0 per cent to 55.0 per cent;
30 µm in diameter, with a finely pitted exine and 6 germinal
- carvacrol: 0.5 per cent to 5.5 per cent;
pores [G].
Be
(0.05 per cent).
STORAGE
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Wild Thyme
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or cut, dried, flowering aerial parts of Thymus
serpyllum L.
Content
Minimum 3.0 mUkg of essential oil (dried drug).
IDENTIFICATION
A. The stem is much branched, up to about 1.5 mm in
diameter, cylindrical or indistinctly quadrangular, green,
reddish or purplish, the older stems brown and woody, the
younger stems pubescent. The leaves are opposite, 3-12 mm
long and up to 4 mm wide, elliptical to ovate-lanceolate with
an obtuse apex, cuneate and shortly petiolate at the base; the
margin is entire and markedly ciliate, especially near the
base; both surfaces are more or less glabrous but distinctly
punctate. The inflorescence is composed of about 6-12
flowers in rounded to ovoid, terminal heads. The calyx is
tubular, 2-lipped with the upper lip dividing to form 3 teeth,
the lower lip with 2 teeth, edged with long hairs; the inner Figure 1891.-1.- Illustration for identification test B of powdered
surfaces are strongly pubescent, the hairs forming a closed herbal drug of wild thyme
tube after flowering. The corolla is purplish-violet or red,
2-lipped, the lower lip with 3 lobes and the upper lip C. Thin-layer chromatography (2.2.27).
notched, the inner surface is strongly pubescent; 4 Test solution To 0.5 g of the powdered herbal drug (355)
epipetalous stamens project from the corolla tube. (2. 9.12) add 5 mL of methanol R. Sonicate for 10 min.
B. Microscopic examination (2.8.23). The powder is greyish- Centrifuge or filter; use the supernatant or the filtrate.
green or greenish-brown. Examine under a microscope using Reference solution Dissolve 1 mg of rutoside trihydrate R and
chloral hydrate solution R. The powder shows the following 1 mg of rosmarinic acid R in 5 mL of methanol R.
diagnostic characters (Figure 1891.-1): fragments of the leaf Plate TLC silica gel F 254 plate R (5-40 µm) [or TLC silica gel
epidermises [A, B, F] covered by a finely striated cuticle and F254 plate R (2-10 µm)].
2023 Tinospora Stem IV-525

(1:1:15 V/V/V).
Tinospora Stem
Application 20 µL [or 5 µL) as bands of 20 mm [or 8 mm). DEFINITION
Development Over a path of 15 cm [or 6 cm]. Dried, whole or fragmented stem of Tinospora cordifolia
(Willd.) Miers. and/or Tinospora sinensis (Lour.) Merr.
Drying In air.
Detection Heat at 100 °C for 3 min; treat the still-hot plate IDENTIFICATION
with a 5 g/L solution of diphenylboric acid aminoethyl ester R in A. Transversely or obliquely cut pieces showing considerable
ethyl acetate R, then treat with a 50 g/L solution of macrogol variation in size, up to about 6 cm in length and 2 cm or
400 R in methylene chloride R; examine in ultraviolet light at more in diameter; more or less cylindrical, sometimes
365 nm. branched; outer surface dark brown, rough, with a thin
papery cork which is sometimes exfoliating; deeply ridged
longitudinally with occasional branch scars and more
numerous small, whitish scars of adventitious roots.
The transversely cut surface shows a wide bark surrounding a
conspicuously radiate and porous xylem with creamish-white,
solution.
narrow medullary rays separating the wedge-shaped strands
Top of the plate
of vascular tissue; the small central pith is slightly depressed.
B. Reduce to a powder (355), Appendix XVII A.
The powder is creamish-brown. Examine under a microscope
using chloral hydrate solution. The powder shows lignified
fibres and crystal fibres containing prisms of calcium oxalate
Rosmarinic acid: a blue fluorescent A blue fluorescent zone (rosmarinic
acid)
from the pericycle; tracheids and vessel elements with
zone
bordered pits from the xylem. Examine under a microscope
-- -- using 50% v/v of glycerol. Simple, ovoid or ovoidelliptical
1 or 2 blue fluorescent zones
starch grains are frequent.
C. Carry out the method for high performance thin-layer
-- -- solutions.
(1) Extract approximately 2 g of the powdered herbal drug
(710), Appendix XVII A with 10 mL of methanol in a
A green or blue fluorescent zone may stoppered conical flask. Shake immediately and then sonicate
be present
for 15 minutes and filter.
Rutoside: an orange-yellow
(2) 0.08% w/v of /3-sitosterol and 0.06% w/v of columbin in
fluorescent zone
methanol.
Reference solution Test solution (3) Dilute 1 volume of solution (2) to 4 volumes with
methanol. Use ~-sitosterol as the intensity marker for zones
(a) to (f) and (h); use columbin as the intensity marker for
TESTS
zone (g).
Maximum 3 per cent, determined on 30 g. (4) 0.06% w/v of columbin and 0.02% w/v of resorcinol in
methanol.
Thymus vulgaris L. or Thymus zygis L
Adulteration with T. vulgaris L. or T. zygis L. is indicated by CHROMATOGRAPHIC CONDITIONS
the presence of acicular to linear-lanceolate leaves with a (a) Use as the coating high performance silica gel F (Merck
strongly bent margin, the adaxial surface showing covering silica gel 60 plates are suitable).
trichomes shaped as pointed teeth with warty walls, the (b) Use the mobile phase as described below.
abaxial surface showing many types of warty covering (c) Apply 10 µL of solution (1) and 2 µL of each of solutions
trichomes: unicellular, straight or slightly curved, bicellular or (2), (3) and (4), as 8 mm bands.
tricellular, often elbow-shaped, and bicellular or tricellular,
more or less straight.
(e) After removal of the plate, dip it in anisaldehyde solution,
dry at 100° for three minutes and examine under white light.
powdered herbal drug (355) (2. 9.12) by drying in an oven at MOBILE PHASE
105 °C for 2 h. 3 volumes of ethyl acetate and 10 volumes of toluene.
Total ash (2.4.16) SYSTEM SUITABILITY
Maximum 10.0 per cent. The test is not valid unless the chromatogram obtained with
Ash insoluble in hydrochloric acid (2.8.1) solution (4) shows two clearly separated zones under white
Maximum 3.0 per cent. light.
ASSAY CONFIRMATION
Essential oil (2. 8.12) The chromatogram obtained with solution (1) shows a
Use 50.0 g of the cut herbal drug, a 1000 mL round- purple zone and a very faint brown to purple zone at the top
bottomed flask and 500 mL of water R as the distillation of the plate, 4 less intense purple zones in the middle of the
liquid. Distil at a rate of 2-3 mUmin for 2 h without xylene R plate, and a green zone may be present in the lower third
in the graduated tube. (indicative of T. cordifolia) or absent (indicative of T. sinensis).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur Other zones may be present.
IV-526 Tolu 2023
Top of the plate Reference solutwn Dissolve 50 mg of benzyl cinnamate R in

methylene chloride R, add 50 µL of benzyl benzoate R and
a faint to intense purple
zone (a)
dilute to 10 mL with methylene chloride R.
a very faint brown or
purple zone (b) Mobile phase light petroleum R, toluene R (5:95 V/V).
- - - Applicatwn 20 µL, as bands.
a faint to intense purple DevelOfJment Over a path of 15 cm.
zone (c) Drying In air.
a faint to equivalent Detection Spray with vanillin reagent R and heat at
purple zone (d)
100-105 °C for 5 min. Examine in daylight.
a purple zone a faint to equivalent
(B-sitosterol) purple zone
(13-sitosterol) (e) chromatograms obtained with the test and reference
solutions. Furthermore, other coloured zones are present in
- - a faint to equivalent
purple zone (f) - the chromatogram obtained with the test solution.
an orange zone
(resorcinol) Top of the plate
a green zone a green zone a very faint green zone Benzyl benzoate: a greyish-blue zone a greyish-blue zone
(columbin) (columbin) (columbin) (g)
Benzyl cinnamate: a greyish-green a greyish-green zone
very faint to equivalent zone
brown and purple
zones (h) Reference solution Test solution
Solution (4) Solutions (2) and (3) Solution (1)

System suitability Intensity marker and Test Solution
test dilution TESTS
Acid value
100 to 160.
TESTS Dissolve 0.5 g of the fragmented drug in 50 mL of alcohol R.
Foreign matter Add 0.5 mL of acid blue 93 solutwn R and 5.0 mL of 0.5 M
Not more than 2.0%, Appendix XI D. alcoholic potassium hydroxide. Stir vigorously and titrate
Loss on drying with 0.5 M hydrochloric acid until the colour changes from
When dried for 2 hours at 105°, loses not more than 11.0% brownish-red to blackish-green (n 1 mL of 0.5 M hydrochloric
of its weight, Appendix IX D. Use 1 g. acid). Carry out a blank test in the same manner (n 2 mL of
Total Ash 0. 5 M hydrochloric acid"). Calculate the acid value in the same
Not more than 11.0%, Appendix XI J. manner as the saponification value (2.5.6).
Matter insoluble in alcohol
Maximum 5 per cent.
Boil 2.0 g of the fragmented drug with 25 mL of alcohol
Tolu Balsam (90 per cent V/V) Rand filter. Wash the residue with alcohol
(90 per cent V/V) R, boiling until completely extracted, then
(Ph. Bur. monograph 1596) dry the residue at 100-105 °C. Weigh the residue.
~~---------------------- Loss on drying (2.2.32)
DEFINITION
fragmented drug by spreading on a flat evaporating dish
Oleo-resin obtained from the trunk of Myroxylon
9 cm in diameter and allowing to dry in vacuo for 4 h.
balsamum (L.) Harms var. balsamum.
Total ash (2.4.16)
Content
25.0 per cent to 50.0 per cent of free or combined acids,
expressed as cinnamic acid (C 9 H 8 0 2; Mr 148.2) (dried ASSAY
drug). Boil 1.500 g under a reflux condenser with 25 mL of 0.5 M
alcoholic potassium hydroxide for 1 h. Evaporate the ethanol
CHARACTERS
and heat the residue with 50 mL of water R until the
Appearance
substance is homogeneously distributed. After cooling, add
Hard, friable, brownish to reddish-brown mass; thin
80 mL of water R and a solution of 1.5 g of magnesium
fragments are brownish-yellow when examined against the
sulfate R in 50 mL of water R. Mix, and allow to stand for
light.
10 min. Filter through a pleated filter paper and wash the
Reminiscent odour of vanillin. residue with 20 mL of water R. Combine the filtrate and the
Solubility washings, acidify with hydrochloric acid R and extract with
Practically insoluble in water, very soluble to freely soluble in 4 quantities, each of 40 mL, of ether R. Discard the aqueous
alcohol, practically insoluble in light petroleum. layer. Combine the organic extracts and wash with
2 quantities, each of 20 mL, and with 3 quantities, each of
IDENTIFICATION
10 mL, of a 50 g/L solution of sodium hydrogen carbonate R.
Discard the ether layer. Combine the aqueous extracts,
Test solutwn Stir 0.40 g of the fragmented drug with 10 mL acidify with hydrochloric acid R and stir once with 30 mL,
of methylene chloride R for 5 min and filter. twice with 20 mL and once with 10 mL of methylene
2023 Tormentil IV-527
chloride R. Dry the combined methylene chloride extracts TESTS

over anhydrous sodium sulfate R. Filter through a pleated filter Ethanol content
and wash the residue with I 0 mL of methylene chloride R. 31 to 36% v/v, Appendix VIII F.
Reduce the combined methylene chloride extracts to I 0 mL Weight per mL
by distillation and eliminate the remaining methylene 1.125 to 1.155 g, Appendix VG.
chloride in a current of air. Dissolve the residue with heating
in I 0 mL of alcohol R previously neutralised to phenol red
solutwn R. After cooling, titrate with 0.1 M sodium hydroxide,
using the same indicator. Tolu Syrup
1 mL of 0.1 A1 sodium hydroxide is equivalent to 14.82 mg of DEFINITION
total acids, expressed as cinnamic acid.
Tolu-flavour Solution 100 mL
STORAGE Syrup Sufficient to produce 1000 mL
Do not store in powdered form.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur The syrup complies with the requirements stated under Oral
Liquids and with the following requirement.
Weight per mL
Tolu-flavour Solution
DEFINITION
Paediatric Compound Tolu Linctus
Cinnamic acid of commerce 5.0 g
Paediatric Compound Tolu Oral Solution
Benzoic Acid 2.5 g
Ethyl Cinnamate 0.3 g DEFINITION
Vanillin 0.1 g
Cinnamon Oil 0.02 mL
Paediatric Compound Tolu Linctus is an oral solution
Sucrose 500 g containing 0.6% w/v of Citric Acid Monohydrate in a
Ethanol (96 per cent) 350 mL suitable vehicle with a tolu flavour.
The linctus complies with the requirements stated under Oral
The following directions apply. Content of total acid, calculated as citric acid
monohydrate, C 6H 8 01,H20
Dissolve the Sucrose in 320 mL of Water. Add 250 mL of
0.60 to 0.66% w/v.
the Ethanol (96 per cent), with mixing. Dissolve the
cinnamic acid of commerce, Benzoic Acid, Ethyl Cinnamate, ASSAY
Vanillin and Cinnamon Oil in the remaining 100 mL of To 15 g add 100 mL of water and titrate with 0.lM sodium
Ethanol (96 per cent), add this solution to the sucrose hydroxide VS using phenolphthalein solution Rl as indicator.
solution with mixing, dilute to 1000 mL with Water and mix. Each mL of 0.lM sodium hydroxide VS is equivalent to
Allow to stand for a few hours before use. 7.005 mg of C 6 H 8 0 7 ,H2 0. Determine the weight per mL of
the linctus, Appendix V G, and calculate the content of
IDENTIFICATION
C 6 H 8 0 7 ,H2 0, weight in volume.
Carry out the method for thin-layer chromatography,
(1) The solution being examined.
(2) 0.5% w/v of cinnamic acid, 0.25% w/v of benzoic acid and
Tormentil
0.03% v/v of ethyl cinnamate in ethanol (90%). (Ph. Bur. monograph 1478)
Preparation
(a) Use as the coating silica gel GF254 . Tormentil Tincture
(b) Use the mobile phase as described below. Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
(c) Apply 5 µL of each solution.
DEFINITION
(d) Develop the plate to 15 cm. Whole or cut, dried rhizome, freed from the roots, of
(e) After removal of the plate, dry in air for 15 minutes and Potentilla erecta (L.) Raeusch. (P. tormentilla Stokes).
repeat the development using the same mobile phase.
Content
Remove the plate, allow the solvent to evaporate and
Minimum 7 per cent of tannins, expressed as pyrogallol (C 6
examine under ultraviolet light (254 nm).
H 6 0 3 ; M, 126.l) (dried drug).
MOBILE PHASE
IDENTIFICATION
15 volumes of glacial acetic acid, 25 volumes of hexane and A. The rhizome is cylindrically spindle-shaped, with a very
75 volumes of n-pentane.
irregular appearance, often forming, twisted, knotty tubers,
CONFIRMATION up to 10 cm long and 1-2 cm thick, very hard and scarcely
The spots in the chromatogram obtained with solution (1) branched. The surface is brown to reddish-brown, rugose
are similar in size and position to those in the chromatogram and has remains of roots and transversely elongated
obtained with solution (2). depressed whitish scars from the stems. At the top of the
rhizome the remains of numerous aerial stems may be
present. The fracture is short and granular, dark red to
brownish-yellow.
IV-528 Tormentil Preparations 2023
B. Microscopic examination (2.8.23). The powder is reddish- Total ash (2.4.16)

brown. Examine under a microscope using chloral hydrate Maximum 5.0 per cent.
ASSAY
characters: coarsely serrate cluster crystals of calcium oxalate,
Tannins (2.8.14)
up to 60 µm in diameter; fragments of thin-walled
parenchyma containing reddish-brown tannin; groups of
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
narrow, bordered-pitted vessels with lateral pores; thick-
walled and pitted, polygonal parenchyma; groups and
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 V/V Tormentil Tincture
solution of glycerol R. The powder shows spherical or
elliptical starch granules, up to about 20 µm in length. (Ph. Eur. monograph 1895)
C. Thin-layer chromatography (2.2.27). PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Test solution To 0.5 g of the powdered herbal drug (355) DEFINITION

(2. 9.12) add 10 mL of water R, shake for 10 min and filter. Tincture produced from Tormentil (1478).
Shake the filtrate with 2 quantities, each of 10 mL, of ethyl
acetate R and filter the combined upper phases over 6 g of Content
anhydrous sodium sulfate R. Evaporate the filtrate to dryness Minimum I .5 per cent mlm of tannins, expressed as
under reduced pressure and dissolve the residue in 1.0 mL of pyrogallol (C6H6O3; Mr 126.1).
ethyl acetate R. PRODUCTION
Reference solution Dissolve 1.0 mg of catechin R in 1.0 mL of The tincture is produced from 1 part of comminuted drug
methanol R. and 5 parts of ethanol (70 per cent V/J/) by a suitable
Plate TLC silica gel plate R. procedure.
Mobile phase glacial acetic acid R, ether R, hexane R, ethyl CHARACTERS
acetate R (20:20:20:40 VIVIVIV). Red or reddish-brown liquid.
Application 10 µL as bands. IDENTIFICATION
Development Over a path of 10 cm. Thin-layer chromatography (2.2.27).
Drying In air for 10-15 min. Test solution Mix 1.0 mL of the tincture to be examined
Detection Spray with a freshly prepared 5 g/L solution of fast with 1.0 mL of alcohol (70 per cent Vlv; R.
blue B salt R. Reddish zones appear. Expose the plate to Reference solution Dissolve 1.0 mg of catechin R in 1.0 mL of
ammonia vapour, the zones become more intense turning methanol R.
reddish-brown. Examine in daylight. Plate TLC silica gel plate R.
Results See below the sequence of the zones present in the Mobile phase ether R, glacial acetic acid R, hexane R, ethyl
chromatograms obtained with the reference solution and the acetate R (20:20:20:40 VIVIV/V).
test solution. Furthermore, other fainter zones are present in
Top of the plate Drying In air for 10-15 min.
Detection Spray with a freshly prepared 5 g/L solution of fast
blue B salt R. Reddish zones appear. Expose the plate to
ammonia vapour, the zones become more intense, turning
Catechin: an intense reddish-brown A more intense reddish-brown zone reddish-brown. Examine in daylight.
zone (catechin) Results See below the sequence of the zones present in the
test solution.
A fainter zone
An intense zone Top of the plate
Fainter zones -- - -
Reference solution Test solution Catechin: an intense zone An intense zone (catechin)
-- --
TESTS
A fainter zone
Maximum 3 per cent of root and stems as well as rhizomes An intense zone
with black fracture and maximum 2 per cent of other foreign
Fainter zones
matter.
Cadmium (2.4.27) Reference solution Test solution
Maximum 2.0 ppm.
Loss on drying (2.2.32) TESTS
Maximum 12.0 per cent, determined on 1.000 g of the Ethanol content (2.9.10)
powdered herbal drug (355) (2.9.12) by drying in an oven at 64 per cent V/V to 69 per cent V/V.
105 °C for 2 h.
2023 Tribulus Terrestris Fruit IV-529
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 V/V of methanol and maximum (c) Use the temperature gradient described below.
0.05 per cent V/V of 2-propanol. (d) Inject 1.0 µL 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 chromatogram obtained with
--------------------~~fu solution ( 1).
Time Temperature (0 )
Trachyspermum Ammi (Minutes)
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
It contains not less than 2.5% v/w of essential oil calculated Inject JX)rt 250
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, I to 3 mm in length and 1 to 2.8 mm in The test is not valid unless in the chromatogram obtained
diameter, usually with pedicel attached; styles remaining as a with solution (2), the resolution factor between the peaks due
curved, bifid stylopod at the apex. Each fruit composed of to y-terpinene and p-cymene is at least 2.5.
two mericarps, dorsal surface convex with five distinct ridges, In the chromatogram obtained with solution (3), the peaks
surface warty; commissural surface flat; vittae visible as two elute in the following order: y-terpinene, p-cymene and
darker longitudinal bands. thymol.
B. Reduce to a powder (355). The powder is greenish DETERMINATION OF CONTENT
brown. Examine under a microscope using chloral hydrate Using the retention times determined from the
solution. The powder contains numerous fragments of the chromatogram obtained with solution (3), locate the
papillose epicarp, also showing cuticular striations, with components of solution (3) in the chromatogram obtained
attached or detached whole or fragmented unicellular, warty- with solution (1) and calculate the content of p-cymene,
walled trichomes; parquetry layer of endocarp in surface y-terpinene and thymol by normalisation.
view; endosperm of thick-walled cells containing oil globules
and aleurone grains with embedded microrosette crystals of Limits:
calcium oxalate; fragments of yellowish-brown septate vittae; - p-cymene 10 to 25%,
bicollateral vascular bundles with associated lignified, - y-terpinene 10 to 30%,
reticulate or pitted parenchyma. - thymol 45 to 70%.
C. Examine the chromatograms obtained in the test for Disregard any peak with an area less than the peak in the
Chromatographic profile. The retention times of the principal chromatogram obtained with solution (4).
peaks in the chromatogram obtained with solution (1) are ASSAY
similar to those in the chromatogram obtained with Essential oil
solution (3). Carry out the method for Essential Oils in Herbal Drugs,
TESTS Appendix XI E, using 15 g of the powdered drug with
Water 1000 mL of water as distillation liquid. Distil at a rate of 2 to
Not more than 10% w/w, Appendix IX C. Use 10.0 g. 3 mL per minute for 2 hours using 0.5 mL of toluene in the
graduated tube. Measure the quantity of essential oil distilled
Total Ash and use for the test for Chromatographic profile.
Not more than 10%, Appendix XI J, Method II.
Carry out the method for gas chromatography,
Appendix III B, using the following solutions. Tribulus Terrestris Fruit
(1) Use the essential oil-toluene mixture obtained in the
DEFINITION
determination of essential oil.
Tribulus Terrestris Fruit is the dried, ripe, entire fruit of
(2) 0.4% vlv each of y-terpinene and p-cymene in toluene. Tribulus terrestris L.
(3) 0.1 % v/v each of p-cymene and y-terpinene and 0.1 % w/v
of thymol in toluene. IDENTIFICATION
A. The fruit is pale greenish yellow, stalked, up to 10 mm
(4) 0.01 % vlv of y-terpinene in toluene.
long and 4 to 6 mm broad, with five ribs or angles, and
CHROMATOGRAPHIC CONDITIONS covered with short, pubescent hairs. There are 5
(a) Use a fused silica column (30 m x 0.53 mm) bonded (occasionally 4) pairs of short, stiff, and very sharp spines,
with a 1 µm film thickness and coated with polyethylene glycol each pointing downwards, the tips of each pair almost
20,000 as the bonded phase (DB-Wax is suitable). meeting and together forming a pentagonal framework
around the fruit. The ripe fruit separates into five piano-
IV-530 Javanese Turmeric 2023
convex or half-moon shaped nutlets or cocci, each armed Top of the plate
with a pair of spines and appearing as a single fruit; each
nutlet contains one chamber containing four or more seeds.
B. Reduce to a powder, Appendix XVII A. The powder is Intense purple band
pale greenish brown. Examine under a microscope using
A purple to brown band
chloral hydrate solution. The powder contains numerous (linalyl acetate)
2 purple-grey bands An orange-yellow band (thymol)
unicellular trichomes; small, rectangular epidermal cells of A brown band (carvone)
the cocci; large parenchyma cells of the mesocarp, some
containing rosette crystals of calcium oxalate; small A purple band
A purple band A purple to brown band
parenchyma cells containing prism crystals of calcium A purple band (linalool)
oxalate. Intense purple band
C. Carry out the method for high-performance thin-layer Solution (1) Solutions (2) Solution (7)
chromatography, Appendix XI W, using the following and (3)
solutions.
(1) Mix 0.3 g of freshly powdered drug, Appendix XVII A,
with 3 mL of methanol and sonicate for 15 minutes; filter or TESTS
centrifuge at 5000 rpm for 5 minutes and use the filtrate or Total Ash
the supernatant liquid. Not more than 15.0%, Appendix XI J.
(2) Add 50 µL of linalool to 1 mL of methanol and dilute Acid-insoluble Ash
1 volume of the resulting solution to 40 volumes with Not more than 1.0%, Appendix XI K.
methanol. Foreign matter
(3) Add 50 µL of linalyl acetate to 1 mL of methanol and Not more than 2%, Appendix XI D.
dilute 1 volume of the resulting solution to 20 volumes with
Loss on drying
methanol.
When dried for 2 hours at 105°, loses not more than 10.0%
(4) Mix 1 volume of solution (2) with 3 volumes of methanol. of its weight. Use 1 g of the powdered drug (355).
(5) Mix 1 volume of solution (3) with 3 volumes of methanol.
ANNEX
(6) Add 50 µL of carvone to 1 mL of methanol and dilute This section is non-mandatory.
1 volume of the resulting solution to 100 volumes with
methanol.
A DNA reference sequence for the identity of Tribulus
(7) A 0.05% w/v solution of thymol in solution (6). Terrestris Fruit is published in Supplementary Chapter VII D.
(a) Use high-performance silica gel 60 F 254 plates (Merck
silica gel F 254 HPTLC plates are suitable).
(b) Use the mobile phase as described below. Javanese Turmeric
(c) Apply 5 µL of solution (1) and 2 µLeach of solutions (2)
to (5) and solution (7) as 8 mm-bands.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
(e) Remove the plate, dip in freshly-prepared anisauiehyde DEFINITION
solution, heat at 100° for about 3 minutes and examine in Dried rhizome, cut in slices, of Curcuma zanthorrhiza Roxb.
daylight within 15 minutes of derivatisation. (syn. C. zanthorrhiza D. Dietrich).
MOBILE PHASE Content
A mixture of 5 volumes of ethyl acetate and 95 volumes of - essential oil: minimum 50 mIJkg (anhydrous drug);
toluene. - dicinnamoyl methane derivatives, expressed as curcumin
(C 2 1H20 0 6 ; Mr 368.4): minimum 1.0 per cent
SYSTEM SUITABILITY
(anhydrous drug).
solution (7) shows two clearly separated bands. CHARACTERS
Aromatic odour.
CONFIRMATION
The chromatogram obtained with solution (1) shows one
IDENTIFICATION
intense purple band above and two purple-grey bands below A. Orange-yellow or yellowish-brown or greyish-brown slices,
the band corresponding to linalyl acetate in the mostly peeled 1.5-6 mm thick and 15-50 mm, more rarely
chromatogram obtained with solution (3); a purple-to-intense up to 70 mm, in diameter. Fragments of the brownish-grey
purple band and three purple bands in the lower third of the cork are sporadically present. The transverse surface is yellow
chromatogram. The middle of the three bands corresponds with dark spots in the paler centre. The fracture is short and
in position to the band due to linalool in the chromatogram finely grained.
obtained with solution (2). Other bands may be present. B. Microscopic examination (2.8.23). The powder is orange-
yellow or yellowish-brown. Examine under a microscope
following diagnostic characters (Figure 1441.-1): fragments of
parenchyma [C, E] with large, rounded or ovoid cells
containing orange-yellow or yellowish-brown secretary cells
[Ea]; fragments of spiral [Fa, G] or reticulate [F] vessels; rare
fragments of cork (surface view [BJ, side view [DJ);
2023 Javanese Turmeric IV-531
fragments of epidermis [HJ with fragments of thick-walled Top of the plate

unicellular covering trichomes [Ha] with a pointed tip [Hb].
A faint pink zone
solution of glycerol R. The powder shows numerous ovoid or An intense reddish zone
irregular starch granules (A], about 30-50 µm long and about
A dark zone
10-30 µm wide, with a more or less visible eccentric hilum.
-- - -
Thymol: a dark zone
A pinkish-red zone
Curcuminoids: a brown zone A brown zone (curcuminoids)
-- --
Curcuminoids: 2 yellow zones A yellow zone (curcuminoids)
TESTS
Curcuma longa L
Test solution To 1 g of the freshly powdered herbal drug
(355) (2.9.12) 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 F 254 plate R (5-40 µm) [or TLC silica
gel F2s4 plate R (2-10 µm)].
Mobile phase glacial acetic acid R, toluene R (20:80 V/V).
Drying In air.
Figure 1441. -1. - Illustration for identification test B of powdered Results A The chromatogram obtained with the test solution
herbal drng of Javanese tunneric does not show a very intense greenish fluorescent zone in the
C. Thin-layer chromatography (2.2.27). Examine the lower third.
chromatograms obtained in the test for Curcuma longa L. Detection B Treat with anisaldehyde solution R and heat at
Results A See below the sequence of zones present in the 100-105 °C for 10 min; examine in ultraviolet light at
chromatograms obtained with the reference solution and the 365 nm.
test solution. Furthermore, other faint zones may be present Water (2.2.13)
in the chromatogram obtained with the test solution. Maximum 120 mLJkg, determined on 20.0 g of the
Top of the plate Total ash (2.4.16)
-- --
ASSAY
Curcuminoids: a greenish fluorescent A greenish fluorescent zone
zone (curcuminoids)
Use 2.5 g of the freshly powdered herbal drug (500) (2. 9.12),
-- -- a 2 L round-bottomed flask, 400 mL of water R as the
Curcuminoids: 2 greenish A greenish fluorescent zone distillation liquid and 0.5 mL of xylene R in the graduated
fluorescent zones (curcuminoids) 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. 9.12)
Reference solution Test solution 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.
Results B See below the sequence of zones present in the Cool and filter into a volumetric flask, rinse the round-
chromatograms obtained with the reference solution and the bottomed flask and the filter with ethanol (96 per cent) R and
test solution. Furthermore, other faint zones may be present dilute to 100.0 mL with the same solvent. Dilute 1.0 mL of
in the chromatogram obtained with the test solution. 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.
IV-532 Turmeric Rhizome 2023
Calculate the percentage content of dicinnamoyl methane

derivatives, expressed as curcumin, using the following
expression:
Ax 5000
1607 X m
i.e. taking the specific absorbance of curcumin to be 1607.
A absorbance at 425 nm,

Turmeric Rhizome
DEFINITION
Whole, cured (by boiling or steaming), dried rhizome of
Curcuma longa L. (syn. Curcuma domestica Valeton) with
roots and outer surface removed.
Content
- essential oil: minimum 25 mUkg (anhydrous drug);
- dicinnamoyl methane derivatives, expressed as curcumin
(C 21 H 20O 6 ; Mr 368.4): minimum 2.0 per cent
(anhydrous drug).
CHARACTERS Figure 2543.-1. - Illustration for identification test B of powdered
Spicy odour. herbal drug of turmeric rhizome
IDENTIFICATION
A. The rhizome is ovate, oblong-ovoid, pyriform or Top of the plate
cylindrical, often shortly branched, up to 6 cm long and
15 mm thick. The primary rhizome shows scars from the -- - -
lateral branches. The surface is slightly dusty, spotted and

brownish-yellow, yellow or brownish-grey, finely striated.
The fracture is granular, smooth, non-fibrous, slightly glossy, Curcuminoids: a greenish fluorescent A greenish fluorescent zone
zone (curcuminoids)
uniformly orange-yellow; it shows a narrow cortex that is
darker on the outside. -- --
B. Microscopic examination (2.8.23). The powder is orange- Curcuminoids: 2 greenish 2 greenish fluorescent zones
yellow. Examine under a microscope using chwral hydrate fluorescent zones (curcuminoids)
characters (Figure 2543.-1): fragments of parenchyma
including some secretory cells that contain masses of Reference solution Test solution
brownish-yellow oil [G]; reticulate or pitted vessels [B, DJ;
rare fragments of epidermis (surface view [F]), with cells
whose walls are slightly and irregularly thickened [Fa] and
scars of covering trichomes [Fb]; occasional long and
flexuous, thick-walled, unicellular covering trichomes,
fragmented and free [E] or attached to fragments of
epidermis 01; rare fragments of cork (surface view [A], side
view [H]), sometimes covered by epidermis [Ha]. Examine
g'lycerol R. The powder shows starch granules, free or
included in parenchymatous cells, usually gelatinised and
agglomerated in a starchy paste; occasional ovoid starch
granules, often deformed by curing [C], are also present.
C. Thin-layer chromatography (2.2.27). Examine the
chromatograms obtained in the test for Curcuma zanthorrhiza
Roxb.
2023 Turpentine Oil IV-533
Top of the plate Calculate the percentage content of dicinnamoyl methane

derivatives, expressed as curcumin, using the following
A faint pink zone
expression:
An intense reddish zone
Ax 5000
-- -- 1607 X m
Thymol: a dark zone
i.e. taking the specific absorbance of curcumin to be 1607.
A pinkish-red zone
Curcuminoids: a brown zone A brown zone (curcuminoids) A absorbance at 425 nm;

-- --
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Curcuminoids: 2 yellow zones 2 yellow zones (curcuminoids)

Turpentine Oil
TESTS Turpentine Oil, Pinus Pinaster Type
Curcuma zanthorrhiza Roxb. (Ph. Bur. monograph 162 7)
(syn. Curcuma zanthorrhiza D. Dietr.). Thin-layer
Preparation
chromatography (2.2.27).
White Liniment
Test solutwn To 1 g of the freshly powdered herbal drug ~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
(355) (2.9.12) add 10 mL of ethanol (96 per cent) R, shake,
allow to stand for 30 min with occasional shaking and filter; DEFINITION
use the filtrate. Essential oil obtained by steam distillation, followed by
Reference solutwn Dissolve 20 mg of curcuminoids R and rectification at a temperature below 180 °C, from the
10 mg of thymol R in 10 mL of ethanol (96 per cent) R. oleoresin obtained by tapping Pinus pinaster Aiton and/or
Plate TLC silica gel F 254 plate R (5-40 µm) [or TLC silica Pinus massoniana D.Don. A suitable antioxidant may be
gel F2s4plate R (2-10 µm)].
added.
Mobile phase glacial acetic acid R, toluene R (20:80 V/V). CHARACTERS
Applicatwn 10 µL [or 3 µL] as bands of 10 mm [or 8 mm]. Appearance
Odour reminiscent of a.-pinene and P-pinene.
Drying In air.
Detection A Examine in ultraviolet light at 365 nm. IDENTIFICATION
Detection B Treat with anisaldehyde solutwn R and heat at
100-105 °C for 10 min; examine in ultraviolet light at Second identificatwn: A.
365 nm. A. Thin-layer chromatography (2.2.27).
Results B The chromatogram obtained with the test solution Test solutwn Dilute 1 mL of the oil to be examined in
shows no dark zone just above the zone due to thymol in the 10 mL of toluene R.
chromatogram obtained with the reference solution. Reference solutwn Dissolve 10 µL of /3-caryophyllene R and
Water (2.2.13) 10 mg of caryophyllene oxide R in 10 mL of toluene R.
Maximum 120 mUkg, determined on 15.0 g of the Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
powdered herbal drug (500) (2.9.12). plate R (2-10 µm)].
Total ash (2.4.16) Mobile phase ethyl acetate R, toluene R (5:95 VIV).
Maximum 7 .0 per cent. Applicatwn 10 µL [or 2 µL] as bands of 10 mm [or 8 mm].
ASSAY Development Over a path of 15 cm [or 6 cm].
Essential oil (2. 8.12) Drying In air.
Use 2.5 g of the freshly powdered herbal drug (500) (2. 9.12), Detection Treat with anisaldehyde solution R and heat at
a 2 L round-bottomed flask, 400 mL of water R as the 100-105 °C for 5-10 min; examine in daylight.
tube. Distil at a rate of 2 mUmin for 3 h.
Dicinnamoyl methane derivatives test solution. Furthermore, other faint zones may be present
Disperse 0.500 g of the powdered herbal drug (500) (2. 9.12) below the zone due to caryophyllene oxide in the
in 30 mL of ethanol (96 per cent) R in a 100 mL round- chromatogram obtained with the test solution.
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.
IV-534 Typhae Pollen 2023
Top of the plate Detection Flame ionisation.

~-Caryophyllene: a pink zone A pink zone
Injection 1.0 µL.
-- - -
substances.
Caryophyllene oxide: a pink zone A pink zone (caryophyllene oxide)
- resolution: minimum 1.5 between the peaks due to car-3-
-- -- ene and ~-myrcene.
chromatogram obtained with reference solution (a), locate
the components of reference solution (a) in the
A brownish-violet zone chromatogram obtained with the test solution.
Determine the percentage content of these components.
The limits are within the following ranges:
- a-pinene: 70.0 per cent to 85.0 per cent;
- camphene: 0.5 per cent to 2.0 per cent;
- /3-pinene: 5.0 per cent to 20.0 per cent;
Results The peaks in the chromatogram obtained with the - car-3-ene: maximum 1.0 per cent;
test solution are similar in retention time to those in the
- /3-myrcene: 0.4 per cent to 1.5 per cent;
chromatogram obtained with reference solution (a). - limonene: 1.0 per cent to 7.0 per cent;
TESTS - longi,folene: 0.2 per cent to 4.0 per cent;
Relative density (2.2.5) - /3-caryophyllene: 0.1 per cent to 3.0 per cent;
0.856 to 0.872. - caryophyllene oxide: maximum 1.0 per cent;
Refractive index (2.2.6) - disregard limit: the area of the peak in the chromatogram
1.465 to 1.475. obtained with reference solution (b) (0.05 per cent).
Optical rotation (2.2.7) Residue on evaporation (2.8.9)
-40° to -28°. Maximum 2.5 per cent, determined after heating on a water-
Acid value (2.5.1) bath for 3 h.
Maximum 1.0. STORAGE
Peroxide value (2.5.5, Method B) At a temperature not exceeding 25 °C.
Maximum 20. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhE~
Fatty oils and resinified essential oils (2. 8. 7)
Gas chromatography (2.2.28): use the normalisation Typhae Pollen
procedure.
Test solutwn Dilute 1.0 mL of the oil to be examined in (Ph. Bur. monograph 2937)
heptane Rand dilute to 10.0 mL with the same solvent. Ph Eur - - - - - - - - - - - - - - - - - - - - - ~
Reference solutwn (a) Dissolve 30 µL of a-pinene R, 10 mg
DEFINITION
of camphene R, 20 µL of /3-pinene R, 10 µL of car-3-ene R,
Dried pollen of Typha angustifolia L., Typha orientalis C.Presl
10 µL of /3-myrcene R, 20 µL of limonene R, 10 µL of
or other species of the genus Typha with single pollen grains
longi,folene R, 10 µL of /3-caryophyllene R and 10 mg of
(monads).
caryophyllene oxide R in 1.0 mL of heptane R.
Reference solutwn (b) Dissolve 5 µL of /3-caryophyllene R in Content
heptane Rand dilute to 10.0 mL with the same solvent. Minimum 0.8 per cent of total flavonoids, expressed as
Dilute 0.1 mL of the solution to 1.0 mL with heptane R. typhaneoside (C34H42 0 20; Mr 771) (dried drug).
- material: fused silica; A. Yellow or brownish-yellow, granular powder, mixed with
- size: l = 60 m, 0 = 0.25 mm; small, irregular, mostly translucent, fragments of floral
- statwnary phase: macrogol 20 000 R (film thickness organs. The powder floats on water and disperses rapidly.
0.25 µm). The texture is fine and light, feels silky when rubbed between
Gamer gas helium for chromatography R. the fingers and easily adheres to the skin.
Flow rate 1.0 mUmin. B. Microscopic examination (2.8.23). The powder is yellow
to brownish-yellow. Examine under a microscope using
Split ratw 1:200.
Temperature: diagnostic characters (Figure 2937.-1): single, light yellow
Time Temperature pollen grains (monads), usually rounded, subtriangular or
(min) ("C) subrectangular with numerous intermediary forms, 17-32 µm
Column 0 - 10 60 in diameter, with a single, more or less distinct, germinal
10 - 80 60 - 200 pore and a finely reticulate exine; a few collapsed grains [F].
80 - 120 200 The following fragments of floral organs may be present:
Injection port 200 fragments of stamens, either containing pollen grains [C] or
Detector 250 showing the endothecium [G]; the endothecium is covered
2023 Typhae Pollen IV-535
by a cuticle and, in side view [DJ, shows cells with walls fluorescence and the upper zone (chlorogenic acid)
thickened on 3 sides, whereas in surface view or from an shows a light blue fluorescence.
angle [Ga, HJ, the cell walls appear as wave-shaped or Results See below the sequence of zones present in the
punctiform thickenings; fragments of bracts consisting of chromatograms obtained with reference solution (a) and the
either slightly lignified, fusiform cells [AJ or slightly test solution. Furthermore, in the chromatogram obtained
thickened, narrow, elongated, rectangular cells sometimes with the test solution, other faint to very faint blue, green
containing fine annular vessels [EJ; raphides of calcium and/or orange fluorescent zones may be present.
oxalate, either free m or included in rounded, thin-walled,
parenchymatous cells; styloid crystals of calcium oxalate, Top of the plate
usually broken [BJ.
-- --
Isorhamnetin-3-0-neohesperidoside: A green fluorescent zone, intense

a green fluorescent zone, intense (isorhamnetin-3-0-neohesperidoside)
A yellow fluorescent zone, faint
-- --
Typhaneoside: a green fluorescent A green fluorescent zone, intense
zone, intense (typhaneoside)
A yellow fluorescent zone, faint
TESTS
Foreign matter
Weigh 10 g of the herbal drug onto a sieve (125) (2.1.4) and
Figure 2937.-1. - Illustration for identification test B of powdered sieve gently with light tapping. The material remaining on
herbal drug of typhae pollen the sieve weighs a maximum of 1.0 g.
C. High-performance thin-layer chromatography (2.8.25). Loss on drying (2.2.32)
(2.9.12) add 5.0 mL of methanol Rand sonicate for 15 min.
105 °C for 2 h.
Centrifuge or filter and use the supernatant or the filtrate.
If necessary, filter through a membrane filter (nominal pore Total ash (2.4.16)
size 0.45 µm). Maximum 7 .0 per cent.
Reference solution (a) Dissolve 3.0 mg of isorhamnetin-3-0- Ash insoluble in hydrochloric acid (2.8.1)
neohesperidoside R and 3.0 mg of typhaneoside R in methanol R Maximum 4.0 per cent.
and dilute to 10.0 mL with the same solvent. ASSAY
Reference solution (b) Dilute 2.5 mL of reference solution (a) Liquid chromatography (2.2.29).
to 10.0 mL with methanol R. Test solution Introduce 1.0 g of the sieved herbal drug
Reference solution (c) Dissolve 3 mg of chlorogenic acid R and obtained in the test for foreign maner into a round-bottomed
3 mg of isorhamnetin-3-0-neohesperidoside R in methanol Rand flask and add 50 mL of methanol R. Heat in a water-bath
dilute to 10 mL with the same solvent. under a reflux condenser at 90 °C for 30 min. Allow to cool
Intensity marker Typhaneoside. and filter. Repeat the extraction with a further 50 mL of
Plate TLC silica gel plate R (2-10 µm). methanol R. Allow to cool and filter. Combine the filtrates
and evaporate to dryness under reduced pressure. Dissolve
the residue in methanol Rand dilute to 10.0 mL with the
water R, ethyl acetate R (11:11:27:100 V/VIVIV).
same solvent. Filter through a membrane filter (nominal pore
Application 5 µL as bands of 8 mm. size 0.22 µm).
Development 7 cm from the lower edge of the plate. Reference solution (a) Dissolve 8.0 mg of typhaneoside CRS
Drying In a current of cold air for 5 min. in methanol Rand dilute to 25.0 mL with the same solvent.
Detection Heat at 100 °C for 3 min; treat the warm plate Reference solution (b) Dissolve 0.230 g of typhae pollen dry
with a 5 g/L solution of diphenylboric acid aminoethyl ester R in extract for system suitability HRS in methanol R and dilute to
ethyl acetate R, then with a 50 g/L solution of macrogol 400 R 10 mL with the same solvent. Weigh and sonicate for
in methylene chloride R; examine in ultraviolet light at 366 nm. 10 min. Allow to cool, weigh again and compensate for the
System suitability Reference solution (c): loss of solvent with methanol R. Shake thoroughly and filter
- the chromatogram shows in the middle third 2 distinct through a membrane filter (nominal pore size 0.22 µm).
zones, which may be touching; the lower zone Column:
(isorhamnetin-3-0-neohesperidoside) shows a green - size: l = 0.15 m, 0 = 2.1 mm;
IV-536 Uncaria Stem with Hooks 2023
- statu:mary phase: end-capped octadecylsilyl silica gel for B. Microscopic examination (2.8.23). The powder is reddish-
chromatography R (1.8 µm); brown or yellowish-brown. Examine under a microscope
- temperature: 25 °C. using chloral hydrate solutwn R. The powder shows the
Mobile phase acetonitrile R, 0.1 per cent V/V solution of following diagnostic characters (Figure 2729.-1): brown
anhydrous formic acid R (12.5:87.5 V/V). fragments of the epidermis composed of irregular polygonal
cells (surface view [DJ); fragments of the dermal tissue
Fl,ow rate 0.4 mllmin.
(transverse section [Al) consisting of the epidermis [Ab]
Detection Spectrophotometer at 254 nm. covered by a thick cuticle [Aa] and collenchymatous cells of
Infection 2 µL. the parenchyma [Ac]; fragments of phloem parenchyma with
Run time 30 min. some cells containing sandy crystals of calcium oxalate
Identification of peaks Use the chromatogram supplied with (longitudinal section m); numerous pericyclic fibres, often in
typhae polkn dry extract for system suitability HRS and the groups, with thick unchannelled walls [C]; sclereids of the
chromatogram obtained with reference solution (b) to cortical parenchyma with slightly thickened walls and oblique
identify the peaks due to flavonoids 1, 2, 3, 5 and 6; use the pits, varying greatly in shape and size, sometimes elongated
chromatogram obtained with reference solution (a) to [G], sometimes rectangular [H], isolated or in small groups;
identify the peak due to typhaneoside (flavonoid 4). fragments of the xylem [E, K] consisting of large bordered-
pitted vessels [Ka], bordered-pitted tracheids [Kb], annular
Relative retention With reference to typhaneoside (retention
vessels [Ea], these vessels are often associated with the outer
time= about 16.2 min): flavonoid 1 = about 0.55;
layers of the pith with cells with channelled walls [Eb];
flavonoid 2 = about 0.76; flavonoid 3 = about 0.90;
numerous rectangular cells of the medullary rays
flavonoid 5 = about 1.23; flavonoid 6 = about 1.41.
(longitudinal section [F]) with cells whose walls are regularly
System suitability Reference solution (b): thickened and pitted; fragments of the pith with rounded to
- resolution: minimum 3.3 between the peaks due to ovoid cells with slightly pitted walls [BJ.
flavonoid 3 and typhaneoside.
expressed as typhaneoside, using the following expression:
(2. 9.12) add 1 mL of concentrated ammonia R. After 30 min,
A1 xmzxpx2 add 25 mL of ethyl acetate R and sonicate for 20 min. Filter
and evaporate the filtrate to dryness under reduced pressure.
A2 x m1 x 5
Dissolve the residue in 1 mL of methanol R, centrifuge and
A1 sum of the areas of the peaks due to typhaneoside and use the supernatant.
flavonoids I, 2, 3, 5 and 6 in the chromatogram obtained with Reference solution Dissolve 1 mg of isorhynchophylline R and
the test solution;
A2 area of the peak due to typhaneoside in the chromatogram
1 mg of rhynchophylline R in 2 mL of methanol R.
obtained with reference solution (a); Plate TLC silica gel F254 plate R (2-10 µm).
solution, in grams;
m2 mass of typhaneoside CRS used to prepare reference solution (a), (1:15:125 VIVIV).
in grams; Applicatwn 5 µL as bands of 8 mm.
p percentage content of typhaneoside in ryphaneoside CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur Drying In air.
Uncaria Stem with Hooks test solution. Furthermore, other quenching zones may be
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Top of the plate
DEFINITION A quenching zone

Fragments of dried branch or stem of Uncaria rhynchophylla
(Miq.) Miq. ex Havil., each bearing paired hooks.
Content -- --
Minimum 0.2 per cent of total alkaloids, expressed as Isorhynchophylline: a quenching A quenching zone
isorhynchophylline (C 22H 28N 2 O 4 ; Mr 384.5) (dried drug). zone (isorhynchophylline)
IDENTIFICATION -- --
A. The glabrous, reddish-brown fragments of the stem are A quenching zone
about 2-3 cm long and bear paired hooks. The stem is
cylindrical or sub-square, 2-5 mm in diameter, with fine Rhynchophylline: a quenching zone A quenching zone (rhynchophylline)
longitudinal striations. The hooks, rounded and curved
downwards, are paired, opposite to each other on the stem;
occasionally there is only 1 hook; the hooks are about 1-5 cm A quenching zone
long, with an acute apex and a relatively broad base. Petiole Reference solution Test solution
scars and arc-shaped stipule scars are visible on the stem,
below the hook. A transverse section of the stem shows either
a central cavity or a spongy, whitish pith.
2023 Valerian IV-537
- mobile phase B: acetonitrile for chromatography R;

OooC:8w
Ab~~gv(
Aa
00a~r
Q Time
(min)
0- 3
Mobile phase A
(per cent V/Jl)
75
Mobile phase B
(per cent V/Jl)
25
3 - 15 75 -> 70 25 -> 30
DiS
15 - 25 70 -> 55 30 -> 45
25 - 35 55 45
35 - 40 55 _, I 0 45 -, 90

F . 1, \
•Jo
0
O Vr
0
0 0
0 0
Injection 10 µL.
'
0
uncaria stem with hooks dry extract for system suitability HRS
' and the chromatogram obtained with reference solution (b)
to identify the peaks due to alkaloids 1, 2, 3, 4, 6, 7, 8 and
\t
9; use the chromatogram obtained with reference solution (a)
to identify the peak due to isorhynchophylline.
Relative retention With reference to isorhynchophylline
(retention time = about 28 min): alkaloid 1 = about 0.50;
Ea
=
alkaloid 4 about 0.97; alkaloid 6 about 1.05; =
alkaloid 9 = about 1.27. Additional peaks may be present.
alkaloid 4 and isorhynchophylline.
Figure 2729 .-1. - Illustration for identification test B of powdered
herbal drug of uncaria stem with hooks Calculate the percentage content of total alkaloids expressed
as isorhynchophylline using the following expression:
TESTS
(LA1) x m2 xp
Maximum 10.0 per cent, determined on 1.000 g of the A2 xm 1 x25
powdered herbal drug (355) (2.9.12) by drying in an oven at rA 1 sum of the areas of the peaks due to alkaloids I, 2, 3, 4, 6, 7, 8,
105 °C for 2 h. 9 and the peak due to isorhynchophylline, in the chromatogram
Total ash (2.4.16) A2 area of the peak due to isorhynchophylline in the chromatogram
Maximum 3.0 per cent. obtained with reference solution (a);
ASSAY
solution, in grams;
Liquid chromatography (2.2.29). Use freshly prepared solutions. m2 mass of isorhynchophylline CRS used to prepare reference
Store and inject them at 5 °C, using a cooled autosampler. solution (a), in grams;
p percentage content of isorhynchophylline in
isorhynchophylline CRS.
(2.9.12) add 3.0 mL of concentrated ammonia R. After
30 min, add 50.0 mL of a mixture of equal volumes of
methanol R and methylene chloride R. Sonicate for 30 min.
Filter and evaporate the filtrate to dryness under reduced
pressure. Dissolve the residue in methanol R and dilute to
Valerian
isorhynchophylline CRS in a mixture of equal volumes of (Valerian Root, Ph. Bur. monograph 0453)
methanol R and methylene chloride Rand dilute to 25.0 mL
with the same mixture of solvents. Dilute 1.0 mL of the Preparations
solution to 10.0 mL with methanol R. Valerian Dry Extract
Reference solution (b) Dissolve 5.0 mg of uncaria stem with Valerian Dry Hydroalcoholic Extract
hooks dry extract for system suitability HRS in 1.0 mL of Valerian Tincture
methanol R. Filter through a membrane filter (nominal pore When Powdered Valerian is prescribed or demanded,
size 0.45 µm). material complying with the appropriate requirements below
Column: shall be dispensed or supplied.
- size: l = 0.15 m, 0 = 4.6 mm; Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
chromatography with extended pH range R (5 µm). DEFINITION
Dried, whole or fragmented underground parts of Valeriana
Mobile phase:
officinalis L. s. l., including the rhizome surrounded by the
- mobile phase A: dilute 0.15 mL of diethylamine R to 1 L
roots and stolons.
with water for chromatography R;
IV-538 Valerian 2023
Content polygonal cells with irregularly thickened walls [F]; a few

- essential oil: minimum 4 mUkg (dried drug); groups of sclereids with thick walls and a narrow lumen [El
- sesquiterpenic acids: minimum 0.17 per cent mlm, expressed from the pith of the rhizome. Examine under a microscope
as valerenic acid (C 15 H 220 2 ; Mr 234.3) (dried drug). using a 50 per cent V/V solution of glycerol R. The powder
IDENTIFICATION shows numerous starch granules, simple or 2- to
6-compound, but frequently separated, rounded or irregular
A. The rhizome is yellowish-grey or pale brownish-grey,
and up to about 15 µm in diameter; most of the granules
obconical or cylindrical, up to about 50 mm long and 30 mm
show a rather indistinct cleft or radiate hilum [C].
in diameter; the base is elongated or compressed, usually
entirely covered by numerous roots. The apex usually C. Thin-layer chromatography (2.2.27).
exhibits a cup-shaped scar from the aerial parts; stem bases Test solution Suspend 1 g of the powdered herbal
are rarely present. When cut longitudinally, the pith exhibits drug (355) (2. 9.12) in 10 mL of methanol R and sonicate for
a central cavity transversed by septa. The roots are 10 min. Filter the supernatant through a membrane filter
numerous, almost cylindrical, of the same colour as the (nominal pore size 0.45 µm). Use the filtrate.
rhizome, 1-3 mm in diameter and sometimes more than Reference solution Dissolve 5 mg of acetoxyvalerenic acid R
100 mm long. A few filiform fragile secondary roots are and 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 µm) [or TLC silica gel
nodes separated by longitudinally striated internodes, each plate R (2-10 µm)].
20-50 mm long, with a fibrous fracture.
Mobile phase glacial acetic acid R, ethyl acetate R,
cydohexane R (2:38:60 V/V/V).
Drying In air.
test solution. Furthermore, other violet zones may be present
Top of the plate
-- --
Valerenic acid: a violet zone A violet zone (valerenic acid)
Acetoxyvalerenic acid: a violet zone A violet zone (acetoxyvalerenic acid)
-- --
2 faint or very faint violet zones
TESTS
Maximum 12.0 per cent, determined on 1.000 g ofwell-
Figure 0453.-1. - Illustration for identification test B of powdered homogenised powdered herbal drug (355) (2.9.12) by drying
herbal drng of valerian root in an oven at 105 °C for 2 h.
Total ash (2.4.16)
yellowish-grey or pale greyish-brown. Examine under a
microscope using chloral hydrate solution R. The powder Ash insoluble in hydrochloric acid (2.8.1)
shows the following diagnostic characters (Figure 0453.-1): Maximum 5.0 per cent.
occasional groups of rectangular sclereids with moderately ASSAY
thickened walls and a large lumen, from the stem base [H]; Essential oil (2.8.12)
very numerous fragments of parenchyma with large ovoid Use 40.0 g of freshly powdered herbal drug (500) (2. 9.12), a
cells (longitudinal section [K], transverse section [J]); spiral, 2000 mL flask, 500 mL of water R as the distillation liquid
reticulate or pitted lignified vessels, isolated or in small and 0.50 mL of 1,2,4-trimethylbenzene R in the graduated
groups [D, G]; thin-walled, elongated cells of the piliferous tube. Distil at a rate of 3-4 mUmin for 4 h.
layer (surface view [A], transverse section [B]), some with
root hairs [Aa, Ba] or their scars [Ab]; the piliferous layer is Sesquiterpenic acids
usually accompanied by an underlying layer of cells with Liquid chromatography (2.2.29).
slightly thickened and elongated walls [Ac, Bb]; fragments of Test solutwn Place 1.50 g of the powdered herbal drug
dermal tissue from the rhizome composed of 1 or 2 layers of (710) (2.9.12) in a 100 mL round-bottomed flask with a
2023 Valerian IV-539
ground-glass neck. Add 20 mL of methanol Rl. Mix and heat

on a water-bath under a reflux condenser for 30 min. Allow
Cut Valerian
to cool and filter. Place the filter with the residue in the (Valerian Root, Cut Ph. Bur. monograph 2526)
100 mL round-bottomed flask. Add 20 mL of methanol Rl
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
and heat on a water-bath under the reflux condenser for
15 min. Allow to cool and filter. Combine the filtrates and DEFINITION
dilute to 50.0 mL with methanol Rl, rinsing the round- Dried, cut underground parts of Valeriana officinalis L. s.l.,
bottomed flask and the filter. including the rhizome, roots and stolons.
Reference solution Dissolve an amount of valerian dry It is produced from Valerian root (0453) for the purpose of
extract HRS corresponding to 1.0 mg of valerenic acid in being used in herbal teas.
Sonicate for 10 min and filter through a membrane filter Content
(nominal pore size 0.45 µm). - essential oil: minimum 3 mL/kg (dried drug);
- sesquiterpenic acids: minimum 0.10 per cent mlm expressed
Column: as valerenic acid (C 15 H 22 0 2 ; Mr 234.3) (dried drug).
- size: l = 0.25 m, 0 = 4.6 mm;
- stationary phase: octadecylsilyl silica gel for chromatography R IDENTIFICATION
(5 µm). A. Microscopic examination (2.8.23). The powder is pale
Mobile phase: yellowish-grey or pale greyish-brown. Examine under a
- mobile phase A: acetonitrile Rl, 5 g/L solution of phosphoric microscope using chloral hydrate solution R. The powder
acid R (20:80 V/V); shows the following diagnostic characters (Figure 2526.-1):
- mobile phase B: 5 g/L solution of phosphoric acid R, occasional groups of rectangular sclereids with moderately
acetonitrile Rl (20:80 V/V); thickened walls and a large lumen, from the stem base [H];
very numerous fragments of parenchyma with large ovoid
Time Mobile phase A Mobile phase B cells (longitudinal section [K], transverse section [J]); spiral,
(min) (per cent V/V) (per cent V/V) reticulate or pitted lignified vessels, isolated or in small
0- 5 55 45 groups [D, G]; thin-walled, elongated cells of the piliferous
5 - 18 55 ➔ 20 45 ➔ 80 layer (surface view [A], transverse section [BJ), some with
18 - 22 20 80 root hairs [Aa, Ba] or their scars [Ab]; the piliferous layer is
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
Detection Spectrophotometer at 220 nm. polygonal cells with irregularly thickened walls [F]; a few
Injection 20 µL. groups of sclereids with thick walls and a narrow lumen [E]
Peak identification V se the chromatogram supplied with from the pith of the rhizome. Examine under a microscope
valerian dry extract HRS and the chromatogram obtained with using a 50 per cent V/V solution of grycerol R. The powder
the reference solution to identify the peaks due to shows numerous starch granules, simple or 2- to
acetoxyvalerenic acid and valerenic acid. 6-compound, but frequently separated, rounded or irregular
System suitability Reference solution: and up to about 15 µm in diameter; most of the granules
- relative retention with reference to valerenic acid (retention show a rather indistinct cleft or radiate hilum [CJ.
time = about 19 min): acetoxyvalerenic acid = about 0.5. B. Thin-layer chromatography (2.2.27).
Calculate the percentage content of sesquiterpenic acids, Test solution Suspend 1 g of the powdered herbal
expressed as valerenic acid, using the following expression: drug (355) (2. 9.12) in 10 mL of methanol R and sonicate for
10 min. Filter the supernatant through a membrane filter
(A1+A2)xm2xpx5 (nominal pore size 0.45 µm). Use the filtrate.
A3 xm1 Reference solution Dissolve 5 mg of acetoxyvalerenic acid R
and 5 mg of valerenic acid R in 20 mL of methanol R.
area of the peak due to acetoxyvalerenic acid in the
chromatogram obtained with the test solution; Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
area of the peak due to valerenic acid in the chromatogram plate R (2-10 µm)].
obtained with the test solution; Mobile phase glacial acetic acid R, ethyl acetate R,
area of the peak due to valerenic acid in the chromatogram
cyclohexane R (2:38:60 V/V/V).
mass of the herbal drug to be examined used to prepare the test Application 20 µL [or 5 µL] as bands of 10 mm [or 8 mm].
solution, in grams;
mass of valerian dry extract HRS used to prepare the reference
solutioni in grams; Drying In air.
p percentage content of valerenic acid in val.erian dry exiract HRS.
----------------------~&
test solution. Furthermore, other violet zones may be present
IV-540 Valerian 2023
Sesquiterpenic acids
(710) (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 water-bath under a reflux condenser for 30 min. Allow
to cool and filter. Place the filter with the residue in the
100 mL round-bottomed flask. Add 20 mL of methanol Rl
and heat on a water-bath under the reflux condenser for
15 min. Allow to cool and filter. Combine the filtrates and
dilute to 50.0 mL with methanol Rl, rinsing the round-
bottomed flask and the filter.
Reference solution Dissolve an amount of valerian dry
extract HRS corresponding to 1.0 mg of valerenic acid in
Sonicate for 10 min and filter through a membrane filter
Column:
- size: l = 0.25 m, 0 = 4.6 mm;
(5 µm).
Mobile phase:
- mobile phase A: acetonitrile Rl, 5 g/L solution of phosphoric
acid R (20:80 V/V);
- mobile phase B: 5 g/L solution of phosphoric acid R,
acetonitrile Rl (20:80 V/V);

(min) (per cent V/JI) (per cent VIJI)
Figure 2526.-1. - Illustration for identification test A of powdered 0-5 55 45
herbal drug of cut valerian root 5 - 18 55 ➔ 20 45 ➔ 80
18 - 22 20 80
Top of the plate

Flow rate 1.5 mlJmin.
-- -- Detection Spectrophotometer at 220 nm.
Valerenic acid: a violet zone A violet zone (valerenic acid) Injection 20 µL.
Peak identification Use the chromatogram supplied with
Aceroxyvalerenic acid: a violet zone A violet zone (acetoxyvalerenic acid)
valerian dry extract HRS and the chromatogram obtained with
-- -- the reference solution to identify the peaks due to
acetoxyvalerenic acid and valerenic acid.
Reference solution Test solution - 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,
Foreign matter (2.8.2) expressed as valerenic acid, using the following expression:
of other foreign matter, determined on the herbal drug prior (A1 +A2) x m2 xpx 5
to cutting. A3 xm1
Loss on drying (2.2.32) area of the peak due to acetoxyvalerenic acid in the
Maximum 12.0 per cent, determined on 1.000 g ofwell- chromatogram obtained with the test solution;
homogenised powdered herbal drug (355) (2.9.12) by drying area of the peak due to valerenic acid in the chromatogram
in an oven at 105 °C for 2 h. area of the peak due to valerenic acid in the chromatogram
Total ash (2.4.16) obtained with the reference solution;
Maximum 12.0 per cent. mass of the herbal drug to be examined used to prepare the test
solution, in grams;
Ash insoluble in hydrochloric acid (2. 8.1) mass of valerian dry extract HRS used to prepare the reference
Maximum 5.0 per cent. solution, in grams;
p percentage content of valerenic acid in valerian dry extract HRS.
ASSAY
Essential oil (2.8.12) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
2000 mL flask, 500 mL of water R as the distillation liquid
and 0.50 mL of 1,2,4-trimethylbenzene R in the graduated
tube. Distil at a rate of 3-4 mlJmin for 4 h.
2023 Valerian Preparations IV-541
swirling. Add 40 mL of metharwl R and swirl for 1 h at

Valerian Dry Aqueous Extract 200 r/min. Filter the suspension into a volumetric flask and
(Ph. Bur. monograph 2400) rinse the conical flask with 3 quantities, each of 5 mL, of the
solvent mixture. Dilute to 100.0 mL with the solvent
mixture.
DEFINITION Reference solution (a) Dissolve a quantity of valerian dry
Extract produced from Valerian root (0453). extract HRS corresponding to 1.0 mg of valerenic acid in
Content methanol R and dilute to 10.0 mL with the same solvent.
Minimum 0.02 per cent of sesquiterpenic acids, expressed as Sonicate for 10 min and filter through a membrane filter
valerenic acid (C 15 H 22 0 2 ; Mr 234.3) (dried extract). (nominal pore size 0.45 µm).
PRODUCTION
Column:
procedure using water at not less than 60 °C.
- size: l = 0.25 m, 0 = 4 mm;
CHARACTERS - stationary phase: octadecylsilyl silica gel for chromatography R
Appearance (5 µm).
Brown or brownish, hygroscopic powder. Mobile phase:
IDENTIFICATION - mobile phase A: acetonitrile Rl, 5 g/L solution of phosphoric
Thin-layer chromatography (2.2.21). acid R (20:80 V/V);
Test solution Suspend 1.0 g of the extract to be examined in - mobile phase B: 5 g/L solution of phosphoric acid R,
10 mL of methanol R and sonicate for 10 min. Filter the acetonitrile Rl (20:80 V/V);
supernatant through a membrane filter (nominal pore size
0.45 µm). Use the filtrate as the test solution. (rnin) (per cent V/J,') (per cent V/J,')
Reference solution Dissolve 5 mg of acetoxyvalerenic acid R 0-5 55 45
and 5 mg of valerenic acid R in 20 mL of methanol R. 5 - 18 55 ➔ 20 45 ➔ 80
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel 18 - 22 20 80
Mobile phase glacial acetic acid R, ethyl acetate R, Flow rate 1.5 mUmin.
cyclohexane R (2:38:60 V/V/V). Detection Spectrophotometer at 220 nm.
Application 20 µL [or 5 µL] as bands of 10 mm [or 8 mm]. Injection 20 µL.
Development Over a path of 10 cm [or 6 cm]. Identification of peaks Use the chromatogram supplied with
Drying In air. valerian dry extract HRS and the chromatogram obtained with
Detection Spray with anisaldehyde solution R and heat at reference solution (a) to identify the peaks due to
100-105 °C for 5-10 min; examine in daylight. acetoxyvalerenic acid and hydroxyvalerenic acid.
Results See below the sequence of zones present in the Relative retention With reference to valerenic acid (retention
chromatograms obtained with the reference solution and the time= about 19 min): hydroxyvalerenic acid = about 0.2;
test solution. A faint violet zone due to valerenic acid may be acetoxyvalerenic acid = about 0.5.
present in the chromatogram obtained with the test solution. Calculate the percentage content of sesquiterpenic acids,
Furthermore, other zones may be present in the expressed as valerenic acid, using the following expression:
(A1 +A2)xm2xp x0.2
Top of the plate A3 xm1
- -
-- A1 area of the peak due to hydroxyvalerenic acid in the
Valerenic acid: a violet zone
A2 area of the peak due to acetoxyvalerenic acid in the
Acetoxyvalerenic acid: a violet zone A violet zone (acetoxyvalerenic acid) chromatogram obtained with the test solution;
A3 area of the peak due to valerenic acid in the chromatogram
-- --
A violet zone (hydroxyvalerenic acid) solution, in grams;
m2 mass of valerian dry extract HRS used to prepare reference
TESTS
ASSAY
Solvent mixture methanol R, water R (50:50 V/V).
Test solutwn In a 300 mL conical flask suspend 1.00 g of
the extract to be examined in 40 mL of water R whilst
IV-542 Valerian Preparations 2023
Reference solution Dissolve a quantity of valerian dry

Valerian Dry Hydroalcoholic extract HRS corresponding to 0.5 mg of valerenic acid in
Extract methanol Rl and dilute to 10.0 mL with the same solvent.
Sonicate for 10 min and filter through a membrane filter
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Column:
DEFINITION =
- size: l 0.25 m, 0 =
4.6 mm;
Extract produced from Valerian root (0453). - stationary phase: octadecylsilyl silica gel for chromatography R
(5 µm).
Content
Minimum 0.25 per cent m/m of sesquiterpenic acids, Mobile phase:
expressed as valerenic acid (C 15H 220 2 ; Mr 234.3) - mobile phase A: acetonitrile Rl, 5 g/L solution of phosphoric
(anhydrous extract). acid R (20:80 VIV);
- mobile phase B: 5 g/L solution of phosphoric acid R,
PRODUCTION acetonitrile Rl (20:80 V/V);
procedure using ethanol (30-90 per cent V/V) or methanol Time Mobile phase A Mobile phase B
(40-55 per cent V/V). (min) (per cent VIV) (per cent V/V)
CHARACTERS 0-5 55 45
5 - 18 55 ➔ 20 45 ➔ 80
Appearance
Brown, hygroscopic powder. 18 - 22 20 80
IDENTIFICATION
Test solution Suspend 1 g of the extract to be examined in
10 mL of methanol R and sonicate for 10 min. Filter the Injection 20 µL.
supernatant through a membrane filter (nominal pore size Identification of peaks Use the chromatogram supplied with
0.45 µm). valerian dry extract HRS and the chromatogram obtained with
Reference solution Dissolve 5 mg of acetoxyvalerenic acid R the reference solution to identify the peaks due to
and 5 mg of valerenic acid R in 20 mL of methanol R. hydroxyvalerenic acid, acetoxyvalerenic acid and valerenic
acid.
plate R (2-10 µm)]. System suitability Reference solution:
- relative retention with reference to valerenic acid (retention
Mobile phase glacial acetic acid R, ethyl acetate R,
time = about 19 min): hydroxyvalerenic acid = about 0.2;
cyclohexane R (2:38:60 VIVIV).
acetoxyvalerenic acid = about 0.5.
Calculate the percentage content of sesquiterpenic acids,
Development Over a path of 10 cm [or 6 cm]. expressed as valerenic acid, using the following expression:
Drying In air.
Detection Treat with anisaldehyde solution R and heat at (A1 +A2 +A3) x m 2 xp x 5
100-105 °C for 5-10 min; examine in daylight. A4xm1
area of the peak due to hydroxyvalerenic acid in the
chromatograms obtained with the reference solution and the chromatogram obtained with the test solution;
test solution. Furthermore, other violet zones may be present area of the peak due to acetoxyvalerenic acid in the
in the chromatogram obtained with the test solution. chromatogram obtained with the test solution;
Top of the plate area of the peak due to valerenic acid in the chromatogram
- - -- mass of the extract to be examined used to prepare the test
solution, in grams;
Valerenic acid: a violet zone A violet zone (valerenic acid) mass of valerian dry extract HRS used to prepare the reference
solution, in grams;
- - --
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
TESTS Valerian Tincture

Water (2.5.12) (Ph. Bur. monograph 1899)
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
ASSAY
Test solution Suspend 1.00 g of the extract to be examined Tincture produced from Valerian root (0453).
in 50.0 mL of methanol Rl, sonicate for 10 min and filter Content
through a membrane filter (nominal pore size 0.45 µm). Minimum 0.015 per cent m/m ofsesquiterpenic acids,
expressed as valerenic acid (C 15H 220 2 ; Mr 234.3).
2023 Verbena Herb IV-543
PRODUCTION Time Mobile phase A Mobile phase B

(min) (per cent V/Jl) (per cent V/Jl)
of ethanol (60 to 80 per cent V/V) by an appropriate 0- 5 55 45
5 - 18 SS ➔ 20 45 ➔ 80
procedure.
18 - 22 20 80
CHARACTERS
Appearance
Fww rate 1.5 mL/min.
Brown liquid.
IDENTIFICATION
Injection 20 µL.
Peak identificatwn Use the chromatogram supplied with
Test solutwn Dilute 5 mL of the tincture to be examined
valerian dry extract HRS and the chromatogram obtained with
with 5 mL of ethanol (70 per cent VIV,) R.
the reference solution to identify the peaks due to
Reference solutwn Dissolve 5 mg of acetoxyvalerenic acid R acetoxyvalerenic acid and valerenic acid.
and 5 mg of valerenic acid R in 20 mL of methanol R.
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel - relative retention with reference to valerenic acid (retention
plate R (2-10 µm)]. =
time about 19 min): acetoxyvalerenic acid about 0.5. =
Mobile phase glacial acetic acid R, ethyl acetate R, Calculate the percentage content of sesquiterpenic acids,
cycwhexane R (2:38:60 VIVIV). expressed as valerenic acid, using the following expression:
Development Over a path of 10 cm [or 6 cm]. (A1+A2)xm2xpx5
Drying In air. A 3 xm 1
Detection Spray with anisaldehyde solutwn R and heat at A, area of the peak due to acetoxyvalerenic acid in the
100-105 °C for 5-10 min; examine in daylight. chromatogram obtained with the test solution;
chromatograms obtained with the reference solution and the area of the peak due to valerenic acid in the chromatogram
test solution. Furthermore, other violet zones may be present obtained with the reference solution;
in the chromatogram obtained with the test solution. mass of the tincture to be examined used to prepare the test
solution, in grams;
mass of valerian dry extract HRS used to prepare the reference
Top of the plate solution, in grams;
- - --
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Valerenic acid: a violet zone A violet zone (valerenic acid)
-- --
2 faint or very faint violet zones Verbena Herb

PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
TESTS DEFINITION
Ethanol (2.9.10) Whole or fragmented, dried aerial parts of Verbena
95 per cent to 105 per cent of the quantity stated on the officinalis L. collected during flowering.
label.
Content
ASSAY Minimum 1.5 per cent ofverbenalin (C17H24010; Mr 388.4)
Liquid chromatography (2.2.29). (dried drug).
Test solutwn Dilute 10.0 g of the tincture to be examined to IDENTIFICATION
50.0 mL with methanol RJ. A. The stem is greenish-brown, quadrangular, longitudinally
Reference solutwn Dissolve an amount of valerian dry extract grooved and roughly hairy, especially on the angles.
HRS corresponding to 1.0 mg of valerenic acid in The larger leaves are petiolate and deeply pinnately lobed,
methanol Rl and dilute to 10.0 mL with the same solvent. with bluntly dentate margins, the smaller leaves are sessile,
Sonicate for 10 min and filter through a membrane filter not lobed, with crenate or dentate margins; the surfaces are
(nominal pore size 0.45 µm). rough and covered with bristly hairs, particularly over the
Column: veins, which are prominent on the lower surface. The flowers
- size: l = 0.25 m, 0 = 4.6 mm; are numerous, arranged in a slender spike in the axils of leaf-
- stationary phase: octadecylsilyl silica gel for chromatography R like bracts; the tubular calyx has 5 acutely pointed lobes with
(5 µm). the pale pink or lilac corolla forming a tube about twice as
Mobile phase: long as the calyx.
- mobile phase A: acetonitrile Rl, 5 g/L solution of phosphoric B. Microscopic examination (2.8.23). The powder is
acid R (20:80 V/V); greenish-brown. Examine under a microscope using chloral
- mobile phase B: 5 g/L solution of phosphoric acid R, hydrate solution R. The powder shows the following diagnostic
acetonitrile Rl (20:80 V/V); characters (Figure 1854.-1): fragments of the leaves, which in
surface view [C] show sinuous-walled epidermal cells [Ca]
with anisocytic [Cb] or anomocytic [Cc] stomata (2.8.3),
IV-544 Verbena Herb 2023
more numerous on the lower epidermis; fragments of stem Top of the plate
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 µm A brown or green zone

long, wide at the base and arising from the centre of a single
Arbutin: a blue or brown zone
ring of domed, spherical epidermal cells (surface view [B],
side view [D]); occasional glandular trichomes of 2 types: (a) An intense brownish-grey zone
long stalk with a flattened head about 35 µm in diameter and
Rutoside: a dark brownish-yellow
consisting of 4-8 radiating cells in side view [E] or in surface zone
view of the head [G], and (b) short unicellular stalk and an
enlarged ovate head composed of 4 radiating cells (surface -- --
view [Cd], transverse section [K]); triangular-ovoid or Reference solution Test solution
rounded pollen grains about 30 µm in diameter, with 3 pores
and a smooth exine m; many fragments of stems [F]
TESTS
consisting of groups of fibres [Fb], vessels [Fa] and
fragments of parenchyma [Fe]; isolated fragments of Aloysia citrodora
A. A lemon-like odour indicates the presence of Aloysia
fibres [H].
citrodora.
B. 1bin-layer chromatography (2.2.27).
(2.9.12) add 5 mL of methanol R. Heat in a water-bath at
60 °C for 10 min. Cool and filter.
Reference solution Dissolve 10 mg of arbutin R and 10 mg of
rutoside trihydrate R in methanol R and dilute to 10 mL with
the same solvent.
water R, ethyl acetate R (11 :11:27:100 V/VIVIV).
Drying In air.
100-105 °C for about 10 min; examine in daylight.
shows no intense blue or violet zone approximately at the
position of rutoside in the chromatogram obtained with the
reference solution.
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
herbal drug of verbena herb Liquid chromatography (2.2.29).
C. Examine the chromatograms obtained in test B for Aloysia Internal standard solution Dissolve 10.0 mg offerulic acid R
citrodora. in ethanol (60 per cent V/V) Rand dilute to 100.0 mL with
Results See below the sequence of zones present in the the same solvent.
chromatograms obtained with the reference solution and the Test solution To 1.00 g of the powdered herbal drug (710)
test solution. Furthermore, other zones may be present in the (2.9.12) add 50.0 mL of the internal standard solution and
chromatogram obtained with the test solution. stir with a magnetic stirrer for 2 h. Centrifuge for 15 min and
filter the supernatant using a membrane filter (nominal pore
size 0.45 µm).
Reference solution Dissolve the contents of a vial of
verbena/in CRS in the internal standard solution and dilute to
5.0 mL with the same solution.
Precolumn:
- size: l = 0.01 m, 0 = 4.0 mm;
(5 µm).
2023 Vitex Negundo Leaf IV-545
0 5 10 15 20 25 30 35 40 45 min
I . verbenalin 2. ferulic acid 3. unknown substance (may be absent) 4. acteoside
Figure 1854.-2. - Chromatogram for the assay of verbena herb: test solution
Column:
- size: l = 0.25 m, 0 = 4.0 mm;
Vitex Negundo Leaf
- stationary phase: octadecylsilyl silica gel for chromatography R DEFINITION
(5 µm); Vitex Negundo Leaf is the dried leaf of Vitex negundo L.
IDENTIFICATION
Mobile phase: A. Leaf usually broken coriaceous, compound, palmate,
- mobile phase A: 0.3 per cent V/V solution of phosphoric usually with 5 lanceolate leaflets but occasionally 3, leaf
acid R; margins vary from entire to serrate or dentate, petiole
- mobile phase B: acetonitrile R; 2.5-3.5 cm long. The middle leaflet is longer than the others,
Time Mobile phase A Mobile phase B up to 10 cm. In the pentafoliate leaf, the inner 3 leaflets are
(min) (per cent V/JI} (per cent V/J/) petiolulate up to 1 cm, and in the trifoliate leaf, only the
0 - 20 93--> 83 7 ➔ 17 inner leaflet is petiolate; in both the two outer leaflets are
20 - 30 83 17 subsessile. The upper surface of the leaf is glabrous whereas
30 - 35 83 ➔ 75 17 ➔ 25 the lower surface is densely tomentose.
B. Reduce to a powder. Examine under a microscope using
Flow rate 1.0 mUmin. chloral hydrate solution. Leaf epidermal cells cubical to
ovoid, those on the upper surface slightly larger than on the
lower, generally elongated over the veins, with straight to
Injection 20 µL. slightly wavy anticlinal walls. Paracytic stomata are present
System suitability Test solution: on the lower surface of the leaf only, which is also densely
- the chromatogram obtained is similar to the covered with trichomes. Uni to tricellular covering trichomes
chromatogram shown in Figure 1854.-2; abundant, the bi-cellular type being the most frequent.
- resolution: minimum 3.5 between the peaks due to ferulic Glandular trichomes have a uni-, bi- or tricellular stalk and
acid and acteoside. spherical unicellular head. Petiole epidermis similar to that of
Calculate the percentage content of verbenalin using the the leaf, with few stomata; glandular and non- glandular
following expression: trichomes are common. Chlorenchyma of the hypodermis
and thick walled parenchyma cells from the cortex of the
A1 xA 4 x m2 x 1000 petiole may be present.
A2 xA3 xm1 C. Carry out the method for thin-layer chromatography,
area of the peak due to verbenalin in the chromatogram Appendix III A, using the following solutions.
obtained with the test solution; (1) Reduce to a powder (425). To 0.5 g of powdered sample
area of the peak due to verbenalin in the chromatogram add 5 mL of methanol. Mix thoroughly and heat using a
area of the peak due to ferulic acid in the chromatogram water bath at 58° for 10 minutes and centrifuge or filter, use
obtained with the test solution; the supernatant or filtrate.
area of the peak due to ferulic acid in the chromatogram (2) 0.05% w/v each of hyperoside and rosmarinic acid in
mass of the herbal drug used to prepare the test solution, in
methanol.
grams; (3) Dilute 1 volume of solution (2) to 4 volumes with
mass of verbenalin in the reference solution, in grams. methanol.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
IV-546 Willow Bark 2023
(4) 0.05% w/v of hyperoside and 0.02% chlorogenic acid in quenching zone which may be intense, is present as seen in
methanol. the table and further quenching zones may be present.
CHROMATOGRAPHIC CONDITIONS When examined under UV light at 366 nm the
(a) Use high-performance silica gel 60 F 254 plates (Merck chromatogram obtained with solution (1) shows the
silica gel 60 F 254 HPTLC plates are suitable). fluorescent zones as shown in the table. Other fluorescent
zones may be present.
(c) Apply 2 µL of each solution as 8 mm bands. TESTS
Loss on drying
When dried for 2 hours at 105°, loses not more than 10.0%
(e) After the removal of the plate, dry in air for 5 minutes of its weight, Appendix IX D. Use 1 g.
and examine under UV light at 254 nm. Heat the plate to
100° for 3 minutes and dip the plate in a 0.5% w/v solution Total Ash
of 2-aminoethyl diphenylborinate in ethyl acetate. Dry the plate Not more than 8.0%, Appendix XI J, Method II.
and dip the plate in a 5% w/v solution of polyethylene glycol Foreign Matter
400 in dichloromethane. Examine under UV light at 366nm. Not more than 2%, Appendix XI D.
MOBILE PHASE
1 volume of water, 1 volume of formic acid and 8 volumes of
ethyl acetate.
Willow Bark
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with (Ph. Bur. monograph 1583)
solution (4) shows two clearly separated bands. Preparation
Willow Bark Dry Extract
Visualisation at 254nm
Top of the plate PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
A quenching zone or intense
quenching zone Rosmarinic Acid (A quenching Whole or fragmented dried bark of young branches or whole
zone)
dried pieces of current-year twigs of various species of genus
Salix including S. purpurea L., S. daphnoides Vill.
and S. fragilis L.
Content
Minimum 1.5 per cent of total salicylic derivatives, expressed
A quenching zone or faint
quenching zone Hyperoside (A quenching Hyperoside (A quenching as salicin (C 13H 18 O 7 ; Mr 286.3) (dried drug).
zone) zone)
A quenching zone or intense
quenching zone
Chlorogenic Acid (A very faint
quenching zone)
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-
Solution (1) Solutions (2) and (3) Solution (4) 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
Visualisation at 366nm short in the outer part and coarsely fibrous in the inner
Top oflhe plate region. The diameter of current-year twigs is not greater than
2 red or faint red fluorescent
10 mm. The wood is white or pale yellow.
zones
A faint light blue fluorescent
Rosmarinic Acid (A light blue
fluorescent zone}
yellow, greenish-yellow or light brown. Examine under a
zone microscope using chloral hydrate solution R. The powder
A faint or very faint yellow bundles [B, C] of narrow fibres [Ba, Ca], up to about
fluorescent zone
600 µm long, with very thick walls and surrounded by a
A faint or very faint light blue
fluorescent zone crystal sheath containing prisms of calcium oxalate [Bb, Cb];
A faint light blue fluorescent
Hyperoside (An orange Hyperos1de (An orange parenchymatous cells of the cortex [D, TI, with thick, pitted
zone
fluorescent zone) fluorescent zone)
Chlorogenic Acid (A faint blue
and deeply beaded walls [Da], and containing large cluster
A yellow fluorescent zone fluorescent zone) crystals of calcium oxalate [Ga, Ja]; some parenchyma cells
are collenchymatous [G]; uniseriate medullary rays
(tangential section [Db]); thickened cork cells (surface
Solution (1) Solutions (2) and (3) Solution (4) view [F]); numerous scattered prism crystals [E] and cluster
crystals [A] of calcium oxalate; fragments of brownish
collenchyma from the buds may also be present. Twigs show,
CONFIRMATION additionally, wood fragments [HJ composed of lignified
When examined under UV light at 254 nm the fibres [Ha] and vessels [Hb], sometimes accompanied by
chromatogram obtained with solution (1) shows two medullary rays [He].
quenching zones visible at a similar Rf to the quenching zone
due to hyperoside in the chromatogram obtained with
solution (2), the upper of the two quenching zones may be
faint, the lower of the zones may be intense. Another
2023 Willow Bark IV-54 7
Top of the plate
- - --
Several reddish-violet
zones may be present
Salicin: a reddish-violet A weak reddish-violet A reddish-violet zone

zone zone (salicin) (salicin)
-- --
Chlorogenic acid: a
brown zone
Reference solution Test solution (a) Test solution (b)
TESTS
Maximum 3 per cent of twigs with a diameter greater than
1O mm and maximum 2 per cent of other foreign matter.
Cadmium (2.4.27)
Maximum 2.0 ppm.
Maximum 11 per cent, determined on 1.000 g of the
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Figure 1583.-1. - Illustration for identification test B of powdered (2. 9.12) add 40 mL of methanol R and 40.0 mL of a 4.2 g/L
herbal drug of willow bark
solution of sodium hydroxide R. Heat in a water-bath at about
C. Thin-layer chromatography (2.2.27). 60 °C under a reflux condenser, with frequent shaking, for
about I h. After cooling, add 4.0 mL of a 103.0 g/L solution
Test solution (a) To 1.0 g of the powdered herbal drug of hydrochloric acid R. Filter the suspension into a 100 mL
(355) (2. 9.12) add 10 mL of methanol R. Heat on a water-
volumetric flask, wash and dilute to 100.0 mL with a mixture
bath at about 50 °C, with frequent shaking, for 10 min. Cool
of equal volumes of methanol R and water R. Filter through a
and filter.
Test solution (b) To 5.0 mL of test solution (a) add 1.0 mL
Reference solution Dissolve 5.0 mg of picein R in 25.0 mL of
of a 50 g/L solution of anhydrous sodium carbonate R and heat
a mixture of 20 volumes of water R and 80 volumes of
in a water-bath at about 60 °C for 10 min. Cool and filter if
methanol R (solution A). Dissolve 15.0 mg of salicin CRS in
necessary.
25 mL of a mixture of 20 volumes of water R and
Reference solution Dissolve 2 mg of salicin R and 2 mg of 80 volumes of methanol R; add 5.0 mL of solution A and
chlorogenic acid R in 1.0 mL of methanol R. dilute to 50.0 mL with water R.
plate R (2-10 µm)]. =
- size: l 0.10 m, 0 =
4.6 mm;
Mobile phase water R, methanol R, ethyl acetate R - stationary phase: octadecylsilyl silica gel for chromatography R
(8:15:77 VIVIV). (3 µm).
Application 10 µL [or 2 µL) as bands. Mobile phase:
Development Over a path of 15 cm [or 6 cm]. - mobile phase A: tetrahydrofuran R, 0.5 per cent V/V
solution of phosphoric acid R (1.8:98.2 V/V);
- mobile phase B: tetrahydrofuran R;
Detection Treat with a mixture of 5 volumes of sulfuric
acid R and 95 volumes of methanol R. Heat at I 00-105 °C
for 5 min and examine in daylight. (min) (per cent VIV) (per cent VIV)
Results See below the sequence of zones present in the 0 - 15 100 0
chromatograms obtained with the reference solution and test 15 - 17 100 ➔ 90 0 ➔ 10
solutions (a) and (b). Furthermore, other zones may be 17 - 23 90 10
present in the chromatograms obtained with test solutions (a)
and (b).
Injection I O µL.
Retention time Salicin = about 6.4 min; picein = about
7.7 min.
IV-548 Willow Preparations 2023
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.
Calculate the percentage content of total salicylic derivatives, Top of the plate
expressed as salicin, using the following expression:
-- --
Several reddish-violet
zones may be present
A1 area of the peak due to salicin in the chromatogram obtained Salicin: a reddish-violet A weak reddish-violet A reddish-violet zone
with the test solution; zone zone (salicin) (salicin)
A2 area of the peak due to salicin in the chromatogram obtained
with the reference solution; -- --
m1 mass of the herbal drug to be examined used to prepare the test Chlorogenic acid: a
solution, in grams; brown zone
m2 mass of salicin CRS used to prepare the reference solution, in
grams; Reference solution Test solution (a) Test solution (b)
p percentage content of salicin in salicin CRS.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur ASSAY
40 mL of methanol R and 40.0 mL of 0.1 M sodium
Willow Bark Dry Extract hydroxide. Heat in a water-bath at about 60 °C under a reflux
condenser, with frequent shaking, for about 1 h. After
(Ph. Bur. monograph 2312) cooling, add 4.0 mL of 1 M hydrochloric acid. Filter the
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ suspension into a 100 mL volumetric flask, then wash and
dilute to 100.0 mL with a mixture of equal volumes of
DEFINITION water R and methanol R. Filter through a membrane filter
Dry extract produced from Willow bark (1583). (nominal pore size 0.45 µm).
Content Reference solution Dissolve 5.0 mg of picein R in 25.0 mL of
Minimum 5.0 per cent of total salicylic derivatives, expressed a mixture of 20 volumes of water R and 80 volumes of
as salicin (C 13 H 180 7 ; Mr 286.3) (dried extract). methanol R (solution A). Dissolve 15.0 mg of salicin CRS in
PRODUCTION 25 mL of a mixture of 20 volumes of water R and
The extract is produced from the herbal drug by a suitable 80 volumes of methanol R. Add 5.0 mL of solution A and
procedure using either water or a hydroalcoholic solvent dilute to 50.0 mL with water R.
equivalent in strength to a maximum of 80 per cent V/V Column:
ethanol. =
- size: l 0.10 m, 0 4.6 mm; =
CHARACTERS
(3 µm).
Appearance
Yellowish-brown amorphous powder. Mobile phase:
- mobile phase A: tetrahydrofuran R, 0.5 per cent V/V
IDENTIFICATION solution of phosphoric acid R (1.8:98.2 V/V);
Thin-layer chromatography (2.2.27). - mobile phase B: tetrahydrofuran R;
Test solution (a) To 0.200 g of the extract to be examined
add 5 mL of methanol R. Sonicate for 5 min, filter and dilute Time Mobile phase A Mobile phase B
to 10 mL with methanol R. (min) (per cent V/Jl) (per cent V/Jl)
Test solution (b) To 5.0 mL of test solution (a) add 1.0 mL 0 - 15 100 0
15 - 17 100 ➔ 90 0 ➔ 10
of a 50 g/L solution of anhydrous sodium carbonate Rand heat
in a water-bath at about 60 °C for 10 min. Cool and filter if 17 - 23 90 10
23 - 25 90 ➔ 100 10 ➔ 0
necessary.
25 - 40 100 0
Reference solution Dissolve 2.0 mg of salicin Rand 2.0 mg of
chlorogenic acid R in 1.0 mL of methanol R.
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel Flow rate 1.0 mLJmin.
plate R (2-10 µm)]. Detection Spectrophotometer at 270 nm.
Mobile phase water R, methanol R, ethyl acetate R Injection 10 µL.
(8:15:77 VIVIV). Retention time Salicin = about 6.4 min;
Application 10 µL [or 2 µL] as bands. picein = about 7. 7 min.
Development Over a path of 15 cm [or 6 cm]. System suitability Reference solution:
Drying In a current of warm air. - resolution: minimum 1.5 between the peaks due to salicin
and picein.
Detection Spray with a mixture of 5 volumes of sulfuric
acid Rand 95 volumes of methanol R. Heat at 100-105 °C Calculate the percentage content of total salicylic derivatives,
for 5 min and examine in daylight. expressed as salicin, from the following expression:
Results See below the sequence of the zones present in the Ai xm2 xpx2
chromatograms obtained with the reference solution and test A2 xm1
solutions (a) and (b). Furthermore, other zones may be
2023 Withania Somnifera Root IV-549
area of the peak due to salicin in the chromatogram obtained (d) Develop the plate to 8 cm.
area of the peak due to salicin in the chromatogram obtained (e) After removal of the plate, dry in air and examine under
with the reference solution; ultraviolet light (254 nm). Immerse the plate in 5% v/v of
mass of the extract to be examined used to prepare the test methanolic sulfuric acid for 1 second, allow to dry in air, heat
solution, in grams;
at ll0° for 2 minutes and examine immediately in daylight.
mass of salicin CRS used to prepare the reference solution, in
grams; Examine the derivatised plate under ultraviolet light (366 nm).
p percentage content of salicin in salicin CRS. MOBILE PHASE
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
5 volumes of anhydrous formic acid, 15 volumes of ethyl acetate
and 50 volumes of toluene.
SYSTEM SUITABILITY
Withania Somnifera Root solution (2) shows three clearly separated bands.
DEFINITION CONFIRMATION
Withania Somnifera Root consists of the dried mature roots The bands with Rf values of approximately 0.1 (withaferin
of Withania somnifera (L.) Dunal. A), 0.26 (withanolide B) and 0.57 (P-sitosterol) in the
lt contains not less than 0.01 % withaferin A (C 28 H 38 0 6 ) and chromatogram obtained with solution (1) correspond in
itot less than 0.01 % withanolide A (C 29 H 42 0 7 ). colour and position to those in the chromatogram obtained
with solution (2).
PRODUCTION
TESTS
It is collected in winter, washed, dried and cut into short
pieces. Absence of Withania coagulans
In Identification test C, the derivatised plate under ultraviolet
IDENTIFICATION light (366 nm) shows no orange band with an Rf of
A. Pieces of root, cut into lengths of up to 8 cm, varying in approximately 0.2 in the chromatogram obtained with
diameter from 2 mm to 1 cm, with some narrower pieces of solution (1).
rhizome, often cut at the transition zone. Outer surface pale
Ash
greyish-brown, somewhat darker brown in larger specimens.
Fracture short, showing a whitish interior. The cut surface of
the root may show a distinction between the xylem and other Acid-insoluble ash
tissues marked by a faint yellow-green cambial ring. Not more than 1.0%, Appendix XI K.
B. Reduce to a powder (355). Examine under a microscope Loss on drying
using chloral hydrate solution. Cork cells in surface view When dried for 2 hours at 105°, loses not more than 12.0%
polygonal, in sectional view rectangular, thin-walled, of its weight. Use 1 g.
yellowish brown, often broken. Parenchymatous cells in ASSAY
groups, elongated, rectangular, or oval to round, filled with Carry out the method for liquid chromatography,
starch; some pitted, lightly lignified, found alongside vascular Appendix III D, using the following solutions.
fragments; parenchyma of the medullary rays, one or two
(1) Extract 1 g of the powdered drug with 3.0 mL of
cells wide shown crossing xylem elements at right angles;
methanol with the aid of ultrasound for 10 minutes, centrifuge
Occasional fragments of spiral, scalariform or pitted vessels
at 3000 rpm for 5 minutes and retain the supernatant
with broad lumen; tracheids and vessels usually heavily
extract. The extraction is repeated twice as described.
lignified, reticulate or bordered pitted, single or in small
Combine the three supernatant extracts, adjust the total
groups. Fibres often accompanying vessels, thick walled,
volume of the combined extracts to 20.0 ml with methanol
heavily pitted, and lignified; others less pitted and lignified,
and filter through a 0.45-µm filter.
thin walled, either found singly or in groups of two or three.
Microcrystals of calcium oxalate scattered or occasionally in (2) 0.02% w/v each of withaferin A CRS and
idioblasts. Examine under a microscope using a 50% v/v withanolide A CRS and 0.01 % w/v of withanolide B CRS in
solution of glycerol. Starch granules abundant, simple or 2 to methanol.
4 compound, round to oval, with a point, stellate or cleft CHROMATOGRAPHIC CONDITIONS
hilum. (a) Use a stainless steel column (15 cm x 4.6 mm) packed
C. Carry out the method for thin-layer chromatography, with dodecylsilyl silica gel for chromatography (4 µm)
Appendix III A, using the following solutions. (Phenomenex Synergi Max-RP soA is suitable).
(1) Shake 0.5 g of freshly powdered (355) drug with 1 mL of (b) Use gradient elution and the mobile phases described
dilute ammonia R4, add 10 mL of methanol, sonicate for below. Equilibrate the column with a mixture of 65% mobile
10 seconds, heat on a water-bath for 3 minutes, cool, filter, phase A and 35% mobile phase B for at least 15 minutes.
evaporate the filtrate to dryness at 60° and dissolve the dried (c) Use a flow rate of 1 mL per minute.
residue in 1 mL of methanol. Filter through a 0.45 µm filter. (d) Use a column temperature of 50°.
(2) 0.1 % w/v each of withaferin A CRS, withanolide B CRS (e) Use a detection wavelength of 230 nm.
and P-sitosterol in methanol.
MOBILE PHASE
(a) Use a silica gel F254 precoated high performance plate
(Merck silica gel F 254 HP1LC plates are suitable). Mobile phase A water.
(b) Use the mobile phase described below. Mobile phase B Equal volumes of ethanol (96%) and
methanol.
(c) Apply 2 µL of each of solution, as 6 mm bands.
IV-550 Wormwood 2023
Time
(Minutes)
Mobile phase A
% vlv
Mobile phase B
% vlv
Comments
Wormwood
0-5 65 35 isocratic (Ph. Bur. monograph 1380)
5-30 65 ➔ 55 35➔45 linear gradient Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
30-31 55 ➔ 0 45➔ 100 linear gradient
DEFINITION
31-36 0 100 isocratic
Basal leaves or slightly leafy, flowering tops, or mixture of
36-37 0➔65 100 ➔ 35 linear gradient these dried, whole or cut organs of Artemisia absinthium L.
37-45 65 35 re-equilibration Content
Minimum 2 mIJkg of essential oil (dried drug).
SYSTEM SUITABILITY IDENTIFICATION
The test is not valid unless, in the chromatogram obtained A. The leaves are greyish or greenish, densely tomentose on
with solution (2), the resolution facwr between the first two both surfaces. The basal leaves, with long petioles, have
main peaks, ofwithaferin A and withanolide A is at least 5.0 triangular or oval bipinnatisect or tripinnatisect lamina, with
and the symmetry facwr for both peaks is less than 1.3. rounded or Janceolate segments. The cauline leaves are less
segmented and the apical leaves are lanceolate. The stem of
the flower-bearing region is greenish-grey, tomentose, up to
Withaferin A Using the retention time and peak area of the 2.5 mm in diameter and usually with 5 flattened longitudinal
peak due to withaferin A in the chromatogram obtained with grooves. The capitula are arranged as loose, axillary panicles,
solution (2), locate and integrate the peak due to withaferin inserted at the level of the lanceolate or slightly pinnatisect
A in the chromatogram obtained with solution (1). leaves; they are spherical or flattened hemispherical, 2-4 mm
Calculate the content of withaferin A in the sample using the in diameter and consist of a grey, tomentose involucre, the
declared content withaferin A (C 28 H 38 0 6) in outer bracts linear, inner layer ovate, blunt at the apices with
withaferin A CRS and the following expression: scarious margins, a receptacle with very long paleae up to
1 mm or more long, numerous yellow, tubular,
hermaphroditic florets about 2 mm long and few yellow, ray
A, m, V, 100
-x-x-xpx--- florets.
Ai V2 mi 100-d B. Microscopic examination (2.8.23). The powder is
A1 Area of the peak due to withaferin A in the chromatogram obtained characters (Figure 1380.-1.): many T-shaped trichomes [A]
with solution (!). with a short uniseriate stalk consisting of 1-5 small cells,
A2 Area of the peak due to withaferin A in the chromatogram obtained
perpendicularly capped by a very long, undulating terminal
with solution (2).
m1 Weight of the drug being examined in mg. cell tapering at the ends; fragments of epidermises (surface
m2 Weight of withaferin A CRS in mg. view [D]) with sinuous or wavy walls, anomocytic stomata
VI Dilution volume of solution (I) in mL. (2.8.3) [Da], covering trichomes [Db] and glandular
V2 Dilution volume of solution (2) in mL.
trichomes containing oil [De] or not containing oil [Dd],
p Percentage content of withaferin A in withaferin A CRS.
d Percentage loss on drying of the herbal drug being examined. each with a short, biseriate, 2-celled stalk and a biseriate
head with 2-4 cells; free glandular trichomes (side view [Cl);
Withanolide A Using the retention time and peak area of fragments of the corollas of the tubular and ray florets, some
the peak due to withanolide A in the chromatogram obtained containing small cluster crystals of calcium oxalate [H];
with solution (2), locate and integrate the peak due to numerous paleae each composed of a small cell forming a
withanolide A in the chromatogram obtained with stalk and a very long, cylindrical and thin-walled terminal cell
solution ( 1). about 1-1.5 mm long, either whole [E] or limited to the
Calculate the content of withanolide A in the sample using distal part [BJ; spheroidal pollen grains, about 30 µm in
diameter, with 3 pores and a finely warty exine [G];
the declared content of withanolide A (C 29H 42 0 7 ) in
withanolide A CRS and the following expression: fragments of vascular tissue from the leaves [F] or the
stems fJ] consisting of vessels with spiral or annular
thickenings [Fa], or with bordered pits CTa], fibres [Fb, Jb]
A, m, V, 100 and parenchymatous cells with pitted, moderately thickened
-x-x-xpx--- walls [Fe, Jc].
A2 V, mi 100-d
Test solution Place 2 g of the powdered herbal drug (355)
Area of the peak due to withanolide A in the chromatogram (2. 9.12) in 50 mL of boiling water R and allow to stand for
obtained with solution (I). 5 min, shaking the flask several times. After cooling, add
Area of the peak due to withanolide A in the chromatogram 5 mL of a 100 g/L solution of lead acetate R. Mix and filter.
Rinse the flask and the residue on the filter with 20 mL of
Weight of the drug in mg.
Weight of withanolide A CRS in mg. water R. Shake the filter with 50 mL of methylene chloride R.
Dilution volume of solution (I) in mL. Separate the organic layer, dry over anhydrous sodium
Dilution volume of solution (2) in mL. sulfate R, filter and evaporate the filtrate to dryness on a
Percentage content withanolide A in withanolide A CRS.
water-bath. Dissolve the residue in 0.5 mL of ethanol
(96 per cent) R.
STORAGE Reference solution Dissolve 2 mg of methyl red R and 2 mg of
resorcinol R in 10.0 mL of methanol R.
Withania Somnifera Root should be protected from moisture.
2023 Yarrow IV-551
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 Ras the distillation liquid. Add 0.5 mL
of xylene R in the graduated tube. Distil at a rate of
2-3 mIJmin for not less than 3 h.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Yarrow
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Whole or cut, dried flowering tops of Achillea millefolium L.
Content
- essential oil: minimum 2 mIJkg (dried drug);
- proazulenes, expressed as chamazulene (C 14H 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
herbal drug of wormwood
the end of the stem. Each capitulum, 3-5 mm in diameter,
Plate TLC silica gel plate R. consists of the receptacle, usually 4-5 ligulate ray-florets and
Mobile phase acetone R, glacial acetic acid R, toluene R, 3-20 tubular disk-florets. The involucre consists of 3 rows of
methylene chloride R (10:10:30:50 VIVIVIV). 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
Drying In air. tubular disk-florets with a radial, five-lobed, yellowish or light
Detection A Spray with acetic anhydride - sulfuric acid brownish corolla. The pubescent green, partly brown or
solution R and examine in daylight. violet stems are longitudinally furrowed, up to 3 mm thick
Results A The chromatogram obtained with the test solution with a light-coloured medulla.
shows a blue zone due to artabsin shortly above a red zone B. Microscopic examination (2.8.23). The powder is green or
due to methyl red in the chromatogram obtained with the greyish-green. Examine under a microscope using chloral
reference solution. hydrate solution R. The powder shows the following diagnostic
Detection B Examine in daylight while heating at characters (Figure 1382.-1): fragments of the stem epidermis
100-105 °C for 5 min. (surface view [K]), with cells having a smooth cuticle and
Results B The chromatogram obtained with the reference anomocytic stomata (2.8.3); fragments of leaf and bract
solution shows in the middle third a red zone due to methyl epidermises (surface view [B]), with cells having wavy and
red and below it a light pink zone due to resorcinol. irregularly thickened walls, a finely striated cuticle and
The chromatogram obtained with the test solution shows an anomocytic stomata (2.8.3); very rare glandular trichomes
intense red or brownish-red zone due to absinthin with a with a short stalk and a head formed of 2 rows of 3-5 cells
similar Rp value to that of the zone due to resorcinol in the enclosed in a bladder-like membrane [HJ; uniseriate, whole
chromatogram obtained with the reference solution. Other or fragmented covering trichomes [A] consisting of 4-6 small,
zones are visible, but less intense than that due to absinthin. more or less isodiametric cells at the base and a thick-walled,
often somewhat tortuous terminal cell, about 400 µm to
TESTS greater than 1000 µm long; fragments of the ligulate corolla
Foreign matter (2.8.2) with papillary epidermal cells [D]; fragments of the corolla
Maximum 5 per cent of stems with a diameter greater than tubes, with sinuous epidermal cells, covered by a thin striated
4 mm and maximum 2 per cent of other foreign matter. cuticle (surface view [F]); small-celled parenchyma from the
Bitterness value (2. 8.15) corolla tubes containing cluster crystals of calcium
Minimum 10 000. oxalate [E]; groups of lignified and pitted cells from the
Loss on drying (2.2.32) bracts [G]; spherical pollen grains, about 30 µm in diameter,
Maximum 10.0 per cent, determined on 1.000 g of the with 3 germinal pores and a spiny exine [C]; groups of
powdered herbal drug (355) (2. 9.12) by drying in an oven at sclerenchymatous fibres and small vessels with spiral or
105 °C for 2 h. annular thickening, from the stem W.
IV-552 Zanthoxylum Bungeanum Pericarp 2023
obtained with the reference solution. Furthermore, other faint

zones may be present.
TESTS
3 mm and maximum 2 per cent of other foreign matter.
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
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 grycol R as the distillation liquid.
Add 0.50 mL of 1,2,4-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,
Figure 1382.-1. - Illustration for identification test B of powdered transfer the blue mixture of essential oil and
herbal drug of yarrow 1,2,4-trimethylbenzene obtained in the assay of essential oil
into a 50 mL volumetric flask with the aid of small portions
C. To 2.0 g of the powdered herbal drug (710) (2.9.12) add
of xylene R, rinsing the graduated tube of the apparatus with
25 mL of ethyl acetate R, shake for 5 min and filter.
xylene R, and dilute to 50.0 mL with the same solvent.
Evaporate to dryness on a water-bath and dissolve the
Measure the absorbance (2.2.25) at 608 nm using xylene Ras
residue in 0.5 mL of toluene R (solution A). To 0.1 mL of
the compensation liquid.
this solution add 2.5 mL of dimethylaminobenzaldehyde
solution RB and heat on a water-bath for 2 min. Allow to Calculate the percentage content of proazulenes, expressed as
cool. Add 5 mL of light petroleum R and shake the mixture chamazulene, using the following expression:
vigorously. The aqueous layer shows a blue or greenish-blue
A x2.l
colour.
m
D. Thin-layer chromatography (2.2.27).
Test solutwn Use solution A prepared in identification i.e. taking the specific absorbance of chamazulene to be 23.8.
test C.
Reference solution Dissolve 10 mg of cineole R and 10 mg of A absorbance at 608 nm;
rn mass of the herbal drug to be examined, in grams.
guaiazulene R in 20 mL of toluene R.
Plate TLC silica gel plate R. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Drying In air. Zanthoxylum Bungeanum Pericarp
Detection Treat with anisaldehyde solution R, heat at (Ph. Bur. monograph 2656)
100-105 °C for 5-10 min and examine in daylight.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Results The chromatogram obtained with the reference
solution shows in the upper part a red zone (guaiazulene) DEFINITION
and in the middle part a blue or greyish-blue zone (cineole). Dried pericarp of the ripe fruit, with seeds removed, of
The chromatogram obtained with the test solution shows a Zanthoxylum bungeanum Maxim.
violet zone a little above the zone due to guaiazulene in the
Content
chromatogram obtained with the reference solution; below Minimum 15 mL/kg of essential oil (anhydrous drug).
this zone a reddish-violet zone; below which, 1-2 not clearly
separated greyish-violet or greyish zones (which changes to IDENTIFICATION
greenish-grey after a few hours) and a reddish-violet zone a A. Mostly single, dehisced spherical follicles, split along the
little above the zone due to cineole in the chromatogram ventral suture; the dehisced fruit is 4-6 mm in diameter, one
half-follicle is 3.0-4.5 mm in diameter; 1-3 smaller, aborted
2023 Zanthoxylum Bungeanum Pericarp IV-553
fruits are often present near the base of the dehisced, ripe Test solution To 0.5 g of the powdered herbal drug (355)
fruit(s); the outer surface is dark red or brownish-red, with (2. 9.12) add 5 mL of methanol R. Sonicate for 10 min.
numerous convex, translucent, often yellowish-brown Centrifuge or filter. Use the supernatant or filtrate.
verrucose oil dots; the inner surface is light yellow and Reference solution Dissolve 1 mg of chlorogenic acid R and
smooth; the endocarp is mostly separated from the mesocarp 1 mg of emodin R in 1 mL of methanol R.
at the base and is rolled up. Remains of the narrow pedicel Plate TLC silica gel plate R (2-10 µm).
are often present.
B. Microscopic examination (2.8.23). The powder is (10:20:80 VIVIV).
yellowish-brown or reddish-brown. Examine under a
microscope using chloral hydrate solution R. The powder Application 5 µL as bands of 8 mm.
shows the following diagnostic characters (Figure 2656.-1): Development Over a path of 6 cm.
fragments of the epicarp [A], covered by a striated cuticle, Drying In air.
consisting of polygonal cells with rigid walls (surface view Detection Examine in ultraviolet light at 366 nm.
[Aa], transverse section [Ba, Gal) and anomocytic stomata Results See below the sequence of zones present in the
(2.8.3) with 5-7 subsidiary cells [Ab], some cells of the
epicarp contain orange-yellow granular contents; fragments of
the mesocarp (surface view [F]) and of the mesocarp
associated with epicarp cells covered with a striated cuticle solution.
(transverse section [B, G]), consisting of parenchymatous
cells [Bb, Fa, Gb] with some cells containing one [Be, Fb,
Top of the plate
Ge] or more [Fe] calcium oxalate cluster crystals; rare
fragments of oil glands with elongated cells containing
droplets of essential oil [Bd]; vascular bundles with spiral
vessels [C, E] accompanied by fibres with thickened, pitted Emodin: a yellow fluorescent zone A green fluorescent zone
walls and pointed ends [Ca, Ea], sometimes bifurcated [Cb];
occasional prisms of calcium oxalate [Eb]; fragments of - - --
endocarp consisting of several layers of narrow, elongated, A red fluorescent zone may be
thick-walled, pitted cells in a parquetry arrangement [DJ, in present
transverse section the inner cell layers are perpendicular to A faint green fluorescent zone
the outer cell layers.
2 green fluorescent zones
-- --
Chlorogenic acid: a blue fluorescent A blue fluorescent zone

zone
TESTS
Maximum 5 per cent of seeds and maximum 2 per cent of
Water (2.2.13)
Total ash (2.4.16)
ASSAY
Essential oil (2. 8. 12)
Use 15.0 g of the freshly powdered herbal drug (710)
(2.9.12), a 500 mL round-bottomed flask, 250 mL of water R
herbal drng of zanthoxylum bungeanum pericarp
as the distillation liquid and 0.50 mL of xylene R in the
C. Thin-layer chromatography (2.2.27). graduated tube. Distil at a rate of 2-3 mUmin for 2 h.
~ - - - - - - - - - - - - - - Ph Eur
Monographs
Materials for use in the Manufacture of

Homoeopathic Preparations
2023 Homoeopathic Preparations IV-557
Mother tinctures comply with the requirements of the

Homoeopathic Preparations monograph Mother tinctures for homoeopathic
preparations (2029).
Glycerol macerates are liquid preparations obtained from raw
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
materials of botanical, zoological or human origin by using
glycerol or a mixture of glycerol and either ethanol of a
DEFINITION
suitable concentration or a solution of sodium chloride of a
Homoeopathic preparations are prepared from substances,
suitable concentration.
products or preparations called stocks, in accordance with a
homoeopathic manufacturing procedure. A homoeopathic Potentisation
preparation is usually designated by the Latin name of the Dilutions and triturations are obtained from stocks by a
stock, followed by an indication of the degree of dilution process of potentisation in accordance with a homoeopathic
and/or potentisation, if applicable. manufacturing procedure: this means successive dilutions and
succussions, or successive appropriate triturations, or a
Raw materials
combination of the 2 processes.
Raw materials for the production of homoeopathic
preparations may be of natural or synthetic origin. The potentisation steps are usually one of the following:
- 1 part of the stock plus 9 parts of the vehicle; they may be
For raw materials of zoological or human origin, adequate
designated as 'D', 'DH' or 'X' (decimal);
measures are taken to minimise the risk of agents of
- 1 part of the stock plus 99 parts of the vehicle; they may
infection, including viruses (5. J. 7), in the homoeopathic
be designated as 'C' or 'CH' (centesimal).
preparations. For this purpose, it is demonstrated that:
- the method of production includes a step or steps that The number of potentisation steps defines the degree of
have been shown to remove or inactivate agents of dilution; for example, 'D3', '3 DH' or '3X' means 3 decimal
infection; potentisation steps, and 'C3', '3 CH' or '3C' means
- where applicable, raw materials of zoological origin 3 centesimal potentisation steps.
comply with the monograph Products with nsk of 'LM' potencies are manufactured according to a specific
transmitting agents of animal spongiform procedure with a 50 000 dilution factor by alternate steps of
encephalopathies (1483); liquid dilution and impregnation of pillules. The number of
- where applicable, the animals and the tissues used to potentisation steps defines the degree of dilution, for
obtain the raw materials comply with the health example, 3rd LM means 3 successive LM dilutions.
requirements of the competent authorities for animals for Dosage forms
human consumption; A dosage form of a homoeopathic preparation complies with
- for materials of human origin, the donor follows the any relevant dosage form monograph in the European
recommendations applicable to human blood donors and Pharmacopoeia, and with the following:
to donated blood (see Human plasma for - for the purpose of dosage forms for homoeopathic use,
fractionation (0853)), unless otherwise justified and 'active substances' are considered to be 'dilutions or
authorised. triturations of homoeopathic stocks' or 'homoeopathic
A raw material of botanical, zoological or human origin may stocks' (in case of a mother tincture or a glycerol
be used either in the fresh state or in the dried state. Where macerate);
appropriate, fresh material may be kept deep-frozen. - these dosage forms can contain one or more 'active
Raw materials of botanical origin comply with the substances';
requirements of the monograph Herbal drugs for homoeopathic - they are prepared using appropriate excipients.
preparations (2045). Homoeopathic dosage form 'pillule'
Where justified and authorised for transportation or storage Pillules for homoeopathic use are solid preparations obtained
purposes, fresh plant material may be kept in ethanol from sucrose, lactose or other suitable excipients. Pillules for
(96 per cent) or in ethanol of a suitable concentration, homoeopathic preparations (2153) are intended for
provided the whole material including the storage medium is impregnation or coating with one or more homoeopathic
used for processing. preparations. The impregnated pillules comply with the
Raw materials comply with any requirements of the relevant requirements of the monograph Homoeopathic pillules,
monographs of the European Pharmacopoeia. impregnated (2079). The coated pillules comply with the
Vehicles requirements of the monograph Homoeopathic pillules, coated
Vehicles are excipients used for the preparation of certain (2786). Both are intended for sublingual or oral use.
stocks or for the potentisation process. They may include, for Homoeopathic dosage form 'tablet'
example: purified water, ethanol of a suitable concentration, Tablets for homoeopathic use are solid preparations obtained
glycerol and lactose. from sucrose, lactose or other suitable excipients according to
Vehicles comply with any requirements of the relevant the monograph Tablets (0478). They may be prepared either
monographs of the European Pharmacopoeia. by compressing one or more 'active substances' with the
Stocks excipients or by impregnating preformed tablets with one or
Stocks are substances, products or preparations used as more liquid 'active substances'. The preformed tablets for
starting materials for the production of homoeopathic impregnation are obtained from sucrose, lactose or other
preparations. A stock is usually one of the following: a suitable excipients according to the monograph
mother tincture or a glycerol macerate, for raw materials of Tablets (0478). Tablets for homoeopathic use are intended
botanical, zoological or human origin, or the substance itself, for sublingual or oral use.
for raw materials of chemical or mineral origin. Homoeopathic dosage forms 'parenteral preparation', 'eye
preparation', 'nasal preparation'
IV-558 Homoeopathic Preparations 2023
For the last potentisation step(s), an ethanol-free vehicle is exudates that have not been subjected to a specific treatment
used to minimise the content of ethanol in the final are also considered to be herbal drugs for homoeopathic
preparation. preparations. Herbal drugs for homoeopathic preparations
The residual ethanol content (2. 9.10) is not greater than are precisely defined by the botanical scientific name of the
1 per cent V/V unless otherwise justified and authorised. source species according to the binomial system (genus,
Homoeopathic dosage form 'liquid preparation for oral use' species, variety and author).
If necessary, for the last potentisation step (s), purified water
Whole Describes a herbal drug for homoeopathic
is used to minimise the content of ethanol in the final preparations that has not been reduced in size and is
preparation. presented, dried or undried, as harvested.
Manufacturing methods Fragmented Describes a herbal drug for homoeopathic
preparations that has been reduced in size after harvesting to
Homoeopathic preparations are produced using the methods permit ease of handling, drying and/or packaging.
described in the monograph Methods of preparation of
homoeopathic stocks and potentisation (2371). The preparations Broken Describes a herbal drug for homoeopathic
obtained using the manufacturing methods listed in preparations in which the more fragile parts of the plant have
Table 1038.-1 are restricted to the production of the broken during drying, packaging or transportation.
corresponding dosage forms indicated in the right-hand For dried herbal drugs for homoeopathic preparations, cut
column of the table. describes size reduction, other than powdering, that reduces
The competent authority has the right to accept or reject the particle size below that which is described in the
particular combinations of manufacturing method and macroscopic identity of the herbal drug for homoeopathic
preparations.
substance.
PRODUCTION
Table 1038.-1. Herbal drugs for homoeopathic preparations are obtained
Manufacturing methods Dosage forms from cultivated or wild plants. Suitable collection, cultivation,
harvesting, sorting, drying, fragmentation and storage
1.4.1, 1.4.2, 1.4.3, 1.4.4 Eye drops
conditions are essential to guarantee the quality of herbal
Coated homoeopathic pillules
Solutions for injection drugs for homoeopathic preparations.
Herbal drugs for homoeopathic preparations are, as far as
2.1.2 Eye drops
Solutions for injection
possible, free from impurities such as soil, dust, dirt and
Nasal preparations other contaminants such as fungal, insect and other animal
contaminants. They do not present signs of decay.
2.2.1, 2.2.2, 2.2.3 Eye drops
Coated homoeopathic pil!ules If a decontaminating treatment has been used, it is necessary
Solutions for injection to demonstrate that the constituents of the plant are not
Nasal preparations affected and that no harmful residues remain. The use of
Ointments, creams and gels ethylene oxide is prohibited for the decontamination of
Oral powders (triturations)
Suppositories
herbal drugs for homoeopathic preparations.
Fresh herbal drugs are processed as rapidly as possible after
2.2.4 Solutions for injection harvesting. Where justified and authorised for transportation
3.1.2, 3.2.2 Eye drops or storage purposes, fresh plant material may be deep-frozen;
Coated homoeopathic pillules it may also be kept in ethanol (96 per cent) or in ethanol of a
Solutions for injection suitable concentration, provided the whole material including
Nasal preparations
the storage medium is used for processing.
Ointments, creams and gels
Suppositories Adequate measures have to be taken in order to ensure that
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.
Herbal Drugs for Homoeopathic IDENTIFICATION

Herbal drugs for homoeopathic preparations are identified
Preparations using their macroscopic and, where necessary, microscopic
Herbal Drugs for Homoeopathic Use descriptions and any further tests that may be required (for
example, thin-layer chromatography).
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ TESTS
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
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
member state. In the absence of such a monograph, the state Where a dried plant is used as a starting material for the
in which the herbal drug is used has to be defined. Certain manufacture of homoeopathic preparations, carry out a test
for foreign matter, unless otherwise prescribed in the

individual monograph. The content of foreign matter is not
Methods of Preparation of
more than 2 per cent mlm, unless otherwise prescribed or Homoeopathic Stocks and
justified and authorised.
Potentisation
Adulteration
A specific appropriate test may apply to herbal drugs for (Ph. Bur. monograph 2371)
homoeopathic preparations liable to be falsified.
Loss on drying (2.2.32) Homoeopathic stocks are prepared, using suitable methods,
Carry out a test for loss on drying on dried herbal drugs for from raw materials that comply with the requirements of the
homoeopathic preparations. monograph Homoeopathic preparations (1038). The methods
If a fresh plant is processed more than 24 h after harvesting, described below, combined with established methods for
a test for loss on drying should be carried out. The minimum potentisation, are examples of methods, but other methods
limit is indicated in the individual monograph. described in an official national pharmacopoeia of a Member
Water (2.2.13) State may equally be used.
A determination of water is carried out on herbal drugs for Where material of animal or human origin is to be used,
homoeopathic preparations with a high essential oil content. particular reference is made to the requirements concerning
Pesticides (2. 8.13) the use of such raw material in the monograph Homoeopathic
Herbal drugs for homoeopathic preparations comply with the preparations (1038).
requirements for pesticide residues. The requirements take In the preparation of hydroalcoholic liquid dilutions, the
into account the origin and the nature of the plant, where concentration of ethanol prescribed in the method may, if
necessary the preparation in which the plant might be used necessary, be replaced by ethanol (36 per cent V/V) or
and, where available, knowledge of the complete record of ethanol (18 per cent V/V).
treatment of the batch of the plant. Where justified, the test When the individual monograph allows that the mother
for pesticides may be performed on the mother tincture tincture be prepared from more than one plant species, the
according to the requirements of the general monograph mother tincture can be prepared from the specified parts of
Mother tinctures for homoeopathic preparations (2029). an individual plant species or from any mixture thereof. If for
If appropriate, herbal drugs for homoeopathic preparations comply the preparation of a mother tincture the loss on drying has to
with other tests, such as the following, for example. be determined, the herbal drug or mixture of herbal drug
Total ash (2.4.16) with ethanol has to be processed immediately after the value
of the loss on drying has been determined.
Bitterness value (2. 8. 15)
Unless otherwise stated in the individual monographs:
Heavy metals (2.4.27) - for the preparation of stocks, the term 'part(s)' denotes
Unless otherwise stated in an individual monograph or unless 'mass part(s)';
otherwise justified and authorised: - for potentisation, the term 'part(s)' denotes 'mass part(s)',
- cadmium: maximum 1.0 ppm; except for Methods 1.1.10, 1.1.11, 2.1.3 and 3.2.3, where
- lead: maximum 5.0 ppm; 'part(s)' denotes 'volume part(s)' or 'mass part(s)'.
- mercury: maximum 0.1 ppm.
1. MOTHER TINCTURES AND LIQUID
If justified by the nature or origin of the herbal drug or if
POTENTISATIONS
required by the competent authority, suitable limits for the
METHOD 1.1. HYDROALCOHOLIC MOTHER
content of other heavy metals such as arsenic or nickel are
TINCTURES PREPARED WITHOUT HEATING
defined.
Unless otherwise stated, mother tinctures are prepared by
Where justified, the test for heavy metals may be performed maceration lasting 10-30 days.
on the mother tincture according to the requirements of the
Maceration may be replaced by long maceration (maximum
general monograph Mother tinctures for homoeopathic
60 days) or very long maceration (maximum 180 days),
provided it is demonstrated that the quality of the resulting
Aflatoxin B 1 (2. 8. 18) mother tincture is the same as that of the mother tincture
Where appropriate, limits for aflatoxins may be required. prepared by maceration.
Ochratoxin A (2.8.22) Unless otherwise stated in the method, the maximum
Where appropriate, a limit for ochratoxin A may be required. temperature during the preparation is 25 °C.
Radioactive contamination METHOD 1.1.1 (HOMOOPATHISCHES ARZNEIBUCH (HAB) lA:
In some specific circumstances, the risk of radioactive MOTHER TINCTURES AND LIQUID DILUTIONS)
contamination is to be considered. Method 1.1.1 is used for fresh herbal drugs containing
ASSAY generally more than 70 per cent of expressed juice and no
Where applicable, herbal drugs for homoeopathic essential oil or resin or mucilage. Mother tinctures prepared
preparations are assayed by an appropriate method. according to Method 1.1.1 are mixtures of equal parts of
expressed juices and ethanol (90 per cent V/V).
STORAGE
Express the comminuted herbal drug. Immediately mix the
Store dried herbal drugs protected from light.
expressed juice with an equal mass of ethanol
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur (90 per cent V/V). Allow to stand in a closed container for
not less than 5 days, then filter.
Adjustment to any value specified in the individual - 7 parts of ethanol (36 per cent V/V).
monograph The 2nd decimal dilution (D2) is made from:
Determine the percentage dry residue (2. 8.16) or, where - 1 part of the 1st 'decimal' dilution;
prescribed, the percentage assay content of the above- - 9 parts of ethanol (18 per cent V/V).
mentioned filtrate. Calculate the amount (A 1), in kilograms, Subsequent decimal dilutions are produced as stated for D2.
of ethanol (50 per cent V/V) required, using the following
METHOD 1.1.3 (HAB 2A: MOTHER TINCTURES AND LIQUID
expression:
DILUTIONS)
mx (Nx -No) Method 1.1.3 is used for fresh herbal drugs containing
No generally less than 70 per cent of expressed juice and more
than 60 per cent moisture (determined by loss on drying)
m mass of filtrate, in kilograms; and no essential oil or resin.
N0 percentage dry residue or percentage assay content as required
in the individual monograph; Mother tinctures prepared according to Method 1.1.3
Nx percentage dry residue or percentage assay content of the (ethanol content approximately 50 per cent V/V) are
filtrate. prepared by maceration as described below.
Comminute appropriately the herbal drug.
Mix the filtrate with the calculated amount of ethanol
(50 per cent V/V). Allow to stand for not less than 5 days, Take a sample and determine the loss on drying (2.2.32).
then filter if necessary. Unless otherwise prescribed, determine the loss on drying on
2.00-5.00 g of the comminuted herbal drug and dry at
Potentisation 105 °C for 2 h.
The 1st 'decimal' dilution (D 1) is made from:
- 2 parts of the mother tincture; To the comminuted herbal drug immediately add not less
- 8 parts of ethanol (50 per cent V/V). than half the mass of ethanol (90 per cent V/V) and allow to
stand in well-closed containers.
The 2nd decimal dilution (D2) is made from:
- 1 part of the 1st 'decimal' dilution; Use the following expression to calculate the amount (A 2), in
- 9 parts of ethanol (50 per cent VIV). kilograms, of ethanol (90 per cent V/V) required for the mass
(m) of raw material, then subtract the amount of ethanol
Subsequent decimal dilutions are produced as stated for D2. (90 per cent V/V) already added and add the difference to
The 1st 'centesimal' dilution (C 1) is made from: the mixture.
- 2 parts of the mother tincture;
- 98 parts of ethanol (50 per cent V/V). mxT
The 2nd centesimal dilution (C2) is made from: 100
- 1 part of the I st 'centesimal' dilution; m mass of raw material, in kilograms;
- 99 parts of ethanol (50 per cent V/V). T percentage loss on drying of the sample.
Subsequent centesimal dilutions are produced as stated for
C2. Allow to stand for not less than 10 days, swirling from time
METHOD 1.1.2 (HAB 18: MOTHER TINCTURES AND LIQUID to time, then express the mixture and filter the resulting
DILUTIONS) liquid.
Method 1.1.2 is used where the fresh latex of a herbal drug Adjustment to any value specified in the individual
is to be processed. monograph
Mother tinctures prepared according to Method 1.1.2 are Determine the percentage dry residue (2.8.16) or, where
mixtures of fresh plant latex with ethanol (36 per cent V/V). prescribed, the percentage assay content of the above-
Mix the fresh latex with 2 parts by mass of ethanol mentioned filtrate. Calculate the amount (A 1), in kilograms,
(36 per cent VIV) and filter. of ethanol (50 per cent V/V) required, using the following
expression:
Adjustment to any value specified in the individual
monograph mx(Nx-No)
Determine the percentage dry residue (2. 8.16) or, where No
prescribed, the percentage assay content of the above-
mentioned filtrate. Calculate the amount (A 1 ), in kilograms, m mass of filtrate, in kilograms;
of ethanol (36 per cent VIV) required, using the following N0 percentage dry residue or percentage assay content as required
in the individual monograph;
expression: Nx percentage dry residue or percentage assay content of the
filtrate.
mx(Nx-No)
No Mix the filtrate with the calculated amount of ethanol
(50 per cent V/V). Allow to stand for not less than 5 days,
m mass of filtrate, in kilograms;
NO percentage dry residue or percentage assay content as required then filter if necessary.
in the individual monograph; Potentisation
Nx percentage dry residue or percentage assay content of the
filtrate.
The 1st 'decimal' dilution (Dl) is made from:
Mix the filtrate with the calculated amount of ethanol - 8 parts of ethanol (50 per cent V/V).
(36 per cent V/V). Allow to stand for not less than 5 days, The 2nd decimal dilution (D2) is made from:
then filter if necessary. - 1 part of the 1st 'decimal' dilution;
Potentisation - 9 parts of ethanol (50 per cent V/V).
The 1st 'decimal' dilution (D 1) is made from: Subsequent decimal dilutions are produced as stated for D2.
- 3 parts of the mother tincture; The 1st 'centesimal' dilution (C 1) is made from:
- 2 parts of the mother tincture; METHOD 1.1.S (HAB 3A: MOTHER TINCTURES AND LIQUID
- 98 parts of ethanol (50 per cent V/V). DILUTIONS)
The rd centesimal dilution (C2) is made from: Method 1.1.5 is used for fresh herbal drugs containing
- 1 part of the 1st 'centesimal' dilution; essential oil or resin, or generally less than 60 per cent
- 99 parts of ethanol (50 per cent V/V). moisture.
Subsequent centesimal dilutions are produced as stated for Mother tinctures prepared according to Method 1.1. 5
C2. (ethanol content approximately 65 per cent V/V) are
METHOD 1.1.4 (HAB 2B: MOTHER TINCTURES AND LIQUID prepared by maceration as described below.
DILUTIONS) Comminute appropriately the herbal drug.
Method 1. 1.4 is used for fresh herbal drugs containing Determine the water content (2.2.13) or loss on drying
generally less than 70 per cent of expressed juice and more (2.2.32). Unless otherwise prescribed, determine the loss on
than 60 per cent moisture (determined by loss on drying) drying on 2.00-5.00 g of the comminuted herbal drug and
and no essential oil or resin. dry at 105 °C for 2 h.
Mother tinctures prepared according to Method 1. 1.4 To the comminuted herbal drug immediately add not less
(ethanol content approximately 36 per cent V/V) are than half the mass of ethanol (90 per cent V/V) and allow to
prepared by maceration as described below. stand in well-closed containers.
Comminute appropriately the herbal drug. Use the following expression to calculate the amount (A 3), in
Take a sample and determine the loss on drying (2.2.32). kilograms, of ethanol (90 per cent V/V) required for the mass
Unless otherwise prescribed, determine the loss on drying on (m) of raw material, then subtract the amount of ethanol
2.00-5.00 g of the comminuted herbal drug and dry at (90 per cent V/V) already added and add the difference to
105 °C for 2 h. the mixture.
To the comminuted herbal drug immediately add not less 2xmx T
than half the mass of ethanol (70 per cent V/V) and allow to 100
stand in well-closed containers.
m mass of raw material} in kilograms;
Use the following expression to calculate the amount (A 2 ), in T percentage loss on drying or water content of the sample.
kilograms, of ethanol (70 per cent VIV) required for the mass
(m) of raw material, then subtract the amount of ethanol Allow to stand for not less than 10 days, swirling from time
(70 per cent V/V) already added and add the difference to to time, then express the mixture and filter the resulting
the mixture. liquid.
mxT Adjustment to any value specified in the individual
100 monograph
Determine the percentage dry residue (2. 8.16) or, where
m mass of raw material, in kilograms;
T percentage loss on drying of the sample. prescribed, the percentage assay content of the above-
mentioned filtrate. Calculate the amount (A 1), in kilograms,
Allow to stand for not less than 10 days, swirling from time of ethanol (70 per cent V/V) required, using the following
to time, then express the mixture and filter the resulting expression:
liquid.
mx (Nx-No)
Adjustment to any value specified in the individual No
monograph
Determine the percentage dry residue (2. 8.16) or, where m mass of filtrate, in kilograms;
mentioned filtrate. Calculate the amount (A 1 ), in kilograms, Nx percentage dry residue or percentage assay content of the
of ethanol (36 per cent V/V) required, using the following filtrate.
expression:
Mix the filtrate with the calculated amount of ethanol
m x (Nx - No) (70 per cent V/V). Allow to stand for not less than 5 days,
No then filter if necessary.
m mass of filtrate, in kilograms; Potentisation
N0 percentage dry residue or percentage assay content as required The Jst 'decimal' dilution (D 1) is made from:
filtrate. - 7 parts of ethanol (70 per cent V/V).
Mix the filtrate with the calculated amount of ethanol - 1 part of the 1st 'decimal' dilution;
(36 per cent V/V). Allow to stand for not less than 5 days, - 9 parts of ethanol (70 per cent V/V).
then filter if necessary. Subsequent decimal dilutions are produced as stated for D2.
Potentisation Use ethanol (50 per cent V/V) for dilutions from D4
The 1st 'decimal' dilution (Dl) is made from: onwards.
- 2 parts of the mother tincture; The 1st 'centesimal' dilution (Cl) is made from:
- 8 parts of ethanol (36 per cent V/V). - 3 parts of the mother tincture;
The 2nd decimal dilution (D2) is made from: - 97 parts of ethanol (70 per cent V/V).
- 1 part of the 1st 'decimal' dilution; The 2nd centesimal dilution (C2) is made from:
- 9 parts of ethanol (18 per cent V/V). - 1 part of the 1st 'centesimal' dilution;
Subsequent decimal dilutions are produced as stated for D2. - 99 parts of ethanol (50 per cent V/V).
Subsequent centesimal dilutions are produced as stated for METHOD 1.1.7 (HAB 3C: MOTHER TINCTURES AND LIQUID
C2. DILUTIONS)
METHOD 1.1.6 (HAB 38: MOTHER TINCTURES AND LIQUID Method 1.1. 7 is used for fresh herbal drugs containing
DILUTIONS) generally less than 60 per cent moisture (determined by loss
Method 1.1.6 is used for fresh herbal drugs containing on drying).
essential oils or resins or generally Jess than 60 per cent Mother tinctures prepared according to Method 1.1. 7
moisture determined by loss on drying (2.2.32) or water (ethanol content approximately 35 per cent V/V) are
content (2.2. 13). prepared by maceration as described below.
Mother tinctures prepared according to Method 1.1.6 Comminute appropriately the herbal drug.
(ethanol content approximately 57 per cent V/V) are Take a sample and determine the loss on drying (2.2.32).
prepared by maceration as described below. Unless otherwise prescribed, determine the loss on drying on
Comminute appropriately the herbal drug. 2.00-5.00 g of the comminuted herbal drug and dry at
Determine the water content (2.2.13) or loss on drying 105 °C for 2 h.
(2.2.32). Unless otherwise prescribed, determine the Joss on To the comminuted herbal drug immediately add not less
drying on 2.00-5.00 g of the comminuted herbal drug and than half the mass of ethanol (50 per cent V/V) and allow to
dry at 105 °C for 2 h. Unless otherwise prescribed, stand in well-closed containers.
determine the water content on 2.00-5.00 g of the Use the following expression to calculate the amount (A 3 ), in
comminuted herbal drug. kilograms, of ethanol (50 per cent V/V) required for the mass
To the comminuted herbal drug immediately add not less (m) of raw material, then subtract the amount of ethanol
than half the mass of ethanol (80 per cent VIV) and allow to (50 per cent V/Jl) already added and add the difference to
stand in well-closed containers. the mixture.
Use the following expression to calculate the amount (A 3 ), in 2xmx T
kilograms, of ethanol (80 per cent V/V) required for the mass
100
(m) of raw material, then subtract the amount of ethanol
(80 per cent V/V) already added and add the difference to m mass of raw material, in kilograms;
T percentage loss on drying of the sample.
the mixture.
2xmx T Allow to stand for not less than 10 days, swirling from time
100 to time, then express the mixture and filter the resulting
liquid.
T percentage loss on drying of the sample. Adjustment to any value specified in the individual
monograph
Allow to stand for not less than 10 days, swirling from time Determine the percentage dry residue (2. 8.16) or, where
to time, then express the mixture and filter the resulting prescribed, the percentage assay content of the above-
liquid. mentioned filtrate. Calculate the amount (A 1), in kilograms,
Adjustment to any value specified in the individual of ethanol (36 per cent V/V) required, using the following
monograph expression:
Determine the percentage dry residue (2.8.16) or, where mx (Nx -No)
No
of ethanol (50 per cent V/V) required, using the following m mass of filtrate, in kilograms;
expression: N0 percentage dry residue or percentage assay content as required
N, percentage dry residue or percentage assay content of the
mx (Nx -No)
filtrate.
No
m mass of filtrate, in kilograms; Mix the filtrate with the calculated amount of ethanol
N0 percentage dry residue or percentage assay content as required (36 per cent V/V). Allow to stand for not less than 5 days,
in the individual monograph; then filter if necessary.
Nx = percentage dry residue or percentage assay content of the
filtrate. Potentisation
Mix the filtrate with the calculated amount of ethanol - 3 parts of the mother tincture;
(50 per cent V/V). Allow to stand for not Jess than 5 days, - 7 parts of ethanol (36 per cent VIV).
then filter if necessary. The 2nd decimal dilution (D2) is made from:
Potentisation - 1 part of the 1st 'decimal' dilution;
The 1s t 'decimal' dilution (D 1) is made from: - 9 parts of ethanol ( 18 per cent V/V).
- 3 parts of the mother tincture; Subsequent decimal dilutions are produced as stated for D2.
- 7 parts of ethanol (50 per cent V/V). METHOD 1.1.8 (HAB 4A: MOTHER TINCTURES AND LIQUID
The 2nd decimal dilution (D2) is made from: DILUTIONS)
- 1 part of the 1st 'decimal' dilution; Method 1. 1.8 is generally used for dried herbal drugs.
- 9 parts of ethanol (36 per cent V/V). Mother tinctures prepared according to Method 1.1.8 are
The 3rd decimal dilution (D3) is made from: prepared by maceration or percolation as described below,
- 1 part of the 2nd decimal dilution; using 1 part of dried herbal drug and 10 parts of ethanol of
- 9 parts of ethanol (18 per cent VIV). the appropriate concentration (anhydrous, 96 per cent V!V,
Subsequent decimal dilutions are produced as stated for D3. 90 per cent VIV, 80 per cent V!V, 70 per cent VIV,
50 per cent V/V, 36 per cent VIV, 18 per cent VIV), unless Production by maceration Unless otherwise prescribed,
otherwise prescribed in the individual monograph. comminute the raw material, mix thoroughly with ethanol of
Production by maceration Unless otherwise prescribed, the appropriate concentration and allow to stand in a closed
comminute the herbal drug, mix thoroughly with ethanol of container for an appropriate time. Separate the residue from
the appropriate concentration and allow to stand in a closed the ethanol and, if necessary, press out. In the latter case,
container for an appropriate time. Separate the residue from combine the 2 liquids obtained.
the ethanol and, if necessary, press out. In the latter case, Production by percolation If necessary, comminute the raw
combine the 2 liquids obtained. material. Mix thoroughly with a portion of ethanol of the
Production by percolation If necessary, comminute the herbal appropriate concentration and allow to stand for an
drug. Mi.x thoroughly with a portion of ethanol of the appropriate time. Transfer to a percolator and allow the
appropriate concentration and allow to stand for an percolate to flow slowly at room temperature, making sure
appropriate time. Transfer to a percolator and allow the that the raw material to be extracted is always covered with
percolate to flow slowly, at room temperature, making sure the remaining ethanol. The residue may be pressed out and
that the herbal drug to be extracted is always covered with the expressed liquid combined with the percolate.
the remaining ethanol. The residue may be pressed out and If adjustment to a given concentration is necessary, calculate
the expressed liquid combined with the percolate. the amount (A 1), in kilograms, of ethanol of the appropriate
If adjustment to a given concentration is necessary, calculate concentration required to obtain the concentration specified
the amount (A 1 ), in kilograms, of ethanol of the appropriate or used for production, using the following expression:
concentration required to obtain the concentration specified
or used for production, using the following expression: m x (Nx -No)
No
mX (Nx - No)
m mass of percolate or macerate, in kilograms;
No N0 percentage dry residue or percentage assay content as required
m mass of percolate or macerate, in kilograms; Nx percentage dry residue or percentage assay content of the
N0 percentage dry residue or percentage assay content as required percolate or macerate.
percolate or macerate. Mix the macerate or percolate with the calculated amount of
ethanol of the appropriate concentration. Allow to stand for
Mix the macerate or percolate with the calculated amount of not less than 5 days, then filter if necessary.
ethanol of the appropriate concentration. Allow to stand for Potentisation
not less than 5 days, then filter if necessary. The mother tincture corresponds to the 1st 'decimal' dilution
Potentisation (0 = DI).
The mother tincture corresponds to the 1st 'decimal' dilution The 2nd decimal dilution (D2) is made from:
(0 = Dl). - 1 part of the mother tincture (DI);
The 2nd decimal dilution (D2) is made from: - 9 parts of ethanol of the same concentration.
- l part of the mother tincture (Dl); The 3rd decimal dilution (D3) is made from:
- 9 parts of ethanol of the same concentration. - 1 part of the 2nd decimal dilution;
The 3rd decimal dilution (D3) is made from: - 9 parts of ethanol of the same concentration.
- l part of the 2nd decimal dilution; Unless a different ethanol concentration is specified, use
- 9 parts of ethanol of the same concentration. ethanol (50 per cent V/V) for subsequent decimal dilutions
Unless a different ethanol concentration is specified, use from D4 onwards and proceed as stated for D3.
ethanol (50 per cent V/V) for subsequent decimal dilutions The 1st 'centesimal' dilution (Cl) is made from:
from D4 onwards and proceed as stated for D3. - 10 parts of the mother tincture (D1);
The 1st 'centesimal' dilution (Cl) is made from: - 90 parts of ethanol of the same concentration.
- 10 parts of the mother tincture (D1); The 2nd centesimal dilution (C2) is made from:
- 90 parts of ethanol of the same concentration. - 1 part of the 1st 'centesimal' dilution;
The 2nd centesimal dilution (C2) is made from: - 99 parts of ethanol (50 per cent VIV), unless a different
- 1 part of the 1st 'centesimal' dilution; ethanol concentration is specified.
- 99 parts of ethanol (50 per cent VIV), unless a different Subsequent centesimal dilutions are produced as stated for
ethanol concentration is specified. C2.
Subsequent centesimal dilutions are produced as stated for METHOD 1.1.10 (FRENCH PHARMACOPOEIA)
C2. Method I. 1.10 is generally used for herbal drugs. The state
METHOD 1.1.9 (HAB 4B: MOTHER TINCTURES AND LIQUID of the herbal drug, fresh or dried, is specified in the
DILUfIONS) individual monograph.
Method l. l. 9 is generally used for raw materials of animal Mother tinctures prepared according to Method 1.1.10 are
origin. Pathological material is excluded. prepared by maceration.
Mother tinctures prepared according to Method 1.1. 9 are Comminute appropriately the herbal drug. Take a sample
prepared by maceration or percolation as described below, and determine the loss on drying at 105 °C for 2 h (2.2.32)
using 1 part of raw material and 10 parts of ethanol of the or the water content (2.2.13). Taking this value into account,
appropriate concentration (anhydrous, 96 per cent V/V, calculate and add to the herbal drug the quantities of ethanol
90 per cent V/V, 80 per cent VIV, 70 per cent VIV, of the appropriate concentration required to produce, unless
50 per cent V/V, 36 per cent VIV, 18 per cent VIV), unless otherwise prescribed, a 1 in 10 mother tincture ( 1: 10 mother
otherwise prescribed in the individual monograph.
tincture) with a suitable ethanol content. Allow to macerate and more than 60 per cent moisture (loss on drying) and no
for at least 10 days, with sufficient shaking. essential oil or resin.
Separate the residue from the ethanol and strain under Mother tinctures prepared according to Methods 1.2.1
pressure if necessary. Allow the combined liquids to stand for (ethanol content approximately 50 per cent V/V) and 1.2.2
48 h and filter. For mother tinctures with a required assay (ethanol content approximately 36 per cent V/V) are
content, adjustment may be carried out, if necessary, by ethanolic digestions prepared by heat treatment and
adding ethanol of the same concentration as used for the additional maceration as described below.
preparation of the tincture. Comminute appropriately the herbal drug.
Potentisation Take a sample and determine the loss on drying (2.2.32).
The 1st 'decimal' dilution (D 1) is made from: Unless otherwise prescribed, determine the loss on drying on
- 1 part of the mother tincture; 2.00-5.00 g of the comminuted herbal drug and dry at
- 9 parts of ethanol of the appropriate concentration. 105 °C for 2 h.
The 2nd decimal dilution (D2) is made from: To the comminuted herbal drug immediately add not less
- 1 part of the 1st decimal dilution; than half the mass of ethanol of the concentration prescribed
- 9 parts of ethanol of the appropriate concentration. below and allow to stand in well-closed containers:
Subsequent decimal dilutions are produced as stated for D2, - Method 1.2.1: ethanol (90 per cent V/V);
using ethanol of the appropriate concentration. - Method 1.2.2: ethanol (70 per cent VIV).
The 1st 'centesimal' dilution (Cl) is made from: Use the following expression to calculate the amount (A 2 ), in
- 1 part of the mother tincture; kilograms, of ethanol of the appropriate concentration
- 99 parts of ethanol of the appropriate concentration. required for the mass (m) of raw material, then subtract the
The 2nd centesimal dilution (C2) is made from: amount of ethanol of the appropriate concentration already
- 1 part of the 1st centesimal dilution; added and add the difference to the mixture.
- 99 parts of ethanol of the appropriate concentration.
mxT
Subsequent centesimal dilutions are produced as stated
for C2, using ethanol of the appropriate concentration. 100
METHOD 1.1.11 (FRENCH PHARMACOPOEIA) m mass of raw material, in kilograms;
Method 1.1.11 is generally used for raw materials of animal
origin. Pathological material is excluded.
Warm the mixture containing the total amount of ethanol of
Mother tinctures prepared according to Method 1.1.11 are the appropriate concentration to 37 °C in a covered
prepared by maceration. container and maintain at this temperature for 1 h, swirling
The mass ratio of raw material to mother tincture is usually from time to time. Cool, allow to stand for not less than
1 to 20. To the raw material, appropriately comminuted, add 10 days, swirling from time to time, then express the mixture
the quantiry of ethanol of the appropriate concentration and filter the resulting liquid.
required to produce a 1 in 20 mother tincture. Allow to Adjustment to any value as specified in the individual
macerate for at least 10 days, with sufficient shaking. Decant monograph
and filter. Allow to stand for 48 h and filter again. Determine the percentage dry residue (2. 8.16) or, where
For mother tinctures with a required assay content, prescribed, the percentage assay content of the above-
adjustment may be carried out, if necessary, by adding mentioned filtrate. Calculate the amount (A 1 ), in kilograms,
ethanol of the same concentration as used for the preparation of ethanol of the appropriate concentration required, using
of the tincture. the following expression:
Potentisation
The 1st decimal dilution (D 1) is made from: mx (N, -No)
- 1 part of the mother tincture; No
m mass of filtrate, in kilograms;
The 2nd decimal dilution (D2) is made from: N0 percentage dry residue or percentage assay content as required
- 1 part of the 1st decimal dilution; in the individual monograph;
- 9 parts of ethanol of the appropriate concentration. Nx percentage dry residue or percentage assay content of the
filtrate.
Subsequent decimal dilutions are produced as stated for D2,
using ethanol of the appropriate concentration.
Mix the filtrate with the required amount of ethanol of the
The 1st centesimal dilution (Cl) is made from: concentration prescribed below:
- 1 part of the mother tincture; - Method 1.2.1: ethanol (50 per cent V/V);
- 99 parts of ethanol of the appropriate concentration. - Method 1.2.2: ethanol (36 per cent V/V).
The 2nd centesimal dilution (C2) is made from: Allow to stand for not less than 5 days, then filter if
- 1 part of the 1st centesimal dilution; necessary.
Potentisation
Subsequent centesimal dilutions are produced as stated The 1st 'decimal' dilution (D1) is made from:
for C2, using ethanol of the appropriate concentration. - 2 parts of the mother tincture;
METHOD 1.2. HYDROALCOHOLIC MOTHER - 8 parts of ethanol (50 per cent V/V) (Method 1.2.1) or
TINCTURES PREPARED WITH HEATING ethanol (36 per cent V/V) (Method 1.2.2).
METHODS 1.2.1, 1.2.2. ETHANOLIC DIGESTIONS (HAB 18A, 18B: The 2nd decimal dilution (D2) is made from:
HEAT-TREATED MOTHER TINCTURES) - 1 part of the 1st 'decimal' dilution;
Methods 1.2.1 and 1.2.2 are used for fresh herbal drugs - 9 parts of ethanol (36 per cent V/V) (Method 1.2.1) or
containing generally less than 70 per cent of expressed juice ethanol (18 per cent V/V) (Method 1.2.2).
The 3 rd decimal dilution (D3) is made from: Allow to stand for not less than 5 days, then filter if
- 1 part of the 2nd decimal dilution; necessary.
- 9 parts of ethanol (18 per cent V/V). Potentisation
Subsequent decimal dilutions are produced as stated for D3. The 1st 'decimal' dilution (Dl) is made from:
METHODS 1.2,3, 1.2.4, 1.2.5. ETHANOLIC DIGESTIONS (HAB !SC, - 3 parts of the mother tincture;
18D, 18E: HEAT-TREATED MOTHER TINCTURES) - 7 parts of ethanol (70 per cent V/V) (Method 1.2.3),
Methods 1.2.3, 1.2.4 and 1.2.5 are used for fresh herbal ethanol (50 per cent V/V) (Method 1.2.4) or ethanol
drugs containing essential oil or resin or generally less than (36 per cent V/V) (Method 1.2.5).
60 per cent moisture. The 2nd decimal dilution (D2) is made from:
Mother tinctures prepared according to Methods 1.2.3 - l part of the 1st 'decimal' dilution;
(ethanol content approximately 65 per cent VIV), 1.2.4 - 9 parts of ethanol (50 per cent V/V) (Method 1.2.3),
(ethanol content approximately 57 per cent VIV) and 1.2.5 ethanol (36 per cent V/V) (Method 1.2.4) or ethanol
(ethanol content approximately 35 per cent V/V) are (18 per cent V/V) (Method 1.2.5).
ethanolic digestions prepared by heat treatment and Subsequent decimal dilutions are produced accordingly.
additional maceration as described below. In this process the ethanol concentration is reduced with
Comminute appropriately the herbal drug. each step, according to the sequence 70 per cent VIV -
50 per cent VIV - 36 per cent V/V - 18 per cent V/V.
Determine the water content (2.2.13) or loss on drying
METHOD 1.2.6. ETHANOLIC DIGESTIONS (HAB 18F: HEAT-
(2.2.32). Unless otherwise prescribed, determine the loss on
drying on 2.00-5.00 g of the comminuted herbal drug and TREATED MOTHER TL"ICTURES)
dry at 105 °C for 2 h. Method 1.2.6 is used for dried herbal drugs.
To the comminuted herbal drug immediately add not less Mother tinctures prepared according to Method 1.2.6 are
than half the mass of ethanol of the concentration prescribed ethanolic digestions prepared by heat treatment and
below and allow to stand in well-closed containers: additional maceration as described below, using 1 part of
- Method 1.2.3: ethanol (90 per cent V/V); dried herbal drug and 10 parts of ethanol of the appropriate
- Method 1.2.4: ethanol (80 per cent V/V); concentration (96 per cent VIV, 90 per cent VIV,
- Method 1.2.5: ethanol (50 per cent V/V). 80 per cent V!V, 70 per cent V/V, 50 per cent VIV,
Use the following expression to calculate the amount (A 3 ), in 36 per cent VIV, 18 per cent VIV), unless otherwise
kilograms, of ethanol of the appropriate concentration prescribed in the individual monograph.
required for the mass (m) of raw material, then subtract the Unless otherwise prescribed, comminute appropriately the
amount of ethanol of the appropriate concentration already herbal drug, mix thoroughly with the total amount of ethanol
added and add the difference to the mixture. of the appropriate concentration, warm the mixture to 3 7 °C
in a covered container and maintain at this temperature for
2xmx T 1 h, swirling from time to time. Cool, then allow to stand in
100 a closed container for an appropriate time. After
sedimentation, decant the supernatant and, if necessary, press
m mass of raw material, in kilograms; out. In the latter case, combine the 2 liquids obtained. Filter
the resulting liquid.
Warm the mixture containing the total amount of ethanol of Adjustment to any value as specified in the individual
the appropriate concentration to 37 °C in a covered monograph
container and maintain at this temperature for 1 h, swirling Determine the percentage dry residue (2. 8.16) or, where
from time to time. Cool, allow to stand for not less than prescribed, the percentage assay content of the above-
10 days, swirling from time to time, then express the mixture mentioned filtrate. Calculate the amount (A 1), in kilograms,
and filter the resulting liquid. of ethanol of the appropriate concentration required to obtain
the concentration specified or used for production, using the
Adjustment to any value as specified in the individual
monograph
Determine the percentage dry residue (2. 8.16) or, where m x (Nx - No)
No
mentioned filtrate. Calculate the amount (A 1 ), in kilograms,
of ethanol of the appropriate concentration required, using m mass of the filtrate, in kilograms;
.t-,10 percentage dry residue or percentage assay content as required
the following expression:
percentage dry residue or percentage assay content of the
mx(Nx-No) filtrate.
No
Mix the filtrate with the calculated amount of ethanol of the
m mass of filtrate, in kilograms; appropriate concentration. Allow to stand for not less than
5 days, then filter if necessary.
'lv.\ percentage dry residue or percentage assay content of the Potentisation
filtrate. The mother tincture corresponds to the 1st 'decimal' dilution
(0 = Dl).
concentration prescribed below:
- 1 part of the mother tincture (Dl);
- Method 1.2.3: ethanol (70 per cent V/V);
- 9 parts of ethanol of the same concentration.
- Method 1.2.5: ethanol (36 per cent V/V). Subsequent decimal dilutions are produced accordingly.
In this process the ethanol concentration is reduced with
each step, according to the sequence 96 per cent V/V - - 2 parts of the mother tincture;
90 per cent V/V - 80 per cent V/V - 70 per cent VIV - - 8 parts of ethanol (50 per cent VIV) (Method 1.2.7) or
50 per cent VIV - 36 per cent V/V - 18 per cent VIV. ethanol (36 per cent V/V) (Method 1.2.8).
METHODS 1.2.7, 1.2.8. ETHANOLIC DECOCTIONS (HAB 19A, 19B: The 2nd decimal dilution (D2) is made from:
HEAT-TREATED MOTHER TINCTURES) - 1 part of the 1st 'decimal' dilution;
Methods 1.2.7 and 1.2.8 are used for fresh herbal drugs - 9 parts of ethanol (36 per cent V/V) (Method 1.2.7) or
containing generally less than 70 per cent of expressed juice ethanol (18 per cent V/V) (Method 1.2.8).
and more than 60 per cent moisture (loss on drying) and no The 3rd decimal dilution (D3) is made from:
essential oil or resin. - 1 part of the 2nd decimal dilution;
Mother tinctures prepared according to Methods 1.2.7 - 9 parts of ethanol (18 per cent V/V).
(ethanol content approximately 50 per cent V/V) and 1.2.8 Subsequent decimal dilutions are produced as stated for D3.
(ethanol content approximately 36 per cent V/V) are METHODS 1.2.9, 1.2.10, 1.2.11. ETHANOLIC DECOCTIONS (HAB 19C,
ethanolic decoctions prepared by heat treatment and 19D, 19E: HEAT-TREATED MOTHER TINCTURES)
additional maceration as described below.
Methods 1.2.9, 1.2.10 and 1.2.11 are used for fresh herbal
Comminute appropriately the herbal drug. drugs containing essential oil or resin or generally less than
Take a sample and determine the loss on drying (2.2.32). 65 per cent moisture.
Unless otherwise prescribed, determine the loss on drying on Mother tinctures prepared according to Methods 1.2.9
2.00-5.00 g of the comminuted herbal drug and dry at (ethanol content approximately 65 per cent VIV), 1.2.10
105 °C for 2 h. (ethanol content approximately 57 per cent V/V) and 1.2.11
To the comminuted herbal drug immediately add not less (ethanol content approximately 35 per cent V/V) are
than half the mass of ethanol of the concentration prescribed ethanolic decoctions prepared by heat treatment and
below and allow to stand in well-closed containers: additional maceration as described below.
- Method 1.2.7: ethanol (90 per cent VIV); Comminute appropriately the herbal drug.
- Method 1.2.8: ethanol (70 per cent V/V).
Determine the water content (2. 2.13) or loss on drying
Use the following expression to calculate the amount (A 2), in (2.2.32). Unless otherwise prescribed, determine the loss on
kilograms, of ethanol of the appropriate concentration drying on 2.00-5.00 g of the comminuted herbal drug and
required for the mass of raw material, then subtract the dry at 105 °C for 2 h.
amount of ethanol of the appropriate concentration already
To the comminuted herbal drug immediately add not less
added and add the difference to the mixture.
than half the mass of ethanol of the concentration prescribed
mxT below and allow to stand in well-closed containers:
100
- Method 1.2.10: ethanol (80 per cent VIV);
m mass of raw material, in kilograms; - Method 1.2.11: ethanol (50 per cent VIV).
Use the following expression to calculate the amount (A 3 ), in
kilograms, of ethanol of the appropriate concentration
Heat the mixture containing the total amount of ethanol of
required for the mass of raw material, then subtract the
the appropriate concentration to boiling under reflux, and
amount of ethanol of the appropriate concentration already
maintain for 30 min, unless otherwise specified in the
added and add the difference to the mixture.
individual monograph. Cool or allow to cool, allow to stand
in a closed container for 12-36 h, then express the mixture 2xmxT
and filter the resulting liquid.
100
monograph T percentage loss on drying of the sample.
Determine the percentage dry residue (2.8. 16) or, where
prescribed, the percentage assay content of the above- Heat the mixture containing the total amount of ethanol of
mentioned filtrate. Calculate the amount (A 1 ), in kilograms, the appropriate concentration to boiling under reflux, and
of ethanol of the appropriate concentration required, using maintain for 30 min, unless otherwise specified in the
the following expression: individual monograph. Cool or allow to cool, then allow to
stand in a closed container for 12-36 h, express the mixture
mx (Nx-No)
and filter the resulting liquid.
No
m mass of filtrate, in kilograms; monograph
N0 percentage dry residue or percentage assay content as required Determine the percentage dry residue (2. 8.16) or, where
filtrate. mentioned filtrate. Calculate the amount (A 1 ), in kilograms,
of ethanol of the appropriate concentration required, using
Mix the filtrate with the required amount of ethanol of the the following expression:
concentration prescribed below:
- Method 1.2.7: ethanol (50 per cent V/V); mx(Nx-No)
- Method 1.2.8: ethanol (36 per cent V/V). No
Allow to stand for not less than 5 days, then filter if m mass of filtrate, in kilograms;
necessary. No percentage dry residue or percentage assay content as required
Potentisation N, percentage dry residue or percentage assay content of the
The 1st 'decimal' dilution (Dl) is made from: filtrate.
Potentisation
Mix the filtrate with the required amount of ethanol of the The mother tincture corresponds to the I st 'decimal' dilution
concentration prescribed below: (0 = DI).
- Method 1.2.9: ethanol (70 per cent VIV); The 2nd decimal dilution (D2) is made from:
- Method 1.2.10: ethanol (50 per cent VIV); - I part of the mother tincture (DI);
- Method 1.2.11: ethanol (36 per cent VIV). - 9 parts of ethanol of the same concentration.
Allow to stand for not less than 5 days, then filter if Subsequent decimal dilutions are produced accordingly.
necessary. In this process the ethanol concentration is reduced with
each step, according to the sequence 96 per cent VIV -
Potentisation
The 1st 'decimal' dilution (DI) is made from: 90 per cent V/V - 80 per cent VIV - 70 per cent VIV -
50 per cent V/V - 36 per cent V/V - 18 per cent V/V.
- 7 parts of ethanol (70 per cent V/V) (Method 1.2.9), METHOD 1.2.13. ETHANOLIC INFUSIONS (HAB 20: HEAT-
ethanol (50 per cent V/V) (Method 1.2.10) or ethanol TREATED MOTHER TINCTURES)
(36 per cent V/V) (Method 1.2.11). Method 1.2.13 is used for dried herbal drugs.
The 2nd decimal dilution (D2) is made from: Mother tinctures prepared according to Method 1.2.13 are
- I part of the 1st 'decimal' dilution; ethanolic infusions prepared by heat treatment and additional
- 9 parts of ethanol (50 per cent V/V) (Method 1.2.9), maceration as described below using I part of dried herbal
ethanol (36 per cent V/V) (Method 1.2.10) or ethanol drug and 10 parts of ethanol of the appropriate concentration
(18 per cent V/V) (Method 1.2.11). (90 per cent VIV, 80 per cent VIV, 70 per cent VIV,
Subsequent decimal dilutions are produced accordingly. 50 per cent V/V, 36 per cent V/V, 18 per cent VIV), unless
In this process the ethanol concentration is reduced with otherwise prescribed in the individual monograph.
each step, according to the sequence 70 per cent V/V - The quantities of ethanol (96 per cent V/V) and purified
50 per cent V/V - 36 per cent V/V - 18 per cent V/V. water required to achieve the specified ethanol concentration
are added separately as described below.
METHOD 1.2.12. ETHANOLIC DECOCTIONS (HAB 19F: HEAT-
TREATED MOTHER TINCTURES) Unless otherwise prescribed, comminute appropriately the
herbal drug, mix thoroughly with the total amount of ethanol
Method 1.2.12 is used for dried herbal drugs.
(96 per cent VIV), cover and allow to stand for 15 min.
Mother tinctures prepared according to Method 1.2.12 are Add the purified water (heated to boiling) and heat the
ethanolic decoctions prepared by heat treatment and mixture to boiling under reflux for 5 min. Cool, or allow to
additional maceration as described below, using I part of cool, then allow to stand in a closed container for 12-36 h.
dried herbal drug and 10 parts of ethanol of the appropriate After sedimentation, decant the supernatant and, if necessary,
concentration (96 per cent V/V, 90 per cent VIV, press out. In the latter case, combine the 2 liquids obtained.
80 per cent V/V, 70 per cent VIV, 50 per cent VIV, Filter the resulting liquid.
36 per cent V/V, 18 per cent VIV), unless otherwise
prescribed in the individual monograph. Adjustment to any value as specified in the individual
monograph
Unless otherwise prescribed, comminute appropriately the
Determine the percentage dry residue (2. 8.16) or, where
herbal drug, mix thoroughly with the total amount of ethanol
of the appropriate concentration, heat to boiling under reflux
and maintain for 30 min. Cool or allow to cool, then allow to
of ethanol of the appropriate concentration required to obtain
stand in a closed container for 12-36 h. After sedimentation,
decant the supernatant and, if necessary, press out. In the
latter case, combine the 2 liquids obtained. Filter the
resulting liquid. mx (Nx-No)
Adjustment to any value as specified in the individual No
monograph
Determine the percentage dry residue (2. 8.16) or, where m mass of filtrate, in kilograms;
prescribed, the percentage assay content of the above- N0 percentage dry residue or percentage assay content as required
mentioned filtrate. Calculate the amount (A 1), in kilograms, Nx percentage dry residue or percentage assay content of the
of ethanol of the appropriate concentration required to obtain filtrate.
following expression: Mix the filtrate with the required amount of ethanol of the
appropriate concentration. Allow to stand for not less than
mx (Nx -No) 5 days, then filter if necessary.
No Potentisation
mass of filtrate, in kilograms;
The mother tincture corresponds to the I st 'decimal' dilution
percentage dry residue or percentage assay content as required (0 = Dl).
in the individual monograph; The 2nd decimal dilution (D2) is made from:
percentage dry residue or percentage assay content of the
- I part of the mother tincture (DI);
filtrate.
- 9 parts of ethanol of the same concentration.
appropriate concentration. Allow to stand for not less than
5 days, then filter if necessary.
Subsequent decimal dilutions are produced accordingly. for 1 h, swirling from time to time. Express the mixture and
In this process the ethanol concentration is reduced with filter the resulting liquid. The mother tincture is used
each step, according to the sequence 90 per cent V/V - immediately, unless otherwise justified.
80 per cent VIV - 70 per cent VIV - 50 per cent VIV - Potentisation
36 per cent VIV - 18 per cent V/V. The 1st 'decimal' dilution (D 1) is made from:
METHOD 1.3. AQUEOUS MOTHER TINCTURES - 4 parts of the mother tineture;
PREPARED WITHOUT HEATING - 6 parts of water for injection.
METHOD 1.3.1. AQUEOUS MACERATES (HAB 49: AQUEOUS The 2nd decimal dilution (D2) is made from:
MOTHER TINCTURES) - 1 part of the 1st 'decimal' dilution;
Method 1.3.1 is used for fresh herbal drugs. - 9 parts of water for injection.
Mother tinctures prepared according to Method 1.3.1 are Subsequent decimal dilutions are produced as stated for D2.
aqueous macerates prepared by short maceration with water METHOD 1.4.2. AQUEOUS DECOCTIONS (HAB 23B: HEAT-
as described below. This method is used solely in the TREATED AQUEOUS MOTHER TINCTURES)
manufacture of injections and eye preparations. Method 1.4.2 is used for fresh herbal drugs.
Comminute appropriately the herbal drug. Mother tinctures prepared according to Method 1.4.2 are
Take a sample and determine the loss on drying (2.2.32). aqueous decoctions prepared by heat treatment with water as
Unless otherwise prescribed, determine the loss on drying on described below. This method is solely used in the
2.00-5.00 g of the comminuted herbal drug and dry at preparation of injections, eye preparations and coated
105 °C for 2 h. homoeopathic pillules.
Use the following expression to calculate the amount, in Comminute appropriately the herbal drug.
kilograms, of water required for the mass of raw material: Take a sample and determine the loss on drying (2.2.32).
m x (300 - T) Unless otherwise prescribed, determine the loss on drying on
100 2.00-5.00 g of the comminuted herbal drug and dry at
105 °C for 2 h.
T percentage loss on drying of the sample. Use the following expression to calculate the amount, in
kilograms, of water required for the mass of raw material:
Add the comminuted herbal drug to the calculated amount
of water. Allow to stand for 2 h, then express the mixture m x (300- T)
and filter the resulting liquid. The mother tincture is used 100
immediately, unless otherwise justified.
Potenti.sation T percentage loss on drying of the sample.
The 1st 'decimal' dilution (DI) is made from:
- 3 parts of the mother tincture; Heat the calculated amount of water to above 90 °C and add
- 7 parts of water for injection. the comminuted herbal drug. Maintain the mixture at this
The 2nd decimal dilution (D2) is made from: temperature under reflux for 30 min, then express the
- 1 part of the 1st 'decimal' dilution; mixture and filter the resulting liquid. The mother tineture is
- 9 parts of water for injection. used immediately, unless otherwise justified.
Subsequent decimal dilutions are produced as stated for D2. Potentisati.on
METHOD 1.4. AQUEOUS MOTHER TINCTURES - 3 parts of the mother tincture;
PREPARED WITH HEATING - 7 parts of water for injection.
METHOD 1.4.1. AQUEOUS DIGESTIONS (HAB 24B: HEAT-
TREATED AQUEOUS MOTHER TINCTURES) The 2nd decimal dilution (D2) is made from:
- 1 part of the 1st 'decimal' dilution;
Method 1.4.1 is used for fresh herbal drugs.
- 9 parts of water for injection.
Mother tinctures prepared according to Method 1.4.1 are
Subsequent decimal dilutions are produced as stated for D2.
aqueous digestions prepared by heat treatment with water as
METHOD 1.4.3. AQUEOUS DECOCTIONS (HAB 23A: HEAT-
described below. This method is solely used in the
TREATED AQUEOUS MOTHER TINCTURES)
preparation of injections, eye preparations and coated
homoeopathic pillules. Method 1.4.3 is used for dried herbal drugs.
Comminute appropriately the herbal drug. Mother tinctures prepared according to Method 1.4.3 are
Take a sample and determine the loss on drying (2.2.32). aqueous decoctions prepared by heat treatment with water as
Unless otherwise prescribed, determine the loss on drying on described below. This method is solely used in the
2.00-5.00 g of the comminuted herbal drug and dry at preparation of injections, eye preparations and coated
105 °C for 2 h. homoeopathic pillules.
Use the following expression to calculate the amount, in Comminute appropriately the herbal drug.
kilograms, of water required for the mass of raw material: Mix 1 part of the comminuted dried herbal drug with
10 parts of boiling water and boil under reflux for 30 min,
mx (400- T) unless otherwise specified in the individual monograph. Filter
100 while hot. If gentle pressure applied to the residue does not
m mass of raw material, in kilograms; achieve a final mass of mother tincture equal to 10 parts,
T percentage loss on drying of the sample. pour a sufficient amount of boiling water over the residue
and express gently. Use the resulting extract to make the
Heat the mixture containing the total amount of water in a mother tincture up to 10 parts. Filter the combined liquid.
covered container to 37 °C and maintain at this temperature
The filtrate is the mother tincture. The mother tincture is harvesting, subject the expressed juice to the 2 x 12 h hot-
used immediately, unless otherwise justified. cold ("day-night") rhythm ('Rh') starting with the first cold
For starch-containing material process 1 part of herbal drug period at 2-6 °C described below until fermentation is
with 100 parts of water. In such cases the mother tincture complete.
corresponds to the 2nd decimal dilution (0 = D2). Warm the expressed liquid to 35-39 °C over a period of
Potentisation 30-90 min and maintain it at this temperature. After 12 h,
The mother tincture corresponds to the 1si 'decimal' dilution cool the expressed liquid to 2-6 °C over a period of
(0 = D1). 30-90 min and maintain it at this temperature. After a
further 12 h, repeat the cycle by warming the liquid again as
The 2 nd decimal dilution (D2) is made from:
described above.
- 1 part of the mother tincture (D1);
- 9 parts of water for injection. Generate a marked vortex by gently stirring the liquid for
180-200 min during both temperature phases at the
beginning of the process, gradually decreasing the stirring
METHOD 1.4.4. AQUEOUS INFUSIONS (HAB 24A: HEAT-TREATED time over a period of 5 weeks to 10 min by the end of the
AQUEOUS MOTHER TINCTURES) process. An example of how this can be achieved is provided
Method 1.4.4 is used for dried herbal drugs. in Table 2371.-1. If the pH prescribed in the individual
Mother tinctures prepared according to Method 1.4.4 are monograph has not been reached after 35 days, continue the
aqueous infusions prepared by heat treatment with water and fermentation process until the prescribed pH is reached,
additional maceration as described below. This method is without exceeding a maximum fermentation time of 55 days.
solely used in the preparation of injections, eye preparations Filter (nominal pore size not greater than 15 µm) to remove
and coated homoeopathic pillules. the sediment. The filtrate is the mother tincture.
Comminute appropriately the herbal drug. Method 1.5.2
1 part of dried herbal drug is extracted with 10 parts of Comminute appropriately the plants within 12 h of
water. harvesting. Expose the plant material to 35-39 °C for 12 h,
Grind 1 part of the comminuted herbal drug in a mortar then cool to 2-6 °C for 12 h for 10-14 days. Express the
thoroughly with 3-5 parts of water, allow to stand for plant material. Transfer the expressed juice into glass
15 min, then add the remainder of the water, which has been containers, designed to largely keep out air and to allow for
heated to boiling point. Place the mixture in a water-bath heating and cooling. Subject the expressed juice to the
and maintain at a temperature above 90 °C for 5 min, 2 x 12 h hot-cold ("day-night") rhythm ('Rh') according to
swirling from time to time. Cover, allow to cool, then Method 1.5.1 and described below for minimum 35 days or
separate the residue from the liquid. If gentle pressure until fermentation is complete (maximum 55 days).
applied to the residue does not achieve a final mass of Warm the expressed liquid to 35-39 °C over a period of
mother tincture equal to 10 parts, pour a sufficient amount 30-90 min and maintain at this temperature. After 12 h, cool
of cold water over the residue and express gently. Use the the expressed liquid to 2-6 °C over a period of 30-90 min
resulting extract to make the mother tincture up to 10 parts. and maintain at this temperature. After a further 12 h, repeat
Filter the combined liquid. The filtrate is the mother the cycle by warming the liquid again as described above.
tincture. The mother tincture is used immediately, unless Generate a marked vortex by gently stirring the liquid for
otherwise justified. 180-200 min during both temperature phases at the
Potentisation beginning of the process, gradually decreasing the stirring
The mother tincture corresponds to the 1si 'decimal' dilution time over a period of 5 weeks down to 10 min at the end of
(0 = D1). the process. An example of how this can be achieved is
The 2nd decimal dilution (D2) is made from: provided in Table 2371.-1. If the pH prescribed in the
- 1 part of the mother tincture (D1); individual monograph has not been reached after 35 days,
- 9 parts of water for injection. continue the fermentation process until the prescribed pH is
reached (maximum 55 days). Filter (nominal pore size not
Subsequent decimal dilutions are produced as stated for D2. greater than 15 µm) to remove the sediment. The filtrate is
METHOD 1.5. MOTHER TINCTURES OBTAINED BY the mother tincture.
FERMENTATION (RHYTHMIC CONDITIONS)
Potentisation
METHODS 1.5.1 AND 1.5.2 (HAB 21, 22: RH MOTHER TINCTURES)
Methods 1.5.1 and 1.5.2
Methods 1.5.1 and 1.5.2 are used for fresh (harvested and The 1' 1 'decimal' dilution (D1) is made from:
processed within less than 12 h) herbal drugs generally - 1 part of the Rh mother tincture;
containing more (Method 1.5.1) or less (Method 1.5.2) than - 9 parts of water for injections.
50 per cent (mlm) of expressed juice, respectively. Mother
tinctures prepared according to Methods 1.5.1 and 1.5.2 are Subsequent decimal dilutions are produced as described for
obtained by fermentation under specified rhythmic conditions DL
without adding excipients, as described below. Where If the contents of the containers exceed 20 mL, suitable
appropriate, e.g. for roots, the fresh herbal drug is first measures must be taken to guarantee the microbiological
washed with water and rinsed with purified water. These quality of the mother tincture after opening. Addition of
methods are used in the preparation of injections, eye antimicrobial preservatives is not allowed.
preparations and aqueous dilutions. Storage
Method 1.5.1 Store the mother tinctures at 2 °C to 15 °C, in an airtight
Express the appropriately comminuted herbal drug within container, protected from light.
12 h of harvesting. Immediately transfer the expressed juice Labelling
into glass containers, designed to largely keep out air and to 'Rh' is an abbreviation for the 'rhythm' described above.
allow for heating and cooling. Beginning within 12 h of
Mother tinctures prepared according to Methods 1. 5 .1 METHOD 2.1.3 (FRENCH PHARMACOPOEIA)

and 1.5.2 and any preparations thereof are labelled 'Rh'. Fresh or dried raw materials of herbal or animal origin are
used. Pathological material is excluded.
Table 2371.-1. - Example of a stepwise stirring time reduction
plan Maceration
Comminute the raw material appropriately. Take a sample
Step Duration of Stirring time Stirring time and determine the loss on drying at 105 °C for 2 h (2.2.32)
step (= after 12 h)
or the water content (2.2.13). Taking this value into account,
Step 1 9 days 180 min 180 min calculate and add to the raw material the quantity of the
ethanoVglycerol mixture of the appropriate concentration to
Step 2 8 days 80 min 80 min
produce, unless otherwise prescribed, a 1 in 20 glycerol
Step 3 7 days 30min 30 min macerate. Allow to macerate for at least 21 days, with
sufficient shaking. Decant and strain under pressure if
Step 4 6 days 15 min 15 min
necessary. Allow the combined liquids to stand for 48 hand
Step 5 5 days 10 min 10 min filter.
Potentisation
2. GLYCEROL MACERATES The l st decimal dilution (D 1) is made from:
METHOD2.1 - 1 part of the glycerol macerate;
Method 2.1 is used for maceration of raw materials of animal - 9 parts of a water/ethanol/glycerol mixture of appropriate
or herbal origin in glycerol (85 per cent) or glyceroVethanol concentration.
mixtures of appropriate concentration. The 2nd decimal dilution (D2) is made from:
The raw materials are finely minced before use, where - 1 part of the 1st decimal dilution;
appropriate. - 9 parts of a water/ethanol/glycerol mixture of appropriate
concentration.
METHODS 2.1.1, 2.1.2 (HAB 42A AND 42B: MOTHER TINCTURES
AND LIQUID DILUTIONS THEREOF)
Subsequent decimal dilutions are produced as stated for D2
or using another appropriate vehicle.
Raw materials of animal origin - freshly killed animals or
parts thereof - are used. Animals are processed immediately The 1st centesimal dilution (Cl) is made from:
after being killed. - 1 part of the glycerol macerate;
- 99 parts of a water/ethanoVglycerol mixture of appropriate
Maceration concentration.
Disperse 1 part of finely minced animal material in:
The 2nd centesimal dilution (C2) is made from:
- 9 parts (decimal dilutions) or 99 parts (centesimal
dilutions) of glycerol (85 per cent) for Method 2.1.1; or - 1 part of the 1st centesimal dilution;
- 99 parts of a water/ethanoVglycerol mixture of appropriate
- 2.1 parts of glycerol (85 per cent) for Method 2.1.2.
concentration.
Allow to macerate for at least 2 h, then succuss. Filter when
Subsequent centesimal dilutions are produced as stated
necessary.
for C2 or using another appropriate vehicle.
Where justified, 1 part of glycerol (85 per cent) may be
added to 1 part of animal material before mincing. Where METHOD2.2
METHODS 2.2.1, 2.2.2, 2.2.3, 2.2.4 (HAB 41A, 418, 41C AND 41D:
very small amounts of animal material are used, the dilution
GLYCEROL MOTHER TINCTURES AND LIQUID DILUTIONS
may be prepared by dispersing 1 part of finely minced animal
THEREOF)
material in 99 parts of glycerol (85 per cent) (Cl or 'D2' if
to be used for further decimal dilutions). Method 2.2 is used for maceration of raw materials of animal
origin in a glycerol solution containing sodium chloride.
Potentisation
Pathological material is excluded.
Method 2.1.1
Raw materials from freshly killed animals, parts or secretions
thereof are used in Methods 2.2.1, 2.2.2 and 2.2.3. Lower
- 1 part of the glycerol macerate DI;
- 9 parts of glycerol (85 per cent) or ethanol
animals are killed with carbon dioxide in a covered vessel.
(18 per cent V/V). All animals are processed immediately after being killed.
Blood components from live horses are used in
Subsequent decimal dilutions are produced as stated for D2
method 2.2.4.
but with ethanol (18 per cent V/V) as the vehicle.
The 2nd centesimal dilution (C2) is made from: Sample collection and/or pre-treatment
- 1 part of the glycerol macerate Cl; The raw materials used in Methods 2.2.1, 2.2.2 and 2.2.3
- 99 parts of ethanol (18 per cent V/V). are finely minced before use, where appropriate.
Subsequent centesimal dilutions are produced as stated for The blood used in Method 2.2.4 is collected by a
C2. veterinarian. Blood obtained from animals killed by bleeding
must not be used. Take 200 mL of this blood and add 15 IU
Method 2.1.2
of heparin sodium and 0.625 mL of a 9 g/kg solution of
The 1st 'decimal' dilution (DI) is made from: sodium chloride per millilitre. Separate the blood
- 3 parts of the glycerol macerate; components by fractional centrifugation and resuspend each
- 7 parts of water for injections. individual cell sediment in 1.1 mL of a 9 g/kg solution of
The 2nd decimal dilution (D2) is made from: sodium chloride. These cell suspensions are processed into
- 1 part ofDl; the glycerol macerate.
- 9 parts of water for injections. Maceration
Subsequent decimal dilutions are produced as stated for D2. Mix 1 part of finely minced animal material, secretions or
blood cell suspensions, according to the method used, with 5
parts of a sodium chloride solution of the appropriate Table 2371.-3

concentration (see Table 2371.-2) and 95 parts of glycerol. Method 3.1.1 Method 3.1.2
Allow to stand protected from light for at least 7 days, then
decant. If necessary for Methods 2.2.1, 2.2.2 and 2.2.3, Anhydrous ethanol Water for injections
centrifuge before decanting, then filter the supernatant if
Ethanol (96 per cent V/V) Purified water
necessary. The decanted liquid or the filtrate respectively is
the glycerol macerate. Ethanol (90 per cent V/V)
Any sediment present must be resuspended before processing Ethanol (80 per cent V/V)
the glycerol macerate.
Ethanol (70 per cent V/V)
Table 2371.-2 Ethanol (SO per cent V/V)
Methods 2.2.1 and
Method 2.2.2 Method 2.2.3 Ethanol (36 per cent V/V)
2.2.4
Ethanol ( 18 per cent V/V)
15 g/kg solution of 40 g/kg solution of 80 g/kg solution of
sodium chloride in sodium chloride in sodium chloride in Purified water
purified water purified water purified water
Glycerol (85 per cent)
Vehicle for potentisation

0.2 parts of sodium hydrogen carbonate and 8.8 parts of For Method 3.1.1, if ethanol (18 per cent V/V) is used, the
sodium chloride in 991 parts of water for injections or starting material may be dissolved in 7 .58 parts of purified
purified water as appropriate. water and the ethanol concentration adjusted by adding
1.42 parts of ethanol (96 per cent V/V) to the solution, for
Potentisation
decimal dilutions. For centesimal dilutions, use 83.4 parts of
The glycerol macerate corresponds to the 2nd decimal
purified water for 15.6 parts of ethanol (96 per cent V/V).
dilution ('D2') or the 1st centesimal dilution (Cl).
For Method 3.1.2, if the starting material is not stable and/or
The 3rd decimal dilution (D3) is made from:
soluble in water, glycerol (85 per cent) may be added at a
- 1 part of the 2nd decimal dilution;
concentration of not more than 35 per cent of the vehicle, for
- 9 parts of the appropriate vehicle.
potentisation up to D4.
Potentisation
Where appropriate, the 4th decimal dilution (D4) is made Unless otherwise specified, the 2nd decimal dilution (D2) is
from 1 part of the 3rd decimal dilution, 5.6 parts of the made from:
vehicle and 3.4 parts of water for injections. - 1 part of the 1st decimal dilution (Dl);
The 2nd centesimal dilution (C2) is made from: - 9 parts of ethanol (50 per cent V/V) for Method 3.1.1 or
- 1 part of the 1st centesimal dilution; 9 parts of water for injections (or purified water, as
- 99 parts of the appropriate vehicle. appropriate) for Method 3.1.2.
Subsequent centesimal dilutions are produced as stated Subsequent decimal dilutions are produced as stated for D2.
for C2. Unless otherwise specified, the 2nd centesimal dilution (C2)
3. DILUTIONS is made from:
METHODJ.1 - 1 part of the 1st centesimal dilution (Cl);
Methods 3.1.1, 3.1.2 and 3.1.3 are used for dissolution of - 99 parts of ethanol (50 per cent V/V) for Method 3.1.1 or
any suitable inorganic or organic starting material, for 99 parts of water for injections (or purified water, as
example minerals or venoms. appropriate) for Method 3.1.2.
Unless otherwise specified, dissolve 1 part of the starting Subsequent centesimal dilutions are produced as stated
material in 9 parts (DI) or 99 parts (Cl) of the liquid vehicle for C2.
and succuss. Additives
Where justified and authorised, in case of insufficient For Method 3 .1.1, if a reaction such as precipitation is
solubility of the starting material in the specified vehicle, observed in the final dilution, the following additives may be
directly produce the first possible dilution. For example, if used to enhance stability and/or solubility, unless otherwise
the starting material is slightly soluble, dissolve 1 part of the specified:
starting material in 99 parts of the vehicle (Cl or 'D2' if to - glacial acetic acid;
be used for further decimal dilutions). - concentrated hydrochloric acid;
METHODS 3.1.1, 3.1.2 (HAB SA, 58: SOLUTIONS, AQUEOUS - lactic acid;
SOLUTIONS) - sodium hydroxide.
Vehicles Where solutions or dilutions have been pH-adjusted, they
The vehicles in Table 2371.-3 may be used. must not be potentised further.
METHOD 3.1.3
Vehicles
Suitable vehicles, for example, ethanol of an appropriate
concentration, glycerol or purified water may be used alone
or combined.
Potentisation
Unless otherwise specified, the 2nd decimal dilution (D2) is
made from:
- 1 part of the 1st decimal dilution (D1); - 1 part of the centesimal trituration Cn-2;
- 9 parts of the appropriate vehicle. - 99 parts of purified water or of another suitable vehicle in
Subsequent decimal dilutions are produced as stated for D2. appropriate proportions.
Unless otherwise specified, the 2nd centesimal dilution (C2) The following centesimal dilution (Cn) is made from:
is made from: - 1 part of the first liquid centesimal dilution Cn-1;
- 1 part of the 1st centesimal dilution (C 1); - 99 parts of a suitable vehicle.
- 99 parts of the appropriate vehicle. Subsequent centesimal dilutions are produced as stated
Subsequent centesimal dilutions are produced as stated for Cn.
for C2. 4. TRITURATIONS
METHODJ.2 METHOD4.1
Method 3.2 is generally used to produce liquid dilutions of Method 4.1 is used for triturations, that is solid dilutions, of
triturations of substances that for the most part are sparingly raw materials or of triturations prepared according to
soluble to practically insoluble. Methods 4.2.1 or 4.2.2. The duration and intensity of the
METHODS 3.2.1, 3.2.2 (HAB SA, 8B: LIQUID PREPARATIONS MADE trituration are such that homogeneity and potentisation are
FROM TRITURATIONS, AQUEOUS PREPARATIONS MADE FROM achieved.
TRITURATIONS) Vehicle
Preparations made according to Method 3.2.1 and Unless otherwise specified, lactose monohydrate is used.
Method 3.2.2 are produced from triturations D4, D5 and D6 METHOD 4.1.1 (HAB 6: TRITURATIONS)
or from triturations C4, CS and C6, prepared according to Triturations are prepared manually or mechanically.
method 4.1.1 by at least 2 potentisation steps. Mechanical trituration must be used for quantities exceeding
Vehicles 1 kg. The resulting particle size of the raw material in the
The vehicles in Table 2371.-4 may be used. first decimal or centesimal dilution does not exceed 100 µm,
unless otherwise prescribed in the individual monograph.
Table 2371.-4
Ratios of raw material to vehicle
Method 3.2.1 Method 3.2.2
I st potentisation: Purified water All potentisations: Water for Decimal triturations Centesimal triturations
injections Purified water
The 1 decimal trituration (DI) is
st The I st centesimal trituration (C I) is
2nd potentisation: Ethanol made from: made from:
(36 per cent VIV)
I part of the raw material I part of the raw material
Further potentisations: Ethanol
(50 per cent V/V) 9 parts of the vehicle 99 parts of the vehicle
Subsequent decimal Subsequent centesimal

triturations (Dn) are produced as triturations (Cn) are produced as
Potentisation
stated for DI, using I part of the stated for C I, using I part of the
For the first liquid potentisation, dissolve 1 part of the previous trituration (Dn-1). previous trituration (Cn-1).
trituration in 9 parts (decimal dilutions) or 99 parts
(centesimal dilutions) of the specified vehicle (see
Table 2371.-4) and succuss. For further potentisations, Where fresh plant material is used, the quantity of vehicle
proceed in the same manner with 1 part of the previous added is such so as to obtain 10 parts of the trituration
dilution. (decimal trituration) or 100 parts of the trituration
(centesimal trituration) from 1 part of the raw material
The D6, D7, C6 and C7 dilutions produced by the above
(replace the mass of water lost from the fresh plant by an
method are not to be used for the preparation of further
equivalent amount of the vehicle). A suitable gentle drying
dilutions. For the preparation of higher dilutions use the D8
process may need to be applied to the solid dilution.
or CS dilution.
Where justified and authorised, it may be necessary to
METHOD 3.2.3
directly produce a C 1 or 'D2' if to be used for further
Preparations made according to Method 3.2.3 are produced decimal triturations as the first solid trituration, made from
from triturations D2 onwards and from triturations C 1, C2, 1 part of raw material and 99 parts of vehicle.
C3 and C4, prepared according to method 4.1.2.
Trituration
Vehicles Unless otherwise justified and authorised, the method
Suitable vehicles such as ethanol of an appropriate consists of dividing the vehicle into 3 equal parts and adding
concentration or purified water may be used. the raw material to the first part, then adding the second and
Potentisation third part of the vehicle, thoroughly triturating after each
Unless otherwise specified, the first liquid decimal addition.
dilution (Dn-1) is made from: For mechanical trituration, use a machine allowing the
- 1 part of the decimal trituration Dn-2; requirements for particle size of the first decimal or
- 9 parts of purified water or of another suitable vehicle in centesimal solid trituration to be met. A machine fitted with
appropriate proportions. a scraping device may be used to ensure even trituration.
The following decimal dilution (Dn) is made from: The time required to prepare one trituration is at least 1 h,
- 1 part of the first liquid decimal dilution Dn-1; unless otherwise justified and authorised.
- 9 parts of a suitable vehicle. For manual trituration, divide the vehicle into 3 equal parts
Subsequent decimal dilutions are produced as stated for Dn. and briefly triturate the first part in a porcelain mortar.
Unless otherwise specified, the first liquid centesimal Add the raw material, triturate the mixture for 6 min, scrape
dilution (Cn-1) is made from: down for 4 min with an appropriate non-metallic device (for
example, a porcelain spatula). Triturate for a further 6 min, Table 2371.-5

scrape down again for a further 4 min, then add the second Centesimal triturations
Decimal triturations
part of the vehicle and continue as above. Proceed in the
same manner with the rest of the vehicle. The minimum time Mother tinctures prepared according to Methods 1.1.1, 1.1.3 and 1.1.4
required for the whole process is thus 1 h. Carry out the
The 1st 'decimal' trituration (DI) is The I" 'centesimal' trituration (Cl)
whole process again for each subsequent solid dilution. made from: is made from:
Triturations from D5 or CS onwards may also be prepared
2 parts of the mother tincture 2 parts of the mother tincture
by intense mechanical treatment by a suitable mixing
machine as follows: add the solid trituration to one third of maximum I O parts of the vehicle, maximum I 00 parts of the vehicle,
the vehicle and mix. Add the second third of the vehicle, mix taking the mass of the dry residue taking the mass of the dry residue
into consideration into consideration
and proceed in the same manner with the last third of the
vehicle. The whole process lasts minimum 1 hour, unless Mother tinctures prepared according to Methods 1.1.2, 1.1.5, 1.1.6
otherwise justified and authorised. and 1.1.7
It is possible to change to a liquid medium from the 4'\ 5th The 1st 'decimal' trituration (DI) is The 1st 'centesimal' trituration (Cl)
and 6th decimal or centesimal triturations, as described in made from: is made from:
Methods 3.2.1 and 3.2.2.
3 parts of the mother tincture 3 parts of the mother tincture
METHOD 4.1.2 (FRENCH PHARMACOPOEIA)
maximum 10 parts of the vehicle, maximum I 00 parts of the vehicle,
Trituration taking the mass of the dry residue taking the mass of the dry residue
Triturations are prepared as follows: into consideration into consideration
Decimal tn'turations
Mother tinctures prepared according to Methods 1.1.8 and 1.1.9
Reduce 1 part of the homoeopathic stock to a powder.
The mother tincture corresponds to the 1" decimal dilution (DJ)
Triturate carefully with a small quantity of the vehicle.
Add the vehicle in small quantities until 9 parts of this The znd decimal trituration (DZ) is The I" 'centesimal' trituration (Cl)
vehicle have been used. The resulting trituration is the 1st made from: is made from:
decimal trituration (DI). I part of the mother tincture I O parts of the mother tincture
Triturate as described above 1 part of this trituration with 9
maximum 10 parts of the vehicle, maximum 100 parts of the vehicle,
parts of the vehicle. The resulting trituration is the 2nd taking the mass of the dry residue taking the mass of the dry residue
decimal trituration (D2). into consideration into consideration
In all cases, it is possible to change to a liquid medium after
Solutions prepared according to Method 3.1.l or liquid dilutions,
the 7th decimal trituration (D7) as described in mixtures and co-potentised mixtures
Method 3.2.3.
Decimal trituration n+ 1 (Dn+ 1) is Centesimal trituration n+ 1 (Cn+ I)
Centesimal triturations
made from: is made from:
Proceed in the same manner but following a centesimal
I part of the dilution (Dn) I part of the dilution (Cn)
senes.
In all cases, it is possible to change to a liquid medium after maximum 10 parts of the vehicle, maximum I 00 parts of the vehicle,
the 3rd centesimal trituration (C3) as described in taking the mass of the dry residue taking the mass of the dry residue
into consideration into consideration
Method 3.2.3.
METHOD4.2
Method 4.2 is used for triturations, that is solid dilutions, of METHOD 4.2.2
liquid preparations such as mother tinctures and solutions, Ratios of starting material to vehicle
their dilutions, mixtures and co-potentised mixtures.
Gradually impregnate the total amount of vehicle, gently dry Mother tinctures prepared according to Methods 1. 1. 10 and 1.1.11
the moist mixture, mill and sieve if necessary, then mix and
The 1" decimal trituration (DI) is The 1" centesimal trituration (Cl) is
triturate until homogeneity and potentisation are achieved. made from: made from:
Trituration is further carried out as described for
Method 4.1.1 or Method 4.1.2. I part of the mother tincture l part of the mother tincture
Vehicle I O parts of the vehicle I 00 parts of the vehicle

Unless otherwise specified, lactose monohydrate is used.
METHOD 4.2.1 (HAB 7: TRITURATIONS)
5. OTHER PREPARATIONS
Ratios of starting material to vehicle METHOD5.1
The quantity of vehicle added must always be such so as to Method 5.1 is used for preparing homoeopathic preparations
obtain 10 parts of the trituration (decimal trituration) or by co-potentising 2 or more stocks and/or dilutions thereof,
100 parts of the trituration (centesimal trituration) from the where co-potentisation consists of mixing several stocks or
required number of parts of the liquid preparation (see dilutions of stocks then potentising them together in one or
Table 2371.-5), taking the mass of the dry residue into more potentisation steps.
consideration. Where the dry residue is considered negligible,
METHODS 5.1.1, 5.1.2, 5.1.3 (HAB 40A, 40B, 40C: CO-POTENTISED
the quantity of vehicle added is 10 parts (decimal trituration)
MIXTURES)
or 100 parts (centesimal trituration), for 1 part of the liquid
preparation. The stocks and/or dilutions in Table 2371.-6 may be used.
Table 2371.-6 proportion of the active substances

[i.e. 100 per cent -(1 per cent x the number of active
Method 5.1.1 Method 5.1.2 Method 5.1.3
substances)]. The same procedure applies, in the appropriate
Stocks Aqueous preparations Triturations proportions, when co-potentising decimal dilutions.
Solutions Glycerol macerates and METHODS.2
aqueous dilutions Method 5.2 is used to prepare potencies with a 50 000
thereof dilution factor (LM) by alternate steps of liquid dilution and
Triturations Triturations impregnation of sucrose pillules (category 1, unless otherwise
authorised).
Liquid dilutions
Solid and liquid potencies
Mother tinctures whose Use a dropper (2.1.1).
method of production
specifies a 1/10 To prepare pillules of 1st LM potency, a C3 trituration of the
(or 1/100) dilution substance to be potentised is processed as follows: dissolve
60 mg of the C3 trituration in a volume of ethanol
(18 per cent VIV) or another authorised concentration,
Vehicles corresponding to 500 drops. To 1 drop of this solution add a
The choice of the vehicle is determined by and must comply volume of ethanol (90 per cent VIV) or another authorised
with any special requirement for the particular stock as well concentration, corresponding to 100 drops and succuss at
as the dosage form (see table Table 2371.-7). least 100 times. Use all ofthis solution to impregnate a mass
of pillules corresponding to 50 000 pillules, then allow to dry
Table 2371.-7
m arr.
Method 5.1.1 Method 5.1.2 Method 5.1.3
To prepare pillules of 2nd LM potency, process pillules of
Ethanol (96 per cent V/V) Water for injections Lactose 1st LM potency as follows: dissolve 1 pillule of 1st LM
monohydrate potency in 1 drop of purified water, add a volume of ethanol
Ethanol (90 per cent V/V) Purified water (90 per cent VIV) or another authorised concentration,
corresponding to 100 drops and succuss at least 100 times.
Ethanol (80 per cent V/V) Sugar syrup (sucrose,
Use all of this solution to impregnate a mass of pillules
purified water
(64:36)) corresponding to 50 000 pillules, then allow to dry in air.
Subsequent solid potencies are prepared as for the 2nd LM
Ethanol (70 per cent VIV)
potency.
Ethanol (50 per cent V/V) Liquid potencies (HAB 17)
Ethanol (36 per cent VIV) To prepare a liquid LM potency, dissolve 1 pillule of the
required potency in 10.0 mL of ethanol (18 per cent V/V) or
Ethanol (18 per cent V/V)
another authorised concentration. The potency of the
solution corresponds to the potency of the pillule dissolved
For Method 5.1.1, when starting from a trituration and therein.
where justified, purified water is used for the 1st potentisation _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
step.
For Method 5.1.2, when starting from a glycerol macerate
containing sodium chloride, unless otherwise justified and
authorised, the following vehicle is used: 0.2 parts of sodium
hydrogen carbonate and 8.8 parts of sodium chloride in
Mother Tinctures for
991 parts of water for injections. Homoeopathic Preparations
Potentisation (Ph. Bur. monograph 2029)
For each potentisation step, combine and succuss or triturate PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
1 part of the given mixture with 9 parts (decimal dilutions)
or 99 parts (centesimal dilutions) of the appropriate vehicle. DEFINITION
METHOD 5.1.4 Mother tinctures for homoeopathic preparations are liquid
preparations obtained by the solvent action of a suitable
Vehicles vehicle upon raw materials. The raw materials are usually in
Ethanol of an appropriate concentration, purified water or
the fresh form but may be dried. Mother tinctures for
lactose monohydrate may, for example, be used.
homoeopathic preparations may also be obtained from plant
Potentisation juices, with or without the addition of a vehicle. For some
Potentisation may be performed as prescribed for preparations, the matter to be extracted may undergo a
Methods 5.1.1, 5.1.2 and 5.1.3, either on the last step or on preliminary treatment.
several successive steps.
PRODUCTION
METHOD 5.1.5
Mother tinctures for homoeopathic preparations are prepared
Vehicle by maceration, percolation, digestion, infusion, decoction,
Ethanol of an appropriate concentration, purified water or fermentation or as described in the individual monographs,
lactose monohydrate may, for example, be used. usually using ethanol of suitable concentration.
Potentisation Mother tinctures for homoeopathic preparations are obtained
For a co-potentisation of centesimal dilutions, each using a fixed proportion of raw material to solvent, taking the
dilution ( Cn-1) represents 1 per cent of the final product and moisture content of the raw material into account, unless
the proportion of vehicle to be added is reduced by the otherwise justified and authorised.
If fresh plants are used, suitable procedures are used to herbal drug. The dilution factor of the mother tincture and
ensure freshness. The competent authorities may require that the limit of quantification of the method are also taken into
the freshness is demonstrated by means of a suitable test. account when setting these limits.
Where the mother tincture contains ethanol, it is tested for Heavy metals (2.4.27)
2-propanol (2.9.11), with a maximum limit of Justification is provided in cases where the test for heavy
0.05 per cent V/V, unless assurance of compliance with this metals is performed on the mother tincture, instead of on the
limit is provided by a detailed knowledge of the ethanol herbal drug according to the requirements of the general
supply chain and the mother tincture manufacturing process. monograph Herbal drugs for homoeopathic preparations (2045).
Mother tinctures for homoeopathic preparations are usually Limits are set, taking into consideration the nature and the
clear. A slight sediment may form on standing and that is origin of the herbal drug. The dilution factor of the mother
acceptable as long as the composition of the tincture is not tincture and the limit of quantification of the method are also
changed significantly. taken into account when setting 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
Production by maceration be defined.
Unless otherwise prescribed, reduce the matter to be Aflatoxin B 1 (2.8.18)
extracted to pieces of suitable size, mix thoroughly and Where appropriate, limits for aflatoxins may be required.
extract according to the prescribed extraction method with Justification is provided in cases where the test for aflatoxin
the prescribed extraction solvent. Allow to stand in a closed B 1 is performed on the mother tincture, instead of on the
vessel for the prescribed time. The residue is separated from herbal drug. Limits are set, taking into consideration the
the extraction solvent and, if necessary, pressed out. In the nature and the origin of the herbal drug. The dilution factor
latter case, the 2 liquids obtained are combined. of the mother tincture and the limit of quantification of the
Adjustment of the contents method are also taken into account when setting these limits.
Adjustment of the content of constituents may be carried out ASSAY
if necessary, either by adding the extraction solvent of Where applicable, an assay with quantitative limits is
suitable concentration, or by adding another mother tincture performed.
for homoeopathic preparations of the vegetable or animal
STORAGE
matter used for the preparation.
Protected from light. A maximum storage temperature may
CHARACTERS be specified.
Where appropriate, the appearance and odour of the mother
LABELLING
tincture are determined.
The label states:
IDENTIFICATION - that the product is a mother tincture for homoeopathic
Where applicable, at least 1 chromatographic identification preparations (designated as 'TM' or '0');
test is carried out. - the name of the raw material using the Latin title of the
TESTS European Pharmacopoeia monograph where one exists;
The limits in an individual monograph are set to include - the method of preparation;
official methods of production. Specific limits will apply to - the ethanol content or other solvent content,
each defined method of production. in per cent V/V, in the mother tincture;
- the ratio of raw material to mother tincture;
If the test for relative density is carried out, the test for ethanol - where applicable, the storage conditions.
need not be carried out, and vice versa.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
The mother tincture for homoeopathic preparations complies
with the limits prescribed in the monograph.
Ethanol (2. 9. 10) Coated Homoeopathic Pillules
The ethanol content complies with that prescribed in the
monograph. (Ph. Bur. monograph 2786)
Methanol (2.9.11) Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Maximum 0.05 per cent V/V, unless otherwise prescribed or
justified and authorised. DEFINITION
Solid preparations prepared from sucrose Pillules for
homoeopathic preparations (2153) (category 5), by coating with
Where applicable, the mother tincture for homoeopathic
a syrup made from homoeopathic preparations either
preparations complies with the limits prescribed in the
potentised or mixed with sucrose syrup. Triturations can be
monograph.
incorporated separately. Coated homoeopathic pillules
Pesticides (2. 8.13) possess a suitable mechanical strength to resist handling
Where applicable, the mother tincture for homoeopathic without crumbling or breaking. They are intended for
preparations complies with the test. This requirement is met sublingual or oral use. Coated homoeopathic pillules may
if the herbal drug has been shown to comply with the test. also be called 'globuli velati'.
Justification is provided in cases where the test for pesticides PRODUCTION
is performed on the mother tincture, instead of on the herbal
In the manufacture, packaging, storage and distribution of
drug according to the requirements of the general monograph
coated homoeopathic pillules, suitable measures are taken to
Herbal drugs for homoeopathic preparations (2045). Limits are
ensure their microbiological quality; recommendations on this
set, taking into consideration the nature and the origin of the
aspect are provided in general chapter 5.1.4. Microbiological

quality of non-sterile phannaceutical preparations and substances
Impregnated Homoeopathic
for phannaceutical use. Pillules
For coating, use homoeopathic stocks (in case of a mother (Ph. Bur. monograph 2079)
tincture or a glycerol macerate) and/or decimal dilutions
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
thereof or decimal triturations and a syrup made from 64
parts of sucrose and 36 parts of purified water. DEFINITION
The homoeopathic preparations used are prepared according Solid preparations obtained from sucrose, lactose or other
to the general monograph Methods of preparation of suitable excipients. They possess a suitable mechanical
homoeopathic stocks and potentisation (2371). strength to resist handling without crumbling or breaking.
To prepare 100 parts of coated homoeopathic pillules, the Impregnated homoeopathic pillules are prepared by
pillules are coated with the syrup using one of the following impregnation of Pillules for homoeopathic preparations (2153)
procedures, depending upon the type of homoeopathic with one or more liquid homoeopathic preparations. They
preparation used. are intended for sublingual or oral use.
Aqueous dilutions PRODUCTION
Mix 1 part of an aqueous preparation with 9 parts of the The impregnation process takes place using liquid
syrup and potentise by succussion; evenly apply the mixture preparations containing ethanol usually at a concentration of
to (100 - x) parts of pillules (where x is the mass of sucrose at least 68 per cent V/V according to either of the following
in the syrup). The potency of the coated homoeopathic approaches:
pillules corresponds to the potency of the liquid preparation - in proportions of 1 mass part of liquid to 100 mass parts
used for coating. of pillules;
Triturations - in proportions of 1 volume part of liquid to 100 mass
Mix 10 parts of a trituration with 20 parts of the syrup; parts of pillules.
evenly apply the mixture to (100 - x- y) parts of pillules In the manufacture, packaging, storage and distribution of
(where x is the mass of sucrose in the syrup and y is the homoeopathic pillules, suitable measures are taken to ensure
mass of lactose monohydrate in the incorporated trituration). their microbiological quality; recommendations on this aspect
The potency of the coated homoeopathic pillules corresponds are provided in general chapter 5.1. 4. Microbiological quality of
to the potency of the trituration used. non-sterile phannaceutical preparations and substances for
Mixtures phannaceutical use.
Unless otherwise justified and authorised, prepare a mixture CHARACTERS
obtained from 10 parts of a liquid preparation (obtained Appearance
from aqueous preparations, triturations or aqueous dilutions White, almost white or slightly coloured spheroids.
of glycerol macerates) and 90 parts of the syrup, adding
sufficient purified water to evenly apply the mixture to
Solubility
Usually freely soluble in water.
(100 - x - y) parts of pillules (where x is the mass of sucrose
in the syrup and y is the mass of lactose monohydrate in the TESTS
incorporated triturations). The potency/potencies of the Microbial contamination
coated homoeopathic pillules correspond to the Unless otherwise justified, authorised and labelled, the
potency/potencies of the individual preparations used in the pillules are intended for sublingual administration and the
mixture. following acceptance criteria apply.
TESTS TAMC: acceptance criterion 102 CFU/g (2.6.12).
Uniformity of mass. TYMC: acceptance criterion 10 1 CFU/g (2.6.12).
Carry out the test using 20 coated homoeopathic pillules to Absence of Staphywcoccus aureus (2. 6.13).
constitute 1 unit. Weigh individually 20 units taken at Absence of Pseudomonas aernginosa (2.6.13).
random and determine the individual and average masses.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Not more than 2 of the individual masses deviate from the
average mass by more than 10 per cent and none deviate by
more than 20 per cent.
Microbial contamination.
Unless otherwise justified, authorised and labelled, coated Pillules for Homoeopathic
homoeopathic pillules are intended for sublingual Preparations
administration and the following acceptance criteria apply.
TAMC: acceptance criterion 102 CFU/g (2.6.12).
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
TYMC: acceptance criterion 10 1 CFU/g (2.6.12).
Absence of Staphylococcus aureus (2.6.13). DEFINITION
Absence of Pseudomonas aerngi,nosa (2. 6.13). Solid preparations obtained from sucrose, lactose or other
suitable excipients. They possess a suitable mechanical
---------------------~& strength to resist handling without crumbling or breaking.
They are intended for impregnation or coating with one or
more homoeopathic preparations. The impregnated pillules
comply with the requirements of the monograph
Homoeopathic pillules, impregnated (2079). The coated pillules
comply with the requirements of the monograph
Homoeopathic pillules, coated (2786).
PRODUCTION methylene blue impregnation solution to achieve a content of

In the manufacture, packaging, storage and distribution of 10 µL of impregnation solution per gram of pillules.
pillules for homoeopathic preparations, suitable measures are Test solution Dissolve 5.00 g of the impregnated pillules in
taken to ensure their microbiological quality; water R and dilute to 25.0 mL with the same solvent.
recommendations on this aspect are provided in general Reference solution Dilute 1.0 mL of the methylene blue
chapter 5.1. 4. Microbiological quality of non-steril,e impregnation solution to 100.0 mL with water R. To 5.0 mL
pharmaceutical preparations and substances for pharmaceutical of this solution add 5.00 g of pillules for homoeopathic
use. preparations, dissolve in water Rand dilute to 25.0 mL with
Ifa system of sizing is used, the indications in Table 2153.-1 the same solvent.
are used. Measure the absorbance (2. 2. 25) of the test solution and the
Table 2153.-1. - Classification of pillules according to their mass reference solution at 665 nm. Calculate the percentage of
and size impregnation of the pillules for homoeopathic preparations
Category Number of pillules for Mass Fineness
homoeopathic (g) (µm)
preparations
470 - 530 1.0 1000 - 1600
2 160 - 333 1.0 1400 - 2000
absorbance of the test solution;
3 110 - 130 1.0 1800 - 2500 absorbance of the reference solution;
4 70 - 90 1.0 2000 - 2800 mass of the impregnated pillules used to prepare the test
5 40 - 50 1.0 2500 - 3350 solution, in grams.
6 16 - 30 1.0 3150 - 4500
7 10 0.9 - 1.1 4000 - 5600 METHOD B - CAFFEINE
8 5 0,9 - I.I 5600 - 6700 As the test results are particularly influenced by the
9 3 0.9 - I.I 7100 - 8000 procedures for impregnation and drying of the pillules, the
10 2 0.9 - 1.1 8000 - 9500 parameters of these procedures are defined and stated with
NOTE: for categories 7-10, the mass is obtained by weighing the specified the result.
number of pillules. Caffeine impregnation solution Prepare a 15 g/L solution of
caffeine R in ethanol (70 per cent V/V) R.
CHARACTERS
Impregnation Impregnate a suitable quantity of pillules for
Appearance
homoeopathic preparations with a suitable quantity of the
White or almost white spheroids.
caffeine impregnation solution to achieve a content of 10 µL
Solubility of impregnation solution per gram of pillules.
Usually freely soluble in water. During the preparation of the following solutions, if the pillules are
IDENTIFICATION not fully dissolved, sonicate the dispersion for 30 min and cool to
The excipients used for the manufacture of pillules for 20 °C. Before measuring the absorbance, centrifuge the dispersion
homoeopathic preparations are identified by one or more and filter the supernatant through a membrane filter (nominal
suitable test(s). pore size 0.45 µm).
TESTS Test solution To 5.00 g of the impregnated pillules add
If the test for fineness is carried out, the test for uniformity of mass 30 mL of water R. Shake manually then sonicate until
need not be carried out, and vice versa. complete dissolution of the pillules. Dilute to 50.0 mL with
water R.
Uniformity of mass
Reference solution Dilute 1.0 mL of the caffeine
Carry out the test using 20 pillules to constitute 1 unit.
impregnation solution to 100.0 mL with water R. To 5.0 mL
Weigh individually 20 units taken at random and determine
of this solution add 5.00 g of pillules for homoeopathic
the individual and average masses. Not more than 2 of the
preparations and 30 mL of water R. Shake manually then
individual masses deviate from the average mass by more
sonicate until complete dissolution of the pillules. Dilute to
than 10 per cent and none deviate by more than 20 per cent.
Fineness (2.9.35)
Compensation l¼uid To 5.00 g of pillules for homoeopathic
Not less than 90 per cent m/m of the pillules are between the
preparations add 30 mL of water R. Shake manually then
lower and upper limits of the corresponding category as
sonicate until complete dissolution of the pillules. Dilute to
indicated in Table 2153.-1.
Uniformity of impregnation Measure the absorbance (2. 2. 25) of the test solution and the
None of the individual values deviate by more than reference solution at 273 nm by comparison with the
10 per cent relative from the average of 10 determinations. compensation liquid. Calculate the percentage of
Determine the uniformity of impregnation using either impregnation of the pillules for homeopathic preparations
method A or method B. Carry out 10 individual using the following expression:
determinations.
METHOD A - METHYLENE BLUE
Methylene blue impregnation solution Use a freshly prepared
solution. Dissolve 1.000 g of methylene blue R in 50 mL of
absorbance of the test solution;
ethanol (70 per cent V/V) Rand dilute to 1000.0 mL with the absorbance of the reference solution;
same solvent. mass of the impregnated pillules used to prepare the test
solution, in grams.
Impregnation Impregnate a suitable quantity of pillules for
homoeopathic preparations with a suitable quantity of the
IV-5 7 8 Homoeopathic Preparations 2023
Microbial contamination Dilute 10 mL of solution S to 15 mL with water R and filter

TAMC: acceptance criterion 102 CFU/g (2.6.12). if necessary.
TYMC: acceptance criterion 10 1 CFU/g (2.6.12). Sulfates (2.4.13)
Absence of Staphylococcus aureus (2.6.13). Maximum 0.1 per cent.
Absence of Pseudomonas aeruginosa (2. 6.13). Dilute 3 mL of solution S to 15 mL with distilled water R and
filter if necessary.
LABELLING
The label states: ASSAY
- the composition of the pillules; Dissolve 0.200 g of the dried substance to be examined in
- where applicable, the size of the pillules. 100 mL of water R. Titrate with 0.1 M sodium hydroxide,
using 0.2 mL of phenol red solution Ras indicator.
- - - - - - - - - - - - - - - - - - - - - PhEur
1 mL of 0.1 M sodium hydroxide is equivalent to 22.91 mg of
C5H3N301.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Acidum Picrinicum for

(Ph. Bur. monograph 2695) Acidum Succinicum for
118.1 110-15-6
229.1 88-89-1 Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
DEFINITION Butanedioic acid (succinic acid).
2,4,6-Trinitrophenol (picric acid).
Content
Content 99.0 per cent to 101.0 per cent.
98.5 per cent to 101.5 per cent.
CHARACTERS
The commercial substance is supplied immersed in water.
Appearance
CAUTION: dry picric acid may explode if subjected to shock or
White or almost white, crystalline powder or colourless
excessive heat. Appropriate precautions must be taken and only crystals.
very small quantities handled.
Solubility
CHARACTERS Soluble in water and in ethanol (96 per cent), sparingly
Appearance of picric acid Light yellow. soluble in acetone.
IDENTIFICATION IDENTIFICATION
A. Melting point (2.2.14): 121 °C to 123 °C, determined on First identification C.
the moistened substance to be examined dried in a desiccator Second identification A, B, D.
to constant mass.
A. Solution S (see Tests) is strongly acid (2.2.4).
B. Suspend 0.1 g of the moistened substance to be examined
B. Melting point (2.2.14): 184 °C to 189 °C.
in 1 mL of water R. Add 1 mL of sodium sulfide solution R.
A red colour is produced. C. Infrared absorption spectrophotometry (2.2.24).
Comparison succinic acid CRS.
TESTS
Dry 5. 0 g of the moistened substance to be examined in a D. Neutralise 3 mL of solution S with 1 M sodium hydroxide,
desiccator to constant mass and use it to prepare solution S and to then add 1 mL of silver nitrate solution R2. A white precipitate
carry out the tests and the assay. is formed.
Solution S TESTS
To 1.5 g of the dried substance to be examined add 30 mL Solution S
of distilled water R, heat to boiling, allow to cool and filter. Dissolve 5.0 g in distilled water R and dilute to 100 mL with
Dilute to 30 mL with distilled water R. the same solvent.
Appearance of solution Chlorides (2. 4. 4)
The solution is not more opalescent than reference Maximum 100 ppm.
suspension II (2. 2.1). Dilute 10 mL of solution S to 15 mL with water R.
Dissolve 0 .1 g of the dried substance to be examined in Sulfates (2. 4.13)
10 mL of distilled water Rand heat to 50 °C. Maximum 200 ppm, determined on solution S.
Chlorides (2.4. 4) Oxalates
Maximum 100 ppm. Neutralise 5 mL of solution S with dilute ammonia Rl, using
0 .1 mL of phenolphthalein solution R as indicator. Add 0 .1 mL
of acetic acid R and 5 mL of calcium sulfate solution R. Content

The solution remains clear for at least 20 min. 0.01 per cent mlm to 0.05 per cent mlm of total cardenolic
ASSAY glycosides, expressed as cymarin (C 30H 44 0 9 ; Mr 548.7).
Dissolve 0.500 g in 50 mL of water R. Titrate with 1 M PRODUCTION
sodium hydroxide, determining the end-point The mother tincture is prepared from the fresh flowering
potentiometrically (2.2.20) or using 0.2 mL of phenolphthalein aerial parts of Adonis vernalis L. according to the following
solution R as indicator. methods as prescribed in the monograph Methods of
1 mL of 1 M sodium hydroxide is equivalent to 59.05 mg of preparation of homoeopathic stocks and potentisation (2371):
C4H504. - method 1.1.3;
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur - method 1.1.10, using ethanol (45 per cent VIV) and a
maceration time of about 3 weeks.
CHARACTERS
Appearance
Adonis Vernalis for Homoeopathic Brown liquid.
Preparations IDENTIFICATION
(Ph Bur. monograph 2832)
Solvent mixture ethyl acetate R, methanol R (50:50 VIV).
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Test solution To 10 mL of the mother tincture to be
DEFINITION examined add 10 mL of ethanol (50 per cent V/V) Rand
Fresh aerial parts of Adonis vernalis L., collected during 10 mL of lead acetate solution R. Boil for 2 min, then cool
flowering. and centrifuge. Collect the supernatant and shake with
2 quantities, each of 15 mL of ethyl acetate R, centrifuging if
IDENTIFICATION an emulsion forms. Dry the combined organic phases over
The pithy, light green stem grows to a height of 10-40 cm, anhydrous sodium sulfate R and filter. Evaporate the filtrate to
very occasionally up to 60 cm, and is up to 5 mm in dryness and dissolve the residue in 0.5 mL of the solvent
diameter, erect, usually unbranched, round, often flattened mixture.
towards the top, with longitudinal furrows. Younger shoots
Reference solution Dissolve 10 mg of convallatoxin R and
are slightly hairy but soon become completely glabrous.
10 mg of cymarin R in the solvent mixture and dilute to
The alternate and sessile leaves sheath the stem and are 10 mL with the solvent mixture.
closer together in the upper part of the stem. They are
Plate TLC silica gel plate R (5-40 µm) [ or TLC silica gel
glabrous or sparsely haired, 2- to 4-pinnate, and narrowly
linear to filiform with entire margins, with downward-turned
tips that are approximately 1 mm across. Brownish-black Mobile phase water R, methanol R, ethyl acetate R
scale-like leaves are present at the base of the stem. (8:11:81 VIVIV).
The single, erect or slightly overhanging, terminal flowers are Application 25 µL [or 20 µL] as bands of 20 mm [or
normally 40-70 mm in size. The calyx has 5 greenish sepals 10 mm].
with a covering of fine hairs on the outside. The sepals are Development Over a path of 10 cm [or 6 cm].
broadly oval, half as long as the petals and pressed to the Drying In air until the solvents have evaporated.
latter. The corolla consists of 10-20, normally 12, distinctly Detection Treat with a mixture of equal volumes of dilute
lustrous, deep yellow petals that are 20-40 mm in length, sodium hydroxide solution R and dinitrobenzoic acid solution R;
6-10 mm wide with entire margins, elongated and tapering to examine in daylight.
a finely toothed point, and glabrous with clearly visible
venation. The numerous stamens are an intense yellow.
The numerous, rounded, apocarpous ovaries are covered all
over with hairs; they are attached to a conical, convex
receptacle and have a short, hooked style.
TESTS
Maximum 5 per cent.
Minimum 60.0 per cent, determined on 5.0 g of the
comminuted herbal drug by drying in an oven at 105 °C
for 2 h.
MOTHER TINCTURE
The mother tincture complies with the requirements of the
DEFINITION
The mother tincture is prepared from the fresh aerial parts of
Adonis vernalis L., collected during flowering.
Top of the plate maximum absorbance of the reference solution;

mass of the mother tincture to be examined used to prepare the
mass of cymarin R used to prepare the reference solution, in
grams;
p percentage content of cymarin in cymarin R.
-- --
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhE~
Cymarin: a violet zone A violet zone
A violet zone Agaricus Phalloides for

Homoeopathic Preparation
A violet zone
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
_,_._, ____
--
DEFINITION
Convallatoxin: a violet zone
Whole, fresh mushroom (fruiting body) Amanita phalloides
(Vaill. ex Fr.) Link.
A yellow or violet zone IDENTIFICATION
A. In young specimens, the cap, 50-150 mm in diameter, is
hemispherical to campanulate with margins rolled inwards
and is still covered by the white universal veil; in mature
specimens, the cap is expanded, umbrella-like, convex to
nearly plane and occasionally depressed; its colour is pale
green along the margin and elsewhere greyish-green to
TESTS yellowish-green, typically with fine grey streaks growing
Relative density (2.2.5) thicker towards the centre; the cuticle is peelable up to the
0.930 to 0.956, where method 1.1.3 is used. middle of the cap as a fine membrane; the underlying flesh
Ethanol (2. 9.10) appears greenish-yellow, becoming more intense towards the
40 per cent V/V to 50 per cent V!V, where method 1.1.10 is centre of the cap, while in the other parts of the mushroom
used. the flesh is uniformly whitish; lamellae are free, closely
packed, white with a slight yellowish tinge; lamellulae are
Dry residue (2.8.16) truncate; the stipe, about 10 cm high and 2 cm in diameter,
Minimum 3.6 per cent (where method 1.1.3 is used), is thin and solid, with a whitish, membranous, striate annulus
minimum 1.0 per cent (where method 1.1.10 is used). and an enlarged and bulbous base that almost always shows
ASSAY a tough, white, membranous sac-like volva torn into irregular
Test solution To 20.00 g of the mother tincture to be lobes at the top. In old specimens, the stipe is hollow,
examined, add 12 mL of lead subacetate solution R. Shake and whitish and often covered with small greenish scales around
centrifuge. Collect the supernatant. Repeat the centrifugation the annulus. The spores are white.
twice using 5 mL of water R each time and collect the B. Examined under a lens (xlO), the upper surface is shiny,
supernatant, then dilute to 50.0 mL with water R. dry, appearing somewhat uneven, with no remains of the veil.
To 10.0 mL of this solution add 10.0 mL of a 100 g/L C. Examine under a microscope using a solution containing
solution of anhydrous sodium sulfate R. Filter. To 10.0 mL of 1. 5 g of iodine R, 5 g of potassium iodide R and 100 g of
the filtrate add 2.0 mL of dinitrobenzoic acid solution Rand chloral hydrate R in 100 mL of water R. The spores are
1.0 mL of 1 M sodium hydroxide. blackish-blue (starch reaction), short elliptical to
Reference solution Dissolve 10.0 mg of cymarin R in 20 mL subspherical, 8-11 µm long and 7-9 µm in diameter.
of ethanol (96 per cent) Rand dilute to 100.0 mL with
TESTS
water R. Dilute 5.0 mL of the solution to 20.0 mL with
water R. To 10.0 mL of this solution, add 2.0 mL of
Maximum 5 per cent.
dinitrobenzoic acid solution Rand 1.0 mL of 1 M sodium
hydroxide. Loss on drying (2.2.32)
Measure the absorbance (2. 2. 25) of the test solution and the Minimum 85.0 per cent, determined on 5.0 g of the finely
reference solution at 540 nm several times during the first cut drug by drying in an oven at 105 °C for 2 h.
12 min following the end of solution preparation until the Other Amanita species
maximum absorbance is reached, using a mixture of 10.0 mL Veil remnants on caps are typical for most Amanita species,
of water R, 2.0 mL of dinitrobenzoic acid solution Rand but not for A. phalloides. Therefore all mushrooms with veil
1.0 mL of 1 M sodium hydroxide as the compensation liquid. remnants on the cap have to be discarded. The presence of
Calculate the percentage content mlm of total cardenolic veil remnants (patches) on the cap indicates adulteration with
glycosides, expressed as cymarin, using the following A. citrina (Schaeff.) Pers. (whitish-yellow cap with whitish to
expression: brownish patches) or with A. muscaria (L.: Fr.) Lamarck, A.
caesarea (Scop.: Fr.) Pers., or A. rubescens Pers. (orange to
A1 xm2 xpx0.25 bright red cap with white patches); a brownish cap indicates
A2xm1 adulteration with A. pantherina (DC.) Krombh.; a greenish-
white cap and a white stipe with a labile annulus indicates
A1 maximum absorbance of the test solution; adulteration with A. verna (Bull.: Fr.) Lamarck.
MOTHER TINCTURE Top of the plate
The mother tincture complies with the requirements of the -- -- --

Rutoside: a quenching Rutoside: a yellow zone
preparations (2029). zone
DEFINITION A violet zone
Content
0.001 per cent mlm to 0.010 per cent mlm for the sum of
O'.-amanitine and ~-amanitine (C 39H 54N 10 O 14 S; M, 919). -- -- --
PRODUCTION Sennoside B: a A violet zone
The mother tincture is prepared according to the following quenching zone
methods as prescribed in the monograph Methods of
A violet zone
preparation of homoeopathic stocks and potentisation (2371):
- method 1.1.5; 2 faint greyish-violet
- method 1.1.10, using 5 parts of the cut drug for 100 parts zones may be present
of ethanol (45 per cent V/V) and maceration for 3 weeks.

CHARACTERS Reference solution Reference solution Test solution
Appearance (detection A) (detection B) (detection B)
Brownish-yellow, yellowish or green liquid.
IDENTIFICATION B. Thin-layer chromatography (2.2.27).
Carry out test A when method 1.1.5 is used and carry out Test solution The mother tincture to be examined.
test B when method 1.1.10 is used. Reference solution Dissolve 2 mg of gramine R and 2 mg of
A. Thin-layer chromatography (2.2.27). rutoside trihydrate R in methanol R and dilute to 10 mL with
Test solution Evaporate to dryness 20 mL of the mother the same solvent.
tincture under vacuum at about 40 °C. Dissolve the residue Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel F 254
in 1 mL of water R and add 1 mL of methanol R. Filter plate R (2-10 µm)].
immediately. Mobile phase glacial acetic acid R, water R, methanol R,
Reference solution Dissolve 10 mg of rutoside trihydrate R and methylene chloride R (4:6:30:60 VIVIVIV).
10 mg of sennoside B R in methanol R and dilute to 40 mL Application 40 µL [or 10 µL] as bands.
F 254 plate R (2-10 µm)].
Detection Treat with a 5 per cent V/V solution of cinnamic
Mobile phase glacial acetic acid R, water R, butanol R
aldehyde R in methanol R and expose to hydrochlonc acid R
(17:17:66 VIVIV).
vapour for 30 min; examine in daylight.
Development Over a path of 10 cm [or 6 cm]. chromatograms obtained with the reference solution and the
Drying In a current of warm air. test solution. Furthermore, other faint zones may be present
Detection A Examine in ultraviolet light at 254 nm. in the chromatogram obtained with the test solution.
Results A Locate a quenching zone (sennoside B) in the
lower third and a quenching zone (rutoside) in the middle Top of the plate
third of the chromatogram obtained with the reference
-- --
solution.
Gramine: a yellow zone
Detection B Treat immediately with a 1 per cent V/V
solution of cinnamic aldehyde R in methanol R and allow to Rutoside: a yellow zone
dry; treat with hydrochwric acid R; examine in daylight.
A pink zone
chromatograms obtained with the reference solution and the A pink zone
-- --
An orange-yellow zone
TESTS
0.895 to 0.915 when method 1.1.5 is used.
Ethanol (2.9.10)
40 per cent V/V to 50 per cent V/V when method 1.1.10 is
used.
Mother tincture of Agaricus muscarius A, area of the peak due to tryptophan in the chromatogram
area of the peak due to o:-amanitine in the chromatogram
Test solution The mother tincture to be examined. obtained with the test solution;
area of the peak due to ~-amanitine in the chromatogram
Reference solution Dissolve 10 mg of leucine R and 10 mg of
threonine R in 5 mL of water R and dilute to 20 mL with mass of trypwphan CRS used to prepare the reference
ethanol (96 per cent) R. solution, in grams;
mass of the mother tincture to be examined used to
prepare the test solution, in grams;
plate R (2-10 µm)]. p percentage content of tryptophan in trypiophan CRS;
Mobile phase glacial acetic acid R, water R, acetone R, K correction factor between ct-amanitine, ~-amanitine and
butanol R (10:20:35:35 VIVIVIV). tryptophan (0.1).

- - - - - - - - - - - - - - - - - - - - - - Ph Eur
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 Allium Sativum for Homoeopathic
daylight.
Results The presence of noticeable zones in the
Preparations
chromatogram obtained with the test solution, in the same Garlic for Homoeopathic Preparations
position as the zones due to leucine and threonine in the (Ph. Bur. monograph 2023)
chromatogram obtained with the reference solution, indicates PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
adulteration with mother tincture of Agaricus muscarius.
DEFINITION
ASSAY Fresh bulb of Allium sativum L.
CHARACTERS
Test solution Evaporate 2.000 g of the mother tincture to be
examined to dryness. Dissolve the residue in 2.0 mL of water It has a characteristic odour after cutting.
for chromatography R. Filter through a membrane filter IDENTIFICATION
(nominal pore size 0.45 µm). The bulb is generally 3 cm to 5 cm broad and almost
Reference solution Dissolve 10.0 mg of tryptophan CRS in spherical; the flat base bears the remnants of numerous short
mobile phase A and dilute to 20.0 mL with mobile phase A. greyish-brown adventitious roots. The bulb consists of about
Column: 10 daughter bulbs (cloves) arranged roughly in a circle
- size: l = 0.10 m, 0 = 4.6 mm; around a central axis. Individual daughter bulbs are 1 cm to
- stationary phase: end-capped solid core octadecylsilyl silica gel 3 cm long, laterally compressed and convex on the dorsal
for chromatography R (2.6 µm); side. Each daughter bulb has a tough, white or reddish skin
- temperature: 35 °C. around a fleshy tubular leaf, investing a more or less rounded
elongated cone of leaf primordia and vegetative apex.
Mobile phase:
- mobile phase A: dissolve 1.54 g of ammonium acetate R in TESTS
900 mL of water for chromatography R, adjust to pH 5.0 Water (2.2.13)
with glacial acetic acid R and dilute to 1 L with water for Minimum 55.0 per cent, determined on 10.0 g of the finely
chromatography R; cut drug, if performed to demonstrate the freshness of the
- mobile phase B: acetonitrile for chromatography R; drug.
Time Mobile phase A Mobile phase B MOTHER TINCTURE

(min) (per cent V/J.') (per cent V/J.')
0-2 95 5
2 - 17 95 ➔ 80 5 ➔ 20
17 - 22 80 ➔ 50 20 ➔ 50
PRODUCTION
Flow rate 1.0 mUmin. The mother tincture of Allium sativum L. is prepared by
Detection Spectrophotometer at 303 nm. maceration of the cut drug using alcohol of a suitable
concentration.
Injection 10 µL.
Relative retention With reference to tryptophan (retention CHARACTERS
time =about 3 min): ~-amanitine =about 2.9; Appearance
o:-amanitine = about 3.2. Brownish-yellow liquid.
System suitability Test solution: It has a peculiar and unpleasant aromatic odour.
- resolution: minimum 2.0 between the peaks due to IDENTIFICATION
~-amanitine and o:-amanitine. A. To 2 mL of the mother tincture to be examined, add
Calculate the percentage content mlm of o:-amanitine and 0.2 mL of dilute sodium hydroxide solution R. A yellowish-
~-amanitine using the following expression: white precipitate develops.
B. Thin-layer chromatography (2. 2. 27).
Test solution Extract 5 mL of the mother tincture to be
examined with 2 quantities, each of 10 mL, of ether R.
Combine the ether layers and dry over anhydrous sodium
2023 Ammonium Carbonicum for Homoeopathic Preparations IV-583
sulfate R. Filter and evaporate the filtrate in a water-bath at

low temperature. Dissolve the residue in 0.4 mL of
Ammonium Carbonicum for
methanol R. Homoeopathic Preparations
Reference solution Dissolve 10 mg of resorcinol R, 10 mg of (Ph. Bur. monograph 2916)
thymol R and 30 mg of gallic acid R in 10 mL of methanol R.
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Plate TLC silica gel F2s4 plate R.
Mobile phase anhydrous formic acid R, toluene R, di-isopropyl DEFINITION
ether R (10:40:50 V/V/V). Mixture of varying proportions of diammonium carbonate
((NH 4) 2 CO3 ; Mr 96.1), ammonium hydrogen carbonate
(NH 4 HCO 3 ; Mr 79.1) and ammonium carbamate
reference solution.
(NH 2 COONH 4 ; Mr 78.1).
Content
Drying In air.
30.0 per cent to 37.0 per cent of NH 3 (Mr 17.03).
Detection Examine in ultraviolet light at 254 nm and
identify gallic acid; spray with anisaldehyde solution R, heat to CHARACTERS
105-110 °C for 5-10 min. Examine in daylight within Appearance
10 min. White or almost white, translucent masses or white or almost
white, crystalline powder, with a strong smell of ammonia.
chromatograms obtained with the reference solution and the Solubility
test solution. Other zones may also be visible in the Freely soluble in water, practically insoluble in ethanol
chromatogram obtained with the test solution. (96 per cent).
IDENTIFICATION
Top of the plate A. Dissolve 1 g in water R and dilute to 20 mL with the
same solvent. The solution is slightly alkaline (2.2.4).
An intense reddish-violet zone
B. It gives the reaction of carbonates and bicarbonates
Thymol: an orange-red zone
(2.3.1).
An intense reddish-violet zone C. It gives the reaction of ammonium salts and salts of
volatile bases (2. 3.1).
A violet zone
TESTS
A yellowish or greenish zone
Solution S
- - - - Dissolve 10.0 gin 100 mL of distilled water R. Boil until the
Resorcinol: an intense orange-red
volume is reduced to about 10 mL. Verify that the emitted
zone vapours no longer make barium hydroxide solution R cloudy.
Cool and dilute to 50 mL with distilled water R.
-- --
Appearance of solution
Gallic acid: a yellow zone A violet zone Solution Sis clear (2.2.1) and colourless (2.2.2, Method l).
(UV at 254 nm: a fluorescent A greenish-yellow zone Chlorides (2.4.4)
quenching zone) Maximum 20 ppm.
A violet zone may be present Dissolve 2.5 g by gradually adding a mixture of 5 mL of
nitric acid R and 10 mL of water R.
Sulfates (2. 4.13)
Maximum 60 ppm, determined on solution S.
TESTS ASSAY
Relative density (2.2.5) Dissolve 2.5 gin water Rand dilute to 100 mL with the
0.885 to 0.960. same solvent. To 10.0 mL of the solution add 10.0 mL of
Ethanol (2. 9.10) 1 M hydrochloric acid. Shake continuously until the
50 per cent V/V to 70 per cent V/V. effervescence ceases. Add 40 mL of water R. Titrate the
Dry residue (2.8.16) excess of acid with 1 M sodium hydroxide, determining the
Minimum 4.0 per cent. end-point potentiometrically (2.2.20).
STORAGE 1 mL of 1 M hydrochloric acid is equivalent to 17.03 mg of
NH3.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur STORAGE
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Anacardium for Homoeopathic Top of the plate
Preparations
Oriental Cashew for Homoeopathic Preparations -- --

PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION ~-naphthol: a violet zone A broad, intense violet zone (at the
Dried fruit of Semecarpus anacardium L.f. (Anacardium same level or slightly higher than
orientate L.). ~-naphthol)
1-2 violet zones, sometimes faint
Content
Minimum 6.0 per cent mlm of total phenol derivatives - - --
expressed as eugenol (C 10H 12 0 2 ; Mr 164.2) (dried drug).
IDENflFICATION
A violet zone, sometimes faint
A. The dried fruit is oval and more or less cordate; about
2 cm long, nearly 2 cm wide and 0.5 cm thick. Its surface is
smooth, shiny and blackish. A transverse section shows a
rather well developed, tough pericarp riddled with rather
wide lacunae containing an abundant thick reddish-brown Resorcinol: an orange zone
juice. The pericarp covers a white kernel under a reddish An orange zone, sometimes faint
skin. The fruit may include the blackish, fleshy, wrinkled,
cupuliferous receptacle.
B. Thin-layer chromatography (2.2.27). Reference solution Test solution
Test solutwn To 1.0 g of the suitably cut herbal drug, add
10 mL of ethanol (90 per cent V/V) R. Heat under reflux on a
water-bath at 60 °C for 15 min. Allow to cool and filter. TESTS
Anacardium occidentale L
Reference solutwn Dissolve 5 mg of resorcinol R and 10 mg of
Fruits of Anacardium occidentale L. are not present. These are
/3-naphthol R in methanol R and dilute to 10 mL with the
up to 35 mm long, 30 mm wide, 20 mm thick, light brown
same solvent.
and distinctly kidney-shaped. The pericarp is smooth or
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel slightly crinkled with dark marbling in places.
plate R (2-10 µm)).
Mobile phase methanol R, toluene R (15:85 VIV).
Maximum 12.0 per cent, determined on 1.00 g of the finely
Application 10 µL [or 5 µL], as bands of 20 mm [or 8 mm]. cut herbal drug by drying in an oven at 105 °C for 2 h.
Development Over a path of 15 cm [or 6 cm]. Total ash (2.4.16')
Drying In air. Maximum 5.0 per cent.
Detection Treat with anisaldehyde solution R, heat at ASSAY
100-105 °C for 5-10 min and examine in daylight within Total phenol derivatives
10 min. Ultraviolet and visible absorption spectrophotometry (2.2.25).
Stock solutwn Place 4.50 g of the crushed herbal drug in a
chromatograms obtained with the reference solution and the flask. Add 200 mL of ethanol (90 per cent V/V) R. Boil in a
test solution. Furthermore, other fainter zones may be water-bath under reflux for 4 h. Cool the flask.
present in the chromatogram obtained with the test solution. Quantitatively transfer into a volumetric flask. Dilute to
250.0 mL with ethanol (90 per cent VIV) R. Filter the liquid
through a paper filter 125 mm in diameter. Discard the first
50 mL of the filtrate. Dilute 5 .0 mL of filtrate to 50.0 mL
with ethanol (90 per cent VIV,) Rand shake. Dilute 5.0 mL of
this solution to 10.0 mL with ethanol (90 per cent V/V) Rand
shake.
Test solutwn To 2.0 mL of the stock solution add 1.0 mL of
phosphomolybdotungstic reagent R and 10 mL of water R, mix
and dilute to 25.0 mL with a 290 g/L solution of sodium
carbonate R. Wait exactly 3 min then filter the solution
through a fibre-glass filter with a I µm mesh aperture,
discarding the first 5 mL.
Reference solution Dissolve 80.0 mg of eugenol R in ethanol
(90 per cent V/V) Rand dilute to 250.0 mL with the same
solvent. Dilute 5.0 mL of the solution to 25.0 mL with
ethanol (90 per cent VIV) R. To 2.0 mL of this solution add
1.0 mL of phosphomolybdotungstic reagent Rand 10 mL of
water R, mix and dilute to 25.0 mL with a 290 g/L solution
of sodium carbonate R. Wait exactly 3 min then filter the
solution through a fibre-glass filter with a 1 µm mesh Calculate the percentage content mlm of total phenol
aperture, discarding the first 5 mL. derivatives expressed as eugenol, using the following
Measure the absorbance (2.2.25) of the test solution and the expression:
reference solution at 755 nm after 30 min using water Ras
the compensation liquid.
Calculate the percentage content mlm of total phenol
derivatives, expressed as eugenol, using the following A1 absorbance of the test solution;
expression: A2 absorbance of the reference solution;
m, mass of the mother tincture to be examined, in milligrams;
mass of eugenol in the reference solution, in milligrams.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur

A2 absorbance of the reference solution;
m1 mass of the herbal drug to be examined, in milligrams;
m2 mass of eugenol in the reference solution, in milligrams.
Apis for Homoeopathic
Preparations
MOTHER TINCTURE
Honey Bee for Homoeopathic Preparations
The mother tincture complies with the requirements of the (Ph. Bur. monograph 2024)
preparations (2029). ~~----------------------
DEFINITION DEFINITION
The mother tincture of Anacardium is prepared from the Live worker honey bee (Apis mellifera L.).
dried fruit of Semecarpus anacardium L.f. (Anacardium CHARACTERS
orientate L.) . Characters described under Identification.
Content PRODUCTION
0.5 per cent mlm to 1.0 per cent mlm of total phenol If the bee has been exposed to treatment to prevent or cure
derivatives, expressed as eugenol. diseases, appropriate measures are taken to ensure that the
PRODUCTION levels of residues are as low as possible.
The mother tincture is prepared according to the following IDENTIFICATION
methods prescribed in the general monograph Methods of The body is about 15 mm long, black, with a silky sheen,
preparation of homoeopathic stocks and potentisation (2371): and covered with red hairs with a touch of grey. The broad
- method 1.1.8, using the crushed drug (2000) (2.9.12) and tibiae are without spines. The posterior margins of the
ethanol (90 per cent VIV); use ethanol (70 per cent V/V) segments and legs are brown, with gradual transition to
to prepare the 4th decimal dilution, and use ethanol orange-red. The claws are two-membered, the maxillary
(50 per cent V/V) for subsequent dilutions; palps single-membered. On the hind legs are baskets or
- method 1.1.10, using ethanol (90 per cent V/V). scoops invested with bristles. The wings have 3 complete
CHARACTERS cubital cells, with the radial cell twice as long as it is wide;
Appearance the 3 cells on the lower margin and the 3 middle cells are
Yellowish-brown or reddish-brown liquid. closed. A duct connects the barbed sting with the poison sac.
IDENTIFICATION
Thin-layer chromatography (2.2.27) as described in MOTHER TINCTURE
identification test B for the herbal drug with the following The mother tincture complies with the requirements of the
modification. general monograph Mother tinctures for homoeopathic
Test solution The mother tincture to be examined. preparations (2029).
Results See identification test B for the herbal drug. PRODUCTION
TESTS The mother tincture of Apis mellifera L. is prepared by
Relative density (2.2.5) maceration using alcohol of a suitable concentration.
0.815 to 0.845. CHARACTERS
Ethanol (2. 9. 10) Pale yellow liquid that may darken on storage.
85 per cent V/V to 95 per cent VIV. IDENTIFICATION
Dry residue (2. 8. 16) Thin-layer chromatography (2.2.27).
Minimum 1.50 per cent. Test solution The mother tincture to be examined.
ASSAY Reference solution Dissolve 12 mg of 4-aminobutanoic
Total phenol derivatives acid R, 12 mg of leucine R and 12 mg of proline R in 5 mL of
Absorption spectrophotometry (2.2.25) as described in the water R and dilute to 50 mL with alcohol R.
assay of the herbal drug with the following modification. Plate TLC silica gel plate R.
Stock solution Place 8.00 g of the mother tincture to be Mobile phase water R, ethanol R (17:63 V/V).
examined in a volumetric flask and dilute to 250.0 mL with Application20 µL, as bands.
ethanol (90 per cent VIJ:,] R. Dilute 5.0 mL of this solution to
20.0 mL with ethanol (90 per cent VIJ:,] R.
Drying In air.
Detection Spray with ninhydrin solution R and heat at greenish colour. Add 0.25 mL of 0.05M iodine and shake.
100-105 °C for 10 min; examine in daylight. The precipitate becomes a greyish-green colour. Collect the
Results See below the sequence of the zones present in the precipitate. The precipitate dissolves in ether giving a purple
chromatograms obtained with the reference and test solution, dissolves in dichloromethane giving a violet-blue
solutions. Other zones may also be visible. solution and dissolves in alcohol giving a blue solution.
B. To 2 mL of solution S add 0.1 mL of nitric acid, mix and
Top of the plate filter. The filtrate yields reaction A characteristic of chlorides,
Appendix VI.
- - --
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
80° for 10 minutes. A red colour develops.
Leucine: a pink zone A pink zone
ASSAY
A pink zone
Disperse 2.5 g of the substance being examined in a mixture
A pink zone of 5.0 mL of 0.01M hydrochloric acid and 50 mL of ethanol
(96%). Carry out the method for potentiometric titration,
- - - -
Appendix VIII B, using 0.lM sodium hydroxide. Measure the
Praline: an orange-yellow zone An orange-yellow zone titrant between the first 2 points of inflexion. Each mL of
4-Aminobutanoic acid: a pink zone A pink zone
0.lM sodium hydroxide is equivalent to 30.38 mg of
C17H 17NO 2,HCI.

Arsenious Trioxide for
TESTS
Relative density (2.2.5) (Arsenicum Album for Homoeopathic Preparations,
0.890 to 0.910. Ph. Bur. monograph 1599)
Ethanol (2.9.10) 197.8 1327-53-3
60 per cent V/V to 70 per cent V/V. ~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Dry residue (2.8.16) DEFINITION

Minimum 0.30 per cent. Content
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur 99.5 per cent to 100.5 per cent of As2 O 3 .
CHARACTERS
Appearance
White or almost white powder.
Apomorphine Hydrochloride for Solubility
Homoeopathic Preparations Practically insoluble to sparingly soluble in water. It dissolves
Apomorphinum Muriaticum for Homoeopathic Preparations in solutions of alkali hydroxides and carbonates.
Apomorphine Hydrochloride for Homoeopathic Preparations A. Dissolve 20 mg in 1 mL of dilute hydrochloric acid R, add
contains Apomorphine Hydrochloride Hemihydrate. 4 mL of water Rand 0.1 mL of sodium sulfide solution R.
The resulting yellow precipitate is soluble in dilute
PRODUCTION OF STOCK ammonia Rl.
The first trituration of Apomorphine Hydrochloride for
B. Dissolve 20 mg in 1 mL of hydrochloric acid Rl, add 5 mL
Homoeopathic Preparations is prepared using a suitable
of hypophosphorous reagent R and heat for 15 min on a water-
quantity of Lactose Monohydrate or Lactose as the vehicle
bath. A black precipitate develops.
and a validated trituration method that ensures homogeneity
is achieved. The vehicle complies with the statement under TESTS
Vehicles in the monograph for Homoeopathic Preparations. Appearance of solution
Content of apomorphine hydrochloride, C 17H 17NO2 , The solution is clear (2.2.1) and colourless (2.2.2,
HCl Method II).
The first decimal trituration contains 9.5% to 10.5% of Prepare a 100 g/L solution in dilute ammonia Rl, heating if
C 17H 17NO2,HCl (dried substance). necessary.
CHARACTERS Sulfides
The first decimal trituration is a white powder. Maximum 20 ppm.
Dissolve 1.0 gin 10.0 mL of dilute sodium hydroxide
IDENTIFICATION
solution R. Add 0.05 mL of lead acetate solution R. Any colour
Dissolve 2.5 g of the substance being examined without
in the test solution is not more intense than that in a
heating in water and dilute to 25 mL with the same solvent standard prepared at the same time and in the same manner
(solution S).
using a mixture of 10.0 mL of a 0.015 g/L solution of sodium
A. To 5 mL of solution S add a few millilitres of sodium sulfide R in dilute sodium hydroxide solution Rand 0.05 mL of
hydrogen carbonate solution until a permanent, white lead acetate solution R.
precipitate is formed. The precipitate slowly becomes a
ASSAY A large number of immature pollen grains are present,

Dissolve 40.0 mg in a mixture of 10 mL of dilute sodium forming elongated, closely packed masses.
hydroxide solution R and 10 mL of water R. Add 10 mL of C. Carry out the method for thin-layer chromatography,
dilute hydrochloric acid R and 3 g of sodium hydrogen Appendix III A, using the following solutions.
carbonate R and mix. Add 1 mL of starch solution R and (1) Add 10 mL of ethanol (90%) to 1 g of the coarsely
titrate with 0. 05 M iodine. powdered drug, stir for one hour and filter.
1 mL of 0.05 M iodine is equivalent to 4.946 mg of As 2 0 3 . (2) 0.1 % w/v each of santonin and cineole in methanol.
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
(a) Use as the coating silica gel H.
Artemisia Cina for Homoeopathic (c) Apply 10 µL of each solution.
Preparations (e) After removal of the plate, allow to dry in air, spray with
DEFINITION ethanolic phosphomolybdic acid solution, heat at 100° to 105° for
Artemisia Cina for Homoeopathic Preparations is the dried, about 5 minutes and examine in daylight.
unexpanded flower heads of Seriphidium cinum (Berg ex
MOBILE PHASE
Poljakov) Poljakov (Syn Artemisia cina Berg ex Poljakov)
Artemisia cina O.C.Berg et C.F. Schmidt. 5 volumes of glacial acetic acid, 45 volumes of hexane and
50 volumes ethyl acetate.
It contains not less than 1.0% of santonin (C 15H 1s03)
calculated with reference to the dried material. SYSTEM SUITABILITY
IDENTIFICATION The test is not valid unless the chromatogram obtained with
solution (2) shows the grey-blue santonin band just between
A. The dried, slightly shiny capitula are conical to elongated-
the lower third and middle third and the grey-blue cineole
ovoid, 2 to 4 mm long and 1 to 2 mm wide, yellow-green to
band in the upper third.
brownish and composed of 3 to 6 hermaphrodite florets
enclosed in an involucre of 14 to 20 imbricated ovate to CONFIRMATION
lanceolate bracts. Each bract has a distinct keel which is most The chromatogram obtained with solution (1) shows a grey-
pronounced in the ovate outer bracts near the base; the keel blue band below the band obtained for santonin in solution
forms the midrib and it branches freely, the veinlets (2), a strong grey-blue band level with that of santonin in
becoming contorted and frequently anastomising. The outer solution (2) and one or two grey-blue bands just above
surface of the bracts is covered with glistening glandular santonin; one or two grey-blue bands between the bands
hairs. The florets are about 1 mm long and 0.5 mm wide; obtained for santonin and cineole in solution (2) and a
the corolla is contracted at the base and divides at the apex strong grey-blue band level with the band obtained with
into 5 short, triangular teeth. cineole.
B. Reduce to a powder and examine under a microscope
using chloral hydrate solution. Abundant fragments of the Top of the plate
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 A grey-blue band Cineole: a grey-blue band
from the central region of the bracts show polygonal,
1 or 2 a grey-blue bands
isodiametric epidermal cells with thickened and beaded walls;
fairly numerous anomocytic stomata are present on the outer
surface only. Very small cluster crystals of calcium oxalate 1 or 2 grey-blue bands
may be present in the parenchymatous cells underlying the a grey-blue band Santonin: a grey-blue band
a grey-blue band
epidermis.
Groups of sclereids from the central region of the bracts
show: individual cells varying in shape but usually
considerably elongated; the ends are square or bluntly Solution (1) Solution (2)
tapering or, occasionally, somewhat enlarged; the walls are
strongly thickened and have scattered pits. Small groups of
TESTS
these sclereids are occasionally found attached to fragments
Foreign matter
of the epidermis of the bracts.
Not more than 5% of sections of stem and pieces ofnarrow-
The unicellular covering trichomes are nearly always found linear hairy leaves; not more than 2% of other foreign matter,
detached; they are usually very thin-walled although slight Appendix XI D.
thickening may occur in the basal region; some of these
trichomes are very long and can be found in groups forming Ash
loosely felted, cottony masses. The typically labiate glandular Not more than 11.0%, Appendix XI J, Method II.
trichomes are abundant on the bracts and are also found Loss on drying
detached; each has a short, biseriate stalk, and a biseriate Not more than 10.0%, Appendix IX D.
head of two or four cells around which the cuticle is raised to ASSAY
form a bladder-like covering. Carry out the method for luiuid chromatography,
The abundant pollen grains are fairly small, spherical, with Appendix III D, using the following solutions.
three pores and three furrows; the exine is finely warted. (1) To 1.0 g of the powdered herbal drug add 50 mL of
methanol and stir for 2hours. Filter the solution using a dry
filter paper into a 100 mL volumetric flask, wash the filtrate CHARACTERISTICS
with methanol, add the washings to the filtrate and dilute to The mother tincture is a golden yellow to greenish liquid.
100 mL with methanol and mix. Weigh approximately 5 g
IDENTIFICATION
(6.5 mL) of the solution and add 20 mL of methanol in a
The mother tincture complies with Identification test C
50 mL volumetric flask and dilute to volume with water.
above using the mother tincture as solution (1).
(2) 0.005% w/v of santonin BPCRS prepared by dissolving
100 mg santonin BPCRS in 100 ml methanol and diluting TESTS
5 mL of the resulting solution to 100 mL with the mobile Ethanol
phase. 40% to 46% w/w (47% to 54% v/v), Appendix VIII F.
(3) 0.005% w/v each of santonin BPCRS and methyl Dry residue
4-hydroxybenzoate in the mobile phase. Not less than 1.8% w/w, Appendix XI P.
CHROMATOGRAPHIC CONDITIONS Relative density
(a) Use a stainless steel column (15 cm x 4.6 mm) packed 0.835 to 0.855, Appendix V G.
with octadecylsilyl silica gel for chromatography (5 µm) ASSAY
(Kromasil Cl8 is suitable) fitted with a stainless steel guard Carry out the method for liquid chromatography,
column packed with the same material. Appendix III D, as described for the herbal drug using as
(b) Use isocratic elution and the mobile phase described solution (1) the mother tincture.
below. DETERMINATION OF CONTENT
(c) Use a flow rate of 1.0 mL per minute. Calculate the content of C 15 H 18 0 3 in the mother tincture
(d) Use a column temperature of 25°. using the declared content of C 15H 18 0 3 in santonin BPCRS
(e) Use a detection wavelength of 236 nm. using the following expression:
MOBILE PHASE A m V,
_1 x - 2 x - 1 xp
Equal volumes of methanol and water.
~ Vi "1i
SYSTEM SUITABILITY
with solution (3), the resolution factor between the peaks due area of the peak due to santonin in the chromatogram
to methyl 4-hydroxybenzoate and santonin is not less than 2.0. obtained with solution (!);
area of the peak due to santonin in the chromatogram
DETERMINATION OF CONTENT obtained with solution (2);
Calculate the content of C 15 H 18 0 3 in the herbal drug using weight of the herbal drug being examined in mg;
the declared content of C 15 H 180 3 in santonin BPCRS using weight of sanwnin BPCRS in mg;
dilution volume of solution (1) in mL;
the following expression: dilution volume of solution (2) in mL;
percentage content of santonin (C 15 H 18O 3 ) in
santtmin BPCRS.

Barium Chloride Dihydrate for
obtained with solution(!);
obtained with solution (2); (Barium Chloratum for Homoeopathic Preparations,
weight of the herbal drug being examined in mg;
Ph. Bur. monograph 2142)
weight of santom·n BPCRS in mg;
dilution volume of solution (1) in mL; 244.3 10326-27-9
dilution volume of solution (2) in mL;
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
percentage content of santonin (C 15 H 18O 3 ) in
sanwnin BPCRS;
d percentage loss on drying of the herbal drug being
DEFINITION
examined. Content
99.0 per cent to 101.0 per cent of BaC12 ,2H2 0.
CHARACTERS
MOTHER TINCTURE Appearance
The mother tincture complies with the requirements stated under White or almost white, crystalline powder or colourless
Mother Tinctures for Homoeopathic Preparations and with the crystals.
following requirements. Solubility
Freely soluble in water, very slightly soluble or practically
DEFINITION
insoluble in ethanol (96 per cent).
It contains not less than 0.1 % of santonin (C 15H 18 0 3).
IDENTIFICATION
PRODUCTION
A. Dissolve 0.1 gin 1 mL of water R. Add 0.3 mL of dilute
The mother tincture of Artemisia cina is prepared from the
sulfuric acid R. A white precipitate is formed; it is insoluble in
powdered drug using Method 1.1. 8 described in the
dilute hydrochloric acid R and in dilute nitric acid R.
monograph for Methods of Preparation of Homoeopathic
Stocks and Potentisation. Use 86% w/w (90% v/v) of ethanol. B. It gives reaction (a) of chlorides (2.3. 1).
TESTS MOTHER TINCTURE

Solution S
Dissolve 10.0 gin water Rand dilute to 100 mL with the
same solvent.
Solution Sis clear (2.2.1) and colourless (2.2.2, Method II). DEFINITION
Content
Acidity or alkalinity 0.020 per cent mlm to 0.050 per cent mlm of hyoscyamine
To 10 mL of solution S add 0 .1 mL of phenolphthalein (C11H2 3N03; Mr 289.4).
solution R. Not more than 0.2 mL of 0.01 M hydrochloric acid
or 0.011vI sodium hydroxide is required to change the colour PRODUCTION
of the indicator. The mother tincture is prepared from the comminuted herbal
drug according to the following methods prescribed in the
ASSAY monograph Methods of preparation of homoeopathic stocks and
Dissolve 0.200 gin 100 mL of water R. Add 100 mL of potentisation (2371):
methanol R, 10 mL of concentrated ammonia R and 2 mg of - method 1.1.3;
phthalein purple R. Titrate with 0.1 M sodium edetate until the - method 1. 1.10, using ethanol (45 per cent VIV) and a
colour changes from violet to colourless. maceration time of 3-5 weeks.
1 mL of 0.1 M sodium edetate is equivalent to 24.43 mg
of BaC12 ,2H2 O. CHARACTERS
Appearance
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Brown liquid.
IDENTIFICATION
Belladonna for Homoeopathic atropine.
Detection A Spray with potassium iodabismuthate solution R2
Preparations until orange or brown zones become visible against a yellow
(Ph. Bur. monograph 2489) background. Examine in daylight.
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Results A See below the sequence of zones present in the
DEFINITION test solution. Furthermore, other faint zones may be present,
Whole, fresh, flowering plant of Atropa belladanna L., in particular in the middle of the chromatogram obtained
harvested at the end of flowering, with the ligneous base of with the test solution.
the stems removed.
Top of the plate
IDENTIFICATION
The root crown is short, thick, cylindrical, with one or more
heads, and with many branching roots extending from it;
An orange or brown zone, sometimes
these are grey or brown, fleshy, cylindrical or fusiform, very Hyoscine: an orange or brown zone very faint or absent (hyoscine)
rarely twisted. The erect stem of the youngest part is
-- --
cylindrical and pubescent. The petiolate leaves are alternate,
simple, oval, attenuated at both ends, lacking stipules, with -- --
pinnate venation, up to 20 cm long and 10 cm wide; on the
Hyoscyamine: an orange or brown An orange or brown zone
flowering stems they are paired and unequal in size. zone (hyoscyamine)
The drooping, pedunculate, solitary flowers appear between
the petioles of the paired leaves; they are actinomorphous,
pentameric, with superior ovaries; they have a gamosepalous, Reference solution Test solution
glandular, green calyx, which grows larger after flowering.
The corolla, 2-3 cm long, is campanulate, gamopetalous, The zone due to hyoscine in the chromatogram obtained
greenish-yellow with violet veins in the upper part; it is hairy with the reference solution and that with the same Rp in the
and deciduous, divided into 5 narrow, rounded lobes. The 5 chromatogram obtained with the test solution are located at
alternipetalous stamens, attached at the base of the corolla, the limit between the middle third and the upper third and
have long filaments which are hairy in the lower part; they they may therefore appear in either part of the
bear bilocular, introrse anthers which dehisce longitudinally. chromatogram.
The superior ovary consists of 2 carpels with axile
placentation. The style is simple, ending in a slightly TESTS
cordiform stigma, extending beyond the stamens. The fruit is Relative density (2.2.5)
a spherical, fleshy berry, the size of a cherry, green then black 0.932 to 0.947 when method 1.1.3 is used.
and shiny when ripe. The seeds are numerous, reniform with Ethanol (2.9.10)
a testa showing markings. 40 per cent V/Vto 50 per cent V/Vwhen method 1.1.10 is
TESTS used.
Foreign matter (2.8.2) Dry residue (2.8.16)
Maximum 5 per cent. Minimum 1.4 per cent.
Loss on drying (2.2.32) Mother tincture of Hyoscyamus niger - mother
Minimum 75.0 per cent, determined on 5.0 g of the finely tincture of Datura stramonium.
cut drug by drying in an oven at 105 °C for 2 h. Concentrate 10 mL of the mother tincture to be examined
on a water bath until a volume of 5 mL is obtained.
Add 10 mL of water R and filter. Shake the filtrate with Reference solutwn (c) Mix 3.0 mL of reference solution (a)
5 mL of methylene chloride R and collect the organic layer. and 2.5 mL of reference solution (b) and dilute to 25.0 mL
Filter and evaporate the filtrate to dryness under reduced with mobile phase A.
pressure. Take up the residue with 10 mL of hot water R, Column:
and add 0.1 mL of dilute ammonia Rl. A greenish-blue - size: l = 0.15 m, 0 = 4.6 mm;
fluorescence appears when examined under ultraviolet light at - statwnary phase: end-capped octadecylsilyl silica gel for
365 nm. chromatography R (3 µm);
The absence of greenish-blue fluorescence indicates - temperature: 30 °C.
substitution by the mother tincture of Hyoscyamus niger Mobile phase:
and/or by the mother tincture of Datura stramonium. - mobile phase A: triethylamine R, acetonitrile for
Atropine chromatography R, phosphate buffer solution pH 7.0 R6
Thin-layer chromatography (2.2.27). (0.01 :5:95 VIVIV);
Test solutwn Evaporate 10 mL of the mother tincture to be - mobile phase B: triethylamine R, phosphate buffer solution
examined on a water-bath. Take up the residue with 5 mL of pH 7.0 R6, acetonitrile for chromatography R
dilute sulfuric acid Rl and filter. Make alkaline with (0.01 :40:60 V/V/V);
concentrated ammonia R and extract with 15 mL of
1,1-dimethylethyl methyl ether R. Dry the ether layer over Time Mobile phase A Mobile phase B
anhydrous sodium sulfate R and filter. Evaporate to dryness on
0-5 90 10
a water-bath and dissolve the residue in 1 mL of methanol R.
5 - 20 90 -, 10 10-> 90
Reference solutwn Dissolve 50 mg of hyoscyamine sulfate R in
9 mL of methanol R (solution A). Dissolve 15 mg of hyoscine
hydrobromide R in 10 mL of methanol R (solution B). Flow rate 1.0 mUmin.
To 8 mL of solution A add 1.8 mL of solution B and dilute Detection Spectrophotometer at 210 nm.
to 10 mL with methanol R. Injection 20 µL of the test solution and reference
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel solution (c).
plate R (2-10 µm)]. Retention time Hyoscyamine = about 12 min;
Mobile phase concentrated ammonia R, water R, acetone R hyoscine = about 13.5 min.
(3:7:90 VIVIV). System suitability Reference solution (c):
Applicatwn 20 µL [or 5 µL] as bands. - resolution: minimum 5 between the peaks due to
Development Over a path of 10 cm [or 6 cm]. hyoscyamine and hyoscine;
- symmetry factor. maximum 2.0 for the peak due to
Drying At 105 °C for 15 min; allow to cool.
hyoscyamine.
Detection B After detection A (see Identification), spray with
Calculate the percentage content m/m of hyoscyamine using
sodium nitrite solutwn R until the yellow background
the following expression:
disappears. Examine in daylight.
Result B No greyish-blue zone (atropine) appears in place of
the brown or reddish-brown zone due to hyoscyamine in the A2 x m1 x 16.667
A, area of the peak due to hyoscyamine in the chromatogram
A2 area of the peak due to hyoscyamine in the chromatogram
Test solutwn Introduce 1.500 g of the mother tincture to be m1 mass of the mother tincture to be examined used to prepare the
examined into a volumetric flask and dilute to 10.0 mL with test solution, in grams;
water for chromatography R. Apply the mixture to a cartridge m2 mass of hyoscyamine sulfate CRS used to prepare the reference
solution, in grams;
filled with a cation-exchange material (60 µm; 60 mg), pre- p assigned percentage content of anhydrous hyoscyamine in
conditioned with 2 mL of methanol R2 and then 2 mL of hyoscyamine sulfate CRS.
water for chromatography R. Rinse the volumetric flask with
1 mL of a mixture of 2 volumes of anhydrous formic acid R - - - - - - - - - - - - - - - - - - - - - - Ph Eur
and 98 volumes of water for chromatography R, and apply the
rinsings to the cartridge. Wash the cartridge with 2 mL of a
mixture of 2 volumes of anhydrous formic acid R and
98 volumes of water for chromatography R and then with
2 mL of methanol R2. Dry the cartridge with the aid of a
Cadmium Sulfate Hydrate for
vacuum and elute with 3.5 mL of a mixture of 2 volumes of Homoeopathic Preparations
concentrated ammonia R and 98 volumes of methanol R2. (Cadmium Sulfuricum for Homoeopathic Preparations,
Expel the solvent remaining in the cartridge into the eluate, Ph. Bur. monograph 2143)
add 40 µL of glacial acetic acid Rand dilute to 5.0 mL with
water for chromatography R. CdSO4,8/3H 2O 256.5
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Reference solutwn (a) Dissolve 10.0 mg of hyoscyamine
sulfate CRS in methanol R2 and dilute to 10.0 mL with the DEFINITION
same solvent. Content
Reference solutwn (b) Dissolve 10.0 mg of hyoscine 98.0 per cent to 102.0 per cent (anhydrous substance).
hydrobromide R in methanol R2 and dilute to 10.0 mL with
CHARACTERS
the same solvent.
Appearance
White or almost white, crystalline powder.
Solubility
Freely soluble in water, practically insoluble in ethanol
Calcium Fluoratum for
(96 per cent). Homoeopathic Preparations
IDENTIFICATION (Ph. Bur. monograph 2996)
A. It gives reaction (a) of sulfates (2.3. 1). 7789-75-5
CaFz 78.1
B. To 2 mL of solution S (see Tests) add 2 mL of sodium PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
sulfide solution R. A yellow precipitate is formed.
DEFINITION
TESTS
Content
Solution S
98.0 per cent to 102.0 per cent of CaF2 •
Dissolve 5.0 gin carbon dioxide-free water Rand dilute to
50 mL with the same solvent. CHARACTERS
Appearance of solution Appearance
Solution S is clear (2.2. 1) and colourless (2.2.2, Metlwd II). Fine, white or almost white powder.
Acidity or alkalinity Solubility
To 10 mL of solution S add 0.3 mL of methyl orange Practically insoluble in water, slightly soluble in mineral
solution R. Not more than 0.5 mL of 0. 01 M hydrochloric acid acids.
or 0.01 M sodium hydroxide is required to change the colour IDENTIFICATION
of the indicator. A. To 0.80 g add 20 mL of hydrochloric acid Rand heat to
Nitrates boiling under a reflux condenser until complete dissolution
Maximum 100 ppm. (about 30 min). After cooling, add 0.1 mL of phenolphthalein
Dissolve 1.0 g in water R and dilute to 20.0 mL with the solution R, and then concentrated ammonia R until a pink
same solvent. To 1.0 mL of this solution add 0.2 mL of a colour is obtained. Add glacial acetic acid R until the solution
10 gJL solution of sulfanilic acid R in acetic acid Rand 0.2 mL is decolourised, then add 1 mL in excess. Filter and dilute to
of a recently prepared 3 gJL solution of naphthylamine R in 40 mL with water R. Dilute 1 mL of the solution obtained to
acetic acid R. Add a turning of zinc R. A pink colour is 5 mL with distilled water R and add 2 mL of ammonium
produced within 5 min. It is not more intense than that of a oxalate solution R. A white precipitate is formed which
mixture of 0.5 mL of nitrate standard solution dissolves in 2 mL of dilute hydrochloric acid R.
(10 ppm NOJ) Rand 0.5 mL of water R, prepared at the B. In a lead or platinum crucible, mix 10 mg with 20 mg of
same time. anhydrous colloidal silica R and a few drops of sulfuric acid R,
with the aid of a copper wire, in order to give a thin slurry.
Zinc sulfate, alkaline-earth sulfates, rare-earth sulfates
Cover the crucible with a thin, transparent plate of plastic
Dissolve 1.0 g in 17 mL of water R. Add 0.5 mL of
under which a drop of water R is suspended, and warm
hydrochloric acid R and 1 g of thioacetamide R. Heat in a
gently. A white ring is rapidly formed around the drop of
water-bath for 10 min. Dilute to 20.0 mL with water Rand
filter. Evaporate 10.0 mL of this solution to dryness in an water.
oven. Ignite the residue at about 800 ± 50 °C to constant TESTS
mass. The residue weighs a maximum of 2 mg. Free acid
Arsenic (2.4.2, Method A) Shake 5.0 g with 2 g of calcium chloride Rand 100 mL of
Maximum 2 ppm, determined on 5 mL of solution S. water R for 5 min. Heat to 70 °C and filter. To 40 mL of the
filtrate, maintained at 70 °C, add 0.1 mL of methyl red
Water (2.5.12)
solution R. Not more than 1.0 mL of 0.1 M sodium hydroxide
16.0 per cent to 20.0 per cent, determined on 80 mg. Shake
is required to change the colour of the indicator to yellow.
for 10 min before carrying out the determination.
ASSAY
ASSAY
Introduce 0.150 g into a 500 mL conical flask and add 8 mL
Dissolve 0.200 g in 50 mL of water R. Add 10 mL of
of hydrochloric acid R. Boil for 3-4 min on a preheated hot
ammonium chloride buffer solution pH 10. 0 R and 50 mg of
plate and allow to cool. Add 300 mL of water R, followed by
mordant black 11 triturate Rl. Titrate with 0.1 M sodium
strong sodium hydroxide solution R until the first appearance of
edetate until the colour changes from red to green.
persistent opalescence (about pH 14). Add 0.13 g of
1 mL of 0.1 M sodium edetate is equivalent to 20.85 mg calconecarboxylic acid tn"turate R and titrate with 0.1 M sodium
of CdSO 4 • edetate until the colour changes from red-violet to pure blue.
- - - - - - - - - - - - - - - - - - - - - PhEur The opalescence caused by the strong sodium hydroxide
solution disappears during the course of the titration. If still
visible at the end of the titration, it can be dissolved by
adding a few drops of hydrochloric acid R.
1 mL of 0.1 M sodium edetate is equivalent to 7 .81 mg of
CaF2 •
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Calcium Iodide Tetrahydrate for Calcium Phosphate for Homoeopathic

Homoeopathic Preparations Preparations
(Calcium Iodatum for Homoeopathic Preparations, Calcium Phosphoricum for Homoeopathic Preparations
Ph. Bur. monograph 2144) DEFINITION
366.0 13640-62-5 Calcium Phosphate for Homoeopathic Preparations contains
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Calcium Phosphate.
PRODUCTION OF STOCK
DEFINITION
The first trituration of Calcium Phosphate for Homoeopathic
Content
Preparations is prepared using a suitable quantity of a
97.0 per cent to 102.0 per cent ofCalz (anhydrous
vehicle, such as Lactose Monohydrate, Lactose or Sucrose,
substance).
and a validated method for trituration that ensures
CHARACTERS homogeneity is achieved. The vehicle complies with the
Appearance statement under Vehicles in the monograph for
White or almost white, very hygroscopic powder. Homoeopathic Preparations.
Solubility Content of calcium Ca
Very soluble or freely soluble in water and in ethanol The first decimal trituration contains 3.5% to 4.0% of Ca.
(96 per cent).
CHARACTERISTICS
IDENTIFICATION The first decimal trituration is a white powder.
A. Solution S (see Tests) gives reaction (a) of calcium
IDENTIFICATION
(2.3.1).
Wash 5 g of the first decimal trituration of the substance
B. Solution S (see Tests) gives reaction (b) of iodides (2.3.1). being examined with three 10-mL quantities of water.
TESTS The dried residue complies with the following tests.
Solution S A. Dissolve 0.1 g of the dried residue in 5 mL of a 25% v/v
Dissolve 10.0 gin distilled water Rand dilute to 100.0 mL solution of nitric acid. The resulting solution yields reaction B
with the same solvent. of phosphates, Appendix VI.
Appearance of solution B. The dried residue yields reaction B characteristic of
Solution S is clear (2. 2.1) and not more intensely coloured calcium salts, Appendix VI. Filter before adding potassium
than reference solution GY5 (2.2.2, Method 11). f errocyanide solution.
Free iodine, iodates C. The dried residue complies with the limits of the Assay.
To 5 mL of solution S add 2 mL of methylene chloride R. D. If the preparation includes Lactose Monohydrate as the
Shake and allow to stand. The organic layer is colourless vehicle, it complies with the following test. Dissolve 0.25 g in
(2.2.2, Method I) (free iodine). Add 0.2 mL of duute sulfuric 5 mL of water. Add 5 mL of ammonia and heat in a water-
acid R. Shake and allow to stand. The organic layer remains bath at 80° for 10 minutes. A red colour develops.
colourless (2.2.2, Method I) (iodates). E. If the preparation includes Sucrose as the vehicle, it
Sulfates (2.4.13) complies with the following test. Dissolve 5.0 gin carbon
Maximum 150 ppm. dioxide-free water and dilute to 10 mL with the same solvent.
Dilute 10 mL of solution S to 15 mL with distuled water R. Dilute 1 mL of the solution to 100 mL with water. To 5 mL
of the solution add O.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 S.
solution. The solution is blue and clear and remains so after
Water (2.5.12) boiling. To the hot solution add 4 mL of dilute hydrochloric
18.0 per cent to 22.0 per cent, determined on 0.100 g. acid and boil for 1 minute. Add 4 mL of di/,ute sodium
ASSAY hydroxide solution. An orange precipitate is formed
Dissolve 0.300 g in 50 mL of water R. Add 5 mL of dilute immediately.
nitric acid Rand 25.0 mL of 0.1 M suver nitrate. Shake. ASSAY
Add 2 mL of ferric ammonium sulfate solution R2 and titrate Dissolve 0.2 g of the residue in a mixture of 1.0 mL of
with 0.1 M ammonium thiocyanate until the colour changes to hydrochloric acid Rl and 5 mL of water. Add 25.0 mL of
reddish-yellow. 0.1M disodium edetate and dilute to 200 mL with water.
1 mL of 0.1 M suver nitrate is equivalent to 14. 70 mg Adjust to about pH 10 with concentrated ammonia.
of Cal2. Add 10 mL of ammonia buffer pH 10. 0 and a few milligrams
STORAGE of mordant black 11 triturate. Titrate the excess disodium
edetate with 0.lM zinc sulfate until the colour changes from
blue to violet. Each mL of 0.lM sodium hydroxide is
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
equivalent to 4.008 mg of Ca.
SYSTEM SUITABILITY
Cineraria Maritima for Homoeopathic The test is not valid unless the chromatogram obtained with
Preparations solution (2) shows three fluorescent bands: an orange
DEFINITION fluorescent band with a low Rf value (rutin), an orange
fluorescent band with an Rf value in the middle region
Cineraria Maritima for Homoeopathic Preparations is the
(hyperoside) and a blue fluorescent band with an Rf value in
fresh aerial parts of Cineraria maritima L. harvested before
the upper region (scopoletin).
flowering.
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.
Top of the plate
The under surface is covered with a dense white felt, the
upper surface is green with scattered 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 with the requirements stated under
Mother Tinctures for Homoeopathic Preparations and with the
fol/,owing 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% w/w (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 water B. Carry out the method for thin-layer chromatography,
and transfer to a cartridge containing octadecyl-bonded silica Appendix III 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 Rl
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 F2s4. (a) Use as the coating silica gel F254.
(b) Use the mobile phase described below. (b) Use the mobile phase described below.
(c) Apply 30 µL of solution (1) and 10 µL of solution (2) as (c) Apply 30 µL of solution (1) and 20 µL of solution (2) as
10 mm bands. 10 mm bands.
(d) Develop the plate to 15 cm. (d) Develop the plate to 10 cm.
(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
ultraviolet light (365 nm). MOBILE PHASE
MOBILE PHASE
10 volumes of methanol and 90 volumes of chloroform.
10 volumes of water, 10 volumes of formic 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.
CONFIRMATION distinct pitted circular to oval areas, lignified, spirally or

The chromatogram obtained with solution (1) shows a series annularly thickened vessels.
of violet bands between the line of application and Rf value C. Carry out the method for thin-layer chromatography,
0.65, one pink band at Rf value 0.75 and one red band Appendix III A, using the following solutions.
at Rf value 0.90. (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
5 mL.
A red band (2) 0.1 % w/v each of caffeine, coumarin and resorcinol in
methanol.
Reserpine: a blue
A pink band band
(a) Use as the coating silica gel F254 •
A series of violet (c) Apply 20 µL of each solution.
bands between the (d) Develop the plate to 10 cm.
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 solution (2) shows three clearly separated bands
Ethanol (approximate Rf values: resorcinol 0.31, caffeine 0.67 and
25 to 35% w/w (31 to 42% v/v), Appendix VIII F. coumarin 0.87).
Dry residue CONFIRMATION
Not less than 1.0%, determined on 2 mL, Appendix XI P. The chromatogram obtained with solution (1) shows two
Relative density dark bands at Rf values of 0.08 and 0.1 respectively between
0.957 to 0.977, Appendix VG. 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 O. 7 8 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
Coumarin: a dark band
Citrullus Colocynthis Fruit for Homoeopathic Preparations is
the dried, peeled fruits of Citrnllus 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
A dark band
and the placentae. The external surface is marked by spiral,
flattish, knife marks where the peel has been removed.
In cross section, three conspicuous fissures can be seen
Resorcinol: a dark
radiating from the centre and dividing the fruit into three
parts. Each part contains two groups of seeds near the band
periphery, the remaining space being filled with pithy
A dark band
parenchyma. Each fruit contains 200 to 300 seeds.
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. Solution ( 1) Solution (2)
Examine under a microscope using chloral hydrate solution.
The powder shows abundant, large, partly lignified, thin-
walled, finely pitted, usually fragmented parenchyma; smaller
cells with slightly collenchymatous thickening and more
TESTS endocarp projects. Cut longitudinally, the endosperm shows

Foreign matter the presence of 2 narrow cavities in each of which is enclosed
Not more than 2.0% of the outer part of the pericarp; not 1 of the foliaceous cotyledons.
more than 5.0% of seeds; not more than 2.0% of other B. Microscopic examination (2.8.23). Reduce to a
foreign matter, Appendix XI D. powder (710) (2.9.12). The powder is brown. Examine
Loss on drying under a microscope using chloral hydrate solution R.
When dried at 100° to 105° for 2 hours, loses not more than The powder shows the following diagnostic characters
22.0% of its weight. Use 1 g. (Figure 2486.-1): fragments of the epicarp (surface view [D])
consisting of thin-walled, polygonal cells, about 30-50 µm in
Total ash
diameter [Da], anomocytic stomata (2.8.3) [Db], and cells in
a pattern consisting of a cell with slightly thickened walls,
pitted at the centre, surrounded by 4-6 cells [De]; fragments
MOTHER TINCTURE of the epicarp and outer layers of the mesocarp (transverse
The mother tincture complies with the requirements stated under section [CJ) showing the epicarp covered by a fine cuticle
Mother Tinctures for Homoeopathic Preparations and with the [Ca] and cells of the mesocarp, ovoid or rounded, some
following requirements. containing prisms of calcium oxalate [Cb]; numerous
fragments of the inner layers of the mesocarp and of the
PRODUCTION endocarp [A] consisting of sclereids [Aa] and short fibres
The mother tincture of Citrullus colocynthis (L.) Schrad. with pitted walls [Ab]; fragments of the endocarp consisting
is prepared from the powdered drug using Method 4a of layers of variously oriented fibres (surface view [E]);
described in the monograph for Methods of Preparation of sclereids and isolated fibres [BJ; vascular bundles
Homoeopathic Stocks and Potentisation. Use 86% w/w (longitudinal section [F]), accompanied by fibres [Fa];
(90% v/v) ethanol. fragments of the endosperm [G, HJ containing very
CHARACTERISTICS numerous small acicular crystals.
The mother tincture is a light yellow to yellow liquid. C. Thin-layer chromatography (2.2.27).
IDENTIFICATION Test solution To 2.00 g of the powdered herbal drug (710)
(2. 9.12) add 20 mL of ethanol (90 per cent VIV) R, shake for
The mother tincture complies with Identification test C
above using the mother tincture as solution (1). 2 hand then centrifuge (1000 g).Use the supernatant.
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% w/w (86% to 94% v/v), Appendix VIII F.
Dry residue plate R (2-10 µm)].
1.0% to 2.5% w/w, Appendix XI P.
Mobile phase methanol R, ethyl acetate R, heptane R
Relative density (10:40:50 VIVIV).
0.830 to 0.850, Appendix V G. Application 40 µL [or 10 µL] as bands of 20 mm [or
10 mm].
Drying In air.
Cocculus lndicus for
Detection Spray with anisaldehyde solution R, heat at
Homoeopathic Preparations 100-105 °C for 5-10 min and examine immediately in
(Cocculus for Homoeopathic Preparations, Ph. Bur. daylight.
monograph 2486) Results See below the sequence of zones present in the
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ chromatograms obtained with the reference solution and the
test solution. Above the zone due to picrotoxinin, several
DEFINITION pink or violet zones may also be visible in the chromatogram
Dried, ripe fruit of Anamirta cocculus (L.) Wight & Arn. (syn. obtained with the test solution.
A. paniculata Colebr.).
Content Top of the plate
Minimum 0.80 per cent ofpicrotoxinin (C 15 H 16O 6;
-- --
Picrotoxinin: a blue zone A blue zone (picrotoxinin)
IDENTIFICATION
First identification: A, B, D. - -
--
Second identification: A, B, C. 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
long; the outer surface is irregularly wrinkled with a ridge
about 4-6 mm long running between the pale, circular scar D. Examine the chromatograms obtained in the assay.
left by the stalk and the small beak of the remains of the Results The peaks due to picrotoxinin and picrotin in the
stigma. The pericarp is hard, about 1 mm thick and the chromatogram obtained with the test solution are similar in
inner surface is brownish-grey, hard and woody. retention time to the corresponding peaks in the
Cut transversely, the fruit shows a single, cup-shaped seed chromatogram obtained with the reference solution.
into the hollow of which an ingrowth of the mesocarp and
Calculate the percentage content of picrotoxinin using the

A1 area of the peak due to picrotoxinin in the chromatogram

A2 area of the peak due to picrotoxinin in the chromatogram
solution, in grams;
m2 mass of picrotoxinin CRS used to prepare the reference solution,
in grams;
p assigned percentage content of picrotoxinin in picrotoxinin CRS.
MOTHER TINCTURE
DEFINITION
Content
0.07 per cent mlm to 0.15 per cent mlm of picrotoxinin
(C1sH16O6)-
PRODUCTION
The mother tincture is prepared from the dried, ripe fruit of
Figure 2486.-1. - Illustration for identificatwn test B of powdered A. cocculus (L.) Wight & Am. according to the following
herbal drug of Cocculus methods prescribed in the monograph Methods of preparation
of homoeopathic stocks and potentisation (2371):
TESTS - method 1.1.8 using the powdered herbal drug (710)
Loss on drying (2.2.32) (2. 9.12) and ethanol (90 per cent V/V); use ethanol
Maximum 10.0 per cent, determined on 1.000 g of the (70 per cent V/V) to prepare the 4th decimal dilution and
powdered herbal drug (710) (2. 9.12) by drying in an oven at ethanol (50 per cent V/V) for subsequent dilutions;
105 °C for 2 h. - method 1.1.10 using the crushed drug in fragments of
about 2-3 mm, ethanol (90 per cent V/V) and a
Total ash (2.4.16) maceration time of about 3 weeks.
CHARACTERS
ASSAY
Appearance
Yellow or dark yellow liquid.
(2.9.12) 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 the mobile phase identification test C for the herbal drug with the following
and filter through a membrane filter (nominal pore size modification.
0.45 µm). Test solutwn The mother tincture to be examined.
Reference solutwn Dissolve 5.0 mg of picrotin CRS and B. Examine the chromatograms obtained in the assay.
5.0 mg of picrotoxinin CRS in 10.0 mL of acetonitrue R. Results The peaks due to picrotoxinin and picrotin in the
Dilute 2.0 mL of the solution to 20.0 mL with the mobile chromatogram obtained with the test solution are similar in
phase. retention time to the corresponding peaks in the
Column: chromatogram obtained with the reference solution.
- size: l = 0.125 m, 0 = 4.0 mm; TESTS
- statwnary phase: octadecylsilyl silica gel for chromatography R Relative density (2.2.5)
(5 µm).
Mobile phase acetonitrue Rl, water R (30:70 V/V).
Ethanol (2. 9.10)
Flow rate 0.5 mL'min. 85 per cent V/V to 95 per cent V/V when method 1.1.10 is
Detection Spectrophotometer at 200 nm. used.
Injection 10 µL. 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 solutwn Dilute 0.500 g of the mother tincture to be
and picrotoxinin. examined to 10.0 mL with the mobile phase and filter using
a membrane filtre (nominal pore size 0.45 µm).
Calculate the percentage content of picrotoxinin using the Test solution Dissolve 1.00 g in 5 mL of nitric acid R and
following expression: dilute to 50.0 mL with water R.
Reference solutions Prepare the reference solutions using iron
standard solution (20 ppm Fe) R, diluted as necessary with a
1 per cent V/V solution of nitric acid R.
A, area of the peak due to picrotoxinin in the chromatogram Source Iron hollow-cathode lamp.
obtained with the test solution; Wavelength 248.3 nm.
A, area of the peak due to picrotoxinin in the chromatogram
obtained with the reference solution; Flame Air-acetylene.
m, mass of the mother tincture to be examined used to prepare the Lead
Maximum 100 ppm.
mass of picrotoxinin CRS used to prepare the reference solution,
in grams; Atomic absorption spectrometry (2.2.23, Method[).
p assigned percentage content of picrotoxinin in picrotoxinin CRS.
Test solution Use the test solution prepared for the test for
iron.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Reference solutions Prepare the reference solutions using lead
standard solution (0.1 per cent Pb) R, diluted as necessary with
a 1 per cent V/V 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. Bur. Maximum 50 ppm.
monograph 1610) Atomic absorption spectrometry (2.2.23, Method[).
Cu 63.5 7440-50-8 Test solution Use the test solution prepared for the test for
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ iron.
DEFINITION Reference solutions Prepare the reference solutions using zinc
standard solution (100 ppm Zn) R, diluted as necessary with a
Content
98.0 per cent to 102.0 per cent of Cu. 1 per cent V/V solution of nitric acid R.
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 nitric acid, Dissolve 0.100 gin 5 mL of nitric acid R. Heat to expel the
practically insoluble in ethanol (96 per cent). nitrous fumes. Add 200 mL of water Rand neutralise (2.2.3)
with dilute ammonia Rl. Add 1 g of ammonium chloride R and
IDENTIFICATION
3 mg of murexide R. Titrate with 0.1 M sodium edetate until
A. To 2 mL of solution S (see Tests) add 0.5 mL of
the colour changes from green to violet.
potassium ferrocyanide solution R. A reddish-brown precipitate
is formed. 1 mL of 0.1 M sodium edetate is equivalent to 6.354 mg of
Cu.
B. To 5 mL of solution S add 0.6 mL of ammonia R. A blue
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
precipitate is formed. Add 2 mL of ammonia R.
The precipitate disappears; the solution has an intense blue
colour.
TESTS
Solution S
Copper Acetate Monohydrate for
Dissolve 2.0 g in 10 mL of nitric acid R. After nitrous fumes Homoeopathic Preparations
are no longer evolved, dilute to 60 mL with distilled water R.
(Cuprum Aceticum for Homoeopathic Preparations,
Acidity or alkalinity Ph. Bur. monograph 2146)
To 5.0 g add 20 mL of carbon dioxide-free water R. Boil for
Cu(C2H3O2)2,H2O 199.7 6046-93-1
1 min. Cool. Filter and dilute to 25.0 mL with carbon
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
dioxide-free water R. To 10 mL of the solution add 0.1 mL of
bromothymol blue solution Rl. Not more than 0.5 mL of DEFINITION
0.01 M hydrochloric acid or 0.01 M sodium hydroxide is Content
required to change the colour of the indicator. 99.0 per cent to 101.0 per cent of Cu(C 2 H 3 O2 )z,H 2 O.
Chlorides (2.4. 4)
CHARACTERS
Appearance
Sulfates (2. 4.13) Greenish-blue crystals or green powder.
Solubility
Iron Soluble in water, slightly soluble or very slightly soluble in
Maximum 50 ppm. ethanol (96 per cent).
Atomic absorption spectrometry (2.2.23, Method[).
IDENTIFICATION
A. It gives reaction (a) of acetates (2.3.1).
Crocus for Homoeopathic
B. Dissolve 0.1 gin 10 mL of water Rand add dilute Preparations
ammonia Rl dropwise. A dark blue colour is produced. Saffron for Homoeopathic Use
TESTS Saffron for Homoeopathic Preparations
Solution S (Ph. Bur. monograph 1624)
Dissolve 3.0 gin a mixture of 40 mL of distilled water Rand Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
0.6 mL of glacial acetic acid R, with heating at 70 °C. Cool
and dilute to 45 mL with distilled water R. DEFINITION
Appearance of solution Dried stigmas of Crocus sativus L. usually joined by the base
Solution S is clear (2.2.1). to a short style.
Impurities not precipitating with hydrogen sulfide CHARACTERS

Maximum 0.1 per cent, calculated as sulfates. Characteristic, aromatic odour.
To 2.000 g add 92 mL of water R and 8.0 mL of dilute IDENTIFICATION
suljuric 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
Chlorides (2. 4. 4) and not more than 5 mm long.
Maximum 50 ppm, determined on solution S. B. Examine under a microscope using chloral hydrate
Sulfates (2.4.13) solution R. It shows the following diagnostic characters:
Maximum 150 ppm, determined on solution S. elongated epidermal cells, frequently with a short, central
papilla; in water they release a yellow colouring matter; the
Iron (2.4.9) upper border of the stigma has finger-shaped papillae, up to
Maximum 20 ppm. 150 µm long; between them are single, globular pollen
Dissolve 0.500 gin 10 mL of water R. Transfer to a grains, about 100 µm wide, with a finely pitted exine,
separating funnel. Add 20 mL of hydrochloric acid Rl and vascular bundles with small spirally thickened vessels and no
10 mL of methyl isoburyl 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
suljuric acid R and 17 .0 mL of water R. To 4.0 mL of this
5 mL of methanol R and add a solution of 5 mg of Sudan red
solution add 4.0 mL of water R, 5.0 mL of bromine water R,
G R in 5 mL of methylene chloride R.
7 .0 mL of dilute ammonia Rl and 3.0 mL of a 10 g/L
solution of dimethylglyoxime R in ethanol (90 per cent VIV) R. Plate TLC silica gel F 254 plate R.
This solution is not more intensely coloured within I min Mobile phase water R, 2-propanol R, ethyl acetate R
than a solution prepared as follows: mix 4.0 mL of a I ppm (10:25:65 VIVIV).
solution of nickel (Ni) prepared from nickel standard solution Application 10 µL of the test solution and 5 µL of the
(10 ppm Ni) R, 4.0 mL of water Rand 5.0 mL of bromine reference solution as bands.
water R; carefully add 7 .0 mL of dilute ammonia Rl 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
Dissolve 0.400 gin water Rand dilute to 50 mL with the
same solvent. Add 6.0 mL of glacial acetic acid R, 10.0 g of
test solution.
potassium iodide R and I 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
Cu(C2H3O2)2,H2O. A red zone
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
A yellow zone
2 yellow zones
An intense yellow zone (crocine)


Cydonia Oblonga for Homoeopathic
chromatograms obtained with the reference solution and the Preparations
test solution. DEFINITION
Cydonia Oblonga for Homoeopathic Preparations is the
Top of the plate seeds of Cydonia oblonga Mill.
A red zone 1 or 2 quenching zones IDENTIFICATION
A yellow zone A quenching zone
The seeds are 6 to 7 mm long, reddish-brown to dark-
brown, frequently cohering by a white mucilage appearing in
Reference solution Test solution flakes on the surface and in the spaces between the seeds;
four-sided, one arched, one often distinctly ridged and two
Detection C Treat with anisaldehyde solution R and examine larger and flattened; pointed at one end, where the hilum
in daylight while heating at 100-105 °C for 5-10 min. occurs as a paler spot, obtuse at the other extremity, where
the chalaza is situated. Cut transversely, the seed shows a
very narrow endosperm surrounding two yellowish-white
cotyledons.
test solution.
TESTS
Top of the plate Total ash
Not more than 5%, Appendix XI J, Method II.
A red zone 1 or 2 red to reddish-violet zones
A blue to bluish-green zone A red to reddish-violet zone MOTHER TINCTURE

2 blue to bluish-green zones The mother tincture complies with the requirements stated under
An intense blue to bluish-green zone Mother Tinctures for Homoeopathic Preparations and with the
(crocine) following requirements.
Reference solution Test solution PRODUCTION
The mother tincture of Cydonia oblonga Mill. is prepared
from the powdered drug using Method 1.1. 8 described in the
E. Dilute 0.1 mL of the test solution (see Identification D)
with 1 mL of methanol R. Deposit O.1 mL of this solution on
Stocks and Potentisation. Use glycerol.
a filter paper, allow to dry and spray with a 10 g/L solution
of diphenylboric acid aminoethyl ester R in methanol R. Examine CHARACTERISTICS
in ultraviolet light at 365 nm. The spot shows an intense The mother tincture is a pale yellow, clear or slightly turbid
orange-yellow fluorescence. viscous liquid.
TESTS IDENTIFICATION
Colouring intensity Carry out the method for thin-layer chromatography,
Introduce 0.10 g into a 5 mL volumetric flask and dilute to Appendix III A, using the following solutions.
5.0 mL with distilled water R. Close the flask and shake every ( 1) Dilute 5 mL of the mother tincture with 5 mL of water,
30 min for 8 h. Then allow to stand for 16 h. Dilute 1.0 mL mix thoroughly and transfer the diluted tincture to a
to 500.0 mL with distilled water R. The absorbance (2.2.25) cartridge containing octadecyl-bonded silica sorbent (a Sep-pak
measured at 440 nm using distilled water R as the CIS cartridge is suitable) previously washed with 10 mL of
compensation liquid, is not less than 0.44. methanol followed by 10 mL of water. Wash the cartridge
Foreign matter with 15 mL of water and elute with 10 mL of methanol.
Examine the herbal drug microscopically. No parts with Evaporate the eluant to dryness using a rotary evaporator.
rough walls, no crystals and no pollen grains containing Dissolve the residue in 0.5 mL of methanol.
3 germinal pores are present. (2) 0.1 % w/v of hyperoside, 0.1 % w/v of rutin and 0.01 % w/v
Loss on drying (2.2.32) of scopoletin in methanol.
Maximum 10.0 per cent, determined on 0.200 g by drying in CHROMATOGRAPHIC CONDITIONS
an oven at 105 °C. (a) Use as the coating silica gel 60 F 254•
Total ash (2.4.16) (b) Use the mobile phase as described below.
Maximum 7.0 per cent, determined on the residue obtained (c) Apply 40 µL of solution (1) and 10 µL of solution (2), as
in the test for loss on drying. 12 mm bands.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
with a 1% w/v solution of diphenylboric acid aminoethyl ester in
methanol, and then with a 5% w/v solution of polyethylene
glycol 400 in methanol and examine under ultraviolet light
(365 nm).
MOBILE PHASE
15 volumes of anhydrous formic acid, 15 volumes of water and
70 volumes of ethyl acetate.
SYSTEM SUITABILITY B. From non-flowering plants, take a fragment of the lower

The test is not valid unless the chromatogram obtained with epidermis of the leaf. Examine under a microscope using
solution (2) shows two clearly separated orange fluorescent chloral hydrate solution R. The leaf has a smooth cuticle and
bands and one blue fluorescent band at a higher Rf value. shows the following diagnostic characters: epidermal cells
In order of increasing Rf value the bands are: rutin, 30-75 µm long, with distinctly sinuous anticlinal walls;
hyperoside and scopoletin. numerous anomocytic stomata (2.8.3); covering trichomes
often articulated and bent at right angles, uniseriate, usually
CONFIRMATION
with 3-5 cells, sometimes collapsed, and a terminal cell
The chromatogram obtained with solution (1) shows three covered in a sometimes smooth but usually verrucose or
yellow fluorescent bands in the lower third, a blue fluorescent slightly striated cuticle; glandular trichomes with unicellular
band just below the rutin standard, a blue fluorescent band stalks and globular bicellular heads; glandular trichomes with
just below the hyperoside standard and a blue fluorescent multicellular, uniseriate stalks and unicellular heads.
band with the same Rf value of the scopoletin standard.
Other bands may be present. TESTS
Top of the plate Maximum 5 per cent.
Digitalis lanata
A blue fluorescent band Scopolelin: a blue The presence of brownish-yellow flowers, of oval lanceolate,
fluorescent band
narrow leaves, with entire margins or with dentate margins
only near the apex, the existence of a few glandular
trichomes of epidermal cells with very characteristic beaded
Hyperoside: an orange
fluorescent band walls, and the absence of covering trichomes, indicates
A blue fluorescent band adulteration with Digitalis lanata Ehrh.
Digitalis lutea
The presence of brownish-yellow flowers, of sessile,
Rutin: an orange lanceolate, denticulate, almost glabrous leaves and the
A blue fluorescent band fluorescent band
A yellow fluorescent band
scarcity of smooth covering trichomes, indicates adulteration
A yellow fluorescent band with Digitalis lutea L.
IA yellow fluorescent band Digitalis grandiflora
The presence of brownish-yellow flowers, of oblong or oval
Solution (1) Solution (2) leaves with serrate margins, non-reticulate venation and veins
bearing rare, very large covering trichomes with large pits,
indicates adulteration with Digitalis grandijiora All.
TESTS
Refractive index
Minimum 70.0 per cent, determined on 5.0 g of the
1.468 to 1.475, Appendix VE. comminuted herbal drug by drying in an oven at 105 °C
for 2 h.
MOTHER TINCTURE
Digitalis for Homoeopathic
Preparations general monograph Mother tinctures for homoeopathic
(Ph. Bur. monograph 2705) preparations (2029).
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ DEFINITION
The mother tincture is prepared from the fresh leaf of
DEFINITION
Digitalis purpurea L, collected just before or during flowering.
Fresh leaf of Digitalis purpurea L, collected just before or
during flowering. Content
0.003 per cent mlm to 0.013 per cent m/m of digitoxin
IDENTIFICATION (C41H640 13; M, 765).
A. The flower is light to dark violet. The leaf is variable in
size, usually 10-50 cm long and 4-15 cm wide, simple, entire, PRODUCTION
lanceolate, oblong, and ending in a subacute apex. The mother tincture is prepared from the comminuted herbal
The margins of the lamina are crenate or dentate. It is thick drug according to the following methods prescribed in the
with a velvety or rough texture. The lamina is decurrent, monograph Methods of preparation of homoeopathic stocks and
attenuated along the midrib, the whole forming a winged, potentisation (23 71):
triangular petiole, with purple-pink spots at the base. - method 1.1.3;
The venation is pinnate, with the lateral veins leaving the - method 1.1.10, using ethanol (65 per cent V/V) and a
midrib at about 45°; they anastomose near the leaf margin maceration time of 3-5 weeks.
forming arcs, and are connected to each other by an CHARACTERS
extensive network of tertiary veinlets. The upper surface is Appearance
greyish-green and pubescent, but sometimes almost glabrous. Light greenish-brown or brown liquid.
The veins are sunken, forming depressed lines around
bulging areas in the lamina. The lower surface is paler and IDENTIFICATION
very tomentose; the whitish veins are prominent giving the Thin-layer chromatography (2.2.27).
surface a honeycomb-like appearance. Test solution Evaporate 10 mL of the mother tincture to be
examined to dryness under reduced pressure. Take up the
residue in 1 mL of a mixture of equal volumes of ethyl Column:

acetate R and methanol R. =
- size: l 0.15 m, 0 =
4.6 mm;
Reference solution Dissolve 5 mg of digitoxin R and 2 mg of - stationary phase: end-capped octadecylsilyl silica gel for
gitoxin R in a mixture of equal volumes of ethyl acetate R and chromatography R (3 µm);
methanol R, and dilute to 10 mL with the same mixture of - temperature: 30 "C.
solvents. Mobile phase:
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel - mobi7e phase A: water for chromatography R;
plate R (2-10 µm)]. - mobile phase B: acetonitrile for chromatography R;
(7.5:10:75 VIVIV). (min) (per cent V/V) (per cent V/1')
Applicatwn 20 µL [or 10 µL] as bands. 0-2 80 20
Development Over a path of 10 cm [or 6 cm]. 2 - 32 80-, 40 20-, 60
Drying In air. 32 - 33 40-, 80 60-> 20
Detection Treat with a mixture of 2 volumes of a 10 g/L 33 - 38 80 20
solution of chloramine R and 8 volumes of a 250 g/L solution
of trichloroacetic acid R in ethanol (96 per cent) R, then heat at Flow rate 1.0 mIJmin.
100-105 °C for 10 min; examine in ultraviolet light at Detection Spectrophotometer at 220 nm.
365 nm. Injection 10 µL.
Results See below the sequence of zones present in the Retention time Digitoxin = about 25 min.
System suitabi7iry Reference solution (b):
lanatoside C and digoxin.
Calculate the percentage content of digitoxin using the
Top of the plate
-- --
Digitoxin: a bluish-green fluorescent A bluish-green fluorescent zone

zone (digitoxin)
Gitoxin: a light blue fluorescent zone A light blue fluorescent zone AI area of the peak due to digitoxin in the chromatogram obtained
(gitoxin) with the test solution;
A2 area of the peak due to digitoxin in the chromatogram obtained
-- -- with reference solution (a);
m1 mass of the mother tincture to be examined used to prepare the
A faint brownish-yellow fluorescent test solution, in grams;
zone may be present m2 mass of digiwxin for LG assay GRS used to prepare reference
A faint light blue fluorescent zone solution (a), in grams;
may be present p percentage content of digiwxin for LG assay GRS.

- - - - - - - - - - - - - - - - - - - - - - Ph Eur
TESTS
0.935 to 0.955 when method 1.1.3 is used. Hedera Helix for Homoeopathic
Ethanol (2.9.10) Preparations
60 per cent V/Vto 70 per cent V/Vwhen method 1.1.10 is
used.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Minimum 2.5 per cent. DEFINITION
ASSAY Fresh, young, fully developed but not yet lignified branch of
Liquid chromatography (2.2.29). Hedera helix L., harvested immediately before or at the
beginning of flowering.
Test solution Dilute 8.0 g of the mother tincture to be
examined to 10.0 mL with water for chromatography Rand IDENTIFICATION
filter through a membrane filter (nominal pore size 0.45 µm). The fresh, young branches of Hedera helix L. are thin and
Reference solutwn (a) Dissolve 10.0 mg of digitoxinfor LG flexible, climbing; they cling to their support by stem-roots.
assay CRS in methanol R2 and dilute to 10.0 mL with the The leaves are alternate, simple and petiolate. The petiole
same solvent. Dilute 2.00 mL of the solution to 20.0 mL shows a cylindrical section. The upper surface of the leaves is
with a mixture of equal volumes of methanol R2 and water for glabrous and shiny, darker than the lower surface.
chromatography R. The lamina is usually divided into 3-5 more or less deeply
cut lobes on sterile branches; it is oval, with a pointed apex
Reference solutwn (b) Dissolve 10.0 mg of lanatoside CR and
10.0 mg of digoxin R in methanol R2 and dilute to 20.0 mL on fertile branches. The inflorescences are arranged in a
simple semi-globular corymb and grouped in terminal
with the same solvent. Dilute 1.0 mL of the solution to
20.0 mL with a mixture of equal volumes of methanol R2 and clusters. The pedicels of the umbel are covered in whitish
water for chromatography R. hairs. Each flower shows 5 small teeth formed by the upper
part of the sepals and 5 petals covered in very small inverted
hairs.
TESTS Dry residue (2.8.16)

Foreign matter (2.8.2) Minimum 2.0 per cent.
If required by the competent authority, maximum 5 per cent. ASSAY
Loss on drying (2.2.32) Liquid chromatography (2.2.29).
If required by the competent authority, minimum Test solution In a 20.0 mL volumetric flask, dilute 3.000 g
50 per cent, determined on 5.0 g of the finely cut drug by of the mother tincture to be examined to 20.0 mL with the
drying in an oven at 105 °C for 2 h. mobile phase.
Reference solution In a 50.0 mL volumetric flask, dissolve
MOTHER TINCTURE 20.0 mg of hederacoside CR in the mobile phase and dilute to
The mother tincture complies with the requirements of the 50.0 mL with the mobile phase.
general monograph Mother tinctures for homoeopathic Column:
preparations (2029). =
- size: l 0.25 m, 0 4 mm; =
PRODUCTION - stationary phase: octadecylsilyl silica gel for chromatography R
The mother tincture of Hedera helix L. is prepared by (5 µm).
maceration using ethanol of a suitable concentration. Mobile phase Mix 35 volumes of water R, adjusted to pH 3
with phosphoric acid R, and 65 volumes of methanol R.
Content
Minimum 0.15 per cent mlm ofhederacoside C (C 59 H 96 0 26 ; Flow rate 1 mUmin.
Mr 1221). Detection Spectrophotometer at 205 nm.
CHARACTERS Injection 20 µL.
Appearance Retention time Hederacoside C = about 8 min.
Dark greenish-brown liquid. Calculate the percentage content m/m of hederacoside C
IDENTIFICATION using the following expression:
A 1 xm 2 xCx0.4
Test solution The mother tincture to be examined.
A2xm1
Reference solution Dissolve 1 mg of r1.-hederin R and 1 mg of
hederacoside C R in methanol R and dilute to 2 mL with the area of the peak due to hederacoside C in the chromatogram
same solvent. obtained with the test solution;
Plate TLC silica gel plate R. obtained with the reference solution;
Mobile phase glacial acetic acid R, water R, butanol R mass of the mother tincture in the test solution, in grams;
mass of hederacoside C R in the reference solution, in grams;
(1:1:4 VIVIV). percentage content of hederacoside C R.
Development Over half of the plate. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Drying In air.
Detection Spray with a 10 per cent V/V solution of sulfuric
acid R in methanol Rand heat at 100-105 °C for 10 min.
Examine in daylight. Histaminum for Homoeopathic
Results See below the sequence of the zones present in the Preparations
test solution. Other faint zones may also be present in the
Top of the plate
-- --
111.1 51-45-6
o:-Hederin: a violet zone A violet zone
(o:-hederin) Ph Eur - - - - - - - - - - - - - - - - - - - - - ~
Hederacoside C: a brown zone A brown zone DEFINITION

(hederacoside C) 2-( lH-Imidazol-4-yl) ethan-1-amine.
-- A greyish-brown zone -- Content
A yellow zone 97.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
Appearance
Reference solution Test solution Slightly yellow, deliquescent crystals.
Solubility
TESTS Soluble in water and in ethanol (96 per cent).
Relative density (2.2.5) IDENTIFICATION
0.890 to 0.925. A. Melting point (2.2.14): 82 °C to 85 °C.
Ethanol (2.9.10) B. Infrared absorption spectrophotometry (2.2.24).
Comparison histamine CRS.
TESTS
Solution S
Hydrastis Canadensis for
Dissolve 0.3 gin 2.75 mL of 2 M hydrochloric acid Rand Homoeopathic Preparations
dilute to 10 mL with distilled water R. (Ph. Bur. monograph 2500)
Solution S is clear (2.2.1) and not more intensely coloured
than reference solution Y3 (2.2.2, Method[). The herbal drug complies with the requirements of the
Histidine monograph Goldenseal rhizome (1831).
Test solutwn Solution S. MOTHER TINCTURE
Reference solutwn (a) Dissolve 25 mg of histidine The mother tincture complies with the requirements of the
hydrochloride monohydrate CRS in water R and dilute to general monograph Mother tinctures for homoeopathic
50 mL with the same solvent. preparations (2029).
Reference solutwn (b) Mix 1 mL of the test solution and DEFINITION
1 mL ofreference solution (a). The mother tincture is prepared from the whole or cut, dried
Plate TLC silica gel G plate R (5-40 µm) [or TLC silica gel rhizome and roots of Hydrastis canadensis L.
plate R (2-10 µm)]. Content
Mobile phase concentrated ammonia R, water R, acetonitrile R - hydrastine (C 21 H 21 N06 ; M, 383.4): 0.10 per cent to
(5:20:75 V/V/V). 0.40 per cent;
Applicatwn I µL of the test solution and reference - berberine (C 20 H 18N04 ; M, 336.4): 0.20 per cent to
solution (a); 2 µL ofreference solution (b). 0.50 per cent.
Development Over a path of 10 cm [or 7 cm]. PRODUCTION
Drying At 100-105 °C for 15 min. The mother tincture is prepared by the following methods
Detection Spray with ninhydrin solutwn Rl and heat at prescribed in the monograph Methods of preparation of
110 °C for 10 min. homoeopathic stocks and potentisation (2371):
- Method 1.1.8, using the powdered herbal drug (710)
(2. 9.12) and ethanol (70 per cent V/V) [or ethanol
- the chromatogram shows 2 clearly separated spots.
(62 per cent m/m)];
Limit: - Method 1.1.10, using the fragmented herbal drug (pieces
- histidine: any spot due to histidine in the chromatogram about I cm in diameter), ethanol (65 per cent V/V) and
obtained with the test solution is not more intense than maceration for 3-5 weeks.
the corresponding spot in the chromatogram obtained
with reference solution (a) (1.7 per cent). CHARACTERS
Appearance
Sulfates (2.4.13)
Yellowish-brown liquid.
Dilute 5 mL of solution S to 15 mL with distilled water R. IDENTIFICATION
Water (2.5.12)
Maximum 2.0 per cent, determined on 0.300 g. Test solution The mother tincture to be examined.
Sulfated ash (2. 4.14) Reference solutwn Immediately before use, dissolve 5 mg of
Maximum 0.2 per cent, determined on 0.5 g. hydrastine hydrochloride R and 5 mg of berberine chloride R in
ASSAY Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
Dissolve 50.0 mg in 5 mL of anhydrous formic acid R and add plate R (2-10 ~tm)].
20 mL of anhydrous acetic acid R. Titrate with 0.1 M
perchloric acid, determining the end-point potentiometrically
(10:10:80 V/V/V).
(2.2.20). Carry out a blank titration.
I mL of 0.1 M perchloric acid is equivalent to 5.557 mg of
CsH9N3. Development Over a path of 15 cm [or 6 cm].
Drying In air.
STORAGE
In an airtight container, protected from light, at a Detection Examine in ultraviolet light at 365 nm.
temperature of 2 °C to 8 °C. Results See below the sequence of fluorescent zones present
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur in the chromatograms obtained with the reference solution
test solution.
Top of the plate p percentage content of hydrastine hydrochlori<k CRS or percentage

content of berberine chlori<k CRS.
-- -- _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Berberine: a bright yellow A bright yellow fluorescent zone

Hydrastine: a deep blue fluorescent A deep blue fluorescent zone

zone (hydrastine) Hyoscyamus for Homoeopathic
-- -- Preparations
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
TESTS DEFINITION
Relative density (2.2.5) Whole, fresh flowering plant of Hyoscyamus niger L.
IDENTIFICATION
Ethanol (2.9.10) Hyoscyamus is an annual or biennial plant, with a well
60 per cent V/Vto 70 per cent V/Vwhen method 1.1.10 is developed taproot. The robust, erect stem is hollow and
used. subcylindrical and up to 80 cm long. The soft, viscid, dull
Dry residue (2.8.16) dark-green leaves are densely pubescent on both surfaces,
Minimum 1.2 per cent. especially on the veins. The lower leaves are petiolate and are
arranged in a rosette; the lower cauline leaves are semi-
ASSAY
amplexicaul and the upper ones are completely amplexicaul.
The lamina, up to 25 cm long, is oblong to ovate with 2 to 5
Test solution Dilute about 1.000 g, accurately weighed, of broadly dentate lobes on each side. The midrib is well
the mother tincture to be examined to 20.0 mL with the developed. The secondary veins arise at a wide angle from
mobile phase. the midrib and terminate in the apices of the lobes.
Reference solution Immediately before use, dissolve 10.0 mg The flowering tops are densely pubescent and form a short
of hydrastine hydrochloride CRS and 10.0 mg of berberine drooping cluster. Each flower arises in the axils of a large
chloride CRS in methanol Rand dilute to 100.0 mL with the bract. The gamosepalous calyx is covered with dense cotton-
same solvent. like hairs and has 5 triangular-ovate lobes, each ending in a
Column: short point that becomes spiny. The gamopetalous corolla,
=
- size: l 0.125 m, 0 4 mm; = with 5 nearly equal lobes, is yellowish and with a delicate,
- stationary phase: end-capped octadecylsilyl silica gel for brown to blackish-violet venation. The fruit, sometimes
chromatography R (5 µm). present at the base of the inflorescences, is a pyxis distinctly
Mobile phase Dissolve 9.93 g of potassium dihydrogen swollen at the base.
phosphate R in 730 mL of water R, add 270 mL of TESTS
acetonitrile R and mix. Foreign matter (2.8.2)
Flow rate 1.2 mUmin. If required by the competent authority, maximum 5 per cent.
Detection Spectrophotometer at 235 nm. Loss on drying (2.2.32)
Injection 10 µL. If required by the competent authority, minimum
Elution order Hydrastine, berberine. 50 per cent, determined on 5.0 g of the finely cut drug by
drying in an oven at 105 °C for 2 h.
the reference solution to identify the peaks due to hydrastine Hyoscyamus albus L
and berberine. The presence of middle and upper leaves with a petiole and
of fruits barely swollen at the base indicates adulteration by
Hyoscyamus albus L.
hydrastine and berberine.
Calculate the percentage content m/m of hydrastine using the MOTHER TINCTURE
following expression: The mother tincture complies with the requirements of the
A1 xm2 xpx0.913 preparations (2029).
A2 xm1 x 5
PRODUCTION
Calculate the percentage content m/m of berberine using the The mother tincture of Hyoscyamus niger L. is prepared by
following expression: maceration of the drug, using ethanol of a suitable
concentration.
A1 x m2 xp x 0.905 Content
A 2 x m1 x 5 0.002 per cent m/m to 0.01 per cent mlm of total alkaloids,
area of the peak due to hydrastine or to berberine in the
expressed as hyoscyamine (C 17 H 23N0 3 ; Mr 289.4).
chromatogram obtained with the test solution; CHARACTERS
area of the peak due to hydrastine or to berberine in the
chromatogram obtained with the reference solution; Appearance
mass of the mother tincture to be examined used to prepare the Dark greenish-brown liquid.
mass of hydrastine hydrochloride CRS or mass of berben'ne IDENTIFICATION
chloride CRS used to prepare the reference solution, in grams; Thin-layer chromatography (2.2.27).
Test solution Evaporate 10 mL of the mother tincture to be about 10 g is obtained. Quantitatively transfer the residue to
examined in a water-bath at 40 °C, under reduced pressure. a separating funnel using a few millilitres of ethanol
Take up the residue with 1 mL of ammonia R, and shake (70 per cent Vlv,) R. Add 5 mL of concentrated ammonia R
with 2 quantities, each of 10 mL, of ether R. Combine the and 25 mL of water R. Extract with successive fractions of a
ether layers, dry over anhydrous sodium sulfate R and filter. mixture of 1 volume of chloroform R and 3 volumes of
Evaporate on a water-bath and dissolve the residue in peroxide-free ether R until the alkaloids are completely
0.50 mL of methanol R. extracted. Evaporate to dryness a few millilitres of the last
Reference solution (a) Dissolve 50 mg of hyoscyamine organic fraction. Take up the residue in 0.25 M sulfuric acid
sulfate R in 10 mL of methanol R (solution A). Dissolve and verify the absence of alkaloids using potassium
15 mg of hyoscine hydrobromide R in 10 mL of methanol R tetraiodomercurate solution R. Combine the organic layers and
(solution B). Mix 4 mL of solution A and 2 mL of extract several times with 0.25 M sulfu1ic acid. Separate the
solution B and dilute to 10 mL with methanol R. layers by centrifugation if necessary and transfer the acid
Reference solution (b) Dissolve 20 mg of atropine sulfate R in layers to a second separating funnel. Make the acid layer
alkaline with ammonia R and shake with at least 3 quantities,
methanol Rand dilute to 10 mL with the same solvent.
each of 30 mL, of chloroform R. Combine the chloroform
Plate TLC silica gel plate R. layers, add 4 g of anhydrous sodium sulfate R and allow to
Mobile phase concentrated ammonia R, water R, acewne R stand for 30 min with occasional shaking. Decant the
(3:7:90 VIVIV). chloroform and wash the anhydrous sodium sulfate with
Application 20 µL, as bands. 3 quantities, each of 10 mL, of chloroform R. Combine the
Development Over a path of 10 cm. chloroform fractions, evaporate to dryness on a water-bath
and dry in an oven at 100-105 °C for 15 min. Dissolve the
residue in a few millilitres of chloroform R, add 10.0 mL of
Detection A Spray with dilute potassium iodobismuthate 0. 005 M sulfuric acid and remove the chloroform by
solution R until orange zones become visible. Examine in evaporation on a water-bath. Titrate the excess of acid with
daylight. 0.01 M sodium hydroxide using methyl red mixed solution R as
Results A See below the sequence of the zones present in indicator.
the chromatograms obtained with the reference solutions and Calculate the percentage content mlm of total alkaloids,
the test solution. Other faint zones may be present in the expressed as hyoscyamine, from the expression:
0.2894(10 - n)
Top of the plate m
Hyoscine: an orange An orange zone n volume of 0.01 M sodium hydroxide used, in millilitres;
zone (hyoscine) m mass of the mother tincrure used~ in grams.
-- -- --
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
Hyoscyamine: an Atropine: an orange A orange zone
orange zone zone (hyoscyamine/atropine)
-- -- --
Faint orange zones Hypericum for Homoeopathic
(line of application)
Preparations
Reference Reference Test solution
solution (a) solution (b)
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Detection B Subsequently spray with sodium nitrite solution R DEFINITION

until the yellow background disappears. Examine in daylight Whole, fresh plant of Hypericum perforatum L., at the
after 15 min. beginning of the flowering period.
Results B See test for atropine. IDENTIFICATION
TESTS The perennial plant consists of a spindle-shaped root and a
Relative density (2.2.5) branched rhizome, giving rise to long, decumbent runners.
0.930 to 0.960. The cylindrical, erect stem is woody at the base, 0.2 m to
1 m long, branched in the upper part, with 2 raised
Atropine
longitudinal lines.
identification. The leaves are opposite, sessile, exstipulate, oblong-oval and
15 mm to 30 mm long. The leaf margins show black
Results The zone due to hyoscyamine in the chromatogram
obtained with the test solution changes from orange to glandular dots, and many small translucent oil glands are
present on the entire surface and are visible by transmitted
reddish-brown but not to greyish-blue (atropine).
light.
Ethanol (2. 9.10)
The flowers are regular and form corymbose clusters at the
apex of the stem. They have 5 green, lanceolate sepals with
Dry residue (2.8.16) acuminate apices, and black oil glands near the entire
Minimum 1.2 per cent. margins; 5 orange-yellow petals, much longer than the sepals,
ASSAY with black oil glands near the terminal margins only;
Evaporate 100. 0 g of the mother tincture to be examined, at 3 stamina! blades, each divided into many orange-yellow
a low temperature under reduced pressure, until a residue of stamens and 3 carpels surmounted by red styles. Each petal
is asymmetrically linear-ovate in shape, with one of the TESTS

margin entire and the other dentate. Relative density (2.2.5)
TESTS 0.900 to 0.920.
Foreign matter (2.8.2) Ethanol (2. 9.10)
Maximum 4 per cent of fruits and maximum 1 per cent of 60 per cent V/V to 75 per cent V/V.
other foreign matter. Dry residue (2.8.16)
Loss on drying (2.2.32) Minimum 1.3 per cent.
If performed to demonstrate the freshness of the drug, _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
minimum 55 per cent, determined on 5.0 g of finely cut drug
by drying in an oven at 105 °C.
MOTHER TINCTURE lgnatia for Homoeopathic

Preparations
preparations (2029). (Ph. Bur. monograph 2513)
PRODUCTION
The mother tincture of Hypericum perforatum L. is prepared DEFINITION
by maceration using alcohol of a suitable concentration. Dried, ripe seed of Strychnos ignatii P.J.Bergius.
CHARACTERS Content
Dark red to brownish red liquid. Minimum 1.80 per cent for the sum of the contents of
IDENTIFICATION brucine (C23H2 6N 2O 4 ; M, 394.5) and strychnine
Thin-layer chromatography (2.2.27). (C 21 H 22N 2O 2; M, 334.4), of which minimum 65 per cent
consists of strychnine (dried drug).
Reference solution Dissolve 5 mg of rutoside trihydrate R, IDENTIFICATION
1 mg of hypericin R and 5 mg of hyperoside R in methanol R A. The seed is grey, brown and dull, up to 3 cm long and
and dilute to 5 mL with the same solvent. 10-25 mm thick. It is irregular, with 3-5 distinct sides: one of
these is usually wider, convex and glabrous; the others are
angular and flattened and show the remains of testa hairs
Mobile phase anhydrous formic acid R, water R, ethyl acetate R forming lighter zones in the depressions. The stony granular
(6:9:90 V/V/V). texture resembles that of pebbles from a river bed; the hilum
Application I O µL of the test solution and 5 µL of the is found on the most rounded end and forms a small, light
reference solution, as 10 mm bands. brown depression. The fracture shows a compact, semi-
Development Over a path of 10 cm. translucent, horny endosperm; the embryo is located in the
Drying At 100-105 °C for 10 min. centre and is about 10-15 mm long, with a foliaceous
cotyledon.
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 B. CAUTION: take all necessary handl,ing precautions when
macrogol 400 R in methanol R. Examine the plates after reducing this toxic herbal drug to a powder.
30 min in ultraviolet light at 365 nm. Wash the herbal drug rapidly in cold water, then expose to
Results See below the sequence of the zones present in the steam; once sufficiently softened, cut into thin slices and
chromatograms obtained with the reference solution and the crush in a suitable apparatus. Allow to dry, finish reducing to
test solution. In the chromatogram obtained with the test a powder (710) (2. 9.12) and pass through a covered sieve.
solution, the zone due to rutoside may be weak or even Microscopic examination (2.8.23). The powder is light
absent. The chromatogram obtained with the test solution brown. Examine under a microscope using chloral hydrate
shows a group of zones that may be blue or yellow, with a Rp solution R. The powder shows the following diagnostic
similar to that of the zone due to hyperoside in the characters (Figure 2513.-1): oil droplets [D]; fragments of
chromatogram obtained with the reference solution. Other endosperm [B, C, F] consisting of thick-walled cells of
weak zones may also be visible. various sizes, the smallest located at the periphery of the
endosperm [Cb] and the largest towards the centre of the
Top of the plate seed [F]; a few fragments of the outer layer of the endosperm
A yellow to blue zone
(surface view m, transverse section [Cal), with polygonal
cells sometimes associated with the inner layer of the testa,
Hypericin: a red zone 2 red zones composed of cells with indistinct walls (surface view [E],
transverse section [Cd]); sclerified covering trichomes [A, K],
-- -- sheared off, not enlarged at the base [Aa] and with walls
Several zones composed of small, oblique, sclerified strips, tightly fused
longitudinally [Ab, Ka]; numerous fragments of strips [G, HJ
-- --
and rare rounded tips of covering trichomes [K].
Hyperoside: a yellow to orange zone Blue or yellow zones
Rutoside: a yellow to orange zone A yellow to orange zone
(2. 9.12) add 20 mL of ethanol (70 per cent VIV) R, allow to
macerate for 15 min at room temperature, with stirring, and
Reference solution Test solution centrifuge. Use the supernatant.
TESTS
CAUTION: when the powdered herbal drug (710) (2.9.12) is
used, take the necessary precautions as indicated under
Identification B.
Strychnos nux-vomica L
The presence of flattened discoid seeds and the presence in
the powdered herbal drug, examined under a microscope, of
testa cells transformed into hairs, with a sclerified base and a
lignified tip, bent at a right angle and with 7-10 lignified
ridges, and of numerous sclerified rods, indicate adulteration
with Strychnos nux-vomica L.
105 °C for 2 h.
Total ash (2.4.16)
Aflatoxins (2. 8.18)
Maximum 2 µg/kg (aflatoxin B 1) and maximum 4 µg/kg
(sum of aflatoxins B1, B2 , G 1 and G 2 ).
ASSAY
(2. 9.12) add 10.0 mL of ethanol (60 per cent V!v,> R. Boil
gently, with stirring, under a reflux condenser. After 30 min,
cool and filter into a 20.0 mL volumetric flask. Wash the
Figure 2513.-1. - Illustration for identification test B of powdered filter with ethanol (60 per cent V/V) R and dilute to 20.0 mL
herbal drug of Ignatia with the same solvent. Dilute 1.0 mL of the solution to
20.0 mL with mobile phase A.
Reference solution Dissolve 10 mg of brucine R and 10 mg of
Reference solution Dissolve 10.0 mg of brucine CRS and
strychnine R in 10 mL of ethanol (96 per cent) R.
10.0 mg of strychnine CRS in acetonitrile Rand dilute to
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel 10.0 mL with the same solvent. Dilute 1.0 mL of the
plate R (2-10 µm)]. solution to 20.0 mL with mobile phase A.
Mobile phase concentrated ammonia R, methanol R, methylene Column:
chloride R (1:5:95 V/V/V); use the lower layer. =
- size: l 0.15 m, 0 = 4.6 mm;
Application 10 µL [or 5 µL] as bands. - stationary phase: end-capped ethylene-bridged octadecylsilyl
Development Over a path of 15 cm [or 6 cm]. silica gel for chromatography (hybrid material) R (3.5 µm);
Drying In air, then in an oven at 105-110 °C for 15 min;
allow to cool. Mobile phase:
- mobz1e phase A: triethylamine R, acetonitrile for
Detection Spray with iodoplatinate reagent R and examine
immediately in daylight. chromatography R, methanol R2, tris(hydroxymethyl)
aminomethane buffer solution pH 9. 0 R
(0.1:7.5:7.5:85 VIVIVIV);
chromatograms obtained with the reference solution and the - mobile phase B: triethylamine R, tris(hydroxymethyl)
aminomethane buffer solution pH 9. 0 R, acetonitrile for
in the chromatogram obtained with the test solution. chromatography R, methanol R2
(0.1: 15:42.5:42.5 VIVIVIV);
Top of the plate
-- --
-- -- 0-5 100 0
5 - 25 100 ➔ 70 0 ➔ 30
Strychnine: a violet zone A violet zone (strychnine)
25 - 30 70 ➔ 65 30 ➔ 35
Bru.cine: a blue zone A blue zone (brucine) 30 - 31 65 ➔ 0 35 ➔ 100
31 - 32 0 100

Injection 10 µL.
Elution order Brucine, strychnine.

- resolution: minimum 3.0 between the peaks due to brucine
and strychnine.
area of the peak due to brucine or strychnine in the
Calculate the percentage contents of brucine and strychnine chromatogram obtained with the test solution;
using the following expression: area of the peak due to brucine or strychnine in the
mass of brucine CRS or strychnine CRS used to prepare the
area of the peak due to brucine or strychnine in the p assigned percentage content of brucine in brucine CRS or
chromatogram obtained with the test solution; strychnine in strychnine CRS.
area of the peak due to brucine or strychnine in the
chromatogram obtained with the reference solution; _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
solution, in grams;
mass of brucine CRS or strychnine CRS used to prepare the
p assigned percentage content of brucine in brucine CRS or
strychnine in strychnine CRS. Iron for Homoeopathic
Preparations
MOTHER TINCTURE Iron for Homoeopathic Use
The mother tincture complies with the requirements of the (Ferrum Metallicum for Homoeopathic Preparations, Ph. Bur.
monograph 2026)
preparations (2029). Fe 55.85 7439-89-6
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
The mother tincture is prepared from the dried, ripe seed of DEFINITION
Strychnos ignatii P.J.Bergius. Iron obtained by reduction or sublimation as a fine blackish-
Content grey powder.
0.18 per cent mlm to 0.36 per cent m!m for the sum of the Content
contents of brucine (C 23 H 26N 2 O 4 ; Mr 394.5) and strychnine 97.5 per cent to 101.0 per cent.
(C 21 H 22N 2 O 2; Mr 334.4), of which minimum 65 per cent is
strychnine. CHARACTERS
Appearance
PRODUCTION Fine, blackish-grey powder, without metallic lustre.
The mother tincture is prepared by the following methods
Solubility
prescribed in the general monograph Methods of preparation of
Practically insoluble in water and in ethanol (96 per cent).
homoeopathic stocks and potentisation (2371):
It dissolves with heating in dilute mineral acids.
- method 1.1.8, using the powdered herbal drug (710)
(2.9.12) and ethanol (70 per cent V/V); IDENTIFICATION
- method 1.1.10, using the powdered herbal drug, ethanol Dissolve 50 mg in 2 mL of dilute suljuric acid R and dilute to
(65 per cent V/V) and a maceration time of 3-5 weeks. 10 mL with water R. The solution gives reaction (a) of iron
(2.3.1).
CHARACTERS
Appearance TESTS
Brownish-yellow liquid. Solution S
To 10.0 g add 40 mL of water R. Boil for 1 min. Cool, filter
IDENTIFICATION
and dilute to 50.0 mL with water R.
Thin-layer chromatography (2.2.27) as described in
identification test C for the herbal drug with the following Alkalinity
modification. To 10 mL of solution S add 0.1 mL of bromothymol blue
Test solution The mother tincture to be examined. solution Rl. Not more than 0.1 mL of 0.01 M hydrochloric
acid is required to change the colour of the indicator to
TESTS yellow.
Relative density (2.2.5) Substances insoluble in hydrochloric acid
0.890 to 0.904 when method 1.1.8 is used. Dissolve 2.00 g in 40 mL of hydrochloric acid R. Heat on a
Ethanol (2. 9.10) water-bath. As soon as fumes are no longer evolved, filter
60 per cent V/V to 70 per cent V!V when method 1. 1.10 is through a sintered-glass filter (16) (2. 1.2). Rinse with
used. water R. Dry the residue in an oven at 100-105 °C for 1 h.
Dry residue (2.8.16) The residue weighs a maximum of 20 mg (1.0 per cent).
Minimum 1.2 per cent. Substances soluble in water
ASSAY Evaporate 10.0 mL of solution S on a water-bath and dry at
Liquid chromatography (2.2.29) as described in the assay of 100-105 °C for 1 h. The residue weighs a maximum of 2 mg
the herbal drug with the following modification. (0.1 per cent).
Test solution Dilute 2.000 g of the mother tincture to be Chlorides (2.4. 4')
examined to 20.0 mL with ethanol (60 per cent V/V) R. Maximum 50 ppm.
Calculate the percentage contents of brucine and strychnine 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
Hydrated lron(m) Phosphate for
dilute hydrochloric acid R. Within 30 s lead acetate paper R Homoeopathic Preparations
moistened with water R and placed over the mouth of the
222.8 10045-86-0
flask is not coloured more intensely than light brown by the
(anhydrous)
resulting fumes.
Arsenic (2.4.2) DEFINITION
Maximum 5 ppm. Hydrated Iron(m) Phosphate for Homoeopathic Preparations
contains hydrated iron(m) phosphate. It contains not less
Boil 0.2 gin 25 mL of dilute hydrochloric acid R until than 96.0% and not more than 106.5% ofFePO 4,4H 2 O.
completely dissolved. The solution complies with limit test A.
CHARACTERISTICS
Copper
Maximum 50 ppm. A yellow to pale ochre powder.
Atomic absorption spectrometry (2.2.23, Method[). Insoluble in water; soluble in dilute mineral acids.
Test solution Dissolve 1.00 g in a mixture of 60 mL of dilute IDENTIFICATION
hydrochloric acid R and 10 mL of dilute hydrogen peroxide Dissolve 0.5 g of the substance being examined in 5 mL of
solution R. Reduce to a volume of 5 mL and dilute to dilute hydrochloric acid, with warming. Dilute the resulting
50.0 mL with water R. solution to 35 mL with water and filter if necessary
Reference solutions Prepare the reference solutions using (solution S).
copper standard solution (0.1 per cent Cu) R, diluting with a A. Solution S yields reactions B and C characteristic of iron
1 per cent V/V solution of hydrochloric acid R. and iron salts, Appendix VI.
Source Copper hollow-cathode lamp. B. Solution S yields reaction B characteristic of phosphates,
Wavelength 324.8 nm. Appendix VI.
Flame Air-acetylene. TESTS
Lead Clarity of solution
Maximum 50 ppm. Solution S is clear, Appendix IV A, Method II.
Atomic absorption spectrometry (2.2.23, Method[). Chloride
To 0.05 g of the substance being examined add 1 mL of
Test solution In a separating funnel, place 20 mL of the test
dilute nitric acid. Heat, dilute with 14 mL of water and filter.
solution prepared for the test for copper. Add 25 mL of lead-
The filtrate complies with the limit test for chlorides,
free hydrochloric acid R. Stir with 3 quantities, each of 25 mL,
Appendix VII (0.1%).
of di-isopropyl ether R. Collect the aqueous layer. Add 0.10 g
of sodium sulfate decahydrate R. Evaporate to dryness. Take Heavy metals
up the residue with 1 mL of lead-free nitric acid R and dilute Dissolve 1.0 g of the substance being examined in 20 mL of
to 20 mL with water R. hydrochloric acid if necessary with heating. Extract the solution
Reference solutions Prepare the reference solutions using lead using five 20-mL quantities of a mixture of 100 mL of
standard solution (0.1 per cent Pb) R, diluting with a freshly distilled methyl isobutyl ketone and 1 mL of hydrochloric
10 per cent V/V solution of nitric acid R containing 5 g/L of acid Rl. Allow to stand, separate the aqueous layer and
sodium sulfate decahydrate R. evaporate to half its volume, allow to cool and dilute to
35 mL with water. Neutralise 7.5 mL of this solution to
Source Lead hollow-cathode lamp.
litmus paper using dilute ammonia Rl and dilute to 15 mL
Wavelength 217 nm. with water. 12 mL of the resulting solution complies with
Flame Air-acetylene. limit test heavy metals, Appendix VII, Method A (70 ppm).
ASSAY Use lead standard solution (1 ppm Pb) to prepare the standard.
Stir for 10 min 0.100 gin a hot solution of 1.25 g of copper Loss on drying
sulfate pentahydrate R in 20 mL of water Rina 100 mL When dried to constant weight at 200°, loses not less than
conical flask with a ground-glass stopper. Filter rapidly and 28% and not more than 33% of its weight, Appendix IX D.
wash the filter. Combine the filtrate and the washings, acidify Use 1 g.
with dilute sulfuric acid Rand titrate with 0.02 M potassium ASSAY
permanganate until a pink colour is obtained. Dissolve 0.45 g in 3 mL of hydrochloric acid Rl in an iodine
1 mL of 0. 02 M potassium permanganate is equivalent to flask, add 10 mL of water and 6.0 g of potassium iodide, close
5.585 mg of Fe. the flask and allow to stand protected from light for
LABELLING 30 minutes. Add 100 mL of water and 1 mL of starch solution
The label indicates whether the substance is obtained by and titrate with 0. lM sodium thiosulfate VS. Each mL of
reduction or sublimation. 0.IM sodium thiosulfate VS is equivalent to 22.29 mg of
FePO 4 ,4H2 O.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
PRODUCTION OF STOCK
The first decimal trituration of Hydrated Iron(m) Phosphate
for Homoeopathic Preparations is prepared using a suitable
quantity of Lactose Monohydrate or Lactose as the vehicle
and a validated trituration method that ensures homogeneity
is achieved. The vehicle complies with the statement under
Vehicles in the monograph for Homoeopathic Preparations.
Content of hydrated iron(IIIJ phosphate FePO4 , 4H2 O Sulfates

The first decimal trituration contains 9.0% to 11.0% of Dissolve 0.25 g of the substance being examined in water.
FePO 4 , 4H 2 O. Add 3 mL of dilute hydrochloric acid and dilute to 15 mL with
water. The resulting solution complies with the limit test for
CHARACTERISTICS
sulfates, Appendix VII.
The first decimal trituration is a yellowish powder.
ASSAY
IDENTIFICATION
Dissolve 0.3 g of the substance being examined in 3 mL of
Dissolve, with warming, 1.5 g of the first decimal trituration
orthophosphoric acid and 10 mL of a 14% v/v solution of
in a mixture of 1.5 mL of dilute hydrochloric acid and 9 mL of
sulfuric acid in water. Add 100 mL of water and titrate with
water (solution Sl).
0.lM potassium permanganate. Each mL of 0.lM potassium
A. Solution Sl yields reactions B and C characteristic of iron permanganate VS is equivalent to 27.925 mg of Fe 2+.
and iron salts, Appendix VI.
PRODUCTION OF STOCK
B. Solution Sl yields reaction B characteristic of phosphates,
Appendix VI. The first decimal trituration of Hydrated Iron(n) and Iron(III)
Phosphate for Homoeopathic Preparations is prepared using
C. Dissolve 0.25 g of the substance being examined in 5 mL a suitable quantity of Lactose Monohydrate or Lactose as the
of water. Add 5 mL of ammonia and heat in a water-bath at vehicle and a validated trituration method that ensures
80° for 10 minutes. A red colour develops. homogeneity is achieved. The vehicle complies with the
ASSAY statement under Vehicles in the monograph for
Dissolve 4.0 g of the first decimal trituration in 3 mL of Homoeopathic Preparations.
hydrochloric acid Rl in an iodine flask, add 10 mL of water Content of hydrated iron(II) and iron(III) phosphate
and 8.0 g of potassium iodide, close the flask and allow to The first decimal trituration contains 4.5% to 5.0% of
stand protected from light for 30 minutes. Add 100 mL of Fe3 (PO 4 h, 8H2 O.
water and 1 mL of starch solutwn and titrate with 0.lM sodium
thiosulfate VS. Each mL of 0.lM sodium thwsulfate VS is CHARACTERISTICS
equivalent to 22.29 mg of FePO 4 ,4H2 O. The first decimal trituration is a light grey powder.
STORAGE IDENTIFICATION
Hydrated Iron(rn) Phosphate for Homoeopathic Preparations A. Yields the reactions characteristic of iron and iron salts,
should be protected from light. Appendix VI.
B. Yields the reactions characteristic of phosphates,
Appendix VI.
Hydrated lron(n) and lron(m) Phosphate C. Dissolve 0.25 g in 5 mL of water. Add 5 mL of ammonia
and heat in a water-bath at 80° for 10 minutes. A red colour
for Homoeopathic Preparations develops.
DEFINITION ASSAY
Hydrated Iron(n) and Iron(m) Phosphate for Homoeopathic Dissolve 3.0 g of the first decimal trituration in 3 mL of
Preparations contains a mixture of hydrated iron(n) orthophosphoric acid and 10 mL of a 14% v/v solution of
phosphate and iron(rn) phosphate and some hydrated oxides suljuric acid in water. Add 100 mL of water and titrate with
of iron. It contains not less than 16.0% ofFe 2+, equivalent to 0.lM potassium permanganate. Each mL of O.lM potassium
not less than 47.9% of Fe3 (PO 4)z,8H2O. permanganate VS is equivalent to 27.925 mg ofFe 2 +.
CHARACTERISTICS STORAGE
A slate blue amorphous powder. Hydrated Iron(n) and Iron(m) Phosphate for Homoeopathic
Insoluble in water; soluble in hydrochloric acid. Preparations should be protected from light.
IDENTIFICATION
Dissolve 0.5 gin 5 mL of dilute hydrochf.oric acid with
warming. Dilute the resulting solution to 35 mL with water
and filter if necessary (solution S). Magnesium Fluoratum for
A. Solution S yields the reactions characteristic of iron and Homoeopathic Preparations
iron salts, Appendix VI.
B. Solution S yields the reactions characteristic of phosphates,
Appendix VI. MgF2 62.3 7783-40-6
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
TESTS
Heavy metals DEFINITION
Dissolve 1.0 g of the substance being examined in 20 mL of Content
hydrochloric acid. Extract the solution using five 20-mL 98.5 per cent to 100.5 per cent of MgF2 •
quantities of a mixture of 100 mL of freshly distilled methyl
CHARACTERS
isoburyl ketone with 1 mL of hydrochloric acid Rl. Allow to
Appearance
stand, separate the aqueous layer and evaporate to half its
White or almost white powder or crystals.
volume, allow to cool and dilute to 35 mL with water.
Neutralise 7.5 mL of this solution to litmus paper using dilute Solubility
ammonia Rl and dilute to 15 mL with water. 12 mL of the Practically insoluble in water, very slightly soluble in dilute
resulting solution complies with limit test A for heavy metals, nitric acid and in concentrated sulfuric acid, practically
Appendix VII (70 ppm). Use lead standard solutwn insoluble in anhydrous acetic acid.
(1 ppm Pb) to prepare the standard.
IDENTIFICATION
A. Mix 0.2 g with 2 g of potassium hydrogen sulfate Rina
Magnesium Phosphate for
platinum crucible and melt at 800 °C. Cool, carefully take up Homoeopathic Preparations
the melt in 20 mL of water R and boil briefly. Cool and (Magnesium Phosphoricum for Homoeopathic
filter. Dilute 0.5 mL of the filtrate to 2 mL with water R. Preparations, Ph. Bur. monograph 2505)
The solution gives the reaction of magnesium (2. 3.1).
MgHPO4,3H2 O 174.3 7782-75-4
B. Mix 0.2 g with 1 g of anhydrous sodium carbonate Rina
platinum crucible and melt at 850 °C. Cool, take up the melt
in 10 mL of dilute acetic acid Rand boil briefly. Cool, filter DEFINITION
and dilute the filtrate to 20 mL with water R. To 0.4 mL of Content
this solution add dropwise a mixture of 0 .1 mL of alizarin S 98.0 per cent to 102.0 per cent of MgHPO 4 ,3H2 O.
solution R and 0.1 mL of zirconyl nitrate solution R.
The colour changes from red to yellow. CHARACTERS
Appearance
TESTS White or almost white powder.
Solution S
Boil 5.0 g with 100.0 mL of distilled water R under a reflux Solubility
condenser for about 5 min. Allow to cool, then filter through Very slightly soluble in water, practically insoluble in ethanol
an ashless filter paper (nominal pore size not greater than (96 per cent). It dissolves in dilute acids.
2 µm). IDENTIFICATION
Appearance of solution A. Dissolve 0 .1 g in a mixture of 2 mL of dilute nitric acid R
Solution S is colourless (2.2.2, Method I[). and 8 mL of water R. The solution gives reaction (b) of
phosphates (2.3.1).
Carbonates
Suspend 0.5 gin 5 mL of carbon dioxide-free water Rand add B. Dissolve 0.1 gin dz7ute hydrochloric acid Rand dilute to
5 mL of dilute acetic acid R. Quickly stopper the test tube 3 mL with the same acid. Add ammonia R until a precipitate
with a stopper fitted with a glass tube bent twice through 90° is formed. Dissolve the precipitate by adding dilute
and which dips at the other end in barium hydroxide hydrochloric acid R dropwise until complete dissolution and
solution R. Warm the mixture. The barium hydroxide dilute to 10 mL with distilled water R. To 2 mL of the
solution remains clear. solution add a mixture of 0.5 mL of titan yellow solution R
and 1.5 mL of dilute sodium hydroxide solution R. A red,
Chlorides (2. 4. 4)
flocculent precipitate is formed.
Maximum 100 ppm.
Dilute 10 mL of solution S to 15 mL with water R. TESTS
Solution S
Sulfates (2.4.13) Dissolve 5.0 gin 30 mL of dilute hydrochloric acid Rand
dilute to 50.0 mL with the same acid.
Water-soluble matter
Arsenic (2.4.2, Method B)
Maximum 5 ppm, determined on 1.0 g.
Boil 2.00 g with 100 mL of water R for 5 min. Filter the hot
Chlorides (2. 4. 4)
solution, then cool the filtrate and dilute to 100 mL with
Maximum 200 ppm.
water R. Evaporate 50 mL of this solution to dryness in an
evaporating dish and dry at 100-105 °C. The residue weighs Dissolve 0.25 g in 5 mL of dilute nitric acid R and dilute to
a maximum of 5 mg. 15 mL with water R.
ASSAY Magnesium dihydrogen phosphate and magnesium
Thoroughly mix 0.100 g with 2 g of potassium hydrogen
phosphate
Dissolve 2.00 g in 30.0 mL of 1 M hydrochloric acid.
sulfate R in a platinum crucible and melt at 800 °C. Allow to
Add 20 mL of water Rand 0.05 mL of methyl orange
cool, carefully take up the melt in 10 mL of dilute hydrochloric
solution R. Titrate the excess of hydrochloric acid with 1 M
acid Rand heat to dissolve. Dilute the solution to 100.0 mL
sodium hydroxide. The volume of 1 M hydrochloric acid used is
with water R. Transfer 50.0 mL of this solution to a 500 mL
between 11.0 mL and 12.5 mL.
conical flask and dilute to 300 mL with water R. Carry out
the complexometric titration of magnesium (2. 5.11). Sulfates (2. 4.13)
1 mL of 0.1 M sodium edetate is equivalent to 6.23 mg of Maximum 300 ppm.
MgFz. Dilute 5 mL of solution S to 15 mL with distilled water R.
- - - - - - - - - - - - - - - - - - - - ~ Ph Eur Iron (2.4.9)
Maximum 50 ppm.
Dilute 2 mL of solution S to 10 mL with water R.
ASSAY
Dissolve 0.280 g in a mixture of 1 mL of hydrochloric acid RI
and 5 mL of water R. Add 25.0 mL of 0.1 M sodium edetate
and dilute to 200 mL with water R. Neutralise with
concentrated ammonia R, add 10 mL of ammonium chloride
buffer solution pH 10. 0 R and about 50 mg of mordant black
11 triturate R. Titrate the excess of 0.1 M sodium edetate with
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
MgHPO 4 ,3H 2 O. Under ultra-violet light, the chromatogram obtained with
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
Medicago Sativa for Homoeopathic by one or two blue bands approximately level with
formononetin and a red band between the bands obtained
Preparations for formononetin and coumarin in solution (2). Other bands
DEFINITION may be present in the chromatogram obtained with
Medicago Sativa for Homoeopathic Preparations is the fresh solution ( 1).
whole flowering plant of Medicago sativa L.
Top of the olate
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. Coumarin: a blue band
Flowers Compact, axillary, racemes of up to 40 flowers,
peduncle up to 3 cm long, typically pubescent; corolla Red fluorescent band
papilionaceous, up to 1 cm long, 5 mm broad, purple to
whitish; calyx tube approximately 5 mm in length, 2 mm in
diameter, 5-lobed, typically glabrous, lobes equal or One or two blue fluorescent
subequal, up to 4 mm long. 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 band
following requirements.
Blue or pink fluorescent band
PRODUCTION
The mother tincture of Medicago sativa L. is prepared from Solution (1) Solution (2)
the herbal drug using method 1.1. 5 described in the
Stocks and Potentisation. Use 86% w/w (90% v/v) of ethanol. The chromatogram obtained with solution (1) sprayed with
the anisaldehyde solution shows the following fluorescent
CHARACTERISTICS bands: a faint blue band close to the origin, followed by a
The mother tincture is a greenish-brown liquid. 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 chromatography, by three yellow to orange bands between the bands obtained
Appendix III A, using the following solutions. for formononetin and coumarin in solution (2) and an orange
band between the band obtained for coumarin and the
(1) The mother tincture.
solvent front. Other bands may be present in the
(2) 0.1 % w/v coumarin BPCRS and 0.05% w/v of chromatogram obtained with solution (1).
formononetin BPCRS in methanol.
(a) Use as the coating silica gel 60 F254 (M.erck silica gel 60
precoated plates are suitable).
(e) After removal of the plate dry in air and examine under
ultra-violet light (365 nm).
(f) Spray the plate with anisaldehyde solution and heat at 105°
for 5 minutes and examine under ultra-violet light (365 nm).
MOBILE PHASE
10 volumes of methanol and 90 volumes of toluene.
SYSTEM SUITABILITY
with solution (2), two clearly separated bands are observed
Top of the plate located in the central cavity, is small (about 6 mm long),
with 2 cotyledons; the radicle is turned towards the
micropyle.
Orange fluorescent band
B. CAUTION: take all necessary handling precautwns 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,
Coumarin: a faint blue band finish reducing to a powder (710) (2.9.12) and pass through
a covered sieve.
Microscopic examination (2.8.23). The powder is grey.
Yellow orange fluorescent
band
Faint orange fluorescent band (Figure 2514.-1): fragments of the outer testa (surface
view [BJ, transverse section [Cl), with cells with an enlarged,
Formononetin: a yellow sclerified, strongly thickened and channelled base [Ba, Cb],
Orange fluorescent band fluorescent 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 µm 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
aleurone grains; a few fragments of the outer layers of the
Faint blue band
endosperm (surface view [E], transverse section [Cl);
in surface view [E] the cells are polyhedral [Ea], sometimes
Solution (1) Solution (2) 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
associated with the pigmented layer [Cd] and the outer testa
The mother tincture is a greenish-brown liquid.
[Cb].
TESTS
Ethanol
Dry residue
Not less than 1.0% w/w, Appendix XI P.
Relative density
Nux-vomica for Homoeopathic

Preparations
DEFINITION
Dried, ripe seed of Strychnos nux-vomica L.
Content
Minimum 1.50 per cent for the sum of the contents of
brucine (C 23H 2 6N204; Mr 394.5) and strychnine
(C 21 H 22 N 2 O 2 ; Mr 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 identificatwn test B of powdered
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
on the edge of the seed (the micropyle). A grey horny C. Thin-layer chromatography (2.2.27).
endosperm makes up most of the seed. The embryo, which is
Test solution To 2.0 g of the powdered herbal drug (710) with the same solvent. Dilute 1.0 mL of the solution to
(2. 9.12) add 20 mL of ethanol (70 per cent V/V) R, allow to 20.0 mL with mobile phase A.
macerate for 15 min at room temperature, with stirring, and Reference solution Dissolve 10.0 mg of brucine CRS and
centrifuge. Use the supernatant. 10.0 mg of strychnine CRS in acetonitrile Rand dilute to
Reference solution Dissolve 10 mg of brucine R and 10 mg of 10.0 mL with the same solvent. Dilute 1.0 mL of the
strychnine R in 10 mL of ethanol (96 per cent) R. solution to 20.0 mL with mobile phase A.
plate R (2-10 µm)]. =
- size: l 0.15 m, 0 4.6 mm; =
Mobile phase concentrated ammonia R, methanol R, methylene - stationary phase: end-capped ethylene-bridged octadecylsilyl
chloride R (1:5:95 VIVIV); use the lower layer. silica gel for chromatography (hybrid material) R (3.5 µm);
Application 10 µL [or 5 µL] 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; chromatography R, methanol R2, tris(hydroxymethyl)
allow to cool. aminomethane buffer solution pH 9.0 R
Detection Spray with iodoplatinate reagent R and examine (0.1:7.5:7.5:85 VIVIVIV);
immediately in daylight. - mobile phase B: triethylamine R, tris(hydroxymethyl)
Results See below the sequence of zones present in the aminomethane buffer solution pH 9. 0 R, acetonitrile for
chromatograms obtained with the reference solution and the 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
0-5 100 0
-- -- 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)
Injection 10 µL.
Elution order Brucine, strychnine.
TESTS System suitability Reference solution:
When the powdered herbal drug (710) (2.9.12) is used, take - resolution: minimum 3.0 between the peaks due to brucine
the necessary precautions as indicated under Identification B. and strychnine.
Foreign matter (2.8.2) Calculate the percentage content of brucine and strychnine
Maximum 1.0 per cent. using the following expression:
Strychnos ignatii P .J. Bergius
The presence of irregularly shaped seeds that are neither
discoid nor flattened and the presence in the powdered
herbal drug, examined under a microscope, of sclerified hairs,
A1 area of the peak due to brucine or to strychnine in the
usually sheared off, not thickened at the base and with walls chromatogram obtained with the test solution;
composed of small, oblique, sclerified strips, tightly fused A2 area of the peak due to brucine or to strychnine in the
longitudinally, indicate adulteration with Strychnos ignatii P.J. chromatogram obtained with the reference solution;
Bergius. m1 mass of the herbal drug to be examined used to prepare the test
solution_, in grams;
Loss on drying (2.2.32) m2 mass of brncine CRS or of strychnine CRS used to prepare the
Maximum 10.0 per cent, determined on 1.00 g of the reference solution, in grams;
p assigned percentage content of brucine in brucine CRS, or
strychnine in strychnine CRS.
105 °C for 2 h.
Total ash (2.4.16)
Maximum 3.0 per cent. MOTHER TINCTURE
Aflatoxins (2. 8.18) The mother tincture complies with the requirements of the
Maximum 2 µglkg (aflatoxin B 1) and maximum 4 µglkg general monograph Mother tinctures for homoeopathic
(sum of aflatoxins B 1, B2, G 1 and G 2). preparations (2029).
ASSAY DEFINITION
Liquid chromatography (2.2.29). The mother tincture is prepared from the dried ripe seed of
Test solution To 1. 000 g of the powdered herbal drug (710) Strychnos nux-vomica L.
(2. 9.12) add 10.0 mL of ethanol (60 per cent V/V) R. Boil Content
gently, under a reflux condenser, with stirring. After 30 min, 0.15 per cent mlm to 0.30 per cent mlm for the sum of the
cool and filter into a 20.0 mL volumetric flask. Wash the contents of brucine (C2 3H 26N 2 O4 ; M, 394.5) and strychnine
filter with ethanol (60 per cent V/V) Rand dilute to 20.0 mL (C 21 H 22N 2 O 2 ; M, 334.4), of which 43 per cent to
67 per cent is strychnine.
PRODUCTION IDENTIFICATION
The mother tincture is prepared by the following methods A. Relative density (see Tests).
prescribed in the general monograph Methods of preparation of B. Refractive index (see Tests).
homoeopathic stocks and potentisation (2371): C. Distillation range (see Tests).
- method 1.1.8, using the powdered herbal drug (710)
(2. 9.12) and ethanol (70 per cent V/V); TESTS
- method 1.1.10, using the powdered herbal drug, ethanol Solution S
(65 per cent V/V) and a maceration time of 3-5 weeks. Dissolve 1 g in 99 g of ethanol (90 per cent Vlv'.) R.
CHARACTERS Appearance of solution
Appearance Solution S is clear (2.2.1) and colourless (2.2.2, Method 11).
Yellow or light brown liquid. Acidity or alkalinity
To 10 mL of the substance to be examined, add 5 mL of
IDENTIFICATION
carbon dioxide-free water R and 0.25 mL of phenol red
Thin-layer chromatography (2.2.27) as described in
solution Rand shake. The aqueous phase is yellow.
identification test C for the herbal drug with the following
Add 0.05 mL of 0.01 M sodium hydroxide and shake.
modification.
The aqueous phase is red.
Test solutwn The mother tincture to be examined.
TESTS 0.752 to 0.762.
Relative density (2.2.5) Refractive index (2.2.6)
0.888 to 0.903 when method 1.1.8 is used. 1.422 to 1.426.
Ethanol (2.9.10) Distillation range (2. 2.11)
60 per cent V/Vto 70 per cent V/Vwhen method 1.1.10 is Minimum 90 per cent m/m distils between 180 °C and
used.
220 °C.
Dry residue (2.8.16) Aromatic hydrocarbons
Minimum 1.0 per cent. The absorbance (2.2.25) of solution S between 250 nm and
ASSAY 400 nm is not greater than 0.100.
Liquid chromatography (2.2.29) as described in the assay of Non-volatile matter
the herbal drug with the following modifications. Maximum 0.04 per cent.
Test solutwn Dilute 2.000 g of the mother tincture to be Evaporate 5.000 g to dryness on a water-bath and dry the
examined to 20.0 mL with ethanol (60 per cent V/v'.) R. residue in an oven at 100-105 °C for 2 h. The residue weighs
Calculate the percentage content of brucine and strychnine a maximum of 2.0 mg.
STORAGE
LABELLING
The label states that the substance is obtained by
area of the peak due to brucine or to strychnine in the
rectification.
area of the peak due to brucine or to strychnine in the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
mass of brucine CRS or of sirychnine CRS used to prepare the
p
assigned percentage content of brucine in brucine CRS, or
Potassium Dichromate for
strychnine in sirychnine CRS. Homoeopathic Preparations
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
(Kalium Bichomicum for Homoeopathic Preparatwns,
294.2 7778-50-9
PhE~ - - - - - - - - - - - - - - - - - - - ~
Petroleum Rectificatum for DEFINITION
Homoeopathic Preparations Content
99.0 per cent to 101.0 per cent ofK2 Cr2 07.
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ CHARACTERS
Appearance
DEFINITION Orange crystals.
Petroleum spirit distilling between 180 °C and 220 °C Solubility
obtained by rectification of crude oil. Freely soluble in water, practically insoluble in ethanol
CHARACTERS (96 per cent).
Appearance IDENTIFICATION
Clear, colourless, highly flammable liquid. A. It gives reaction (b) of potassium (2.3.1).
Solubility B. Dissolve 10 mg in 5 mL of water R. Add 0.25 mL of
Practically insoluble in water, freely soluble in acetone, dilute sulfuric acid R, 0.5 mL of strong hydrogen peroxide
sparingly soluble in ethanol (96 per cent).
solution R and I mL of ether R. Shake. The upper layer is CHARACTERISTICS

blue. The mother tincture is a purple-red to red, clear to slightly
TESTS turbid liquid.
Solution Sl IDENTIFICATION
Dissolve 5.0 gin distilled water Rand dilute to 50.0 mL with Carry out the method for thin-layer chromatography,
the same solvent. Appendix III A, using the following solutions.
Solution S2 (1) Centrifuge 10 mL of the mother tincture at about 3000
To 20.0 mL of solution SI add 20 mL of hydrochloric acid R rpm for 5 minutes. Precondition a cartridge containing
and 50 mL of tributyl phosphate R. Stir for 2 min. Remove octadecyl-bonded silica sorbent (a Sep-pak C18 cartridge is
the lower layer and shake it with 10 mL of ether R. Evaporate suitable) with 10 mL of methanol followed by 10 mL of
the lower layer to dryness under reduced pressure. Dissolve water. Apply 4.5 mL of the clear supernatant to the column,
the residue in 10 mL of distilled water R. Add dilute wash with 5 mL water and elute with 5 mL of methanol.
ammonia Rl until the solution is neutral to blue litmus paper R Evaporate the eluant on a rotary evaporator at 50°. Dissolve
and dilute to 20.0 mL with distilled water R. the residue in 1 mL of methanol.
Appearance of solution (2) 0.025% w/v of chlorogenic acid and 0.005% w/v of
Solution SI is clear (2.2.1). scopoletin in methanol.
Calcium (2.4.3) CHROMATOGRAPHIC CONDITIONS
Maximum 500 ppm. (a) Use as the coating silica gel F2 s4 .
Dilute 2.0 mL of solution S2 to 15 mL with distilled water R. (b) Use the mobile phase described below.
Chlorides (2.4.4) (c) Apply 40 µL of each solution as 12 mm bands.
Maximum 50 ppm. (d) Develop the plate to 15 cm.
Dissolve 1.0 gin 15 mL of dilute nitric acid R. Use 1 mL of (e) After removal of the plate, dry it in air and spray with a
nitric acid R instead of the prescribed dilute nitric acid R. 1% w/v solution of diphenylboric acid aminoethyl ester in
Sulfates (2.4.13) methanol, followed by a 5% w/v solution of polyethylene glycol
Maximum 150 ppm. 400 in methanol and examine under ultraviolet light (365 nm).
Dilute 10 mL of solution S2 to 15 mL with distilled water R. MOBILE PHASE
ASSAY 15 volumes of anhydrous formic acid, 15 volumes of water and
Dissolve 0.100 gin 25 mL of water R. Add 2 g of potassium 70 volumes of ethyl acetate.
iodide R and 25 mL of dilute sulfuric acid R. Allow to stand in SYSTEM SUITABILITY
the dark for 10 min. Add 150 mL of water R. Titrate with The test is not valid unless the chromatogram obtained with
0.1 M sodium thiosulfate until the colour changes from blue to solution (2) shows a yellow-green fluorescent band
green, adding 1 mL of starch solution R near the end of the (chlorogenic acid) in the middle third of the plate and a blue
titration. fluorescent band (scopoletin) in the upper third of the plate.
1 mL of 0.1 M sodium thiosulfate is equivalent to 4.903 mg of
CONFIRMATION
K2Cr2O1.
The chromatogram obtained with solution (1) shows at least
- - - - - - - - - - - - - - - - - - - - - PhEur
one orange fluorescent band below chlorogenic acid, one
yellow-green fluorescent band with the Rf of chlorogenic acid
and one orange fluorescent band below scopoletin.
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
of scopoletin. Two or more orange fluorescent bands may be
DEFINITION 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 A yellow-green Chlorogenic acid: a
thick. The stone bulges slightly and has a sharp edge. fluorescent band yellow-green fluorescent
band
An orange
MOTHER TINCTURE fluorescent band

following requirements.
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 TESTS
monograph for Methods of Preparation of Homoeopathic Ethanol
Stocks and Potentisation. Use 43% w/w (50% v/v) of ethanol. 24 to 34% w/w (29.3 to 40.8% v/v), Appendix VIII F.
Dry residue
Relative density
Sanguinaria for Homoeopathic

Preparations
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Dried rhizome of Sanguinaria canadensis L., collected from
fully developed plants before dormant phase (i.e. in autumn).
Content
Minimum 1.4 per cent for the sum of sanguinarine and
chelerythrine, expressed as chelerythrine (C 21 H 18NO4 +;
IDENTIFICATION
A. The rhizome consists of large, irregular cylindrical, hard,
knotty fragments, 3-10 cm long and 6-12 mm wide, usually
unbranched and with slightly tapered ends; some fragments
are flattened and tortuous, others are tubercle-shaped; the
surface is reddish-brown, sometimes dark, battered, wrinkled
and distinctly annulated. The lower surface shows rounded
scars left by the roots. The fracture is smooth, spongy, light
Figure 2687 .-1. - lllustratwn for identificatwn test B of powdered
brown to orange, with red and brown zones in the middle (in
herbal drug of sanguinaria for homoeopathic preparations
places). The adventitious roots are spindly and about 1 mm
in diameter. Examined under a lens ( x 6), the entire surface Drying In air.
is scattered with glossy red dots, sometimes appearing a dark
reddish-brown all over.
solutwn R. The powder shows the following diagnostic
characters (Figure 2687.-1): numerous fragments of cellulose
test solution.
parenchyma consisting of ovoid cells with spaces between
them [CJ; fragments of parenchyma containing laticiferous
vessels with slightly and regularly thickened cell walls and Top of the plate
reddish granular contents (longitudinal section [BJ);
fragments of reticulate or pitted xylem vessels [D, E], some
of which consist of short elements [F]; rare fragments of cork - - --
consisting of polyhedral cells with dark brown walls (surface Cheleiythrine: a yellow fluorescent A yellow fluorescent zone
view [A]). Examine under a microscope using a zone (cheleiythrine)
Sanguinarine: an orange fluorescent An orange fluorescent zone
very numerous starch granules, either single or 2-3 zone (sanguinarine)
compound, ovoid or rounded, up to 18-20 µm in diameter,
free [G] or within parenchyma cells. - - --

Test solutwn Introduce 1 g of the powdered herbal drug Reference solution Test solution
(355) (2.9.12) into a round-bottomed flask fitted with a
reflux condenser and add 10 mL of ethanol
(65 per cent VIV) R. Heat on a water-bath at 60 °C for TESTS
15 min. Allow to cool and filter. Loss on drying (2.2.32)
Reference solutwn Dissolve 5 mg of chelerythrine chloride R Maximum 11.0 per cent, determined on 1.000 g of the
and 5 mg of sanguinarine chloride R in ethanol (96 per cent) R powdered herbal drug (355) (2.9.12) by drying in an oven at
and dilute to 10 mL with the same solvent. 105 °C for 2 h.
Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel Total ash (2.4.16)
plate R (2-10 µm)]. Maximum 6.0 per cent.
1\1obile phase Jonnie acid R, water R, ethyl acetate R Ash insoluble in hydrochloric acid (2.8.1)
(10:10:80 V/V/V). Maximum 2.0 per cent.
Applicatwn 5 µL [or 2 µL] as bands of 20 mm [or 10 mm]. ASSAY
Development Over a path of 10 cm [or 6 cm]. Liquid chromatography (2.2.29).
Solution A Dissolve 10.0 mg of chelerythrine chloride CRS in MOTHER TINCTURE

Test solution Place 2.500 g of the powdered herbal drug general monograph Mother tinctures for homoeopathic
(355) (2.9.12) in a 250 mL round-bottomed flask. preparations (2029).
Add 40 mL of methanol R and heat under a reflux condenser
on a water-bath at 75 °C for 1 h. Allow to cool. Filter DEFINITION
through a plug of absorbent cotton into a 100 mL volumetric The mother tincture is prepared from the dried rhizome of
flask. Take up the residue with 40 mL of methanol R and Sanguinaria canadensis L., collected from fully developed
heat again under a reflux condenser on a water-bath at 75 °C plants before dormant phase (i.e. in autumn).
for 1 h. Allow to cool. Filter through the same plug of PRODUCTION
absorbent cotton into the volumetric flask. Rinse the round- The mother tincture is prepared according to the following
bottomed flask and the filter with methanol R, add the methods as prescribed in the monograph Methods of
rinsings to the volumetric flask and dilute to 100.0 mL with preparation of homoeopathic stocks and potentisation (2371):
methanol R. Dilute 2.0 mL of the solution to 20.0 mL with - method 1.1.8, using the powdered herbal drug (710)
ethanol (60 per cent V/V) R. (2. 9. 12) and ethanol (70 per cent V/V);
Reference solution (a) Dilute 10.0 mL of solution A to - method 1. 1.10, using the fragmented herbal drug and
20.0 mL with methanol R. ethanol (65 per cent V/V);
Reference solution (b) Dissolve 5 mg of sanguinarine - method 1.2.12, using the powdered herbal drug (710)
chwride R in methanol R and dilute to 100 mL with the same (2. 9.12) and ethanol (70 per cent V/V).
solvent. Dilute 10 mL of the solution to 20 mL with CHARACTERS
solution A. Appearance
Column: Brownish-red liquid.
- size: l = 0.25 m, 0 = 4.6 mm;
- stationary phase: end-capped octadecylsilyl silica gel for IDENTIFICATION
chromatography R (5 µm); Thin-layer chromatography (2. 2. 27) as described in
- temperature: 30 °C. identification test C for the herbal drug with the following
modification.
Mobile phase:
- mobile phase A: 0.2 per cent V/V solution of phosphoric Test solution The mother tincture to be examined.
acid R; TESTS
- mobile phase B: methanol R; Relative density (2.2.5)
0.890 to 0.905 when methods 1.1.8 and 1.2.12 are used.
(min) (per cent VIP) (per cent VIP)
Ethanol (2. 9.10)
60 per cent V/Vto 70 per cent V/Vwhen method 1.1.10 is
0-5 70 30
used.
5 - 25 70---+ 0 30---+ 100
25 - 30 0 100 Dry residue (2.8.16)
Flow rate 1.0 mUmin. Sanguinarine and chelerythrine
Detection Spectrophotometer at 275 nm. Liquid chromatography (2.2.29) as described in the assay of
the herbal drug with the following modifications.
Injection 5 µL.
Test solution Dilute 1.000 g of the mother tincture to be
Relative retention With reference to sanguinarine (retention examined to 20.0 mL with ethanol (60 per cent Vii] R.
time= about 15.7 min): chelerythrine = about 1.1.
Calculate the percentage content of the sum of sanguinarine
System suitability Reference solution (b): and chelerythrine, expressed as chelerythrine, using the
- resolution: minimum 2.0 between the peaks due to following expression:
sanguinarine and chelerythrine;
- symmetry factor. maximum 2.0 for the peaks due to (Ai +A2) x m2 x 0.1 xp
sanguinarine and chelerythrine.
A3 xm1
Calculate the percentage content of the sum of sanguinarine
and chelerythrine, expressed as chelerythrine, using the A1 area of the peak due to sanguinarine in the chromatogram
following expression: obtained with the test solution;
A2 area of the peak due to chelerythtine in the chromatogram
(A1 +A2)xm2x5xp A3 area of the peak due to chelerythrine in the chromatogram
A 3 xm 1 obtained with reference solution (a);
m1 mass of the mother tincture to be examined used to prepare the
area of the peak due to sanguinarine in the chromatogram test solution, in grams;
obtained with the test solurion; m2 mass of chelerythrine chloride CRS used to prepare solution A, in
area of the peak due to chelerythrine in the chromatogram grams;
obtained with the test solution; p percentage content of chelerythtine in chelerythrine chloride CRS.
area of the peak due to chelerythrine in the chromatogram
obtained with reference solution (a); Limit Maximum 0.5 per cent m/m for the sum of
sanguinarine and chelerythrine, expressed as chelerythrine
solution, in grams;
mass of chelerythrine chloride CRS used to prepare solution A, in (C21H1sNO4 +; Mr 348.4).
grams;
LABELLING
p percentage content of chelerythtine in chelerythrine chloride CRS.
When the mother tincture is prepared according to
method 1.2.12 prescribed in the monograph Methods of
preparation of homoeopathic stocks and potentisation (2371), acid R, and add 5.0 mL of a freshly prepared 1 g/L solution
'ethanolicum decoctum' must be mentioned in the Latin title. of 2,3-naphthalenediamine R in 0.1 M hydrochloric acid. Dilute
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur to 50 mL with water Rand allow to stand, protected from
light, for 2 h. Shake the mixture with 10 mL of toluene R for
3 min and allow the layers to separate for 3 min. Isolate the
organic layer and dilute to 10.0 mL with toluene R.
Selenium for Homoeopathic Reference solution Mix 5.0 mL of selenium standard solution
(1 ppm Se) Rand 25 mL of water R, and prepare in the same
Preparations manner as for the filtrate in the test solution.
(Ph. Bur. monograph 2844) Compensation liquid Treat 30 mL of water R in the same
manner as for the filtrate in the test solution.
Se 79.0 7782-49-2
Measure the absorbance (2.2.25) of the test solution and the
reference solution at 380 nm by comparison with the
DEFINITION compensation liquid.
Amorphous selenium. The absorbance of the test solution is not greater than that of
Content the reference solution.
98.0 per cent to 101.0 per cent of Se (dried substance). Iron (2.4. 9)
CHARACTERS Maximum 100 ppm.
Appearance To 5 mL of solution S2 add 5 mL of water R.
Black, reddish-brown or red powder, softens at 50 °C to Loss on drying (2.2.32)
60 °C. Maximum 1.0 per cent, determined on 1.000 g by drying in
Solubility an oven at 105 °C for 1 h.
Practically insoluble in water, soluble in concentrated sulfuric Sulfated ash (2. 4. 14)
acid giving a green solution. Maximum 0.3 per cent, determined on 1.000 g.
IDENTIFICATION ASSAY
A. Warm 5 mL of solution SI (see Tests) on a water-bath Dissolve 0.2000 gin 10 mL of nitric acid R. Warm until the
and add 50 mg of hydrazine sulfate R. A red colour is evolution of brown fumes has ceased and the solution is only
produced. slightly yellow. Cool, add 2 g of sulfamu acid R and dilute to
B. To 5 mL of solution Sl add 0.5 mL of dilute hydrochwric 100.0 mL with water R. To 10.0 mL of this solution add
acid R and 2 mL of a 100 g/1. solution of thiourea R. A red 50 mL of water R and 2.0 g of potassium iodide R. Titrate
precipitate is formed. with 0.1 M sodium thiosulfate, determining the end-point
potentiometrically (2.2.20).
TESTS
Solution Sl 1 mL of 0.1 M sodium thiosuljate is equivalent to 1. 97 4 mg of
Dissolve 20 mg in 1 mL of nitric acid R, warming on a water- Se.
bath. Continue warming until the evolution of brown fumes
has ceased, then cool and add 10 mL of water R.
Solution S2
To the residue obtained in the test for sulfated ash add 2 mL
of nitn"c acid R and 3 mL of hydrochloric acid R and evaporate Sodium Tetrachloroaurate
to dryness on a water-bath. Take up the residue in a mixture
of 1 mL of dilute hydrochwric acid R and 25 mL of hot
Dihydrate for Homoeopathic
water R. Cool, filter and dilute to 50.0 mL by washing the Preparations
filter with water R. (Aurum Chwratum Natronatum for Homoeopathic
Appearance of solution Preparations, Ph. Bur. monograph 2141)
Solution S2 is clear (2.2.1) and colourless (2.2.2, Method II).
397.8 13874-02-7
Sulfur PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
To 0.100 g add 1 mL of nitric acid R dropwise, then add DEFINITION
2 mL of hydrochloric acid R and evaporate to dryness on a Sodium tetrachloroaurate( 1-) dihydrate.
water-bath. Add 2 mL of hydrochloric acid R and evaporate Content
again to dryness on a water-bath. Dissolve the residue in 97.0 per cent to 101.0 per cent ofNa[AuC4l,2H2 0.
6 mL of a mixture of 1 mL of dilute hydrochloric acid R and
CHARACTERS
30 mL of distilled water R, then filter and dilute the filtrate to
Appearance
20 mL by washing the filter with distilled water R. Dilute
Orange-yellow, hygroscopic powder or crystals.
5 mL of this solution to 15 mL with distilled water R.
The solution complies with the limit test for sulfates (2.4.13) Solubility
(0.6 per cent of sulfate corresponds to 0.2 per cent of sulfur). Very soluble or freely soluble in water and in ethanol
(96 per cent).
Water-soluble selenium compounds
Maximum 5 ppm. IDENTIFICATION
Test solution Shake 1.0 g with 20 mL of water R for 30 min A. Dissolve 20 mg in 2.0 mL of 0.1 M nitric acid. Add 0.1 g
and filter, washing the filter with 2 quantities, each of 5 mL, of oxalic acid R and boil in a water-bath for 1 h. A deposit of
of water R. Adjust the filtrate to pH 2 with dilute hydrochloric metallic gold is formed.
B. Solution S (see Tests) gives reaction (a) of chlorides B. Microscopic examination (2.8.23). Reduce to a powder
(2.3.1). (710) (2. 9.12). The powder consists of a mixture of dark
C. Solution S gives reaction (b) of sodium (2.3.1). brown and white particles with oily constituents. Examine
TESTS The powder shows the following diagnostic characters
Solution S (Figure 2289.-1): fragments of the episperm consisting of a
Ignite 0.20 g in a porcelain crucible at 600 °C ± 50 °C for layer of large cells with regularly thickened walls and a wide
30 min. Allow to cool and extract with 3 mL of water R, lumen, elongated (surface view [Al), more or less
heating if necessary. Use the supernatant. isodiametric (transverse section [D]); the outside surface of
Free hydrochloric acid the episperm cells generally exhibits numerous short,
When a glass rod impregnated with concentrated ammonia R is unequal, irregularly bent, cylindrical papillae, often with a
held close to the substance to be examined, no white fumes globoid head, that look like cuticular papillae but are the
are produced. conidiophores of a fungus present in almost every seed
Nitrates [Aa, Da]; underlying the episperm is an intermediate area,
Maximum 200 ppm. partly collapsed, composed of 5-6 layers of tangentially
elongated, colourless, thin-walled cells [Cd, Db]; fragments
Dissolve 0.20 g in 4.0 mL of nitrate-free water R. Add 0.6 g
of the inner testa, usually associated with the endosperm
of zinc R and 10 mL of dilute sulfuric acid R. Heat the
[B, C]; the inner testa comprises brown cells that, in
solution on a water-bath for 30 min, allow to cool and filter.
transverse section, form a layer and are regular, 4-7 µm wide,
Rinse the filter with nitrate-free water R and dilute the filtrate
with numerous U-shaped thickenings [Ca], whereas in
to 20.0 mL with the same solvent. To 5.0 mL of this
surface view [Ba] they are elongated and about 70-100 µm
solution add 0.4 mL of a 100 g/1. solution of potassium
long, with thin, finely striated walls; the endosperm consists
chloride R, 0.1 mL of diphenylamine solution Rand, dropwise
of pentagonal or hexagonal cells, isodiametric or slightly
with shaking, 5.0 mL of nitrogen-free sulfuric acid R. Heat in a
elongated, with straight, thin walls [Bb, Cb], containing
water-bath at 50 °C for 15 min, centrifuge if necessary and
aleurone grains and oil droplets [Be, Cc].
use the clear supernatant. Any blue colour in the solution is
not more intense than that in a reference solution prepared at
the same time and in the same manner using a mixture of <.
1. 0 mL of nitrate standard solution (10 ppm NOJ) R and
, ~.".)
3.0 mL of nitrate-free water R. / .
0
~:. a
•
,,,~ . _, i" -:,
ASSAY /
~v
C o
t. r- C .,
Dissolve 40. 0 mg in 10 mL of potassium iodide solution R. C

';;, "' ,., ~
• .
/'
Allow to stand for 5 min. Titrate with 0.01 M sodium c. J / ,:,
C, \ __ ,· _,,,/ :· .~
thiosulfate until decolourised. Shortly before reaching the .. ,j
endpoint, add 0.5 mL of starch solution R.

1 mL of 0.01 M sodium thiosulfate is equivalent to 1.989 mg
of Na[AuC1 4],2H2O.
STORAGE
In an airtight container, protected from light.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
'p ..
Staphysagria for Homoeopathic D
Preparations
(Ph. Bur. monograph 2289) ~a
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION Figure 2289.-1. - Illustration for identification test B of powdered

Dried, ripe seed of Delphinium staphisagria L. herbal drug of Staphysagria for homoeopathic preparations
Content
Minimum 1.5 per cent of total alkaloids, expressed as C. Thin-layer chromatography (2.2.27).
delphinine (C33H4sNO9; Mr 599.7) (dried drug). Test solution To 1.0 g of the powdered herbal drug (710)
(2. 9.12) add 10 mL of ethanol (90 per cent V/V) R and boil
IDENTIFICATION under a reflux condenser for 15 min. Allow to cool and filter.
A. The isolated seed has an irregular, flattened, tetrahedral
Reference solution Dissolve 10 mg of hyoscine hydrobromide R
shape, measuring 3-7 mm on each side; one of the sides is
and I 0 mg of papaverine hydrochloride R in ethanol
convex; the others are flat or concave. The surface is
(90 per cent VIJ,J R and dilute to 50 mL with the same
generally dark brown, rarely light brown, and shows a raised
solvent.
network defining alveoli; on the surface there are occasionally
greyish spots. The seed coat is brittle and encloses an Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
abundant endosperm, which is whitish to dark brown. plate R (2-10 µm)].
The more or less wrinkled seeds are sometimes found as 5-6 Mobile phase concentrated ammonia R, anhydrous ethanol R,
seeds fitting together into an ovoid mass. acetone R, toluene R (3:7:45:45 VIVIV/V).
Development Over a path of 10 cm [or 6 cm]. PRODUCTION

Drying In air. The mother tincture is prepared from the dried seed of
Detection Treat with dilute potassium iodobismuthate Delphinium staphisagria L. according to the following methods
solution R, then with sodium nitrite solution R until the yellow prescribed in the monograph Methods of preparation of
background disappears; examine in daylight. homoeopathic stocks and potentisation (2371):
- method 1.1.8 using the powdered herbal drug (710)
(2. 9.12) and ethanol (90 per cent VIV) R; use ethanol
(70 per cent V/V) R to prepare the 4th decimal dilution,
and use ethanol (50 per cent VIV) R for subsequent
dilutions;
- method 1. 1.10 using the fragmented herbal drug and
Top of the plate ethanol (65 per cent V/V) R.
CHARACTERS
3-4 orange zones Appearance
Pale yellow liquid.
-- -- IDENTIFICATION
Papaverine: an orange zone Thin-layer chromatography (2.2.27) as described in
identification test C for the herbal drug with the following
modification.
Hyoscine: an orange zone
-- -- Results See below the sequence of zones present in the
Reference solution Test solution chromatograms obtained with the reference solution and the
TESTS
Foreign matter (2.8.2) Top of the plate
Maximum 1 per cent.
Maximum 10.0 per cent, determined on 1.000 g of the 2-4 orange zones
powdered herbal drug (710) (2. 9. 12) by drying in an oven at
105 °C for 2 h.
-- --
Papaverine: an orange zone
Total ash (2.4.16)
ASSAY Hyoscine: an orange zone
20 mL of ethanol (90 per cent V/V) R, and boil under a reflux -- --
condenser for 15 min. Allow to cool and filter through a plug Reference solution Test solution
of absorbent cotton, rinsing the filter with ethanol
(90 per cent VIV) R. Add 2 mL of dilute hydrochloric acid R
and evaporate to about 5 mL under reduced pressure. TESTS
Add 20 mL of water R. Shake with 4 quantities, each of Relative density (2.2.5)
25 mL, of light petroleum R. Discard the organic layers. 0.830 to 0.840 when method 1.1.8 is used.
Add to the aqueous layer 3 mL of dilute ammonia Rl and Ethanol (2.9.10)
shake with 4 quantities, each of 25 ml., of 1,1-dimethylethyl 60 per cent V/Vto 70 per cent V/Vwhen method 1.1.10 is
methyl ether R. Dry the combined organic layers over used.
anhydrous sodium sulfate R, filter and evaporate to dryness Dry residue (2.8.16)
under reduced pressure at about 40 °C. Dissolve the residue Minimum 0. 7 per cent.
in 10 mL of anhydrous acetic acid R. Titrate with 0. 02 M
perchloric acid, determining the end-point potentiometrically ASSAY
(2.2.20). Potentiometric titration (2.2.20) as described in the assay of
1 mL of 0. 02 M perchloric acid is equivalent to 11. 99 mg of the herbal drug with the following modifications.
delphinine (C 33H 4 5NO9). To 20.00 g of the mother tincture to be examined add 2 mL
of dilute hydrochloric acid R and evaporate to about 5 mL
MOTHER TINCTURE under reduced pressure. Continue as described in the assay
of the herbal drug, beginning at the words "Add 20 mL of
The mother tincture complies with the requirements of the water R".
DEFINITION
Content
0.10 per cent m/m to 0.40 per cent mlm of total alkaloids,
expressed as delphinine (C 33H 45 NO 9 ; Mr 599.7).
of water R and 3 mL of nitric acid R to the residue and

Sulfur for Homoeopathic evaporate to dryness. The residue complies with the test.
Preparations Prepare the standard using I mL of arsenic standard solution
(Ph. Bur. monograph 2515) (10 ppm As) R.
S 32.07 7704-34-9
Sulfated ash (2. 4.14)
PhEur - - - - - - - - - - - - - - - - - - - ~
ASSAY
DEFINITION Carry out the oxygen-flask method (2.5.10), using 60.0 mg
Obtained by sublimation. in a 1000 mL combustion flask with a
Content polytetrafluoroethylene joint. Absorb the combustion
99.0 per cent to 101.0 per cent. products in a mixture of 5 mL of dilute hydrogen peroxide
CHARACTERS solution R and 10 mL of water R. Heat to boiling, boil gently
for 2 min and cool. Using 0.2 mL of phenolphthalein
Appearance
solution Ras indicator, titrate with 0.1 M sodium hydroxide
Yellow powder.
until the colour changes from colourless to red. Carry out a
Solubility blank titration under the same conditions.
Practically insoluble in water, soluble in carbon disulfide,
1 mL of 0.1 M sodium hydroxide is equivalent to 1.603 mg
slightly soluble in vegetable oils.
ofS.
mp
About 120 °C. STORAGE
IDENflFICATION
A. Heated in the presence of air, it burns with a blue flame,
emitting sulfur dioxide, which changes the colour of
moistened blue litmus paper R to red.
B. Heat 0.1 g with 0.5 mL of bromine water R until
decolourised. Add 5 mL of water R and filter. The solution
Symphytum Officinale Root for
gives reaction (a) of sulfates (2.3. 1). Homoeopathic Preparations
TESTS DEFINITION
Solution S Symphytum Officinale Root for Homoeopathic Preparations
To 5.0 g add 50 mL of carbon dioxide-free water R prepared is the fresh root of Symphytum officinale L.
from distilled water R. Allow to stand for 30 min with
IDENfIFICATION
frequent shaking and filter.
The strong, spirally-formed rootstock with a smooth, black-
Appearance of solution brown cortex, subdivides at the base. It is about 5 to 8 cm in
Solution S is colourless (2.2.2, Method II). diameter and 17 to 30 cm long, surmounted by several tops,
Odour (2.3.4) close together, consisting of the black residues of the previous
It has no perceptible odour of hydrogen sulfide. year's rosettes of leaves between the current year's new
Acidity or alkalinity growths. A ring of 10 to 15 horizontally-running, secondary,
To 5 mL of solution S add 0.1 mL of phenolphthalein glabrous smooth roots grows from the base of the rosettes,
solution Rl. The solution is colourless. Add 0.2 mL of the roots often reaching a length of more than 50 cm and a
0.01 M sodium hydroxide. The solution is red. Add 0.3 mL of diameter of approximately 1.5 cm. The middle portion of the
0.01 M hydrochloric acid. The solution is colourless. rootstock, which is more or less glabrous, branches at the
Add O.15 mL of methyl red solution R. The solution is orange- lower end into several clearly separated, straight downwards
red. pointing, smooth roots, each about 1.5 cm thick and bearing
a few secondary rootlets 1 to 2 cm long.
Chlorides (2.4. 4)
The rootstock and the thick secondary roots are non-
Maximum 100 ppm.
lignified, succulent and break off easily. In the yellowish-
Dilute 5 mL of solution S to 15 mL with water R. white, glassy, slightly differentiated cross section, there is a
Sulfates (2.4.13) very thin, black rhizodermis, and an easily detachable layer of
Maximum 100 ppm, determined on solution S. cortex, 5-8 mm thick. Within this is a single dark pigmented
Sulfides vascular ring. The cut surface and particularly the richly
To 10 mL of solution S add 2 mL of buffer solution pH 3.5 R exuding mucilagenous substance turn yellow to reckbrown on
and 1 mL of a freshly prepared 1.6 g/L solution of lead exposure.
nitrate R in carbon dioxide-free water R. Shake. After 1 min TESTS
any colour in the solution is not more intense than that in a Foreign matter
reference solution prepared at the same time using 1 mL of Not more than 2%, Appendix XI D.
lead standard solution (10 ppm Pb) R, 9 mL of carbon dioxide-
free water R, 2 mL of buffer solution pH 3.5 R and 1.2 mL of
MOTHER TINCTURE
thwacetamide reagent R.
Arsenic (2.4.2, Method B) The mother tincture complies with the requirements stated under
Maximum 8 ppm. Mother Tinctures for Homoeopathic Preparations and with the
Shake 2.5 g with 50 mL of dilute ammonia Rl for 1 hand following requirements.
filter. Evaporate 25 mL of the filtrate to dryness. Add 2 mL
PRODUCTION growths. A ring of 10 to 15 horizontally-running, secondary,

The mother tincture of Symphytum officinale L., is prepared glabrous smooth roots, grows from the base of the rosettes,
from the herbal drug using Method 1.1.3 described in the the roots often reaching a length of more than 50 cm and a
monograph for Methods of Preparation of Homoeopathic diameter of approximately 1.5 cm. The middle portion of the
Stocks and Potentisation. Use 86% w/w (90% v/v) of ethanol. rootstock, which is more or less glabrous, branches at the
lower end into several clearly separated, straight downwards
CHARACTERISTICS
pointing, smooth roots, each about 1. 5 cm thick and bearing
The mother tincture is a brown liquid.
a few secondary rootlets 1 to 2 cm long.
IDENTIFICATION The rootstock and the thick secondary roots are non-
Cany out the method for thin-layer chromatography, lignified, succulent and break off easily. In the yellowish-
Appendix III A, using the following solutions. white, glassy, slightly differentiated cross section, there is a
(1) The mother tincture. very thin, black rhizodermis, and an easily detachable layer of
(2) 0.1 % w/v of allantoin in ethanol (45%). cortex, 5-8 mm thick. Within this is a single dark pigmented
vascular ring. The cut surface and particularly the richly
exuding mucilagenous substance turn yellow to red-brown on
(a) Use as the coating silica gel. exposure.
(c) Apply 20 µL of solution (1) and 10 µL of solution (2) as MOTHER TINCTURE
10 mm bands.
(e) After removal of the plate, dry in air and examine under following requirements.
ultraviolet light (365 nm).
(f) Spray the plate with a 5% w/v solution of
PRODUCTION
dimethylaminobenzaldehyde in hydrochloric acid and examine in The mother tincture of Symphytum officinale L. is prepared
daylight. by decoction of the herbal drug using 43% w/w (50% v/v) of
ethanol.
MOBILE PHASE
CHARACTERISTICS
The mother tincture is a brown liquid.
SYSTEM SUIT ABILITY IDENTIFICATION
Cany out the method for thin-layer chromatography,
The test is not valid unless under ultraviolet light (365 nm)
the chromatogram of solution (1) shows two bluish
fluorescent bands at Rf 0.25 and Rf 0.55 and one light-green ( 1) The mother tincture.
fluorescent band at the solvent front. (2) 0.1 % w/v of allantoin in ethanol (45%).
CONFIRMATION CHROMATOGRAPHIC CONDITIONS
After spraying, the chromatogram obtained with solution (2) (a) Use as the coating silica gel.
shows the yellow allantoin band at Rf 0.35. (b) Use the mobile phase as described below.
The chromatogram obtained with solution (1) shows one (c) Apply 20 µL of solution (1) and 10 µL of solution (2) as
yellow band with the same Rf of 0.35 as that for allantoin. 10 mm bands.
TESTS (d) Develop the plate to 10 cm.
Ethanol (e) After removal of the plate, dry it in air and examine
35 to 45% w/w (42 to 53% v/v), Appendix VIII F. under ultraviolet light (365 nm).
Dry residue (f) Spray the plate with a 5% w/v solution of
Not less than 1.5% w/w, Appendix XI P. dimethylaminobenzaldehyde in hydrochloric acid and examine in
Relative density daylight.
0.920 to 0.970, Appendix VG. MOBILE PHASE
Symphytum Officinale Root for Ethanol SYSTEM SUITABILITY

The test is not valid unless under ultraviolet light (365 nm),
Decoction for Homoeopathic the chromatogram obtained with solution (1) shows two
Preparations bluish fluorescent bands at Rf value of 0.25 and Rf value of
0.55 and one light-green fluorescent band at the solvent
DEFINITION front.
Symphytum Officinale Root for Ethanol Decoction for
CONFIRMATION
Homoeopathic Preparations is the fresh root of Symphytum
officinale L. After spraying, the chromatogram obtained with solution (1)
shows a yellow band at Rf value of 0.35 corresponding in
IDENTIFICATION position and colour to that obtained with solution (2).
The strong, spirally-formed rootstock with a smooth, black-
brown cortex, subdivides at the base. It is about 5 to 8 cm in TESTS
diameter and 17 to 30 cm long, surmounted by several tops, Ethanol
close together, consisting of the black residues of the previous 25 to 35% w/w (31 to 42% v/v), Appendix VIII F.
year's rosettes of leaves between the current year's new
Dry residue PRODUCTION

Not less than 3.0% w/w, Appendix XI P. The mother tincture is prepared from the comminuted herbal
Relative density drug according to the following methods prescribed in the
0.973 to 0.982, Appendix VG. monograph Methods of preparation of homoeopathic stocks and
potentisation (2371):
- method 1.1.3, using ethanol (90 per cent VIV);
- method 1.1.10, using ethanol (65 per cent V/V).
Toxicodendron Quercifolium for CHARACTERS
Appearance
Homoeopathic Preparations Reddish-brown or greenish-brown liquid.
Rhus Toxicodendron for Homoeopathic
IDENTIFICATION
Preparations
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Reference solution Dissolve 5 mg of quercitrin R and 5 mg of
DEFINITION rutoside trihydrate R in ethanol (96 per cent) R and dilute to
Fresh young branches, not yet lignified, with fully developed 20 mL with the same solvent.
leaves, of Toxicodendron pubescens Mill. (synonyms Rhus Plate TLC silica gel plate R (5-40 µm) [or TLC silica gel
toxicodendron L., Toxicodendron quercifolium (Michx.) Greene) plate R (2-10 µm)].
collected in summer. Mobile phase water R, methanol R, glacial acetic acid R,
IDENTIFICATION methylene chloride R (2:3:8:15 VIVIVIV).
Caution: the yellowish-white milky latex in the branches is a Application 20 µL [or 5 µL] as bands.
strong cutaneous irritant. Take all necessary handling precautions Development Over a path of 10 cm [or 7 cm].
to avoid any contact with the skin or mucous membranes.
Drying In air.
The thin, young branches are covered with a downy or
cottony indumentum. Pointed leaves and buds covered in
aminoethyl ester R in methanol R, then treat with a 50 g/L
brown, woolly hairs may be found at the ends of the
solution of macrogol 400 R in methanol R; allow to dry in air
branches and in the axils of the alternate leaves. The leaves
for about 30 min and examine in ultraviolet light at 365 nm.
are compound imparipinnate, variously shaped, alternate,
with up to 5 leaflets. There are no stipules. The petiole is Results See below the sequence of fluorescent zones present
15-150 mm long and may be covered in stiff or cottony in the chromatograms obtained with the reference solution
hairs. The terminal leaflet is 50-90 mm long and 40-70 mm and the test solution. The intensity of the zones present in
wide and borne on a stalk up to 20 mm long, whereas the the chromatogram obtained with the test solution may vary.
lateral leaflets are smaller and have short stalks or are almost Furthermore, other faint fluorescent zones may be present in
sessile. The leaflets are ovate to oblong. The margin may be the chromatogram obtained with the test solution.
entire, undulate, lobed-dentate or lyriform-pinnatifid, with 3
or more rounded, blunt, or in rare cases, almost pointed Top of the plate
lobes on either side. The lateral leaflets are often only
A blue zone
unilaterally dentate or crenate. The leaflets are rounded or
cuneate at the base and rounded, acute or very occasionally Quercitrin: an orange zone An orange zone (may be missing)
acuminate at the apex. The upper surface of young leaves is (quercitrin)
shiny dark green, the lower surface of the leaves is light green -- --
and with scattered hairs, more densely hairy on the veins. An orange zone (may be missing)
If bruising occurs with harvesting the damaged leaves may
show brown or black spots. Rutoside: an orange zone
TESTS A blue zone

- - --
Maximum 5 per cent, including a maximum of 2.0 per cent
of lignified shoots. A blue zone
Loss on drying (2.2.32) A blue zone

Minimum 55.0 per cent, determined on 5.0 g of the finely
cut herbal drug by drying in an oven at 105 °C for 2 h.
MOTHER TINCTURE TESTS

The mother tincture complies with the requirements of the Relative density (2.2.5)
general monograph Mother tinctures for homoeopathic 0.940 to 0.965 (method 1.1.3).
preparations (2029). Ethanol (2. 9.10)
60 per cent V/Vto 70 per cent V/V (method 1.1.10).
DEFINITION
Mother tincture prepared from the fresh, young, not yet Dry residue (2.8.16)
lignified shoots with fully developed leaves of Toxicodendron - minimum 3.5 per cent mlm (method 1.1.3);
quercifolium (Michx.) Greene (Rhus toxicodendron L.). - minimum 1.25 per cent mlm (method 1.1.10).
Urushiols
Test solution Introduce 10.000 g of the mother tincture to

be examined into a 100 mL flask with a ground-glass neck
Urtica Dioica for Homoeopathic
and evaporate to dryness under reduced pressure in a water- Preparations
bath at 40 °C. Take up the residue with 10 mL of water R,
Common Stinging Nettle for Homoeopathic
and add 10 mL of heptane R. Stopper the flask. Stir
Preparations
vigorously with a magnetic stirrer for 15 min. Allow the
solids to settle. Collect the upper layer using a glass pipette, (Ph. Bur. monograph 2030)
avoiding suspended particles, and filter over anhydrous sodium Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
sulfate R. DEFINITION
Repeat the extraction twice more, using 10 mL of heptane R Whole, fresh, flowering plant of Urtica dioica L.
each time as described above. Discard the remaining aqueous
layer and rinse the flask with 10 mL of heptane R. Combine CHARACTERS
the extracts and washings and filter over anhydrous sodium The plant causes an itching, burning sensation on the skin.
sulfate R. Evaporate the filtrate to dryness under reduced IDENTIFICATION
pressure in a water-bath at 40 °C. Dissolve the residue with A. The perennial plant has a taproot that sends out creeping
2.0 mL of methanol R. subterranean rhizomes, more or less 4-angled in transverse
Reference solution Dissolve 35.0 mg of section, from which extend adventious secondary roots and
4-dodecylresorcinol CRS in methanol Rand dilute to 20.0 mL very numerous brownish hairy rootlets. The stipes are erect,
with the same solvent. generally unbranched, 3-5 mm in diameter and 0.3-1.5 m
Column: high, rarely up to 2.5 m high, 4-angled, greyish-green and
- size: l = 0.25 m, 0 = 4.6 mm; covered in short hairs and stinging hairs.
- stationary phase: end-capped octadecylsilyl silica gel for The decussate leaves are 30-150 mm long and 20-80 mm
chromatography R (5 µm); wide. The petiole is hispid and usually slightly less than one-
- temperature: 30 °C. third the length of the lamina. The leaf blade is ovate,
Mobile phase: acuminate, cordate or rounded at the base, and coarsely
- mobile phase A: 0.2 per cent V/V solution of phosphoric dentate; the apical tooth is distinctly larger than the lateral
acid R; teeth. The upper side of the leaves is dark green and usually
- mobile phase B: methanol R; matt, both sides bear short serried hairs intermingled with
long stinging hairs. The 2 stipules are linear-subulate and
Time Mobile phase A Mobile phase B free. The inflorescences growing from the leaf axils are
(min) (per cent VII') (per cent VII') complex, the flowers unisexual, and, particularly in male
0-2 20 80 plants, generally distinctly longer than the petiole. After
2 - 82 20 ➔ 0 80 ➔ 100 shedding their pollen, male inflorescences are erect at an
oblique angle or horizontal; female inflorescences are pendent
when the fruit is ripe. All flowers have long stalks.
The perianth of the male flowers is divided half-way down
Detection Spectrophotometer at 276 nm. into equal green lobes, widest at their base, with short bristles
Injection 20 µL. and stinging hairs at the margins. The stamens are equal and
Relative retention With reference to urushiol 2 (principal opposite to the perianth segments, each with a long, whitish
peak) (retention time = about 35 min): filament that curves inwards before pollen is shed and
= =
urushiol 1 about 0.8; urushiol 3 about 1.2; urushiol spreads out afterwards. The ovary is rudimentary, button or
4 = about 1.5. cup-shaped. The perianth of the female flowers is downy or
System suitability Reference solution: bristly on the outside and consists of outer, and 2 inner
- Symmetry factor. 0.8 to 1.8 for the peak due to segments; the inner segments are about twice the length of
4-dodecylresorcinol. the outer ones. The hypogynous, ovate, unilocular ovary
bears a large capitate stigma with a brush-like shock of hair.
Calculate the percentage content m/m of urushiols, expressed
As the one-seeded fruit grows ripe, the 2 inner segments of
as 4-dodecylresorcinol, using the following expression:
the perianth fold around it like wings.
LAixmzxpx0.113 B. It complies with the test for Urtica urens (see Tests).
A 2 xm1 TESTS
Urtica urens
L1 1 sum of the areas of the peaks due to urushiols I to 4 in the
chromatogram obtained with the test solution; The margin of the lamina is not serrate with teeth twice as
A2 area of the peak due to 4-dodecylresorcinol in the long as wide. The clusters of flowers in the axils are longer
chromatogram obtained with the reference solution; than the petiole of the leaf. Unisexual, apetalous flowers are
m, mass of the mother tincture used to prepare the test solution, in not together on the same plant and in the same cluster.
grams;
m2 mass of 4-diJdecylresorcinol CRS used to prepare the reference Foreign matter (2.8.2)
solution, in grams; Maximum 5 per cent.
p percentage content of 4-dodecylresorcinol in
4-dodecylresorcinol CRS. Loss on drying (2.2.32)
Minimum 65.0 per cent, determined on 5.0 g of finely cut
Limit: herbal drug by drying in an oven at 105 °C for 2 h, if
- urushiols: maximum 0.05 per cent mlm, expressed as performed to demonstrate the freshness of the herbal drug.
4-dodecylresorcinol.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
MOTHER TINCTURE Urtica Urens Herb for Homoeopathic

Preparations
preparations (2029). DEFINITION
Urtica Urens Herb for Homoeopathic Preparations is the
PRODUCTION
fresh leaves and flowers of Urtica urens L.
The mother tincture of Urtica dioica is prepared by
maceration using ethanol of a suitable concentration. CHARACTERISTICS
The plant produces an itchy, burning sensation.
CHARACTERS
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
Reference 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, margins are entire.
butanol R (10:20:35:35 V/VIVIV). Flowers The complicated inflorescences consist mainly of
Application 20 µL, 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
shorter than the leaf petioles. The perigonium of the male
Drying In air.
flowers is split into four pale green lobes of equal size; each
Detection Spray with a 1 g/L solution of ninhydrin R in one of the four stamens situated in front of one of the
ethanol (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
Top of the plate conspicuous, brushlike stigma. The ripe fruits are
monospermic and enclosed by the two inner segments of the
-- -- perigonium.
Phenylalanine: a violet to reddish- TESTS
brown zone
Urtica dioica
4 red to violet zones The plant is dioecious as follows:
-- --
the male and female flowers occur on separate plants;
the inflorescences are longer than the leaf petioles;
Serine: a reddish-violet zone A violet zone
the leaves, especially those on the lower part of the stem, are
A violet zone longer than their petioles.
MOTHER TINCTURE
TESTS The mother tincture complies with the requirements stated under
Relative density (2.2.5) Mother Tinctures for Homoeopathic Preparations and with the
0.930 to 0.950. following requirements.
Ethanol (2. 9.10) PRODUCTION
40 per cent V/V to 56 per cent V/V. The mother tincture of Urtica urens L., is prepared from the
Methanol (2. 9.11) herbal drug using Method 2b described in the monograph for
Maximum 0.10 per cent V/V. Methods of Preparation of Homoeopathic Stocks and
Potentisation. Use 62% w/w (70% v/v) of ethanol.
Minimum 1.1 per cent. 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.
C. Carry out the method for thin-layer chromatography, Relative density

Appendix III A, using the following solutions. 0.956 to 0.968, Appendix V G.
( 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 µL 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-1-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-1-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

observed
TESTS
Ethanol
Dry residue
Monographs
2023 Blood-related Products IV-631
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
Solutions for Blood of water produces cloudiness. Dry the plate in a current of
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
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ thymol R in a mixture of 5 mL of sulfuric acid R and 95 mL
of ethanol (96 per cent) R. Heat at 130 °C for 10 min.
DEFINITION The principal spot in the chromatogram obtained with the
Anticoagulant and preservative solutions for human blood are test solution is similar in position, colour and size to the
sterile and pyrogen-free solutions prepared with water for principal spot in the chromatogram obtained with reference
injections, filtered, distributed in the final containers and solution (a). The test is not valid unless the chromatogram
sterilised. The content of sodium citrate obtained with reference solution (b) shows 4 clearly
(C 6 H 5Na 3 O 7,2H2O), glucose monohydrate (C5H12O5,H2O) separated spots.
or glucose (C 6 H 12 O 6) and sodium dihydrogen phosphate B. To 2 mL add 5 mL of cupri-citric solution R. Heat to
dihydrate (NaH2PO 4,2H2O) is not less than 95.0 per cent boiling. An orange precipitate is formed and the solution
and not more than 105. 0 per cent of that stated in the becomes yellow.
formulae below. The content of citric acid monohydrate
C. To 2 mL (for formula A) add 3 mL of water R or to
(C 6H 8 O 7 ,H 2 O) or citric acid (C 6H 8 O 7 ) is not less than
4 mL (for formula B) add 1 mL of water R. The solution
90.0 per cent and not more than 110.0 per cent of that
gives the reaction of citrates (2.3.1).
stated in the formulae below. Subject to agreement by the
competent authority, other substances, such as red-cell D. 0.5 mL gives reaction (b) of sodium (2.3.1).
preservatives, may be included in the formula provided that TESTS
their name and concentration are stated on the label. pH (2.2.3)
Anticoagulant and preservative solutions for human blood are The pH of the solution to be examined is 4.7 to 5.3.
presented in airtight, tamper-evident containers of glass Hydroxymethylfurfural
(3. 2.1) or plastic (3. 3. 4). To 2.0 mL add 5.0 mL of a 100 g/L solution of p-toluidine R
in 2-propanol R containing 10 per cent V/V of glacial acetic
ANTICOAGULANT ACID-CITRATE- acid R and 1.0 mL of a 5 g/L solution of barbituric acid R.
GLUCOSE SOLUTIONS (ACD) The absorbance (2.2.25), determined at 550 nm after
allowing the mixture to stand for 2 min to 3 min, is not
A B greater than that of a standard prepared at the same time in
Sodium citrate (0412) 22.0 g 13.2 g the same manner using 2.0 mL of a solution containing
Citric acid monohydrate (0456) 8.0 g 4.8 g
or Citric acid (0455) 7.3 g 4.4 g
5 ppm of hydroxymethylfuifural R for formula A or 3 ppm of
Glucose monohydrate (0178)* 24.5 g 14.7 g hydroxymethylfuifural R for formula B.
or Glucose (0177)* 22.3 g 13.4 g Sterility (2. 6.1)
Water for irifections (0169) to 1000.0 mL 1000.0 mL
Volume to be used per 100 mL of blood 15.0 mL 25.0 mL
They comply with the test for sterility.
*The competent authority may require that the substances comply with the Pyrogens (2.6.8)
test for pyrogens given in the monographs on Glucose monohydrate (0178) and They comply with the test for pyrogens. Dilute with a
Glucose (0177), respectively.
pyrogen-free, 9 g/L solution of sodium chloride R to obtain a
solution containing approximately 5 g/L of sodium citrate.
CHARACTERS Inject 10 mL of the diluted solution per kilogram of the
A colourless or faintly yellow, clear liquid, practically free rabbit's mass.
from particles.
ASSAY
IDENTIFICATION Citric acid
A. Examine by thin-layer chromatography (2.2.27), using To 10.0 mL (for formula A) or to 20.0 mL (for formula B)
silica gel G R as the coating substance. add 0.1 mL of phenolphthalein solution Rl. Titrate with 0.2 M
Test solution Dilute 2 mL of the solution to be examined sodium hydroxide until a pink colour is obtained.
(for formula A) or 3 mL (for formula B) to 100 mL with a 1 mL of 0.2 M sodium hydroxide is equivalent to 14.01 mg of
mixture of 2 volumes of water R and 3 volumes of C 6H 8O 7,H 2 O or to 12.81 mg of C6HsO1.
methanol R.
Sodium citrate
Reference solution (a) Dissolve 10 mg of glucose Prepare a chromatography column 0.10 m long and 10 mm
monohydrate CRS in a mixture of 2 volumes of water R and
in internal diameter and filled with strongly acidic ion-exchange
3 volumes of methanol R and dilute to 20 mL with the same resin R (300 µm to 840 µm). Maintain a 1 cm layer ofliquid
mixture of solvents. above the resin at all times. Wash the column with 50 mL of
Reference solution (b) Dissolve 10 mg of glucose R, 10 mg of de-ionised water R at a flow rate of 12-14 mIJmin.
fructose R, 10 mg of lactose monohydrate R and 10 mg of Dilute 10.0 mL of the solution to be examined (for
sucrose R in a mixture of 2 volumes of water R and 3 volumes formula A) or 15.0 mL (for formula B) to about 40 mL with
of methanol R and dilute to 20 mL with the same mixture of de-ionised water R in a beaker and transfer to the column
solvents. reservoir, washing the beaker 3 times with a few millilitres of
Apply separately to the plate 2 µL of each solution and de-ionised water R. Allow the solution to run through the
thoroughly dry the points of application. Develop over a path column at a flow rate of 12-14 mIJmin and collect the
of 15 cm using a mixture of 10 volumes of water R, eluate. Wash the column with 2 quantities, each of 30 mL,
15 volumes of methanol R, 25 volumes of anhydrous acetic and with one quantity of 50 mL, of de-ionised water R.
IV-632 Blood-related Products 2023
The column can be used for 3 successive determinations ANTICOAGULANT CITRATE-

before regeneration with 3 times its volume of dilute PHOSPHATE-GLUCOSE SOLUTION (CPD)
hydrochloric acid R. Titrate the combined eluate and washings
(about 150 mL) with 0.2 M sodium hydroxide, using 0.1 mL Sodium citrate (0412) 26.3 g
of phenolphthalein solution Rl as indicator. Citric acid monohydrate (0456) 3.27 g
or Cim·c acid (0455) 2.99 g
Calculate the content of sodium citrate in grams per litre
Glucose monohydrate (0178)* 25.5 g
from the following expressions: or Glucose (0177)* 23.2 g
Sodium dihydrogen phosphate dihydrate (0194) 2.51 g
Water for injections (OJ 69) to I 000.0 mL
For formula A: l.96ln- l.40C Volume to be used per 100 mL of blood 14.0 mL
or 1.961n - l.53C' *The competent authority may require that the substances comply with the
test for pyrogens given in the monographs on Glucose monohydrate (0178) and
Glucose (0177), respectively.
For formula B: 1.307 n - I .40C

CHARACTERS
A colourless or faintly yellow, clear liquid, practically free
or 1.307n - 1.53c'
from particles.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using
n number of miUilitres of 0.2 M sodium hydroxide used in the silica gel G R as the coating substance.
titration;
C content of citric acid monohydrate in grams per litre determined
Test solution Dilute 2 mL of the solution to be examined to
as prescribed above; 100 mL with a mixture of 2 volumes of water R and
C' content of citric acid in grams per litre determined as prescribed 3 volumes of methanol R.
above.
Reference solution (a) Dissolve 10 mg of glucose
monohydrate CRS in a mixture of 2 volumes of water R and
Reducing sugars 3 volumes of methanol R and dilute to 20 mL with the same
Dilute 5.0 mL (for formula A) or 10.0 mL (for formula B) to mixture of solvents.
100.0 mL with water R. Introduce 25.0 mL of the solution
into a 250 mL conical flask with ground-glass neck and add Reference solution (b) Dissolve 10 mg of glucose R, 10 mg of
25.0 mL of cupri-citric solution Rl. Add a few pieces of fructose R, 10 mg of lactose monohydrate R and 10 mg of
sucrose R in a mixture of 2 volumes of water R and 3 volumes
porous material, attach a reflux condenser, heat so that
boiling begins within 2 min and boil for exactly 10 min. Cool of methanol R and dilute to 20 mL with the same mixture of
and add 3 g of potassium iodide R dissolved in 3 mL of solvents.
water R. Add 25 mL of a 25 per cent mlm solution of sulfuric Apply separately to the plate 2 µL of each solution and
acid R with caution and in small quantities. Titrate with thoroughly dry the starting points. Develop over a path of
0.1 M sodium thiosulfate using 0.5 mL of starch solution R, 15 cm using a mixture of 10 volumes of water R, 15 volumes
added towards the end of the titration, as indicator (n 1 mL). of methanol R, 25 volumes of anhydrous acetic acid R and
Carry out a blank titration using 25.0 mL of water R 50 volumes of ethylene chloride R. The volumes of solvents
(n 2 mL). have to be measured accurately since a slight excess of water
Calculate the content of reducing sugars as glucose or as produces cloudiness. Dry the plate in a current of warm air.
glucose monohydrate, as appropriate, from Table 0209.-1. Repeat the development immediately, after renewing the
mobile phase. Dry the plate in a current of warm air and
Table 0209.-1 spray evenly with a solution of 0.5 g of thymol R in a mixture
Volume of0.1 M sodium Glucose Glucose monohydrate
of 5 mL of suifuric acid R and 95 mL of ethanol
thiosulfate (mg) (mg) (96 per cent) R. Heat at 130 °C for 10 min. The principal
(n2-n 1 mL) spot in the chromatogram obtained with the test solution is
8 19.8 21.6 similar in position, colour and size to the principal spot in
9 22.4 24.5 the chromatogram obtained with reference solution (a).
JO 25.0 27.2 The test is not valid unless the chromatogram obtained with
II 27.6 30.2 reference solution (b) shows 4 clearly separated spots.
12 30.3 33.1 B. To 2 mL add 5 mL of cupri-citric solution R. Heat to
13 33.0 36.1 boiling. An orange precipitate is formed and the solution
14 35.7 39.0 becomes yellow.
15 38.3 42.1 C. To 2 mL add 3 mL of water R. The solution gives the
16 41.3 45.2 reaction of citrates (2.3.1).
D. 1 mL gives reaction (b) of phosphates (2.3.1).
STORAGE E. 0.5 mL gives reaction (b) of sodium (2.3.1).
Store in an airtight, tamper-evident container, protected from
light.
TESTS
pH (2.2.3)
LABELLING The pH of the solution is 5.3 to 5.9.
The label states:
Hydroxymethylfurfural
- the composition and volume of the solution;
To 2.0 mL add 5.0 mL of a 100 g/L solution of p-toluidine R
- the maximum amount of blood to be collected in the in 2-propanol R containing 10 per cent V/V of glacial acetic
container. acid R and 1.0 mL of a 5 g/L solution of barbituric acid R.
The absorbance (2.2.25), determined at 550 nm after
allowing the mixture to stand for 2 min to 3 min, is not Calculate the content of sodium citrate in grams per litre
greater than that of a standard prepared at the same time in from the following expressions:
the same manner using 2.0 mL of a solution containing
5 ppm of hydroxymethylfuifural R. 1.961n - 1.257P - l.40C
Sterility (2. 6.1)
They comply with the test for sterility. 1.961n - l.257P- 1.53C'
Pyrogens (2. 6. 8)
They comply with the test for pyrogens. Dilute with a n number of millilitres of 0. 2 M sodium hydroxide used in the
titration;
pyrogen-free, 9 g/L solution of sodium chloride R to obtain a P content of sodium dihydrogen phosphate dihydrate in grams per
solution containing approximately 5 g/L of sodium citrate. litre determined as prescribed above;
Inject 10 mL of the diluted solution per kilogram of the C content of citric acid monohydrate in grams per litre determined
rabbit's mass. as prescribed above;
C' content of citric acid in grams per litre determined as prescribed
ASSAY above.
Sodium dihydrogen phosphate
Dilute 10.0 mL to 100.0 mL with water R. To 10.0 mL of Reducing sugars
this solution add 10.0 mL of nitro-molybdovanadic reagent R. Dilute 5.0 mL to 100.0 mL with water R. Introduce 25.0 mL
Mix and allow to stand at 20 °C to 25 °C for 30 min. At the of the solution into a 250 mL conical flask with ground-glass
same time and in the same manner, prepare a reference neck and add 25.0 mL of cupn·-citric solution Rl. Add a few
solution using 10.0 mL of a standard solution containing pieces of porous material, attach a reflux condenser, heat so
0.219 g of potassium dihydrogen phosphate R per litre. Measure that boiling begins within 2 min and boil for exactly 10 min.
the absorbance (2.2.25) of the 2 solutions at 450 nm using as Cool and add 3 g of potassium iodide R dissolved in 3 mL of
the compensation liquid a solution prepared in the same water R. Add 25 mL of a 25 per cent mlm solution of sulfuric
manner using 10 mL of water R. Calculate the content of acid R with caution and in small quantities. Titrate with
sodium dihydrogen phosphate dihydrate (P) in grams per 0.1 M sodium thiosulfate using 0.5 mL of starch solutwn R,
litre from the expression: added towards the end of the titration, as indicator (n 1 mL).
Carry out a blank titration using 25.0 mL of water R
ll.46xCxA1 (n 2 mL).
A2 Calculate the content of reducing sugars as glucose or as
C concentration of potassium dihydrogen phosphate R in the glucose monohydrate, as appropriate, from Table 0209.-1.
standard solution in grams per litre;
STORAGE
A2 absorbance of the reference solution. Store in an airtight, tamper-evident container, protected from
light.
Citric acid LABELLING
To 20.0 mL add 0.1 mL ofphenolphthakin solutwn Rl and The label states:
titrate with 0.2 M sodium hydroxide. - the composition and volume of the solution;
Calculate the content of citric acid monohydrate (C), or citric - the maximum amount of blood to be collected in the
acid (C' ), in grams per litre from the equations: container.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
C = 0.7005n - 0.4490P
c' = 0.6404n - 0.4105P
n number of millilitres of 0.2 M sodium hydroxide used in the

Plasma for Fractionation
titration;
p (Human Plasma for Fractwnatwn, Ph. Bur.
content of sodium dihydrogen phosphate dihydrate in grams per
litre determined as prescribed above. monograph 0853)
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Sodium citrate
Prepare a chromatography column 0.10 m long and 10 mm DEFINITION
in internal diameter and filled with strongly acidic wn-exchange Liquid part of human blood remaining after separation of the
resin R (300 µm to 840 µm). Maintain a 1 cm layer of liquid cellular elements from blood collected in a receptacle
above the resin at all times. Wash the column with 50 mL of containing an anticoagulant, or separated by continuous
de-ionised water Rat a flow rate of 12-14 mUmin. filtration or centrifugation of anticoagulated blood in an
apheresis procedure; it is intended for the manufacture of
Dilute 10.0 mL of the solution to be examined to about
plasma-derived products.
40 mL with de-ionised water R in a beaker and transfer to
the column reservoir, washing the beaker 3 times with a few PRODUCTION
millilitres of de-ionised water R. Allow the solution to run DONORS
through the column at a flow rate of 12-14 mUmin and Only a carefully selected, healthy donor who, as far as can be
collect the eluate. Wash the column with 2 quantities, each ascertained after medical examination, laboratory blood tests
of 30 mL, and with one quantity of 50 mL, of de-ionised and a study of the donor's medical history, is free from
water R. The column can be used for 3 successive detectable agents of infection transmissible by plasma-derived
determinations before regeneration with 3 times its volume of products may be used. Recommendations in this field are
dilute hydrochloric acid R. Titrate the combined eluate and made by the Council of Europe [Recommendatwn
washings (about 150 mL) with 0.2 M sodium hydroxide, using No. R (95) 15 on the preparation, use and quality assurance of
0.1 mL of phenolphthalein solution Rl as indicator. blood components, or subsequent revision]; a directive of the
European Union also deals with the matter: Commissum separated from cellular elements and frozen in a chamber at
Directive 2004/33/EC of 22 March 2004 implementing -20 °C or below within 72 h of collection.
Directive 2002/98/EC of the European Parliament and of the It is not intended that the determination of total protein and
Council as regards certain technical requirements for blood and human coagulation factor VIII shown below be carried out on
blood components. each unit of plasma. They are rather given as guidelines for good
Immunisation of donors manufacturing practice, the test for human coagulation factor VIII
Immunisation of donors to obtain immunoglobulins with being relevant for plasma intended for use in the preparation of
specific activities may be carried out when sufficient supplies concentrates of labile proteins.
of material of suitable quality cannot be obtained from The total protein content of a unit of plasma depends on the serum
naturally immunised donors. Recommendations for such protein content of the donor and the degree of dilution inherent in
immunisations are formulated by the World Health the donation procedure. W'hen plasma is obtained from a suitable
Organization (Requirements for the collection, processing and donor and using the intended proportion of anticoagulant solution,
quality control of blood, blood components and plasma derivatives, a total protein content complying with the limit of 50 g/L is
WHO Technical Report Series, No. 840, 1994 or subsequent obtained. If a volume of blood or plasma smaller than intended is
revision). collected into the anticoagulant solution, the resulting plasma is not
Records necessarily unsuitable for pooling for fractionation. The aim of
Records of donors and donations made are kept in such a good manufacturing practice must be to achieve the prescribed limit
way that, while maintaining the required degree of for all normal donations.
confidentiality concerning the donor's identity, the origin of Preservation of human coagulation factor VIII in the donation
each donation in a plasma pool and the results of the depends on the collection procedure and the subsequent handling of
corresponding acceptance procedures and laboratory tests the blood and plasma. With good practice, 0. 7 IU/mL can usually
can be traced. be achieved, but units of plasma with a lower activity may still be
Laboratory tests suitable for use in the production of coagulation factor
Laboratory tests are carried out for each donation to detect concentrates. The aim of all steps taken during production of
the following viral markers: plasma is to obtain plasma of the intended quality and to conserve
labile proteins as much as possible.
1. antibodies against human immunodeficiency virus 1 (anti-
HN-1 ); Total protein
2. antibodies against human immunodeficiency virus 2 (anti- Carry out the test using a pool of not fewer than 10 units.
HN-2); Dilute an appropriate volume of the preparation with a 9 g/L
solution of sodium chloride R to obtain a solution containing
3. hepatitis B surface antigen (HBsAg);
about 15 mg of protein in 2 mL. To 2.0 mL of this solution
4. antibodies against hepatitis C virus (anti-HCV). in a round-bottomed centrifuge tube, add 2 mL of a 75 g/L
The test methods used are of suitable sensitivity and solution of sodium molybdate R and 2 mL of a mixture of
specificity and comply with the regulations in force. If a 1 volume of nitrogen-free sulfuric acid R and 30 volumes of
repeat-reactive result is found in any of these tests, the water R. Shake, centrifuge for 5 min, decant the supernatant
donation is not accepted. and allow the inverted tube to drain on filter paper.
INDIVIDUAL PIASMA 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 Hu.man coagulation factor Vlll (2. 7. 4)
introduction of micro-organisms. No antibacterial or Carry out the test using a pool of not fewer than 10 units.
antifungal agent is added to the plasma. The containers Thaw the samples to be examined, if necessary, at 37 °C.
comply with the requirements for glass containers (3.2. I) or Carry out the assay using a reference plasma calibrated
for plastic containers for blood and blood components against the International Standard for human coagulation
(3.3.4). The containers are closed so as to prevent any factor VIII in plasma. The activity is not less than
possibility of contamination. 0.7 IU/mL.
If 2 or more units are pooled prior to freezing, the operations STORAGE AND TRANSPORT
are carried out using sterile connecting devices or under Frozen plasma is stored and transported in conditions
aseptic conditions and using containers that have not designed to maintain the temperature at or below -20 °C; for
previously been used. accidental reasons, the storage temperature may rise above
When obtained by plasmapheresis or from whole blood (after -20 °C on one or more occasions during storage and
separation from cellular elements), plasma intended for the transport but the plasma is nevertheless considered suitable
recovery of proteins that are labile in plasma is frozen within for fractionation if all the following conditions are fulfilled:
24 h of collection by cooling rapidly in conditions validated - the total period of time during which the temperature
to ensure that a temperature of -25 °C or below is attained exceeds -20 °C does not exceed 72 h;
at the core of each plasma unit within 12 h of placing in the - the temperature does not exceed -15 °C on more than
freezing apparatus. 1 occasion;
When obtained by plasmapheresis, plasma intended solely for - the temperature at no time exceeds -5 °C.
the recovery of proteins that are not labile in plasma is frozen POOLED PIASMA
by cooling rapidly in a chamber at -20 °C or below within During the manufacture of plasma products, the first
24 h of collection. homogeneous pool of plasma (for example, after removal of
When obtained from whole blood, plasma intended solely for cryoprecipitate) is tested for HBsAg and for HN antibodies
the recovery of proteins that are not labile in plasma is using test methods of suitable sensitivity and specificity; the
pool must give negative results in these tests.
The plasma pool is also tested for hepatitis C virus RNA 1.0 x 102 IU of hepatitis C virus RNA per millilitre and, to
using a validated nucleic acid amplification technique test for inhibitors, an internal control prepared by addition of
(2.6.21). A positive control with 100 IU/mL of hepatitis C a suitable marker to a sample of the plasma pool are included
virus RNA and, to test for inhibitors, an internal control in the test. The test is invalid if the positive control is non-
prepared by addition of a suitable marker to a sample of the reactive or if the result obtained with the internal control
plasma pool are included in the test. The test is invalid if the indicates the presence of inhibitors. The pool complies with
positive control is non-reactive or if the result obtained with the test if it is found non-reactive for hepatitis C virus RNA.
the internal control indicates the presence of inhibitors. Hepatitis C virus RNA for NAT testing ERP is suitable for use
The plasma pool complies with the test if it is found non- as a positive control.
reactive for hepatitis C virus RNA.
Hepatitis E virus RNA
Hepatitis C virus RNA for NAT testing ERP is suitable for use The plasma pool is tested using a validated nucleic acid
as a positive control. amplification technique (2.6.21). A positive control with
CHARACTERS 3.2 x 10 2 IU of hepatitis E virus RNA per millilitre and, to
Before freezing: clear or slightly turbid liquid without visible test for inhibitors, an internal control prepared by addition of
signs of haemolysis; it may vary in colour from light yellow to a suitable marker to a sample of the plasma pool are included
green. in the test. The test is invalid if the positive control is non-
reactive or if the result obtained with the internal control
LABELLING indicates the presence of inhibitors. The pool complies with
The label enables each individual unit to be traced to a the test if it is found non-reactive for hepatitis E virus RNA.
specific donor.
Hepatitis E virus RNA for NAT testing ERP is suitable for use
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
as a positive control.
B19 virus DNA
The plasma pool contains not more than 10.0 IU/µL.
To limit the potential burden of B 19 virus in plasma pools,
Plasma (Pooled and Treated for the plasma pool is also tested for BI 9 virus using a validated
Virus Inactivation) nucleic acid amplification technique (2.6.21). A positive
control with I 0.0 IU of B 19 virus DNA per microlitre and,
(Human Plasma (Pooled and Treated for Virus
Inactivation), Ph. Bur. monograph 1646)
to test for inhibitors, an internal control prepared by addition
of a suitable marker to a sample of the plasma pool are
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
included in the test. The test is invalid if the positive control
DEFINITION is non-reactive or if the result obtained with the internal
Sterile, frozen or freeze-dried, non-pyrogenic preparation control indicates the presence of inhibitors.
obtained from human plasma derived from donors belonging E19 virus DNA for NAT testing ERP is suitable for use as a
to the same ABO blood group. The preparation is thawed or positive control.
reconstituted before use to give a solution for infusion. METHOD OF PREPARATION
The human plasma used complies with the monograph The method of preparation is designed to minimise activation
Human plasma for fractionation (0853). of any coagulation factor (to minimise potential
thrombogenicity) and includes a step or steps that have been
PRODUCTION
shown to inactivate known agents of infection; if substances
The units of plasma to be used are cooled to -30 °C or
are used for the inactivation of viruses during production, the
lower within 6 h of separation of cells and always within 24 h
subsequent purification procedure must be validated to
of collection.
demonstrate that the concentration of these substances is
The pool is prepared by mixing units of plasma belonging to reduced to a suitable level and that any residues are such as
the same ABO blood group. not to compromise the safety of the preparation for patients.
PIASMA POOL TESTS Inactivation process
The pool of plasma is tested for hepatitis B surface antigen The solvent-detergent process, which is one of the methods
(HBsAg) and for HN antibodies using test methods of used to inactivate enveloped viruses, uses treatment with a
suitable sensitivity and specificity; the pool must give negative combination of tributyl phosphate and octoxinol I O; these
results in these tests. reagents are subsequently removed by oil extraction or by
Hepatitis A virus RNA solid phase extraction so that the amount in the final product
The plasma pool is tested using a validated nucleic acid is less than 2 µg/mL for tributyl phosphate and less than
amplification technique (2.6.21). A positive control with 5 µg/mL for octoxinol I 0.
1.0 x 102 IU of hepatitis A virus RNA per millilitre and, to No antimicrobial preservative is added.
test for inhibitors, an internal control prepared by addition of The solution is passed through a bacteria-retentive filter,
a suitable marker to a sample of the plasma pool are included distributed aseptically into the final containers and
in the test. The test is invalid if the positive control is non- immediately frozen; it may subsequently be freeze-dried.
reactive or if the result obtained with the internal control
indicates the presence of inhibitors. The pool complies with Plastic containers comply with the requirements for sterile
the test if it is found non-reactive for hepatitis A virus RNA. plastic containers for human blood and blood components
(3.3.4).
Hepatitis A virus RNA for NAT testing ERP is suitable for use
as a positive control. Glass containers comply with the requirements for glass
containers for pharmaceutical use (3.2.1).
Hepatitis C virus RNA
The plasma pool is tested using a validated nucleic acid
amplification technique (2.6.21). A positive control with
CHARACTERS Detection Spectrophotometer at 215 nm.

Appearance Equilibration 15 min.
- frozen preparation: clear or slightly opalescent liquid, free Injection 10 µL.
from solid and gelatinous particles after thawing;
Retention time Citrate = about 10 min.
- freeze-dried preparation: almost white or slightly yellow
powder or friable solid mass. Limit:
- citrate: maximum 25 mmoliL.
Thaw or reconstitute the preparation to be examined as stated on
the label immediately before carrying out the identification, tests Calcium
and assay. Maximum 5.0 mmol/L.
IDENTIFICATION Atomic absorption spectrometry (2.2.23, Method l).
A. Examine by electrophoresis (2.2.31) comparing with Source Calcium hollow-cathode lamp using a transmission
normal human plasma. The electropherograms show the band preferably of 0.5 nm.
same bands. Wavelength 622 nm.
B. It complies with the test for anti-A and anti-B Atomisation device Air-acetylene or acetylene-propane flame.
haemagglutinins (see Tests). Potassium
TESTS Maximum 5.0 mmoliL.
pH (2.2.3) Atomic emission spectrometry (2.2.22, Method l).
6.5 to 7.6. Wavelength 766.5 nm.
psmolality (2.2.35) Sodium
Minimum 240 mosmolikg. Maximum 200 mmoliL.
Total protein Atomic emission spectrometry (2.2.22, Method l).
Minimum 45 g/L. Wavelength 589 nm.
Dilute if necessary with a 9 g/L solution of sodium cldoride R Water
to obtain a protein concentration of about 7.5 mg/mL. Place Determined by a suitable method, such as the semi-micro
2.0 mL of this solution in a round-bottomed centrifuge tube determination of water (2.5.12), loss on drying (2.2.32) or
and add 2 mL of a 75 g/L solution of sodium molybdate Rand near-infrared spectroscopy (2.2.40), the water content is
2 mL of a mixture of 1 volume of nitrogen-free sulfuric acid R within the limits approved by the competent authority
and 30 volumes of water R. Shake, centrifuge for 5 min, (freeze-dried product).
decant the supernatant and allow the inverted tube to drain
on filter paper. Determine the nitrogen in the residue by the Sterility (2. 6.1)
method of sulfuric acid digestion (2. 5. 9) and calculate the It complies with the test.
quantity of protein by multiplying the result by 6.25. Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
Activated coagulation factors (2.6.22) It complies with the test for pyrogens or, preferably and
It complies with the test for activated coagulation factors. where justified and authorised, with a validated in vitro test
Carry out the test with O. I mL of the preparation to be such as the bacterial endotoxin test.
examined instead of 10-fold and 100-fold dilutions. For the pyrogen test, inject 3 mL per kilogram of the rabbit's
The coagulation time for the preparation to be examined is mass.
not less than 150 s. Where the bacterial endotoxin test is used, the preparation to
Anti-A and anti-B haemagglutinins (2.6.20, Method A) be examined contains less than 0.1 IU of endotoxin per
The presence of haemagglutinins (anti-A or anti-B) millilitre.
corresponds to the blood group stated on the label. ASSAY
Hepatitis A virus antibodies Assay of human coagulation factor VIII (2.7.4)
Minimum 0.3 IU/mL, determined by a suitable Use a reference plasma calibrated against the International
immunochemical method (2. 7.1). Standard for blood coagulation factor VIII in plasma.
Human hepatitis A immunoglobulin BRP is suitable for use as a The estimated potency is not less than 0.5 IU/mL.
reference preparation. The confidence limits (P = 0.95) are not less than
Irregular erythrocyte antibodies 80 per cent and not more than 120 per cent of the estimated
The preparation to be examined does not show the presence potency.
of irregular erythrocyte antibodies when examined without Assay of human coagulation factor V
dilution by an indirect antiglobulin test. Carry out the assay of human coagulation factor V described
Citrate below using a reference plasma calibrated against the
Liquid chromatography (2.2.29). International Standard for blood coagulation factor V in
plasma.
Test solution Dilute the preparation to be examined with an
equal volume of a 9 g/L solution of sodium cldoride R. Filter Using imidazole buffer solution pH 7.3 R, prepare at least
through a membrane filter (nominal pore size 0.45 µm). 3 twofold dilutions of the preparation to be examined,
preferably in duplicate, from 1 in 10 to 1 in 40. Test each
Reference solution Dissolve 0.300 g of sodium citrate R in
dilution as follows: mix 1 volume of plasma substrate deficient
water Rand dilute to 100.0 mL with the same solvent.
in factor V R, 1 volume of the dilution to be examined,
Column: 1 volume of thromboplastin Rand 1 volume of a 3.5 g/L
- size: l =0.3 m, 0 =7.8 mm; solution of calcium chloride R; measure the coagulation times,
- stationary phase: cation-exchange resin R (9 µm). i.e. the interval between the moment at which the calcium
Mobile phase 0.51 g/L solution of sulfuric acid R. chloride solution is added and the 1st indication of the
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 (for
example, 5.3).
The estimated potency is not less than 0.5 IU/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
Use a reference plasma calibrated against the International Human Albumin
Standard for blood coagulation factor XI in plasma. (Human Albumin Solution, Ph. Bur. monograph 0255)
The estimated potency is not less than 0.5 IU/mL.
80 per cent and not more than 125 per cent of the estimated DEFINITION
potency. Sterile liquid preparation of a plasma protein fraction
Coagulation f acwrs V, VIII, XI and XIII plasma ERP is containing human albumin. It is obtained from plasma that
suitable for use as a reference preparation in the above complies with the monograph Human plasma for fractionation
assays. (0853). The preparation may contain excipients such as
sodium caprylate (sodium octanoate) or N-acetyltryptophan
Assay of human protein C (2. 7.30) or a combination of the two.
Use a reference plasma calibrated against the International
Standard for human protein C in plasma. PRODUCTION
The estimated potency is not less than 0.7 IU/mL. Separation of the albumin is carried out under controlled
The confidence limits (P = 0.95) are not less than conditions, particularly of pH, ionic strength and temperature
80 per cent and not more than 120 per cent of the estimated so that in the final product not less than 95 per cent of the
potency. total protein is albumin. Human albumin solution is prepared
as a concentrated solution containing 150-250 g/L of total
Assay of human protein S (2.7.31) protein or as an isotonic solution containing 35-50 g/L of
Use a reference plasma calibrated against the International total protein. No antimicrobial preservative or antibiotic is
Standard for human protein S in plasma. added. The solution is passed through a bacteria-retentive
The estimated potency is within the limits approved for the filter and distributed aseptically into sterile containers which
particular product. The confidence limits (P = 0.95) are not are then closed so as to prevent contamination. The solution
less than 80 per cent and not more than 120 per cent of the in its final container is heated to 60 ± 1.0 °C and
estimated potency. maintained at this temperature for not less than 10 h.
Assay of human plasmin inhibitor (2.7.25) The containers are then incubated at 30-32 °C for not less
(Cl'.2 -antiplasmin) than 14 days or at 20-25 °C for not less than 4 weeks and
Use a reference plasma calibrated against human normal examined visually for evidence of microbial contamination.
plasma. CHARACTERS
1 unit of human plasmin inhibitor is equal to the activity of Appearance
1 mL of human normal plasma. Human normal plasma is Clear, slightly viscous liquid, almost colourless, yellow, amber
prepared by pooling plasma units from not fewer than or green.
30 donors and storing at -30 °C or lower.
IDENTIFICATION
The estimated potency is not less than 0.2 units/mL. Examine by a suitable immunoelectrophoresis technique.
The confidence limits (P = 0.95) are not less than Using antiserum to normal human serum, compare normal
80 per cent and not more than 120 per cent of the estimated human serum and the preparation to be examined, both
potency. diluted to contain 10 g/L of protein. The main component of
Activated partial thromboplastin time (APTI) the preparation to be examined corresponds to the main
Use an apparatus suitable for measurement of coagulation component of normal human serum. The preparation may
times or perform the assay with incubation tubes maintained show the presence of small quantities of other plasma
in a water-bath at 37 °C. Place in each tube 0.1 mL of the proteins.
preparation to be examined and 0.1 mL of a suitable APTT
TESTS
reagent (containing phospholipid and contact activator), both
previously heated to 37 °C, and incubate the mixture for a pH (2.2.3)
recommended time at 37 °C. To each tube add 0.1 mL of a 6.7 to 7.3.
3.7 g/L solution of calcium chloride R previously heated to Dilute the preparation to be examined with a 9 g/L solution
37 °C. Using a timer, measure the coagulation time, of sodium chloride R to obtain a solution containing 10 g/L of
i.e. the interval between the moment of the addition of the protein.
calcium chloride and the 1st indication of the formation of Total protein
fibrin, which may be observed visually or by means of a If necessary, dilute an accurately measured volume of the
suitable apparatus. The volumes given above may be adapted preparation to be examined with a 9 g/L solution of sodium
to the APTT reagent and apparatus used. The coagulation chloride R to obtain a solution containing about 15 mg of
protein in 2 mL. To 2.0 mL of this solution in a round- Mobile phase Dissolve 1. 7 41 g of sodium dihydrogen phosphate
bottomed centrifuge tube add 2 mL of a 75 g/L solution of monohydrate R, 4.873 g of disodium hydrogen phosphate
sodium molybdate R and 2 mL of a mixture of 1 volume of dihydrate Rand 11.688 g of sodium chloride R in water for
nitrogen-free sulfuric acid R and 30 volumes of water R. Shake, chromatography R and dilute to 1000 mL with the same
centrifuge for 5 min, decant the supernatant and allow the solvent.
inverted tube to drain on filter paper. Determine the nitrogen Flow rate 0.5 mLJmin.
in the residue by the method of sulfuric acid digestion (2. 5. 9)
and calculate the quantity of protein by multiplying by 6.25.
The protein content is not less than 95 per cent and not Injection 20 µL.
more than 105 per cent of the stated content. Run time Twice the retention time of the monomer.
Protein composition Identification of peaks Use the chromatogram supplied with
Zone electrophoresis (2.2.31). human albumin (molecular size) ERP and the chromatogram
obtained with reference solution (a) to identify the peaks due
Use strips of suitable cellulose acetate gel or agarose gel as
to the monomer and the dimer.
the supporting medium and barbital buffer solution pH 8. 6 Rl
as the electrolyte solution. Relative retention With reference to the monomer (retention
If cellulose acetate is the supporting material, the method
= =
time about 17 min): dimer about 0.88.
described below can be used. If agarose gels are used, and System suitability:
because they are normally part of an automated system, the - the chromatogram obtained with reference solution (a) is
manufacturer's instructions are followed instead. qualitatively similar to the chromatogram supplied with
human albumin (molecular size) ERP,
Test solutwn Dilute the preparation to be examined with a
- signal-to-noise ratio: minimum 10 for the peak due to the
9 g/L solution of sodium chloride R to obtain a protein
monomer in the chromatogram obtained with reference
concentration of 20 g/L.
solution (b);
Reference solutwn Dilute human albumin for - resolution: minimum 1.5 between the peaks due to the
electrophoresis ERP with a 9 g/L solution of sodium chloride R dimer and the monomer in the chromatogram obtained
to obtain a protein concentration of 20 g/L. with reference solution (a).
To a strip apply 2.5 µL of the test solution as a 10 mm band Limit:
or apply 0.25 µL per millimetre if a narrower strip is used. - peak due to the polymers and aggregates eluted with the void
To another strip, apply in the same manner the same volume volume: maximum 10 per cent;
of the reference solution. Apply a suitable electric field such - reporting threshold: 0.5 per cent (peak due to the monomer
that the most rapid band migrates at least 30 mm. Treat the in the chromatogram obtained with reference
strips with amido black 10B solutwn R for 5 min. Decolourise solution (b)).
with a mixture of 10 volumes of glacial acetic acid R and
90 volumes of methanol R until the background is just free of Haem
colour. Develop the transparency of the strips with a mixture Dilute the preparation to be examined using a 9 g/L solution
of 19 volumes of glacial acetic acid R and 81 volumes of of sodium chloride R to obtain a solution containing 10 g/L of
methanol R. Measure the absorbance of the bands at 600 nm protein. The absorbance (2.2.25) of the solution measured at
in an instrument having a linear response over the range of 403 nm using water R as the compensation liquid is not
measurement. Calculate the result as the mean of greater than 0.15.
3 measurements of each strip. Prekallikrein activator (2. 6.15)
System suitability In the electropherogram obtained with the Maximum 35 IU/mL.
reference solution on cellulose acetate or on agarose gels, the Aluminium
proportion of protein in the principal band is within the Maximum 200 µg/L.
limits stated in the leaflet accompanying the reference Atomic absorption spectrometry (2.2.23, Method I or II).
preparation. Use a furnace as atomic generator.
Results In the electropherogram obtained with the test Use plastic containers for preparation of the solutions and use
solution on cellulose acetate or on agarose gels, not more plastic equipment where possible. Wash glassware (or equipment)
than 5 per cent of the protein has a mobility different from in nitric acid (200 g/L HNO:,) before use.
that of the principal band.
Test solutwn Use the preparation to be examined, diluted if
Molecular-size distribution necessary.
Size-exclusion chromatography (2.2.30): use the
Reference solutions Prepare at least 3 reference solutions in a
normalisation procedure.
range spanning the expected aluminium concentration of the
Test solutwn Dilute the preparation to be examined with the test solution, for example by diluting aluminium standard
mobile phase to obtain a protein concentration of I O g/L. solution (10 ppm AV R with a 1 g/L solution of octoxinol 10 R.
Reference solution (a) Dilute human albumin (molecular Monitor solution Add aluminium standard solution
size) ERP with the mobile phase to obtain a protein (10 ppm AV R or a suitable certified reference material to the
concentration of 10 g/L. test solution in a sufficient amount to increase the aluminium
Reference solution (b) Dilute 0.25 mL of reference concentration by 20 µg/L.
solution (a) to 50.0 mL with the mobile phase. Blank solution 1 g/L solution of octoxinol 10 R.
Column: Wavelength 309.3 nm or other suitable wavelength.
- size: l =0.30 m, 0 = 7.8 mm;
Slit width 0.5 nm.
- stationary phase: hydrophilic silica gel for chromatography R
(5 µm) with a pore size of 25-30 nm and of a grade Tube Pyrolytically coated, with integrated platform.
suitable for fractionation of globular proteins in the Background corrector Off.
relative molecular mass range of 10 000 to 500 000.
Awmisation device Furnace; fire between readings. - the content of protein expressed in grams per litre;
The operating conditions in Table 0255.-1 are cited as an - the content of sodium expressed in millimoles per litre;
example of conditions found suitable for a given apparatus; - that the product is not to be used if it is cloudy or if a
they may be modified to obtain optimum conditions. deposit has formed;
- the name and quantity of any added substance.
Table 0255.-1. - Operating conditions found suitable, cited as an _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
example
Step Final Ramp time Hold time Gas
temperature (s) (s)
('C)
120 10 80 argon
Antithrombin Ill Concentrate
2 200 5 20 argon
(Human Antithrombin 111 Concentrate, Ph. Eur.
3 650 5 10 argon
monograph 0878)
4 1300 5 10 argon
5 1300 1 10 no gas Action and use
6 2500 0,7 4 no gas Anticoagulant factor.
7 2600 05 3 argon PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
8 20 12.9 3 no gas
DEFINITION
Injection Each of the following solutions 3 times: blank Sterile, freeze-dried preparation of a plasma glycoprotein
solution, reference solutions, test solution and monitor fraction that inactivates thrombin in the presence of an excess
solution. of heparin. It is obtained from human plasma that complies
with the monograph on Human plasma for
System suitabi7ity:
fractionation (0853). The preparation may contain excipients
- the recovery of aluminium added in preparation of the
such as stabilisers.
monitor solution is within the range 80-120 per cent.
When reconstituted in the volume of solvent stated on the
Prepare a calibration curve from the mean of the readings
label, the potency is not less than 25 IU of antithrombin III
obtained with the reference solutions and determine the
per millilitre.
aluminium content of the preparation to be examined using
the calibration curve. PRODUCTION
Potassium The method of preparation is designed to maintain
Maximum 0.05 mmol of K per gram of protein. functional integrity of antithrombin III. It includes a step or
steps that have been shown to remove or to inactivate known
Atomic emission spectrometry (2.2.22, Method 1).
agents of infection; if substances are used for inactivation of
Wavelength 766.5 nm. viruses during production, the subsequent purification
Sodium procedure must be validated to demonstrate that the
Maximum 160 mmol/L and 95 per cent to 105 per cent of concentration of these substances is reduced to a suitable
the content of Na stated on the label. level and any residues are such as not to compromise the
Atomic emission spectrometry (2.2.22, Method 1). safety of the preparation for patients.
Wavelength 589 nm. The specific activity is not less than 3 IU of antithrombin III
per milligram of total protein, excluding albumin.
Sterility (2. 6.1)
It complies with the test. The antithrombin III is purified and concentrated.
No antimicrobial preservative or antibiotic is added.
Pyrogens (2. 6. 8) or Bacterial endotoxins (2. 6.14) The antithrombin III concentrate is passed through a
It complies with the test for pyrogens or, preferably and bacteria-retentive filter, distributed aseptically into its final,
where justified and authorised, with a validated in vitro test
sterile containers and immediately frozen. It is then freeze-
such as the bacterial endotoxin test.
dried and the containers are closed under vacuum or in an
For the pyrogen test, for a solution with a protein content of atmosphere of inert gas.
35-50 g/L, inject 10 mL per kilogram of the rabbit's mass;
It shall be demonstrated that the manufacturing process
for a solution with a protein content of 150-250 g/L, inject
yields a product with a consistent fraction of antithrombin III
5 mL per kilogram of the rabbit's mass.
able to bind to heparin. It is evaluated by a suitable analytical
Where the bacterial endotoxin test is used, the preparation to procedure which is determined during process development,
be examined contains less than 0.5 IU of endotoxin per such as:
millilitre for solutions with a protein content not greater than Heparin-binding fraction Examine by agarose gel
50 g/L, less than 1.3 IU of endotoxin per millilitre for
electrophoresis (2.2.31). Prepare a 10 g/L solution of agarose
solutions with a protein content greater than 50 g/L but not for electrophoresis R containing 15 IU of heparin R per millilitre
greater than 200 g/L, and less than 1. 7 IU of endotoxin per in barbital buffer solution pH 8. 4 R. Pour 5 mL of this solution
millilitre for solutions with a protein content greater than onto a glass plate 5 cm square. Cool at 4 °C for 30 min.
200 g/L but not greater than 250 g/L.
Cut 2 wells 2 mm in diameter 1 cm and 4 cm from the side
STORAGE of the plate and 1 cm from the cathode. Introduce into one
Protected from light. well 5 µL of the preparation to be examined, diluted to an
activity of about 1 IU of antithrombin III per millilitre.
LABELLING
Introduce into the other well 5 µL of a solution of a marker
The label states:
dye such as bromophenol blue R. Allow the electrophoresis to
- the name of the preparation;
proceed at 4 °C, using a constant electric field of 7 V/cm,
- the volume of the preparation;
until the dye reaches the anode.
Cut across the agarose gel 1.5 cm from that side of the plate reference preparation of heparin calibrated in International
on which the preparation to be examined was applied and Units.
remove the larger portion of the gel leaving a band 1.5 cm The International Unit is the activity contained in a stated
wide containing the material to be examined. Replace the amount of the International Standard, which consists of a
removed portion with an even layer consisting of 3.5 mL of a quantity of freeze-dried heparin sodium from pork intestinal
10 g/L solution of agarose for electrophoresis R in barbital buffer mucosa. The equivalence in International Units of the
solution pH 8. 4 R, containing a rabbit anti-human International Standard is stated by the World Health
antithrombin III antiserum at a suitable concentration, Organization.
previously determined, to give adequate peak heights of at Heparin sodium ERP is calibrated in International Units by
least 1.5 cm. Place the plate with the original gel at the comparison with the International Standard by means of the
cathode so that a 2nd electrophoretic migration can occur at assay given below.
right angles to the 1st. Allow this 2nd electrophoresis to
proceed using a constant electric field of 2 V/cm for 16 h. Carry out the assay using one of the following methods for
Cover the plates with filter paper and several layers of thick determining the onset of clotting and using tubes and other
lint soaked in a 9 g/L solution of sodium chloride R and equipment appropriate to the chosen method:
compress for 2 h, renewing the saline several times. Rinse a) direct visual inspection, preferably using indirect
with water R, dry the plates and stain with acid blue 92 illumination and viewing against a matt black background;
solution R. b) spectrophotometric recording of the change in optical
Calculate the fraction of antithrombin III bound to heparin, density at a wavelength of approximately 600 nm;
which is the peak closest to the anode, with respect to the c) visual detection of the change in fluidity on manual tilting
total amount of antithrombin III, by measuring the area of the tubes;
defined by the 2 precipitation peaks. d) mechanical recording of the change in fluidity on stirring,
The fraction of antithrombin III able to bind to heparin is care being taken to cause the minimum disturbance of the
not less than 60 per cent. solution during the earliest phase of clotting.
CHARACTERS It is necessary to validate the method for assay of heparin for
Appearance each preparation to be examined to allow for interference by
White or almost white, hygroscopic, friable solid or powder. antithrombin III.
Reconstitute the preparation to be examined as stated on the label ASSAY PROCEDURE
immediately before canying out the identification, tests (except The volumes are given as examples and may be adapted to the
those for solubility, total protein and water) and assay. apparatus used provided that the ratios between the different
volumes are respected.
IDENTIFICATION
It complies with the limits of the assay. Dilute heparin sodium ERP with a 9 g/L solution of sodium
chloride R to contain a precisely known number of
TESTS International Units per millilitre and prepare a similar
Solubility solution of the preparation to be examined which is expected
To a container of the preparation to be examined add the to have the same activity. Using a 9 g/L solution of sodium
volume of liquid stated on the label at the recommended chloride R, prepare from each solution a series of dilutions in
temperature. The preparation dissolves completely under geometric progression such that the clotting time obtained
gentle swirling within 10 min in the volume of the solvent with the lowest concentration is not less than 1.5 times the
stated on the label, forming a clear or slightly turbid, blank recalcification time, and that obtained with the highest
colourless or almost colourless solution. concentration is such as to give a satisfactory log dose-
pH (2.2.3) response curve, as determined in a preliminary test.
6.0 to 7.5. Place 12 tubes in a bath of iced water, labelling them in
Osmolality (2.2.35) duplicate: T 1, T 2 and T 3 for the dilutions of the preparation
Minimum 240 mosmol/kg. to be examined and S 1, S2 and S3 for the dilutions of the
reference preparation. To each tube add 1.0 mL of thawed
Total protein
plasma substrate Rl and 1.0 mL of the appropriate dilution of
If necessary, dilute an accurately measured volume of the
the preparation to be examined or the reference preparation.
reconstituted preparation to obtain a solution containing
After each addition, mix but do not allow bubbles to form.
about 15 mg of protein in 2 mL. To 2.0 mL of the solution
Treating the tubes in the order S 1, S2, S3, T 1, T 2 , T3,
in a round-bottomed centrifuge tube add 2 mL of a 75 g/L
transfer each tube to a water-bath at 37 cc, allow to
solution of sodium molybdate R and 2 mL of a mixture of
equilibrate at 37 cc for about 15 min and add to each tube
1 volume of nitrogen-free sulfuric acid R and 30 volumes of
1 mL of a suitable APTT (Activated Partial Thromboplastin
water R. Shake, centrifuge for 5 min, decant the supernatant
Time) reagent containing phospholipid and a contact
and allow the inverted tube to drain on filter paper.
activator, at a dilution giving a suitable blank recalcification
Determine the nitrogen in the residue by the method of
time not exceeding 60 s. After exactly 2 min add 1 mL of a
sulfuric acid digestion (2.5.9) and calculate the amount of
3.7 g/L solution of calcium chloride R previously heated to
protein by multiplying the result by 6.25.
37 °C and record as the clotting time the interval in seconds
Heparin between this last addition and the onset of clotting
Maximum 0.1 IU of heparin per International Unit of determined by the chosen technique. Determine the blank
antithrombin III. recalcification time at the beginning and at the end of the
The anticoagulant activity of heparin is determined in vitro by procedure in a similar manner, using 1 mL of a 9 g/L
comparing its ability in given conditions to delay the clotting solution of sodium chloride R in place of one of the heparin
of recalcified citrated sheep plasma with the same ability of a dilutions; the 2 blank values obtained should not differ
significantly. Transform the clotting times to logarithms,
using the mean value for the duplicate tubes. Repeat the coagulation factor VII and may also contain small amounts
procedure using fresh dilutions and carrying out the of the activated form, the 2-chain derivative human
incubation in the order T 1, T 2, T 3, Si, S 2 , S3 . Calculate the coagulation factor VIia. It may also contain human
results by the usual statistical methods (5.3). coagulation factors II, IX and X, protein C and protein S.
Carry out not fewer than 3 independent assays. For each It is obtained from human plasma that complies with the
such assay prepare fresh solutions of the reference monograph on Human plasma for fractionation (0853).
preparation and the preparation to be examined and use The preparation may contain excipients such as stabilisers,
another, freshly thawed portion of plasma substrate. heparin and antithrombin.
Calculate the potency of the preparation to be examined, The potency of the preparation, reconstituted as stated on
combining the results of these assays, by the usual statistical the label, is not less than 15 IU of human coagulation
methods (5.3). When the variance due to differences between factor VII per millilitre.
assays is significant at P = 0.01, a combined estimate of PRODUCTION
potency may be obtained by calculating the non-weighted GENERAL PROVISIONS
mean of potency estimates. The method of preparation is designed to maintain
Water functional integrity of human coagulation factor VII and to
Determined by a suitable method, such as semi-micro minimise activation of any coagulation factor (to minimise
determination of water (2.5.12), loss on drying (2.2.32) or potential thrombogenicity). It includes a step or steps that
near-infrared spectroscopy (2.2.40), the water content is have been shown to remove or to inactivate known agents of
within the limits approved by the competent authority. infection; if substances are used for inactivation of viruses
Sterility (2. 6.1) during production, the subsequent purification procedure
It complies with the test. must be validated to demonstrate that the concentration of
these substances is reduced to a suitable level and that any
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14) residues are such as not to compromise the safety of the
It complies with the test for pyrogens or, preferably and preparation for patients.
such as the bacterial endotoxin test. The specific activity is not less than 2 IU of human
coagulation factor VII per milligram of total protein, before
For the pyrogen test, inject per kilogram of the rabbit's mass
the addition of any protein stabiliser.
a volume equivalent to 50 IU of antithrombin III.
The human coagulation factor VII fraction is dissolved in a
Where the bacterial endotoxin test is used, the preparation to suitable liquid. No antimicrobial preservative or antibiotic is
be examined contains less than 0.1 IU of endotoxin per added. The solution is passed through a bacteria-retentive
International Unit of antithrombin III. filter, distributed aseptically into the final containers and
ASSAY immediately frozen. It is subsequently freeze-dried and the
Human antithrombin III (2. 7.17) containers are closed under vacuum or under an inert gas.
The estimated potency is not less than 80 per cent and not CONSISTENCY OF THE METHOD OF
more than 120 per cent of the stated potency. PRODUCTION
The confidence limits (P = 0.95) are not less than It shall be demonstrated that the manufacturing process
90 per cent and not more than 110 per cent of the estimated yields a product with consistent activities of human
potency. coagulation factors II, IX and X, expressed in International
STORAGE Units relative to the activity of human coagulation factor VII.
Protected from light, in an airtight container. This is evaluated by suitable analytical procedure(s) that is
(are) determined during process development.
LABELLING
It shall be demonstrated that the manufacturing process
The label states:
yields a product with a consistent activity of human
- the number of International Units of antithrombin III in
coagulation factor VIia. This is evaluated by suitable
the container;
analytical procedure(s) that is (are) determined during
- the name and volume of the liquid to be used for
process development.
reconstitution;
- where applicable, the amount of albumin added as a Activity of human coagulation factor VIia
stabiliser. It may be determined, for example, using a recombinant
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
soluble tissue factor that does not activate human coagulation
factor VII but possesses a cofactor function specific for
human coagulation factor VIia; after incubation of a mixture
of the recombinant soluble tissue factor with phospholipids
reagent and the dilution of the test sample in human
Dried Factor VII Fraction coagulation factor VII-deficient plasma, calcium chloride is
added and the clotting time determined; the clotting time is
(Human Coagulation Factor VII, Ph. Bur. monograph
inversely related to the human coagulation factor VIia activity
1224)
of the test sample.
Action and use CHARACTERS
Coagulation factor VII substitute.
Appearance
PhEur - - - - - - - - - - - - - - - - - - - - - ~ White or almost white, pale yellow, green or blue,
hygroscopic powder or friable solid.
DEFINITION
Rec1J11stitute the preparation to be examined as stated on the label
Sterile, liquid or freeze-dried preparation of a plasma protein
immediately before carrying out the identification, tests (except
fraction containing the single-chain glycoprotein human
those for solubility and water) and assay.
IDENTIFICATION Water
It complies with the limits of the assay. Determined by a suitable method, such as the semi-micro
determination of water (2. 5. 12), loss on drying (2. 2. 32) or
TESTS
near-infrared spectrometry (2.2.40), the water content is
Solubility
within the limits approved by the competent authority.
To a container of the preparation to be examined add the
volume of liquid stated on the label at the recommended Sterility (2. 6.1)
temperature. The preparation dissolves completely with It complies with the test.
gentle swirling within 10 min, giving a clear or slightly Pyrogens (2. 6. 8) or Bacterial endotoxins (2. 6.14)
opalescent solution that may be coloured. It complies with the test for pyrogens or, preferably and
pH (2.2.3) where justified and authorised, with a validated in vitro test
6.5 to 7.5. such as the test for bacterial endotoxins.
Osmolality (2.2.35) For the pyrogen test, inject per kilogram of the rabbit's mass
Minimum 240 mosmol/kg. a volume equivalent to not less than 30 IU of human
coagulation factor VII.
Total protein
Where the test for bacterial endotoxins is used, the
preparation to be examined contains less than 0.1 IU of
reconstituted preparation with a 9 g/L solution of sodium
endotoxin per International Unit of human coagulation
chloride R to obtain a solution containing about 15 mg of
protein in 2 mL. To 2.0 mL of the solution in a round- factor VII.
bottomed centrifuge tube, add 2 mL of a 75 giL solution of ASSAY
sodium molybdate R and 2 mL of a mixture of 1 volume of Human coagulation factor VD (2. 7.10)
nitrogen-free suljuric acul R and 30 volumes of water R. Shake, The estimated potency is not less than 80 per cent and not
centrifuge for 5 min, decant the supernatant and allow the more than 125 per cent of the stated potency.
inverted tube to drain on filter paper. Determine the nitrogen The confidence limits (P = 0.95) are not less than
in the residue by the method of sulfuric acid digestion (2.5.9) 80 per cent and not more than 125 per cent of the estimated
and calculate the amount of protein by multiplying the result potency.
by 6.25.
STORAGE
Activated coagulation factors (2.6.22) In an airtight container, protected from light.
For each of the dilutions, the coagulation time is not less
than 150 s. LABELLING
The label states:
Heparin (2.7.12) - the number oflnternational Units of human coagulation
If heparin has been added, the preparation to be examined
factor VII per container;
contains not more than the amount of heparin stated on the - the maximum content of human coagulation factor II,
label and in any case not more than 0.5 IU of heparin per human coagulation factor IX and human coagulation
International Unit of human coagulation factor Vll. factor X per container, in International Units;
Thrombin - the amount of protein per container;
If the preparation to be examined contains heparin, - the name and quantity of any added substances,
determine the amount present as described in the test for including, where applicable, heparin;
heparin and neutralise the heparin by addition of protamine - the name and volume of the liquid to be used for
sulfate R (10 µg of protamine sulfate neutralises 1 IU of reconstitution;
heparin). In each of 2 test-tubes, mix equal volumes of the - that the transmission of infectious agents cannot be totally
reconstituted preparation and of a 3 giL solution of excluded when medicinal products prepared from human
fibrinogen R. Keep one of the tubes at 3 7 °C for 6 h and the blood or plasma are administered.
other at room temperature for 24 h. In a 3rd tube, mix equal _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
volumes of the fibrinogen solution and of a solution of
human thrombin R (I IU/mL) and place the tube in a water-
bath at 37 °C. No coagulation occurs in the tubes containing
the preparation to be examined. Coagulation occurs within
30 s in the tube containing throm~in.
Human coagulation factor II (:i.7.18)
The estimated content is not more than J 25 per cent of the
stated content. The confidence Iuhits (P = 0.95) are not less
than 90 per cent and not more than 111 per cent of the
estimated potency.
Human coagulation factor IX (2. 7.11)
The estimated content is not more than 125 per cent of the
stated content. The confidence limits (P = 0.95) are not less
estimated potency.
Human coagulation factor X (2.7.19)
The estimated content is not more than 125 per cent of the
stated content. The confidence limits (P = 0.95) are not less
estimated potency.
Host-cell- and vector-derived DNA

Factor VIia (rDNA) Concentrated The limit is approved by the competent authority.
Solution Glycan analysis
(Human Coagulation Facwr Vlla (rDNA) Use a suitable method developed according to general
Concentrated Solution, Ph. Bur. monograph 2534) chapter 2.2.59. Glycan analysis of glycoproteins.
Glycan analysis includes the following steps:
light chain
- after desalting, release of the glycans (see 2.2.59
ANAFLEELRP GSLERECKEE QCSFgARE:_I FKDAE:_RTKLF 40
section 2-3);
WISYSDGDQC ASSPCQNGGS CKQQLQSYIC FCLPAFEGRN 80
CETHKDDQLI CVNENGGCEQ YCSDHTGTKR SCRCHEGYSL 120
- labelling of the glycans with a suitable fluorescent label
LADGVSCTF':' VEYPCGKIPI LEKRNASKPQ GR 152
(Table 2.2.59.-2);
heavy chain
- analysis of the labelled glycans by liquid chromatography
IVGGKVCP 160
(2.2.29) with fluorometric detection.
KGECPWQVLL LVNGAQLCGG TLINTIWVVS AAHCFDKIKN 200 The following procedures may be used.
WRNLIAVLGE HDLSEHDGDE QSRRVAQVII PSTYVPGTTN 240
Test solution Dilute the preparation to be examined in
HDIALLRLHQ PVVLTDHVVP LCLPERTFSE RTLAFVRFSL 280
water R to obtain a concentration of about 1.5 mglmL.
VSGWGQLLDR GATALELMVL NVPRLMTQDC LQQSRKVGDS 320
PNITEYMFCA GYSDGSKDSC KGDSGGPHAT HYRGTWYLTG 360
Reference solution Dissolve human coagulation facwr VIIa
IVSWGQGCAT VGHFGVYTRV SQYIEWLQKL MRSEPRPGVL 400 (rDNA) CRS in water R to obtain a concentration of
LRAPFP 406 1.5 mglmL.
DESALTING
disulfide bridges
17-22. 50-61, 55-70, 72-81, 91-102, 98-112, 114-127, 135-262, 159-164. Desalt the test solution and the reference solution as
178-194, 310-329, 340-368 described under Identification B. The buffer used for
glycosylation sites: desalting and elution is a 1.21 g/L solution of
52, 60, 145, 322
tris(hydroxymethyl)aminomethane R, adjusted to pH 7.5 with
modified residues:
!; (4-carboxyGlu) at position 6, 7, 14, 16. 19, 20, 25, 26, 29, 35
hydrochloric acid R. After desalting, the concentration of the
solutions is about 1.0 mglmL.
potentially modified residue:
Q ((3R)-3-hydroxyAsp) at position 63 SELECTIVE RELEASE OF GLYCANS
H NH2 Transfer 500 µL of the desalted test solution and 500 µL of
H02C H NH 2 H02C, .X_ the desalted reference solution to separate centrifuge tubes,
H02C ~ C02H
f_ 'C02H and add 10 µL of a 200 U/mL solution of peptide
HO H
N-glycosidase FR. Cap the tubes and incubate for 16-24 hat
g = 4-carboxyGlu Q = (3R)-3-hydroxyAsp
37 °C. Remove the protein fraction by adding 1.5 mL of
ethanol (96 per cent) R at -20 °C to the tubes. Mix and allow
C19s2H3os4N55oO61sS2s Mr approx. 50 000 to stand at -20 °C for 20-30 min. Centrifuge the tubes at
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 10 000 r/min for 10 min. Collect the supernatant and
evaporate to dryness, using for example a rotary evaporator.
DEFINITION
Solution containing closely related glycoproteins, which have LABELLING OF GLYCANS
the same amino acid sequence (406 amino acids) and Label the liberated glycans with 2-aminobenzamide using a
disulfide bridges as the naturally occurring analogue (plasma- suitable procedure. The procedure employs a combination of
derived activated coagulation factor VII). Human coagulation reagents optimised and validated for the efficient labelling of
factor VIia (rDNA) (eptacog alfa, activated) is a 2-chain glycans, and for the subsequent extraction and recovery of
molecule, obtained by proteolytic cleavage of the peptide the labelled glycans from the reaction.
bond between Arg 152 and lle 153 of single-chain LIQUID CHROMATOGRAPHY (2.2.29)
coagulation factor VII, consisting of a 20 kDa light chain (N- Precolumn:
terminal) and a 30 kDa heavy chain CC-terminal) connected - size: l = 0.05m, 0 = 4.0 mm;
by a disulfide bond. - stationary phase: strongly basic anion-exchange resin for
Human coagulation factor VIia (rDNA) is distinguishable chromatography R.
from the naturally occurring analogue in terms of its post- Column:
translational modifications, including glycosylation pattern. - size: l = 0.25 m, 0 = 4.0 mm;
Content - stationary phase: strongly basic anion-exchange resin for
1.11 mg to 1. 78 mg of protein per millilitre. chromatography R;
Potency
44 000 IU to 64 000 IU per milligram of protein. Mobile phase:
- mobile phase A: 6 g/L solution of sodium hydroxide R;
PRODUCTION - mobile phase B: solution containing 6 g/L of sodium
Human coagulation factor VIia (rDNA) is produced in hydroxide Rand 40.8 g/L of sodium acetate R;
mammalian cells by a method based on recombinant DNA
technology (rDNA). Time Mobile phase A Mobile phase B
Prior to release, the following tests are carried out on each (min) (per cent VIP) (per cent VIP)
batch of human coagulation factor VIia (rDNA) 0 - 52 100 ➔ 35 0 ➔ 65
concentrated solution, unless exemption has been granted by 52.0 - 52.1 35 ➔ 0 65 ➔ 100
the competent authority. 52.1 - 65 0 100
65 - 65.1 0 ➔ 100 100 ➔ 0
Host-cell-derived proteins
65.1-90 100 0
The limit is approved by the competent authority.
Fww rate 0.5 mIJmin. Mobile phase:

Detection Fluorimeter at 330 run for excitation and 420 nm - mobile phase A: add 0.65 mL of trifluoroacetic acid R to
for emission. 1000 mL of water for chromatography R and degas;
Injection l 00 µL, using an automatic injector maintained at - mobile phase B: mix 0.5 mL of trifluoroacetic acid R,
2-8 °C. 100 mL of water for chromatography R and 900 mL of
acetonitrile Rl and degas;
- the chromatogram obtained is qualitatively similar to the Time Mobile phase A Mobile phase B
chromatogram shown in Figure 2534.-1; peaks 1 to 12 (min) (per cent V/Jl) (per cent V/Jl)
are clearly visible; 0 - 100 100 ➔ 50 0-+ 50
- peak width at half-height: maximum 30 s for peak 8. 100 - 105 50-+ 0 50--, JOO
Calculate the percentage content of charged glycans in the 105 - 110 0 100
reference solution using the following expression: 110 - 110.l 0-+ JOO 100 __, 0
110.1 - 125 JOO 0
A
A+B x 100 Fww rate 1.0 mIJmin.
Detection Spectrophotometer at 215 run.
A sum of the areas of the peaks due to charged glycans (peaks
6 to 12);
Injection 25 µL.
B sum of the areas of the peaks due to uncharged glycans (peaks System suitability The chromatogram obtained with the
I to 5). reference solution is qualitatively similar to the chromatogram
supplied with human coagulation factor Vila (rDNA) CRS.
Calculate the percentage content of charged glycans in the
Results The profile of the chromatogram obtained with the
test solution accordingly.
test solution corresponds to that of the chromatogram
Limit: obtained with the reference solution:
- percentage of charged glycans: as authorised by the - all major peaks identified in the chromatogram
competent authority. obtained with the reference solution are present in the
CHARACTERS chromatogram obtained with the test solution;
Appearance - no new major peaks are observed in the
Colourless liquid. chromatogram obtained with the test solution in
comparison with the chromatogram obtained with the
IDENTIFICATION reference solution.
A. It forms a clot when examined in the conditions described
under Assay (Potency).
glycan analysis.
B. Peptide mapping (2.2.55).
Results The profile of the chromatogram obtained with the
SELECTIVE CLEAVAGE OF THE PEPTIDE BONDS test solution corresponds to that of the chromatogram
Solutwn A Dissolve 0.74 g of calcium chwride Rand 6.06 g obtained with the reference solution.
of tris (hydroxymethyl) aminomethane R in 1000 mL of water R
and adjust to pH 7 .5 with hydrochwric acid R.
TESTS
Degraded heavy chain and oxidised forms of human
Test solutwn Dilute the preparation to be examined with coagulation factor Vila (rDNA)
solution A to obtain a concentration of about 1.5 mg/mL. Liquid chromatography (2.2.29): use the normalisation
Desalt a volume of this solution by a suitable method (for procedure.
example using a suitable centrifugal filter unit or gel-filtration
column with solution A as elution buffer). After desalting, Test solutwn Dilute the preparation to be examined in
the concentration should be about 1.0 mg/mL. Transfer the water R to obtain a concentration of about 1.5 mg/mL.
desalted solution to a polypropylene tube. Prepare a Reference solutwn Dissolve human coagulation factor VIIa
1 mg/mL solution of trypsin for peptide mapping R and add (rDNA) CRS in water R to obtain a concentration of
10 µL to 1 mL of the desalted solution. Cap the tube and 1.5 mg/mL.
mix gently by inversion. Incubate at 37 °C for 24 h. At time Column:
5. 5 h, add 10 µL of the trypsin solution. Remove the sample - size: l = 0.25 m, 0 = 4.0 mm;
from the incubator, place it at room temperature, add 9 µL - statwnary phase: end-capped butylsilyl silica gel for
of glacial acetic acid R and mix by inversion. Maintain the chromatography R (5 µm) with a pore size of 30 nm;
solution at -15 °C or below until chromatographic - temperature: 60-70 °C.
separation; if analysed immediately using an automatic Mobile phase:
injector, maintain at 2-8 °C. - mobile phase A: mix 1 mL of trifluoroacetic acid R and
Reference solutwn Dissolve human coagulatwn factor VIia 999 mL of water for chromatography R and degas;
(rDNA) CRS in solution A to obtain a concentration of - mobile phase B: mix 1 mL of trifluoroacetic acid R, 200 mL
1.5 mg/mL. Desalt and digest at the same time and in the of water for chromatography R and 800 mL of
same manner as for the test solution. acetonitrile Rl and degas;
CHROMATOGRAPHIC SEPARATION Mobile phase A
Time Mobile phase B
Liquid chromatography (2.2.29). (min) (per cent V/Jl) (per cent V/Jl)
Column: 0 - 30 54-+ 41 46--, 59
- size: l = 0.25 m, 0 = 4.0 mm; 30 - 33 41--, 0 59 --, 100
- stationary phase: octadecylsilyl silica gel for 33 - 38 0 100
chromatography R (5 µm) with a pore size of 30 run; 38 - 40 0--, 54 JOO --, 46
6 7 8
11
12
10
I I
10 15 20 25 30 35 40 45 50 55 min
Peak Charged Structure Peak Charged Structure

I. No Core fucosylated biantennaty - non sialylated 7. Yes Core fucosylated biantennacy -
(2 N-acerylgalactosamine terminals) monosialylated (and I galactose terminal)
2. No Core fucosylated biantennacy - non sialylated (N- 8. Yes Core fucosylated biantennacy -
acetylgalactosamine and galactose terminals) bisialylated
3. No Structure not determined 9. Yes High-mannose structure with
I phosphate group
4. No Core fucosylated biantennaty - non sialylated 10. Yes Core fucosylated triantennacy -
(galactose and N-acetylglucosamine terminals) trisialylated
5. No Core fucosylated biantennaty - non sialylated 11. Yes Core fucosylated triantennaty -
(2 galactose terminals) trisialylated
6. Yes Core fucosylated biantennaty - monosialylated 12. Yes Structure not determined
(and I N-acetylgalactosamine terminal)
Figure 2534.-1. - Chromatogram for the test for glycan analysis of human coagulation factor Vila (rDNA): reference solution
Flow rate 1.0 mIJmin. Gla-domainless human coagulation factor VIia (rDNA)
Detection Spectrophotometer at 214 run. (gamma-carboxylation)
Injection About 20 µL, using an automatic injector Liquid chromatography (2.2.29): use the normalisation
maintained at 2-8 °C. procedure.
Retention time Human coagulation factor Vila Test solution Dilute the preparation to be examined in
(rDNA) = about 26 min. water R to obtain a concentration of about 1.5 mg/mL.
System suitability: Reference solution Dissolve human coagulation factor VIIa
- the chromatogram obtained with the reference solution is (rDNA) CRS in water R to obtain a concentration of
qualitatively similar to the chromatogram shown in 1.5 mg/mL.
Figure 2534.-2; peaks 1 to 10 are clearly visible; Precolumn:
- peak-to-valley ratio: minimum 1.5, where Hp = height - stationary phase: styrene-divinylbenzene copolymer R with
above the baseline of peak 6 and Hv = height above the iminodiacetic groups, for removal of calcium.
baseline of the lowest point of the curve separating this Column:
peak from peak 7. - size: l = 0.25 m, 0 = 4.0 mm;
Results: - stationary phase: strongly basic anion-exchange resin for
- the profile of the chromatogram obtained with the test chromatography Rl;
solution corresponds to that of the chromatogram - temperature: 25 °C.
obtained with the reference solution. Mobile phase:
Calculate the individual percentage area (relative to the total - mobile phase A: solution containing 1.2 g/L of
peak area) of the peaks due to the degraded heavy chain tris(hydroxymethy0aminomethane Rand 2.8 g/L of bis-tris
human coagulation factor Vila (rDNA) (peaks 1, 2 and 6) propane R, adjusted to pH 9.4 with glacial acetic acid R
and oxidised forms of human coagulation factor Vila and degassed;
(rDNA) (peaks 3, 4 and 5). - mobile phase B: solution containing 1.2 g/L of
Limits: tris(hydroxymethyQaminomethane R, 2.8 g/L of bis-tris
- sum of degraded heavy chain forms: maximum 11 per cent; propane Rand 107.9 g/L of ammonium acetate R, adjusted
- sum of oxidised forms: maximum 2.2 per cent. to pH 9.4 with concentrated ammonia Rand degassed;
I I I I I
0 5 10 15 20 25 30 min
I. degraded heavy chain 3. oxidised form 5. oxidised form 7. human coagulation factor Vila (rDNA) 9. unknown
2. degraded heavy chain 4. oxidised form 6. degraded heavy chain 8. 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
Time Mobile phase A Mobile phase B Reference solutwn Dissolve human coagulation factor VIIa
(min) (per cent V/V) (per cent VIV)
(rDNA) CRS in water R to obtain a concentration of
0 - 2.5 100 0 1.5 mg/mL.
2.5 - 27.5 100 ➔ 0 0 ➔ 100
Column:
27.5 - 30.5 0 ➔ 100 100---+ 0
- size: l = 0.3 m, 0 = 7.5 mm;
- stationary phase: hydrophilic silica gel for chromatography R
Flow rate l .O mUmin. (10 µm) of a grade suitable for fractionation of globular
Detection Spectrophotometer at 280 nm. proteins in the relative molecular mass range of 10 000 to
Injection About 100 µL, using an automatic injector 500 000;
maintained at 2-8 °C. - temperature: 21-25 °C.
Relative retention With reference to human coagulation Mobile phase Dissolve 26.4 g of ammonium sulfate R in
factor VIia (rDNA) (retention time = about 14 min): Gla- approximately 900 mL of water for chromatography R. Adjust
domainless human coagulation factor VIia first to pH 2.5 with phosphoric acid Rand then to pH 7.0
(rDNA) = about 0.7. with triethylamine R. Add 50 mL of 2-propanol R and dilute
to 1000 mL with water for chromatography R.
- resolution: baseline separation between the peak due to Flow rate 0.5 mUmin.
Gla-domainless human coagulation factor VIia (rDNA) Detection Spectrophotometer at 215 nm.
and the peak cluster due to human coagulation factor Injection 20 µL, using an automatic injector maintained at
VIia (rDNA). 2-8 °C.
Limit: System suitability Reference solution:
- Gla-domainless human coagulation factor VIia (rDNA): - the chromatogram obtained is qualitatively similar to the
maximum 6.1 per cent. chromatogram supplied with human coagulation factor Vila
Integrate the peak cluster to baseline. (rDNA) CRS;
Dimers and related substances of higher molecular - symmetry factor. maximum 1.3 for the peak due to the
mass monomer;
Size-exclusion chromatography (2.2.30): use the - peak-to-valley ratw: minimum 1.1, where Hp = height
normalisation procedure. above the baseline of the peak due to dimers and
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to the
water R to obtain a concentration of about 1.5 mg/mL.
monomer.
2023 Blood-related Products IV-64 7
Limit: - the correlation coefficient calculated for the standard

- sum of the areas of the peaks with a retention time less than curve (r2) is not less than 0.990.
that of the monomer: maximum 2. 7 per cent. Potency
Non-activated single-chain factor VII (rDNA) The principle of the assay is to measure the ability of a
Polyacrylamide gel electrophoresis (2.2.31): use the factor VIIa preparation to reduce the prolonged coagulation
normalisation procedure. time of factor VII-deficient plasma.
Gel dimenswns 1 mm thick. The biological activity is assessed by comparing the dose-
Resolving gel 12 per cent acrylamide. response curve of the preparation to be examined to that of a
reference preparation calibrated in International Units.
Sample buffer (reducing conditions) concentrated SDS-PAGE
The International Unit is the activity contained in a stated
sample buffer for reducing conditions R containing dithiothreitol R
amount of the International Reference Preparation.
as the reducing agent.
The equivalence in International Units of the International
Reference Preparation is stated by the World Health
water R to obtain a concentration of about 800 µg/mL.
Organization.
Mix equal volumes of this solution and the sample buffer
(reducing conditions). Method.
Reference solution (a) Dissolve human coagulation factor VIia Use a suitable coagulation analyser or cany out the assay with
(rDNA) CRS in water R to obtain a concentration of about incubatwn tubes and reagents maintained in a water-bath at
800 µg/mL. Mix equal volumes of this solution and the 37 °C.
sample buffer (reducing conditions). Solution A Prepare a solution containing 15.12 g/L of
Reference solution (b) Solution of molecular mass markers 1,4-piperazinediethanesulfonic acid R, 5.73 g/L of sodium
suitable for calibrating SDS-polyacrylamide gels in the range chloride R, 0.74 g/L of sodium edetate Rand 10 g/L of bovine
of 10-70 kDa. albumin R; adjust to pH 7 .2 with sodium hydroxide R.
Sample treatment Boil for 5 min or heat at 73 ± 3 °C for Prepare 3 different solutions of the preparation to be
10 min. examined and of the reference preparation, by diluting with
solution A, to obtain concentrations within the linearity range
Applicatwn 10 µL.
(0.002-0.15 IU/mL). Prepare in duplicate and use the
Detection By Coomassie staining. solutions immediately.
Quantification Integrating densitometer.
To 40 µL of each solution, add 40 µL of factor VII-deficient
System suitability: plasma R, incubate for an appropriate time at 37 °C, and add
- the principal bands in the electropherogram obtained with 40 µL of human tissue /actor solution R.
the test solution correspond in position to the principal Measure the coagulation time, i.e. the interval between the
bands in the electropherogram obtained with reference addition of the human tissue factor solution and the first
solution (a) (30 kDa, heavy chain and 20-25 kDa, light indication of the formation of fibrin.
chain);
The volumes given above and sequence of reagents may be
- reference solution (b): the validation criteria are met
(2.2.31); adapted to the human tissue factor solution and apparatus used.
- a band corresponding to non-activated single-chain Calculate the activity in International Units per millilitre
factor VII (rDNA) (molecular mass of 51 kDa) is visible using an appropriate statistical method, for example the
in the electropherogram obtained with reference parallel-line assay (5.3).
solution (a). The confidence limits (P = 0.95) are not less than
Limit: 80 per cent and not more than 125 per cent of the estimated
- non-activated single-chain factor VII (rDNA): maximum potency.
3 per cent. LABELLING
Bacterial endotoxins (2. 6.14) The label states:
Less than 10 IU/mL. - the content of human coagulation factor VIIa (rDNA), in
ASSAY
- the specific activity, in International Units per milligram
Protein
of protein.
Size-exclusion chromatography (2.2.30) as described in the
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
test for dimers and related substances of higher molecular
mass with the following modifications.
Injection I O µL, 20 µL and 30 µL of the reference solution.
Plot peak areas against injected protein content and perform
a linear regression to create a standard curve. Dried Factor VIII (rDNA)
Calculate the content of human coagulation factor VIIa (Human Coagulatwn Factor VIII (rDNA), Ph. Bur.
(rDNA) using the monomer peak area in the chromatogram monograph 1643)
obtained with the test solution and taking into account the
assigned content of human coagulation factor VIIa Action and use
(rDNA) CRS. Coagulation factor VIII substitute.
System suitability: PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
2.0 per cent after 5 injections of 20 µL of the reference
solution; Human coagulation factor VIII (rDNA) is a freeze-dried
preparation of glycoproteins having the same activity as
coagulation factor VIII in human plasma. It acts as a cofactor peptide mapping, Western blots, HPLC, gel electrophoresis,
of the activation of factor X in the presence of factor IXa, capillary electrophoresis, mass spectrometry or other
phospholipids and calcium ions. techniques to monitor integrity and purity. The protein
Human coagulation factor VIII circulates in plasma mainly as composition is comparable to that of the manufacturer's
a two-chain glycosylated protein with 1 heavy (relative reference preparation.
molecular mass of about 200 000) and 1 light (relative Molecular size distribution
molecular mass 80 000) chain held together by divalent Using size-exclusion chromatography (2.2.30), the molecular
metal ions. Human coagulation factor VIII (rDNA) is size distribution is comparable to that of the manufacturer's
prepared as full-length factor VIII (octocog alfa), or as a reference preparation.
shortened two-chain structure (relative molecular mass Peptide mapping (2.2.55)
90 000 and 80 000), in which the B-domain has been There is no significant difference between the test protein
deleted from the heavy chain (moroctocog alfa). and the manufacturer's reference preparation.
Full-length human rDNA coagulation factor VIII contains
Carbohydrates/sialic acid
25 potential N-glycosylation sites, 19 in the B domain of the
To monitor batch-to-batch consistency, the monosaccharide
heavy chain, 3 in the remaining part of the heavy chain
content and the degree of sialylation or the oligosaccharide
(relative molecular mass 90 000) and 3 in the light chain
profile are monitored and correspond to those of the
(relative molecular mass 80 000). The different products are
manufacturer's reference preparation.
characterised by their molecular size and post-translational
modification and/or other modifications. FINAL LOT
It complies with the requirements under Identification, Tests
PRODUCTION and Assay.
Human coagulation factor VIII (rDNA) is produced by
recombinant DNA technology in mammalian cell culture.
Excipients
80 per cent to 120 per cent of the stated content, determined
It is produced under conditions designed to minimise
by a suitable method, where applicable.
microbial contamination.
Purified bulk factor VIII (rDNA) may contain added human CHARACTERS
albumin and/or other stabilising agents, as well as other Appearance
auxiliary substances to provide, for example, correct pH and White or slightly yellow powder or friable mass.
osmolality. IDENTIFICATION
The specific activity is not less than 2000 IU of factor VIII:C A. It complies with the limits of the assay.
per milligram of total protein before the addition of any B. The distribution of characteristic peptide bands
protein stabiliser, and varies depending on purity and the corresponds with that of the manufacturer's reference
type of modification of molecular structure of factor VIII. preparation (SDS-PAGE or Western blot).
The quality of the bulk preparation is controlled using one or
TESTS
more manufacturer's reference preparations as reference.
Reconstitute the preparation as stated on the label immediately
MANUFACTURER'S REFERENCE PREPARATIONS before carrying out the tests (except those for solubility and water)
During development, reference preparations are established and assay.
for subsequent verification of batch consistency during
production, and for control of bulk and final preparation. Solubility
They are derived from representative batches of purified bulk It dissolves within 5 min at 20-25 °C, giving a clear or
factor VIII (rDNA) that are extensively characterised by tests slightly opalescent solution.
including those described below and whose procoagulant and pH (2.2.3)
other relevant functional properties have been ascertained 6.5 to 7.5.
and compared, wherever possible, with the International Osmolality (2.2.35)
Standard for factor VIII concentrate. The reference Minimum 240 mosmol/kg.
preparations are suitably characterised for their intended
Water
purpose and are stored in suitably sized aliquots under
Determined by a suitable method, such as the semi-micro
conditions ensuring their stability.
determination of water (2.5.12), loss on drying (2.2.32) or
PURIFIED BULK FACTOR VIII (RDNA) near-infrared spectroscopy (2.2.40), the water content is
The purified bulk complies with a suitable combination of the within the limits approved by the competent authority.
following tests for characterisation of integrity of the factor VIII
Sterility (2. 6.1)
(rDNA). Where any substance added during preparation of the
It complies with the test for sterility.
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
factor VIII activity.
Specific biological activity or ratio of factor VIII
activity to factor VIII antigen ASSAY
Carry out the assay of human coagulation factor VIII (2. 7. 4). Carry out the assay of human coagulation factor VIII (2. 7. 4).
The protein content, or where a protein stabiliser is present, The estimated potency is not less than 80 per cent and not
the factor VIII antigen content, is determined by a suitable more than 125 per cent of the stated potency.
method and the specific biological activity or the ratio of The confidence limits (P = 0.95) are not less than
factor VIII activity to factor VIII antigen is calculated. 80 per cent and not more than 120 per cent of the estimated
Protein composition potency.
The protein composition is determined by a selection of
appropriate characterisation techniques which may include
STORAGE using a normal human plasma pool as reference.

Protected from light. Visualisation of the multimeric pattern may be performed
using, for example, an immunoenzymatic technique and
LABELLING
quantitative evaluation may be carried out by densitometric
The label states: analysis.
- the factor VIII content in International Units,
- the name and amount of any excipient, Products that show flakes or particles after reconstitution for use
- the composition and volume of the liquid to be used for If a few small flakes or particles remain when the preparation
reconstitution. is reconstituted, it shall be demonstrated during validation
studies that the potency is not significantly affected after
- - - - - - - - - - - - - - - - - - - - - - PhE~ passage of the preparation through the filter provided.
CHARACTERS
Appearance
White or pale yellow, hygroscopic powder or friable solid.
Dried Factor VIII Fraction
Reconstitute the preparation to be examined as stated on the label
(Human Coagulation Factor VIII, Ph. Bur. immediately before carrying out the identification, tests (except
monograph 0275) those for solubility and water) and assay.
Action and use IDENTIFICATION
Coagulation factor VIII substitute. It complies with the limits of the assay.
~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ TESTS
Solubility
DEFINITION To a container of the preparation to be examined, add the
Sterile, freeze-dried preparation of a plasma protein fraction volume of the liquid stated on the label at the recommended
containing the glycoprotein human coagulation factor VIII temperature. The preparation dissolves completely with
together with varying amounts of human von Willebrand gentle swirling within 10 min, giving a clear or slightly
factor, depending on the method of preparation. It is opalescent, colourless or slightly yellow solution.
prepared from human plasma that complies with the
Where the label states that the product may show a few small
monograph on Human plasma for fractionation (0853).
flakes or particles after reconstitution, reconstitute the
The preparation may contain excipients such as stabilisers.
preparation as described on the label and pass it through the
The potency of the preparation, reconstituted as stated on filter provided: the filtered solution is clear or slightly
the label, is not less than 20 IU of factor VIII:C per millilitre. opalescent.
PRODUCTION pH (2.2.3)
GENERAL PROVISIONS 6.5 to 7.5.
The method of preparation is designed to maintain
Osmolality (2.2.35)
functional integrity of human coagulation factor VIII and to
Minimum 240 mosmol/kg.
minimise potential neoantigenicity. It includes a step or steps
that have been shown to remove or to inactivate known Total protein
agents of infection; if substances are used for the inactivation If necessary, dilute an accurately measured volume of the
of viruses, the subsequent purification procedure must be reconstituted preparation with a 9 g/L solution of sodium
validated to demonstrate that the concentration of these chloride R to obtain a protein concentration of about
substances is reduced to a suitable level and that any residues 7.5 mgimL. Place 2.0 mL of this solution in a round-
are such as not to compromise the safety of the preparation bottomed centrifuge tube and add 2 mL of a 75 giL solution
for patients. of sodium molybdate R and 2 mL of a mixture of I volume of
nitrogen-free suljuric acid R and 30 volumes of water R. Shake,
The specific activity is not less than 1 IU of factor VIll:C per
centrifuge for 5 min, decant the supernatant and allow the
milligram of total protein before the addition of any protein
inverted tube to drain on filter paper. Determine the nitrogen
stabiliser.
The human coagulation factor VIII fraction is dissolved in a and calculate the amount of protein by multiplying the result
suitable liquid. No antimicrobial preservative or antibiotic is by 6.25. For some products, especially those without a protein
added. The solution is passed through a bacteria-retentive stabiliser such as albumin, this method may not be applicable and
filter, distributed aseptically into the final containers and another validated method for protein determination must therefore
immediately frozen. It is subsequently freeze-dried and the be peeformed.
containers are closed under vacuum or under an inert gas.
Anti-A and anti-B haemagglutinins (2.6.20, Method A)
CONSISTENCY OF THE METHOD OF The 1 to 64 dilution does not show agglutination. Dilute the
PRODUCTION reconstituted preparation with a 9 g/L solution of sodium
Products stated to have human van Willebrand factor activity chloride R to contain 3 IU of factor VIII:C per millilitre.
(products intended for treatment of von Willebrand's disease).
It shall be demonstrated by suitable analytical procedures Water
Determined by a suitable method, such as semi-micro
determined during process development that the
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 (2.2.40), 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 Sterility (2. 6.1)
Willebrand factor multimers may be determined by sodium It complies with the test.
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)

It complies with the test for pyrogens or, preferably and
Factor IX (rDNA) Concentrated
where justified and authorised, with a validated in vitro test Solution
such as the test for bacterial endotoxins.
(Human Coagulation Factor IX (rDNA) Concentrated
For the pyrogen test, inject per kilogram of the rabbit's mass Solution, Ph. Bur. monograph 2522)
a volume equivalent to not less than 50 IU of factor VIll:C.
Where the test for bacterial endotoxins is used, the YNSGKLEEFV QGNL!lR!lCM!l -EKCSFEEARE
- - VF!lNT!lRTT!l 40
preparation to be examined contains less than 0.03 IU of FWKQYVDGDQ CESNPCLNGG SCKI:>DIN§_YE CWCPFGFEGK 80
endotoxin per International Unit of factor VIlI:C. NCELDVTCNI KNGRCEQFCK NSADNKVVCS CTEGYRLAEN 120
QKSCEPAVPF PCGRVSVSQT SKLTRAEAVF PDVD}'VN§_TE 160
ASSAY
AETILDNITQ STQSFNDFTR VVGGEDAKPG QFPWQVVLNG 200
Human coagulation factor VIII (2. 7. 4)
KVDAFCGGSI VNEKWIVTAA HCVETGVKIT VVAGEHNIEE 240
The estimated potency is not less than 80 per cent and not
TEHTEQKRNV IRIIPHHNYN AAINKYNHDI ALLELDEPLV 280
more than 120 per cent of the stated potency.
LNSYVTPICI ADKEYTNIFL KFGSGYVSGW GRVFHKGRSA 320
LVLQYLRVPL VDRATCLRST KFTIYNNMFC AGFHEGGRDS 360
CQGDSGGPHV TEVEGTSFLT GIISWGEECA MKGKYGIYTK 400
potency.
VSRYVNWIKE KTKLT 415
Human von Willebrand factor (2.7.21)
If preparations are intended for the treatment of von disulfide bridges:
18-23, 51-62, 56-71, 73-82, 88-99, 95-109, 111-124, 132-289,
Willebrand's disease, the estimated potency is not less than 206-222, 336-350, 361-389
60 per cent and not more than 140 per cent of the stated
glycosylation sites:
potency. Ser-53, Ser-61, Asn-157, Thr-159, Asn-167, Thr-169
Pending the availability of an International Standard for human modified residues:
von Wille brand factor concentrate calibrated for use in the f (4-carboxyGlu): 7, 8, 15, 17, 20, 21, 26, 27, 30, 33, 36, 40
collagen-binding assay, only the ristocetin cofactor assay may be Q ((3R)-3-hydroxyAsp): 64
)2 (03-phosphoSer): 68, 158
used.
Y. (04-sulfoTyr): 155
STORAGE H _NH2
In an airtight container, protected from light. H02C H NH 2 H02C,
j(_
x_'C02H
LABELLING H02C~C02H HO H
The label states: f = 4-carboxyGlu Q = (3R)-3-hydroxyAsp
- the number of International Units of factor VIll:C and, H _NH2
where applicable, of human von Willebrand factor in the 0 "'P/ 0 ~ c o H
H03S/O~ H. _...NH 2
container; I \ 2
HO OH ~C02H
- the amount of protein in the container;
~ = (03-phosphoSer) Y. = (04-sulfoTyr)
- the name and quantity of any added substance;
reconstitution; C20s3H311,,NsssO614P2S26 Mr approx. 55 000
- where applicable, that the preparation may show the Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
presence of a few small flakes or particles after
reconstitution; DEFINITION
- that the transmission of infectious agents cannot be totally Solution containing closely related glycoproteins, which have
excluded when medicinal products prepared from human the same amino acid sequence (415 amino acids) as the
blood or plasma are administered. naturally occurring Ala 148 allelic form analogue (plasma-
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur derived coagulation factor IX). It is a single-chain
glycoprotein with structural and functional characteristics
similar to those of the endogenous factor IX. It may contain
buffer salts and/or non-proteinaceous stabilisers.
Content
Minimum 150 IU per millilitre.
Potency
200 to 360 IU per milligram of protein.
PRODUCTION
Human coagulation factor IX (rDNA) is produced in
mammalian cells by a method based on recombinant DNA
technology (rDNA). The method of preparation is designed
to maintain the functional integrity of factor IX, and to
minimise the activation of human coagulation factor IX
(rDNA) (to minimise potential thrombogenicity).
No antibiotic or antimicrobial preservative is added.
Prior to release, the following tests are carried out on each
batch of human coagulation factor IX (rDNA) concentrated
solution, unless exemption has been granted by the
competent authority.
Host-cell-derived proteins Detection Fluorimeter at 330 nm for excitation and 420 nm

The limit is approved by the competent authority. for emission.
Host-cell- and vector-derived DNA Injection 20 µL, using an automatic injector maintained at
The limit is approved by the competent authority 2-8 °C.
Glycan analysis If carry-over of material is observed, running a blank gradient
Use a suitable method developed according to general after each injection may be appropriate.
chapter 2.2.59. Glycan analysis of glycoproteins, Section 2-3. Identification of peak groups Use the chromatogram in Figure
- Release the glycans using one of the agents described in 2522.-1 to identify the 5 groups of oligosaccharides
Table 2.2.59.-1, for example peptide N-glycosidase F corresponding to PO neutral, PI mono-, P2 di-, P3 tri- and
(PNGase F). P4 tetrasialylated oligosaccharides. Record the retention
- Label the released glycans with one of the fluorescent times of the most prominent peaks in groups PO to P4.
labelling agents described in Table 2.2.59.-2, for example Calculate the relative retentions of the most prominent peaks
2-aminobenzamide. in groups PO to P3 with reference to the most prominent
- Analyse the labelled glycans by liquid chromatography peak in group P4.
(2.2.29) using a high-pH-resistant column with Calculate the tetrasialylated peak area ratio for the test
fluorescence detection. solution using the following expression:
The following procedure is given as an example.
Test solution Dilute the preparation to be examined with the
formulation buffer (see Tests) to obtain a concentration of
about 2 mgimL. Use 50 µL of this solution to proceed to
glycan release and labelling. Resuspend or dilute the labelled peak area of group P4;
peak area of groups PO to P3.
glycans in 200 µL of water R.
Reference solution (a) Dissolve the contents of a vial of System suitability:
human coagulation f acwr IX (rDNA) CRS in the formulation - the chromatogram obtained with reference solution (a) is
buffer (see Tests) to obtain a concentration of about qualitatively similar to the chromatogram supplied with
2 mgimL. Use 50 µL of this solution to proceed to glycan human coagulation facwr IX (rDNA) CRS; 5 groups of
release and labelling. Resuspend or dilute the labelled glycans oligosaccharide peaks corresponding to PO neutral, Pl
in 200 µL of water R. mono-, P2 di-, P3 tri- and P4 tetrasialylated
Reference solution (b) Use a suitable human coagulation oligosaccharides are present; group P4 includes the
factor IX (rDNA) in-house reference preparation shown to highest peak, and P3 the second-highest peak;
be representative of batches tested clinically and batches used - no significant peaks are observed in regions PO to P4 in
to demonstrate consistency of production. Dilute with the the chromatogram obtained with the blank solution.
formulation buffer to obtain a concentration of about Results:
2 mgimL. Use 50 µL of this solution to proceed to glycan - the profile of the chromatogram obtained with the test
release and labelling. Resuspend or dilute the labelled glycans solution corresponds to that of the chromatogram
in 200 µL of water R. obtained with reference solution (b);
Blank solution Use 50 µL of the formulation buffer to - the relative retentions of the most prominent peaks in
proceed to glycan release and labelling. groups PO to P3 in the chromatogram obtained with the
Analyse the labelled glycans by liquid chromatography test solution correspond to those in the chromatogram
(2.2.29). obtained with reference solution (b);
- the tetrasialylated peak area ratio for the test solution is
Precolumn:
within the limits authorised by the competent authority.
=
- size: l O.OI m, 0 = 4.6 mm;
- stationary phase: polyamine grafted poly(vinyl alcohol) CHARACTERS
copolymer R. Appearance
Column: Clear, colourless liquid.
- size: l = 0.25 m, 0 = 4.6 mm; IDENTIFICATION
- stationary phase: polyamine grafted poly(vinyl alcohol) A. It forms a clot when examined in the conditions described
copolymer R (5 µm). under Assay (Potency).
B. Peptide mapping (2.2.55).
Mobile phase:
SELECTIVE CLEAVAGE OF THE PEPTIDE BONDS
- mobile phase A: water for chromatography R, glacial acetic
acid R, acewnitrile R (1:2:97 V!VIV); Solution A Dissolve 143.3 g of guanidine hydrochloride R,
- mobile phase B: concentrated ammonia R, glacial acetic 9.086 g of tris(hydroxymethyl)aminomethane Rand 0.931 g of
acid R, water for chromawgraphy R (1:3:96 VIVIV); sodium edetate R in 250 mL of water R and adjust to
pH 8.0 ± 0.1 with hydrochloric acid R.
Time Mobile phase A Mobile phase B Test solution Dilute the preparation to be examined with the
(min) (per cent V/V) (per cent V/V) formulation buffer (see Tests) to obtain a concentration of
0-2 70 30 about 1.5 mgimL.
2 - 67 70 ➔ 0 30 ➔ 100 Reference solution Dissolve the contents of a vial of human
67 - 70 0 100 coagulation factor IX (rDNA) CRS in the formulation buffer
70 - 70.1 0 ➔ 70 JOO ➔ 30 (see Tests) to obtain a concentration of about 1.5 mgimL.
70.1 - 95 70 30
Reduction and alkylation To 67 µL of the test solution add
28 µL of water R, I 00 µL of solution A, then 5 µL of a
Flow rate 0.5 mUmin. 30.85 g/L solution of dithiothreiwl R, mix well and centrifuge
P4
P3
P2
P1
n
n n
PO
I I I
0 10 20 30 40 50 60 70 80 90 100 110 min
Figure 2522.-1. - Chromatogram for the test for glycan analysis of human coagulation factor IX (rDNA)
briefly. Overlay with nitrogen. Incubate in a water-bath at Time Mobile phase A Mobile phase B
40 °C for 1 h. Add 6.6 µL of a freshly prepared 115.04 g/L (min) (per cent V/J/) (per cent V/J/)
solution of iodoacetic acid R, mix well and centrifuge briefly. 0-5 97 3
Overlay with nitrogen. Incubate at room temperature for 1 h 5 - 35 97 ➔ 85 3 ➔ 15
protected from light. Add 5.3 µL of a 30.85 g/L of solution 35 - 60 85 ➔ 81 15 ➔ 19
of dithwthreitol Rand mix well. Add 188.1 µL of water R. 60 - 81 81 ➔ 74 19 ➔ 26
81 - IOI 74 ➔ 71 26 ➔ 29
Digestwn To the reduced solution prepared previously, add
IOI - 135 71 ➔ 60 29 ➔ 40
10 µL of a freshly prepared 3.4 U/mL solution of lysyl
135 - 140 60 ➔ 0 40 ➔ 100
endopeptidase R, mix well and centrifuge briefly. Overlay with
140 - 150 0 100
nitrogen. Incubate at 30 °C for 4 h. Mix 90 µL of the
150 - 150.01 0 ➔ 97 100 ➔ 3
digested solution and 180 µL of a 33.22 g/L solution of
150.01 - 190 97 3
sodium edetate R.
190 - 191 97-+ 50 3 ➔ 50
Carry out the reduction/alkylation and digestion steps for the 191 - 251 50 50
reference solution in the same manner as for the test
solution.
CHROMATOGRAPHIC SEPARATION
Injection 240 µL, using an automatic injector maintained at
Column:
2-8 °C.
- size: l = 0.25 m, 0 = 2.1 mm;
- stationary phase: end-capped octadecylsilyl silica gel for System suitability:
chromatography R (5 µm) with a pore size of 30 nm; - the chromatogram obtained with the reference
- temperature: 25 °c. solution is qualitatively similar to the chromatogram
supplied with human coagulatwn factor IX
Mobile phase:
(rDNA) CRS;
- mobile phase A: add 0.5 mL of trifiuoroacetic acid R to
- all peaks identified in the chromatogram supplied with
1000 mL of water for chromatography R and degas;
human coagulatwn factor IX (rDNA) CRS are visible in
- mobile phase B: mix 0.5 mL of trifiuoroacetic acid R,
the chromatogram obtained with the reference
50 mL of water for chromatography R and 950 mL of
solution.
acetonitrile Rl and degas;
Results:
- the profile of the chromatogram obtained with the test
solution corresponds to that of the chromatogram
- no new major peaks are observed in the
chromatogram obtained with the test solution in
comparison to the chromatogram obtained with the Injection 50 µL; perform at least 3 injections using an
reference solution. automatic injector maintained at 2-8 °C.
C. Polyacrylamide gel electrophoresis (2.2.31). Relative retention With reference to human coagulation
Examine the electropherograms obtained in the test for factor IX (rDNA) containing 12 Gia residues per molecule
impurities with molecular masses differing from that of (12 Gia, retention time = about 25 min): 9Gla = 0.60;
human coagulation factor IX (rDNA). l0Gla = 0.75; l lGla = 0.85. NOTE: molecular species
Calculate the relative mobility (in per cent) of the main band containing 9 or fewer Gia residues per molecule of human
in the electropherogram obtained with the test solution with coagulation factor IX (rDNA) may not be present in the
reference to the mobility of the main band in the preparation.
electropherogram obtained with reference solution (a), using System suitability Reference solution:
the following expression: - repeatability: maximum relative standard deviation of
3 per cent for the total area of the peak due to human
coagulation factor IX (rDNA), determined on 3 injections
performed immediately before the run;
- the 10Gla peak is visible and is similar to the
M1 molecular mass of the main band in the electropherogram corresponding peak in the chromatogram supplied with
obtained with the test solution; human coagulation factor IX (rDNA) CRS;
M2 molecular mass of the main band in the electropherogram
obtained with reference solution (a). - peak-to-valley ratw: minimum 1.2, where Hp = height
above the baseline of the peak due to 11 Gia, and
Results: Hv = height above the baseline of the lowest point of the
- the electropherogram obtained with the test solution is curve separating this peak from the peak due to 12Gla.
similar to the electropherogram obtained with Results:
reference solution (a); - repeatability: maximum relative standard deviation of
- the mobility of the main band in the electropherogram 3 per cent for the total area of the peak due to human
obtained with the test solution is within 10 per cent of coagulation factor IX (rDNA), determined on 3 injections
that of the main band in the electropherogram of the test solution;
obtained with reference solution (a). - the profile of the chromatogram obtained with the test
solution corresponds to that of the chromatogram
TESTS
Formulation buffer
Dissolve 19.53 g of glycine R, 1.55 g of histidine Rand Calculate the total Gia content using the following
10.00 g of sucrose R in 1000 mL of water R. Add 50 µL of expression:
polysorbate 80 R and adjust to pH 6.8 with hydrochloric acid R.
A
Gamma-carboxyglutamic acid (Gla) L total peak
12
i= 9
Pi
area
X i Gia.marl
Liquid chromatography (2.2.29): use the normalisation
procedure.
Test solution Dilute the preparation to be examined with the - Ap;: area of the concerned peak (9Gla, lOG!a, llGla or
formulation buffer to obtain a concentration of about 12Gla); any shoulder appearing on the descending part of
1 mglmL. the 12Gla peak is included in the area of the 12Gla peak;
- total peak area: sum of the areas of peaks 9Gla to 12Gla;
Reference solution Dissolve the contents of a vial of human
- i Gla.nwT 1 : 9, 10, 11 or 12, corresponding to the
coagulation factor IX (rDNA) CRS in the formulation buffer
theoretical number of Gia residues per mole of human
to obtain a concentration of about 1 mglmL.
coagulation factor IX (rDNA) for the concerned peak.
Blank solution The formulation buffer.
Limit:
Column: - 11.0 to 12.0 moles of Gia per mole of human coagulation
=
- size: l 0.05 m, 0 =
5 mm; factor IX (rDNA).
- stationary phase: strongly basic anion-exchange resin for
chromatography R (10 µm). Related proteins and impurities
Mobile phase:
- mobile phase A: solution containing 2.42 glL of Test solutwn Dilute the preparation to be examined with the
tris(hydroxymethyl)aminomethane R, adjusted to pH 9.0 formulation buffer to obtain a concentration of about
with hydrochloric acid R; 0.5 mglmL.
- mobile phase B: solution containing 2.42 glL of Reference solutwn Dissolve the contents of a vial of human
tris(hydroxymethyl)aminomethane Rand 58.45 g/L of coagulation factor IX (rDNA) CRS in the formulation buffer
sodium chloride R, adjusted to pH 9.0 with hydrochloric to obtain a concentration of about 0.5 mglmL.
acid R; Column:
= =
- size: l 0.10 m, 0 4.6 mm;
Time Mobile phase A Mobile phase B - stationary phase: styrene-divinylbenzene copolymer R (10 µm)
(min) (per cent V/J') (per cent V/J') with a pore size of 400 nm;
0 - 40 70 ➔ 60 30 ➔ 40 - temperature: 37 °C.
40 - 49 60 ➔ 0 40 ➔ 100 Mobile phase:
49 - 50 0 ➔ 70 100 ➔ 30 - mobile phase A: add 1 mL of trifiuoroacetic acid R to
50 - 71 70 30 1000 mL of water for chromatography R;
- mobile phase B: mix 1 mL of trifiuoroacetic acid R, 200 mL
Fbw rate 0. 75 mUmin. of water for chromatography R and 800 mL of
Detection Spectrophotometer at 214 nm. acetonitrile R 1.
Time Mobile phase A Mobile phase B - 1.5 volumes of tetramethylethylenediamine R;

(min) (per cent V/J') (per cent V/J')
- 33 volumes of a 150 mg/mL solution of ammonium
0 - 0.5 75 25 persulfate R.
0.5 - 30 75 ➔ 20 25 ➔ 80
Load the compartments of the gradient-forming apparatus
30 - 31 20 ➔ 0 80 ---+ JOO
with the acrylamide solutions and proceed as per the
31 - 33 0 100
instructions of the equipment supplier to obtain the
polymerised gradient gel.
Flow rate 2.0 mUmin. After polymerisation is completed, rinse the gradient gel with
Detection Spectrophotometer at 214 run. water R. Remove any excess liquid. Pour the stacking gel
Infection 100 µL; perform at least 3 injections using an solution into the equipment, insert a clean comb and allow
automatic injector. for polymerisation.
Relative retention With reference to the 2nd peak of the Alternatively, commercially available gradient gels may be
double peak due to human coagulation factor IX (rDNA) used.
(retention time= about 12-14 min): related Test solution Dilute the preparation to be examined with the
protein A= about 0.75; related protein B = about 0.78; formulation buffer to obtain a concentration of about
related protein C = about 0.80; related protein D = about 1 mg/mL.
0.85; related protein E = about 0.93. Reference solution (a) Dissolve the contents of a vial of
System suitability Reference solution: human coagulation factor IX (rDNA) CRS in the formulation
- the chromatogram obtained is qualitatively similar to the buffer to obtain a concentration of about 1 mg/mL.
chromatogram supplied with human coagulation factor IX Reference solution (b) 0.01 mg/mL solution of bovine
(rDNA) CRS; albumin R in the formulation buffer.
Reference solution (c) A solution of molecular mass markers
3 per cent for the total area of the peak due to human
suitable for calibrating SDS-polyacrylamide gels in the range
coagulation factor IX (rDNA) after 3 injections performed
of 5-200 kDa.
immediately before the run;
- peak-to-valley ratio: minimum 1.2, where Hp = height Sample buffer Concentrated SDS-PAGE sample buffer for
above the baseline of the peak due to related protein E reducing conditions R containing dithiothreitol as the reducing
and Hv = height above the baseline of the lowest point of agent.
the curve separating this peak from the peak due to Sample treatment Incubate in a water-bath for 5 min.
human coagulation factor IX (rDNA). Application 35 µL.
Report individual relative peak areas considering the peak Use SDS-PAGE running buffer R for running the gel.
area of the entire chromatogram. Individual relative per cent For each gel, run 1 lane with reference solution (b), 2 lanes
peak areas are calculated as the average of the 3 injections of with reference solution (c), 2 lanes with the incubated
the test solution. reducing buffer (as blank) and at least 1 lane with reduced
Results: reference solution (a); use the remaining lanes for reduced
- the profile of the chromatogram obtained with the test test solutions.
solution corresponds to that of the chromatogram Detection By Coomassie staining.
obtained with the reference solution, except for minor
Identification of bands Human coagulation factor IX (rDNA)
peaks due to impurities, that may be absent in the
has an approximate molecular mass of 55 kDa, and related
protein bands with molecular masses of approximately
Limits: 54 kDa, 44 kDa, 29-32 kDa, 27 kDa and 14 kDa are
- related protein C: maximum 0.6 per cent; present.
- total impurities (all peaks not eluted at the positions expected
System suitability:
for human coagulation factor IX (rDNA) and its related
proteins): maximum 1.0 per cent. - a clear background is obtained after destaining;
- the band in the electropherogram obtained with reference
Impurities with molecular masses differing from that solution (b) is clearly visible;
of human coagulation factor IX (rDNA) - all expected bands in the electropherogram obtained with
Polyacrylamide gel electrophoresis (2.2.31) using a gradient reference solution (c) are visible;
gel with the following modifications. - the bands in the electropherogram obtained with reference
Gel dimensions 1.0 mm thick. solution ( c) are clearly separated;
Resolving gel 3-15 per cent acrylamide gradient gel. - no band is visible in the blank lanes.
Acrylamide solution 30 per cent acrylamide/bisacrylamide Results:
(36. 5: 1) solution R. - the electropherogram obtained with the test solution is
To prepare a 15 per cent acrylamide solution, add sucrose R similar to the electropherogram obtained with reference
to obtain a concentration of 142 g/L. solution (a);
- no new band in the electropherogram obtained with the
The following elements are provided as examples for loading
test solution has an intensity greater than that of the band
the gel forming apparatus to prepare the stacking gel.
in the electropherogram obtained with reference
Stacking gel The stacking gel solution is prepared by mixing: solution (b).
- 100 volumes of 30 per cent acrylamidelbisacrylamide
Impurities with molecular masses greater than that of
(36. 5: 1) solution R;
human coagulation factor IX (rDNA)
- 125 volumes of 1 M tris-hydrochloride buffer solution
pH 6.8 R;
- 740.5 volumes of water R;
Test solution Dilute the preparation to be examined with the Microbial contamination (2. 6.12)
formulation buffer to obtain a concentration of about Maximum 10 CFU/mL.
400 µg/mL. Bacterial endotoxins (2. 6.14)
Reference solution Dissolve the contents of a vial of human Less than 1 IU per 100 IU of factor IX activity.
ASSAY
to obtain a concentration of about 400 µg/mL.
The specific biological activity of the substance is determined
Resolution solution Dissolve the contents of a vial of human before the addition of any protein stabiliser.
to obtain a concentration of about 400 µg/mL. Desalt and Protein
concentrate the preparation to be examined using a suitably Size-exclusion chromatography (2.2.30) as described in the
validated procedure. Reconstitute the recovered material in test for impurities with molecular masses greater than that of
0.1 M phosphate buffer solution pH 8.0 R to obtain a human coagulation factor IX (rDNA) with the following
concentration of 400 µg/mL. To 500 µL of this solution add modifications.
1.4 µL of a 250 mg/L solution of glutaraldehyde R. Mix and Prepare triplicate dilutions of the test solution.
incubate at 37 °C for 120 min. Reference solutions Dissolve the contents of a vial of human
Blank solution The formulation buffer. coagulation factor IX (rDNA) CRS in the formulation buffer
Precolumn: to obtain a concentration of 1 mg/mL. Further dilute this
- size: l = 0.04 m, 0 = 6 mm; solution to prepare a standard curve with concentrations in
- stationary phase: hydrophilic silica gel for chromatography R the range of 100-800 µg/mL (5 concentrations, typically
(5 µm) of a grade suitable for the fractionation of globular 100 µg/mL, 200 µg/mL, 400 µg/mL, 600 µg/mL,
proteins in the relative molecular mass range of 10 000 to 800 µg/mL).
500 000. Plot peak areas versus injected protein content and perform
Column:
linear regression to create a standard curve.
=
- size: l 0.30 m, 0 =
7.8 mm; System suitability (in addition to those described in the test
- stationary phase: hydrophilic silica gel for chromatography R for impurities with molecular masses greater than that of
(5 µm) of a grade suitable for the fractionation of globular human coagulation factor IX (rDNA)):
proteins in the relative molecular mass range of 10 000 to - the correlation coefficient (r2) calculated for the standard
500 000. curve is not less than 0.995.
Mobile phase Dissolve 7 .10 g of anhydrous disodium hydrogen Calculate the protein concentration of each replicate of the
phosphate Rand 8.77 g of sodium chloride R in 1000 mL of preparation to be examined using the standard curve and the
water for chromatography R. Adjust to pH 7 .00 ± 0.05 with assigned content in human coagulation factor IX (rDNA) CRS.
phosphoric acid R. Potency
Flow rate 1.0 mUmin. Assay of human coagulation factor IX (2. 7.11).
Detection Spectrophotometer at 214 nm. The estimated potency is not less than 80 per cent and not
Injection 50 µL; perform 3 injections using an automatic
injector maintained at 2-8 °C.
Retention time Human coagulation factor IX potency.
(rDNA) = about 9 min.
Human coagulation factor IX concentrate ERP is suitable for
System suitability: use as a reference preparation.
- the chromatogram obtained with the reference solution is
qualitatively similar to the chromatogram supplied with STORAGE
human coagulation factor IX (rDNA) CRS; In an airtight container, under approved conditions.
- peak-to-valley ratio: minimum 2.0, where Hp = height LABELLING
above the baseline of the peak due to the high molecular The label states the factor IX content in International Units
mass species and Hv = height above the baseline of the per millilitre and in International Units per milligram of
lowest point of the curve separating this peak from the protein.
peak due to human coagulation factor IX (rDNA) in the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph£~
chromatogram obtained with the resolution solution.
Calculate the relative area (in per cent) of the sum of the
peaks with retention times less than that of human
coagulation factor IX (rDNA), with reference to the area of
the peak due to human coagulation factor IX (rDNA). Dried Factor IX Fraction
Any shoulder appearing on the descending part of the peak (Human Coagulation Factor IX, Ph. Bur. monograph
due to human coagulation factor IX (rDNA) is included in
1223)
its area.
Result: Action and use
- the profile of the chromatogram obtained with the test Coagulation factor IX substitute.
solution corresponds to that of the chromatogram PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Limit:
- sum of the peaks eluted before the principal peak: maximum
Sterile freeze-dried preparation of a plasma protein fraction
1.3 per cent. containing coagulation factor IX. It is obtained from human
plasma that complies with the monograph on Human plasma
for fractionation (0853), by a method that effectively separates
human coagulation factor IX from other prothrombin Total protein

complex factors (human coagulation factors II, VII and X). If necessary, dilute an accurately measured volume of the
The preparation may contain excipients such stabilisers, reconstituted preparation with a 9 g/L solution of sodium
heparin and antithrombin. chloride R to obtain a solution containing about 15 mg of
The potency of the preparation, reconstituted as stated on protein in 2 mL. To 2.0 mL of the solution in a round-
the label, is not less than 20 IU of human coagulation bottomed centrifuge tube, add 2 mL of a 75 g/L solution of
factor IX per millilitre. sodium molybdate R and 2 mL of a mixture of 1 volume of
nitrogen-free suljuric acid R and 30 volumes of water R. Shake,
PRODUCTION centrifuge for 5 min, decant the supernatant and allow the
GENERAL PROVISIONS inverted tube to drain on filter paper. Determine the nitrogen
The method of preparation is designed to maintain in the residue by the method of sulfuric acid digestion (2.5. 9)
functional integrity of human coagulation factor IX and to and calculate the amount of protein by multiplying the result
minimise activation of any coagulation factor (to minimise by 6.25. For some products, especially those without a protein
potential thrombogenicity). It includes a step or steps that stabiliser such as albumin, this method may not be applicable.
have been shown to remove or to inactivate known agents of Another validated method for protein determination must therefore
infection; if substances are used for inactivation of viruses be performed.
during production, the subsequent purification procedure
must be validated to demonstrate that the concentration of Activated coagulation factors (2.6.22)
these substances is reduced to a suitable level and that any If necessary, dilute the reconstituted preparation to contain
residues are such as not to compromise the safety of the 20 IU of human coagulation factor IX per millilitre. For each
preparation for patients. of the dilutions, the coagulation time is not less than 150 s.
The specific activity is not less than 50 IU of human Heparin (2.7.12)
coagulation factor IX per milligram of total protein, before If heparin has been added, the preparation to be examined
the addition of any protein stabiliser. contains not more than the amount of heparin stated on the
label and in all cases not more than 0.5 IU of heparin per
The human coagulation factor IX fraction is dissolved in a
International Unit of human coagulation factor IX.
suitable liquid. No antimicrobial preservative or antibiotic is
added. The solution is passed through a bacteria-retentive Water
filter, distributed aseptically into the final containers and Determined by a suitable method, such as semi-micro
immediately frozen. It is subsequently freeze-dried and the determination of water (2.5.12), loss on drying (2.2.32) or
containers are closed under vacuum or under an inert gas. near-infrared spectroscopy (2.2.40), the water content is
CONSISTENCY OF THE METHOD OF
PRODUCTION Sterility (2. 6.1)
It shall be demonstrated that the manufacturing process It complies with the test.
yields a product having a consistent composition. This is Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14')
evaluated by suitable analytical procedures that are It complies with the test for pyrogens or, preferably and
determined during process development and that normally where justified and authorised, with a validated in vitro test
include: such as the test for bacterial endotoxins.
- assay of human coagulation factor IX; For the pyrogen test, inject per kilogram of the rabbit's mass
- determination of activated coagulation factors; a volume equivalent to not less than 50 IU of human
- determination of activities of human coagulation coagulation factor IX.
factors II, VII and X, which shall be shown to be not
more than 5 per cent of the activity of human coagulation
preparation to be examined contains less than 0.03 IU of
factor IX.
endotoxin per International Unit of human coagulation
CHARACTERS factor IX.
Appearance
ASSAY
White or pale yellow, hygroscopic powder or friable solid.
Human coagulation factor IX (2. 7.11)
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 canying out the identification, tests (except more than 125 per cent of the stated potency.
those for solubility and water) and assay. The confidence limits (P = 0.95) are not less than
IDENTIFICATION 80 per cent and not more than 125 per cent of the estimated
It complies with the limits of the assay. potency.
TESTS STORAGE
Solubility In an airtight container, protected from light.
To a container of the preparation to be examined add the LABELLING
volume of the liquid stated on the label at the recommended The label states:
temperature. The preparation dissolves completely with - the number oflnternational Units of human coagulation
gentle swirling within 10 min, giving a clear or slightly factor IX per container;
opalescent, colourless solution. - the amount of protein per container;
pH (2.2.3) - the name and quantity of any added substances including,
6.5 to 7.5. where applicable, heparin;
Osmolality (2.2.35) - the name and volume of the liquid to be used for
Minimum 240 mosmol/kg. reconstitution;
- that the transmission of infectious agents cannot be totally of sodium molybdate R and 2 mL of a mixture of 1 volume of
excluded when medicinal products prepared from human nitrogen-free sulfuric acid R and 30 volumes of water R. Shake,
blood or plasma are administered. centrifuge for 5 min, decant the supernatant and allow the
and calculate the amount of protein by multiplying the result
by 6.25.
Dried Factor XI Fraction Activated coagulation factors (2.6.22)
For each of the dilutions, the coagulation time is not less
(Human Coagulation Facwr XI, Ph. Bur. monograph than 150 s.
1644)
Heparin (2. 7.12)
Action and use If heparin has been added, the preparation to be examined
Coagulation factor XI substitute. contains not more than the amount of heparin stated on the
label and in all cases not more than 0.5 IU of heparin
per unit of factor XI.
DEFINITION Antithrombin III (2. 7.17)
Sterile plasma protein fraction containing coagulation If antithrombin III has been added, the preparation to be
factor XI. It is prepared from Human plasma for examined contains not more than the amount of
fractionation (0853). The preparation may contain excipients antithrombin III stated on the label.
such as heparin, Cl-esterase inhibitor and antithrombin III.
Cl-esterase inhibitor (2. 7.34)
The potency of the preparation, reconstituted as stated on If C 1-esterase inhibitor has been added, the preparation to
the label, is not less than 50 units per millilitre. be examined contains not more than the amount of
PRODUCTION C 1-esterase inhibitor stated on the label.
The method of preparation is designed to maintain Anti-A and anti-B haemagglutinins (2.6.20, Method A)
functional integrity of human coagulation factor XI and to The 1 to 64 dilution does not show agglutination.
minimise activation of any coagulation factor (to minimise
Water
potential thrombogenicity). It includes a step or steps that
have been shown to remove or to inactivate known agents of
infection; if substances are used for inactivation of viruses
near-infrared spectroscopy (2.2.40), the water content is
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 It complies with the test.
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 bacterial endotoxin test.
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 100 IU of factor XI.
CHARACTERS Where the bacterial endotoxin test is used, the preparation to
Appearance be examined contains less than 0.1 IU of endotoxin per
White or almost white powder or friable solid. International Unit of factor XI.
Reconstitute the preparation to be examined as stated on the label ASSAY
immediately before canying out the identification, tests (except Carry out the assay of human coagulation factor XI (2.7.22).
those for solubility and water) and assay. The estimated potency is not less than 80 per cent and not
IDENTIFICATION more than 120 per cent of the stated potency.
It complies with the limits of the assay. The confidence limits (P = 0.95) are not less than
TESTS
potency.
Solubility
To a container of the preparation to be examined, add the STORAGE
volume of liquid stated on the label at room temperature. Protected from light, at a temperature of 2 °C to 8 °C.
The preparation dissolves completely with gentle swirling LABELLING
within 10 min. The label states:
pH (2.2.3) - the number of units per container;
6.8 to 7.4. - the maximum amount of protein per container;
Osmolality (2.2.35) - where applicable, the amount of heparin per container;
Minimum 240 mosmol/kg. - where applicable, the amount of antithrombin III per
container;
Total protein
- where applicable, the amount of C 1-esterase inhibitor per
container;
preparation to be examined with a 9 g/L solution of sodium
chloride R to obtain a protein concentration of about
reconstitution.
7.5 mg/mL. Place 2.0 mL of this solution in a round-
bottomed centrifuge tube and add 2 mL of a 7 5 g/L solution
TESTS
Dried Prothrombin Complex Solubility
(Human Prothrombin Complex, Ph. Bur. monograph To a container of the preparation to be examined add the
0554) volume of the liquid stated on the label at the recommended
temperature. The preparation dissolves completely with
Action and use gentle swirling within 10 min, giving a clear solution that
Coagulation factor IX substitute. Preparations with may be coloured.
appropriate activity may be used to correct deficiencies of
pH (2.2.3)
coagulation factors II or X.
6.5 to 7.5.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Osmolality (2.2.35)
DEFINITION Minimum 240 mosmol/kg.
Sterile plasma protein fraction containing human coagulation Total protein
factor IX together with variable amounts of human If necessary, dilute an accurately measured volume of the
coagulation factors II, VII and X; the presence and reconstituted preparation with a 9 g/L solution of sodium
proportion of these additional factors depends on the method chloride R to obtain a solution containing about 15 mg of
of fractionation. It is obtained from human plasma that protein in 2 mL. To 2.0 mL of the solution in a round-
complies with the monograph on Human plasma for bottomed centrifuge tube add 2 mL of a 75 g/L solution of
fractionation (0853). The preparation may contain excipients sodium molybdate R and 2 mL of a mixture of 1 volume of
such as stabilisers, heparin and antithrombin. nitrogen-free suljuric acid R and 30 volumes of water R. Shake,
The potency of the preparation, reconstituted as stated on centrifuge for 5 min, decant the supernatant and allow the
the label, is not less than 20 IU of human coagulation inverted tube to drain on filter paper. Determine the nitrogen
factor IX per millilitre. in the residue by the method of sulfuric acid digestion (2. 5. 9)
If the content of any of the factors is stated as a single value,
and calculate the amount of protein by multiplying the result
by 6.25.
the estimated potency is not less than 80 per cent and not
more than 125 per cent of the stated potency; if the content Activated coagulation factors (2. 6.22)
of any of the factors is stated as a range, the estimated If necessary, dilute the reconstituted preparation to contain
potency is not less than the lower limit and not greater than 20 IU of human coagulation factor IX per millilitre. For each
the upper limit of the stated range. of the dilutions, the coagulation time is not less than 150 s.
PRODUCTION Heparin (2. 7.12)
The method of preparation is designed to maintain If heparin has been added during preparation, the
functional integrity of the relevant coagulation factors it preparation to be examined contains not more than the
contains and to minimise activation of any coagulation factor amount of heparin stated on the label and in all cases not
(to minimise potential thrombogenicity). It includes a step or more than 0.5 IU of heparin per International Unit of human
steps that have been shown to remove or to inactivate known coagulation factor IX.
agents of infection; if substances are used for inactivation of Thrombin
viruses during production, the subsequent purification If the preparation to be examined contains heparin,
procedure must be validated to demonstrate that the determine the amount present as described in the test for
concentration of these substances is reduced to a suitable heparin and neutralise it by addition of protamine sulfate R
level and that any residues are such as not to compromise the (10 µg ofprotamine sulfate neutralises 1 IU of heparin).
safety of the preparation for patients. In each of 2 test-tubes, mix equal volumes of the
The specific activity is not less than 0.6 IU of human reconstituted preparation and of a 3 g/L solution of
coagulation factor IX per milligram of total protein, before fibrinogen R. Keep one of the tubes at 37 °C for 6 hand the
the addition of any protein stabiliser. other at room temperature for 24 h. In a 3rd tube, mix equal
volumes of the fibrinogen solution and of a solution of
The prothrombin complex fraction is dissolved in a suitable
human thrombin R (1 IU/mL) and place the tube in a water-
liquid. No antimicrobial preservative or antibiotic is added.
bath at 37 °C. No coagulation occurs in the tubes containing
The solution is passed through a bacteria-retentive filter,
the preparation to be examined. Coagulation occurs within
distributed aseptically into the final containers and
immediately frozen. It is subsequently freeze-dried and the 30 s in the tube containing thrombin.
containers are closed under vacuum or under an inert gas. Water
CHARACTERS
Appearance near-infrared spectrometry (2.2.40), the water content is
White or slightly coloured, very hygroscopic powder or friable within the limits approved by the competent authority.
solid.
Sterility (2. 6.1)
immediately before carrying out the identification, tests ( except
those for solubility and water) and assay. Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
IDENTIFICATION where justified and authorised, with a validated in vitro test
It complies with the limits of the assays for human such as the bacterial endotoxin test.
coagulation factors IX and II and, where applicable, those for
human coagulation factors VII and X.
a volume equivalent to not less than 30 IU of human
coagulation factor IX.
Where the bacterial endotoxin test is used, the preparation to When reconstituted as stated on the label, the solution
be examined contains less than 0.05 IU of endotoxin per contains not less than 10 g/L of fibrinogen.
International Unit of human coagulation factor IX. PRODUCTION
ASSAY The method of preparation is designed to maintain
Human coagulation factor IX (2. 7. J J) functional integrity of human fibrinogen. It includes a step or
The estimated potency is not less than 80 per cent and not steps that have been shown to remove or to inactivate known
more than 125 per cent of the stated potency. agents of infection; if substances are used for inactivation of
The confidence interval (P = 0.95) is not greater than viruses during production, the subsequent purification
80 per cent to 125 per cent of the estimated potency. procedure must be validated to demonstrate that the
Human coagulation factor II (2. 7.18) concentration of these substances is reduced to a suitable
The estimated potency is not less than 80 per cent and not level and any residues are such as not to compromise the
more than 125 per cent of the stated potency. safety of the preparation for patients.
The confidence interval (P = 0.95) is not greater than The specific activity (fibrinogen content with respect to total
90 per cent to 111 per cent of the estimated potency. protein content) is not less than 80 per cent before addition
The estimated human coagulation factor II potency is not of any protein stabiliser. The fibrinogen content is
less than 70 per cent and not more than 165 per cent of the determined by a suitable method such as that described
estimated human coagulation factor IX potency. under Assay, and the total protein content is determined by a
suitable method such as that described under Total protein
Human coagulation factor VII (2. 7. J0)
in Human albumin solution (0255). Albumin may also be
If the label states that the preparation contains human obtained with fibrinogen during fractionation, in which case a
coagulation factor VII, the estimated potency is not less than specific determination of albumin is carried out by a suitable
80 per cent and not more than 125 per cent of the stated immunochemical method (2. 7. J) and the quantity of albumin
potency. The confidence interval (P = 0.95) is not greater determined is subtracted from the total protein content for
than 80 per cent to 125 per cent of the estimated potency. the calculation of the specific activity.
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 stated 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 VII 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.
- - - - - - - - - - - - - - - - - - - - - - PhEur Osmolality (2.2.35)
Minimum 240 mosmollkg.
Stability of solution
No gel formation appears at 20-25 °C within 60 min
Dried Fibrinogen following reconstitution.
(Human Fibri,wgen, Ph. Bur. monograph 0024) Water
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
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. J)
transformed to fibrin on the addition of thrombin. It is It complies with the test.
obtained from human plasma that complies with the
monograph on Human plasma for fractionation (0853).
The preparation may contain excipients such as salts, buffers
and stabilisers.
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14) concentration of these substances is reduced to a suitable
It complies with the test for pyrogens or, preferably and level and any residues are such as not to compromise the
where justified and authorised, with a validated in vitro test safety of the preparation for patients.
such as the test for bacterial endotoxins. The production method is shown to yield consistent levels of
For the pyrogen test, inject per kilogram of the rabbit's mass human coagulation factor XIII.
a volume equivalent to not less than 30 mg of fibrinogen. The constituents or mixtures of constituents are dissolved in
Where the test for bacterial endotoxins is used, the a suitable liquid. No antimicrobial preservative or antibiotic is
preparation to be examined contains less than 0.03 IU of added. Constituents or mixtures of constituents are passed
endotoxin per milligram of fibrinogen. through a bacteria-retentive filter and distributed aseptically
ASSAY into sterile containers. Containers of freeze-dried constituents
are closed under vacuum or filled with a suitable inert gas,
Mix 0.2 mL of the reconstituted preparation with 2 mL of a
such as oxygen-free nitrogen, before being closed.
suitable buffer solution (pH 6.6-6.8) containing sufficient
thrombin (approximately 3 IU/mL) and calcium If the label states that human coagulation factor XIII is an
(0.05 mol/L). Maintain at 37 °C for 20 min, separate the active substance in component 1, the assay of human
precipitate by centrifugation (5000 g, 20 min) and wash coagulation factor XIII is carried out.
thoroughly with a 9 g/L solution of sodium chloride R. CHARACTERS
Determine the nitrogen content by sulfuric acid digestion Appearance
(2.5.9) and calculate the fibrinogen (clottable protein) - freeze-dried constituents: white or pale yellow, hygroscopic
content by multiplying the result by 6.0. The content is not powder or friable solid;
less than 70 per cent and not more than 130 per cent of the - frozen constituents: colourless or pale yellow, opaque solid;
stated content of fibrinogen. - liquid constituents: colourless or pale yellow liquid.
STORAGE For the freeze-dried or frozen constituents, reconstitute or thaw as
In an airtight container, protected from light. stated on the label immediately before carrying out the
identification and the tests, except those for solubility and water.
LABELLING
The label states:
- the content of fibrinogen in the container; COMPONENT 1 (FIBRINOGEN
- the name and volume of the liquid to be used for CONCENfRATE)
reconstitution; IDENTIFICATION
- where applicable, the name and amount of protein A. It complies with the limits of the assay of fibrinogen.
stabiliser added in the preparation.
B. It complies with the limits of the assay of human
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
coagulation factor XIII (where its content is stated on the
label).
TESTS
Solubility
Fibrin Sealant Kit Freeze-dried concentrates dissolve within 20 min in the
(Ph. Eur. monograph 0903) volume of liquid and at the temperature stated on the label,
forming an almost colourless, clear or slightly turbid solution.
pH (2.2.3)
DEFINITION 6.5 to 8.0.
Sterile, freeze-dried, frozen or liquid preparation of plasma Stability of solution
protein fractions containing essentially 2 components, namely No gel formation appears at room temperature during
fibrinogen concentrate (component 1), a protein fraction 120 min following thawing or reconstitution.
containing human fibrinogen, and a preparation containing
human thrombin (component 2). A fibrin clot is rapidly Water
formed when the 2 thawed or reconstituted components are Determined by a suitable method, such as semi-micro
mixed. Other ingredients (for example, human coagulation determination of water (2.5.12), loss on drying (2.2.32) or
factor XIII, a fibrinolysis inhibitor or calcium ions) and near-infrared spectroscopy (2.2.40), the water content is
stabilisers (for example, Human albumin solution (0255)) may within the limits approved by the competent authority.
be added. Sterility (2. 6.1)
Human constituents are obtained from plasma that complies It complies with the test.
with the monograph Human plasma for fractionation (0853). ASSAY
When thawed or reconstituted as stated on the label, Fibrinogen (clottable protein)
component 1 contains not less than 40 g/L of clottable Mix 0.2 mL of the reconstituted concentrate with 2 mL of a
protein; the thrombin activity of component 2 varies over a suitable buffer solution (pH 6.6-7 .4) containing sufficient
wide range (approximately 4-1000 IU/mL). human thrombin R (approximately 3 IU/mL) and calcium
(0.05 mol/L). Maintain at 37 °C for 20 min, separate the
PRODUCTION
precipitate by centrifugation at 5000 g for 20 min, wash
The method of preparation is designed to maintain
thoroughly with a 9 g/L solution of sodium chloride R and
functional integrity of the components. It includes a step or
determine the protein as nitrogen by sulfuric acid digestion
steps that have been shown to remove or to inactivate known
(2.5.9). Calculate the clottable protein content by multiplying
agents of infection; if substances are used for inactivation of
the result by 6.0. The estimated content in milligrams of
viruses during production, the subsequent purification
clottable protein is not less than 70 per cent and not more
procedure must be validated to demonstrate that the
than 130 per cent of the stated content. If for a particular
preparation this method cannot be applied, use another measurement of the clotting time immediately. Repeat the
validated method for determination of fibrinogen. procedure with each of at least 3 dilutions, in the range
Human coagulation factor XIII stated above, of a reference preparation of thrombin,
Use a reference plasma calibrated against the International calibrated in International Units.
Standard for blood coagulation factor XIII in plasma. If the Calculate the activity of the test preparation by the usual
label states that human coagulation factor XIII is present as statistical methods (5.3, for example). The estimated activity
an active substance, the estimated potency is not less than is not less than 80 per cent and not more than 125 per cent
80 per cent and not more than 120 per cent of the stated of the stated activity. For a component with a low thrombin
potency if the factor XIII potency is stated as a single value, concentration and a nominal value of approximately
or is within the stated range if the factor XIII potency is 4 IU/mL, the estimated activity is not less than 50 per cent
stated as a range. and not more than 150 per cent of the stated activity.
Make at least 3 suitable dilutions of thawed or reconstituted The confidence limits (P = 0.95) are not less than
concentrate and of the reference preparation using human 80 per cent and not more than 125 per cent of the estimated
coagulation factor XIII-deficient plasma or another suitable activity.
diluent. Coagulation factors V, VIII, XI and XIII plasma ERP The following sections apply to 2-component fibn·n sealant.
is suitable for use as a reference preparation. Add to each STORAGE
dilution suitable amounts of the following reagents: Protected from light and, for freeze-dried components, in an
- activator reagent, containing bovine or human thrombin, airtight container.
a suitable buffer, calcium chloride and a suitable inhibitor
such as Gly-Pro-Arg-Pro-Ala-NH 2 which inhibits clotting LABELLING
of the sample but does not prevent human coagulation The label states:
factor XIII activation by thrombin; - the amount of fibrinogen (milligrams of clottable protein)
- detection reagent, containing a suitable factor XIIIa- and thrombin (International Units) per container, and the
specific peptide substrate, such as Leu-Gly-Pro-Gly-Glu- content of human coagulation factor XIII (International
Ser-Lys-Val-Ile-Gly-NH2 and glycine ethyl ester as 2nd Units per millilitre), if the latter is present as an active
substrate in a suitable buffer solution; substance;
- NADH reagent, containing glutamate dehydrogenase, - where applicable, the name and volume of the liquid to
a-ketoglutarate and NADH in a suitable buffer solution. be used to reconstitute the components.
After mixing, the absorbance changes (M/min) are measured _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
at a wavelength of 340 nm, after the linear phase of the

reaction is reached.
Calculate the potency of the test preparation by the usual
statistical methods (5.3, for example). The confidence limits Human Haematopoietic Stem Cells
(P = 0.95) are not less than 80 per cent and not more than
125 per cent of the estimated potency. (Ph. Bur. monograph 2323)
COMPONENT 2 (THROMBIN This monograph provides a standard for the preparation and
PREPARATION) control of human haematopoietic stem cells for use in therapy.
IDENTIFICATION It does not exclude the use of alternative preparation and control
It complies with the limits of the assay of thrombin. methods that are acceptable to the competent authority.
TESTS DEFINITION
Solubility Human haematopoietic stem cells are primitive multipotent
Freeze-dried preparations dissolve within 5 min in the cells capable of self-renewal as well as differentiation and
volume of liquid stated on the label, forming a colourless, maturation into all haematopoietic lineages. They are found
clear or slightly turbid solution. in small numbers in bone marrow, in the mononuclear cell
fraction of circulating blood and in umbilical cord blood.
pH (2.2.3)
The preparation also contains haematopoietic progenitor
5.0 to 8.0.
cells, which are capable of differentiation but not self-
Water renewal. The numbers of haematopoietic stem cells and
Determined by a suitable method, such as semi-micro haematopoietic progenitor cells are correlated.
This monograph applies to haematopoietic stem cells that
have not undergone expansion or genetic modification, and
that are intended to provide a successful engraftment leading
Sterility (2. 6.1) to a permanent restoration of all lineages of blood cell
It complies with the test. 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 R. 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
are able to give rise to colonies in the presence of various Transmissible spongiform encephalopathies (5.2.8)
growth factors. The membrane marker CD34 is commonly A risk assessment of the product with respect to transmissible
used for the successful isolatioru'purification of spongiform encephalopathies is carried out, and suitable
haematopoietic stem cells from crude preparations and as an measures are taken to minimise any such risk.
indicator of haematopoietic stem cell content in routine Water
quality control. Water used in the preparation of cellular products complies
PRODUCTION with the relevant monograph (Water for injections (0169),
DONORS Purified water (0008)). Water incorporated into the final
Where allogeneic cells are used, they are derived from product complies with the section on Water for injections in
carefully selected donors in accordance with donor selection bulk in the monograph Water for injections (0169), and in
criteria. Directive 2004/23/EC of the European Union deals addition is sterile.
with the criteria for donor selection. TESTS
COLLECTION Target specifications are established for the different tests, but these
Peripheral blood stem cells These are collected by cytapheresis are not used as rigid acceptance criteria.
after mobilisation from the bone marrow by administration of Tests carried out include the following (further tests, such as
growth factors and/or treatment of autologous donors with purging, cell depletion, allogeneic application, may be
cytotoxic substances. The cells may be processed to select a necessary depending on any treatment applied to the cells
population of interest and may be cryopreserved. and on the intended recipient):
Bone marrow Bone marrow is harvested by aspirating the Nucleated cell count (2.7.29)
cells from the cavities of hollow bones, then removing bone
fragments by filtration and, if necessary, separating the buffy Viability (2. 7. 29)
coat cells after centrifugation or with commercial kits based Viability is assessed for products that are not infused within
on the cytapheresis principle. The cells may be processed to 24 h of collection.
select a population of interest and may be cryopreserved. CD34+ cell count
Umbilical cord blood Placental blood haematopoietic cells are For peripheral blood stem cells, CD34+ cell count is
collected from placentae via the vein of the umbilical cord. determined using a validated automated apparatus to analyse
The cells are then cryopreserved. cells labelled with anti-CD34 antibodies. The apparatus and
method employed must be able to determine the number of
CRYOPRESERVATION CD34+ cells with a sensitivity, accuracy and reproducibility
Cryopreservation allows storage for long periods. The cells comparable with those of immunophenotyping (2. 7.23),
are suspended in a validated medium containing a suitable where cells are labelled using anti-CD34 and anti-CD45
cryoprotectant (for example, dimethyl sulfoxide) and antibodies conjugated to a fluorochrome and analysed by
macromolecules (for example, autologous plasma/albumin) flow cytometry (2.7.24).
and are frozen in cryobags in a manner designed to maintain
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 any 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
of CD34+ cells is made to the protocol for packaging or
SUBSTANCES USED IN PRODUCTION mobilisation, it is carried out on a suitable number of units.
The quality of substances used in production may be critical
with respect to the quality, safety and efficacy of the final Microbiological control
product, particularly for substances of biological origin. This Examine as prescribed in general chapter 2.6.27.
is of particular importance for: Microbiological examination of cell-based preparations. Where
- proteins, including enzymes and antibodies; justified, the product may be released before completion of
- cryopreservation reagents; the test.
- purification reagents. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Quality assurance
All substances must be produced within a recognised quality
management system using suitable production facilities.
Quality specifications Normal lmmunoglobulin for
A suitable quality specification must be presented for each Intramuscular Administration
substance, including notably:
Normal Immunoglobulin
- identity;
- potency (where applicable); Normal Immunoglobulin Injection
- purity; (Human Normal Immunoglobulin for Intramuscular
- determination of bacterial endotoxins (2.6.14) (where Administration, Ph. Bur. monograph 0338)
applicable); PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
- microbiological quality (total viable count, tests for
specified micro-organisms); DEFINITION
- sterility (2. 6. 1) (where applicable). Sterile liquid or freeze-dried preparation containing
immunoglobulins, mainly immunoglobulin G (lgG). 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 corresponds to the IgG component of normal human serum.
normal subjects and is intended for intramuscular The preparation to be examined may show the presence of
administration. The preparation may contain excipients such small quantities of other plasma proteins; if human albumin
as stabilisers. Multidose preparations contain an antimicrobial has been added as a stabiliser, it may be seen as a
preservative. component.
This monograph does not apply to products intentionally TESTS
prepared to contain fragments of IgG or chemically modified Solubility
IgG. For the freeze-dried preparation, to a container of the
Human normal immunoglobulin for intramuscular preparation to be examined add the volume of the liquid
administration is obtained from plasma that complies with stated on the label at the recommended temperature.
the monograph Human p/,asma for fractionation (0853). The preparation dissolves completely within 20 min at
PRODUCTION 20-25 °C.
The method of preparation includes a step or steps that have pH (2.2.3)
been shown to remove or to inactivate known agents of 5.0 to 7.2.
infection; if substances are used for inactivation of viruses, it Dilute the preparation to be examined with a 9 g/L solution
shall have been shown that any residues present in the final of sodium chloride R to obtain a protein concentration of
product have no adverse effects on the patients treated with 10 g/L.
the immunoglobulin. Total protein
The product shall have been shown, by suitable tests in The preparation has a protein concentration of not less than
animals and evaluation during clinical trials, to be well 100 g/L and not more than 180 g/L and contains not less
tolerated when administered intramuscularly. than 90 per cent and not more than 110 per cent of the
Any antimicrobial preservative or stabilising agent used shall quantity of protein stated on the label.
have been shown to have no deleterious effect on the final Dilute the preparation to be examined with a 9 g/L solution
product in the amount present. of sodium chloride R to obtain a protein concentration of
Human normal immunoglobulin for intramuscular about 7.5 mg/mL. Place 2.0 mL of this solution in a round-
administration is prepared from pooled material from not bottomed centrifuge tube and add 2 mL of a 75 g/L solution
fewer than 1000 donors by a method that has been shown to of sodium molybdate R and 2 mL of a mixture of 1 volume of
yield a product that: nitrogen-free sulfuric add R and 30 volumes of water R. Shake,
- does not transmit infection; centrifuge for 5 min, decant the supernatant and allow the
- at a protein concentration of 50 g/L, contains at least 2 inverted tube to drain on filter paper. Determine the nitrogen
antibodies ( 1 viral and 1 bacterial) for which an in the centrifugation residue by the method of sulfuric acid
International Standard or reference preparation is digestion (2.5. 9) and calculate the content of protein by
available, the concentration of such antibodies being at multiplying the result by 6.25.
least 3 times that in the initial pooled material;
Protein composition
- has a defined distribution of IgG subclasses.
Zone electrophoresis (2.2.31).
Human normal immunoglobulin for intramuscular
Use strips of suitable cellulose acetate gel or suitable agarose
administration is prepared as a stabilised solution, for
gel as the supporting medium and barbital buffer solution
example in a 9 g/L solution of sodium chloride, a 22.5 g/L
pH 8. 6 Rl as the electrolyte solution.
solution of glycine or, if the preparation is to be freeze-dried,
a 60 g/L solution of glycine. No antibiotic is added to the If cellulose acetate is the supporting material, the analytical
plasma used. Single-dose preparations do not contain an procedure described below can be used. If agarose gels are
antimicrobial preservative. The solution is passed through a used, and because they are normally part of an automated
bacteria-retentive filter. The preparation may subsequently be system, the manufacturer's instructions are followed instead.
freeze-dried and the containers closed under vacuum or Test solution Dilute the preparation to be examined with a
under an 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
electrophoresis BRP and dilute with a 9 g/L solution of sodium
CHARACTERS
chloride R to obtain a protein concentration of 30 g/L.
Appearance
To a strip apply 4.0 µL of the test solution as a 10 mm band
- liquid preparation: clear or slightly opalescent, colourless or
or apply 0.4 µL per millimetre if a narrower strip is used.
pale-yellow or light-brown liquid; during storage it may
To another strip apply in the same manner the same volume
show formation of slight turbidity or a small amount of
of the reference solution. Apply a suitable electric field such
visible particulate matter;
that the albumin band of normal human serum applied on a
- freeze-dried preparation: white or slightly yellow powder or
control strip migrates at least 30 mm. Stain the strip with
solid friable mass, hygroscopic.
amido black 10B solution R for 5 min. Decolourise with a
For the freeze-dried preparation, reconstitute as stated on the label mixture of 10 volumes of glacial acetic add R and 90 volumes
immediately before carrying out the identification and the tests, of methanol R so that the background is just free of colour.
except those for solubz7ity and water. Develop the transparency of the strips with a mixture of
IDENTIFICATION 19 volumes of glacial acetic add R and 81 volumes of
Examine by a suitable immunoelectrophoresis 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 Determine the antibody content by comparison with a
reference solution, the proportion of protein in the principal reference preparation calibrated in International Units, using
band is within the limits stated in the leaflet accompanying an immunoassay of suitable sensitivity and specificity (2. 7.1).
the reference preparation. The International Unit is the activity contained in a stated
Results In the eiectropherogram obtained with the test amount of the International Standard for anti-hepatitis A
solution, not more than 10 per cent of protein has a mobility immunoglobulin. The equivalence in International Units of
different from that of the principal band. This limit is not the International Standard is stated by the World Health
applicable if albumin has been added to the preparation as a Organization.
stabiliser; for such preparations, a test for protein Human hepatitis A immunoglobulin ERP is calibrated in
composition is carried out during manufacture before International Units by comparison with the International
addition of the stabiliser. Standard.
Molecular-size distribution The stated potency is not less than 100 IU/mL.
Size-exclusion chromatography (2.2.30): use the The estimated potency is not less than the stated potency.
normalisation procedure. The confidence limits (P = 0.95) are not less than
Test solution Dilute the preparation to be examined with a 80 per cent and not more than 125 per cent of the estimated
9 g/L solution of sodium ch/,oride R to a concentration suitable potency.
for the chromatographic system used. A concentration in the
Immunoglobulin A
range of 4-12 g/L and injection of 50-600 µg of protein are
As determined by a suitable immunochemicai method
usually suitable.
(2. 7.1), the content of immunoglobulin A is not greater than
Reference solution Dilute human immunoglobulin (molecular the maximum content stated on the label.
size) ERP with a 9 g/L solution of sodium chloride R to the
same protein concentration as the test solution. Water
Column:
determination of water (2.5. 12), loss on drying (2.2.32) or
- size: l = 0.6 m, 0 =7.5 mm [or l = 0.3 m, 0 = 7.8 mm];
- stationary phase: hydrophilic silica gel for chromatography R,
of a grade suitable for fractionation of globular proteins
with relative molecular masses in the range 10 000 to Sterility (2. 6.1)
500 000. It complies with the test.
Mobile phase Dissolve 4.873 g of disodium hydrogen phosphate Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
dihydrate R, 1. 741 g of sodium dihydrogen phosphate It complies with the test for pyrogens or, preferably and
monohydrate R, 11.688 g of sodium chloride Rand 50 mg of where justified and authorised, with a validated in vitro test
sodium azide R in 1 L of water R. such as the bacterial endotoxin test.
Flow rate 0.5 mIJmin. For the pyrogen test, inject 1 mL per kilogram of the rabbit's
Detection Spectrophotometer at 280 nm. mass.
Identification of peaks In the chromatogram obtained with Where the bacterial endotoxin test is used, the preparation to
the reference solution, the principal peak corresponds to the be examined contains less than 5 IU of endotoxin per
IgG monomer and there is a peak corresponding to the millilitre.
dimer with a relative retention to the principal peak of STORAGE
about 0.85. Identify the peaks in the chromatogram obtained In an airtight, colourless glass container, protected from light,
with the test solution by comparison with the chromatogram at the temperature stated on the label.
obtained with the reference solution; any peak with a
retention time less than that of the dimer corresponds to LABELLING
polymers and aggregates; any peak with a retention time The label states:
greater than that of the monomer corresponds to fragments. - for liquid preparations, the volume of the preparation in
Results In the chromatogram obtained with the test the container and the protein content expressed in grams
solution: per litre;
- retention time: for the monomer and for the dimer, the - for freeze-dried preparations:
retention time relative to the corresponding peak in the - the quantity of protein in the container;
chromatogram obtained with the reference solution is - the name or composition and the volume of the
1 ± 0.02; reconstituting liquid to be added;
- peak area: the sum of the peak areas of the monomer and - the route of administration;
the dimer represent not less than 85 per cent of the total - the distribution of subclasses of IgG present in the
area of the chromatogram and the sum of the peak areas preparation;
of polymers and aggregates represents not more than - where applicable, that the preparation is suitable for use
10 per cent of the total area of the chromatogram. This in the prophylaxis of hepatitis A infection;
requirement is not applicable if albumin has been added - where applicable, the anti-hepatitis A virus activity in
as a stabiliser; for such preparations, a test for molecular- International Units per millilitre;
size distribution is carried out during manufacture before - where applicable, the amount of albumin added as a
addition of the stabiliser. stabiliser;
Antibody to hepatitis B surface antigen - where applicable, the name and amount of antimicrobial
Minimum 0.5 IU per gram of immunoglobulin, determined preservative in the preparation;
- the maximum content of immunoglobulin A.
by a suitable immunochemicai method (2.7.1).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Antibody to hepatitis A virus
If intended for use in the prophylaxis of hepatitis A, it
complies with the following additional requirement.
CHARACTERS
Normal lmmunoglobulin for Appearance
Intravenous Use - liquid preparation: clear or slightly opalescent, colourless or
pale yellow liquid;
(Human Normal Immunoglobulin for Intravenous
Administration, Ph. Bur. monograph 0918) - freeze-dried preparatwn: hygroscopic, white or slightly
yellow powder or solid friable mass.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
For the freeze-dried preparation, reconstitute as stated on the label
DEFINITION immediately before carrying out the identification and the tests,
Human normal immunoglobulin for intravenous except those for solubility and water.
administration is a sterile liquid or freeze-dried preparation IDENTIFICATION
containing immunoglobulins, mainly Examine by a suitable immunoelectrophoresis technique.
immunoglobulin G (IgG). Other proteins may be present. Using antiserum to normal human serum, compare normal
Human normal immunoglobulin for intravenous human serum and the preparation to be examined, both
administration contains the IgG antibodies of normal diluted to contain 10 g/L of protein. The main component of
subjects. This monograph does not apply to products the preparation to be examined corresponds to the IgG
intentionally prepared to contain fragments or chemically component of normal human serum. The preparation to be
modified IgG. examined may show the presence of small quantities of other
Human normal immunoglobulin for intravenous plasma proteins; if human albumin has been added as a
administration is obtained from plasma that complies with stabiliser, it may be seen as a major component.
the monograph Human plasma for fractionation (0853).
TESTS
The preparation may contain excipients such as stabilisers.
Solubility
PRODUCTION For the freeze-dried preparation, add to the container the
The method of preparation includes a step or steps that have volume of the liquid stated on the label at the recommended
been shown to remove or to inactivate known agents of temperature. The preparation dissolves completely within
infection; if substances are used for inactivation of viruses, it 30 min at 20-25 °C.
shall have been shown that any residues present in the final
pH (2.2.3)
product have no adverse effects on the patients treated with
4.0 to 7.4.
the immunoglobulin. The method of preparation also
includes a step or steps that have been shown to remove Dilute the preparation to be examined with a 9 g/L solution
thrombosis-generating agents. Emphasis is given to the of sodium chloride R to obtain a solution containing 10 g/L of
identification of activated coagulation factors and their protein.
zymogens and process steps that may cause their activation. Osmolality (2.2.35)
Consideration is also to be given to other procoagulant Minimum 240 mosmol/kg.
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 ofthis 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 suljuric 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 (2.2.31).
- complies with the test for Fe function of immunoglobulin
Use strips of suitable cellulose acetate gel or suitable agarose
(2.7.9);
gel as the supporting medium and barbital buffer solutwn
- 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 analytical
administration is prepared as a stabilised solution or as a
procedure described below can be used. If agarose gels are
freeze-dried preparation. In both cases the preparation is
used, and because they are normally part of an automated
passed through a bacteria-retentive filter. The preparation
system, the 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 Test solutwn Dilute the preparation to be examined with a
to the plasma used. No antimicrobial preservative is added 9 g/L solution of sodium chloniie R to an immunoglobulin
either during fractionation or at the stage of the final bulk concentration of 30 g/L.
solution. Reference solution Reconstitute human immunoglobulin for
The stability of the preparation is demonstrated by suitable electrophoresis BRP and dilute with a 9 g/L solution of sodium
tests carried out during development studies. chloride R to a protein concentration of 30 g/L.
To a strip apply 4.0 µL of the test solution as a 10 mm band chromatogram obtained with the reference solution is
or apply 0.4 µL per millimetre if a narrower strip is used. 1 ± 0.02;
To another strip apply in the same manner the same volume - peak area: the sum of the peak areas of the monomer and
of the reference solution. Apply a suitable electric field such the dimer represent not less than 90 per cent of the total
that the albumin band of normal human serum applied on a area of the chromatogram and the sum of the peak areas
control strip migrates at least 30 mm. Stain the strips with of polymers and aggregates represents not more than
amido black 10B solution R for 5 min. Decolourise with a 3 per cent of the total area of the chromatogram. This
mixture of 10 volumes of glacial acetic acid Rand 90 volumes requirement does not apply to products where albumin
of methanol R so that the background is just free of colour. has been added as a stabiliser; for products stabilised with
Develop the transparency of the strips with a mixture of albumin, a test for distribution of molecular size is carried
19 volumes of glacial acetic acid Rand 81 volumes of out during manufacture before addition of the stabiliser.
methanol R. Measure the absorbance of the bands at 600 nm Anticomplementary activity (2. 6.17)
in an instrument having a linear response over the range of The consumption of complement is not greater than
measurement. Calculate the result as the mean of 50 per cent (1 CH 50 per milligram of immunoglobulin).
3 measurements of each strip.
Prekallikrein activator (2. 6.15)
System suitability In the electropherogram obtained with the Maximum 35 IU/mL, calculated with reference to a dilution
reference solution, the proportion of protein in the principal of the preparation to be examined containing 30 g/L of
band is within the limits stated in the leaflet accompanying immunoglobulin.
the reference preparation.
Anti-A and anti-B haemagglutinins (2.6.20, Method B)
Results In the electropherogram obtained with the test
It complies with the test for anti-A and anti-B
solution, not more than 5 per cent of protein has a mobility
haemagglutinins (direct method).
different from that of the principal band. This limit is not
applicable if albumin has been added to the preparation as a Anti-D antibodies (2.6.26)
stabiliser; for such preparations, a test for protein It complies with the test for anti-D antibodies in human
composition is carried out during manufacture before immunoglobulin.
addition of the stabiliser. Antibody to hepatitis B surface antigen
Molecular size distribution Minimum 0.5 IU per gram of immunoglobulin, determined
Size exclusion chromatography (2.2.30): use the by a suitable immunochemical method (2.7.1).
normalisation procedure. Immunoglobulin A.
Test solutwn Dilute the preparation to be examined with a As determined by a suitable immunochemical method
9 g/L solution of sodium chloride R to a concentration suitable (2. 7. 1), the content of immunoglobulin A is not greater than
for the chromatographic system used. A concentration in the the maximum content stated on the label.
range of 4-12 g/L and injection of 50-600 µg of protein are Water
usually suitable. Determined by a suitable method, such as the semi-micro
Reference solutwn Dilute human immunoglobulin (molecular determination of water (2.5.12), loss on drying (2.2.32) or
size) ERP with a 9 g/L solution of sodium chloride R to the near-infrared spectroscopy (2.2.40), the water content is
same protein concentration as the test solution. within the limits approved by the competent authority.
Column: Sterility (2. 6.1)
- size: l = 0.6 m, 0 = 7.5 mm, or l = 0.3 m, 0 = 7.8 mm; It complies with the test.
- statwnary phase: hydrophilic silica gel for chromatography R Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
of a grade suitable for fractionation of globular proteins It complies with the test for pyrogens or, preferably and
with relative molecular masses in the range 10 000 to where justified and authorised, with a validated in vitro test
500 000. such as the bacterial endotoxin test.
Mobile phase Dissolve 4.873 g of disodium hydrogen phosphate For the pyrogen test, inject per kilogram of the rabbit's mass
dihydrate R, 1. 7 41 g of sodium dihydrogen phosphate a volume equivalent to 0.5 g of immunoglobulin, but not
monohydrate R, 11.688 g of sodium chloride Rand 50 mg of more than 10 mL per kilogram of the rabbit's mass.
sodium azide R in 1 L of water R.
Where the bacterial endotoxin test is used, the preparation to
Flow rate 0.5 mUmin. be examined contains less than 0.5 IU of endotoxin per
Detection Spectrophotometer at 280 nm. millilitre for solutions with a protein content not greater than
Identification of peaks In the chromatogram obtained with 50 g/L, and less than 1.0 IU of endotoxin per millilitre for
the reference solution, the principal peak corresponds to the solutions with a protein content greater than 50 g/L but not
IgG monomer and there is a peak corresponding to the greater than 100 g/L.
dimer with a relative retention to the principal peak of STORAGE
about 0.85; identify the peaks in the chromatogram obtained Liquid preparation: in a colourless glass container, protected
with the test solution by comparison with the chromatogram from light, at the temperature stated on the label.
obtained with the reference solution; any peak with a
Freeze-dried preparation: in an airtight colourless glass
retention time shorter than that of the dimer corresponds to
polymers and aggregates; any peak with a retention time container, protected from light, at a temperature not
greater than that of the monomer corresponds to fragments. exceeding 25 °C.
Results In the chromatogram obtained with the test LABELLING
solution: The label states:
- retention time: for the monomer and for the dimer, the - for liquid preparations, the volume of the preparation in
retention time relative to the corresponding peak in the the container and the protein content expressed in grams
per litre;
- for freeze-dried preparations: - has a defined distribution of lgG subclasses;

- the quantity of protein in the container; - complies with the test for Fe function of immunoglobulin
- the name or composition and the volume of the (2. 7. 9);
reconstituting liquid to be added; - does not exhibit thrombogenic (procoagulant) activity.
- the amount of immunoglobulin in the container; Human normal immunoglobulin for subcutaneous
- the route of administration; administration is prepared as a stabilised solution, for
- the distribution of subclasses of immunoglobulin G example in a 9 g/L solution of sodium chloride, a 22.5 g/L
present in the preparation; solution of glycine or, if the preparation is to be freeze-dried,
- where applicable, the amount of albumin added as a a 60 g/L solution of glycine. No antibiotic is added to the
stabiliser; plasma used. Preparations do not contain an antimicrobial
- the maximum content of immunoglobulin A. preservative. The solution is passed through a bacteria-
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur retentive filter. The preparation may subsequently be freeze-
dried and the containers closed under vacuum or under an
inert gas.
The stability of the preparation is demonstrated by suitable
Normal lmmunoglobulin for tests carried out during development studies.
Subcutaneous Administration CHARACTERS

Appearance
(Human Normal Immunoglobulin for Subcutaneous - liquid preparation: clear or slightly opalescent, colourless or
Administration, Ph. Bur. monograph 2788)
pale-yellow or light-brown liquid; during storage it may
show formation of slight turbidity or a small amount of
visible particulate matter;
DEFINITION
Sterile liquid or freeze-dried preparation containing
solid friable mass, hygroscopic.
immunoglobulins, mainly immunoglobulin G (lgG). Other
proteins may be present. Human normal immunoglobulin for For the freeze-dried preparation, reconstitute as stated on the label
subcutaneous administration contains the IgG antibodies of immediately before canying out the identification and the tests,
normal subjects and is intended for subcutaneous except those for solubility and water.
administration. The preparation may contain excipients such IDENTIFICATION
as stabilisers. Examine by a suitable immunoelectrophoresis technique.
This monograph does not apply to products intentionally Using antiserum to normal human serum, compare normal
prepared to contain fragments of IgG or chemically modified human serum and the preparation to be examined, both
IgG. diluted to obtain a protein concentration of 10 g/L.
Human normal immunoglobulin for subcutaneous The main component of the preparation to be examined
administration is obtained from plasma that complies with corresponds to the IgG component of normal human serum.
the monograph Human plasma for fractionation (0853). The preparation to be examined may show the presence of
small quantities of other plasma proteins; if human albumin
PRODUCTION has been added as a stabiliser, it may be seen as a
The method of preparation includes a step or steps that have component.
been shown to remove or to inactivate known agents of
infection; 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
stated on the label at the recommended temperature.
The method of preparation also includes a step or steps that
The preparation dissolves completely within 20 min at
have been shown to remove thrombosis-generating agents.
20-25 °C.
Emphasis is given to the identification of activated
coagulation factors and their zymogens and process steps that pH (2.2.3)
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 Dilute the preparation to be examined with a 9 g/L solution
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 Total protein
tolerated when administered subcutaneously. Any stabilising 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. than 90 per cent and not more than 110 per cent of the
Human normal immunoglobulin for subcutaneous quantity of protein stated on the label.
administration is prepared from pooled material from not Dilute the preparation to be examined with a 9 g/L solution
fewer than 1000 donors by a method that has been shown to of sodium chloride R to obtain a protein concentration of
yield a product that: 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 nitrogen-free sulfuric acid R and 30 volumes of water R. Shake,
International Standard or reference preparation is centrifuge for 5 min, decant the supernatant and allow the
available, the concentration of such antibodies being at inverted tube to drain on filter paper. Determine the nitrogen
least 3 times that in the initial pooled material;
in the centrifugation residue by the method of sulfuric acid monohydrate R, 11.688 g of sodium chloride R and 50 mg of
digestion (2. 5. 9) and calculate the content of protein by sodium azide R in 1 L of water R.
multiplying the result by 6.25. Flow rate 0.5 mL'min.
Protein composition Detection Spectrophotometer at 280 nm.
Zone electrophoresis (2.2.31). Identification of peaks In the chromatogram obtained with
Use strips of suitable cellulose acetate gel or suitable agarose the reference solution, the principal peak corresponds to the
gel as the supporting medium and barbital buffer solution IgG monomer and there is a peak corresponding to the
pH 8.6 Rl as the electrolyte solution. dimer with a relative retention to the principal peak of
If cellulose acetate is the supporting material, the analytical about 0.85. Identify the peaks in the chromatogram obtained
procedure described below can be used. If agarose gels are with the test solution by comparison with the chromatogram
used, and because they are normally part of an automated obtained with the reference solution; any peak with a
system, the manufacturer's instructions are followed instead. retention time less than that of the dimer corresponds to
Test solution Dilute the preparation to be examined with a polymers and aggregates; any peak with a retention time
9 g/L solution of sodium chloride R to obtain a protein greater than that of the monomer corresponds to fragments.
concentration of 30 g!L. Results In the chromatogram obtained with the test
Reference solution Reconstitute human immunoglobulin for solution:
electrophoresis BRP and dilute with a 9 glL solution of sodium - retention time: for the monomer and for the dimer, the
chloride R to obtain a protein concentration of 30 g!L. retention time relative to the corresponding peak in the
chromatogram obtained with the reference solution is
To a strip apply 4.0 µL of the test solution as a 10 mm band
1 ± 0.02;
or apply 0.4 µL per millimetre if a narrower strip is used.
- peak area: the sum of the peak areas of the monomer and
To another strip apply in the same manner the same volume
the dimer represent not less than 85 per cent of the total
of the reference solution. Apply a suitable electric field such
area of the chromatogram and the sum of the peak areas
that the albumin band of normal human serum applied on a
of polymers and aggregates represents not more than
control strip migrates at least 30 mm. Stain the strip with
10 per cent of the total area of the chromatogram. This
amido black 10B solution R for 5 min. Decolourise with a
requirement is not applicable if albumin has been added
mixture of 10 volumes of glacial acetic acid R and 90 volumes
as a stabiliser; for such preparations, a test for molecular-
of methanol R so that the background is just free of colour.
size distribution is carried out during manufacture before
Develop the transparency of the strips with a mixture of
addition of the stabiliser.
19 volumes of glacial acetic acid R and 81 volumes of
methanol R. Measure the absorbance of the bands at 600 nm Anti-A and anti-B haemagglutinins (2.6.20, Method B)
in an instrument having a linear response over the range of It complies with the test.
measurement. Calculate the result as the mean of Anti-D antibodies (2. 6.26)
3 measurements of each strip. It complies with the test.
System suitability In the electropherogram obtained with the Antibody to hepatitis B surface antigen
reference solution, the proportion of protein in the principal Minimum 0.5 IU per gram of immunoglobulin, determined
band is within the limits stated in the leaflet accompanying by a suitable immunochemical method (2.7.1).
the reference preparation.
Immunoglobulin A
Results In the electropherogram obtained with the test As determined by a suitable immunochemical method
solution, not more than 10 per cent of protein has a mobility (2. 7.1), the content of immunoglobulin A is not greater than
different from that of the principal band. This limit is not the maximum content stated on the label.
applicable if albumin has been added to the preparation as a
Water
stabiliser; for such preparations, a test for protein
composition is carried out during manufacture before Determined by a suitable method, such as the semi-micro
addition of the stabiliser.
Molecular-size distribution within the limits approved by the competent authority.
Sterility (2. 6.1)
Test solution Dilute the preparation to be examined with a
9 g/L solution of sodium chloride R to a concentration suitable Pyrogens (2. 6. 8) or Bacterial endotoxins (2. 6. 14')
for the chromatographic system used. A concentration in the It complies with the test for pyrogens or, preferably and
range of 4-12 glL and injection of 50-600 µg of protein are where justified and authorised, with a validated in vitro test
usually suitable. such as the bacterial endotoxin test.
Reference solution Dilute human immunoglobulin (molecular For the pyrogen test, inject 1 mL per kilogram of the rabbit's
size) BRP with a 9 glL solution of sodium chloride R to the mass.
same protein concentration as the test solution. Where the bacterial endotoxin test is used, the preparation to
Column: be examined contains less than 5 IU of endotoxin per
- size: l = 0.6 m, 0 = 7.5 mm [or l = 0.3 m, 0 = 7.8 mm]; millilitre.
- stationary phase: hydrophilic silica gel for chromatography R, STORAGE
of a grade suitable for fractionation of globular proteins In an airtight, colourless glass container, protected from light,
with relative molecular masses in the range 10 000 to at the temperature stated on the label.
500 000.
Mobile phase Dissolve 4.873 g of disodium hydrogen phosphate
dihydrate R, 1. 7 41 g of sodium dihydrogen phosphate
LABELLING have been formulated by the World Health Organization

The label states: (Requirements for the collection, processing and quality control of
- for liquid preparations, the volume of the preparation in blood, blood components and plasma derivatives, WHO
the container and the protein content expressed in grams Technical Report Series, No. 840, 1994 or subsequent
per litre; revision).
- for freeze-dried preparations: POOLED PIASMA
- the quantity of protein in the container; To limit the potential Bl 9 virus burden in plasma pools used
- the name or composition and the volume of the for the manufacture of anti-D immunoglobulin, the plasma
reconstituting liquid to be added; pool is tested for B 19 virus using validated nucleic acid
- the route of administration; amplification techniques (2.6.21).
- the distribution of subclasses of IgG present in the
B19 virus DNA
preparation;
Maximum 10.0 IU/µL.
- where applicable, the amount of albumin added as a
stabiliser; A positive control with 10.0 IU of Bl 9 virus DNA per
- the maximum content of immunoglobulin A. microlitre and, to test for inhibitors, an internal control
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
positive control is non-reactive or if the result obtained with
the internal control indicates the presence of inhibitors.
B19 virus DNA for NAT testing ERP is suitable for use as a
Anti-D {RhO) lmmunoglobulin positive control.
(Human Anti-D Immunoglobulin, Ph. Eur. If Human normal immunoglobulin for intramuscular
monograph 0557) administration (0338) and/or Human albumin solution (0255)
are added to the preparation, the plasma pool or pools from
which they are derived comply with the above requirement
DEFINITION for B19 virus DNA.
Sterile liquid or freeze-dried preparation containing POTENCY
immunoglobulins, mainly immunoglobulin G. Human anti-D immunoglobulin (2.7.13)
The preparation is intended for intramuscular administration. The estimated potency is not less than 90 per cent of the
It contains specific antibodies against erythrocyte D-antigen stated potency. The confidence limits (P = 0.95) are not less
and may also contain small quantities of other blood-group than 80 per cent and not more than 120 per cent of the
antibodies. Human normal immunoglobulin for intramuscular estimated potency.
administration (0338) and/or Human albumin solution (0255)
may be added. STORAGE
See Human normal immunoglobulin for intramuscular
It complies with the monograph Human normal
administration (0338).
immunoglobulin for intramuscular administration (0338), except
for the minimum number of donors and the minimum total LABELLING
protein content. See Human normal immunoglobulin for intramuscular
The assay of human anti-D immunoglobulin (2.7.13) is administration (0338).
carried out, as prescribed below under Potency. The label states the number of International Units per
For products prepared by a method that eliminates container.
immunoglobulins with specificities other than anti-D, where _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
authorised, the test for antibodies to hepatitis B surface
antigen is not required.
PRODUCTION
Human anti-D immunoglobulin is preferably obtained from Anti-D lmmunoglobulin for
the plasma of donors with a sufficient titre of previously
acquired anti-D antibodies. Where necessary, in order to
Intravenous Use
ensure an adequate supply of human anti-D (Human Anti-D Immunoglobulin for Intravenous
immunoglobulin, it is obtained from plasma derived from Administration, Ph. Eur. monograph 1527)
donors immunised with D-positive erythrocytes that are PhEur - - - - - - - - - - - - - - - - - - - - - ~
compatible in relevant blood group systems in order to avoid
formation of undesirable antibodies. DEFINITION
During product development, the integrity and stability of
immunoglobulins, mainly immunoglobulin G. It contains
the Fe region of the anti-D antibodies must be demonstrated
specific antibodies against erythrocyte D-antigen and may
using a suitable method specific for anti-D antibodies.
also contain small quantities of other blood-group antibodies.
ERYTHROCYTE DONORS Human normal immunoglobulin for intravenous
Erythrocyte donors comply with the requirements for donors administration (0918) and/or Human albumin solution (0255)
prescribed in the monograph Human plasma for may be added.
fractionation (0853).
It complies with the monograph Human normal
IMMUNISATION immunoglobulin for intravenous administration (0918), except
Immunisation of the plasma donor is carried out under for the minimum number of donors, the minimum total
proper medical supervision. Recommendations concerning protein content, the limit for osmolality and the limit for
donor immunisation, including testing of erythrocyte donors, prekallikrein activator.
The test for anti-D antibodies (2.6.26) prescribed in the STORAGE

monograph Human normal immunoglobulin for intravenous See Human normal immunogwbulin for intravenous
administration (0918) is not carried out, since it is replaced by administration (0918).
the assay of human anti-D immunoglobulin (2.7.13) as
LABELLING
prescribed below under Potency.
See Human normal immunogwbulin for intravenous
For products prepared by a method that eliminates administration (0918).
immunoglobulins with specificities other than anti-D, where
The label states the number of International Units per
authorised, the test for antibodies to hepatitis B surface
container.
antigen is not required; a suitable test for Fe function is
carried out instead of that described in general chapter 2. 7. 9,
which is not applicable to such a product.
PRODUCTION
Human anti-D immunoglobulin is preferably obtained from
the plasma of donors with a sufficient titre of previously Hepatitis A lmmunoglobulin
acquired anti-D antibodies. Where necessary, in order to
(Human Hepatitis A Immunogwbulin, Ph. Bur.
ensure an adequate supply of human anti-D
monograph 0769)
immunoglobulin, it is obtained from plasma derived from
donors immunised with D-positive erythrocytes that are
compatible in relevant blood group systems in order to avoid DEFINITION
formation of undesirable antibodies. Sterile liquid or freeze-dried preparation containing
During product development, the integrity and stability of immunoglobulins, mainly immunoglobulin G.
the Fe region of the anti-D antibodies must be demonstrated The preparation is intended for intramuscular administration.
using a suitable method specific for anti-D antibodies. It is obtained from plasma from selected donors having
ERYTHROCYTE DONORS antibodies against hepatitis A virus. Human normal
Erythrocyte donors comply with the requirements for donors immunoglobulin for intramuscular administration (0338) may be
prescribed in the monograph Human plasma for added.
fractionation (0853). It complies with the monograph on Human normal
IMMUNISATION immunogwbulin for intramuscular administration (0338), except
Immunisation of the plasma donor is carried out under for the minimum number of donors and the minimum total
proper medical supervision. Recommendations concerning protein content.
donor immunisation, including testing of erythrocyte donors, POTENCY
have been formulated by the World Health Organization The potency is determined by comparing the antibody titre
(Requirements for the collectwn, processing and quality control of of the immunoglobulin to be examined with that of a
bwod, bwod components and plasma derivatives, WHO reference preparation calibrated in International Units, using
Technical Report Series, No. 840, 1994 or subsequent an immunoassay of suitable sensitivity and specificity (2. 7.1).
revision).
POOLED PLASMA amount of the International Standard for anti-hepatitis A
To limit the potential B19 virus burden in plasma pools used immunoglobulin. The equivalence in International Units of
for the manufacture of anti-D immunoglobulin, the plasma the International Standard is stated by the World Health
pool is tested for B 19 virus using validated nucleic acid Organization.
amplification techniques (2.6.21). Human hepatitis A immunogwbulin ERP is calibrated in
B19 virus DNA International Units by comparison with the International
Maximum 10.0 IU/µL. Standard.
A positive control with 10.0 IU ofB19 virus DNA per The stated potency is not less than 600 IU/mL.
microlitre and, to test for inhibitors, an internal control The estimated potency is not less than the stated potency.
prepared by addition of a suitable marker to a sample of the The confidence limits (P = 0.95) are not less than
plasma pool are included in the test. The test is invalid if the 80 per cent and not more than 125 per cent of the estimated
positive control is non-reactive or if the result obtained with potency.
the internal control indicates the presence of inhibitors.
STORAGE
B19 virus DNA for NAT testing BRP is suitable for use as a See Human normal immunogwbulin for intramuscular
positive control. administration (0338).
If Human normal immunoglobulin for intravenous
LABELLING
administration (0918) and/or Human albumin solution (0255)
are added to the preparation, the plasma pool or pools from See Human normal immunogwbulin for intramuscular
which they are derived comply with the above requirement administration (0338).
for Bl 9 virus DNA. The label states the number of International Units per
container.
ASSAY
Human anti-D hnmunoglobulin (2.7.13)
The estimated potency is not less than 90 per cent of the
stated potency. The confidence limits (P = 0.95) are not less
estimated potency.
2023 Blood-related Products IV-6 71
Hepatitis B lmmunoglobulin Hepatitis B lmmunoglobulin for

(Human Hepatitis B Immunoglobu/,in, Ph. Eur. Intravenous Use
monograph 0722) (Human Hepatitis B Immunoglobulin for Intravenous
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Administratwn, Ph. Eur. monograph 1016)
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Sterile liquid or freeze-dried preparation containing DEFINITION
immunoglobulins, mainly immunoglobulin G. Sterile liquid or freeze-dried preparation containing
The preparation is intended for intramuscular or immunoglobulins, mainly immunoglobulin G. It is obtained
subcutaneous administration. It is obtained from plasma from plasma from selected and/or immunised donors having
from selected and/or immunised donors having antibodies antibodies against hepatitis B surface antigen. Human normal
against hepatitis B surface antigen. Human normal immunoglobulin for intravenous administration (0918) may be
immunoglobulin for intramuscular administration (0338) or added.
Human normal immunoglobulin for subcutaneous It complies with the monograph Human normal
administration (2788) may be added. immunoglobulin for intravenous administration (0918), except
Depending on the route of administration, it complies with for the minimum number of donors, the minimum total
the monograph on Human normal immunoglobulin for protein content and the limit for osmolality.
intramuscular administration (0338) or Human normal
POTENCY
immunoglobulin for subcutaneous administratwn (2788), except
The potency is determined by comparing the antibody titre
for the minimum number of donors and the minimum total
protein content. of the immunoglobulin to be examined with that of a
reference preparation calibrated in International Units, using
POTENCY an immunoassay (2. 7.1) of suitable sensitivity and specificity.
The potency is determined by comparing the antibody titre The International Unit is the activity contained in a stated
of the immunoglobulin to be examined with that of a amount of the International Standard for anti-hepatitis B
reference preparation calibrated in International Units, using immunoglobulin. The equivalence in International Units of
an immunoassay of suitable sensitivity and specificity (2. 7.1). the International Standard is stated by the World Health
The International Unit is the activity contained in a stated Organization.
amount of the International Standard for anti-hepatitis B Human hepatitis B immunoglobulin BRP is calibrated in
immunoglobulin. The equivalence in International Units of International Units by comparison with the International
the International Standard is stated by the World Health Standard.
Organization.
The stated potency is not less than 50 IU/mL. The estimated
Human hepatitis B immunoglobulin BRP is calibrated in potency is not less than the stated potency. The confidence
International Units by comparison with the International limits (P = 0.95) are not less than 80 per cent and not more
Standard. than 125 per cent of the estimated potency.
The stated potency is not less than 100 IU/mL.
STORAGE
The estimated potency is not less than the stated potency.
See Human normal immunoglobulin for intravenous
potency. LABELLING
STORAGE See Human normal immunoglobulin for intravenous
administration (0338) or Human normal immunoglobulin for The label states the minimum number of International Units
subcutaneous administratwn (2788). of hepatitis B immunoglobulin per container.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
LABELLING
administration (0338) or Human normal immunoglobu/,in for
subcutaneous administration (2788).
The label states the number of International Units per Measles lmmunoglobulin
container.
(Human Measles Immunoglobulin, Ph. Eur.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur monograph 0397)
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
immunoglobulins, mainly immunoglobulin G.
The preparation is intended for intramuscular administration.
It is obtained from plasma containing specific antibodies
against measles virus. Human normal immunoglobulin for
intramuscular administration (0338) may be added.
It complies with the monograph on Human normal
immunoglobulin for intramuscular administratwn (0338), except
for the minimum number of donors and the minimum total It is obtained from human plasma that complies with the
protein content. monograph Human plasma for fractionation (0853), using a
POTENCY suitable fractionation process and further purification steps.
Other plasma proteins may be present. The preparation may
The potency of the liquid preparation and of the freeze-dried
contain excipients such as stabilisers.
preparation after reconstitution as stated on the label is not
less than 50 IU per millilitre of neutralising antibody against PRODUCTION
measles virus. GENERAL PROVISIONS
The potency is determined by comparing the antibody titre The method of preparation is designed to maintain
of the immunoglobulin to be examined with that of a functional integrity of c,:-1-proteinase inhibitor. It includes a
reference preparation calibrated in International Units, using step or steps that have been shown to remove or to inactivate
a challenge dose of measles virus in a suitable cell culture known agents of infection. The subsequent purification
system. An alternative method may be used providing that procedure must be validated to demonstrate that the
the competent authority is satisfied that it correlates with the concentration of any substances used for inactivation of
determination of neutralising activity for the measles virus by viruses during production is reduced to a suitable level and
comparison with the reference preparation. that any residues are such as not to compromise the safety of
The International Unit is the specific neutralising activity for the preparation for patients.
measles virus contained in a stated amount of the The specific activity is not less than 0.35 mg of active human
International Standard for human anti-measles serum. a.-1-proteinase inhibitor per milligram of total protein.
The equivalence in International Units of the International The ratio of human r1,-l proteinase inhibitor activity to
Reference Preparation is stated by the World Health human c,:-1-proteinase inhibitor antigen is not less than 0.7.
Organization. No antimicrobial preservative or antibiotic is added.
Method The solution is passed through a bacteria-retentive filter and
Prepare serial 2-fold dilutions of the immunoglobulin to be distributed aseptically into the final containers. It may be
examined and of the reference preparation. Mix each dilution subsequently freeze-dried.
with an equal volume of a suspension of measles virus CONSISTENCY OF THE METHOD OF
containing about 100 CCID 50 in 0.1 mL and incubate PRODUCTION
protected from light at 37 °C for 2 h. Using not fewer than 6 It shall be demonstrated that the manufacturing process
cell cultures per mixture, inoculate 0.2 mL of each mixture yields a product with a consistent composition. It is evaluated
into each of the cell cultures allocated to that mixture and by suitable analytical procedures that are determined during
incubate for not less than 10 days. Examine the cultures for process development, and which include:
viral activity and compare the dilution containing the smallest - assay of human c,:-1-proteinase inhibitor activity;
quantity of the immunoglobulin which neutralises the virus - determination of specific human r1,-l-proteinase inhibitor
with that of the corresponding dilution of the reference activity, expressed as the ratio of active human c,:-1-
preparation. proteinase inhibitor to total protein;
Calculate the potency of the immunoglobulin to be examined - characterisation of isoform composition and protein
in International Units per millilitre of neutralising antibody structure by suitable methods such as isoelectric focusing
against measles virus. (2.2.54), spectrometric methods (for example, mass
spectrometry) or capillary electrophoresis (2.2.47);
STORAGE - determination of the ratio of human c,:-1-proteinase
See Human normal immunoglobulin for intramuscular inhibitor activity to human C1.-l-proteinase inhibitor
administration (0338). antigen;
LABELLING - characterisation of accompanying plasma proteins that
See Human normal immunoglobulin for intramuscular might be present, by a set of suitable methods such as
administration (0338). SDS-PAGE, cellulose acetate electrophoresis or capillary
zone electrophoresis (2.2.31) and quantitative
determination of relevant accompanying plasma proteins;
container.
- determination of molecular-size distribution, used to
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
quantify the polymeric forms of human a.-1-proteinase
inhibitor; consideration is given to the potential presence
of accompanying proteins that might affect the results.
CHARACTERS
Human rt-1-Proteinase Inhibitor Appearance
(Ph. Bur. monograph 2387) - liquid preparations: clear or slightly opalescent, colourless
or pale yellow or pale green or pale brown;
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
- 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 canying out the
(also known as human c,:-1-antitrypsin or c,:-1-antiproteinase). identification, tests ( except those for solubility and water) and
Human c,:-1-proteinase inhibitor is a glycoprotein existing in assay.
isoforms with different isoelectric points and is the most IDENTIFICATION
abundant multifunctional serine proteinase inhibitor in It complies with the limits of the assay.
human plasma.
TESTS
pH (2.2.3)
Human C1-Esterase Inhibitor
6.5 to 7.8. (Ph. Bur. monograph 2818)
Solubility
Action and use
For freeze-dried preparations, add to a container of the Prophylaxis and treatment of hereditary angioedema.
preparation to be examined the volume of the liquid stated
on the label at room temperature. The preparation dissolves PhEur
completely, giving a clear, colourless or pale green or pale
DEFINITION
yellow or pale brown solution.
Plasma protein fraction containing mainly human C 1-esterase
Osmolality (2.2.35) inhibitor (also known as Cl-inhibitor or Cl-INH). Human
Minimum 210 mosmoVkg. C 1-esterase inhibitor is a soluble, single-chain glycoprotein
Total protein containing 478 amino-acid residues. Human Cl-esterase
Dilute the preparation to be examined with a 9 g/L solution inhibitor belongs to the group of serine protease inhibitors.
of sodium chloride R to obtain a protein concentration of It is obtained from human plasma that complies with the
about 7.5 mg/mL. To 2.0 mL ofthis solution in a round- monograph Human plasma for fractionation (0853), using a
bottomed centrifuge tube, add 2 mL of a 75 g/L solution of suitable fractionation process and further purification steps.
sodium molybdate R and 2 mL of a mixture of I volume of Other plasma proteins may be present.
nitrogen-free sulfuric acid R and 30 volumes of water R. Shake,
PRODUCTION
centrifuge for 5 min, decant the supernatant and allow the
The method of preparation includes a step or steps that have
been shown to remove or to inactivate known agents of
infection; if substances are used for inactivation of viruses
and calculate the protein content by multiplying by 6.25.
Water must be validated to demonstrate that the concentration of
Determined by a suitable method, such as the semi-micro these substances is reduced to a suitable level and any
determination of water (2.5.12), loss on drying (2.2.32) or residues are such as not to compromise the safety of the
near-infrared spectroscopy (2.2.40), the water content is preparation for patients.
within the limits approved by the competent authority. Human C 1-esterase inhibitor is purified. The method of
Sterility (2. 6.1) preparation is designed to maintain the functional integrity of
It complies with the test. human C 1-esterase inhibitor. Buffers and other auxiliary
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14) substances such as a stabiliser may be included. The solution
It complies with the test for pyrogens or, preferably and is passed through a bacteria-retentive filter, distributed
where justified and authorised, with a validated in vitro test aseptically into the final containers and immediately frozen.
such as the bacterial endotoxin test. It is subsequently freeze-dried and the containers are closed
For the pyrogen test, inject per kilogram of the rabbit's mass under vacuum or under an inert gas. No antimicrobial
a volume equivalent to not less than 60 mg of human a-1- preservative is added at any stage of production.
proteinase inhibitor. Where the bacterial endotoxin test is CONSISTENCY OF THE METHOD OF
used, the preparation to be examined contains less than PRODUCTION
0.08 IU of endotoxin per milligram of human a-1-proteinase It shall be demonstrated that the manufacturing process
inhibitor. yields a product having a consistent composition. This is
evaluated by suitable analytical procedures that are
ASSAY
determined during process development and that normally
Assay of human a-1-proteinase inhibitor (2. 7. 32)
include:
- assay of human Cl-esterase inhibitor (2.7.34);
more than 120 per cent of the stated potency. - determination of specific human C 1-esterase inhibitor
The confidence limits (P = 0.95) are not less than activity, expressed as the ratio of active human
80 per cent and not more than 120 per cent of the estimated C I -esterase inhibitor content to total protein content;
potency. - determination of molecular-size distribution by size-
STORAGE exclusion chromatography (2.2.30);
In an airtight and sterile container, at a temperature not - molecular identification of human Cl-esterase inhibitor,
exceeding 25 °C, unless otherwise justified and authorised. characterisation of accompanying plasma proteins that
might be present, by a set of suitable methods such as
LABELLING
SDS-PAGE, cellulose acetate electrophoresis or capillary
The label states:
zone electrophoresis (2.2.31, 2.2.47) and quantitative
- the potency of active (functional) human a-1-proteinase
determination of relevant accompanying plasma proteins.
inhibitor per container;
- the name and quantity of any added substances; CHARACTERS
- the quantity of protein in the container; Appearance
- the route of administration; White, pale yellow, pale blue or greenish, hygroscopic
- where applicable, the name and volume of the liquid to powder or friable solid.
be used for reconstitution; Reconstitute the preparation to be examined as stated on the label
- that the transmission of infectious agents cannot be totally immediately before canying out the identification, tests (except
excluded when medicinal products prepared from human those for solubility and water) and assay.
blood or plasma are administered.
IDENTIFICATION
PhEur
It complies with the limits of the assay.
TESTS - the quantity of protein in the container;

Solubility - the name and quantity of any added substance;
To the contents of a container of the preparation to be - the name and volume of the solvent to be used for
examined add the volume of the solvent stated on the label reconstitution;
at the recommended temperature. The preparation dissolves - that the transmission of infectious agents cannot be totally
completely with gentle swirling within the limits approved by excluded when medicinal products prepared from human
the competent authority, giving a clear, colourless, pale blood or plasma are administered.
yellow, pale blue or greenish solution. - - - - - - - - - - - - - - - - - - - - - - Ph Eur
pH (2.2.3)
6.5 to 7.5.
Osmolality (2.2.35)
Minimum 200 mosmol/kg. Rabies lmmunoglobulin
Total protein
If necessary, dilute an accurately measured volume of the (Human Rabies Immunoglobulin, Ph. Bur. monograph
reconstituted preparation to be examined with a 9 g/L 0723)
solution of sodium chloride R to obtain a protein concentration Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
of about 7.5 mg/mL. Place 2.0 mL of this solution in a
round-bottomed centrifuge tube and add 2 mL of a 75 g/L DEFINITION
solution of sodium molybdate R and 2 mL of a mixture of Sterile liquid or freeze-dried preparation containing
1 volume of nitrogen-free sulfuric acid R and 30 volumes of immunoglobulins, mainly immunoglobulin G.
water R. Shake, centrifuge for 5 min, decant the supernatant The preparation is intended for intramuscular administration.
and allow the inverted tube to drain on filter paper. It is obtained from plasma from donors immunised against
Determine the nitrogen in the residue by the method of rabies. It contains specific antibodies neutralising the rabies
sulfuric acid digestion (2. 5. 9) and calculate the protein virus. Human normal immunoglobulin for intramuscular
content by multiplying the result by 6.25. The protein administration (0338) may be added.
content is within the limits approved by the competent It complies with the monograph on Human normal
authority. immunoglobulin for intramuscular administration (0338), except
Specific activity for the minimum number of donors and the minimum total
Minimum 4 IU/mg. protein content.
Calculate the ratio of active human C 1-esterase inhibitor POTENCY
content (as determined under Assay) to total protein content. The potency is determined by comparing the dose of
Water immunoglobulin required to neutralise the infectivity of a
Determined by a suitable method, such as the semi-micro rabies virus suspension with the dose of a reference
determination of water (2.5.12), loss on drying (2.2.32) or preparation, calibrated in International Units, required to
near-infrared spectroscopy (2.2.40), the water content is produce the same degree of neutralisation (2. 7.1). The test is
within the limits approved by the competent authority. performed in sensitive cell cultures and the presence of
unneutralised virus is revealed by immunofluorescence.
Sterility (2. 6.1)
It complies with the test. The International Unit is the specific neutralising activity for
rabies virus in a stated amount of the International Standard
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14) for anti-rabies immunoglobulin. The equivalence in
It complies with the test for pyrogens or, preferably and International Units of the International Standard is stated by
where justified and authorised, with a validated in vitro test the World Health Organization.
such as the bacterial endotoxin test.
Human rabies immunoglobulin BRP is calibrated in
For the pyrogen test, inject per kilogram of the rabbit's mass International Units by comparison with the International
a volume equivalent to 50 IU of human C 1-esterase Standard.
inhibitor.
Method
Where the bacterial endotoxin test is used, the preparation to
Carry out the test in suitable sensitive cells. It is usual to use
be examined contains less than 0.25 IU of endotoxin per
the BHK-21 cell line, grown in the medium described below,
International Unit of human Cl-esterase inhibitor.
between the 18th and 30th passage levels counted from the
ASSAY ATCC seed lot. Harvest the cells after 2 to 4 days of growth,
Carry out the assay of human C 1-esterase inhibitor (2. 7. 34). treat with trypsin and prepare a suspension containing
The estimated potency is not less than 75 per cent and not 500 000 cells per millilitre (cell suspension). 10 min before
more than 125 per cent of the stated potency. using this suspension add 10 µg of diethylaminoethyldextran R
The confidence limits (P = 0.95) are not less than per millilitre, if necessary, to increase the sensitivity of the
80 per cent and not more than 120 per cent of the estimated cells.
potency. Use a fixed virus strain grown in sensitive cells, such as the
STORAGE CVS strain of rabies virus adapted to growth in the BHK-21
In an airtight and sterile container, protected from light, at a cell line (seed virus suspension). Estimate the titre of the seed
temperature not exceeding 25 °C, unless otherwise justified virus suspension as follows.
and authorised. Prepare a series of dilutions of the viral suspension. In the
chambers of cell-culture slides (8 chambers per slide), place
LABELLING 0 .1 mL of each dilution and O.1 mL of medium and add
The label states: 0.2 mL of the cell suspension. Incubate in an atmosphere of
- the number oflnternational Units of human Cl-esterase carbon dioxide at 37 °C for 24 h. Carry out fixation,
inhibitor per container;
immunofluorescence staining and evaluation as described The confidence limits (P = 0.95) are not less than
below. Determine the end-point titre of the seed virus 80 per cent and not more than 125 per cent of the estimated
suspension and prepare the working virus dilution potency.
corresponding to 100 CCID50 per 0.1 mL. CULTURE MEDIUM FOR GROWTH OF BHK-21
For each assay, check the amount of virus used by CELLS
performing a control titration: from the dilution Commercially available media that have a slighdy different
corresponding to 100 CCID 50 per 0.1 mL, make 3 tenfold composition from that shown below may also be used.
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 Sodium chloride 6.4 g
suspension. The test is not valid unless the titre lies between Potassium chloride 0.40 g
30 CCID 50 and 300 CCID 50 • Calcium chloride, anhydrous 0.20 g
Magnesium sulfate, heptahydrate 0.20 g
Dilute the reference preparation to a concentration of Sodium dihydrogen phosphate, monohydrate 0.124 g
2 IU/mL using non-supplemented culture medium (stock Glucose monohydrate 4.5 g
reference dilution, stored below -80 'C). Prepare 2 suitable Ferric nitrate, nonahydrate 0.10 mg
L-Arginine hydrochloride 42.0 mg
predilutions (1 :8 and I: I 0) of the stock reference dilution so
L-Cystine 24.0 mg
that the dilution of the reference preparation that reduces the L-Histidine 16.0 mg
number of fluorescent fields by 50 per cent lies within the L-lsoleucine 52.0 mg
4 dilutions of the cell-culture slide. Add 0.1 mL of the L-Leucine 52.0 mg
medium to each chamber, except the first in each of 2 rows, L-Lysine hydrochloride 74.0 mg
L-Phenylalanine 33.0 mg
to which add respectively 0.2 mL of the 2 predilutions of the L- Threonine 48.0 mg
stock reference dilution transferring successively 0.1 mL to L-Tryptophan 8.0mg
the other chambers. L-Tyrosine 36.0 mg
L-Valine 47.0 mg
Dilute the preparation to be examined I in I 00 using non- L-Methionine 15.0 mg
supplemented medium (stock immunoglobulin dilution) - to L-Glutamine 0.292 g
reduce to a minimum errors due to viscosity of the undiluted i-Inositol 3.60 mg
preparation - and make 3 suitable predilutions so that the Choline chloride 2.0 mg
Folic acid 2.0 mg
dilution of the preparation to be examined that reduces the 2.0 mg
Nicotinamide
number of fluorescent fields by 50 per cent lies within the Calcium pantothenate 2.0 mg
4 dilutions of the cell-culture slide. Add 0.1 mL of the Pyridoxal hydrochloride 2.0 mg
medium to all the chambers except the first in each of Thiamine hydrochloride 2.0 mg
Riboflavine 0.2 mg
3 rows, to which add respectively 0.2 mL of the 3
Phenol red 15.0 mg
predilutions of the stock immunoglobulin dilution. Prepare a Sodium hydrogen carbonate 2.75 g
series of 2-fold dilutions transferring successively 0.1 mL to Water to 1000 mL
the other chambers.
To all the chambers containing the dilutions of the reference The medium is supplemented with:
preparation and the dilutions of the preparation to be
examined, add 0 .1 mL of the virus suspension corresponding Foetal calf serum (heated at 56 ··c for 30 min) 10 per cent
Tryptose phosphate broth 10 per cent
to 100 CCID 50 per 0.1 mL (working virus dilution), shake Benzylpenicillin sodium 60 mglL
manually, allow to stand in an atmosphere of carbon dioxide Streptomycin 0.1 glL
at 37 °C for 90 min, add 0.2 mL of the cell suspension,
shake manually and allow to stand in an atmosphere of STORAGE
carbon dioxide at 37 °C for 24 h. See Human normal immunoglobulin for intramuscular
After 24 h, discard the medium and remove the plastic walls. administration (0338).
Wash the cell monolayer with plwsphate buffered saline
pH 7. 4 R and then with a mixture of 20 volumes of water R
LABELLING
and 80 volumes of acetone R and fix in a mixture of See Human normal immunoglobulin for intramuscular
20 volumes of water R and 80 volumes of acetone R at administration (0338).
-20 °C for 3 min. Spread on the slides fiuorescein-conjugated The label states the number of International Units per
rabies antiserum R ready for use. Allow to stand in an container.
atmosphere with a high level of moisture at 37 °C for - - - - - - - - - - - - - - - - - - - - - - PhEur
30 min. Wash with plwsphate buffered saline pH 7.4 Rand
dry. Examine 20 fields in each chamber at a magnification of
250 x , using a microscope equipped for fluorescence
readings. Note the number of fields with at least Rubella lmmunoglobulin
1 fluorescent cell. Check the test dose used in the virus
titration slide and determine the dilution of the reference (Human Rubella Immunoglobulin, Ph. Bur.
preparation and the dilution of the preparation to be monograph 0617)
examined that reduce the number of fluorescent fields by Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
50 per cent, calculating the 2 or 3 dilutions together using
probit analysis. The test is not valid unless the statistical DEFINITION
analysis shows a significant slope of the dose-response curve Sterile liquid or freeze-dried preparation containing
and no evidence of deviation from linearity or parallelism. immunoglobulins, mainly immunoglobulin G.
The preparation is intended for intramuscular administration.
The stated potency is not less than 150 IU/mL.
It is obtained from plasma containing specific antibodies
The estimated potency is not less than-the stated potency
against rubella virus. Human normal immunoglobulin for
and is not greater than twice the stated potency.
intramuscular administration (0338) may be added.
It complies with the monograph on Human nonnal the International Standard is stated by the World Health
immunoglobulin for intramuscular administratwn (0338), except Organization.
for the minimum number of donors and the minimum total Human tetanus immunoglobulin BRP is calibrated in
protein content. International Units by comparison with the International
POTENCY Standard.
The potency is determined by comparing the activity of the Method
preparation to be examined in a suitable haemagglutination- Selection of animals Use mice weighing 16-20 g.
inhibition test with that of a reference preparation calibrated Preparatwn of the test toxin Prepare the test toxin by a
in International Units. suitable method from the sterile filtrate of a culture in liquid
The International Unit is the activity contained in a stated medium of C. tetani. The 2 methods shown below are given
amount of the International Standard for anti-rubella as examples and any other suitable method may be used.
immunoglobulin. The equivalence in International Units of (1) To the filtrate of an approximately 9-day culture, add
the International Reference Preparation is stated by the 1-2 volumes of glycerol Rand store the mixture in the liquid
World Health Organization. state at a temperature slightly below O °C.
The estimated potency is not less than 4500 IU/mL. (2) Precipitate the toxin by addition to the filtrate of
The confidence limits (P = 0.95) of the estimated potency ammonium sulfate R, dry the precipitate in vacuo over
are not less than 50 per cent and not more than 200 per cent diphosphorus pentoxide R, reduce to a powder and store dry,
of the stated potency. either in sealed ampoules or in vacuo over diphosphorus
STORAGE pentoxide R.
See Human nomwl immunoglobulin for intramuscular Detennination of test dose of toxin (Lpl 10 dose) Prepare a
administration (0338). solution of the reference preparation in a suitable liquid such
that it contains 0.5 IU of antitoxin per millilitre. If the test
LABELLING
toxin is stored dry, reconstitute it using a suitable liquid.
See Human nonnal immunoglobulin for intramuscular
Prepare mixtures of the solution of the reference preparation
and the test toxin such that each contains 2.0 mL of the
The label states the number of International Units per solution of the reference preparation, one of a graded series
millilitre. of volumes of the test toxin and sufficient of a suitable liquid
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur to bring the volume to 5.0 mL. Allow the mixtures to stand,
protected from light, for 60 min. Using 6 mice for each
mixture, inject a dose of 0.5 mL subcutaneously into each
mouse. Observe the mice for 96 h. Mice that become
Tetanus lmmunoglobulin paralysed may be euthanised. The test dose of toxin is the
quantity in 0.5 mL of the mixture made with the smallest
(Human Tetanus lmmunoglobulin, Ph. Bur. amount of toxin capable of causing, despite partial
monograph 0398) neutralisation by the reference preparation, paralysis in all
6 mice injected with the mixture, within the observation
PhE~ - - - - - - - - - - - - - - - - - - - - - ~
period.
DEFINITION Detennination of potency of the immunoglobulin Prepare a
Sterile liquid or freeze-dried preparation containing solution of the reference preparation in a suitable liquid such
immunoglobulins, mainly immunoglobulin G. that it contains 0.5 JU of antitoxin per millilitre. Prepare a
The preparation is intended for intramuscular administration. solution of the test toxin in a suitable liquid such that it
It is obtained from plasma containing specific antibodies contains 5 test doses per millilitre. Prepare mixtures of the
against the toxin of Clostridium tetani. Human normal solution of the test toxin and the immunoglobulin to be
immunoglobulin for intramuscular administration (0338) may be examined such that each contains 2.0 mL of the solution of
added. the test toxin, one of a graded series of volumes of the
It complies with the monograph Human normal immunoglobulin to be examined and sufficient of a suitable
immunoglobulin for intramuscular administratwn (0338), except liquid to bring the total volume to 5.0 mL. Also prepare
for the minimum number of donors and the minimum total mixtures of the solution of the test toxin and the solution of
protein content. the reference preparation such that each contains 2.0 mL of
PRODUCTION the solution of the test toxin, one of a graded series
of volumes of the solution of the reference preparation
During development, a satisfactory relationship shall be
centred on that volume (2.0 mL) that contains 1 JU and
established between the potency determined by immunoassay
sufficient of a suitable liquid to bring the total volume to
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 subcutaneously a dose of 0.5 mL into each mouse. Observe
determined by comparing the quantity necessary to protect the mice for 96 h. Mice that become paralysed may be
mice against the paralytic effects of a fixed quantity of euthanised. The mixture that contains the largest volume of
tetanus toxin with the quantity of a reference preparation of immunoglobulin that fails to protect the mice from paralysis
human tetanus immunoglobulin, calibrated in International contains 1 IU. This quantity is used to calculate the potency
Units, necessary to give the same protection. of the immunoglobulin in International Units per millilitre.
The International Unit of antitoxin is the specific neutralising The test is not valid unless 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 with mixtures containing more do not.
POTENCY Apply 100 µL of each of the samples of the dilution series to

The potency is determined by comparing the antibody titre the plate. Incubate for 2 hat 37 °C on a plate shaker set at
of the preparation to be examined with that of a reference 120 r/min and wash the plate 5 times with PBS-T. Apply
preparation calibrated in International Units, using suitable 100 µL of a peroxidase-conjugated anti-human IgG antibody
immunochemical methods (2. 7. 1) such as enzyme-linked diluted to a suitable concentration with PBS-T containing
immunosorbent assay (ELISA) or toxoid inhibition assay 5 g/L of bovine albumin R to each of the wells and incubate
(TIA). for 1 hat 37 °C on a plate shaker set at 120 r/min. Wash the
The International Unit is the activity contained in a stated plate 5 times with PBS-T and apply 100 µL of a suitable
amount of the International Standard for anti-tetanus 3,3 1,5,5 1-tetramethylbenzidine (TMB) substrate to each of
immunoglobulin. The equivalence in International Units of the wells and incubate at room temperature for 10 min in the
the International Standard is stated by the World Health dark. To stop the reaction, add 100 µL of a 196.2 g/L
Organization. solution of sulfuric acid R to each of the wells. Measure the
absorbances at 450 nm and at the reference wavelength of
Human tetanus immunoglobulin ERP is calibrated in
630 nm. Calculate the potencies of the preparations by the
International Units and is suitable for use as a reference
usual statistical methods (5.3).
preparation.
The stated potency is not less than 100 IU/mL of tetanus Method B: indirect determination by toxoid-binding
antitoxin. The estimated potency is not less than the stated inhibition assay
potency. The confidence limits (P = 0.95) are not less than The amount of unbound toxoid in a mixture of toxoid and
80 per cent and not more than 125 per cent of the estimated tetanus immunoglobulin is determined by an enzyme
potency. immunoassay and is inversely proportional to the amount of
tetanus immunoglobulin present. The method is performed
The description of methods A and B below are provided as over 2 consecutive days.
examples.
Materials
Method A: direct enzyme immunoassay - Phosphate-buffered saline pH 7.1 (PBS). See under
The amount of tetanus immunoglobulin bound to tetanus Method A.
toxoid, which is coated to a microtitre plate, is determined by - PBS-T. See under Method A.
means of a peroxidase-conjugated polyclonal anti-human IgG - Carbonate buffer pH 9.6. See under Method A.
antibody. - Tetanus toxoid. See under Method A.
Materials - Mab. Mouse monoclonal tetanus toxoid antibody.
- Phosphate-buffered saline pH 7.1 (PBS). Dissolve 0.2 g of Use according to the instructions. Prepare a suitable
potassium chloride R, 0.2 g of potassium dihydrogen dilution of Mab, e.g. 1/5000, in PBS.
phosphate R, 1.15 g of anhydrous disodium hydrogen - Peroxidase-conjugated antibody. Peroxidase-conjugated anti-
phosphate Rand 8.0 g of sodium chloride R in water Rand mouse IgG (H+L) antibody, affinity-purified F(ab)2
adjust the pH (2.2.3) if necessary. Dilute to 1000 mL fragment without cross-reactivity to human serum
with water R. proteins. Use according to the instructions. Prepare a
- PBS-T. PBS containing 0.05 per cent V/V of suitable dilution of the peroxidase-conjugated antibody in
polysorbate 20 R. PBS-T containing 5 g/L of bovine albumin R.
- Carbonate buffer pH 9. 6. Dissolve 1.4 g of anhydrous - Microtitre plate. Use a round-bottomed microtitre plate
sodium carbonate Rand 3.0 g of sodium hydrogen with medium protein-binding capacity.
carbonate R in water Rand adjust the pH (2.2.3) if - ELISA plate. Use a flat-bottomed microtitre plate with
necessary. Dilute to 1000 mL with water R. high protein-binding capacity.
- Tetanus toxoid. Purified and chemically inactivated tetanus Method
toxin.
Day 1
- Microtitre plate. Use a flat-bottomed microtitre plate with
high protein-binding capacity. To block the protein-binding sites of the microtitre plate, add
200 µL of PBS containing 5 g/L of bovine albumin R to each
Method
of the wells of the microtitre plate and incubate for 1 h at
Distribute 100 µL of a 0.2 LflmL solution of tetanus toxoid 37 °C on a plate shaker set at 120 r/min. Wash the plate
in carbonate buffer pH 9.6 into each of the wells of the 5 times with PBS-T.
microtitre plate. Incubate at 4 °C for approximately 18 h.
Reconstitute the reference preparation and the preparation to
Wash the plate 5 times with PBS-T. To block unbound
be examined according to the instructions. For each
binding sites add 200 µL of PBS containing 5 g/L of bovine
preparation, prepare 2 independent predilutions of
albumin R to each of the wells and incubate for 1 h at 3 7 °C
0.4 IU/mL in PBS by applying several dilution steps. Prepare
on a plate shaker set at 120 r/min. Wash 5 times with PES-
from each predilution a dilution series of dilutions
T.
containing 0.04 IU/mL, 0.10 IU/mL, 0.12 IU/mL,
Reconstitute the reference preparation and the preparation to 0.14 IU/mL, 0.16 IU/mL, 0.18 IU/mL and 0.20 IU/mL.
be examined according to the instructions. For each Prepare each dilution directly from the 0.4 IU/mL
preparation, prepare 2 independent predilutions of predilution.
0.004 IU/mL in PBS by applying several dilution steps.
Transfer 100 µL of each dilution of the dilution series to a
Using PBS, prepare from each predilution 5 serial dilutions
well of the blocked plate and add 50 µL of a O.2 LflmL
with a dilution factor of 1.5 resulting in a dilution series of
solution of tetanus toxoid in carbonate buffer pH 9.6 into
6 dilutions in the range of 0.0005-0.004 IU/mL. Depending
each of the wells. Incubate for approximately 18 h at 37 °C
on the reagents used, a small modification of the dilution
on a plate shaker set at 120 r/min.
series might be necessary to meet the conditions of the
statistical model used. To coat the ELISA plate, distribute 100 µL of a solution of a
human tetanus immunoglobulin diluted to 1 IU/mL in
carbonate buffer pH 9.6 into each of the wells of the ELISA
plate. Incubate for approximately 18 hat 37 °C on a plate The equivalence in International Units of the International
shaker set at 120 r/min. Standard is stated by the World Health Organization.
Day 2 The stated potency is not less than 100 IU/mL.
Wash the coated ELISA plate 5 times with PBS-T. To block The estimated potency is not less than the stated potency.
unbound binding sites add 200 µL of PBS containing 5 g/L The confidence limits (P = 0.95) are not less than
of bovine albumin R to each of the wells and incubate for 1 h 80 per cent and not more than 125 per cent of the estimated
at 37 °C on a plate shaker set at 120 r/min. Wash the plate potency.
5 times with PBS-T. Transfer 100 µL of each mixture of STORAGE
toxoid and tetanus immunoglobulin from the microtitre plate See Human normal immunoglobulin for intramuscular
to the coated ELISA plate and incubate for 2 hours at 37 °C administration (0338).
on a plate shaker set at 120 r/min. Wash the plate 5 times
with PBS-T. Add 100 µL of diluted Mab to each of the LABELLING
wells, incubate the plate for 1 h at 37 °C on a plate shaker See Human normal immunoglobulin for intramuscular
set at 120 r/min and wash the plate 5 times with PBS-T. administration (0338).
Add 100 µL of the diluted peroxidase-conjugated antibody to The label states the number of International Units per
each of the wells, incubate the plate for 1 h at 37 °C on a container.
plate shaker set at 120 r/min and wash the plate 5 times _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
with PBS-T. Apply 100 µL of a suitable 3,3',5,5'-
tetramethylbenzidine (TMB) substrate to each of the wells
and incubate at room temperature for 10 min in the dark.
To stop the reaction, add 100 µL of a 196.2 g/L solution of
sulfuric acid R to each of the wells. Measure the absorbances
Varicella lmmunoglobulin for
at 450 nm and at the reference wavelength of 630 nm. Intravenous Use
Calculate the potencies of the preparations by the usual (Human Varicella lmmunoglobulin for Intravenous
statistical methods (5.3). Administration, Ph. Bur. monograph 1528)
STORAGE Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
administration (0338). DEFINITION
LABELLING
immunoglobulins, mainly immunoglobulin G. It is obtained
See Human normal immunoglobulin for intramuscular from plasma from selected donors having antibodies against
administration (0338). human herpesvirus 3 (varicella-zoster virus 1). Human normal
The label states the number oflnternational Units per immunoglobulin for intravenous administration (0918) may be
container. added.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur It complies with the monograph on Human normal
immunoglobulin for intravenous administration (0918), except
for the minimum number of donors, the minimum total
protein content and the limit for osmolality.
Varicella lmmunoglobulin POTENCY
The potency is determined by comparing the antibody titre
(Human Varicella lmmunoglobulin, Ph. Bur.
of the immunoglobulin to be examined with that of a
monograph 0724)
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ an immunoassay of suitable sensitivity and specificity (2. 7. 1).
DEFINITION The International Unit is the activity contained in a stated
Sterile liquid or freeze-dried preparation containing amount of the International Standard for anti varicella-zoster
immunoglobulins, mainly immunoglobulin G. immunoglobulin. The equivalence in International Units of
The preparation is intended for intramuscular administration. the International Standard is stated by the World Health
It is obtained from plasma from selected donors having Organization.
antibodies against Herpesvirus varicellae. Human normal The stated potency is not less than 25 IU/mL. The estimated
immunoglobulin for intramuscular administration (0338) may be potency is not less than the stated potency. The confidence
added. limits (P = 0.95) are not less than 80 per cent and not more
It complies with the monograph on Human normal than 125 per cent of the estimated potency.
immunoglobulin for intramuscular administration (0338) except STORAGE
for the minimum number of donors, the minimum total See Human normal immunoglobulin for intravenous
protein content and, where authorised, the test for antibody administration (0918).
to hepatitis B surface antigen.
LABELLING
POTENCY See Human normal immunoglobulin for intravenous
The potency is determined by comparing the antibody titre administration (0918).
of the immunoglobulin to be examined with that of a
container.
an immunoassay of suitable sensitivity and specificity (2. 7.1).
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
amount of the International Standard for anti varicella-zoster.
Human von Willebrand factor activity/antigen ratio

von Willebrand Factor Consistency of the manufacturing process with respect to the
(Human von Wille brand factor, Ph. Bur. monograph ratio of human von Willebrand factor activity to human von
2298) Willebrand factor antigen content is demonstrated.
PRODUCTS THAT SHOW PARTICLES AFTER RECONSTITUTION
PhEur - - - ~ · - - - - - - - - - - - - - - - - - - ~
If a few particles remain when the preparation is
DEFINITION reconstituted, it shall be demonstrated during validation
Sterile, freeze-dried preparation of a plasma protein fraction studies that the potency is not significantly affected after
containing the glycoprotein human von Willebrand factor passage of the preparation through the filter to be provided
with varying amounts of human coagulation factor VIII, with the preparation.
depending on the method of preparation. It is prepared from
CHARACTERS
human plasma that complies with the monograph on Human
Appearance
plasma for fractionation (0853). The preparation may contain
Hygroscopic, white or pale yellow, powder or friable solid.
excipients such as stabilisers.
This monograph applies to preparations formulated
immediately before carrying out the identification, tests (except
according to the human von Willebrand factor activity.
those for solubility and water) and assay.
The potency of the preparation, reconstituted as stated on
the label, is not less than 20 IU of human von Willebrand IDENTIFICATION
factor per millilitre. It complies with the limits of the assay.
PRODUCTION TESTS
GENERAL PROVISIONS Solubility
The method of preparation is designed to maintain To a container of the preparation to be examined, add the
functional integrity of human von Willebrand factor. volume of the liquid stated on the label at the recommended
It includes steps that have been shown to remove or to temperature. The preparation dissolves completely with
inactivate known agents of infection; if substances are used gentle swirling within 10 min, forming a clear or slightly
for the inactivation of viruses, the subsequent purification opalescent, colourless or slightly yellow solution.
procedure must be validated to demonstrate that the In addition, where the label states that the product may show
concentration of these substances is reduced to a suitable a few particles after reconstitution, reconstitute the
level and that any residues are such as not to compromise the preparation as described on the label and pass it through the
safety of the preparation for patients. filter provided: the filtered solution is clear or slightly
The specific activity is not less than 1 IU of human von opalescent.
Willebrand factor per milligram of total protein, before the pH (2.2.3)
addition of any protein stabiliser. 6.5 to 7.5.
The human von Willebrand factor fraction is dissolved in a Osmolality (2.2.35)
suitable liquid. No antimicrobial preservative or antibiotic is Minimum 240 mosmol/kg.
added. The solution is passed through a bacteria-retentive Total protein
filter, distributed aseptically into the final containers and If necessary, dilute an accurately measured volume of the
immediately frozen. It is subsequently freeze-dried and the reconstituted preparation with a 9 g/L solution of sodium
containers are closed under vacuum or under an inert gas.
chloride R to obtain a protein concentration of about
CONSISTENCY OF THE METHOD OF 7.5 mg/mL. Place 2.0 mL of this solution in a round-
PRODUCTION bottomed centrifuge tube and add 2 mL of a 75 g/L solution
It shall be demonstrated that the manufacturing process of sodium molybdate R and 2 mL of a mixture of 1 volume of
yields a product having a consistent composition with respect nitrogen-free sulfuric acid R and 30 volumes of water R. Shake,
to human von Willebrand factor, human coagulation centrifuge for 5 min, decant the supernatant and allow the
factor VIII and the proportions of human von Willebrand inverted tube to drain on filter paper. Determine the nitrogen
factor and human coagulation factor VIII. This is evaluated in the residue by the method of sulfuric acid digestion (2. 5. 9)
by suitable analytical procedures that are determined during and calculate the amount of protein by multiplying the result
process development, and that include the following checks: by 6.25. For some products, especially those without a protein
Human von Willebrand factor multimers stabiliser, this method may not be applicable. Another validated
The distribution of the different human von Willebrand method for protein determination must therefore be peiformed.
factor multimers is determined by a suitable method such as Anti-A and anti-B haemagglutinins (2.6.20, Method A)
sodium dodecyl sulfate (SDS) agarose gel electrophoresis The 1 to 64 dilution does not show agglutination. Dilute the
with or without Western blot analysis, using a suitable reconstituted preparation with a 9 g/L solution of sodium
normal human plasma as standard. Visualisation of the chloride R to contain 6 IU of human von Willebrand factor
multimeric pattern may be performed using, for example, an activity per millilitre.
immunoenzymatic technique and quantitative evaluation may
Water
be carried out by densitometric analysis.
Human von Willebrand factor activity (2. 7. 21) determination of water (2.5.12), loss on drying (2.2.32) 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. 6.1)
binding activity using a suitable reference preparation. It complies with the test.
Pyrogens (2. 6. 8) or Bacterial endotoxins (2. 6.14)

such as the test for bacterial endotoxins.
a volume equivalent to not less than 100 JU of human von
Willebrand factor.
preparation to be examined contains less than 0.05 JU of
endotoxin per International Unit of human von Willebrand
factor.
ASSAY
Human von Willebrand factor (2.7.21)
potency.
Pending the availability of an International Standard for human
von Wille brand factor concentrate calibrated for use in the
collagen-binding assay, only the ristocetin cofactor assay may be
used.
Human coagulation factor VIII (2.7.4)
The assay is carried out where the human coagulation
factor VIII content is greater than 10 IU of human
coagulation factor VIII per 100 IU of human von Willebrand
factor activity. The estimated potency is not less than
60 per cent and not more than 140 per cent of the stated
potency. The confidence limits (P = 0.95) are not less than
potency.
STORAGE
In an airtight container, protected from light.
LABELLING
The label states:
- the number oflnternational Units of human von
Willebrand factor in the container;
- the number of International Units of human coagulation
factor VIII in the container, or that the content of human
coagulation factor VIII is less than or equal to 10 IU of
human coagulation factor VIII per 100 IU of human von
Willebrand factor activity;
- the amount of protein in the container;
- the name and quantity of any added substance;
reconstitution;
- where applicable, that the preparation may show the
presence of a few particles after reconstitution;
- that the transmission of infectious agents cannot be totally
excluded when medicinal products prepared from human
blood or plasma are administered.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Monographs
2023 Immunosera IV-683
characterised and the method of immunisation is

Monoclonal Antibodies for Human documented.
Use Process validation
(Ph. Bur. monograph 2031) During development studies, the production method is
validated for the following aspects:
Monoclonal Antibodies for Human Use comply with the - consistency of the production process including cell-
requirements of the European Pharmacopoeia. These requirements culture/fermentation, purification and, where applicable,
are reproduced below. fragmentation method;
The requirements of the monograph for Immunosera do not - removal or inactivation of infectious agents;
necessarz7y apply to the monograph for Monoclonal Antibodies for - adequate removal of product- and process-related
Human Use. impurities (for example, host-cell protein and DNA,
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ protein A, antibiotics, cell-culture components);
- specificity and biological activity of the monoclonal
DEFINITION antibody;
Monoclonal antibodies for human use are preparations of an - absence of non-endotoxin pyrogens, where applicable;
immunoglobulin or a fragment of an immunoglobulin, for - reusability of purification components (for example,
example, F(ab')2, with defined specificity, produced by a column material), limits or acceptance criteria being set as
single clone of cells. They may be conjugated to other a function of the validation;
substances, including for radiolabelling. - methods used for conjugation, where applicable.
They can be obtained from immortalised B lymphocytes that Product characterisation
are cloned and expanded as continuous cell lines or from The product is characterised to obtain adequate information
rDNA-engineered cell lines. including: structural integrity, isotype, amino-acid sequence,
Examined under suitable conditions of visibility, they are secondary structure, carbohydrate moiety, disulfide bridges,
practically free from particles. conformation, specificity, affinity, biological activity and
Currently available rDNA-engineered antibodies include the heterogeneity (characterisation of isoforms).
following antibodies. A battery of suitable analytical techniques is used including
Chimeric monoclonal antibodies The variable heavy- and light- chemical, physical, immunochemical and biological tests (for
chain domains of a human antibody are replaced by those of example, peptide mapping, N- and C-terminal amino-acid
a non-human species that possess the desired antigen sequencing, mass spectrometry, chromatographic,
specificity. electrophoretic and spectroscopic techniques). Additional
Humanised monoclonal antibodies The 3 short hypervariable tests are performed to obtain information on cross-reactivity
sequences (the complementarity-determining regions) of non- with human tissues.
human variable domains for each chain are engineered into For those products that are modified by fragmentation or
the variable domain framework of a human antibody; other conjugation, the influence of the methods used on the
sequence changes may be made to improve antigen binding. antibody is characterised.
Recombinant human monoclonal antibodies The variable Process intermediates
heavy- and light-chain domains of a human antibody are Where process intermediates are stored, an expiry date or a
combined with the constant region of a human antibody. storage period justified by stability data is established for
Monoclonal antibodies obtained from cell lines modified by each.
recombinant DNA technology also comply with the Biological assay
requirements of the monograph Products of recombinant DNA The biological assay is chosen in terms of its correlation with
technology (0784). the intended mode of action of the monoclonal antibody.
This monograph applies to monoclonal antibodies, including Reference preparation
conjugates, for therapeutic and prophylactic use and for use A batch shown to be stable and shown to be suitable in
as in vivo diagnostics. It does not apply to monoclonal clinical trials, or a batch representative thereof, is used as a
antibodies used as reagents in the manufacture of medicinal reference preparation for the identification, tests and assay.
products. Nor does it apply to monoclonal antibodies The reference preparation is appropriately characterised as
produced in ascites, for which requirements are decided by defined under Product characterisation, except that it is not
the competent authority. necessary to examine cross-reactivity for each batch of
PRODUCTION reference preparation.
GENERAL PROVISIONS Definition of a batch
Production is based on a seed-lot system using a master cell Definition of a batch is required throughout the process.
bank and, if applicable, a working cell bank derived from the SOURCE CELLS
cloned cells. The production method is validated during Source cells include fusion partners, lymphocytes, myeloma
development studies in order to prevent transmission of cells, feeder cells and host cells for the expression of the
infectious agents by the final product. All biological materials recombinant monoclonal antibody.
and cells used in the production are characterised and are in The origin and characteristics of the parental cell are
compliance with chapter 5.2.8. Minimising the risk of documented, including information on the health of the
transmitting animal spongiform encephalopathy agents via human donors, and on the fusion partner used (for example,
and veterinary medicinal products. Where monoclonal myeloma cell line, human lymphoblastoid B-cell line).
antibodies for human use are manufactured using materials
of human or animal origin, the requirements of chapter 5.1. 7. Wherever possible, source cells undergo suitable screening for
Viral safety also apply. Where an immunogen is used, it is extraneous agents and endogenous agents. The choice of
viruses for the tests is dependent on the species and tissue of
origin.
IV-684 Immunosera 2023
CELL LINE PRODUCING THE MONOCLONAL time (in accordance with the stability of the cell line).
ANTIBODY Product is harvested in a single operation.
The suitability of the cell line producing the monoclonal Continuous-culture production (multiple harvest)
antibody is demonstrated by: Cells are continuously cultivated for a defined period (in
- documentation on the history of the cell line including accordance with the stability of the system and production
description of the cell fusion, immortalisation or consistency). Monitoring is necessary throughout the life of
transfection and cloning procedure; the culture; the required frequency and type of monitoring
- characterisation of the cell line (for example, phenotype, will depend on the nature of the production system.
isoenzyme analysis, immunochemical markers and
Each harvest is tested for antibody content, bioburden,
cytogenetic markers);
endotoxin and mycoplasmas. General or specific tests for
- characterisation of relevant features of the antibody;
adventitious viruses are carried out at a suitable stage
- consistency of critical quality attributes for the antibody
depending on the nature of the manufacturing process and
up to or beyond the population doubling level or
the materials used. For processes using production at finite
generation number used for routine production;
passage level (single harvest), at least 3 harvests are tested for
- for recombinant DNA products, consistency of the coding
adventitious viruses using a suitable range of in vitro
sequence of the expression construct in cells cultivated to
methods.
the limit of in vitro cell age for production use or beyond,
by either nucleic acid testing or product analysis. The acceptance criteria for harvests for further processing are
clearly defined and linked to the schedule of monitoring
CELLBANKS applied. If any adventitious viruses are detected, the process
The master cell bank is a homogeneous suspension of the is carefully investigated to determine the cause of the
cell line producing the monoclonal antibody, distributed in contamination and the harvest is not further processed.
equal volumes in a single operation into individual containers Harvests in which an endogenous virus has been detected are
for storage. not used for purification unless an appropriate action plan
A working cell bank is a homogeneous suspension of the cell has been defined to prevent transmission of infectious agents.
material derived from the master cell bank at a finite passage
PURIFICATION
level, distributed in equal volumes in a single operation into
Harvests or intermediate pools may be pooled before further
individual containers for storage.
processing. The purification process includes steps that
Post-production cells are cells cultured up to or beyond the remove and/or inactivate non-enveloped and enveloped
population doubling level or generation number used for viruses. A validated purification process, for which removal
routine production. and/or inactivation of infectious agents and removal of
The following tests are performed on the master cell bank: product- and process-related impurities has been
viability, identity, absence of bacterial, fungal and demonstrated, is used. Defined steps of the process lead to a
mycoplasmal contamination, characterisation of the purified monoclonal antibody (active substance) of consistent
monoclonal antibody produced. Adventitious viral quality and biological activity.
contamination is tested with a suitable range of in viva and in ACTIVE SUBSTANCE
vitro tests. Retrovirus and other endogenous viral The test programme for the active substance depends on the
contamination is tested using a suitable range of in vitro tests. validation of the process, on demonstration of consistency
The following tests are performed on the working cell bank: and on the expected level of product- and process-related
viability, identity, absence of bacterial, fungal and impurities. The active substance is tested for appearance,
mycoplasmal contamination. Adventitious viral identity, bioburden and bacterial endotoxins, product-related
contamination is tested with a suitable range of in viva and in substances, product- and process-related impurities including
vitro tests. For the first working cell bank, these tests are tests for host-cell-derived proteins and host-cell- and vector-
performed on post-production cells, generated from that derived DNA, as well as structural integrity, protein content
working cell bank; for working cell banks subsequent to the and biological activity by suitable analytical methods,
first working cell bank, a single in vitro and in viva test can comparing with the reference preparation where necessary.
be done either directly on the working cell bank or on post- When the active substance is a conjugated or transformed
production cells. antibody, appropriate tests must be performed before and
For the master cell bank and working cell bank, tests for after the antibody conjugation/modification.
specific viruses are carried out when potentially contaminated If storage of intermediates is intended, adequate stability of
biological material has been used during preparation of the these preparations and its impact on quality or shelf-life of
cell banks, taking into account the species of origin of this the finished product are evaluated.
material. This may not be necessary when this material is
FINAL BULK
inactivated using validated procedures.
One or more batches of active substance may be combined
The following tests are performed on the post-production to produce the final bulk. Suitable stabilisers and other
cells: absence of bacterial, fungal and mycoplasmal excipients may be added during preparation of the final bulk.
contamination. Adventitious viral contamination is tested
The final bulk must be stored under validated conditions
with a suitable range of in viva and in vitro tests. Retrovirus
with respect to bioburden and stability.
and other endogenous viral contamination is tested using a
suitable range of in vitro tests. FINAL LOT
The final bulk is sterile-filtered and distributed under aseptic
CULTURE AND HARVEST
conditions into sterile containers, which may subsequently be
Production at finite passage level (single harvest) freeze-dried.
Cells are cultivated up to a defined maximum number of As part of the in-process control each container (vial, syringe
passages or population doublings, or up to a fixed harvest or ampoule) is inspected after filling to eliminate containers
that contain visible particles. During development of the
product it must be demonstrated that either the process will Sterility (2. 6.1)
not generate visible proteinaceous particles in the final lot or It complies with the test for sterility.
such particles are reduced to a low level as justified and Bacterial endotoxins (2. 6.14)
authorised. It complies with the limits approved for the particular
CHARACTERS product.
Liquid 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, 5.3).
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 (2.2.1) and degree of coloration
(2.2.2). They are without visible particles, unless otherwise LABELLING
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 (2.2.3) - for freeze-dried preparations:
It complies with the limits approved for the particular - the name and the volume of the reconstitution liquid
product. to be added;
Osmolality (2.2.35) - 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 the product, where
Extractable volume (2. 9.17) applicable.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
It complies with the test for extractable volume.
Total protein (2.5.33)
It complies with the limits approved for the particular
product.
Molecular-size distribution
lmmunosera
Molecular-size distribution is determined by a suitable Antisera
method, for example size-exclusion chromatography (2.2.30).
It complies with the limits approved for the particular (lmmunosera for Human Use, Animal, Ph. Bur. 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 isofonns, a number of different tests
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
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
Stabiliser and spiders. The preparation is intended for intravenous or
\'\'here applicable, it complies with the limits approved for intramuscular administration, after dilution where applicable.
the particular product. PRODUCTION
Water (2.5.12) GENERAL PROVISIONS
Freeze-dried products comply with the limits approved for The production method shall have been shown to yield
the particular product. consistently immunosera of acceptable safety, potency in man
and stability.
Any reagent of biological origin used in the production of the immunoserum is purified. If the serum or plasma is
immunosera shall be free of contamination with bacteria, stored before further processing, precautions are taken to
fungi and viruses. The general requirements of chapter 5.1. 7. avoid microbial contamination.
Viral safety apply to the manufacture of animal immunosera Several single plasma or serum samples may be pooled before
for human use, in conjunction with the more specific purification. The single or pooled samples are tested before
requirements relating to viral safety in this monograph. purification for the following tests.
The method of preparation includes a step or steps that have
Tests for contaminating viruses
been shown to remove or inactivate known agents of
If an antimicrobial preservative is added, it must be
infection.
neutralised before carrying out the tests, or the tests are
Methods used for production are validated, effective, carried out on a sample taken before addition of the
reproducible and do not impair the biological activity of the antimicrobial preservative. Each pool is tested for
product. 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 into cell
trials, or a batch representative thereof, is used as the cultures capable of detecting a wide range of viruses relevant
reference preparation for the tests for high molecular mass for the particular product.
proteins and purity.
Potency
ANIMALS Carry 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 are tested and shown to used.
be free from a defined list of infectious agents.
The introduction of animals into a closed herd follows Protein content
specified procedures, including definition of quarantine Dilute the product to be examined with a 9 g/L solution of
measures. Where appropriate, additional specific agents are sodium chloride R to obtain a solution containing about 15 mg
considered depending on the geographical localisation of the of protein in 2 mL. To 2 mL of this solution in a round-
establishment used for the breeding and production of the bottomed centrifuge tube add 2 mL of a 7 5 g/L solution of
animals. The feed originates from a controlled source and no sodium molybdate R and 2 mL of a mixture of 1 volume of
animal proteins are added. The suppliers of animals are nitrogen-free sulfuric acid R and 30 volumes of water R. Shake,
certified by the competent authority. centrifuge for 5 min, decant the supernatant and allow the
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
period is imposed between vaccination and collection of PURIFICATION AND VIRAL INACTIVATION
serum or plasma for immunoserum production. The immunoglobulins are concentrated and purified by
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 the 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 are validated to guarantee total
being immunised according to a defined schedule, with 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')z 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.
manner as to maintain sterility of the product. The blood or The bands are compared in intensity and no additional
plasma collection is conducted at a site separate from the bands are found.
area where the animals are kept or bred and the area where
FINAL BULK round-bottomed centrifuge tube add 2 mL of a 75 g/L

The final bulk is prepared from a single intermediate product solution of sodium molybdate R and 2 mL of a mixture of
or from a pool of intermediate products obtained from 1 volume of nitrogen-free sulfuric acid R and 30 volumes of
animals of the same species. Intermediate products with water R. Shake, centrifuge for 5 min, decant the supernatant
different specificities may be pooled. 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 protein by multiplying by 6.25.
preservative in the final bulk. Molecular-size distribution
Only a final bulk that complies with the following Examine by liquid chromatography (2.2.29 or 2.2.30).
requirements may be used in the preparation of the final lot. It complies with the specification approved for the particular
product.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial Antimicrobial preservative
preservative by a suitable physico-chemical method. Where applicable, determine the amount of antimicrobial
It contains not less than 85 per cent and not more than preservative by a suitable physico-chemical method.
115 per cent of the amount stated on the label. The amount is not less than the minimum amount shown to
Sterility (2. 6.1) be effective and is not greater than 115 per cent of that
It complies with the test for sterility. stated on the label.
Phenol (2. 5.15)
FINAL LOT
Maximum 2.5 g/L for preparations containing phenol.
The final bulk of immunoserum is distributed aseptically into
sterile, tamper-evident containers. The containers are closed Stabiliser
so as to prevent contamination. 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 Purity
preservative, stabiliser, purity, foreign proteins and albumin Examine by non-reducing polyacrylamide gel electrophoresis
and the assay have been carried out with satisfactory results (2.2.31), by comparison with the reference preparation.
on the final bulk, they may be omitted on the final lot. 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 Foreign proteins
those for solubility and water) and assay. 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
Unless otherwise prescribed in the monograph, when
CHARACTERS
examined electrophoretically, the content of albumin is not
Immunosera are clear to opalescent and colourless to very greater than the limit approved for the particular product
faintly yellow liquids. They are free from turbidity. Freeze- and, in any case, is not greater than 3 per cent.
dried products are white or slightly yellow powders or solid
friable masses. After reconstitution they show the same Water (2.5.12)
characteristics as liquid preparations. Maximum 3 per cent.
Sterility (2. 6.1)
TESTS
Solubility
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 (2.2.3) 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) Animal tests
Minimum 240 mosmoVkg after dilution, where applicable. In accordance with the provisions of the European
Convention for the Protection of Vertebrate Animals Used
Protein content for Experimental and Other Scientific Purposes, tests must
90 per cent to 110 per cent of the amount stated on the be carried out in such a way as to use the minimum number
label, and, unless otherwise justified and authorised, not of animals and to cause the least pain, suffering, distress or
more than 100 g/L. lasting harm. The criteria for judging tests in monographs
Dilute the preparation to be examined with a 9 g/L solution must be applied in light of this. For example, if it is indicated
of sodium chloride R to obtain a solution containing about that an animal is considered to be positive, infected, etc.
15 mg of protein in 2 mL. To 2 mL of this solution in a when typical clinical signs or death occur, then as soon as
sufficient indication of a positive result is obtained the animal PRODUCTION

in question shall be either euthanised or given suitable GENERAL PROVISIONS
treatment to prevent unnecessary suffering. In accordance The production method has been shown to yield consistently
with the General Notices, alternative test methods may be immunoglobulins of acceptable safety, potency in man and
used to demonstrate compliance with the monograph and the stability.
use of such tests is particularly encouraged when this leads to Any reagent of biological origin used in production shall be
replacement or reduction of animal use or reduction of free of contamination with bacteria, fungi and viruses.
suffering. Guidance on how to substitute in vivo methods by The method of preparation includes a step or steps that have
in vitro methods where a direct head-to-head comparison is been shown to remove or inactivate known agents of
not possible may be found in general chapter 5.2.14. infection.
STORAGE During development studies, it shall be demonstrated that
Protected from light, at the temperature stated on the label. the production method yields a product that:
Do not allow liquid preparations to freeze. - does not transmit infectious agents,
Expiry date The expiry date is calculated from the beginning - is characterised by a defined pattern of immunological
of the assay. activity, notably: antigen binding, complement-dependent
and independent cytotoxicity, cytokine release, induction
LABELLING of T-cell activation, cell death,
The label states: - does not contain antibodies that cross-react with human
- the number oflntemational Units per millilitre, where tissues to a degree that would impair clinical safety,
applicable; - has a defined maximum content of anti-thrombocyte
- the amount of protein per container; antibody activity,
- for freeze-dried preparations: - has a defined maximum content of haemoglobin.
- the name and volume of the reconstituting liquid to
The product has been shown, by suitable tests in animals
be added;
and evaluation during clinical trials, to be well tolerated.
- that the immunoserum is to be used immediately after
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
- the route of administration; demonstrated in clinical trials, or a batch representative
- the storage conditions; thereof.
- the expiry date, except for containers of less than 1 mL ANIMALS
which are individually packed; the expiry date may be The animals used are of a species approved by the competent
omitted from the label on the container, provided it is authority, are healthy and exclusively reserved for production
shown on the package and the label on the package states of anti-T lymphocyte immunoglobulin. They are tested and
that the container must be kept in the package until shown to be free from a defined list of infectious agents.
required for use; The introduction of animals into a closed herd follows
- the animal species of origin; specified procedures, including definition of quarantine
- the name and amount of any antimicrobial preservative, measures. Where appropriate, tests for additional specific
any stabiliser and any other excipient. agents are 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
lmmunoglobulin 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
(Ph. Bur. monograph 1928) period is imposed between vaccination and collection of
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ serum or plasma for immunoglobulin production.
The species, origin and identification number of the animals
DEFINITION
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
related to the immunisation process, it is not used, nor are fractionation (0853). If other human material is used, it is
any other of the animals in the group concerned, unless it is shown by validated methods to be free from relevant blood-
evident that their use will not impair the safety of the borne pathogens, notably HBV, HCV and HIV. If substances
product. are used for inactivation or removal of viruses, it shall have
Human antigens such as continuously growing T-lymphocyte been shown that any residues present in the final product
cell lines or thymocytes are used to immunise the animals. have no adverse effects on the patients treated with the anti-
Cells may be subjected to a sorting procedure. T lymphocyte immunoglobulin.
The immunising antigens are shown to be free from FINALBULK
infectious agents by validated methods for relevant blood- The final bulk is prepared from a single intermediate product
borne pathogens, notably hepatitis B virus (HBV), or from a pool of intermediate products obtained from
hepatitis C virus (HCV) and human immunodeficiency animals of the same species. No antimicrobial preservative is
virus (HIV) and other relevant adventitious agents originating added either during the manufacturing procedure or for
from the preparation of the antigen. The cells used comply preparation of the final bulk solution. During manufacturing,
with defined requirements for purity of the cell population the solution is passed through a bacteria-retentive filter.
and freedom from adventitious agents. FINAL LOT
COLLECTION OF BLOOD OR PLASMA The final bulk of anti-T-lymphocyte immunoglobulin is
Collection of blood is made by venepuncture or distributed aseptically into sterile, tamper-evident containers.
plasmapheresis. The puncture area is shaved, cleaned and The containers are closed as to prevent contamination.
disinfected. The animals may be anaesthetised under Only a final lot that complies with the requirements
conditions that do not influence the quality of the product. prescribed below under Identification, Tests and Assay may
No antimicrobial preservative is added to the plasma and be released for use.
serum samples. The blood or plasma is collected in such a
CHARACTERS
manner as to maintain sterility of the product. The blood or
Appearance
plasma collection is conducted at a site separate from the
- liquid preparation: clear or slightly opalescent, colourless or
area where the animals are kept or bred and the area where
pale yellow liquid;
the immunoglobulin is purified. If the serum or plasma is
stored before further processing, precautions are taken to
solid friable mass, which after reconstitution gives a liquid
avoid microbial contamination.
preparation corresponding to the description above.
Several single plasma or serum samples may be pooled before
purification. The single or pooled samples are tested before IDENfIFICATION
purification for the following tests. A. Using a suitable range of species-specific antisera, carry
out precipitation tests on the preparation to be examined.
Tests for contaminating viruses
It is recommended that the test be carried out using antisera
Each pool is tested for contaminating viruses by suitable in
specific to the plasma proteins of each species of domestic
vitro tests including inoculation to cell cultures capable of
animal commonly used in the preparation of materials of
detecting a wide range of viruses relevant for the particular
biological origin in the country concerned and antisera
product. Where applicable, in vitro tests for contaminating
specific to human plasma proteins. The preparation is shown
viruses are carried out on the adsorbed pool, after the last
to contain proteins originating from the animal used for the
production stage that may introduce viral contaminants.
anti-T lymphocyte immunoglobulin production.
PURIFICATION AND VIRAL INACTIVATION
B. Examine by a suitable immunoelectrophoresis technique.
The immunoglobulins are concentrated and purified by
Using antiserum to normal serum of the animal used for
fractional precipitation, chromatography, immuno-adsorption
production, compare this serum and the preparation to be
or by other suitable chemical or physical methods.
examined, both diluted to a concentration that will allow a
The methods are selected and validated to avoid
clear gammaglobulin precipitation arc to be obtained on the
contamination at all steps of processing and to avoid
gel. The main component of the preparation to be examined
formation of protein aggregates that effect immunobiological
corresponds to the IgG component of normal serum of the
characteristics of the product.
animal used for production.
Unless otherwise justified and authorised, validated
C. The preparation complies with the assay.
procedures are applied for removal and/or inactivation of
VlfUses. TESTS
After purification and treatment for removal and/or Solubility
inactivation of viruses, a stabiliser may be added to the For the freeze-dried preparation, to a container add the
intermediate product, which may be stored for a period volume of the liquid stated on the label. The preparation
defined in the light of stability data. dissolves completely within the time stated on the label.
Only an intermediate product that complies with the Extractable volume (2. 9.17)
following requirements may be used in the preparation of the It complies with the requirement for extractable volume.
final bulk. pH (2.2.3)
If the method of preparation includes a step for adsorption of The pH is within the limits approved for the particular
cross-reacting anti-human antibodies using material from product.
human tissues and/or red blood cells, the human materials Osmolality (2.2.35)
are submitted to a validated procedure for inactivation of Minimum 240 mosmol/kg after dilution, where applicable.
infectious agents, unless otherwise justified and authorised.
Total protein (2.5.33)
If erythrocytes are used for adsorption, the donors for such
90 per cent to 110 per cent of the amount stated on the
materials comply with the requirements for donors of blood
label.
and plasma of the monograph on Human plasma for
Stabiliser Haemolysins
Determine the amount of stabiliser by a suitable physico- Prepare a 1 to 64 dilution of the preparation to be examined,
chemical method. The preparation contains not less than diluted if necessary as stated on the label. Take 6 aliquots of
80 per cent and not more than 120 per cent of the quantity the 1 to 64 dilution. To 1 volume of 3 of the aliquots, add
stated on the label. 1 volume of a 10 per cent V/V suspension of group Al,
Distribution of molecular size group B and group O erythrocytes in a 9 g/L solution of
Size-exclusion chromatography (2.2.30). sodium chloride R, respectively. To 1 volume of the remaining
3 aliquots, add 1 volume of a 10 per cent V/V suspension of
Test solution Dilute the preparation to be examined with a
group Al, group Band group O erythrocytes in a 9 g/L
9 g/L solution of sodium chloride R to a concentration suitable
solution of sodium chloride R, respectively, and to each aliquot
for the chromatographic system used. A concentration in the
1 volume of fresh group AB serum (as a source of
range 2-20 g/L is usually suitable.
complement). Mix and incubate at 37 °C for 1 h. Examine
Reference solution Dilute human immunoglobulin (molecular the supernatant liquids for haemolysis. No signs of
size) ERP with a 9 g/L solution of sodium chloride R to the haemolysis are present.
same protein concentration as the test solution.
Thrombocyte antibodies
Column: Examined by a suitable method, the level of thrombocyte
- size: l = 0.6 m, 0 = 7.5 mm, antibodies is shown to be below that approved for the
- stationary phase: silica gel for size-exclusion specific product.
chromatography R, a grade suitable for fractionation of
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 R, I .7 41 g of sodium dihydrogen phosphate It complies with the test.
monohydrate Rand 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
Flow 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 µg 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
milligrams per millilitre which mediates 50 per cent
System suitability:
cytotoxicity.
- reference solution: the principal peak corresponds to IgG
monomer and there is a peak corresponding to dimer with Lymphocyte separation medium Commercial separation media
a retention time relative to monomer of 0.85 ± 0.05, with low viscosity and a density of 1.077 g/mL.
- test solution: the relative retentions of monomer and dimer Complement Commercial complement is suitable.
are 1 ± 0.05 with reference to the corresponding peaks in Buffered salt solution pH 7.2 Dissolve 8.0 g of sodium
the chromatogram obtained with the reference solution. chloride R, 0.2 g of potassium chloride R, 3.18 g of disodium
Limits: hydrogen phosphate dodecahydrate R and 0.2 g of potassium
- total monomer and dimer: at least 95 per cent of the total dihydrogen phosphate R in water R and dilute to 1000.0 mL
area of the peaks; with the same solvent.
- total polymers and aggregates: maximum 5 per cent of the Buffer solution for flow cytometry Add 40 mL of
total area of the peaks. 0.1 per cent V/V sodium azide R and 10 mL of foetal calf
Purity serum to 440 mL of buffered salt solution pH 7 .2. The foetal
Polyacrylamide gel electrophoresis (2.2.31), under non- calf serum is inactivated at 56 °C for 30 min prior to use.
reducing and reducing conditions. Store at 4 °C.
Resolving gel Non-reducing conditions: 8 per cent Propidium iodide solution Dissolve propidium iodide R in
acrylamide; reducing conditions: 12 per cent acrylamide. buffered salt solution pH 7 .2, to a concentration of
Test solution Dilute the preparation to be examined to a 1 mg/mL. Store this stock solution at 2-8 °C and use within
protein concentration of 0.5-2 mg/mL. 1 month. For the assay, dilute this solution with buffer
solution for flow cytometry, to obtain a concentration of
Reference solution Dilute the reference preparation to the
5 µg/mL. Store at 2-8 °C and use within 3 h.
same protein concentration as the test solution.
Microtitre plates Plates used to prepare immunoglobulin
Application I 0 µL.
dilutions are U- or V-bottomed polystyrene or poly(vinyl
Detection Coomassie staining. chloride) plates without surface treatment.
Results Compared with the electropherogram of the Micronic tubes Suitable for flow cytometry measurement.
reference solution, no additional bands are found in the
Cell suspension Collect blood in anticoagulant from at least
electropherogram of the test solution.
one healthy donor. Immediately isolate the peripheral blood
Anti-A and anti-B haemagglutinins (2. 6.20, Method A) mononuclear cells (PBMC) by gradient centrifugation in
The 1 to 64 dilution does not show agglutination. lymphocyte separation medium so that the PBMC form a
Where applicable, dilute the preparation to be examined as visible clean interface between the plasma and the separation
prescribed for use before preparing the dilutions for the test. medium. Collect the layer containing the cells and dispense
into centrifuge tubes containing buffered salt solution Transfer the suspension into tubes. Incubate at 25 °C for
pH 7.2. Centrifuge at 400 g at 2-8 °C for 10 min. Discard 10 min then place immediately on ice.
the supernatant. Suspend the cell pellet in buffer solution for Proceed with fluorescence measurement in a flow cytometer.
flow cytometry. Repeat the centrifugation and resuspension Define a region including all propidium iodide-positive cells
procedure of the cells twice. After the third centrifugation, on the basis of Forward-Scattered, light (FSC) and
resuspend the cell pellet in 1 mL of buffer solution for flow flourescence (FL2 or FL3 for propidium iodide). Measure
cytometry. Determine the number and vitality of the cells the percentage of propidium iodide-positive cells, without
using a haemocytometer. Cell viability of at least 90 per cent gating but excluding debris. Analyse at least 3000 cells for
is required. Adjust the cell number to 7 x 106 /mL by adding each of the test and reference solutions.
buffer solution for flow cytometry. Store the cell suspension Use the percentages of dead cells to estimate the potency as
at 4 °C and use within 12 h. the concentration in milligrams per millilitre of the
If necessary, the first PBMC pellet may be resuspended in preparation to be examined necessary to induce 50 per cent
buffered salt solution pH 7.2 containing 20 per cent foetal of cytotoxicity by fining a sigmoidal dose response curve to
calf serum and stored overnight at 2 °C. Centrifuge at 400 g the data obtained with the test and the reference preparations
at 2-8 °C for 10 min. Discard the supernatant. Suspend the and by using a 4-parameter logistic model (see, for example,
cell pellet in buffer solution for flow cytometry. Determine chapter 5.3) and suitable software. The test is not valid
the number and vitality of the cells using a haemocytometer. unless the percentage of propidium iodide-positive cells at the
Cell viability of at least 90 per cent is required. Adjust the lower asymptote of the curve is less then 15 per cent and the
cell number to 7 x 106/mL by adding buffer solution for percentage of propidium iodide-positive cells at the upper
flow cytometry. asymptote of the curve is at least 80 per cent.
It is also possible for cells to be immediately frozen and The estimated activity is 70 per cent to 130 per cent of the
stored in nitrogen using the following method. activity approved for the particular product.
Buffer solution for freezing To 20 mL of cell culture medium, The confidence limits (P = 0.95) are not less than
add 25 mL of foetal calf serum and 5 mL of dimethyl 80 per cent and not more than 125 per cent of the estimated
sulfoxide (DMSO). Store this solution at 2-8 °C and use potency.
within 3 h.
STORAGE
20 x 10 6 cells per ampoule are frozen. These ampoules are
Protected from light at the temperature stated on the label.
stored in liquid nitrogen.
Expiry date The expiry date is calculated from the beginning
Buffer solution for thawing To 450 mL of cell culture
of the assay.
medium, add 50 mL of foetal calf serum. Store this solution
at 2-8 °C and use within 3 h. LABELLING
Each ampoule is thawed in a water-bath at 37 °C with The label states:
shaking. Cell suspension is repeated in a buffer solution for - for liquid preparations, the volume of the preparation in
thawing. Centrifuge at 200 g at 2-8 °C for 10 min. Discard the container and the protein content,
the supernatant. Suspend the cell pellet in buffer solution for - for freeze-dried preparations:
flow cytometry. Repeat the procedure for centrifugation and - the name and the volume of the reconstitution liquid
resuspension of cells once. After the second centrifugation, to be added,
resuspend the cells pellet in 1 mL of buffer solution for flow - the quantity of protein in the container,
cytometry. Determine the number and vitality of the cells - that the immunoserum is to be used immediately after
using a haemocytometer. Cell viability of at least 90 per cent reconstitution,
is required. Adjust the cell number to 7 x 106/mL by adding - the time required for complete dissolution,
buffer solution for flow cytometry. Store the cell suspension - the animal species of origin,
at 4 °C and use within 3 h. - the name and amount of stabiliser, where applicable,
- the dilution to be made before use of the product.
Test solutions For freeze-dried preparations, reconstitute as
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
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
according to the instructions for use. Prepare 3 independent Botulinum Antitoxin
dilution series of not fewer than 7 dilutions using buffer
solution for flow cytometry as diluent. (Ph. Bur. monograph 0085)
Distribute 75 µL of each of the dilutions of the test solution The label may state 'Bot/Ser' followed by a letter or letters
or reference solution to each of a series of wells of a indicating the type or types present.
microtitre plate. Add 25 µL of the cell suspension of PBMC When Mixed Botulinum Antitoxin or Botulinum Antitoxin is
into each well. Add 25 µL of rabbit complement to each of prescribed or demanded and the types to be present are not
the wells. Incubate at 37 °C for 30 min. stated, Botulinum Antitoxin prepared from types A, B and E
Centrifuge the plates at 200 g at 4 °C for 8 min, discard the shall be dispensed or supplied.
supernatant and keep the plate on ice. Preparation for flow Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
cytometry measurement is done step-wise by using a certain
number of wells in order to allow labelling with propidium DEFINITION
iodide R solution and measurement within a defined time Liquid or freeze-dried preparation containing purified
period. Resuspend carefully the cell pellet of a certain immunoglobulin fragments obtained from serum or plasma
number of wells with 200 µL of propidium iodide solution. of immunised animals that have the power of specifically
neutralising the toxins formed by Clostridium botulinum
type A, type B or type E, or any mixture of these types.
PRODUCTION Prepare mixtures of the solution of the reference preparation

Botulinum antitoxin is obtained by fractionation from the and the test toxin such that each contains 2.0 mL of the
serum of horses, or other mammals, that have been solution of the reference preparation, one of a graded series
immunised against C. botulinum type A, type B and type E of volumes of the test toxin and a sufficient volume of a
toxins. suitable liquid to bring the total volume to 5.0 mL. Allow the
mixtures to stand at room temperature, protected from light,
IDENTIFICATION
for 60 min. Using 4 mice for each mixture, inject a dose of
Botulinum antitoxin specifically neutralises the types of
1.0 mL intraperitoneally into each mouse. Observe the mice
C. botulinum toxins stated on the label, rendering them
for 96 h.
harmless to susceptible animals. Alternatively, botulinum
antitoxin can be identified by a suitable immunochemical The test dose of toxin is the quantity in 1.0 mL of the
method (2. 7.1). mixture made with the smallest amount of toxin capable of
causing, despite partial neutralisation by the reference
POTENCY preparation, the death of all 4 mice injected with the mixture
Not less than 500 IU of antitoxin per millilitre for each of within the observation period.
types A and B and not less than 50 IU of antitoxin per Determination of potency of the antitoxin Prepare solutions of
millilitre for type E. each reference preparation in a suitable liquid such that each
In accordance with the provisions of the European contains 0.25 IU of antitoxin per millilitre.
Convention for the Protection of Vertebrate Animals used for Prepare solutions of each test toxin in a suitable liquid such
Experimental and Other Scientific Purposes, tests must be that each contains 2.5 test doses per millilitre.
carried out in such a way as to use the minimum number of
animals and to cause the least pain, suffering, distress or Using each toxin solution and the corresponding reference
lasting harm. The assay described below is associated with preparation in turn, determine the potency of the antitoxin.
severe suffering of animals and manufacturers are strongly Prepare mixtures of the solution of the test toxin and the
encouraged to develop and validate assays that will reduce antitoxin to be examined such that each contains 2.0 mL of
the number of animals used, or refine or replace the test the solution of the test toxin, one of a graded series
procedure with the goal of promoting animal welfare. of volumes of the antitoxin to be examined, and a sufficient
volume of a suitable liquid to bring the total volume to
The potency of botulinum antitoxin is determined by 5.0 mL. Also prepare mixtures of the solution of the test
comparing the dose necessary to protect mice against the toxin and the solution of the reference preparation such that
lethal effects of a fixed dose of botulinum toxin with the each contains 2.0 mL of the solution of the test toxin, one of
quantity of the standard preparation of botulinum antitoxin a graded series of volumes of the solution of the reference
necessary to give the same protection. For this comparison a preparation centred on that volume (2.0 mL) that contains
reference preparation of each type of botulinum antitoxin 0.5 IU, and a sufficient volume of a suitable liquid to bring
calibrated in International Units, and suitable preparatio~s of the total volume to 5.0 mL. Allow the mixtures to stand at
botulinum toxins, for use as test toxins, are required. room temperature, protected from light, for 60 min. Using
The potency of each test toxin is determined in relation to 4 mice for each mixture, inject a dose of 1.0 mL
the specific reference preparation; the potency of the intraperitoneally into each mouse. Observe the mice for 96 h.
botulinum antitoxin to be examined is determined in relation
to the potency of the test toxins by the same method. The mixture that contains the largest volume of antitoxin
that fails to protect the mice from death contains 0.5 IU.
International Units of the antitoxin are the specific This quantity is used to calculate the potency of the antitoxin
neutralising activity for botulinum toxin type A, type B and in International Units per millilitre.
type E contained in stated amounts of the International
Standards that consist of dried immune horse sera of types The test is not valid unless all the mice injected with
A, B and E. The equivalence in International Units of the mixtures containing 2.0 mL or less of the solution of the
International Standard is stated by the World Health reference preparation die and all those injected with mixtures
Organization. containing more survive.
Selection of animals Use mice having body masses such that
After validation with respect to the LD 50 method, the
the difference between the lightest and the heaviest does not product potency may be assessed by other methods, for
exceed 5 g. example mouse bioassays using paralysis or digit abduction as
an end point, ex vivo assays using mouse phrenic nerve
Preparation of test toxins CAUTION: botulinum toxin is
diaphragm, or cell-based or other suitable in vitro methods.
extremely toxic; exceptional care must be taken in any procedure
Potency is calculated with respect to a suitable reference
in which it is employed. Prepare types A, B and E toxins from preparation calibrated in International Units.
sterile filtrates of approximately 7-day cultures in liquid
medium of C. botulinum types A, B and E. To the filtrates LABELLING
add_ 2 volumes of glycerol, concentrate if necessary by dialysis The label states the types of C. botulinum toxin neutralised by
agamst glycerol, and store at or slightly below O c.
0 the preparation.
Selection of test toxins Select toxins of each type for use as - - - - - - - - - - - - - - - - - - - - - - PhE~
test toxins by determining for mice the L+/10 dose and the
LDso, the observation period being 96 h. The test toxins
contain at least 1000 LD 50 in an L+/10 dose.
Dete1;11ination of test doses of the toxins (L+/10 dose). Prepare
solut10ns of the reference preparations in a suitable liquid
such that each contains 0.25 IU of antitoxin per millilitre.
Using each solution in turn, determine the test dose of the
corresponding test toxin.
Detennination of test dose of toxin (lr/100 dose). Prepare a

Diphtheria Antitoxin solution of the reference preparation in a suitable liquid such
(Ph. Bur. monograph 0086) that it contains 0.1 IU of antitoxin per millilitre.
The label may state 'Dip/Ser'. and of the test toxin such that each contains 1.0 mL of the
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
solution of the reference preparation, one of a graded series
DEFINITION of volumes of the test toxin and sufficient of a suitable liquid
Diphtheria antitoxin is a preparation containing antitoxic to bring the total volume to 2.0 mL. Allow the mixtures to
globulins that have the power of specifically neutralising the stand at room temperature, protected from light, for 15 min
toxin formed by Co1ynebacterium diphtheriae. to 60 min. Using two animals for each mixture, inject a dose
of 0.2 mL intracutaneously into the shaven or depilated
PRODUCTION flanks of each animal. Observe the animals for 48 h.
It is obtained by fractionation from the serum of horses, or The test dose of toxin is the quantity in 0.2 mL of the
other mammals, that have been immunised against diphtheria mixture made with the smallest amount of toxin capable of
toxin. causing, despite partial neutralisation by the reference
IDENTIFICATION preparation, a small but characteristic erythematous lesion at
It specifically neutralises the toxin formed by C. diphtheriae, the site of injection.
rendering it harmless to susceptible animals. Detennination of potency of the antitoxin Prepare a solution of
ASSAY the reference preparation in a suitable liquid such that it
Not less than 1000 IU of antitoxin per millilitre for antitoxin contains 0.125 IU of antitoxin per millilitre.
obtained from horse serum. Not less than 500 IU of Prepare a solution of the test toxin in a suitable liquid such
antitoxin per millilitre for antitoxin obtained from the serum that it contains 12.5 test doses per millilitre.
of other mammals. Prepare mixtures of the solution of the test toxin and of the
The potency of diphtheria antitoxin is determined by antitoxin to be examined such that each contains 0.8 mL of
comparing the dose necessary to protect guinea-pigs or the solution of the test toxin, one of a graded series
rabbits against the erythrogenic effects of a fixed dose of of volumes of the antitoxin to be examined and sufficient of a
diphtheria toxin with the quantity of the standard preparation suitable liquid to bring the total volume to 2.0 mL. Also
of diphtheria antitoxin necessary to give the same protection. prepare mixtures of the solution of the test toxin and the
For this comparison a reference preparation of diphtheria solution of the reference preparation such that each contains
antitoxin, calibrated in International Units, and a suitable 0.8 mL of the solution of the test toxin, one of a graded
preparation of diphtheria toxin, for use as a test toxin, are series of volumes of the solution of the reference preparation
required. The potency of the test toxin is determined in centred on that volume (0.8 mL) that contains 0.1 IU and
relation to the reference preparation; the potency of the sufficient of a suitable liquid to bring the total volume to
diphtheria antitoxin to be examined is determined in relation 2.0 mL. Allow the mixtures to stand at room temperature,
to the potency of the test toxin by the same method. protected from light, for 15 min to 60 min. Using two
The International Unit of antitoxin is the specific neutralising animals for each mixture, inject a dose of 0.2 mL
activity for diphtheria toxin contained in a stated amount of intracutaneously into the shaven or depilated flanks of each
the International Standard, which consists of a quantity of animal. Observe the animals for 48 h.
dried immune horse serum. The equivalence in International The mixture that contains the largest volume of antitoxin
Units of the International Standard is stated by the World that fails to protect the guinea-pigs from the erythematous
Health Organization. effects of the toxin contains O.1 IU. This quantity is used to
Preparation of test toxin Prepare diphtheria toxin from calculate the potency of the antitoxin in International Units
cultures of C. diphtheriae in a liquid medium. Filter the per millilitre.
culture to obtain a sterile toxic filtrate and store at 4 °C. The test is not valid unless all the sites injected with mixtures
Selection of test toxin Select a toxin for use as a test toxin by containing 0.8 mL or less of the solution of the reference
determining for guinea-pigs or rabbits the lr/100 dose and the preparation show erythematous lesions and at all those
minimal reacting dose, the observation period being 48 h. injected with mixtures containing more there are no lesions.
The test toxin has at least 200 minimal reacting doses in the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
lr/100 dose.
Minimal reacting dose This is the smallest quantity of toxin
which, when injected intracutaneously into guinea-pigs or
rabbits, causes a small, characteristic reaction at the site of European Viper Venom Antiserum
injection within 48 h.
The test toxin is allowed to stand for some months before
being used for the assay of antitoxin. During this time its The only poisonous snake native to the British Isles is the
toxicity declines and the lr/100 dose may be increased. adder or common viper, Vipera berus. In a geographical
Determine the minimal reacting dose and the lr/100 dose at region where other species of snake (including elapids) are
frequent intervals. When experiment shows that the found, antisera able to neutralise the venoms of the species of
lr/100 dose is constant, the test toxin is ready for use and snake indigenous to the region should be used. When the
may be used for a long period. Store the test toxin in the preparation is intended to neutralise the venom or venoms of
dark at O °C to 5 °C. Maintain its sterility by the addition of one or more snakes other than vipers, the title Snake Venom
toluene or other antimicrobial preservative that does not Antiserum is used.
cause a rapid decline in specific toxicity.
~~---------------------- In each mouse dose of the venom-antiserum mixture at the

end point there is one LD 50 of venom remaining
DEFINTilON
unneutralised by the antiserum and it is this unneutralised
European viper venom antiserum is a preparation containing
venom that is responsible for the deaths of 50 per cent of the
antitoxic globulins that have the power of neutralising the
mice inoculated with the mixture. The amount of venom
venom of one or more species of viper. The globulins are
neutralised by the antiserum is thus one LD 50 less than the
obtained by fractionation of the serum of animals that have
total amount contained in each mouse dose. Therefore, as
been immunised against the venom or venoms.
the potency of the antiserum is defined in terms of the
IDENTIFICATION number of LD 50 of venom that are neutralised. rather than
It neutralises the venom of Vipera ammodytes, or Vipera aspis, the number of LD 50 in each mouse dose, the expression
or Vipera berus, or Vipera ursinii or the mixture of these required in the calculation of potency is Tv - 1 rather than
venoms stated on the label, rendering them harmless to Tv.
susceptible animals. Alternatively, the quantity of test venom in milligrams that is
ASSAY neutralised by 1 mL or some other defined volume of the
Each millilitre of the preparation to be examined contains antiserum to be examined may be calculated.
sufficient antitoxic globulins to neutralise not less than LABELLING
100 mouse LD 50 of Vipera ammodytes venom or Vipera aspis The label states the venom or venoms against which the
venom and not less than 50 mouse LD 50 of the venoms of antiserum is effective.
other species of viper.
CAUTION because of the allergenic properties of viper venoms,
The potency of European viper venom antiserum is inhalation of venom dust should be avoided by suitable
determined by estimating the dose necessary to protect mice precautions.
against the lethal effects of a fixed dose of venom of the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
relevant species of viper.
Selection of test venoms Use venoms which have the normal
physico-chemical, toxicological and immunological
characteristics of venoms from the particular species of
vipers. They are preferably freeze-dried and stored in the
Gas-gangrene Antitoxin (Novyi)
dark at 5 ± 3 °C.
Gas-gangrene Antitoxin (Oedematiens)
Select a venom for use as a test venom by determining the
LD 50 for mice, the observation period being 48 h.
The label may state 'Nov/Ser'.
Determination of the test dose of venom Prepare graded
dilutions of the reconstituted venom in a 9 g/L solution of Preparation
sodium chloride R or other isotonic diluent in such a manner Mixed Gas-gangrene Antitoxin
that the middle dilution contains in 0.25 mL the dose PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
expected to be the LD 50 • Dilute with an equal volume of the DEFINITION
same diluent. Using at least four mice, each weighing 18 g to
Gas-gangrene antitoxin (novyi) is a preparation containing
20 g, for each dilution, inject 0.5 mL intravenously into each
antitoxic globulins that have the power of neutralising the
mouse. Observe the mice for 48 h and record the number of
alpha toxin formed by Clostridium novyi (Former
deaths. Calculate the LD 50 using the usual statistical
nomenclature: Clostridium oedematiens). It is obtained by
methods.
fractionation from the serum of horses, or other mammals,
Determination of the potency of the antiserum to be examined that have been immunised against Cl. novyi alpha toxin.
Dilute the reconstituted test venom so that 0.25 mL contains
the test dose of 5 LD 50 (test venom solution). IDENTIFICATION
It specifically neutralises the alpha toxin formed by Cl. novyi,
Prepare serial dilutions of the antiserum to be examined in a
rendering it harmless to susceptible animals.
9 g/L solution of sodium chloride R or other isotonic diluent,
the dilution factor being 1.5 to 2.5. Use a sufficient number ASSAY
and range of dilutions to enable a mortality curve between Not less than 3750 IU of antitoxin per millilitre.
20 per cent and 80 per cent mortality to be established and The potency of gas-gangrene antitoxin (novyi) is determined
to permit an estimation of the statistical variation. by comparing the dose necessary to protect mice or other
Prepare mixtures such that 5 mL of each mixture contains suitable animals against the lethal effects of a fixed dose of
2.5 mL of one of the dilutions of the antiserum to be Cl. novyi toxin with the quantity of the standard preparation
examined and 2.5 mL of the test venom solution. Allow the of gas-gangrene antitoxin (novyi) necessary to give the same
mixtures to stand in a water-bath at 37 °C for 30 min. Using protection. For this comparison a reference preparation of
not fewer than six mice, each weighing 18 g to 20 g, for each gas-gangrene antitoxin (novyi), calibrated in International
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 PD 50, using the usual statistical methods. determined in relation to the reference preparation; the
At the same time verify the number of LD 50 in the test dose potency of the gas-gangrene antitoxin (novyi) to be examined
of venom, using the method described above. Calculate the is determined in relation to the potency of the test toxin by
potency of the antiserum using the following expression: 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
PDso the International Standard, which consists of a quantity of
T,, number of l.D 50 in the test dose of venom. dried immune horse serum. The equivalence in International
Units of the International Standard is stated by the World

Health Organization.
Gas-gangrene Antitoxin
Selection of animals Use mice having body masses such that (Perfringens)
the difference between the lightest and the heaviest does not (Ph. Bur. monograph 0088)
exceed 5 g.
The label may state 'Perf/Ser'.
Preparation of test toxin Prepare the test toxin from a sterile
filtrate of an approximately 5-day culture in liquid medium of Preparation
Cl. novyi. Treat the filtrate with ammonium sulfate R, collect Mixed Gas-gangrene Antitoxin
the precipitate, which contains the toxin, dry in vacua over PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
diphosphorus pentoxide R, powder and store dry.
DEFINITION
Selection of test toxin Select a toxin for use as a test toxin by Gas-gangrene antitoxin (perfringens) is a preparation
determining for mice the L+ dose and the LD 50 , the containing antitoxic globulins that have the power of
observation period being 72 h. The test toxin has an L+ dose specifically neutralising the alpha toxin formed by Clostridium
of 0.5 mg or less and contains not less than 25 LD 50 in each perjringens. It is obtained by fractionation from the serum of
L+ dose. horses, or other mammals, that have been immunised against
Determination of test dose of toxin (L+ dose) Prepare a Cl. perjringens alpha toxin.
solution of the reference preparation in a suitable liquid such
IDENTIFICATION
that it contains 12.5 IU of antitoxin per millilitre.
It specifically neutralises the alpha toxin formed by
Prepare a solution of the test toxin in a suitable liquid such Cl. perfringens, rendering it harmless to susceptible animals.
that 1 mL contains a precisely known amount such as
10 mg. ASSAY
Prepare mixtures of the solution of the reference preparation Not less than 1500 IU of antitoxin per millilitre.
and the solution of the test toxin such that each contains The potency of gas-gangrene antitoxin (perfringens) is
0.8 mL of the solution of the reference preparation, one of a determined by comparing the dose necessary to protect mice
graded series of volumes of the solution of the test toxin 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. perjringens toxin with the quantity of the standard
2.0 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 0.2 mL intramuscularly into each preparation of gas-gangrene antitoxin (perfringens), calibrated
mouse. Observe the mice for 72 h. in International Units, and a suitable preparation of
The test dose of toxin is the quantity in 0.2 mL of the Cl. perjringens toxin for use as a test toxin are required.
mixture made with the smallest amount of toxin capable of The potency of the test toxin is determined in relation to the
causing, despite partial neutralisation by the reference reference preparation; the potency of the gas-gangrene
preparation, the death of all six mice injected with the antitoxin (perfringens) to be examined is determined in
mixture within the observation period. relation to the potency of the test toxin by the same method.
Determination of potency of the antitoxin Prepare a solution of The International Unit of antitoxin is the specific neutralising
the reference preparation in a suitable liquid such that it activity for Cl. perjringens toxin contained in a stated amount
contains 12.5 IU of antitoxin per millilitre. of the International Standard, which consists of a quantity of
dried immune horse serum. The equivalence in International
Prepare a solution of the test toxin in a suitable liquid such
that it contains 12.5 test doses per millilitre.
Prepare mixtures of the solution of the test toxin and the
Selection of animals Use mice having body masses such that
antitoxin to be examined such that each contains 0.8 mL of
the difference between the lightest and the heaviest does not
the solution of the test toxin, one of a graded series
exceed 5 g.
of volumes of the-antitoxin to be examined and sufficient of a
suitable liquid to bring the total volume to 2.0 mL. Also Preparation of test toxin Prepare the test toxin from a sterile
prepare mixtures of the solution of the test toxin and the filtrate of an approximately 5-day culture in liquid medium of
solution of the reference preparation such that each contains Cl. perjringens. Treat the filtrate with ammonium sulfate R,
0.8 mL of the solution of the test toxin, one of a graded collect the precipitate, which contains the toxin, dry in vacua
series of volumes of the solution of the reference preparation over diphosphorus pentoxide R, powder and store dry.
centred on that volume (0.8 mL) that contains 10 IU and Selection of test toxin Select a toxin for use as a test toxin by
sufficient of a suitable liquid to bring the total volume to determining for mice the L+ dose and the LD 50 , the
2.0 mL. Allow the mixtures to stand at room temperature, observation period being 48 h. The test toxin has an L+ dose
protected from light, for 60 min. Using six mice for each of 4 mg or less and contains not less than 20 LD 50 in each
mixture, inject a dose of 0.2 mL intramuscularly into each L+ dose.
mouse. Observe the mice for 72 h. Determination of test dose of toxin (L+ dose) Prepare a
The mixture that contains the largest volume of antitoxin solution of the reference preparation in a suitable liquid such
that fails to protect the mice from death contains 10 IU. This that it contains 5 IU of antitoxin per millilitre.
quantity is used to calculate the potency of the antitoxin in Prepare a solution of the test toxin in a suitable liquid such
International Units per millilitre. that 1 mL contains a precisely known amount such as
The test is not valid unless all the mice injected with 10 mg.
mixtures containing 0.8 mL or less of the solution of the Prepare mixtures of the solution of the reference preparation
reference preparation die and all those injected with mixtures and the solution of the test toxin such that each contains
containing a larger volume survive. 2.0 mL of the solution of the reference preparation, one of a
~ - - - - - - - - - - - - - - - - - - - Ph Eur graded series of volumes of the solution of the test toxin and
sufficient of a suitable liquid to bring the total volume to ASSAY

5.0 mL. Allow the mixtures to stand at room temperature, Not less than 1500 IU of antitoxin per millilitre.
protected from light, for 60 min. Using six mice for each The potency of gas-gangrene antitoxin (septicum) is
mixture, inject a dose of 0.5 mL intravenously into each determined by comparing the dose necessary to protect mice
mouse. Observe the mice for 48 h. or other suitable animals against the lethal effects of a fixed
The test dose of toxin is the quantity in 0.5 mL of the dose of Cl. septicum toxin with the quantity of the standard
mixture made with the smallest amount of toxin capable of preparation of gas-gangrene antitoxin (septicum) necessary to
causing, despite partial neutralisation by the reference give the same protection. For this comparison a reference
preparation, the death of all six mice injected with the preparation of gas-gangrene antitoxin (septicum), calibrated
mixture within the observation period. in International Units, and a suitable preparation of
Detemiination of potency of the antitoxin Prepare a solution of Cl. septicum toxin for use as a test toxin are required.
the reference preparation in a suitable liquid such that it The potency of the test toxin is determined in relation to the
contains 5 IU of antitoxin per millilitre. reference preparation; the potency of the gas-gangrene
Prepare a solution of the test toxin in a suitable liquid such antitoxin (septicum) to be examined is determined in relation
to the potency of the test toxin by the same method.
that it contains five test doses per millilitre.
Prepare mixtures of the solution of the test toxin and the
activity for Cl. septicum toxin contained in a stated amount of
antitoxin to be examined such that each contains 2.0 mL of
the International Standard, which consists of a quantity of
the solution of the test toxin, one of a graded series of
dried immune horse serum. The equivalence in International
volumes of the antitoxin to be examined and sufficient of a
suitable liquid to bring the total volume to 5.0 mL. Also
prepare mixtures of the solution of the test toxin and the
Selection of animals V se mice having body masses such that
solution of the reference preparation such that each contains
2.0 mL of the solution of the test toxin, one of a graded the difference between the lightest and the heaviest does not
series of volumes of the solution of the reference preparation exceed 5 g.
centred on that volume (2.0 mL) that contains 10 IU and Preparation of test toxin Prepare the test toxin from a sterile
sufficient of a suitable liquid to bring the total volume to filtrate of an approximately 5-day culture in liquid medium of
5.0 mL. Allow the mixtures to stand at room temperature, Cl. septicum. Treat the filtrate with ammonium sulfate R,
protected from light, for 60 min. Using six mice for each collect the precipitate, which contains the toxin, dry in vacuo
mixture, inject a dose of 0.5 mL intravenously into each over diphosphorus pentoxide R, powder and store dry.
mouse. Observe the mice for 48 h. Selection of test toxin Select a toxin for use as a test toxin by
The mixture that contains the largest volume of antitoxin determining for mice the L+ dose and the LD 50 , the
that fails to protect the mice from death contains 10 IU. This observation period being 72 h. The test toxin has an L+ dose
quantity is used to calculate the potency of the antitoxin in of 0.5 mg or less and contains not less than 25 LD 50 in each
International Units per millilitre. L+ dose.
Detemiination of test dose of toxin (L+ dose) Prepare a
The test is not valid unless all the mice injected with
solution of the reference preparation in a suitable liquid such
mixtures containing 2.0 mL or less of the solution of the
that it contains 5 IU of antitoxin per millilitre.
reference preparation die and all those injected with mixtures
containing a larger volume survive. Prepare a solution of the test toxin in a suitable liquid such
that 1 mL contains a precisely known amount such as
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
20mg.
and the solution of the test toxin such that each contains
2.0 mL of the solution of the reference preparation, one of a
Gas-gangrene Antitoxin graded series of volumes of the solution of the test toxin and
(Septicum) sufficient of a suitable liquid to bring the total volume to
5.0 mL. Allow the mixtures to stand at room temperature,
(Ph. Bur. monograph 0089) protected from light, for 60 min. Using six mice for each
The label may state 'Sep/Ser'. mixture, inject a dose of 0.5 mL intravenously into each
Preparation mouse. Observe the mice for 72 h.
Mixed Gas-gangrene Antitoxin The test dose of toxin is the quantity in 0.5 mL of the
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ mixture made with the smallest amount of toxin capable of
causing, despite partial neutralisation by the reference
DEFINITION preparation, the death of all six mice injected with the
Gas-gangrene antitoxin (septicum) is a preparation mixture within the observation period.
containing antitoxic globulins that have the power of Detemiination of potency of the antitoxin Prepare a solution of
specifically neutralising the alpha toxin formed by Cwstridium the reference preparation in a suitable liquid such that it
septicum. It is obtained by fractionation from the serum of contains 5 IU of antitoxin per millilitre.
horses, or other mammals, that have been immunised against
Prepare a solution of the test toxin in a suitable liquid such
Cl. septicum alpha toxin.
that it contains five test doses per millilitre.
IDENTIFICATION Prepare mixtures of the solution of the test toxin and the
It specifically neutralises the alpha toxin formed by antitoxin to be examined such that each contains 2.0 mL of
Cl. septicum, rendering it harmless to susceptible animals. the solution of the test toxin, one of a graded series of
volumes of the antitoxin to be examined and sufficient of a
suitable liquid to bring the total volume to 5.0 mL. Also
prepare mixtures of the solution of the test toxin and the
solution of the reference preparation such that each contains IDENTIFICATION

2.0 mL of the solution of the test toxin, one of a graded It specifically neutralises the toxin formed by Cl. tetani,
series of volumes of the solution of the reference preparation rendering it harmless to susceptible animals.
centred on that volume (2.0 mL) that contains 10 IU and
POTENCY
sufficient of a suitable liquid to bring the total volume to
Not less than 1000 IU of antitoxin per millilitre when
5.0 mL. Allow the mixtures to stand at room temperature,
intended for prophylactic use. Not less than 3000 IU of
protected from light, for 60 min. Using six mice for each
antitoxin per millilitre when intended for therapeutic use.
mixture, inject a dose of 0.5 mL intravenously into each
mouse. Observe the mice for 72 h. The potency of tetanus antitoxin is determined by comparing
the dose necessary to protect guinea-pigs or mice against the
The mixture that contains the largest volume of antitoxin
paralytic effects of a fixed dose of tetanus toxin with the
that fails to protect the mice from death contains 10 IU. This
quantity of the standard preparation of tetanus antitoxin
quantity is used to calculate the potency of the antitoxin in
necessary to give the same protection. In countries where the
International Units per millilitre.
paralysis method is not obligatory the lethal method may be
The test is not valid unless all the mice injected with used. For this method the number of animals and the
mixtures containing 2.0 mL or less of the solution of the procedure are identical with those described for the paralysis
reference preparation die and all those injected with mixtures method but the end-point is the death of the animal rather
containing more survive. than the onset of paralysis and the L+/10 dose is used
- - - - - - - - - - - - - - - - - - - - - - PhEur instead of the Lp/ 10 dose. For this comparison a reference
preparation of tetanus antitoxin, calibrated in International
Units, and a suitable preparation of tetanus toxin, for use as
Mixed Gas-gangrene Antitoxin a test toxin, are required. The potency of the test toxin is
determined in relation to the reference preparation; the
(Ph. Eur. monograph 0090) potency of the tetanus antitoxin to be examined is
determined in relation to the potency of the test toxin by the
The label may state 'Gas/Ser'.
same method.
PhEif _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION activity for tetanus toxin contained in a stated amount of the
Mixed gas-gangrene antitoxin is prepared by mixing gas- International Standard which consists of a quantity of dried
gangrene antitoxin (novyi), gas-gangrene antitoxin immune horse serum. The equivalence in International Units
(perfringens) and gas-gangrene antitoxin (septicum) in of the International Standard is stated by the World Health
appropriate quantities. Organization.
IDENTIFICATION Selection of animals If mice are used, the body masses
It specifically neutralises the alpha toxins formed by should be such that the difference between the lightest and
Clostridium novyi (former nomenclature: Clostridium the heaviest does not exceed 5 g.
oedematiens), Clostridium perfringens and Clostridium septicum, Preparation of test toxin Prepare the test toxin from a sterile
rendering them harmless to susceptible animals. filtrate of an approximately 9-day culture in liquid medium of
ASSAY Cl. tetani. To the filtrate add 1 to 2 volumes of glycerol and
Gas-gangrene antitoxin (novyi), not less than 1000 IU of store slightly below O °C. Alternatively, treat the filtrate with
antitoxin per millilitre; gas-gangrene antitoxin (perfringens), ammonium sulfate R, collect the precipitate, which contains
not less than 1000 IU of antitoxin per millilitre; gas-gangrene the toxin, dry in vacuo over diphosphorus pentoxide R, powder
antitoxin (septicum) not less than 500 IU of antitoxin per and store dry, either in sealed ampoules or in vacuo over
diphosphorus pentoxide R.
millilitre.
Carry out the assay for each component, as prescribed in the Determination of test dose of toxin (Lpl 10 dose) Prepare a
monographs on Gas-gangrene antitoxin (novyi) (0087), Gas- solution of the reference preparation in a suitable liquid such
gangrene antitoxin (perfringens) (0088) and Gas-gangrene that it contains 0.5 IU of antitoxin per millilitre.
antitoxin (septicum) (0089). If the test toxin is stored dry, reconstitute it using a suitable
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur liquid.
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 of volumes of the test toxin and sufficient of a suitable liquid
(Tetanus Antitoxin for Human Use, Ph. Eur. to bring the volume to 5.0 mL. Allow the mixtures to stand
monograph 0091) at room temperature, protected from light, for 60 min. Using
six mice for each mixture, inject a dose of 0.5 mL
The label may state 'Tet/Ser'. subcutaneously into each mouse. Observe the mice for 96 h.
PhEif _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Mice that become paralysed may be euthanised.
DEFINITION The test dose of toxin is the quantity in 0.5 mL of the
Tetanus antitoxin for human use is a preparation containing mixture made with the smallest amount of toxin capable of
antitoxic globulins that have the power of specifically causing, despite partial neutralisation by the reference
neutralising the toxin formed by Clostridium tetani. preparation, paralysis in all six mice injected with the mixture
within the observation period.
PRODUCTION
Determination of potency of the antitoxin Prepare a solution of
It is obtained by fractionation from the serum of horses, or
the reference preparation in a suitable liquid such that it
other mammals, that have been immunised against tetanus
contains O.5 IU of antitoxin per millilitre.
toxin.
IV-698 Vaccines 2023
Prepare a solution of the test toxin in a suitable liquid such Bacterial vaccines containing whole cells Are suspensions of
that it contains five test doses per millilitre. various degrees of opacity in colourless or almost colourless
Prepare mixtures of the solution of the test toxin and the liquids, or may be freeze-dried. They may be adsorbed.
antitoxin to be examined such that each contains 2.0 mL of The concentration of living or inactivated bacteria is
the solution of the test toxin, one of a graded series expressed in terms of International Units of opacity or, where
of volumes of the antitoxin to be examined and sufficient of a appropriate, is determined by direct cell count or, for live
suitable liquid to bring the total volume to 5.0 mL. Also bacteria, by viable count.
prepare mixtures of the solution of the test toxin and the Bacterial vaccines containing bacterial components Are
solution of the reference preparation such that each contains suspensions or freeze-dried products. They may be adsorbed.
2.0 mL of the solution of the test toxin, one of a graded The antigen content is determined by a suitable validated
series of volumes of the solution of the reference preparation assay.
centred on that volume (2.0 mL) that contains 1 IU and Bacterial toxoids Are prepared from toxins by diminishing
sufficient of a suitable liquid to bring the total volume to their toxicity to an acceptable level or by completely
5.0 mL. Allow the mixtures to stand at room temperature, eliminating it by physical or chemical procedures whilst
protected from light, for 60 min. Using six mice for each retaining adequate immunogenic properties. The toxins are
mixture, inject into each mouse subcutaneously a dose of obtained from selected strains of micro-organisms.
0.5 mL. Observe the mice for 96 h. Mice that become The method of production is such that the toxoid does not
paralysed may be euthanised. revert to toxin. The toxoids are purified. Purification is
The mixture that contains the largest volume of antitoxin performed before and/or after detoxification. Toxoid vaccines
that fails to protect the mice from paralysis contains 1 IU. may be adsorbed.
This quantity is used to calculate the potency of the antitoxin Viral vaccines Are prepared from viruses grown in animals,
in International Units per millilitre. in fertilised eggs, in suitable cell cultures or in suitable
The test is not valid unless all the mice injected with tissues, or by culture of genetically engineered cells. They are
mixtures containing 2.0 mL or less of the solution of the liquids that vary in opacity according to the type of
reference preparation show paralysis and all those injected preparation or may be freeze-dried. They may be adsorbed.
with mixtures containing more do not. Liquid preparations and freeze-dried preparations after
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur reconstitution may be coloured if a pH indicator such as
phenol red has been used in the culture medium.
Synthetic antigen vaccines Are generally clear or colourless
liquids. The concentration of the components is usually
Vaccines expressed in terms of specific antigen content.
Combined vaccines Are multicomponent preparations
(Vaccines for Human Use, Ph. Bur. monograph 0153) formulated so that different antigens are administered
Vaccines comply with the requirements of the European simultaneously. The different antigenic components are
Pharmacopoeia monograph for Vaccines for Human Use. These intended to protect against different strains or types of the
requirements are reproduced below. same organism and/or against different organisms.
A combined vaccine may be supplied by the manufacturer
PhEur
either as a single liquid or freeze-dried preparation or as
DEFINITION several constituents with directions for admixture before use.
Vaccines for human use are preparations containing antigens Where there is no monograph to cover a particular
capable of inducing a specific and active immunity in man combination, the vaccine complies with the monograph for
against an infecting agent or the toxin or antigen elaborated each individual component, with any necessary modifications
by it. Immune responses include the induction of the innate approved by the competent authority.
and the adaptive (cellular, humoral) parts of the immune Adsorbed vaccines Are suspensions and may form a sediment
system. Vaccines for human use shall have been shown to at the bottom of the container.
have acceptable immunogenic activity and safety in man with
PRODUCTION
the intended vaccination schedule.
General provisions
Vaccines for human use may contain: whole micro-organisms The production method for a given product must have been
(bacteria, viruses or parasites), inactivated by chemical or shown to yield consistently batches comparable with the
physical means that maintain adequate immunogenic batch of proven clinical efficacy, immunogenicity and safety
properties; whole live micro-organisms that are naturally in man. Product specifications including in-process testing
avirulent or that have been treated to attenuate their should be set. Specific requirements for production including
virulence whilst retaining adequate immunogenic properties; in-process testing are included in individual monographs.
antigens extracted from the micro-organisms or secreted by Where justified and authorised, certain tests may be omitted
the micro-organisms or produced by genetic engineering or where it can be demonstrated, for example by validation
chemical synthesis. The antigens may be used in their native studies, that the production process consistently ensures
state or may be detoxified or otherwise modified by chemical compliance with the test.
or physical means and may be aggregated, polymerised or
Unless otherwise justified and authorised, vaccines are
conjugated to a carrier to increase their immunogenicity.
produced using a seed-lot system. The methods of
Vaccines may contain an adjuvant. Where the antigen is
preparation are designed to maintain adequate immunogenic
adsorbed on a mineral adjuvant, the vaccine is referred to as
properties, to render the preparation harmless and to prevent
'adsorbed'.
contamination with extraneous agents.
Terminology used in monographs on vaccines for human use
Where vaccines for human use are manufactured using
is defined in general chapter 5.2.1.
materials of human or animal origin, the general
2023 Vaccines IV-699
requirements of general chapter 5.1. 7. Viral safety apply in preferable to have a medium free from antibiotics during
conjunction with the more specific requirements relating to production.
viral safety in this monograph, in individual vaccine Propagation and harvest
monographs and in general chapters 5.2.2. Chicken flocks free The seed cultures are propagated and harvested under
from specified pathogens for the production and quality control of defined conditions. The purity of the harvest is verified by
vaccines, 5.2.3. Cell substrates for the production of vaccines for suitable tests as defined in the monograph.
human use and, with the exception of egg-derived inactivated
influenza vaccines, 2. 6.16. Tests for extraneous agents in viral Control cells
For vaccines produced in cell cultures, control cells are
vaccines for human use.
maintained and tested as prescribed. In order to provide a
Unless otherwise justified and authorised, in the production valid control, these cells must be maintained in conditions
of a final lot of vaccine, the number of passages of a virus, or that are essentially equivalent to those used for the
the number of subcultures of a bacterium, from the master production cell cultures, including use of the same batches of
seed lot shall not exceed that used for production of the media and media changes.
vaccine shown to be satisfactory in clinical trials with respect
to safety and efficacy or immunogenicity. Control eggs
For live vaccines produced in eggs, control eggs are
Vaccines are as far as possible free from ingredients known to
incubated and tested as prescribed in the monograph.
cause toxic, allergic or other undesirable reactions in man.
Suitable additives, including stabilisers and adjuvants may be Purification
incorporated. Penicillin and streptomycin are neither used at Where applicable, validated purification procedures may be
any stage of production nor added to the final product; applied.
however, master seed lots prepared with media containing Inactivation
penicillin or streptomycin may, where justified and Inactivated vaccines are produced using a validated
authorised, be used for production. inactivation process whose effectiveness and consistency have
Consistency of production is an important feature of vaccine been demonstrated. Where it is recognised that extraneous
production. Monographs on vaccines for human use give agents may be present in a harvest, for example in vaccines
limits for various tests carried out during production and on produced in eggs from healthy, non-SPF flocks, the
the final lot. These limits may be in the form of maximum inactivation process is also validated with respect to a panel
values, minimum values, or minimum and maximum of model extraneous agents representative of the potential
tolerances around a given value. While compliance with these extraneous agents. A test for effectiveness of the inactivation
limits is required, it is not necessarily sufficient to ensure process is carried out as soon as possible after the
consistency of production for a given vaccine. For relevant inactivation process.
tests, the manufacturer must therefore define for each Carrier proteins
product a suitable action or release limit or limits to be Bacterial polysaccharide antigens may be conjugated with
applied in view of the results found for batches tested carrier proteins to improve their immunogenicity to enable
clinically and those used to demonstrate consistency of the induction of a protective response in infants. Carrier
production. These limits may subsequently be refined on a proteins comply with the relevant requirements of general
statistical basis in light of production data. chapter 5. 2.11. Carrier proteins for the production of conjugated
Substrates for propagation polysaccharide vaccines for human use.
Substrates for propagation comply with the relevant Test for sterility of intermediates prior to final bulk
requirements of the Pharmacopoeia (5.2.2, 5.2.3) or in the Individual monographs on vaccines for human use may
absence of such requirements with those of the competent prescribe a test for sterility for intermediates.
authority. Processing of cell banks and subsequent cell In agreement with the competent authority, replacement of
cultures is done under aseptic conditions in an area where no the sterility test by a bioburden test with a low bioburden
other cells are being handled. Serum and trypsin used in the limit based on batch data and process validation may be
preparation of cell suspensions shall be shown to be free from acceptable for intermediates preceding the final bulk,
extraneous agents. provided that a sterilising filtration is performed later in the
Seed lots/cell banks production process.
The master seed lot or cell bank is identified by historical It is a prerequisite that the intermediate is filtered through a
records that include information on its origin and subsequent bacteria-retentive filter prior to storage, that authorised pre-
manipulation. Suitable measures are taken to ensure that no filtration bioburden limits have been established for this
extraneous agent or undesirable substance is present in a filtration, and that adequate measures are in place to avoid
master or working seed lot or a cell bank. contamination and growth of micro-organisms during storage
Culture media of the intermediate.
Culture media are as far as possible free from ingredients Final bulk
known to cause toxic, allergic or other undesirable reactions The final bulk is prepared by aseptically blending the
in man; if inclusion of such ingredients is necessary, it shall ingredients of the vaccine. For non-liquid vaccines for
be demonstrated that the amount present in the final lot is administration by a non-parenteral route, the final bulk is
reduced to such a level as to render the product safe. prepared by blending the ingredients of the vaccine under
Approved animal (but not human) serum may be used in the suitable conditions.
growth medium for cell cultures but the medium used for
Adjuvants One or more adjuvants may be included in the
maintaining cell growth during virus multiplication shall not
formulation of a vaccine to potentiate and/or modulate the
contain serum, unless otherwise stated. Cell culture media
immune response to the antigen(s). Adjuvants may be
may contain a pH indicator such as phenol red and approved
included in the formulation of the final vaccine or presented
antibiotics at the lowest effective concentration, although it is
separately. Suitable characterisation and quality control of the
adjuvant(s), alone and in combination with the antigen(s), is Except where they are used within a short period of time,
essential for consistent production. Quality specifications are stability studies are carried out on the intermediates in the
established for each adjuvant, alone and in combination with intended storage conditions to establish the expected extent
the antigen(s). of degradation. For final bulk vaccine, stability studies may
Adsorbents as adjuvants Vaccines may be adsorbed on be carried out on representative samples in conditions
aluminium hydroxide, aluminium phosphate, calcium equivalent to those intended to be used for storage. For each
phosphate or other suitable adsorbents. The adsorbents are intermediate (except for seed lots and cell banks), a shelf life
prepared in special conditions that confer the appropriate applicable for the intended storage conditions is established,
physical form and adsorptive properties. where appropriate in light of stability studies.
Where an adsorbent is used as an adjuvant and is generated Final lot
in situ during production of the vaccine, quality specifications The final lot is prepared by aseptically distributing the final
are established for each of the ingredients and for the bulk into sterile, tamper-evident containers, which, after
generated adsorbent in the vaccine. Quality specifications are freeze-drying where applicable, are closed so as to exclude
intended to control, in particular: contamination. For non-liquid vaccines for administration by
- qualitative and quantitative chemical composition; a non-parenteral route, the final lot is prepared by
- physical form and associated adsorptive properties, where distributing the final bulk under suitable conditions into
relevant, and particularly where the adjuvant will be sterile, tamper-evident containers. Where justified and
present as an adsorbent; authorised, certain tests prescribed for the final lot may be
- interaction between adjuvant and antigen; carried out on the final bulk, if it has been demonstrated that
- purity, including bacterial endotoxin content and subsequent manufacturing operations do not affect
microbiological quality; compliance.
- any other parameters identified as being critical for Appearance
functionality. Unless otherwise justified and authorised, each container
The stability of each adjuvant, alone and in combination with (vial, syringe or ampoule) in each final lot is inspected
the antigen(s), particularly for critical parameters, is visually or mechanically for acceptable appearance.
established during development studies. Degree of adsorption For an adsorbed vaccine, unless
Antimicrobial preservatives Antimicrobial preservatives are otherwise justified and authorised, a release specification for
used to prevent spoilage or adverse effects caused by the degree of adsorption is established in light of results
microbial contamination occurring during the use of a found for batches used in clinical trials. From the stability
vaccine. Antimicrobial preservatives are not included in data generated for the vaccine it must be shown that at the
freeze-dried products. For single-dose liquid preparations, end of the shelf life the degree of adsorption is not less than
inclusion of antimicrobial preservatives is not normally for batches used in clinical trials.
acceptable. For multidose liquid preparations, the need for Thermal stabiliry When the thermal stability test is
effective antimicrobial preservation is evaluated taking into prescribed in a monograph for a live attenuated vaccine, the
account likely contamination during use and the maximum test is carried out on the final lot to monitor the lot-to-lot
recommended shelf life after broaching of the container. If an consistency in heat-sensitivity of viral/bacterial particles in the
antimicrobial preservative is used, it shall be shown that it product. Suitable conditions are indicated in the individual
does not impair the safety or efficacy of the vaccine. Addition monograph. The test may be omitted as a routine test for a
of antibiotics as antimicrobial preservatives is not normally given product once the consistency of the production process
acceptable. has been demonstrated, in agreement with the competent
During development studies, the effectiveness of the authority, using relevant parameters, such as consistency in
antimicrobial preservative throughout the shelf life shall be yield, ratio of infectious viruses (viable bacteria) before and
demonstrated to the satisfaction of the competent 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 general chapter 5.1.3. If neither the A criteria Where there is a significant change in the manufacturing
nor the B criteria can be met, then in justified cases the procedure of the antigen(s) or formulation, the need for
following criteria are applied to vaccines for human use: re-introduction of the test is considered.
bacteria, no increase at 24 h and 7 days, 3 log 10 reduction at Stability During development studies, maintenance of
14 days, no increase at 28 days; fungi, no increase at 14 days potency of the final lot throughout the shelf life shall be
and 28 days. 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 bacterial endotoxins determined by a suitable method
component which expires first. (2. 6.14) is less than the limit approved for the particular
The expiry date applies to vaccines stored in the prescribed product.
conditions. STORAGE
Animal tests Store protected from light. Unless otherwise stated, the
In accordance with the provisions of the European storage temperature is 5 ± 3 °C; liquid adsorbed vaccines
Convention for the Protection of Vertebrate Animals Used must not be allowed to freeze.
for Experimental and Other Scientific Purposes, tests must LABELLING
be carried out in such a way as to use the minimum number
The label states:
of animals and to cause the least pain, suffering, distress or
- the name of the preparation;
lasting harm. The criteria for judging tests in monographs
- a reference identifying the final lot;
must be applied in light of this. For example, if it is indicated
- the recommended human dose and route of
that an animal is considered to be positive, infected, etc.
administration;
when typical clinical signs or death occur, then as soon as
- the storage conditions;
sufficient indication of a positive result is obtained the animal
- the expiry date;
in question shall be either euthanised or given suitable
- the name and amount of any antimicrobial preservative;
treatment to prevent unnecessary suffering. In accordance
- the name of any antibiotic, adjuvant, flavour or stabiliser
with the General Notices, alternative test methods may be
present in the vaccine;
used to demonstrate compliance with the monograph and the
- where applicable, that the vaccine is adsorbed;
use of such tests is particularly encouraged when this leads to
- the name of any constituent that may cause adverse
replacement or reduction of animal use or reduction of
reactions and any contra-indications to the use of the
suffering. Guidance on how to substitute in viva methods by
vaccine;
in vitro methods, in cases where a direct head-to-head
- for freeze-dried vaccines:
comparison is not possible, can be found in general chapter
- the name or composition and the volume of the
5.2.14.
reconstituting liquid to be added;
TESTS - the time within which the vaccine is to be used after
Vaccines comply with the tests prescribed in individual reconstitution.
monographs including, where applicable, the following: _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
pH (2.2.3)
Liquid vaccines, after reconstitution where applicable,
comply with the limits for pH approved for the particular
preparation. Anthrax Vaccine for Human Use
Adjuvant
If the vaccine contains an adjuvant, the amount is
(Adsorbed, Prepared from Culture
determined and shown to be within acceptable limits with Filtrates)
respect to the expected amount (see also the tests for (Ph. Bur. monograph 2188)
aluminium and calcium below).
The label may state 'Anthrax'.
Aluminium (2.5.13)
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Maximum 1.25 mg of aluminium (Al) per single human dose
where an aluminium adsorbent has been used in the vaccine, DEFINITION
unless otherwise stated. Anthrax vaccine for human use (adsorbed, prepared from
Calcium (2.5.14) culture filtrates) is a preparation of Bacillus anthracis antigens
Maximum 1.3 mg of calcium (Ca) per single human dose precipitated by aluminium potassium sulfate. The antigens
where a calcium adsorbent has been used in the vaccine, are prepared from a sterile culture filtrate produced by a
unless otherwise stated. non-encapsulated strain, either avirulent or attenuated, of
Free formaldehyde (2.4.18) B. anthracis.
Maximum 0.2 g/L of free formaldehyde in the final product The main virulence components of B. anthracis are the
where formaldehyde has been used in the preparation of the polyglutamic aicd capsule and 2 binary anthrax toxins,
vaccine, unless otherwise stated. namely lethal toxin and oedema toxin, formed from the
Phenol (2.5.15) respective combination of protective antigen (PA) with either
Maximum 2.5 g/L in the final product where phenol has lethal factor (LF) or oedema factor (EF).
been used in the preparation of the vaccine, unless otherwise LF is a zinc-dependent endopeptidase and EF is a potent
stated. calmodulin and calcium-dependent adenylate cyclase. Cell-
free cultures of B. anthracis contain PA and because
Water (2.5.12)
expression of the 3 toxin-component genes is co-ordinately
Maximum 3.0 per cent mlm 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,
Extractable volume (2. 9.17) including membrane proteins, secreted proteins, cytoplasmic
Unless otherwise justified and authorised, it complies with proteins, peptidoglycans, nucleic acids and carbohydrates.
the requirement for extractable volume.
PRODUCTION
Bacterial endotoxins
GENERAL PROVISIONS
Unless otherwise justified and authorised, a test for bacterial Cultures are managed in a seed-lot system. The vaccine
endotoxins is carried out on the final product. Where no
strain is toxigenic but lacks the plasmid with the necessary
limit is specified in the individual monograph, the content of
genes for synthesis of the capsule, an important virulence Antimicrobial preservative

factor. Determine the amount of antimicrobial preservative by a
The production method must be shown to yield a consistent suitable chemical method. The amount is not less than
and active product with a safety and efficacy profile that is 85 per cent and not greater than 115 per cent of the
adequate or equivalent to previous lots. The vaccine must intended content.
show a level of protection against a virulent strain of FINAL BULK VACCINE
B. anthracis, in a suitable animal infection model, that is The purified harvest is diluted aseptically with sterile saline
equal to or greater than that of a reference vaccine. solution to make the final bulk vaccine.
The vaccine must not show a level of toxicity that exceeds Only a final bulk vaccine that complies with the following
that of a reference vaccine. requirement may be used in the preparation of the final lot.
The production method and stability of the final lot and Sterility (2. 6.1)
relevant intermediates are evaluated using one or more Carry out the test for sterility, using 10 mL for each
indicator tests. Such tests include potency and specific medium.
toxicity, and may be supported by tests confirming the
presence of relevant antigens and associated proteins. Release FINAL LOT
and shelf life specifications are established based upon the The final bulk vaccine is distributed aseptically into sterile,
results of stability testing so as to ensure satisfactory product tamper-evident glass ampoules and heat-sealed to prevent
performance during the approved shelf life. contamination.
Only a final lot that is satisfactory with respect to each of the
SEED LOTS
The attenuated non-encapsulated strain of B. anthracis used requirements given below under Identification, Tests and
is identified by historical records that include information on Assay may be released for use. Provided the potency assay,
its origin and subsequent manipulation and the tests used to the specific toxicity (oedema) test and the test for
characterise the strain. These include morphological, cultural, antimicrobial preservative have been carried out with
biochemical and genetic properties of the strain. Only a satisfactory results on the purified harvest, they may be
master seed lot or, where applicable, working seed lots, that omitted on the final lot.
comply with the following requirements may be used. IDENTIFICATION
Identification The presence of B. anthracis protective antigen is confirmed
Each seed lot is identified as containing B. anthracis. by a suitable immunochemical method (2. 7.1).
Phenotypic parameters TESTS
Each seed lot must have a known biochemical and enzymatic Specific toxicity (oedema) test
profile and have a known history of absence of antibiotic Use not fewer than 2 rabbits per test. Prepare serial two-fold
resistance. dilutions of vaccine with normal saline, corresponding to 4,
Microbial purity 2, 1, 0.5 and 0.25 human doses. Inject intradermally 0.1 mL
Each seed lot complies with the requirements for absence of of each dilution of the test and of the reference vaccine into
contaminating organisms. Purity of bacterial cultures is the shaved flanks of 2 rabbits. Each rabbit receives the 10
verified by methods of suitable sensitivity. previously prepared injections (5 dilutions of the test vaccine
and 5 dilutions of the reference vaccine). In one of the
Virulence test rabbits, the lower concentrations are injected at the anterior
The absence of bacterial capsule is demonstrated for each end and the higher concentrations at the posterior end.
seed lot by McFadyean stain and the specific toxicity The reverse is used for the 2nd rabbit. The rabbits are
(oedema) test. monitored for 24 h for signs of oedema at the injection site.
REFERENCE PREPARATION The vaccine complies with the test if the oedematous
The potency and toxicity of the vaccine bulk are verified reaction is not greater than that observed with the reference
using reference standards derived from representative vaccine vaccine.
batches. These batches are extensively characterised for their Alternatively, specific in vitro assays for lethal factor and
intended purpose and are stored in suitably sized aliquots adenylate cyclase activity may be used, subject to validation.
under conditions ensuring their stability.
PROPAGATION AND HARVEST Determine the amount of antimicrobial preservative by a
The attenuated strain is grown using suitable liquid media. suitable chemical method. The content is not less than the
At the end of cultivation, the purity of the culture is tested. minimum amount shown to be effective and is not greater
The culture medium is separated from the bacterial mass by than 115 per cent of the intended content.
filtration. The pH of the filtrate is determined after dilution
Aluminium (2.5.13)
with a 0.9 g/L solution of sodium chloride Rand is shown to
Maximum 1.25 mg per single human dose.
be within limits suitable for stability. A suitable test for
absence of live B. anthracis, including spores, is carried out. Sterility (2. 6. 1)
Aluminium potassium sulfate or an alternative adjuvant may It complies with the test for sterility.
be added at this stage. An antimicrobial preservative may be ASSAY
added to the suspension to form the purified harvest. The potency of the anthrax vaccine is determined by
Only a purified harvest that complies with the following comparing the dose required to protect guinea-pigs against
requirements may be used in the preparation of the final lot. intradermal challenge by a virulent strain of B. anthracis with
Immunological identity the dose of a suitable reference preparation that gives the
Confirm the presence of B. anthracis protective antigen by a same protection. Use 9 groups of not fewer than 16 female
suitable immunochemical method (2.7.1). guinea-pigs, each weighing 250-350 g. Prepare 4 dilutions of
the vaccine and of the reference preparation containing 1.5,
0.5, 0.17 and 0.05 human doses in 0.5 mL. Allocate each
2023 Vaccines IV- 703
dilution to a separate group. The remaining group receives sensitise man to tuberculin and to protect animals against
0.5 mL of saline and is used to verify the challenge dose. tuberculosis, and its relative absence of pathogenicity for man
Inject subcutaneously into each guinea-pig 0.5 mL of the and laboratory animals. The strain used shall be identified by
dilution allocated to its group on each of 2 occasions, 1 week historical records that include information on its origin and
apart. 7 days after the 2 nd injection, inject intradermally into subsequent manipulation.
each guinea-pig 2000 spores of a virulent strain of B. A suitable batch of vaccine is prepared from the first working
anthracis (Vollum) in 0.1 mL. Observe the animals for seed lot and is reserved for use as the comparison vaccine.
10 days and record the number of deaths per group. The test When a new working seed lot is established, a suitable test
is not valid unless all the control animals die within 5 days of for delayed hypersensitivity in guinea-pigs is carried out on a
challenge. Using the proportions of animals that survive in batch of vaccine prepared from the new working seed lot; the
each of the vaccinated groups, calculate the potency of the vaccine is shown to be not significantly different in activity
vaccine relative to the reference preparation using the usual from the comparison vaccine. Antimicrobial agent sensitivity
statistical methods (5.3). The vaccine complies with the test testing is also carried out.
if: Only a working seed lot that complies with the following
- the relative potency estimate exceeds 1.0, or; requirements may be used for propagation.
- the 95 per cent confidence interval for the relative potency
includes 1.0, and the lower 95 per cent confidence limit is Identification
not less than 50 per cent of the relative potency estimate. The bacteria in the working seed lot are identified as
Mycobacterium bovis BCG using microbiological techniques,
LABELLING which may be supplemented by molecular biology techniques
The label states that the vaccine is not to be frozen. (for example, nucleic acid amplification and restriction-
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur fragment-length polymorphism).
Bacterial and fungal contamination
Carry out the test for sterility (2. 6.1), using 10 mL for each
medium. The working seed lot complies with the test for
Bacillus Calmette-Guerin Vaccine sterility except for the presence of mycobacteria.
Virulent mycobacteria
BCG Vaccine
Examine the working seed lot as prescribed under Tests,
(BCG Vaccine, Freeze-dried, Ph. Bur. monograph 0163) using 10 guinea-pigs.
The label may state 'BCG'. PROPAGATION AND HARVEST
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ The bacteria are grown in a suitable medium for not more
DEFINITION than 21 days by surface or submerged culture. The culture
medium does not contain substances known to cause toxic or
Freeze-dried BCG vaccine is a preparation of live bacteria
allergic reactions in humans or to cause the bacteria to
derived from a culture of the bacillus of Calmette and Guerin
become virulent for guinea-pigs. The culture is harvested and
(Mycobacterium bovis BCG) whose capacity to protect against
suspended in a sterile liquid medium that protects the
tuberculosis has been established.
viability of the vaccine as determined by a suitable method of
PRODUCTION viable count.
GENERAL PROVISIONS FINAL BULK VACCINE
BCG vaccine shall be produced by a staff consisting of The final bulk vaccine is prepared from a single harvest or by
healthy persons who do not work with other infectious pooling a number of single harvests. A stabiliser may be
agents; in particular they shall not work with virulent strains added; if the stabiliser interferes with the determination of
of Mycobacterium tuberculosis, nor shall they be exposed to a bacterial concentration in the final bulk vaccine, the
known risk of tuberculosis infection. Staff are 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. 6.1), using 10 mL for each
Production of the vaccine is based on a seed-lot system. medium. The final bulk vaccine complies with the test for
The production method shall have been shown to yield 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 the total bacterial concentration by a suitable
used instead of viable count, the method is validated against method, either directly 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 SEED LOTS bacterial concentration is determined before addition of a
The strain used to establish the master seed lot is chosen for stabiliser, the concentration in the final bulk vaccine is
and maintained to preserve its characteristics, its capacity to
established by calculation. The total bacterial concentration is Bacterial and fungal contamination
within the limits approved for the particular product. The reconstituted vaccine complies with the test for sterility
The ratio of the count of viable units to the total bacterial (2. 6.1) except for the presence of mycobacteria.
concentration is not less than that approved for the particular Excessive dermal reactivity
product. Use 6 healthy, white or pale-coloured guinea-pigs, each
FINAL LOT weighing not less than 250 g and having received no
The final bulk vaccine is distributed into sterile containers treatment likely to interfere with the test. Inject intradermally
and freeze-dried to a moisture content favourable to the into each guinea-pig, according to a randomised plan,
stability of the vaccine; the containers are closed either under 0 .1 mL of the reconstituted vaccine and of 2 tenfold serial
vacuum or under an inert gas. dilutions of the vaccine and identical doses of the comparison
Except where the filled and closed containers are stored at a vaccine. Observe the lesions formed at the site of the
temperature of -20 °C or lower, the expiry date is not later injection for 4 weeks. The vaccine complies with the test if
than 4 years from the date of harvest. the reaction it produces is not markedly different from that
produced by the comparison vaccine.
Only a final lot that complies with the following requirement
for count of viable units and with each of the requirements Water
given below under Identification, Tests and Assay may be Not more than the limit approved for the particular product,
released for use. Provided the test for virulent mycobacteria determined by a suitable method.
has been carried out with satisfactory results on the final bulk ASSAY
vaccine, it may be omitted on the final lot. Provided the test Determine the number of viable units in the reconstituted
for excessive dermal reactivity has been carried out with vaccine by viable count on solid medium using a method
satisfactory results on the working seed lot and on suitable for the vaccine to be examined or by a suitable
5 consecutive final lots produced from it, the test may be validated biochemical method. The number is within the
omitted on the final lot. range stated on the label. Determine the number of
Count of viable units viable units in the comparison vaccine in parallel.
Determine the number of viable units per millilitre of the LABELLING
reconstituted vaccine by viable count on solid medium using The label states:
a method suitable for the vaccine to be examined or by a - the minimum and maximum number of viable units per
suitable biochemical method. The ratio of the count of millilitre in the reconstituted vaccine,
viable units after freeze-drying to that before is not less than - that the vaccine must be protected from direct sunlight.
that approved for the particular product.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Thermal stability
Maintain containers of the final lot of freeze-dried vaccine in
the dry state at 37 ± 1 "C for 4 weeks. Determine the
number of viable units as described under Assay in parallel
for the heated vaccine and for vaccine stored at the BCG for lmmunotherapy
temperature recommended for storage. The number of (Ph. Eur. monograph 1929)
viable units in the heated vaccine is not less than 20 per cent
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
of that in the unheated vaccine.
IDENTIFICATION DEFINITION
BCG vaccine is identified by microscopic examination of the BCG for immunotherapy is a freeze-dried preparation of live
bacilli in stained smears demonstrating their acid-fast bacteria derived from a culture of the bacillus of Calmette
property and by the characteristic appearance of colonies and Guerin (Mycobacterium bovis BCG) whose capacity for
grown on solid medium. Alternatively, molecular biology treatment has been established.
techniques (for example nucleic acid amplification) may be It complies with the monograph Vaccines for human
used. use (0153).
TESTS PRODUCTION
Virulent mycobacteria GENERAL PROVISIONS
Inject subcutaneously or intramuscularly into each of BCG for immunotherapy shall be produced by a staff
6 guinea-pigs, each weighing 250-400 g and having received consisting of healthy persons who do not work with other
no treatment likely to interfere with the test, a quantity of infectious agents; in particular they shall not work with
vaccine equivalent to at least 50 human doses. Observe the virulent strains of Mycobacterium tuberculosis, nor shall they be
animals for at least 42 days. At the end of this period, exposed to a known risk of tuberculosis infection. Staff are
euthanise the guinea-pigs and examine by autopsy for signs examined periodically for tuberculosis. BCG for
of infection with tuberculosis, ignoring any minor reactions at immunotherapy is susceptible to sunlight: the procedures for
the site of injection. Animals that die during the observation production shall be so designed that all products are
period are also examined for signs of tuberculosis. protected from direct sunlight and from ultraviolet light at all
The vaccine complies with the test if none of the guinea-pigs stages of manufacture, testing and storage.
shows signs of tuberculosis and if not more than 1 animal Production is based on a seed-lot system. The production
dies during the observation period. If 2 animals die during method shall have been shown to yield consistently BCG
this period and autopsy does not reveal signs of tuberculosis products that can be used for treatment of superficial bladder
repeat the test on 6 other guinea-pigs. The vaccine complies cancer and are safe. The product is prepared from cultures
with the test if not more than 1 animal dies during the which are separated from the master seed lot by as few
42 days following the injection and autopsy does not reveal subcultures as possible and in any case not more than
any sign of tuberculosis.
8 subcultures. During the course of these subcultures the Carry out the test in parallel on a reference preparation of
preparation is not freeze-dried more than once. the same strain.
If a bioluminescence test or other biochemical method is Bacterial concentration
used instead of viable count, the method is validated against Determine the total bacterial concentration by a suitable
the viable count for each stage of the process at which it is method, either directly by determining the mass of the micro-
used. organisms, or indirectly by an opacity method that has been
SEED LOTS calibrated in relation to the mass of the micro-organisms;
The strain used to establish the master seed lot is chosen for if the bacterial concentration is determined before addition of
and maintained to preserve its characteristics, its capacity to a stabiliser, the concentration in the final bulk is established
treat and prevent superficial bladder cancer, and its relative by calculation. The total bacterial concentration is within the
absence of pathogenicity for man and laboratory animals. limits approved for the particular product.
The strain used shall be identified by historical records that The ratio of the count of viable units to the total bacterial
include information on its origin and subsequent concentration is not less than that approved for the particular
manipulation. product.
Before establishment of a working seed lot a batch is FINAL LOT
prepared and reserved for use as the comparison product. The final bulk is distributed into sterile containers and freeze-
When a new working seed lot is established, a suitable test dried to a moisture content favourable to the stability of the
for delayed hypersensitivity in guinea-pigs is carried out on a product; the containers are closed either under vacuum or
batch of product prepared from the new working seed lot; under an inert gas.
the product is shown to be not significantly different in Except where the filled and closed containers are stored at a
activity from the comparison product. Antimicrobial agent temperature of -20 °C or lower, the expiry date is not later
sensitivity testing is also carried out. than 4 years from the date of harvest.
Only a working seed lot that complies with the following Only a final lot that complies with the following requirement
requirements may be used for propagation. for count of viable units and with each of the requirements
Identification given below under Identification, Tests and Assay may be
The bacteria in the working seed lot are identified as released for use. Provided the test for virulent mycobacteria
Mycobacterium bovis BCG using microbiological techniques, has been carried out with satisfactory results on the final
which may be supplemented by molecular biology techniques bulk, it may be omitted on the final lot.
(for example, nucleic acid amplification and restriction- Count of viable units
fragment-length polymorphism). Determine the number of viable units per millilitre of the
Bacterial and fungal contamination reconstituted product by viable count on solid medium using
Carry out the test for sterility (2. 6.1), using 10 mL for each a method suitable for the product to be examined, or by a
medium. The working seed lot complies with the test for suitable biochemical method. The ratio of the count of
sterility, except for the presence of mycobacteria. viable units after freeze-drying to that before is not less than
Virulent mycobacteria that approved for the particular product.
Examine the working seed lot as prescribed under Tests, IDENTIFICATION
using 10 guinea-pigs. BCG for immunotherapy is identified by microscopic
PROPAGATION AND HARVEST examination of the bacilli in stained smears demonstrating
The bacteria are grown in a suitable medium for not more their acid-fast property and by the characteristic appearance
than 21 days by surface or submerged culture. The culture of colonies grown on solid medium. Alternatively, molecular
medium does not contain substances known to cause toxic or biology techniques (for example, nucleic acid amplification)
allergic reactions in human beings or to cause the bacteria to may be used.
become virulent for guinea-pigs. The culture is harvested and TESTS
suspended in a sterile liquid medium that protects the Virulent mycobacteria
viability of the culture as determined by a suitable method of Inject subcutaneously or intramuscularly into each of
viable count. 6 guinea-pigs, each weighing 250-400 g and having received
FINAL BULK no treatment likely to interfere with the test, a quantity of the
The final bulk is prepared from a single harvest or by pooling product to be examined equivalent to at least 1/25 of
a number of single harvests. A stabiliser may be added; if the I human dose. Observe the animals for at least 42 days.
stabiliser interferes with the determination of bacterial At the end of this period, euthanise the guinea-pigs and
concentration on the final bulk, the determination is carried examine by autopsy for signs of infection with tuberculosis,
out before addition of the stabiliser. ignoring any minor reactions at the site of injection. Animals
Only final bulk that complies with the following requirements that die during the observation period are also examined for
may be used in the preparation of the final lot. signs of tuberculosis. The product complies with the test if
none of the guinea-pigs shows signs of tuberculosis and if not
more than 1 animal dies during the observation period.
Carry out the test for sterility (2. 6.1), using 10 mL of final
If 2 animals die during this period and autopsy does not
bulk for each medium. The final bulk complies with the test
reveal signs of tuberculosis, repeat the test on 6 other guinea-
for sterility, except for the presence of mycobacteria.
pigs. The product complies with the test if not more than
Count of viable units I animal dies during the 42 days following the injection and
Determine the number of viable units per millilitre by viable autopsy does not reveal any sign of tuberculosis.
count on solid medium using a method suitable for the
product to be examined or by a suitable biochemical method. Bacterial and fungal contamination
The reconstituted product complies with the test for sterility
(2. 6.1) except for the presence of mycobacteria.
Water plasmid in the recombinant strain during storage and beyond

Not more than the limit approved for the particular product, the passage level used in production is confirmed.
determined by a suitable method. Characterisation of the rCTB is undertaken using a variety of
ASSAY analytical techniques including determination of molecular
Determine the number of viable units in the reconstituted size, charge and amino acid composition. Techniques
product by viable count on solid medium using a method suitable for such purposes include sodium dodecyl sulfate
suitable for the product to be examined or by a suitable polyacrylamide gel electrophoresis (SDS-PAGE) and
validated biochemical method. The number is within the different liquid chromatographies. The identity of the
range stated on the label. Determine the number of product is confirmed by at least partial N-terminal and
viable units in the comparison control in parallel. C-terminal amino acid sequencing.
Master seed lots are grown on agar plates, which may
LABELLING
contain appropriate antibiotics. Colonies are used to produce
The label states:
working seed lots in liquid media that are free from
- the minimum and the maximum number of viable units
antibiotics. Cultures derived from the working seed lot must
per dose in the reconstituted product;
have the same characteristics as the cultures of the strain
- that the product must be protected from direct sunlight.
from which the master seed lot was derived.
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
Only a seed lot that complies with the following requirements
may be used in the preparation of the monovalent cell
harvest.
Identification
Cholera Vaccine (Inactivated, Oral) Master seed lots are identified by colony morphology, and by
(Ph. Bur. monograph 2327) biochemical characterisation, using suitable molecular assays
or immunoassays. Working seed lots are identified by colony
The label may state 'Cholera (oral)'. morphology and by molecular assays or immunoassays.
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
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 O 1, 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 Cultures are checked at different stages of fermentation
cholera toxin (CTB). Just prior to ingestion, one dose of (subcultures and main culture) for purity, identity, cell
vaccine suspension is mixed with a suitable buffer as stated opacity, pH and biochemical characteristics. Unsatisfactory
on the label. 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 MONO VALENT CELL HARVEST
consistently vaccines comparable with the vaccine of proven Only a monovalent harvest that complies with established
clinical efficacy and safety in man. specifications for the following tests may be used.
The production process must be validated to show that no pH (2.2.3)
clinically significant quantities of active toxin are present in Within the range approved for the particular product.
the product.
Identification
CHOICE OF VACCINE STRAIN Relevant antigenic characteristics are verified by suitable
The vaccine consists of a mixture of epidemic V. cholerae immunological or biochemical assays.
strains inactivated by a suitable method such as heat or
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 (ctxA-). 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 MONO VALENT 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 pH (2.2.3)
subunit B (ctxB) are documented. The stability of the rCTB Within the range approved for the particular product.
Identification FINAL LOT

Verified by slide agglutination. The final bulk is mixed to homogeneity and filled aseptically
Inactivation into suitable containers.
Complete inactivation is verified by a suitable culture Only a final lot that is within the limits approved for the
method. particular product and is satisfactory with respect to each of
Sterility (2. 6.1) the requirements given below under Identification, Tests and
It complies with the test for sterility, carried out using 10 mL Assay may be released for use.
for each medium. IDENTIFICATION
Opacity Serotypes are detected by a suitable immunoassay (2. 7.1) or
The inactivation process may affect the accuracy of opacity molecular assay. rCTB is detected by a suitable immunoassay
measurements. (2. 7. J). The antigen-content assays may also serve as an
identity test.
Purity
Samples of culture are examined by microscopy of Gram- TESTS
stained smears, by inoculation of appropriate culture media pH (2.2.3)
or by another suitable procedure. Within the range approved for the particular product.
Smooth LPS content Sterility (2. 6.1)
Verified by a suitable immunoassay (2. 7.1). It complies with the test for sterility.
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. 7.1) 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 the 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 DNA 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) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
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). Adsorbed Diphtheria Vaccine
Sterility (2. 6. 1)
It complies with the test for sterility, carried out using 10 mL (Diphtheria Vaccine (Adsorbed), Ph. Bur. monograph
for each medium. 0443)
rCTB PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
The amount of rCTB is determined by a suitable
DEFINITION
immunoassay (2. 7.1).
Diphtheria vaccine (adsorbed) is a preparation of diphtheria
FINAL BULK formol toxoid with a mineral adsorbent. The formol toxoid is
The final bulk vaccine is prepared by aseptically mixing a prepared from the toxin produced by the growth of
suitable buffer with monovalent cell bulks. Where used, the Co,ynebacterium diphtheriae.
rCTB bulk is added in appropriate amounts. Preservatives, if
used, may be added at this stage. PRODUCTION
BULK PURIFIED TOXOID
Only a final bulk that complies with the following
For the production of diphtheria toxin, from which toxoid is
prepared, seed cultures are managed in a defined seed-lot
Sterility (2. 6. 1) system in which toxinogenicity is conserved and, where
It complies with the test for sterility, carried out using 10 mL necessary, restored by deliberate reselection. A highly
for each medium. toxinogenic strain of Corynebacterium diphtheriae with known
Antimicrobial preservative origin and history is grown in a suitable liquid medium.
Where applicable, determine the amount of antimicrobial At the end of cultivation, the purity of each culture is tested
preservative by a suitable chemical or physico-chemical and contaminated cultures are discarded. Toxin-containing
method. The amount is not less than 85 per cent and not culture medium is separated aseptically from the bacterial
greater than 115 per cent of the intended amount. mass as soon as possible. The toxin content (U per millilitre)
is checked (2. 7.27) to monitor consistency of production. type, adversely affect the antigenic activity and must not be
Single harvests may be pooled to prepare the bulk purified used.
toxoid. The toxin is purified to remove components likely to Only a final bulk vaccine that complies with the following
cause adverse reactions in humans. The purified toxin is requirements may be used in the preparation of the final lot.
detoxified with formaldehyde by a method that avoids
destruction of the immunogenic potency of the toxoid and
Where applicable, determine the amount of antimicrobial
reversion of the toxoid to toxin, particularly on exposure to
preservative by a suitable chemical method. The amount is
heat. Alternatively, purification may be carried out after
not less than 85 per cent and not greater than 115 per cent
detoxification.
of the intended amount.
Only bulk purified toxoid that complies with the following
requirements may be used in the preparation of the final bulk Sterility (2. 6.1)
Carry out the test for sterility using 10 mL for each medium.
vaccine.
Sterility (2. 6. 1) FINAL LOT
Carry out the test for sterility using 10 mL for each medium. The final bulk vaccine is distributed aseptically into sterile,
tamper-evident containers. The containers are closed so as to
Absence of toxin and irreversibility of toxoid prevent contamination.
Using the same buffer solution as for the final vaccine,
without adsorbent, prepare a solution of bulk purified toxoid
requirements given below under Identification, Tests and
at 100 LflmL. Divide the solution into 2 equal parts.
Assay may be released for use. Provided the test for
Maintain 1 part at 5 ± 3 °C and the other at 37 °C for
antimicrobial preservative and the assay have been carried
6 weeks. Carry out a test in Vero cells for active diphtheria
out with satisfactory results on the final bulk vaccine, they
toxin using 50 µUwell of both samples. The sample should
may be omitted on the final lot.
not contain antimicrobial preservatives and detoxifying agents
should be determined to be below the concentration toxic to Provided the free formaldehyde content has been determined
Vero 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
0.2 g/L, the test for free formaldehyde may be omitted on the
Use freshly trypsinised Vero cells at a suitable concentration,
final lot.
for example 2.5 x 10 5 mL- 1 and a reference diphtheria toxin
diluted in 100 LflmL diphtheria toxoid. A suitable reference IDENTIFICATION
diphtheria toxin will contain either not less than Diphtheria toxoid is identified by a suitable immunochemical
100 LD 50/mL or 67 to 133 lr/100 in 1 Lf and 25 000 to method (2. 7. 1). 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 ERP 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 LflmL diphtheria toxoid to a solution. Maintain at 37 °C for about 16 hand centrifuge
suitable concentration, for example 2 x 10-4 LflmL. Prepare until a clear supernatant is obtained. The clear supernatant
serial twofold dilutions of the diluted diphtheria toxin and reacts with a suitable diphtheria antitoxin, giving a
use undiluted test samples (50 µUwell). Distribute them in precipitate.
the wells of a sterile tissue culture plate containing a medium TESTS
suitable for Vero cells. To ascertain that any cytotoxic effect
Aluminium (2.5.13)
noted is specific to diphtheria toxin, prepare in parallel
Maximum 1.25 mg per single human dose, if aluminium
dilutions where the toxin is neutralised by a suitable
hydroxide or hydrated aluminium phosphate is used as the
concentration of diphtheria antitoxin, for example
absorbent.
100 IU/mL. Include control wells without toxoid or toxin
and with non-toxic toxoid at 100 LflmL 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 Antimicrobial preservative
effect is judged to be present where there is complete Where applicable, determine the amount of antimicrobial
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 LflmL of reference label.
diphtheria toxin in 100 LflmL toxoid has no cytotoxic effect Sterility (2. 6.1)
on Vero cells or if the cytotoxic effect of this amount of toxin The vaccine complies with the test for sterility.
is not neutralised in the wells containing diphtheria antitoxin.
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
diphtheria vaccine (adsorbed) (2. 7. 6).
Antigenic purity (2.7.27)
Not less than 1500 Lf per milligram of protein nitrogen. The lower confidence limit (P = 0. 9 5) of the estimated
potency is not less than 30 IU per single human dose.
FINAL BULK VACCINE
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 those of the phenolic vaccination of children and is not necessarily suitable for
reinforcing doses or for administration to adults;
- the name and the amount of the adsorbent; IDENTIFICATION

- that the vaccine must be shaken before use; Diphtheria toxoid is identified by a suitable immunochemical
- that the vaccine is not to be frozen. method (2. 7.1). The following method, applicable to certain
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur vaccines, is given as an example. Dissolve in the vaccine to
be examined sufficient sodium citrate R to give a 100 g/L
solution. Maintain at 37 °C for about 16 hand centrifuge
until a clear supernatant is obtained. The clear supernatant
reacts with a suitable diphtheria antitoxin, giving a
Diphtheria Vaccine (Adsorbed, precipitate. If a satisfactory result is not obtained with a
Reduced Antigen Content) vaccine adsorbed on aluminium hydroxide, carry out the test
as follows. Centrifuge 15 mL of the vaccine to be examined
Adsorbed Diphtheria Vaccine for Adults and
and suspend the residue in 5 mL of a freshly prepared
Adolescents
mixture of 1 volume of a 56 g/L solution of sodium edetate R
(Ph. Bur. monograph 0646) and 49 volumes of disodium hydrogen phosphate solution R.
For a vaccine for use in the United Kingdom, the amount of Maintain at 37 °C for not less than 6 h and centrifuge.
toxoid used is adjusted so that the final vaccine contains not The clear supernatant reacts with a suitable diphtheria
more than 2.0 flocculation equivalents (2.0 Lt) per dose. antitoxin, giving a precipitate.
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
TESTS
DEFINITION Aluminium (2.5.13)
Diphtheria vaccine (adsorbed, reduced antigen content) is a Maximum 1.25 mg per single human dose, if aluminium
preparation of diphtheria formol toxoid with a mineral hydroxide or hydrated aluminium phosphate is used as the
adsorbent. The formol toxoid is prepared from the toxin adsorbent.
produced by the growth of Corynebacterium diphtheriae. Free formaldehyde (2.4.18)
The amount of diphtheria toxoid per single human dose is Maximum 0.2 g/L.
reduced compared to vaccines generally used for primary
vaccination.
PRODUCTION preservative by a suitable chemical method. The content is
BULK PURIFIED TOXOID not less than the minimum amount shown to be effective and
The bulk purified toxoid is prepared as described in the is not greater than 115 per cent of the quantity stated on the
monograph Diphtheria vaccine (adsorbed) (0443) and complies label.
with the requirements prescribed therein. Sterility (2. 6.1)
FINAL BULK VACCINE The vaccine complies with the test for sterility.
The final bulk vaccine is prepared by adsorption of a suitable
ASSAY
quantity of bulk purified toxoid onto a mineral carrier such
Carry out one of the prescribed methods for the assay of
as hydrated aluminium phosphate or aluminium hydroxide;
diphtheria vaccine (adsorbed) (2.7.6).
the resulting mixture is approximately isotonic with blood.
Suitable antimicrobial preservatives may be added. Certain The lower confidence limit (P = 0.95) of the estimated
antimicrobial preservatives, particularly those of the phenolic potency is not less than 2 IU per single human dose.
type, adversely affect the antigenic activity and must not be LABELLING
used. The label states:
Only a final bulk vaccine that complies with the following - the minimum number of International Units per single
requirements may be used in the preparation of the final lot. human dose;
Antimicrobial preservative - the name and the amount of the adsorbent;
Where applicable, determine the amount of antimicrobial - that the vaccine must be shaken before use;
preservative by a suitable chemical method. The amount is - that the vaccine is not to be frozen.
not less than 85 per cent and not greater than 115 per cent _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
of the intended amount.

Sterility (2. 6.1)
FINAL LOT Adsorbed Diphtheria and Tetanus
The final bulk vaccine is distributed aseptically into sterile, Vaccine
(Diphtheria and Tetanus Vaccine (Adsorbed),
prevent contamination.
requirements given below under Identification, Tests and The label may state 'DT'.
Assay may be released for use. Provided the test for PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
Diphtheria and tetanus vaccine (adsorbed) is a preparation of
may be omitted on the final lot.
diphtheria formol toxoid and tetanus formol toxoid with a
Provided the free formaldehyde content has been determined mineral adsorbent. The formol toxoids are prepared from the
on the bulk purified toxoid or on the final bulk and it has toxins produced by the growth of Corynebacterium diphtheriae
been shown that the content in the final lot will not exceed and Clostridium tetam~ respectively.
final lot.
PRODUCTION Antimicrobial preservative

BULK PURIFIED DIPHTHERIA AND TETANUS Where applicable, determine the amount of antimicrobial
TOXOIDS preservative by a suitable chemical method. The content is
The bulk purified diphtheria and tetanus toxoids are not less than the minimum amount shown to be effective and
prepared as described in the monographs Diphtheria vaccine is not greater than 115 per cent of the quantity stated on the
(adsorbed) (0443) and Tetanus vaccine (adsorbed) (0452) and label.
comply with the requirements prescribed therein. Sterility (2. 6.1)
FINAL BULK VACCINE The vaccine complies with the test for sterility.
The final bulk vaccine is prepared by adsorption of suitable ASSAY
quantities of bulk purified diphtheria toxoid and tetanus
Diphtheria component
toxoid onto a mineral carrier such as hydrated aluminium
phosphate or aluminium hydroxide; the resulting mixture is
diphtheria vaccine (adsorbed) (2. 7. 6).
approximately isotonic with blood. Suitable antimicrobial
preservatives may be added. Certain antimicrobial The lower confidence lin1it (P = 0.95) of the estimated
preservatives, particularly those of the phenolic type, potency is not less than 30 IU per single human dose.
adversely affect the antigenic activity and must not be used. Tetanus 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. tetanus vaccine (adsorbed) (2. 7. 8).
Antimicrobial preservative The lower confidence limit (P = 0. 95) of the estimated
Where applicable, determine the amount of antimicrobial potency is not less than 40 IU per single human dose.
preservative by a suitable chemical method. The amount is LABELLING
not less than 85 per cent and not greater than 115 per cent The label states:
of the intended amount. - the minimum number of International Units of each
Sterility (2. 6.1) component per single human dose;
Carry out the test for sterility using 10 mL for each medium. - the name and the amount of the adsorbent;
- where applicable, that the vaccine is intended for primary
FINAL LOT
vaccination of children and is not necessarily suitable for
The final bulk vaccine is distributed aseptically into sterile,
- that the vaccine must be shaken before use;
- that the vaccine is not to be frozen.
- - - - - - - - - - - - - - - - - - - PhEur
Assay may be released for use. Provided the test for
may be omitted on the final lot. Diphtheria and Tetanus Vaccine
Provided the free formaldehyde content has been determined (Adsorbed, Reduced Antigen(s)
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 Content)
0.2 g/L, the test for free formaldehyde may be omitted on the Adsorbed Diphtheria and Tetanus Vaccine for
final lot. Adults and Adolescents
IDENTIFICATION (Ph. Eur. monograph 064 7)
A. Diphtheria toxoid is identified by a suitable The label may state 'dT'.
immunochemical method (2.7.1). The following method, For a vaccine for use in the United Kingdom, the amount of
applicable to certain vaccines, is given as an example. diphtheria toxoid used is adjusted so that the final vaccine
Dissolve in the vaccine to be examined sufficient sodium contains not more than 2.0 flocculation equivalents (2.0 Lf)
citrate R to give a 100 g/L solution. Maintain at 37 °C for of diphtheria toxoid per dose.
about 16 h and centrifuge until a clear supernatant is Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
obtained. The clear supernatant reacts with a suitable
diphtheria antitoxin, giving a precipitate. DEFINITION
B. Tetanus toxoid is identified by a suitable immunochemical Diphtheria and tetanus vaccine (adsorbed, reduced antigen(s)
method (2. 7.1). The following method, applicable to certain content) is a preparation of diphtheria formol toxoid and
vaccines, is given as an example. The clear supernatant tetanus formol toxoid with a mineral adsorbent. The formol
obtained as described in identification test A reacts with a toxoids are prepared from the toxins produced by the growth
suitable tetanus antitoxin, giving a precipitate. of Corynebacterium diphtheriae and Clostridium tetani,
respectively. The amount of diphtheria toxoid per single
TESTS human dose is reduced compared to vaccines generally used
Aluminium (2.5.13) for primary vaccination; the amount of tetanus toxoid may
Maximum 1.25 mg per single human dose, if aluminium also be reduced.
adsorbent. PRODUCTION
BULK PURIFIED DIPHTHERIA TOXOID AND
Free formaldehyde (2.4.18)
TETANUS TOXOIDS
Maximum 0.2 g/L.
The bulk purified diphtheria and tetanus toxoids are
prepared as described in the monographs Diphtheria vaccine
(adsorbed) (0443) and Tetanus vcu:cine (adsorbed) (0452) and Free formaldehyde (2.4.18)
comply with the requirements prescribed therein. Maximum 0.2 g/L.
FINAL BULK VACCINE Antimicrobial preservative
The vaccine is prepared by adsorption of suitable quantities Where applicable, determine the amount of antimicrobial
of bulk purified diphtheria toxoid and tetanus toxoid onto a preservative by a suitable chemical method. The content is
mineral carrier such as hydrated aluminium phosphate or not less than the minimum amount shown to be effective and
aluminium hydroxide; the resulting mixture is approximately is not greater than 115 per cent of the quantity stated on the
isotonic with blood. Suitable antimicrobial preservatives may label.
be added. Certain antimicrobial preservatives, particularly Sterility (2. 6.1)
those of the phenolic type, adversely affect the antigenic The vaccine complies with the test for sterility.
activity and must not be used.
ASSAY
Only a final bulk vaccine that complies with the following
Antimicrobial preservative diphtheria vaccine (adsorbed) (2.7.6).
The lower confidence limit (P = 0.95) of the estimated
of the intended amount. Tetanus component
Sterility (2. 6.1)
tetanus vaccine (adsorbed) (2. 7. 8).
FINAL LOT potency is not less than 20 IU per single human dose.
tamper-evident containers. The containers are closed so as to LABELLING
prevent contamination. The label states:
Only a final lot that is satisfactory with respect to each of the - the minimum number of International Units of each
requirements given below under Identification, Tests and component per single human dose;
Assay may be released for use. Provided the test for - the name and the amount of the adsorbent;
antimicrobial preservative and the assay have been carried - that the vaccine must be shaken before use;
out with satisfactory results on the final bulk vaccine, they - that the vaccine is not to be frozen.
may be omitted on the final lot. - - - - - - - - - - - - - - - - - - - - - - Ph Eur
Provided the free formaldehyde content has been determined
on the bulk purified toxoids 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 omitted on the Diphtheria, Tetanus and
final lot.
Hepatitis B (rDNA) Vaccine
IDENTIFICATION
A. Diphtheria toxoid is identified by a suitable
(Adsorbed)
immunochemical method (2.7.1). The following method, (Ph. Eur. monograph 2062)
applicable to certain vaccines, is given as an example. Label may state 'DT/HepB'.
Dissolve in the vaccine to be examined sufficient sodium
citrate R to give a 100 g/L solution. Maintain at 37 °C for ~&---------------------~
about 16 hand centrifuge until a clear supernatant is DEFINITION
obtained. The clear supernatant reacts with a suitable Diphtheria, tetanus and hepatitis B (rDNA) vaccine
diphtheria antitoxin, giving a precipitate. If a satisfactory (adsorbed) is a combined vaccine composed of: diphtheria
result is not obtained with a vaccine adsorbed on aluminium formol toxoid; tetanus formol toxoid; hepatitis B surface
hydroxide, carry out the test as follows. Centrifuge 15 mL of antigen (HBsAg); a mineral adsorbent such as aluminium
the vaccine to be examined and suspend the residue in 5 mL hydroxide or hydrated aluminium phosphate.
of a freshly prepared mixture of 1 volume of a 56 g/L The formol toxoids are prepared from the toxins produced
solution of sodium edetate R and 49 volumes of disodium by the growth of Corynebacterium diphtheriae and Clostridium
hydrogen phosphate solution R. Maintain at 37 °C for not less tetani, respectively.
than 6 h and centrifuge. The clear supernatant reacts with a
HBsAg is a component protein of hepatitis B virus; the
suitable diphtheria antitoxin, giving a precipitate.
antigen is obtained by recombinant DNA technology.
B. Tetanus toxoid is identified by a suitable immunochemical
method (2. 7.1). The following method, applicable to certain PRODUCTION
vaccines, is given as an example. The clear supernatant GENERAL PROVISIONS
obtained during identification test A reacts with a suitable The production method shall have been shown to yield
tetanus antitoxin, giving a precipitate. consistently vaccines comparable with the vaccine of proven
clinical efficacy and safety in man.
TESTS
The content of bacterial endotoxins (2. 6.14) in the bulk
Aluminium (2.5.13)
purified diphtheria toxoid and tetanus toxoid is determined
to monitor the purification procedure and to limit the
amount in the final vaccine. For each component, the
adsorbent.
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 obtained. The clear supernatant reacts with a suitable
single human dose. diphtheria antitoxin, giving a precipitate.
Reference vaccine(s) Provided valid assays can be performed, B. Tetanus toxoid is identified by a suitable immunochemical
monocomponent reference vaccines may be used for the method (2. 7.1). The following method, applicable to certain
assays on the combined vaccine. If this is not possible vaccines, is given as an example. The clear supernatant
because of interaction between the components of the obtained during identification test A reacts with a suitable
combined vaccine or because of the difference in composition tetanus antitoxin, giving a precipitate.
between monocomponent reference vaccine and the test C. The assay or, where applicable, the electrophoretic profile,
vaccine, a batch of combined vaccine shown to be effective in serves also to identify the hepatitis B component of the
clinical trials or a batch representative thereof is used as a vaccme.
reference vaccine. For the preparation of a representative
batch, strict adherence to the production process used for the TESTS
batch tested in clinical trials is necessary. The reference Aluminium (2. 5.13)
vaccine may be stabilised by a method that has been shown Maximum 1.25 mg per single human dose, if aluminium
to have no effect on the assay procedure. hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
PRODUCTION OF THE COMPONENTS
The production of the components complies with the Free formaldehyde (2. 4.18)
requirements of the monographs Diphtheria vaccine Maximum 0.2 g/L.
(adsorbed) (0443), Tetanus vaccine (adsorbed) (0452) and Antimicrobial preservative
Hepatitis B vaccine (rDNA) (1056). Where applicable, determine the amount of antimicrobial
FINAL BULK VACCINE preservative by a suitable chemical method. The content is
The final bulk vaccine is prepared by adsorption, separately not less than the minimum amount shown to be effective and
or together, of suitable quantities of bulk purified diphtheria is not greater than 115 per cent of the quantity stated on the
toxoid, tetanus toxoid and HBsAg onto a mineral carrier label.
such as aluminium hydroxide or hydrated aluminium Sterility (2. 6.1)
phosphate. Suitable antimicrobial preservatives may be The vaccine complies with the test for sterility.
added. Pyrogens (2. 6. 8)
Only a final bulk vaccine that complies with the following It complies with the test for pyrogens. Inject the equivalent of
requirements may be used in the preparation of the final lot. 1 human dose into each rabbit.
Antimicrobial preservative ASSAY
Where applicable, determine the amount of antimicrobial Diphtheria component
preservative by a suitable chemical method. The amount is Carry out one of the prescribed methods for the assay of
not less than 85 per cent and not greater than 115 per cent diphtheria vaccine (adsorbed) (2.7.6).
of the intended content.
Sterility (2. 6.1) potency is not less than 30 IU per single human dose.
Tetanus component
FINAL LOT Carry out one of the prescribed methods for the assay of
Only a final lot that is satisfactory with respect to the test for tetanus vaccine (adsorbed) (2.7.8).
osmolality and with respect to each of the requirements given The lower confidence limit (P = 0.95) of the estimated
below under Identification, Tests and Assay may be released potency is not less than 40 IU per single human dose.
for use.
Hepatitis B component
Provided the test for antimicrobial preservative and the assays
It complies with the assay of hepatitis B vaccine (2. 7.15).
for the diphtheria and tetanus components have been carried
out with satisfactory results on the final bulk vaccine, they LABELLING
may be omitted on the final lot. The label states:
Provided the content of free formaldehyde has been - the minimum number of International Units of diphtheria
determined on the bulk purified antigens or on the final bulk and tetanus toxoid per single human dose;
and it has been shown that the content in the final lot will - the amount of HBsAg per single human dose;
not exceed 0.2 g/L, the test for free formaldehyde may be - the type of cells used for production of the HBsAg
omitted on the final lot. component;
- the name and the amount of the adsorbent;
If an in vivo assay is used for the hepatitis B component,
provided it has been carried out with satisfactory results on
vaccination of children and is not necessarily suitable for
the final bulk vaccine, it may be omitted on the final lot.
Osmolality (2.2.35) - that the vaccine must be shaken before use;
The osmolality of the vaccine is within the limits approved - that the vaccine is not to be frozen.
for the particular preparation. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
IDENTIFICATION
immunochemical method (2.7.1). The following method,
applicable to certain vaccines, is given as an example.
citrate R to give a 100 g/L solution. Maintain at 37 °C for
about 16 hand centrifuge until a clear supernatant is

Diphtheria, Tetanus and Pertussis on the bulk purified antigens or on the final bulk and it has
(Whole Cell) Vaccine (Adsorbed) been shown that the content in the final lot will not exceed
Diphtheria, Tetanus and Pertussis Vaccine
final lot.
(Adsorbed)
(Ph. Eur. monograph 0445) IDENTIFICATION
The label may state 'DTwP'. A. Diphtheria toxoid is identified by a suitable
Ph Eur - - - - - - - - - - - - - - - - - - - - - ~ applicable to certain vaccines, is given as an example.
DEFINITION Dissolve in the vaccine to be examined sufficient
Diphtheria, tetanus and pertussis (whole cell) vaccine sodium citrate R to give a 100 g/L solution. Maintain at 37 °C
(adsorbed) is a preparation of diphtheria formol toxoid and for about 16 h and centrifuge until a clear supernatant is
tetanus formol toxoid with a mineral adsorbent to which a obtained. The clear supernatant reacts with a suitable
suspension of inactivated Bordetella pertussis has been added. diphtheria antitoxin, giving a precipitate.
The formol toxoids are prepared from the toxins produced B. Tetanus toxoid is identified by a suitable immunochemical
by the growth of Corynebacterium diphtheriae and Clostridium method (2. 7.1). The following method, applicable to certain
tetani, respectively. vaccines, is given as an example. The clear supernatant
PRODUCTION obtained during identification test A reacts with a suitable
BULK PURIFIED DIPHTHERIA AND TETANUS tetanus antitoxin, giving a precipitate.
TOXOIDS, BULK INACTIVATED B. PERTUSSIS C. The centrifugation residue obtained in identification A
SUSPENSION may be used. Other suitable methods for separating the
The bulk purified diphtheria and tetanus toxoids and the bacteria from the adsorbent may also be used. Identify
inactivated B. pertussis suspension are prepared as described pertussis vaccine by agglutination of the bacteria from the
in the monographs Diphtheria vaccine (adsorbed) (0443), resuspended precipitate by antisera specific to B. pertussis or
Tetanus vaccine (adsorbed) (0452) and Permssis vaccine (whole by the assay of the pertussis component described under
cell, adsorbed) (0161), respectively, and comply with the Assay.
requirements prescribed therein. TESTS
FINAL BULK VACCINE Specific toxicity of the pertussis component
The final bulk vaccine is prepared by adsorption of suitable Use not fewer than 5 mice each weighing 14-16 g for the
quantities of bulk purified diphtheria toxoid and tetanus vaccine group and for the saline control. Use mice of the
toxoid onto a mineral carrier such as hydrated aluminium same sex or distribute males and females equally between the
phosphate or aluminium hydroxide and admixture of an groups. Allow the animals access to food and water for at
appropriate quantity of a suspension of inactivated least 2 h before injection and during the test. Inject each
B. pertussis; the resulting mixture is approximately isotonic mouse of the vaccine group intraperitoneally with O.5 mL,
with blood. The B. pertussis concentration of the final bulk containing a quantity of the vaccine equivalent to not less
vaccine does not exceed that corresponding to an opacity of than half the single human dose. Inject each mouse of the
20 IU per single human dose. If 2 or more strains of B. control group with 0.5 mL of a 9 g/L sterile solution of
pertussis are used, the composition of consecutive lots of the sodium chloride R, preferably containing the same amount of
final bulk vaccine shall be consistent with respect to the antimicrobial preservative as that injected with the vaccine.
proportion of each strain as measured in opacity units. Weigh the groups of mice immediately before the injection
Suitable antimicrobial preservatives may be added to the bulk and 72 h and 7 days after the injection. The vaccine
vaccine. Certain antimicrobial preservatives, particularly those complies with the test if: (a) at the end of 72 h the total mass
of the phenolic type, adversely affect the antigenic activity of the group of vaccinated mice is not less than that
and must not be used. preceding the injection; (b) at the end of 7 days the average
Only a final bulk vaccine that complies with the following increase in mass per vaccinated mouse is not less than
requirements may be used in the preparation of the final lot. 60 per cent of that per control mouse; and (c) not more than
5 per cent of the vaccinated mice die during the test.
The test may be repeated and the results of the tests
combined.
not less than 85 per cent and not greater than 115 per cent Aluminium (2.5.13)
of the intended amount. Maximum 1.25 mg per single human dose, if aluminium
Sterility (2. 6. 1)
adsorbent.
FINAL LOT
Maximum 0.2 g/L.
tamper-evident containers. The containers are closed so as to Antimicrobial preservative
prevent contamination. Where applicable, determine the amount of antimicrobial
preservative by a suitable chemical method. The content is
not less than the minimum amount shown to be effective and
is not greater than 115 per cent of the quantity stated on the
Assay may be released for use. Provided the tests for specific
label.
toxicity of the pertussis component, antimicrobial preservative
and the assay have been carried out with satisfactory results Sterility (2. 6.1)
on the final bulk vaccine, they may be omitted on the final The vaccine complies with the test for sterility.
lot.
ASSAY evidence of protection and freedom from unexpected

Diphtheria component reactions in the target group for which the vaccine is
Carry out one of the prescribed methods for the assay of intended.
diphtheria vaccine (adsorbed) (2.7.6). PRODUCTION
The lower confidence limit (P = 0. 95) of the estimated GENERAL PROVISIONS
potency is not less than 30 IU per single human dose. The production method shall have been shown to yield
Tetanus component consistently vaccines comparable with the vaccine of proven
Carry out one of the prescribed methods for the assay of clinical efficacy and safety in man.
tetanus vaccine (adsorbed) (2. 7. 8). The content of bacterial endotoxins (2. 6.14) in the bulk
If the test is carried out in guinea-pigs, the lower confidence purified diphtheria toxoid, tetanus toxoid and pertussis
limit (P = 0.95) of the estimated potency is not less than components is determined to monitor the purification
40 IU per single human dose; if the test is carried out in procedure and to limit the amount in the final vaccine.
mice, the lower confidence limit (P = 0.95) of the estimated For each component, the content of bacterial endotoxins is
potency is not less than 60 IU per single human dose. less than the limit approved for the particular vaccine and, in
Pertussis component any case, the contents are such that the final vaccine contains
Carry out the assay of pertussis vaccine (whole cell) (2. 7. 7). less than 100 IU per single human dose.
The estimated potency is not less than 4.0 IU per single Reference vaccine(s) Provided valid assays can be performed,
human dose and the lower confidence limit (P = 0. 95) of the monocomponent reference vaccines may be used for the
estimated potency is not less than 2.0 IU per single human assays on the combined vaccine. If this is not possible
dose. because of interaction between the components of the
combined vaccine or because of differences in composition
LABELLING between the monocomponent reference vaccine and the test
The label states: vaccine, a batch of combined vaccine shown to be effective in
- the minimum number of International Units of each clinical trials or a batch representative thereof is used as a
component per single human dose; reference vaccine. For the preparation of a representative
- the name and the amount of the adsorbent; 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 production of the components complies with the
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
requirements of the monographs Diphtheria vaccine
(adsorbed) (0443), Tetanus vaccine (adsorbed) (0452) and
Pertussis vaccine ( acellular, component, adsorbed) (1356).
FINAL BULK VACCINE
Adsorbed Diphtheria, Tetanus and The final bulk vaccine is prepared by adsorption of suitable
Pertussis (Acellular Component) quantities of bulk purified diphtheria toxoid, tetanus toxoid
and pertussis components separately or together onto a
Vaccine mineral carrier such as aluminium hydroxide or hydrated
(Diphtheria, Tetanus and Pertussis (Acellular, aluminium phosphate. Suitable antimicrobial preservatives
Component) Vaccine (Adsorbed), Ph. Bur. monograph may be added.
1931) Only a final bulk vaccine that complies with the following
The label may state 'DTaP'.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Antimicrobial preservative
DEFINITION preservative by a suitable chemical method. The amount is
Diphtheria, tetanus and pertussis (acellular, component) not less than 85 per cent and not greater than 115 per cent
vaccine (adsorbed) is a combined vaccine composed of: of the intended content.
diphtheria formol toxoid; tetanus formol toxoid; individually Sterility (2. 6. 1)
purified antigenic components of Bordetella pertussis; a mineral Carry out the test for sterility using 10 mL for each medium.
adsorbent such as aluminium hydroxide or hydrated
aluminium phosphate. FINAL LOT
Only a final lot that is satisfactory with respect to the test for
The formol toxoids are prepared from the toxins produced
osmolality and with respect to each of the requirements given
by the growth of Corynebacterium diphtheriae and Cwstridium
below under Identification, Tests and Assay may be released
tetani, respectively.
for use.
The vaccine contains either pertussis toxoid (chemically
Provided the tests for free formaldehyde and antimicrobial
detoxified pertussis toxin) or a pertussis toxin-like protein
preservative and the assay have been carried out with
free from toxic properties, produced by expression of a
satisfactory results on the final bulk vaccine, they may be
genetically modified form of the corresponding gene.
omitted on the final lot.
The vaccine may also contain filamentous haemagglutinin,
pertactin (a 69 kDa outer-membrane protein) and other Provided the free formaldehyde content has been determined
defined components of B. pertussis such as fimbrial-2 and on the bulk purified antigens or on the final bulk and it has
fimbrial-3 antigens. The latter 2 antigens may be co-purified. been shown that the content in the final lot will not exceed
The antigenic composition and characteristics are based on
0.2 g/L, the test for free formaldehyde may be omitted on the - the names and amounts of the pertussis components per
final lot. single human dose;
Osmolality (2.2.35) - where applicable, that the vaccine contains a pertussis
The osmolality of the vaccine is within the limits approved toxin-like protein produced by genetic modification;
for the particular preparation. - the name and the amount of the adsorbent;
IDENTIFICATION vaccination of children and is not necessarily suitable for
A. Diphtheria toxoid is identified by a suitable reinforcing doses or for administration to adults;
immunochemical method (2.7.1). The following method, - that the vaccine must be shaken before use;
applicable to certain vaccines, is given as an example. - that the vaccine is not to be frozen.
Dissolve in the vaccine to be examined sufficient sodium _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
about 16 h and centrifuge until a clear supernatant is
diphtheria antitoxin, giving a precipitate.
Diphtheria, Tetanus and Pertussis
method (2. 7.1). The following method, applicable to certain (Acellular Component) Vaccine
vaccines, is given as an example. The clear supernatant
obtained as described in identification test A reacts with a
(Adsorbed, Reduced Antigen(s)
suitable tetanus antitoxin, giving a precipitate. Content)
C. The pertussis components are identified by a suitable (Ph. Bur. monograph 2764)
applicable to certain vaccines, is given as an example. The label may state 'DTaP'.
The clear supernatant obtained as described in identification PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
test A reacts with antisera specific to the pertussis
DEFINITION
components of the vaccine.
Diphtheria, tetanus and pertussis (acellular, component)
TESTS vaccine (adsorbed, reduced antigen(s) content) is a combined
Aluminium (2.5.13) vaccine containing: diphtheria formol toxoid; tetanus formol
Maximum 1.25 mg per single human dose, if aluminium toxoid; individually purified antigenic components of
hydroxide or hydrated aluminium phosphate is used as the Bordetella pertussis; a mineral adsorbent such as aluminium
adsorbent. hydroxide or hydrated aluminium phosphate.
Free formaldehyde (2.4.18) The formol toxoids are prepared from the toxins produced
Maximum 0.2 g/L. by the growth of Corynebacterium diphtheriae and Clostridium
Antimicrobial preservative tetani respectively.
Where applicable, determine the amount of antimicrobial The amount of diphtheria toxoid per single human dose is
preservative by a suitable chemical method. The content is reduced compared to vaccines generally used for primary
not less than the minimum amount shown to be effective and vaccination; the amounts of tetanus toxoid and pertussis
is not greater than 115 per cent of the quantity stated on the components may also be reduced.
label. The vaccine contains either pertussis toxoid (chemically
Sterility (2. 6.1) detoxified pertussis toxin) or a pertussis toxin-like protein
The vaccine complies with the test for sterility. free from toxic properties, produced by expression of a
ASSAY The vaccine may also contain filamentous haemagglutinin,
Diphtheria component pertactin (a 69 kDa outer-membrane protein) and other
Carry out one of the prescribed methods for the assay of defined components of B. pertussis such as fimbrial-2 and
diphtheria vaccine (adsorbed) (2.7.6). fimbrial-3 antigens. The latter 2 antigens may be co-purified.
The lower confidence limit (P = 0.95) of the estimated The antigenic composition and characteristics are based on
potency is not less than the minimum potency stated on the evidence of protection and freedom from unexpected
label. reactions in the target group for which the vaccine is
Unless otherwise justified and authorised, the minimum intended.
potency stated on the label is 30 IU per single human dose. PRODUCTION
Tetanus component GENERAL PROVISIONS
Carry out one of the prescribed methods for the assay of The production method shall have been shown to yield
tetanus vaccine (adsorbed) (2. 7. 8). consistently vaccines comparable with the vaccine of proven
The lower confidence limit (P = 0. 95) of the estimated clinical efficacy and safety in man.
potency is not less than 40 IU per single human dose. The content of bacterial endotoxins (2. 6.14) in bulk purified
Pertussis component pertussis components is determined to monitor the
Carry out one of the prescribed methods for the assay of purification procedure and to limit the amount in the final
pertussis vaccine (acellular) (2. 7. 16). The vaccine complies vaccine. For each component, the content of bacterial
with the limit approved for the particular product. endotoxins is less than the limit approved for the particular
vaccine and, in any case, the contents are such that the final
LABELLING vaccine contains less than 100 IU per single human dose.
The label states:
Reference vaccine(s) Provided valid assays can be performed,
- the minimum number of International Units of diphtheria
monocomponent reference vaccines may be used for the
and tetanus toxoid per single human dose;
assays on the combined vaccine. If this is not possible diphtheria antitoxin, giving a precipitate. If a satisfactory
because of interaction between the components of the result is not obtained with a vaccine adsorbed on aluminium
combined vaccine or because of differences in composition hydroxide, carry out the test as follows. Centrifuge 15 mL of
between the monocomponent reference vaccine and the test the vaccine to be examined and suspend the residue in 5 mL
vaccine, a batch of combined vaccine shown to be effective in of a freshly prepared mixture of 1 volume of a 56 g/L
clinical trials or a batch representative thereof is used as a solution of sodium edetate R and 49 volumes of disodium
reference vaccine. For the preparation of a representative hydrogen phosphate solution R. Maintain at 37 °C for not less
batch, strict adherence to the production process used for the than 6 h and centrifuge. The clear supernatant reacts with a
batch tested in clinical trials is necessary. The reference suitable diphtheria antitoxin, giving a precipitate.
vaccine may be stabilised by a method that has been shown B. Tetanus toxoid is identified by a suitable immunochemical
to have no effect on the assay procedure. method (2. 7.1). The following method, applicable to certain
PRODUCTION OF THE COMPONENTS vaccines, is given as an example. The clear supernatant
The production of the components complies with the obtained as described in identification test A reacts with a
requirements of the monographs Diphtheria vaccine (adsorbed) suitable tetanus antitoxin, giving a precipitate.
(0443), Tetanus vaccine (adsorbed) (0452) and Pertussis vaccine C. The pertussis components are identified by a suitable
(acellular, component, adsorbed) (1356). immunochemical method (2.7.1). The following method,
FINAL BULK VACCINE applicable to certain vaccines, is given as an example.
The final bulk vaccine is prepared by adsorption onto a The clear supernatant obtained as described in identification
mineral carrier such as aluminium hydroxide or hydrated test A reacts with antisera specific to the pertussis
aluminium phosphate, separately or together, of suitable components of the vaccine.
quantities of bulk purified diphtheria toxoid, tetanus toxoid TESTS
and acellular pertussis components. Suitable antimicrobial Aluminium (2.5.13)
preservatives may be added. Maximum 1.25 mg per single human dose, if aluminium
Only a final bulk vaccine that complies with the following hydroxide or hydrated aluminium phosphate is used as the
requirements may be used in the preparation of the final lot. adsorbent.
Antimicrobial preservative Free formaldehyde (2.4.18)
Where applicable, determine the amount of antimicrobial Maximum 0.2 g/L.
preservative by a suitable chemical method. The amount is Antimicrobial preservative
not less than 85 per cent and not greater than 115 per cent Where applicable, determine the amount of antimicrobial
of the intended content. preservative by a suitable chemical method. The content is
Sterility (2. 6. 1) not less than the minimum amount shown to be effective and
Carry out the test for sterility using 10 mL for each medium. is not greater than 115 per cent of the quantity stated on the
FINAL LOT label.
The final bulk vaccine is distributed aseptically into sterile, Sterility (2. 6.1)
tamper-evident containers. The containers are closed so as to The vaccine complies with the test for sterility.
ASSAY
Only a final lot that is satisfactory with respect to the test for Diphtheria component
osmolality and with respect to each of the requirements given Carry out one of the prescribed methods for the assay of
below under Identification, Tests and Assay may be released diphtheria vaccine (adsorbed) (2.7.6).
for use.
Provided the test for antimicrobial preservative and the assays potency is not less than 2 IU per single human dose.
for the diphtheria, tetanus and pertussis components have
been carried out with satisfactory results on the final bulk Tetanus component
vaccine, they may be omitted on the final lot. Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2.7.8).
on the bulk purified antigens or on the final bulk and it has The lower confidence limit (P = 0.95) of the estimated
been shown that the content in the final lot will not exceed potency is not less than 20 IU per single human dose.
0.2 g/L, the test for free formaldehyde may be omitted on the Pertussis component
final lot. Carry out one of the prescribed methods for the assay of
Where there is a significant change in the manufacturing pertussis vaccine (acellular) (2.7.16). The vaccine complies
process of the antigens or their formulation, any impact on with the limit approved for the particular product.
the in vivo and in vitro assays must be evaluated, and the LABELLING
need for revalidation considered. The label states:
Osmolality (2.2.35) - the minimum number of International Units of diphtheria
The osmolality of the vaccine is within the limits approved and tetanus toxoid per single human dose;
for the particular preparation. - the names and amounts of the pertussis components per
IDENTIFICATION single human dose;
A. Diphtheria toxoid is identified by a suitable - where applicable, that the vaccine contains a pertussis
immunochemical method (2. 7. 1). The following method, toxin-like protein produced by genetic modification;
applicable to certain vaccines, is given as an example. - the name and the amount of the adsorbent;
Dissolve in the vaccine to be examined sufficient sodium - that the vaccine must be shaken before use;
citrate R to give a 100 g/L solution. Maintain at 37 °C for - that the vaccine is not to be frozen.
about 16 h and centrifuge until a clear supernatant is _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
the haemophilus component is presented in a separate

Adsorbed Diphtheria, Tetanus, container, the contents of the diphtheria, tetanus and
Pertussis (Acellular Component) pertussis antigens are in any case such that the final vial for
these components contains less than 100 IU per single
and Haemophilus Type b human dose.
Conjugate Vaccine During development studies, it shall be demonstrated that
(Diphtheria, Tetanus, Pertussis (Acellular, Component) the vaccine consistently induces a T-cell-dependent B-cell
and Haemophilus Type b Conjugate Vaccine immune response to PRP. If the manufacturing process is
(Adsorbed), Ph. Eur. monograph 1932) modified, it shall be demonstrated by appropriate in vitro
methods that the characteristic properties of the conjugate
The label may state 'DTaP/Hib'. are not affected.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Where the haemophilus component is presented in a separate
DEFINITION container, the production method is validated to demonstrate
that the haemophilus component, if tested, would comply
Diphtheria, tetanus, pertussis (acellular, component) and
with the test for pyrogens (2.6.8), carried out as follows:
haemophilus type b conjugate vaccine (adsorbed) is a
inject per kilogram of the rabbit's mass a quantity of the
combined vaccine composed of: diphtheria formol toxoid;
vaccine equivalent to: 1 µg of PRP for a vaccine with
tetanus formol toxoid; individually purified antigenic
diphtheria toxoid or CRM 197 diphtheria protein as carrier;
components of Bordetella pertussis; polyribosylribitol phosphate
0 .1 µg of PRP for a vaccine with tetanus toxoid as carrier;
(PRP) covalently bound to a carrier protein; a mineral
0.025 µg of PRP for a vaccine with OMP (meningococcal
absorbent such as aluminium hydroxide or hydrated
group B outer membrane protein complex) as carrier.
aluminium phosphate. The product is presented either as a
tetravalent liquid formulation in the same container, or as a Reference vaccine(s) Provided valid assays can be performed,
trivalent liquid formulation with the haemophilus component monocomponent reference vaccines may be used for the
in a separate container, the contents of which are mixed with assays on the combined vaccine. If this is not possible
the other components immediately before use. because of interaction between the components of the
between the monocomponent reference vaccine and the test
by the growth of Corynebacterium diphtheriae and Cwstridium
vaccine, a batch of combined vaccine shown to be effective in
tetani respectively.
clinical trials or a batch representative thereof is used as a
detoxified pertussis toxin) or a pertussis toxin-like protein batch, strict adherence to the production process used for the
free from toxic properties produced by expression of a
batch tested in clinical trials is necessary. The reference
genetically modified form of the corresponding gene. vaccine may be stabilised by a method that has been shown
The acellular pertussis component may also contain
to have no effect on the assay procedure.
filamentous haemagglutinin, pertactin (a 69 kDa outer-
membrane protein) and other defined components of PRODUCTION OF THE COMPONENTS
B. pertussis such as fimbrial-2 and fimbrial-3 antigens. The production of the components complies with the
The latter 2 antigens may be co-purified. The antigenic requirements of the monographs Diphtheria vaccine
composition and characteristics are based on evidence of (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452), Pertussis
protection and freedom from unexpected reactions in the vaccine (acellular, component, adsorbed) (1356) and
target group for which the vaccine is intended. Haemophilus type b conjugate vaccine (1219).
PRP is a linear copolymer composed of repeated units of FINAL BULK VACCINE
3-~-D-ribofuranosyl-(l--+ 1)-ribitol-5-phosphate Different methods of preparation may be used: a final bulk
[(C10H19O11P)nl, with a defined molecular size and derived vaccine may be prepared by adsorption, separately or
from a suitable strain of Haemophilus infiuenzae type b. together, of suitable quantities of bulk purified diphtheria
The carrier protein, when conjugated to PRP, is capable of toxoid, tetanus toxoid, acellular pertussis components and
inducing a T-cell-dependent B-cell immune response to the PRP conjugate onto a mineral carrier such as aluminium
polysaccharide. hydroxide or hydrated aluminium phosphate; or 2 final bulks
may be prepared and filled separately, one containing the
PRODUCTION diphtheria, tetanus and pertussis components, the other the
GENERAL PROVISIONS haemophilus component, which may be freeze-dried. Suitable
The production method shall have been shown to yield antimicrobial preservatives may be added.
consistently vaccines comparable with the vaccine of proven
Where the haemophilus component is presented in a separate
container, as part of consistency studies the assays of the Antimicrobial preservative
diphtheria, tetanus and pertussis components are carried out Where applicable, determine the amount of antimicrobial
on a suitable number of batches of vaccine reconstituted as preservative by a suitable chemical method. The amount is
for use. For subsequent routine control, the assays of these not less than 85 per cent and not greater than 115 per cent
components may be carried out without mixing with the of the intended content.
haemophilus component. Sterility (2. 6.1)
The content of bacterial endotoxins (2. 6.14) in bulk purified Carry out the test for sterility using 10 mL for each medium.
diphtheria toxoid, tetanus toxoid, pertussis components and FINAL LOT
bulk PRP conjugate is determined to monitor the purification Only a final lot that is satisfactory with respect to the test for
procedure and to limit the amount in the final vaccine. osmolality shown below and with respect to each of the
For each component, the content of bacterial endotoxins is requirements given below under Identification, Tests and
less than the limit approved for the particular vaccine; where Assay may be released for use.
Provided the test for antimicrobial preservative and the assay or by anion-exchange liquid chromatography (2.2.29) with
have been carried out with satisfactory results on the final pulsed amperometric detection.
bulk vaccine, they may be omitted on the final lot. Aluminium (2.5.13)
Provided the free formaldehyde content has been determined Maximum 1.25 mg per single human dose, if aluminium
on the bulk purified antigens or the final bulk and it has been hydroxide or hydrated aluminium phosphate is used as the
shown that the content in the final lot will not exceed adsorbent.
0.2 g/L, the test for free formaldehyde may be omitted on the Free formaldehyde (2.4. 18)
final lot. Maximum 0.2 g/L.
Osmolality (2.2.35) Antimicrobial preservative
The osmolality of the vaccine, reconstituted where applicable, Where applicable, determine the amount of antimicrobial
is within the limits approved for the particular preparation. preservative by a suitable chemical method. The content is
pH (2.2.3) not less than the minimum amount shown to be effective and
The pH of the vaccine, reconstituted if necessary, is within is not greater than 115 per cent of the quantity stated on the
the range approved for the particular product. label.
FreePRP Water (2.5.12)
Unbound PRP is determined after removal of the conjugate, Maximum 3.0 per cent for the freeze-dried haemophilus
for example by anion-exchange, size-exclusion or component.
hydrophobic chromatography, ultrafiltration or other Sterility (2. 6.1)
validated methods. The amount of free PRP is not greater The vaccine complies with the test for sterility.
than that approved for the particular product.
Bacterial endotoxins (2.6.14)
IDENTIFICATION The content is within the limits approved by the competent
Where the haemophilus component is presented in a separate authority for the haemophilus component of the particular
container: identification tests A, B and C are carried out using the product. If any components of the vaccine prevent the
container containing the diphtheria, tetanus and pertussis determination of endotoxin, a test for pyrogens is carried out
components; identification test D is carried out on the container as described under General provisions.
containing the haemophilus component.
ASSAY
citrate R to give a 100 g/L solution. Maintain at 37 °C for The lower confidence limit (P = 0.95) of the estimated
about 16 h and centrifuge until a clear supernatant is potency is not less than the minimum potency stated on the
obtained. The clear supernatant reacts with a suitable label.
diphtheria antitoxin, giving a precipitate. Unless otherwise justified and authorised, the minimum
B. Tetanus toxoid is identified by a suitable immunochemical potency stated on the label is 30 IU per single human dose.
method (2. 7.1). The following method, applicable to certain Tetanus component
vaccines, is given as an example. The clear supernatant Carry out one of the prescribed methods for the assay of
obtained as described in identification test A reacts with a tetanus vaccine (adsorbed) (2.7.8).
suitable tetanus antitoxin, giving a precipitate. The lower confidence limit (P = 0.95) of the estimated
C. The pertussis components are identified by a suitable potency is not less than 40 IU per single human dose.
immunochemical method (2.7.J). The following method, Pertussis 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 pertussis vaccine (acellular) (2.7.16). The vaccine complies
test A reacts with antisera specific to the pertussis with the limit approved for the particular product.
LABELLING
D. The haemophilus component is identified by a suitable
The label states:
immunochemical method (2.7.J) for PRP.
TESTS and tetanus toxoid per single human dose;
W'here the product is presented with the haemophilus component in - the names and amounts of the pertussis components per
a separate container: the tests for aluminium, free formaldehyde, single human dose;
antimicrobial preservative and sterility are carried out on the - where applicable, that the vaccine contains a pertussis
container with the diphtheria, tetanus and pertussis components; toxin-like protein produced by genetic modification;
the tests for PRP content, water (where applicable), sterility and - the number of micrograms of PRP per single human
bacterial endotoxins are carried out on the container with the dose;
haemophilus component. - the type and nominal amount of carrier protein per single
If the haemophilus component is freeze-dried, some tests may be human dose;
carried out on the freeze-dried product rather than on the bulk - the name and the amount of the adsorbent;
conjugate where the freeze-drying process may affect the component - where applicable, that the vaccine is intended for primary
to be tested. vaccination of children and is not necessarily suitable for
PRP
Minimum 80 per cent of the amount of PRP stated on the
label. PRP is determined either by assay of ribose (2.5.31) or
phosphorus (2.5.18), by an immunochemical method (2.7.1) - - - - - - - - - - - - - - - - - - - - - - Ph Eur
(adsorbed) (0443), Tetanus vaccine (adsorbed) (0452), Pertussis

Adsorbed Diphtheria, Tetanus, vaccine (acellular, component, adsorbed) (1356) and Hepatitis B
Pertussis (Acellular Component) vaccine (rDNA) (1056).
and Hepatitis B (rDNA) Vaccine FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption, separately
(Diphtheria, Tetanus, Pertussis (Acellular, Component) or together, of suitable quantities of bulk purified diphtheria
and Hepatitis B (rDNA) Vaccine (Adsorbed), toxoid, tetanus toxoid, acellular pertussis components and
Ph. Bur. monograph 1933) hepatitis B surface antigen onto a mineral carrier such as
The label may state 'DTaP/HepB'. aluminium hydroxide or hydrated aluminium phosphate.
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Suitable antimicrobial preservatives may be added.
DEFINITION requirements may be used in the preparation of the final lot.
Diphtheria, tetanus, pertussis (acellular, component) and
hepatitis B (rDNA) vaccine (adsorbed) is a combined vaccine Antimicrobial preservative
composed of: diphtheria formol toxoid; tetanus formol Where applicable, determine the amount of antimicrobial
toxoid; individually purified antigenic components of preservative by a suitable chemical method. The amount is
Bordetella pertussis; hepatitis B surface antigen; a mineral not less than 85 per cent and not greater than 115 per cent
adsorbent such as aluminium hydroxide or hydrated of the intended content.
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 Corynebacterium diphtheriae and Clostridium FINAL LOT
tetani, respectively. Only a final lot that is satisfactory with respect to the test for
The vaccine contains either pertussis toxoid (chemically osmolality and with respect to each of the requirements given
detoxified pertussis toxin) or a pertussis toxin-like protein below under Identification, Tests and Assay may be released
free from toxic properties, produced by expression of a for use.
genetically modified form of the corresponding gene. Provided the test for antimicrobial preservative and the assays
The vaccine may also contain filamentous haemagglutinin, for the diphtheria, tetanus and pertussis components have
pertactin (a 69 kDa outer-membrane protein) and other been carried out with satisfactory results on the final bulk
defined components of B. pertussis such as fimbrial-2 and vaccine, they may be omitted on the final lot.
fimbrial-3 antigens. The latter 2 antigens may be co-purified. Provided the content of free formaldehyde has been
The antigenic composition and characteristics are based on determined on the bulk purified antigens or on the final bulk
evidence of protection and freedom from unexpected and it has been shown that the content in the final lot will
reactions in the target group for which the vaccine is not exceed 0.2 g/L, the test for free formaldehyde may be
intended. omitted on the final lot.
Hepatitis B surface antigen is a component protein of If an in vivo assay is used for the hepatitis B component,
hepatitis B virus; the antigen is obtained by recombinant provided it has been carried out with satisfactory results on
DNA technology. the final bulk vaccine, it may be omitted on the final lot.
PRODUCTION Osmolality (2.2.35)
GENERAL PROVISIONS The osmolality of the vaccine is within the limits approved
The production method shall have been shown to yield for the particular preparation.
IDENTIFICATION
The content of bacterial endotoxins (2. 6.14) in the bulk immunochernical method (2.7.1). The following method,
purified diphtheria toxoid, tetanus toxoid and pertussis applicable to certain vaccines, is given as an example.
components is determined to monitor the purification Dissolve in the vaccine to be examined sufficient sodium
procedure and to limit the amount in the final vaccine. citrate R to give a 100 g/L solution. Maintain at 37 °C for
For each component, the content of bacterial endotoxins is about 16 h and centrifuge until a clear supernatant is
less than the limit approved for the particular vaccine. obtained. The clear supernatant reacts with a suitable
Reference vaccine(s) Provided valid assays can be performed, diphtheria antitoxin, giving a precipitate.
monocomponent reference vaccines may be used for the B. Tetanus toxoid is identified by a suitable immunochemical
assays on the combined vaccine. If this is not possible method (2. 7.1). The following method, applicable to certain
because of interaction between the components of the vaccines, is given as an example. The clear supernatant
combined vaccine or because of differences in composition obtained as described in identification test A reacts with a
between the monocomponent reference vaccine and the test suitable tetanus antitoxin, giving a precipitate.
C. The pertussis components are identified by a suitable
immunochernical method (2.7.1). The following method,
batch, strict adherence to the production process used for the
The clear supernatant obtained as described in identification
test A reacts with antisera specific to the pertussis
vaccine may be stabilised by a method that has been shown
D. The assay or, where applicable, the electrophoretic profile,
PRODUCTION OF THE COMPONENTS serves also to identify the hepatitis B component of the
vaccine.
requirements of the monographs Diphtheria vaccine
TESTS
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)
Antimicrobial preservative and Poliomyelitis (Inactivated) Vaccine (Adsorbed),
Where applicable, determine the amount of antimicrobial Ph. Bur. monograph 1934)
preservative by a suitable chemical method. The content is The label may state 'DTaP/IPV'.
PhEur - - - - - - - - - - - - - - - - - - - - - ~
label. DEFINITION
Sterility (2. 6.1) Diphtheria, tetanus, pertussis (acellular, component) and
The vaccine complies with the test for sterility. poliomyelitis (inactivated) vaccine (adsorbed) is a combined
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
Bordetella 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
ASSAY inactivated by a validated method; a mineral adsorbent such
Diphtheria component as aluminium hydroxide or hydrated aluminium phosphate.
Carry out one of the prescribed methods for the assay of The formol toxoids are prepared from the toxins produced
diphtheria vaccine (adsorbed) (2.7.6). by the growth of Corynebacterium diphtheriae and Clostridium
The lower confidence limit (P = 0.95) of the estimated tetani respectively.
potency is not less than the minimum potency stated on the The vaccine contains either pertussis toxoid (chemically
label. detoxified pertussis toxin) or a pertussis toxin-like protein
Unless otherwise justified and authorised, the minimum free from toxic properties produced by expression of a
potency stated on the label is 30 IU per single human dose. genetically modified form of the corresponding gene.
Tetanus component The vaccine may also contain filamentous haemagglutinin,
Carry out one of the prescribed methods for the assay of pertactin (a 69 kDa outer-membrane protein) and other
tetanus vaccine (adsorbed) (2.7.8). defined components of B. pertussis such as fimbrial-2 and
fimbrial-3 antigens. The latter 2 antigens may be co-purified.
The antigenic composition and characteristics are based on
evidence of protection and freedom from unexpected
Pertussis component reactions in the target group for which the vaccine is
Carry out one of the prescribed methods for the assay of intended.
pertussis vaccine (acellular) (2. 7.16). The vaccine complies
with the limit approved for the particular product. PRODUCTION
GENERAL PROVISIONS
Hepatitis B component The production method shall have been shown to yield
The vaccine complies with the assay of hepatitis B vaccine consistently vaccines comparable with the vaccine of proven
(2.7.15). clinical efficacy and safety in man.
LABELLING The content of bacterial endotoxins (2. 6.14) in bulk purified
The label states: diphtheria toxoid, tetanus toxoid, pertussis components and
- the minimum number of International Units of diphtheria purified, inactivated monovalent poliovirus harvests is
and tetanus toxoid per single human dose; determined to monitor the purification procedure and to
- the names and amounts of the pertussis components per limit the amount in the final vaccine. For each component,
single human dose; the content of bacterial endotoxins is less than the limit
- where applicable, that the vaccine contains a pertussis approved for the particular vaccine and, in any case, the
toxin-like protein produced by genetic modification; contents are such that the final vaccine contains less than
- the amount of HBsAg per single human dose; 100 IU per single human dose.
- the type of cells used for production of the hepatitis B Reference vaccine(s) Provided valid assays can be performed,
component; monocomponent reference vaccines may be used for the
- the name and the amount of the adsorbent; assays on the combined vaccine. If this is not possible
- where applicable, that the vaccine is intended for primary because of interaction between the components of the
vaccination of children and is not necessarily suitable for combined vaccine or because of differences in composition
reinforcing doses or for administration to adults; between the monocomponent reference vaccine and the test
- that the vaccine must be shaken before use; vaccine, a batch of combined vaccine shown to be effective in
- that the vaccine is not to be frozen. clinical trials or a batch representative thereof is used as a
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur reference vaccine. For the preparation of a representative
vaccine may be stabilised by a method that has been shown
PRODUCTION OF THE COMPONENTS formulation, any impact on the in vivo and in m·tro assays
The production of the components complies with the must be evaluated, and the need for revalidation considered.
requirements of the monographs Diphtheria vaccine (adsorbed) Osmolality (2.2.35)
(0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine The osmolality of the vaccine is within the limits approved
(acellular, component, adsorbed) (1356) and Polwmyelitis for the particular preparation.
vaccine (inactivated) (0214).
IDENTIFICATION
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption onto a
mineral carrier such as aluminium hydroxide or hydrated
aluminium phosphate, separately or together, of suitable
quantities of bulk purified diphtheria toxoid, tetanus toxoid,
acellular pertussis components and admixture of suitable
quantities of purified monovalent harvests of human
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. B. Tetanus toxoid is identified by a suitable immunochemical
method (2. 7.1). The following method, applicable to certain
Bovine serum albumin suitable tetanus antitoxin, giving a precipitate.
Determined on the poliomyelitis component by a suitable C. The pertussis components are identified by a suitable
immunochemical method (2. 7.1) after virus harvest and immunochemical method (2.7.1). The following method,
before addition of the adsorbent in the preparation of the applicable to certain vaccines, is given as an example.
final bulk vaccine, the amount of bovine serum albumin is The clear supernatant obtained as described in identification
such that the content in the final vaccine will be not more test A reacts with antisera specific to the pertussis
than 50 ng per single human dose. components of the vaccine.
Antimicrobial preservative D. The vaccine is shown to contain human poliovirus
Where applicable, determine the amount of antimicrobial types 1, 2 and 3 by a suitable immunochemical method
preservative by a suitable chemical method. The amount is (2. 7. 1) such as the determination of D-antigen by enzyme-
not less than 85 per cent and not greater than 115 per cent linked immunosorbent assay (ELISA).
TESTS
Sterility (2. 6.1)
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose if aluminium
FINAL LOT hydroxide or hydrated aluminium phosphate is used as the
Only a final lot that is satisfactory with respect to the test for adsorbent.
osmolality and with respect to each of the requirements given
below under Identification, Tests and Assay may be released
Maximum 0.2 g/L.
for use.
Provided the test for antimicrobial preservative and the assays
for the diphtheria, tetanus and pertussis components have Where applicable, determine the amount of antimicrobial
been carried out with satisfactory results on the final bulk preservative by a suitable chemical method. The content is
vaccine, they may be omitted on the final lot. not less than the minimum amount shown to be effective and
Provided the free formaldehyde content has been determined label.
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 Sterility (2. 6.1)
0.2 g/L, the test for free formaldehyde may be omitted on the The vaccine complies with the test for sterility.
final lot. ASSAY
Provided that the determination of D-antigen content has Diphtheria component
been carried out with satisfactory results during preparation Carry out one of the prescribed methods for the assay of
of the final bulk before addition of the adsorbent, it may be diphtheria vaccine (adsorbed) (2.7.6).
omitted on the final lot. The lower confidence limit (P = 0.95) of the estimated
Provided that the in vivo assay for the poliomyelitis potency is not less than the minimum potency stated on the
component has been carried out with satisfactory results on label.
the final bulk vaccine, it may be omitted on the final lot. Unless otherwise justified and authorised, the minimum
The in vivo assay for the poliomyelitis component may be potency stated on the label is 30 IU per single human dose.
omitted once it has been demonstrated for a given product Tetanus component
and for each poliovirus type that the acceptance criteria for Carry out one of the prescribed methods for the assay of
the D-antigen determination are such that it yields the same tetanus vaccine (adsorbed) (2. 7.8).
result as the in vivo assay in terms of acceptance or rejection
of a batch. This demonstration must include testing of
subpotent batches, produced experimentally if necessary, for
example by heat treatment or other means of diminishing the Pertussis component
immunogenic activity. Where there is a significant change in Carry out one of the prescribed methods for the assay of
the manufacturing process of the antigens or their pertussis vaccine (acellular) (2. 7.16). The vaccine complies
with the limit approved for the particular product.
IV- 722 Vaccines 2023
Poliomyelitis component PRODUCTION

D-antigen content As a measure of consistency of GENERAL PROVISIONS
production, determine the D-antigen content for human The production method shall have been shown to yield
poliovirus types 1, 2 and 3 by a suitable immunochemical consistently vaccines comparable with the vaccine of proven
method (2. 7.1) following desorption, using a reference clinical efficacy and safety in man.
preparation calibrated in European Pharmacopoeia Units of The content of bacterial endotoxins (2. 6.14) in bulk purified
D-antigen. For each type, the content, expressed with diphtheria toxoid, tetanus toxoid and inactivated monovalent
reference to the amount of D-antigen stated on the label, is poliovirus harvests is determined to monitor the purification
within the limits approved for the particular product. procedure and to limit the amount in the final vaccine.
Poliomyelitis vaccine (inactivated) ERP is calibrated in For each component, the content of bacterial endotoxins is
European Pharmacopoeia Units and intended for use in the less than the limit approved for the particular vaccine and, in
assay of D-antigen. The European Pharmacopoeia Unit and any case, the contents are such that the final vaccine contains
the International Unit are equivalent. less than 100 IU per single human dose.
In vivo test The vaccine complies with the in viva assay of Reference vaccine(s) Provided valid assays can be performed,
poliomyelitis vaccine (inactivated) (2. 7. 20). monocomponent reference vaccines may be used for the
LABELLING assays on the combined vaccine. If this is not possible
The label states: because of interaction between the components of the
- the minimum number of International Units of diphtheria combined vaccine or because of the difference in composition
and tetanus toxoid per single human dose; between the monocomponent reference vaccine and the test
- the names and amounts of the pertussis components per vaccine, a batch of combined vaccine shown to be effective in
single human dose; clinical trials or a batch representative thereof is used as a
- where applicable, that the vaccine contains a pertussis reference vaccine. For the preparation of a representative
toxin-like protein produced by genetic modification; batch, strict adherence to the production process used for the
- the types of poliovirus contained in the vaccine; batch tested in clinical trials is necessary. The reference
- the nominal amount of poliovirus of each type ( 1, 2 vaccine may be stabilised by a method that has been shown
and 3), expressed in European Pharmacopoeia Units of to have no effect on the assay procedure.
D-antigen, per single human dose; PRODUCTION OF THE COMPONENTS
- the type of cells used for production of the poliomyelitis The production of the components complies with the
component; requirements of the monographs Diphtheria vaccine ( adsorbed)
- the name and the amount of the adsorbent; (0443), Tetanus vaccine (adsorbed) (0452) and Poliomyelitis
- where applicable, that the vaccine is intended for primary vaccine (inactivated) (0214).
vaccination of children and is not necessarily suitable for FINAL BULK VACCINE
reinforcing doses or for administration to adults; The final bulk vaccine is prepared by adsorption onto a
- that the vaccine must be shaken before use; mineral carrier such as aluminium hydroxide or hydrated
- that the vaccine is not to be frozen. aluminium phosphate, separately or together, of suitable
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur quantities of bulk purified diphtheria toxoid and tetanus
toxoid, 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
Diphtheria, Tetanus and monovalent harvests. Suitable antimicrobial preservatives may
be added.
Poliomyelitis (Inactivated) Vaccine Only a final bulk vaccine that complies with the following
(Adsorbed, Reduced Antigen(s) requirements may be used in the preparation of the final lot.
Content) Bovine serum albumin
Determined on the poliomyelitis component by a suitable
(Ph. Bur. monograph 2328) immunochemical method (2. 7.1) after virus harvest and
The label may state 'Td/IPV'. before addition of the adsorbent in the preparation of the
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ final bulk vaccine, the amount of bovine serum albumin is
such that the content in the final vaccine will be not more
DEFINITION than 50 ng per single human dose.
Diphtheria, tetanus and poliomyelitis (inactivated) vaccine
(adsorbed, reduced antigen(s) content) is a combined vaccine
containing: diphtheria formol toxoid; tetanus formol toxoid;
suitable strains of human poliovirus types 1, 2 and 3 grown
in suitable cell cultures and inactivated by a validated
method; a mineral adsorbent such as aluminium hydroxide
or hydrated aluminium phosphate. Sterility (2. 6.1)
by the growth of Corynebacterium diphtheriae and Clostridium FINAL LOT
tetani respectively. The final bulk vaccine is distributed aseptically into sterile,
The amount of diphtheria toxoid per single human dose is tamper-evident containers. The containers are closed so as to
reduced compared to vaccines generally used for primary prevent contamination.
vaccination; the amount of tetanus toxoid may also be Only a final lot that is satisfactory with respect to the test for
reduced. osmolality and with respect to each of the requirements given
below under Identification, Tests and Assay may be released Free formaldehyde (2. 4.18)
for use. Maximum 0.2 g/L.
Provided the test for antimicrobial preservative and the assays Antimicrobial preservative
for the diphtheria and tetanus components have been carried Where applicable, determine the amount of antimicrobial
out with satisfactory results on the final bulk vaccine, they preservative by a suitable chemical method. The content is
may be omitted on the final lot. not less than the minimum amount shown to be effective and
Provided the free formaldehyde content has been determined is not greater than 115 per cent of the quantity stated on the
on the bulk purified antigens or on the final bulk and it has label.
been shown that the content in the final lot will not exceed Sterility (2. 6. 1)
0.2 g/L, the test for free formaldehyde may be omitted on the The vaccine complies with the test for sterility.
final lot.
ASSAY
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.
Provided 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.
Tetanus component
The in vivo assay for the poliomyelitis component may be
omitted once it has been demonstrated for a given vaccine
tetanus vaccine (adsorbed) (2. 7. 8).
and for each poliovirus type that the acceptance criteria for
the D-antigen determination are such that it yields the same The lower confidence limit (P = 0.95) of the estimated
result as the in vivo assay in terms of acceptance or rejection potency is not less than 20 IU per single human dose.
of a batch. This demonstration must include testing of Poliomyelitis component
subpotent batches, produced experimentally if necessary, for D-antigen content As a measure of consistency of
example by heat treatment or other means of diminishing the production, determine the D-antigen content for human
immunogenic activity. Where there is a significant change in poliovirus types 1, 2 and 3 by a suitable immunochemical
the manufacturing process of the antigens or their method (2. 7.1) following desorption, using a reference
formulation, any impact on the in vivo and in vitro assays preparation calibrated in European Pharmacopoeia Units of
must be evaluated, and the need for revalidation considered. D-antigen. For each type, the content, expressed with
Osmolality (2.2.35) reference to the amount of D-antigen stated on the label, is
The osmolality of the vaccine is within the limits approved within the limits approved for the particular product.
for the particular preparation. Poliomyelitis vaccine (inactivated) BRP is calibrated in
European Pharmacopoeia Units and intended for use in the
IDENTIFICATION assay of D-antigen. The European Pharmacopoeia Unit and
A. Diphtheria toxoid is identified by a suitable the International Unit are equivalent.
In vivo test The vaccine complies with the in vivo assay of
poliomyelitis vaccine (inactivated) (2. 7. 20).
citrate R to give a 100 g/L solution. Maintain at 37 °C for LABELLING
about 16 h and centrifuge until a clear supernatant is The label states:
obtained. The clear supernatant reacts with a suitable - the minimum number of International Units of diphtheria
diphtheria antitoxin, giving a precipitate. If a satisfactory and tetanus toxoid per single human dose;
result is not obtained with a vaccine adsorbed on aluminium - the types of poliovirus contained in the vaccine;
hydroxide, carry out the test as follows. Centrifuge 15 mL of - the nominal amount of poliovirus of each type ( 1, 2 and
the vaccine to be examined and suspend the residue in 5 mL 3), expressed in European Pharmacopoeia Units of
of a freshly prepared mixture of 1 volume of a 56 g/L D-antigen, per single human dose;
solution of sodium edetate R and 49 volumes of disodium - the type of cells used for production of the poliomyelitis
hydrogen phosphate solution R. Maintain at 37 cc for not less component;
than 6 h and centrifuge. The clear supernatant reacts with a - the name and the amount of the adsorbent;
suitable diphtheria antitoxin, giving a precipitate. - that the vaccine must be shaken before use;
B. Tetanus toxoid is identified by a suitable immunochemical - that the vaccine is not to be frozen.
method (2. 7.1). The following method, applicable to certain _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
suitable tetanus antitoxin, giving a precipitate.
C. The vaccine is shown to contain human poliovirus
types 1, 2 and 3 by a suitable immunochemical method
(2. 7.1) such as the determination of D-antigen by enzyme-
linked immunosorbent assay (ELISA).
TESTS
Aluminium (2.5.13)
adsorbent.

Diphtheria, Tetanus, Pertussis vaccine may be stabilised by a method that has been shown
(Acellular, Component) and to have no effect on the assay procedure.
Poliomyelitis (Inactivated) Vaccine PRODUCTION OF THE COMPONENTS
(Adsorbed, Reduced Antigen(s) requirements of the monographs Diphtheria vaccine (adsorbed)
Content) (0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine
(acellular, component, adsorbed) (1356) and Polwmyelitis
(Ph. Bur. monograph 2329) vaccine (inactivated) (0214).
The label may state 'dTaP/IPV'. FINAL BULK VACCINE
~~----------------------
The final bulk vaccine is prepared by adsorption onto a
mineral carrier such as aluminium hydroxide or hydrated
DEFINITION aluminium phosphate, separately or together, of suitable
Diphtheria, tetanus, pertussis (acellular, component) and quantities of bulk purified diphtheria toxoid, tetanus toxoid
poliomyelitis (inactivated) vaccine (adsorbed, reduced and acellular pertussis components, and an admixture of
antigen(s) content) is a combined vaccine containing: suitable quantities of purified monovalent harvests of human
diphtheria formol toxoid; tetanus formol toxoid; individually poliovirus types 1, 2 and 3 or a suitable quantity of a
purified antigenic components of Bordetella pertussis; suitable trivalent pool of such purified monovalent harvests. Suitable
strains of human poliovirus types 1, 2 and 3 grown in antimicrobial preservatives may be added.
suitable cell cultures and inactivated by a validated method;
a mineral adsorbent such as aluminium hydroxide or
hydrated aluminium phosphate.
The formol toxoids are prepared from the toxins produced Bovine serwn albumin
by the growth of Corynebacterium diphtheriae and Clostridium Determined on the poliomyelitis component by a suitable
tetani respectively. immunochemical method (2. 7.1) after virus harvest and
before addition of the adsorbent in the preparation of the
The amount of diphtheria toxoid per single human dose is
final bulk vaccine, the amount of bovine serum albumin is
reduced compared to vaccines generally used for primary
such that the content in the final vaccine will be not more
vaccination; the amounts of tetanus toxoid and pertussis
than 50 ng per single human dose.
components may also be reduced.
detoxified pertussis toxin) or a pertussis toxin-like protein
free from toxic properties produced by expression of a
The vaccine may also contain filamentous haemagglutinin,
pertactin (a 69 kDa outer-membrane protein) and other Sterility (2. 6.1)
defined components of B. pertussis such as fimbrial-2 and Carry out the test for sterility using 10 mL for each medium.
fimbrial-3 antigens. The latter 2 antigens may be co-purified. FINAL LOT
The antigenic composition and characteristics are based on The final bulk vaccine is distributed aseptically into sterile,
evidence of protection and freedom from unexpected tamper-evident containers. The containers are closed so as to
reactions in the target group for which the vaccine is prevent contamination.
intended. Only a final lot that is satisfactory with respect to the test for
PRODUCTION osmolality and with respect to each of the requirements given
GENERAL PROVISIONS below under Identification, Tests and Assay may be released
The production method shall have been shown to yield for use.
consistently vaccines comparable with the vaccine of proven Provided the test for antimicrobial preservative and the assays
clinical efficacy and safety in man. for the diphtheria, tetanus and pertussis components have
The content of bacterial endotoxins (2. 6.14) in bulk purified been carried out with satisfactory results on the final bulk
diphtheria toxoid, tetanus toxoid, pertussis components and vaccine, they may be omitted on the final lot.
inactivated monovalent poliovirus harvests is determined to Provided the free formaldehyde content has been determined
monitor the purification procedure and to limit the amount on the bulk purified antigens or on the final bulk and it has
in the final vaccine. For each component, the content of been shown that the content in the final lot will not exceed
bacterial endotoxins is less than the limit approved for the 0.2 g/L, the test for free formaldehyde may be omitted on the
particular vaccine and, in any case, the contents are such that final lot.
the final vaccine contains less than 100 IU per single human Provided the determination of D-antigen content cannot be
dose. carried out on the final lot, it is carried out during
Reference vaccine(s) Provided valid assays can be performed, preparation of the final bulk before addition of the adsorbent.
monocomponent reference vaccines may be used for the Provided the in vivo assay for the poliomyelitis component
assays on the combined vaccine. If this is not possible has been carried out with satisfactory results on the final bulk
because of interaction between the components of the vaccine, it may be omitted on the final lot.
omitted once it has been demonstrated for a given vaccine
the D-antigen determination are such that it yields the same
of a batch. This demonstration must include testing of Tetanus component

subpotent batches, produced experimentally if necessary, for Carry out one of the prescribed methods for the assay of
example by heat treatment or other means of diminishing the tetanus vaccine (adsorbed) (2.7.8).
immunogenic activity. Where there is a significant change in The lower confidence limit (P = 0.95) of the estimated
the manufacturing process of the antigens or their potency is not less than 20 IU per single human dose.
formulation, any impact on the in vivo and in vitro assays
Pertussis component
must be evaluated, and the need for revalidation considered.
Osmolality (2.2.35) pertussis vaccine (acellular) (2.7.16). The vaccine complies
The osmolality of the vaccine is within the limits approved with the limit approved for the particular product.
for the particular preparation.
Poliomyelitis component
IDENTIFICATION D-antigen content As a measure of consistency of
A. Diphtheria toxoid is identified by a suitable production, determine the D-antigen content for human
immunochemical method (2.7.1). The following method, poliovirus types 1, 2 and 3 by a suitable immunochemical
applicable to certain vaccines, is given as an example. method (2. 7.1) following desorption, using a reference
Dissolve in the vaccine to be examined sufficient sodium preparation calibrated in European Pharmacopoeia Units of
citrate R to give a 100 g/L solution. Maintain at 37 °C for D-antigen. For each type, the content, expressed with
about 16 h and centrifuge until a clear supernatant is reference to the amount of D-antigen stated on the label, is
obtained. The clear supernatant reacts with a suitable within the limits approved for the particular product.
diphtheria antitoxin, giving a precipitate. If a satisfactory Poliomyelitis vaccine (inactivated) BRP is calibrated in
result is not obtained with a vaccine adsorbed on aluminium European Pharmacopoeia Units and intended for use in the
hydroxide, carry out the test as follows. Centrifuge 15 mL of assay of D-antigen. The European Pharmacopoeia Unit and
the vaccine to be examined and suspend the residue in 5 mL the International Unit are equivalent.
of a freshly prepared mixture of 1 volume of a 56 g/L In vivo test The vaccine complies with the in vivo assay of
solution of sodium edetate R and 49 volumes of a disodium poliomyelitis vaccine (inactivated) (2. 7.20).
hydrogen phosphate solution R. Maintain at 37 °C for not less
than 6 h and centrifuge. The clear supernatant reacts with a LABELLING
suitable diphtheria antitoxin, giving a precipitate. The label states:
and tetanus toxoid per single human dose;
- the names and amounts of the pertussis components per
single human dose;
- where applicable, that the vaccine contains a pertussis
suitable tetanus antitoxin, giving a precipitate.
toxin-like protein produced by genetic modification;
C. The pertussis components are identified by a suitable - the types of poliovirus contained in the vaccine;
immunochemical method (2. 7.1). The following method, - the nominal amount ofpoliovirus of each type (1, 2 and
applicable to certain vaccines, is given as an example. 3), expressed in European Pharmacopoeia Units of
The clear supernatant obtained as described in identification D-antigen, per single human dose;
test A reacts with antisera specific to the pertussis - the type of cells used for production of the poliomyelitis
components of the vaccine. component;
D. The vaccine is shown to contain human poliovirus types - the name and the amount of the adsorbent;
1, 2 and 3 by a suitable immunochemical method (2. 7.1) - that the vaccine must be shaken before use;
such as the determination of D-antigen by enzyme-linked - that the vaccine is not to be frozen.
immunosorbent assay (ELISA).
- - - - - - - - - - - - - - - - - - - - - - Ph Eur
TESTS
Aluminium (2.5.13)
hydroxide or hydrated aluminium phosphate is used as the Diphtheria, Tetanus, Pertussis
adsorbent.
Free formaldehyde (2. 4.18)
(Acellular, Component),
Maximum 0.2 g/L. Poliomyelitis (Inactivated) and
Antimicrobial preservative Haemophilus Type b Conjugate
preservative by a suitable chemical method. The content is Vaccine (Adsorbed)
not less than the minimum amount shown to be effective and (Ph. Bur. monograph 2065)
The label may state 'DTaP/IPV/Hib'.
label.
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Sterility (2. 6.1)
The vaccine complies with the test for sterility. DEFINITION
Diphtheria, tetanus, pertussis (acellular, component),
ASSAY
poliomyelitis (inactivated) and haemophilus type b conjugate
vaccine (adsorbed) is a combined vaccine composed of:
diphtheria formol toxoid; tetanus formol toxoid; individually
diphtheria vaccine (adsorbed) (2. 7.6).
purified antigenic components of Bordetella pertussis; suitable
The lower confidence limit (P = 0. 95) of the estimated strains of human poliovirus types 1, 2 and 3 grown in
potency is not less than 2 IU per single human dose. suitable cell cultures and inactivated by a suitable method;
polyribosylribitol phosphate (PRP) covalently bound to a 0.025 µg of PRP for a vaccine with OMP (meningococcal
carrier protein; a mineral adsorbent such as aluminium group B outer membrane protein complex) as carrier.
hydroxide or hydrated aluminium phosphate. The product is Reference vaccine(s) Provided valid assays can be performed,
presented either as a pentavalent liquid formulation in the monocomponent reference vaccines may be used for the
same container, or as a tetravalent liquid formulation with assays on the combined vaccine. If this is not possible
the freeze-dried haemophilus component in a separate because of interaction between the components of the
container, the contents of which are mixed with the other combined vaccine or because of differences in composition
components immediately before use. between the monocomponent reference vaccine and the test
The formol toxoids are prepared from the toxins produced vaccine, a batch of combined vaccine shown to be effective in
by the growth of Corynebacterium diphtheriae and Clostridium clinical trials or a batch representative thereof is used as a
tetani respectively. reference vaccine. For the preparation of a representative
The vaccine contains either pertussis toxoid (chemically batch, strict adherence to the production process used for the
detoxified pertussis toxin) or a pertussis toxin-like protein batch tested in clinical trials is necessary. The reference
free from toxic properties produced by expression of a vaccine may be stabilised by a method that has been shown
genetically modified form of the corresponding gene. to have no effect on the assay procedure.
The acellular pertussis component may also contain PRODUCTION OF THE COMPONENTS
filamentous haemagglutinin, pertactin (a 69 kDa outer- The production of the components complies with the
membrane protein) and other defined components of requirements of the monographs Diphtheria vaccine ( adsorbed)
B. pertussis such as fimbrial-2 and fimbrial-3 antigens. (0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine
The latter 2 antigens may be co-purified. The antigenic ( acellular, component, adsorbed) (1356), Poliomyelitis vaccine
composition and characteristics are based on evidence of (inactivated) (0214) and Haemophilus type b conjugate vaccine
protection and freedom from unexpected reactions in the (1219).
target group for which the vaccine is intended. FINAL BULKS
PRP is a linear copolymer composed of repeated units of The final tetravalent bulk of the diphtheria, tetanus, pertussis
3-~-D-ribofuranosyl-( 1 ➔ 1)-ribitol-5-phosphate and poliomyelitis components is prepared by adsorption,
[(C 10H 19OJIP)nl, with a defined molecular size and derived separately or together, of suitable quantities of bulk purified
from a suitable strain of Haemophilus infiuenzae type b. diphtheria toxoid, bulk purified tetanus toxoid and bulk
The carrier protein, when conjugated to PRP, is capable of purified acellular pertussis components onto a mineral carrier
inducing a T-cell-dependent B-cell immune response to the such as aluminium hydroxide or hydrated aluminium
polysaccharide. phosphate, and admixture of suitable quantities of purified,
PRODUCTION monovalent harvests of human poliovirus types 1, 2 and 3 or
GENERAL PROVISIONS a suitable quantity of a trivalent pool of such monovalent
The production method shall have been shown to yield harvests. Suitable antimicrobial preservatives may be added.
consistently vaccines comparable with the vaccine of proven Where the vaccine is presented with all 5 components in the
clinical efficacy and safety in man. same container, the final bulk is prepared by addition of a
The content of bacterial endotoxins (2. 6.14) in bulk purified suitable quantity of the haemophilus bulk conjugate to the
diphtheria toxoid, tetanus toxoid, pertussis components, tetravalent bulk. Where the haemophilus component is
purified, inactivated monovalent poliovirus harvests and bulk presented in a separate container, the final bulk is prepared
PRP conjugate is determined to monitor the purification by dilution of the bulk conjugate with suitable diluents for
procedure and to limit the amount in the final vaccine. freeze-drying. A stabiliser may be added.
For each component, the content of bacterial endotoxins is Only final bulks that comply with the following requirements
less than the limit approved by the competent authority for may be used in the preparation of the final lot.
the particular vaccine. Bovine serum albumin
During development studies, it shall be demonstrated that Determined on the poliomyelitis component by a suitable
the vaccine consistently induces a T-cell-dependent B-cell immunochemical method (2. 7.1) during preparation of the
immune response to PRP. If the manufacturing process is final bulk vaccine, before addition of the adsorbent, the
modified, it shall be demonstrated by appropriate in vitro amount of bovine serum albumin is such that the content in
methods that the characteristic properties of the conjugate the final vaccine will be not more than 50 ng per single
are not affected. 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 carried 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. Carry out the test for sterility using 10 mL for each medium.
FINAL LOT
container, the production method is validated to demonstrate
that the haemophilus component, if tested, would comply
container, the final bulk of the haemophilus component is
with the test for pyrogens (2.6.8), carried out as follows:
freeze-dried.
inject per kilogram of the rabbit's mass a quantity of the
vaccine equivalent to: 1 µg 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 µg of PRP for a vaccine with tetanus toxoid as carrier; requirements given below under Identification, Tests and
Assay may be released for use.
Provided that the test for antimicrobial preservative and the obtained during identification test A reacts with a suitable
assay have been carried out with satisfactory results on the tetanus antitoxin, giving a precipitate.
final bulk vaccine, they may be omitted on the final lot. C. The pertussis components are identified by a suitable
Provided that the free formaldehyde content has been immunochemical method (2.7.1). The following method,
determined on the bulk purified antigens and the purified applicable to certain vaccines, is given as an example.
monovalent harvests or the trivalent pool of polioviruses or The clear supernatant obtained during identification test A
the final bulk and it has been shown that the content in the reacts with antisera specific to the pertussis components of
final lot will not exceed 0.2 g/L, the test for free the vaccine.
formaldehyde may be omitted on the final lot. D. The vaccine is shown to contain human poliovirus
If the in vivo assay for the poliomyelitis component is used, types 1, 2 and 3 by a suitable immunochemical
provided it has been carried out with satisfactory results on method (2. 7.1), such as determination of D-antigen by
the final bulk vaccine, it may be omitted on the final lot. enzyme-linked immunosorbent assay (ELISA).
The in vivo assay for the poliomyelitis component may be E. The haemophilus component is identified by a suitable
omitted once it has been demonstrated for a given product immunochemical method (2.7.1) for PRP.
TESTS
the D-antigen determination are such that it yields the same
container, the tests for aluminium, free formaldehyde,
of a batch. This demonstration must include testing of
antimicrobial, preservative and sterility are carried out on the
subpotent batches, produced experimentally if necessary, for
container with the diphtheria, tetanus, pertussis and poliomyelitis
example by heat treatment or other means of diminishing the
components; the tests for PRP, water, sterility and bacterial,
immunogenic activity. Where there is a significant change in
entodoxins are carried out on the container with the haemophilus
the manufacturing process of the antigens or their
component alone.
formulation, any impact on the in vivo and in vitro assays
must be evaluated, and the need for revalidation considered. Where the haemophilus component is presented in a separate
container, some tests may be carried out on the freeze-dried
Osmolality (2.2.35) product rather than on the bulk conjugate where the freeze-drying
The osmolality of the vaccine, reconstituted where applicable, process may affect the component to be tested.
is within the limits approved for the particular preparation.
PRP
Free PRP Not less than 80 per cent of the amount of PRP stated on
Where the haemophilus component is presented in liquid the label. PRP is determined either by assay of
formulation, the presence of other components may interfere ribose (2.5.31) or phosphorus (2.5.18), by an
in the assay and it may not be possible to separate the PRP immunochemical method (2. 7.1) or by anion-exchange liquid
from the adjuvant. The presence of free PRP may be chromatography (2.2.29) with pulsed amperometric
determined on the bulk conjugate prior to the addition of detection.
other components or on the non-adsorbed fraction in the
final combination. Aluminium (2.5.13)
container, a number of methods have been used to separate
adsorbent.
free PRP from the conjugate, including precipitation, gel
filtration, size-exclusion, anion-exchange and hydrophobic Free formaldehyde (2.4.18)
chromatography, ultrafiltration and ultracentrifugation. Maximum 0.2 g/L.
The free PRP can then be quantified by a range of Antimicrobial preservative
techniques, including high-performance anion-exchange Where applicable, determine the amount of antimicrobial
chromatography with pulsed amperometric detection preservative by a suitable chemical method. The content is
(HPAEC-PAD) and immunoassays with anti-PRP not less than the minimum amount shown to be effective and
antibodies. The amount of free PRP is not greater than that is not greater than 115 per cent of the quantity stated on the
approved for the particular product. label.
IDENTIFICATION Water (2.5.12)
Identification tests A, B, C and D are carried out using the vial, Maximum 3.0 per cent for the freeze-dried haemophilus
containing the diphtheria, tetanus, pertussis and poliomyelitis component.
components; identification test E is carried out either on the vial Sterility (2. 6.1)
containing all 5 components, or on the vial containing the The vaccine complies with the test for sterility.
haemophilus component alone.
Bacterial endotoxins (2. 6.14)
A. Diphtheria toxoid is identified by a suitable The content is within the limits approved by the competent
immunochemical method (2.7.1). The following method, authority for the haemophilus component of the particular
applicable to certain vaccines, is given as an example. product. If any components of the vaccine prevent the
Dissolve in the vaccine to be examined sufficient sodium determination of endotoxin, a test for pyrogens is carried out
citrate R to give a 100 g/L solution. Maintain at 37 °C for as described under General provisions.
obtained. The clear supernatant reacts with a suitable ASSAY
diphtheria antitoxin, giving a precipitate. Diphtheria component
IV- 728 Vaccines 2023
Unless otherwise justified and authorised, the lower

confidence limit (P = 0.95) of the estimated potency is not
Diphtheria, Tetanus, Pertussis
less than 30 IU per single human dose. (Acellular, Component),
Tetanus component Hepatitis B (rDNA), Poliomyelitis
tetanus vaccine (adsorbed) (2.7.8). (Inactivated) and Haemophilus
The lower confidence limit (P = 0.95) of the estimated Type b Conjugate Vaccine
Pertussis component
(Adsorbed)
Carry out one of the prescribed methods for the assay of (Ph. Bur. monograph 2067)
pertussis vaccine (acellular) (2.7.16). The vaccine complies The label may state 'DTaP/HepB/IPV/Hib'.
with the limit approved for the particular product. PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Poliomyelitis component
D-antigen content As a measure of consistency of
DEFINITION
production, determine the D-antigen content for human Diphtheria, tetanus, pertussis (acellular, component),
poliovirus types 1, 2 and 3 by a suitable immunochemical hepatitis B (rDNA), poliomyelitis (inactivated) and
method (2. 7. 1) following desorption, using a reference haemophilus type b conjugate vaccine (adsorbed) is a
preparation calibrated in European Pharmacopoeia Units of combined vaccine composed of: diphtheria formol toxoid;
D-antigen. For each type, the content, expressed with tetanus formal toxoid; individually purified antigenic
reference to the amount of D-antigen stated on the label, is components of Bordetella pertussis; hepatitis B surface antigen
within the limits approved for the particular product. (HBsAg); human poliovirus types 1, 2 and 3 grown in
Polwmyelitis vaccine (inactivated) ERP is calibrated in suitable cell cultures and inactivated by a suitable method;
European Pharmacopoeia Units and intended for use in the polyribosylribitol phosphate (PRP) covalently bound to a
assay of D-antigen. The European Pharmacopoeia Unit and carrier protein. The antigens in the vaccine may be adsorbed
the International Unit are equivalent. on a mineral carrier such as aluminium hydroxide or
hydrated aluminium phosphate. The product is presented
In vivo test The vaccine complies with the in vivo assay of
either as a hexavalent liquid formulation in the same
poliomyelitis vaccine (inactivated) (2. 7. 20).
container, or as a pentavalent liquid formulation with the
LABELLING haemophilus component in a separate container, the contents
The label states: of which are mixed with the other components immediately
- the minimum number of International Units of diphtheria before or during use.
and tetanus toxoid per single human dose; The formal toxoids are prepared from the toxins produced
- the names and amounts of the pertussis components per by the growth of Corynebacterium diphtheriae and Clostridium
single human dose; tetani respectively.
- where applicable, that the vaccine contains a pertussis- The vaccine contains either pertussis toxoid (chemically
toxin-like protein produced by genetic modification; detoxified pertussis toxin) or a pertussis toxin-like protein
- the nominal amount of poliovirus of each type ( 1, 2 free from toxic properties produced by expression of a
and 3), expressed in European Pharmacopoeia Units of genetically modified form of the corresponding gene.
D-antigen, per single human dose; The acellular pertussis component may also contain
- the type of cells used for production of the poliomyelitis filamentous haemagglutinin, pertactin (a 69 kDa outer-
component; membrane protein) and other defined components of
- the number of micrograms of PRP per single human B. pertussis such as fimbrial-2 and fimbrial-3 antigens.
dose; The latter 2 antigens may be co-purified. The antigenic
- the type and nominal amount of carrier protein per single composition and characteristics are based on evidence of
human dose; protection and freedom from unexpected reactions in the
- the name and the amount of the adsorbent; target group for which the vaccine is intended.
vaccination of children and is not necessarily suitable for Hepatitis B surface antigen is a component protein of
reinforcing doses or for administration to adults; hepatitis B virus; the antigen is obtained by recombinant
- that the vaccine must be shaken before use; DNA technology.
- that the vaccine is not to be frozen. PRP is a linear copolymer composed of repeated units of
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur 3-~-o-ribofuranosyl-(1 ➔ 1)-ribitol-5-
phosphate [(C 10H 190 11 P)nl, with a defined molecular size
and derived from a suitable strain of Haemophilus injluenzae
type b. The carrier protein, when conjugated to PRP, is
capable of inducing a T-cell-dependent B-cell immune
response to the polysaccharide.
PRODUCTION
GENERAL PROVISIONS
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
assays of the diphtheria, tetanus, pertussis, hepatitis B and
poliomyelitis components are carried out on a suitable hepatitis B and poliovirus component is prepared by
number of batches of vaccine reconstituted as for use. adsorption, separately or together, of suitable quantities of
For subsequent routine control, the assays of these bulk purified diphtheria toxoid, tetanus toxoid, acellular
components may be carried out without mixing with the pertussis components and hepatitis B surface antigen onto a
haemophilus component. mineral carrier such as aluminium hydroxide or hydrated
The content of bacterial endotoxins (2. 6. 14) in bulk purified aluminium phosphate and admixture of suitable quantities of
diphtheria toxoid, tetanus toxoid and pertussis components, purified and inactivated, monovalent harvests of human
hepatitis B surface antigen, purified, inactivated monovalent poliovirus types 1, 2 and 3 or a suitable pool of such
poliovirus harvests and bulk PRP conjugate is determined to monovalent harvests. This final bulk is filled separately.
monitor the purification procedure and to limit the amount Suitable antimicrobial preservatives may be added. The final
in the final vaccine. For each component, the content of bulk of the haemophilus component is prepared by dilution
bacterial endotoxins is not greater than the limit approved. of the bulk conjugate to the final concentration with a
During development studies and wherever revalidation is suitable diluent. A stabiliser may be added.
necessary, a test for pyrogens in rabbits (2. 6. 8) is carried out Only final bulks that comply with the following requirements
by injection of a suitable dose of the final lot. The vaccine is may be used in the preparation of the final lot.
shown to be acceptable with respect to absence of pyrogenic Bovine serum albumin
activity. Determined on the poliomyelitis component by a suitable
During development studies, it shall be demonstrated that immunochernical method (2. 7.1) after purification of the
the vaccine consistently induces a T-cell-dependent B-cell harvests and before preparation of the final bulk vaccine,
immune response to PRP. If the manufacturing process is before addition of the adsorbent, the amount of bovine
modified, it shall be demonstrated by appropriate in vitro serum albumin is such that the content in the final vaccine
methods that the characteristic properties of the conjugate will be not more than 50 ng per single human dose.
are not affected. Antimicrobial preservative
The stability of the final lot and relevant intermediates is Where applicable, determine the amount of antimicrobial
evaluated using one or more indicator tests. For the preservative by a suitable chemical method. The amount is
haemophilus component, such tests may include not less than 85 per cent and not greater than 115 per cent
determination of molecular size, determination of free PRP in of the intended content.
the conjugate and kinetics of depolymerisation. Taking Sterility (2. 6.1)
account of the results of the stability testing, release Carry out the test for sterility using 10 mL for each medium.
requirements are set for these indicator tests to ensure that
FINAL LOT
the vaccine will be satisfactory at the end of the shelf life.
Where the haemophilus component is in a separate
Reference vaccine(s) Provided valid assays can be performed, container, the final bulk of the haemophilus component is
monocomponent reference vaccines may be used for the freeze-dried. Only a final lot that is satisfactory with respect
assays on the combined vaccine. If this is not possible to the test for osmolality shown below and with respect to
because of interaction between the components of the each of the requirements given below under Identification,
combined vaccine or because of differences in composition Tests and Assay may be released for use.
Provided that the test for osmolality, the test for
antimicrobial preservative and the assays for the diphtheria,
tetanus and pertussis components have been carried out with
satisfactory results on the final bulk vaccine, they may be
vaccine may be stabilised by a method that has been shown Provided the free formaldehyde content has been determined
to have no effect on the assay procedure. on the bulk purified antigens and the purified monovalent
harvests or the trivalent pool of polioviruses or 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
requirements of the monographs Diphtheria vaccine ( adsorbed)
(0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine
(acellular, component, adsorbed) (1356), Hepatitis B vaccine Provided that the test for bovine serum albumin has been
(rDNA) (1056), Poliomyelitis vaccine (inactivated) (0214) and carried out with satisfactory results on the trivalent pool of
Haemophilus type b conjugate vaccine (1219). inactivated monovalent harvests of polioviruses or on the
final bulk vaccine, it may be omitted on the final lot.
FINAL BULKS
Vaccine with all components in the same container The final
If an in viva assay is used for the hepatitis B component,
bulk is prepared by adsorption, separately or together, of provided it has been carried out with satisfactory results on
suitable quantities of bulk purified diphtheria toxoid, tetanus the final bulk vaccine, it may be omitted on the final lot.
toxoid, acellular pertussis components and hepatitis B surface Provided the in viva assay for the poliomyelitis component
antigen onto a mineral carrier such as aluminium hydroxide has been carried out with satisfactory results on the final bulk
or hydrated aluminium phosphate and admixture of a vaccine, it may be omitted on the final lot.
suitable quantity of PRP conjugate and suitable quantities of The in viva assay for the poliomyelitis component may be
purified and inactivated, monovalent harvests of human omitted once it has been demonstrated for a given product
poliovirus types 1, 2 and 3 or a suitable quantity of a and for each poliovirus type that the acceptance criteria for
trivalent pool of such monovalent harvests. Suitable the D-antigen determination are such that it yields the same
antimicrobial preservatives may be added. result as the in viva assay in terms of acceptance or rejection
Vaccine with the haemophilus component in a separate of a batch. This demonstration must include testing of
container The final bulk of diphtheria, tetanus, pertussis, subpotent batches, produced experimentally if necessary, for
example by heat treatment or other means of diminishing the PRP

immunogenic activity. Where there is a significant change in Minimum 80 per cent of the amount of PRP stated on the
the manufacturing process of the antigens or their label, for a vaccine with the haemophilus component in a
formulation, any impact on the in viva and in vitro assays separate container.
must be evaluated, and the need for revalidation considered. For a vaccine with all components in the same container: the
FreePRP PRP content determined on the non-absorbed fraction is not
For vaccines with all components in the same container, the less than that approved for the product.
free PRP content is determined on the non-absorbed PRP is determined either by assay of ribose (2.5.31) or
fraction. Unbound PRP is determined on the haemophilus phosphorus (2.5.18), by an immunochemical method (2.7.1)
component after removal of the conjugate, for example by or by anion-exchange liquid chromatography (2.2.29) with
anion-exchange, size-exclusion or hydrophobic pulsed amperometric detection.
chromatography, ultrafiltration or other validated methods. Aluminium (2.5.13)
The amount of free PRP is not greater than that approved Maximum 1.25 mg per single human dose, if aluminium
for the particular product. hydroxide or hydrated aluminium phosphate is used as the
Bacterial endotoxins (2. 6.14) adsorbent.
Less than the limit approved for the product concerned. Free formaldehyde (2.4.18)
Osmolality (2.2.35) Maximum 0.2 g/L of free formaldehyde per single human
The osmolality of the vaccine, reconstituted where applicable, dose.
is within the limits approved for the particular preparation. Antimicrobial preservative
IDENTIFICATION Where applicable, determine the amount of antimicrobial
If the vaccine is presented with the haemophilus component in a preservative by a suitable chemical method. The content is
separate container: identification tests A, B, C, D and E are not less than the minimum amount shown to be effective and
carried out using the container with the diphtheria, tetanus, is not greater than 115 per cent of the quantity stated on the
pertussis, hepatitis B and polwmyelitis components; identificatwn label.
test F is carried out on the container with the haemophilus Water (2.5.12)
components. Maximum 3.0 per cent for the freeze-dried haemophilus
A. Diphtheria toxoid is identified by a suitable component.
immunochemical method (2.7.1). The following method is Sterility (2. 6.1)
given as an example. Dissolve in the vaccine to be examined The vaccine complies with the test for sterility.
sufficient sodium citrate R to give a 100 g/L solution.
Maintain at 37 °C for about 16 h and centrifuge until a clear ASSAY
supernatant is obtained. The clear supernatant reacts with a Diphtheria component
suitable 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 is given as an The lower confidence limit (P = 0.95) of the estimated
example. The clear supernatant obtained during potency is not less than the minimum potency stated on the
identification test A reacts with a suitable tetanus antitoxin, label.
giving a precipitate. Unless otherwise justified and authorised, the minimum
C. The clear supernatant obtained during identification potency stated on the label is 30 IU per single human dose.
test A reacts with antisera specific to the pertussis Tetanus component
components of the vaccine when examined by suitable Carry out one of the prescribed methods for the assay of
immunochemical methods (2. 7.1). tetanus vaccine (adsorbed) (2. 7. 8).
D. The hepatitis B component is identified by a suitable The lower confidence limit (P = 0. 95) of the estimated
immunochemical method (2. 7.1), for example the in vitro potency is not less than 40 IU per single human dose.
assay, or by a suitable electrophoretic method (2.2.31). Pertussis 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 pertussis vaccine (acellular) (2.7.16). The vaccine complies
method (2. 7.1), such as determination of D-antigen by with the limit approved for the particular product.
enzyme-linked immunosorbent assay (ELISA).
F. The PRP and its carrier protein are identified by a suitable The vaccine complies with the assay of hepatitis B
immunochemical method (2. 7.1). vaccine (2.7.15).
TESTS Poliomyelitis component
If the product is presented with the haemophilus component in a D-antigen content As a measure of consistency of
separate container, the tests for free formaldehyde, aluminium, production, determine the D-antigen content for human
antimicrobial preservative and sterility are carried out on the poliovirus types 1, 2 and 3 by a suitable immunochemical
container with the diphthen·a, tetanus, pertussis, polwmyelitis and method (2. 7.1) following desorption, using a reference
hepatitis B components; the tests for PRP, water, antimicrobial preparation calibrated in European Pharmacopoeia Units of
preservative (where applicable), aluminium (where applicable) D-antigen. For each type, the content, expressed with
and sterility are carried out on the container with the haer,wphilus reference to the amount of D-antigen stated on the label, is
component. within the limits approved for the particular product.
Some tests for the haemophilus component are carried out on the Polwmyelitis vaccine (inactivated) BRP is calibrated in
freeze-dried product rather than on the bulk conjugate where the European Pharmacopoeia Units and intended for use in the
freeze-drying process may affect the component to be tested.
assay of D-antigen. The European Pharmacopoeia Unit and PRP is a linear copolymer composed of repeated units of
the International Unit are equivalent. 3-~-D-ribofuranosyl-( 1-+ 1)-ribitol-5-phosphate
In viva test The vaccine complies with the in viva assay of [(C10H19O11P)nl, with a defined molecular size and derived
poliomyelitis vaccine (inactivated) (2.7.20). from a suitable strain of Haemophilus infiuenzae type b.
The carrier protein, when conjugated to PRP, is capable of
LABELLING inducing a T-cell-dependent B-cell immune response to the
The label states: polysaccharide.
and tetanus toxoid per single human dose; PRODUCTION
- the names and amounts of the pertussis components per GENERAL PROVISIONS
single human dose; The production method shall have been shovm to yield
- where applicable, that the vaccine contains a pertussis consistently vaccines comparable with the vaccine of proven
toxin-like protein produced by genetic modification; clinical efficacy and safety in man.
- the amount of HBsAg per single human dose; During development studies, it shall be demonstrated that
- the nominal amount of poliovirus of each type (1, 2 and the vaccine consistently induces a T-cell-dependent B-cell
3), expressed in European Pharmacopoeia Units of immune response to PRP. If the manufacturing process is
D-antigen, per single human dose; modified, it shall be demonstrated by appropriate in vitro
- the types of cells used for production of the poliomyelitis methods that the characteristic properties of the conjugate
and the hepatitis B components; are not affected.
- the number of micrograms of PRP per single human As part of consistency studies the assays of the diphtheria,
dose; tetanus, pertussis and poliomyelitis components are carried
- the type and nominal amount of carrier protein per single out on a suitable number of batches of vaccine reconstituted
human dose; as for use. For subsequent routine control, the assays of these
- the name and the amount of the adsorbent; components may be carried out without mixing with the
- where applicable, that the vaccine is intended for primary haemophilus component.
vaccination of children and is not necessarily suitable for For the haemophilus component, the production method is
reinforcing doses or for administration to adults; validated to demonstrate that the haemophilus component, if
- that the vaccine must be shaken before use; tested, would comply with the test for pyrogens (2. 6. 8),
- that the vaccine is not to be frozen. carried out as follows: inject per kilogram of the rabbit's mass
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur a quantity of the vaccine equivalent to: 1 µg of PRP for a
vaccine with diphtheria toxoid or CRM 197 diphtheria
protein as carrier; 0 .1 µg of PRP for a vaccine with tetanus
toxoid as carrier; 0.025 µg of PRP for a vaccine with OMP
Diphtheria, Tetanus, Pertussis (meningococcal group B outer membrane protein complex)
as earner.
(Whole Cell), Poliomyelitis Reference vaccine(s) Provided valid assays can be performed,
(Inactivated) and Haemophilus monocomponent reference vaccines may be used for the
assays on the combined vaccine. If this is not possible
Type b Conjugate Vaccine because of interaction between the components of the
(Adsorbed) combined vaccine or because of the difference in composition
Diphtheria, Tetanus, Pertussis, Poliomyelitis between monocomponent reference vaccine and the test
(Inactivated) and Haemophilus Type b Conjugate
Vaccine (Adsorbed) clinical trials or a batch representative thereof is used as a
(Ph. Bur. monograph 2066) batch, strict adherence to the production process used for the
The label may state 'DTwP/IPV/Hib'. batch tested in clinical trials is necessary. The reference
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ vaccine may be stabilised by a method that has been shown
DEFINITION
Diphtheria, tetanus, pertussis (whole cell), poliomyelitis PRODUCTION OF THE COMPONENTS
(inactivated) and haemophilus type b conjugate vaccine The production of the components complies with the
(adsorbed) is a combined vaccine composed of: diphtheria requirements of the monographs Diphtheria vaccine
formol toxoid; tetanus formol toxoid; an inactivated (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452), Pertussis
suspension of Bordetella pertussis; suitable strains of human vaccine (whole cell, adsorbed) (0161), Poliomyelitis vaccine
poliovirus types 1, 2 and 3 grown in suitable cell cultures and (inactivated) (0214) and Haemophilus type b conjugate
inactivated by a suitable method; polyribosylribitol phosphate vaccine (1219).
(PRP) covalently bound to a carrier protein; a mineral FINAL BULKS
adsorbent such as aluminium hydroxide or hydrated The final bulk of the diphtheria, tetanus, pertussis and
aluminium phosphate. The product is presented with the poliomyelitis components is prepared by adsorption,
haemophilus component in a separate container, the contents separately or together, of suitable quantities of bulk purified
of which are mixed with the other components immediately diphtheria toxoid, and bulk purified tetanus toxoid onto a
before use. mineral carrier such as aluminium hydroxide or hydrated
The formol toxoids are prepared from the toxins produced aluminium phosphate and admixture of suitable quantities of
by the growth of Corynebacterium diphtheriae and Clostridium an inactivated suspension of B. pertussis and of purified,
tetani respectively. monovalent harvests of human poliovirus types 1, 2 and 3 or
a suitable quantity of a trivalent pool of such monovalent
harvests. Suitable antimicrobial preservatives may be added.
The final bulk of the haemophilus component is prepared by IDENTIFICATION

dilution of the bulk conjugate to the final concentration with Identification tests A, B, C and Dare 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 A. Diphtheria toxoid is identified by a suitable
Determined on the poliomyelitis component by a suitable immunochemical method (2.7.J). The following method,
immunochemical method (2. 7. J) 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. The clear supernatant reacts with a suitable
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. J). 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
tetanus antitoxin, giving a precipitate.
Sterility (2. 6. J)
Carry out the test for sterility using 10 mL for each medium. C. The centrifugation residue obtained in identification A
may be used. Other suitable methods for separating the
FINAL LOT 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. penussis 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. J), such as determination of D-antigen by enzyme-linked
the diphtheria, tetanus and pertussis components have been immunosorbent assay (ELISA).
carried 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.J) for PRP.
TESTS
on the bulk purified antigens, the inactivated B. pertussis
The tests for specific toxicity of the penussis component,
suspension and the purified monovalent harvests or the
aluminium, free formaldehyde, antimicrobial preservative and
trivalent pool of polioviruses or on the final bulk and it has
sterility are carried out on the container with diphtheria, tetanus,
been shown that the content in the final lot will not exceed
penussis and poliomyelitis components; the tests for PRP, water,
sterility and bacterial endotoxins are carried out on the container
final lot.
with the haemophilus component.
Provided the in vivo assay for the poliomyelitis component
Some tests for the haemophilus component may be carried out on
the freeze-dried product rather than on the bulk conjugate where
vaccine, it may be omitted on the final lot.
the freeze-drying process may affect the component to be tested.
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 that 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 viva 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
antimicrobial preservative as that injected with the vaccine.
Osmolality (2.2.35) 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
FreePRP 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.
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 (2.5.31) or - the minimum number of International Units of diphtheria
phosphorus (2.5.18), 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 (2.5.13) - 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
component;
Free formaldehyde (2. 4.18)
- 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 - the name and the amount of the adsorbent;
not less than the minimum amount shown to be effective and - where applicable, that the vaccine is intended for primary
is not greater than 115 per cent of the quantity stated on the vaccination of children and is not necessarily suitable for
label. reinforcing doses or for administration to adults;
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)
The vaccine complies with the test for sterility.
The content is within the limits approved by the competent
authority for the haemophilus component of the particular Diphtheria, Tetanus, Pertussis
product. If any components of the vaccine prevent the (Whole Cell) and Poliomyelitis
determination of endotoxin, a test for pyrogens is carried out
as described under General provisions. (Inactivated) Vaccine (Adsorbed)
ASSAY Diphtheria, Tetanus, Pertussis and Poliomyelitis
Diphtheria component (Inactivated) Vaccine (Adsorbed)
Carry out one of the prescribed methods for the assay of (Ph. Bur. monograph 2061)
diphtheria vaccine (adsorbed) (2. 7. 6). The label may state 'DTwP/IPV'.
potency is not less than 30 JU per single human dose.
DEFINITION
Tetanus component
Diphtheria, tetanus, pertussis (whole cell) and poliomyelitis
(inactivated) vaccine (adsorbed) is a combined vaccine
tetanus vaccine (adsorbed) (2.7.8).
containing: diphtheria formol toxoid; tetanus formol toxoid;
If the test is carried out in guinea-pigs, the lower confidence an inactivated suspension of Bordetella pertussis; suitable
limit (P = 0.95) of the estimated potency is not less than strains of human poliovirus types 1, 2 and 3 grown in
40 JU per single human dose; if the test is carried out in suitable cell cultures and inactivated by a validated method;
mice, the lower confidence limit (P = 0.95) of the estimated a mineral adsorbent such as aluminium hydroxide or
potency is not less than 60 JU per single human dose. hydrated aluminium phosphate.
Pertussis component The formol toxoids are prepared from the toxins produced
Carry out the assay of pertussis vaccine (whole cell) (2. 7. 7). by the growth of Corynebacten·um diphtheriae and Clostridium
The estimated potency is not less than 4.0 JU per single tetani respectively.
human dose and the lower confidence limit (P = 0.95) of the PRODUCTION
estimated potency is not less than 2.0 JU per single human
GENERAL PROVISIONS
dose.
Poliomyelitis component consistently vaccines comparable with the vaccine of proven
D-antigen content As a measure of consistency of clinical efficacy and safety in man.
production, determine the D-antigen content for human Reference vaccine(s) Provided valid assays can be performed,
poliovirus types 1, 2 and 3 by a suitable immunochemical monocomponent reference vaccines may be used for the
method (2. 7.1) following desorption using a reference assays on the combined vaccine. If this is not possible
preparation calibrated in European Pharmacopoeia Units of because of interaction between the components of the
D-antigen. For each type, the content, expressed with combined vaccine or because of the difference in composition
reference to the amount of D-antigen stated on the label, is between monocomponent reference vaccine and the test
within the limits approved for the particular product. vaccine, a batch of combined vaccine shown to be effective in
Poliomyelitis vaccine (inactivated) ERP is calibrated in clinical trials or a batch representative thereof is used as a
European Pharmacopoeia Units and intended for use in the reference vaccine. For the preparation of a representative
assay of D-antigen. The European Pharmacopoeia Unit and batch, strict adherence to the production process used for the
the International Unit are equivalent. batch tested in clinical trials is necessary. The reference
Jn vivo test The vaccine complies with the in vivo assay of
poliomyelitis vaccine (inactivated) (2. 7.20).
vaccine may be stabilised by a method that has been shown formulation, any impact on the in vivo and in vitro assays
to have no effect on the assay procedure. 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 carrier 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
Bovine serum albumin may be used. Other suitable methods for separating the
Determined on the poliomyelitis component 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
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
Provided that the tests for specific toxicity of the pertussis than half the single human dose. Inject each mouse of the
component and antimicrobial preservative, and the assays for control group with 0.5 mL of a 9 g/L sterile solution of
the diphtheria, tetanus and pertussis components have been sodium chloride R, preferably containing the same amount of
carried out with satisfactory results on the final bulk vaccine, antimicrobial preservative as that injected with the vaccine.
they may be omitted on the final lot. 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
Provided that the in vivo assay for the poliomyelitis combined.
component has been carried out with satisfactory results on
Aluminium (2.5.13)
the final bulk vaccine, it may be omitted on the final lot.
The in vivo assay for the poliomyelitis component may be hydroxide or hydrated aluminium phosphate is used as the
omitted once it has been demonstrated for a given product adsorbent.
the D-antigen determination are such that it yields the same Free formaldehyde (2. 4.18)
result as the in vivo assay in terms of acceptance or rejection Maximum 0.2 g/L.
of a batch. This demonstration must include testing of Antimicrobial preservative
subpotent batches, produced experimentally if necessary, for Where applicable, determine the amount of antimicrobial
example by heat treatment or other means of diminishing the preservative by a suitable chemical method. The content is
immunogenic activity. Where there is a significant change in not less than the minimum amount shown to be effective and
the manufacturing process of the antigens or their
label.
Haemophilus Type b Conjugate
Sterility (2. 6.1) Vaccine
The vaccine complies with the test for sterility. (Ph. Bur. monograph 1219)
ASSAY The label may state 'Hib'.
Diphtheria component ~& _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
diphtheria vaccine (adsorbed) (2.7.6). DEFINITION
The lower confidence limit (P = 0.95) of the estimated Haemophilus type b conjugate vaccine is a liquid or freeze-
potency is not less than 30 IU per single human dose. dried preparation of a polysaccharide, derived from a suitable
strain of Haemophilus influenzae type b, covalently bound to a
Tetanus component carrier protein. The polysaccharide, polyribosylribitol
Carry out one of the prescribed methods for the assay of phosphate, referred to as PRP, is a linear copolymer
tetanus vaccine (adsorbed) (2. 7.8). composed of repeated units of 3-~-o-ribofuranosyl-(l-> 1)-
If the test is carried out in guinea pigs, the lower confidence ribitol-5-phosphate [(C 10 H 190 12P)nl, with a defined
limit (P = 0.95) of the estimated potency is not less than molecular size. The carrier protein, when conjugated to PRP,
40 IU per single human dose; if the test is carried out in is capable of inducing a T-cell-dependent B-cell immune
mice, the lower confidence limit (P = 0. 9 5) of the estimated response to the polysaccharide.
PRODUCTION
Pertussis component GENERAL PROVISIONS
Carry out the assay of pertussis vaccine (whole cell) (2. 7. 7). The production method shall have been shown to yield
The estimated potency is not less than 4.0 IU per single consistently haemophilus type b conjugate vaccines of
human dose and the lower confidence limit (P = 0.95) of the adequate safety and immunogenicity in man. The production
estimated potency is not less than 2.0 IU per single human of PRP and of the carrier protein are based on seed-lot
dose. systems.
Poliomyelitis component The production method is validated to demonstrate that the
D-antigen content As a measure of consistency of product, if tested, would comply with the test for pyrogens
production, determine the D-antigen content for human (2. 6. 8), carried out as follows: inject per kilogram of the
poliovirus types 1, 2 and 3 by a suitable immunochemical rabbit's mass a quantity of the vaccine equivalent to: 1 µg of
method (2. 7.1) following desorption, using a reference PRP for a vaccine with diphtheria toxoid or CRM 197
preparation calibrated in European Pharmacopoeia Units of diphtheria protein as carrier; 0.1 µg of PRP for a vaccine
D-antigen. For each type, the content, expressed with with tetanus toxoid as carrier; 0.025 µg of PRP for a vaccine
reference to the amount of D-antigen stated on the label, is with OMP (meningococcal group B outer membrane protein
within the limits approved for the particular product. complex) as carrier.
Poliomyelitis vaccine (inactivated) ERP is calibrated in During development studies, it shall be demonstrated that
European Pharmacopoeia Units and intended for use in the the vaccine consistently induces a T-cell-dependent B-cell
assay of D-antigen. The European Pharmacopoeia Unit and immune response to PRP. If the manufacturing process is
the International Unit are equivalent. modified, it shall be demonstrated by appropriate in vitro
In vivo test The vaccine complies with the in vivo assay of methods that the characteristic properties of the vaccine are
poliomyelitis vaccine (inactivated) (2. 7. 20). not affected.
LABELLING The stability of the final lot and relevant intermediates is
The label states: evaluated using one or more indicator tests. Such tests may
- the minimum number of International Units of diphtheria include determination of molecular size, determination of free
and tetanus toxoid per single human dose; PRP in the conjugate and the immunogenicity test in mice.
- the minimum number of International Units of pertussis Taking account of the results of the stability testing, release
vaccine per single human dose; requirements are set for these indicator tests to ensure that
- the nominal amount of poliovirus of each type (1, 2 and the vaccine will be satisfactory at the end of shelf life.
3), expressed in European Pharmacopoeia Units of BACTERIAL SEED LOTS
D-antigen, per single human dose; The seed lots of H. infiuenzae type b are shown to be free
- the type of cells used for production of the poliomyelitis from contamination by methods of suitable sensitivity. These
component; may include inoculation into suitable media, examination of
- the name and the amount of the adsorbent; colony morphology, microscopic examination of Gram-
- where applicable, that the vaccine is intended for primary stained smears and culture agglutination with suitable specific
vaccination of children and is not necessarily suitable for antisera.
reinforcing doses or for administration to adults; No complex products of animal origin are included in the
- that the vaccine must be shaken before use; medium used for preservation of strain viability, either for
- that the vaccine is not to be frozen. freeze-drying or for frozen storage.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
It is recommended that PRP produced by the seed lot be
characterised using nuclear magnetic resonance
spectrometry (2. 2. 33).
H. INFLUENZAE TYPE B POLYSACCHARIDE (PRP)
H. infiuenzae Type b is grown in a liquid medium that does
not contain high-molecular-mass polysaccharides; if any
ingredient of the medium contains blood-group substances,
the process shall be validated to demonstrate that after the to conjugation. As a measure of consistency, the extent of
purification step they are no longer detectable. The bacterial derivatisation is monitored. The conjugate is obtained by the
purity of the culture is verified by methods of suitable covalent binding of PRP and the appropriate carrier protein.
sensitivity. These may include inoculation into suitable Where applicable, unreacted but potentially reactogenic
media, examination of colony morphology, microscopic functional groups are made unreactive by means of capping
examination of Gram-stained smears and culture agents; the conjugate is purified to remove reagents.
agglutination with suitable specific antisera. The culture may Only a bulk conjugate that complies with the following
be inactivated. PRP is separated from the culture medium requirements may be used in the preparation of the final bulk
and purified by a suitable method. Volatile matter, including vaccine. For each test and for each particular product, limits
water, in the purified polysaccharide is determined by a of acceptance are established and each batch of conjugate
suitable method; the result is used to calculate the results of must be shown to comply with these limits. For a freeze-
certain tests with reference to the dried substance, as dried vaccine, some of the tests may be carried out on the
prescribed below. final lot rather than on the bulk conjugate where the freeze-
Only PRP that complies with the following requirements may drying process may affect the component being tested.
be used in the preparation of the conjugate. PRP
Identification The PRP content is determined either by assay of
PRP is identified by an immunochemical method (2. 7.1) or ribose (2.5.31) or phosphorus (2.5.18), by an
other suitable method, for example 1H nuclear magnetic immunochemical method (2. 7.1) or by anion-exchange liquid
resonance spectrometry (2.2.33). chromatography (2.2.29) with pulsed amperometric
Molecular-size or molecular-mass distribution detection.
Molecular-size or molecular-mass distribution is determined Protein
by size-exclusion chromatography (2.2.30) combined with an The protein content is determined by a suitable chemical
appropriate detection system. Where applicable, the method (for example, 2. 5.16).
molecular-size distribution is also determined after chemical PRP-to-protein ratio
modification of the polysaccharide. An acceptable value is Determine the ratio by calculation.
established for the PRP polysaccharide. Each batch must be
shown to comply with this limit.
Molecular-size or molecular-mass distribution
Molecular-size or molecular-mass distribution is determined
Ribose (2.5.31) by size-exclusion chromatography (2.2.30) combined with an
Within the limits approved by the competent authority for appropriate detection system. An acceptable value is
the particular product, calculated with reference to the dried established for the bulk conjugate. Each batch must be
substance. shown to comply with this limit.
Phosphorus (2.5.18) FreePRP
Within the limits approved by the competent authority for A number of methods are used to separate free PRP from
the particular product, calculated with reference to the dried the conjugate, including precipitation, gel filtration, size-
substance. exclusion, anion-exchange and hydrophobic chromatography,
Protein (2.5.16) ultrafiltration and ultracentrifugation. The free PRP can then
Maximum 1.0 per cent, calculated with reference to the dried be quantified by a range of techniques, including high-
substance. Use sufficient PRP to allow detection of proteins performance anion-exchange chromatography with pulsed
at concentrations of 1 per cent or greater. amperometric detection (HPAEC-PAD) and immunoassays
Nucleic acid (2.5.17) with anti-PRP antibodies.
Maximum 1.0 per cent, calculated with reference to the dried Free carrier protein
substance. Determine the content by a suitable method, either directly
Bacterial endotoxins (2.6.14) or by deriving the content by calculation from the results of
Less than 10 IU per microgram of PRP. other tests. The amount is within the limits approved for the
particular product.
Residual reagents
Where applicable, tests are carried out to determine residues Unreacted functional groups
of reagents used during inactivation and purification. No unreacted functional groups are detectable in the bulk
An acceptable value for each reagent is established for the conjugate unless process validation has shown that unreacted
particular product and each batch of PRP must be shown to functional groups detectable at this stage are removed during
comply with this limit. Where validation studies have the subsequent manufacturing process (for example, owing to
demonstrated removal of a residual reagent, the test on PRP short half-life).
may be omitted. Residual reagents
CARRIER PROTEIN Removal of residual reagents such as cyanide, EDAC
The production and characteristics of the carrier proteins are (ethyldimethylaminopropylcarbodiimide) and phenol is
described in general chapter 5.2.11. Carrier proteins for the confirmed by suitable tests or by validation of the process.
production of conjugated polysaccharide vaccines for human use. Sterility (2. 6.1)
Only a carrier protein that complies with the requirements of Carry out the test using for each medium 10 mL or the
this chapter may be used in the preparation of the conjugate. equivalent of 100 doses, whichever is less.
BULK COl',{JUGATE FINAL BULK VACCINE
PRP is chemically modified to enable conjugation; it is An adjuvant, an antimicrobial preservative and a stabiliser
usually partly depolymerised either before or during this may be added to the bulk conjugate before dilution to the
procedure. Reactive functional groups or spacers may be final concentration with a suitable diluent.
introduced into the appropriate carrier protein or PRP prior
Only a final bulk vaccine that complies with the following LABELLING
requirements may be used in the preparation of the final lot. The label states:
Antimicrobial preservative - the number of micrograms of PRP per single human
Where applicable, determine the amount of antimicrobial dose;
preservative by a suitable chemical or physico-chemical - the type and nominal amount of carrier protein per single
method. The content is not less than 85 per cent and not human dose.
greater than 115 per cent of the intended amount. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
Sterility (2. 6. 1)
It complies with the test for sterility, carried out using 10 mL
for each medium.
FINAL LOT Haemophilus Type band
Only a final lot that is satisfactory with respect to each of the Meningococcal Group C
following requirements and the requirements given below
under Identification and Tests may be released for use. Conjugate Vaccine
Provided the test for antimicrobial preservative has been (Ph. Eur. monograph 2622)
carried out on the final bulk vaccine, it may be omitted on
the final lot.
pH (2.2.3) DEFINITION
The pH of the vaccine, reconstituted if necessary, is within Haemophilus type b and meningococcal group C conjugate
the limits approved for the particular product. vaccine is a combined vaccine composed of a preparation of
a polysaccharide derived from a suitable strain of Haemophilus
Free PRP
inftuenzae type b covalently bound to a carrier protein and a
A number of methods are used to separate free PRP from
preparation of purified capsular polysaccharide derived from
the conjugate, including precipitation, gel filtration, size-
a suitable strain of Neisseria meningituiis group C covalently
exclusion, anion-exchange and hydrophobic chromatography,
bound to a carrier protein. The haemophilus type b
ultrafiltration and ultracentrifugation. The free PRP can then
polysaccharide, polyribosylribitol phosphate (referred to as
be quantified by a range of techniques, including HPAEC-
PRP), is a linear copolymer composed of repeating units of
PAD and immunoassays with anti-PRP antibodies.
3-~-D-ribofuranosyl-(l-+ 1)-ribitol-5-phosphate
The amount of free PRP is not greater than that approved
[(C10H19O12P)nl, with a defined molecular size.
for the particular product.
Meningococcal group C polysaccharide consists of partly
IDENTIFICATION O-acetylated or O-deacetylated repeating units of sialic acids,
The vaccine is identified by a suitable immunochemical linked with 2a-+9 glycosidic bonds. The carrier proteins,
method (2.7.1) for PRP. when conjugated to PRP and group C polysaccharide, are
TESTS capable of inducing a T-cell-dependent B-cell immune
response to the polysaccharide.
PRP
Minimum 80 per cent of the amount of PRP stated on the PRODUCTION
label. PRP is determined either by assay of ribose (2.5.31) or GENERAL PROVISIONS
phosphorus (2.5.18), by an immunochemical method (2.7.1) The production method shall have been shown to
or by anion-exchange liquid chromatography (2.2.29) with consistently yield combined haemophilus type b and
pulsed amperometric detection. meningococcal group C conjugate vaccines of adequate safety
Aluminium (2.5.13) and immunogenicity in man. The production of PRP,
Maximum 1.25 mg per single human dose, if aluminium group C polysaccharide and the carrier protein(s) are based
hydroxide or hydrated aluminium phosphate is used as the on seed-lot systems.
adsorbent. During development studies and wherever revalidation is
Antimicrobial preservative necessary, a test for pyrogens (2.6.8) is carried out by
Where applicable, determine the amount of antimicrobial injection of a suitable dose of the final lot as follows: inject,
preservative by a suitable chemical or physico-chemical per kilogram of the rabbit's mass, a quantity of the vaccine
method. The content is not less than the minimum amount equivalent to 0.1 µg of PRP. The vaccine is shown to be
shown to be effective and not greater than 115 per cent of acceptable with respect to absence of pyrogenic activity.
the quantity stated on the label. During development studies and wherever revalidation of the
manufacturing process is necessary, it shall be demonstrated
Water (2.5.12)
by tests in animals that the vaccine consistently induces a
Maximum 3.0 per cent for freeze-dried vaccines.
T-cell-dependent B-cell immune response.
Sterility (2. 6.1)
The stability of the final lot and relevant intermediates is
evaluated using 1 or more indicator tests. Such tests may
Bacterial endotoxins (2.6.14) include determination of molecular size, determination of free
The content is within the limits approved by the competent polysaccharide in the conjugate and the immunogenicity test
authority for the particular product. If any components of the in mice.
vaccine prevent the determination of endotoxin, a test for
BACTERIAL SEED LOTS
pyrogens is carried out as described under General
The bacterial strains used for master seed lots shall be
provisions.
identified by historical records that include information on
their origin and the tests used to characterise the strain.
Cultures from the working seed lot shall have the same
characteristics as the strain that was used to prepare the Bacterial endotoxins (2.6.14)
master seed lot. The content is within the limits approved by the competent
The seed lots are shown to be free from contamination by authority for the particular product.
methods of suitable sensitivity. These may include ASSAY
inoculation into suitable media, examination of colony PRP
morphology, microscopic examination of Gram-stained Minimum 80 per cent of the amount of PRP stated on the
smears and culture agglutination with suitable specific label. PRP is determined either by assay of ribose (2.5.31) or
antisera. phosphorus (2.5.18), by an immunochernical method (2.7.1)
No complex products of animal origin are included in the or by anion-exchange liquid chromatography (2.2.29) with
medium used for preservation of strain viability, either for pulsed amperometric detection.
freeze-drying or for frozen storage. Saccharide
It is recommended that PRP produced by the seed lot be Minimum 80 per cent of the amount of group C
characterised using nuclear magnetic resonance spectrometry polysaccharide stated on the label. The group C
(2.2.33). polysaccharide content is determined by a suitable validated
PRODUCTION OF THE COMPONENTS assay, for example sialic acid assay (2.5.23) or anion-
The production of the bulks and bulk conjugate components exchange liquid chromatography (2.2.29) with pulsed
complies with the requirements of the monographs amperometric detection.
Haemophilus type b conjugate vaccine (1219), Meningococcal LABELLING
group C conjugate vaccine (2112) and the general chapter The label states:
5.2.11. Carrier proteins for the production of conjugated - the number of micrograms of PRP and group C
polysaccharide vaccines for human use. polysaccharide per single human dose;
FINAL BULK VACCINE - the type and nominal amount of carrier protein per single
Only a final bulk vaccine that complies with the following human dose.
requirement and is within the limits approved for the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
particular product may be used in the preparation of the final
lot.
Sterility (2. 6.1)
It complies with the test for sterility, carried out using 10 mL Inactivated Hepatitis A Vaccine
for each medium.
FINAL LOT (Hepatitis A Vaccine (Inactivated, Adsorbed),
Only a final lot that is satisfactory with respect to each of the Ph. Bur. monograph 1107)
requirements given below under Identification, Tests and The label may state 'HepA'.
Assay may be released for use. Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
IDENTIFICATION
DEFINITION
Both antigens are identified by a suitable immunochemical
Hepatitis A vaccine (inactivated, adsorbed) is a suspension
method (2. 7.1) for PRP and group C polysaccharide or other
consisting of a suitable strain of hepatitis A virus grown in
suitable method.
cell cultures, inactivated by a validated method and adsorbed
TESTS on a mineral carrier.
pH (2.2.3)
PRODUCTION
The pH of the vaccine, reconstituted if necessary, is within
GENERAL PROVISIONS
the limits approved for the particular product.
Production of the vaccine is based on a virus seed-lot system
Water (2.5.12) and a cell-bank system. The production method shall have
Maximum 3.0 per cent for freeze-dried vaccines. been shown to consistently yield vaccines that comply with
Free PRP the requirements for immunogenicity, safety and stability.
A number of methods are used to separate free PRP from Unless otherwise justified and authorised, the virus in the
the conjugate, including precipitation, gel filtration, size- final vaccine shall not have undergone more passages from
exclusion, anion-exchange and hydrophobic chromatography, the master seed lot than were used to prepare the vaccine
ultrafiltration and ultracentrifugation. The free PRP can then shown in clinical studies to be satisfactory with respect to
be quantified by a range of techniques, including high- safety and efficacy.
performance anion-exchange chromatography with pulsed Reference preparation A part of a batch shown to be at least
amperometric detection (HPAEC-PAD) and immunoassays as immunogenic in animals as a batch that, in clinical studies
(2. 7.1) with anti-PRP antibodies. The amount of free PRP is in young healthy adults, produced not less than 95 per cent
not greater than that approved for the particular product. seroconversion, corresponding to a level of neutralising
Free saccharide antibody accepted to be protective, after a full-course primary
Unbound group C polysaccharide is determined after immunisation is used as a reference preparation. An antibody
removal of the conjugate, for example by anion-exchange level of 20 mIU/mL determined by enzyme-linked
liquid chromatography, size-exclusion or hydrophobic immunosorbent assay is recognised as being protective.
chromatography, ultrafiltration or other validated methods. SUBSTRATE FOR VIRUS PROPAGATION
The amount of free group C polysaccharide is not greater The virus is propagated in a human diploid cell line (5.2.3)
than that approved for the particular product. or in a continuous cell line approved by the competent
Sterility (2. 6.1) authority.
SEED LOTS PURIFICATION AND PURIFIED HARVEST

The strain of hepatitis A virus used to prepare the master The harvest, which may be a pool of several single harvests,
seed lot shall be identified by historical records that include is purified by validated methods. If continuous cell lines are
information on the origin of the strain and its subsequent used for production, the purification process shall have been
manipulation. shown to reduce consistently the level of host-cell DNA.
Only a seed lot that complies with the following requirements Only a purified harvest that complies with the following
may be used for virus propagation. requirements may be used in the preparation of the
Identification inactivated harvest.
Each master and working seed lot is identified as hepatitis A Virus concentration
virus using specific antibodies. The concentration of infectious virus in the purified harvest
Virus concentration is determined by a suitable cell culture method to monitor
The virus concentration of each master and working seed lot production consistency and as a starting point for monitoring
is determined to monitor consistency of production. the inactivation curve.
Extraneous agents Antigen:total protein ratio
The working seed lot complies with the requirements for Determine the hepatitis A virus antigen content by a suitable
seed lots for virus vaccines (2. 6.16). In addition, if primary immunochemical method (2.7.J). Determine the total protein
monkey cells have been used for isolation of the strain, by a validated method. The ratio of hepatitis A virus antigen
measures are taken to ensure that the strain is not content to total protein content is within the limits approved
contaminated with simian viruses such as simian for the particular product.
immunodeficiency virus and filoviruses. Bovine serum albumin
VIRUS PROPAGATION AND HARVEST Not more than 50 ng in the equivalent of a single
All processing of the cell bank and subsequent cell cultures is human dose, determined by a suitable immunochemical
done under aseptic conditions in an area where no other cells method (2. 7. J). Where appropriate in view of the
are being handled. Animal serum (but not human serum) manufacturing process, other suitable protein markers may
may be used in the cell culture media. Serum and trypsin be used to demonstrate effective purification.
used in the preparation of cell suspensions and media are Residual host-cell DNA
shown to be free from extraneous agents. The cell culture If a continuous cell line is used for virus propagation, the
media may contain a pH indicator, such as phenol red, and content of residual host-cell DNA, determined using a
antibiotics at the lowest effective concentration. Not less than suitable method, is not greater than I 00 pg in the equivalent
500 mL of the cell cultures employed for vaccine production of a single human dose.
is set aside as uninfected cell cultures (control cells). Multiple Residual chemicals
harvests from the same production cell culture may be If chemical substances are used during the purification
pooled and considered as a single harvest. process, tests for these substances are carried out on the
Only a single harvest that complies with the following purified harvest (or on the inactivated harvest), unless
requirements may be used in the preparation of the vaccine. validation of the process has demonstrated total clearance.
When the determination of the ratio of virus concentration to The concentration must not exceed the limits approved for
antigen content has been carried out on a suitable number of the particular product.
single harvests to demonstrate production consistency, it may INACTIVATION AND INACTIVATED HARVEST
subsequently be omitted as a routine test. Several purified harvests may be pooled before inactivation.
Identification In order to avoid interference with the inactivation process,
The test for antigen content also serves to identify the single virus aggregation must be prevented or aggregates must be
harvest. removed immediately before and/or during the inactivation
Bacterial and fungal contamination process. The virus suspension is inactivated by a validated
The single harvest complies with the test for sterility (2. 6. J), method; the method shall have been shown to be consistently
carried out using I O mL for each medium. capable of inactivating hepatitis A virus without destroying
the antigenic and immunogenic activity; for each inactivation
Mycoplasmas (2.6.7)
procedure, an inactivation curve is plotted representing
The single harvest complies with the test for mycoplasmas,
residual live virus concentration measured at not fewer than
carried out using I mL for each medium.
3 points in time (for example, on days 0, 1 and 2 of the
Control cells inactivation process). If formaldehyde is used for inactivation,
The control cells of the production cell culture comply with a the presence of excess free formaldehyde is verified at the
test for identification and the requirements for extraneous end of the inactivation process.
agents (2.6.16).
Only an inactivated harvest that complies with the following
Antigen content requirements may be used in the preparation of the final bulk
Determine the hepatitis A antigen content by a suitable vaccine.
immunochemical method (2. 7. J) to monitor production Inactivation
consistency; the content is within the limits approved for the
Carry out an amplification test for residual infectious
particular product.
hepatitis A virus by inoculating a quantity of the inactivated
Ratio of virus concentration to antigen content harvest equivalent to 5 per cent of the batch or, if the harvest
The consistency of the ratio of the concentration of infectious contains the equivalent of 30 000 doses or more, not less
virus, determined by a suitable cell culture method, to than 1500 doses of vaccine into cell cultures of the same type
antigen content is established by validation on a suitable as those used for production of the vaccine; incubate for a
number of single harvests. total of not less than 70 days making not fewer than one
passage of cells within that period. At the end of the
IV-7 40 Vaccines 2023
incubation period, carry out a test of suitable sensitivity for ASSAY

residual infectious virus. No evidence of hepatitis A virus The vaccine complies with the assay of hepatitis A vaccine
multiplication is found in the samples taken at the end of the (2.7.14).
inactivation process. Use infectious virus inocula concurrently
LABELLING
as positive controls to demonstrate cellular susceptibility and
The label states the biological origin of the cells used for the
absence of interference.
preparation of the vaccine.
Sterility (2. 6.1) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
The inactivated viral harvest complies with the test for
sterility, carried out using 10 mL for each medium.
Less than 2 IU in the equivalent of a single human dose.
Hepatitis A Vaccine (Inactivated
Antigen content
Determine the hepatitis A virus antigen content by a suitable Virosome)
immunochemical method (2. 7.1). (Ph. Bur. monograph 1935)
Residual chemicals The label may state 'HepA'.
See under Purification and purified harvest. PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
FINAL BULK VACCINE
The final bulk vaccine is prepared from one or more DEFINITION
inactivated harvests. Approved adjuvants, stabilisers and Hepatitis A vaccine (inactivated, virosome) is a suspension of
antimicrobial preservatives may be added. a suitable strain of hepatitis A virus grown in cell cultures
and inactivated by a validated method. Virosomes composed
of influenza proteins of a strain approved for the particular
product and phospholipids are used as adjuvants.
Sterility (2. 6.1)
The final bulk vaccine complies with the test for sterility, PRODUCTION
carried out using 10 mL for each medium. GENERAL PROVISIONS
preservative by a suitable chemical or physico-chemical
method. The amount is not less than 85 per cent and not Reference preparation A reference preparation of inactivated
greater than 115 per cent of the intended amount. hepatitis A antigen is calibrated against a batch of hepatitis A
vaccine (inactivated, virosome) that, in clinical studies in
FINAL LOT young healthy adults, produced not less than 95 per cent
The final bulk vaccine is distributed aseptically into sterile seroconversion, corresponding to a level of neutralising
containers. The containers are then closed so as to avoid antibody accepted to be protective, after a full-course primary
contamination. immunisation. An antibody level not less than 20 mIU/mL
Only a final lot that complies with each of the requirements determined by enzyme-linked immunosorbent assay is
given below under Identification, Tests and Assay may be recognised as being protective.
released for use. Provided that the tests for free formaldehyde
PREPARATION OF HEPATITIS A ANTIGEN
(where applicable) and antimicrobial preservative content
Production of the hepatitis A antigen is based on a virus
(where applicable) have been carried out on the final bulk
seed-lot system and a cell-bank system. The production
vaccine with satisfactory results, these tests may be omitted
method shall have been shown to consistently yield vaccines
on the final lot. If the assay is carried out using mice or other
that comply with the requirements for immunogenicity, safety
animals, then provided it has been carried out with
and stability.
satisfactory results on the final bulk vaccine, it may be
omitted on the final lot. Unless otherwise justified and authorised, the virus in the
final vaccine shall not have undergone more passages from
IDENTIFICATION the master seed lot than were used to prepare the vaccine
The assay (2. 7.14) serves also to identify the vaccine. shown in clinical studies to be satisfactory with respect to
TESTS safety and efficacy.
Aluminium (2.5.13) SUBSTRATE FOR PROPAGATION OF HEPATITIS A
Maximum 1.25 mg per single human dose, if aluminium VIRUS
hydroxide or hydrated aluminium phosphate is used as the The virus is propagated in a human diploid cell line (5.2.3).
adsorbent. SEED LOTS OF HEPATITIS A VIRUS
Free formaldehyde (2.4.18) The strain of hepatitis A virus used to prepare the master
Maximum 0.2 g/L. seed lot shall be identified by historical records that include
Antimicrobial preservative information on the origin of the strain and its subsequent
Where applicable, determine the amount of antimicrobial manipulation.
preservative by a suitable chemical or physico-chemical Only a seed lot that complies with the following requirements
method. The amount is not less than the minimum amount may be used for virus propagation.
shown to be effective and is not greater than 115 per cent of Identification
that stated on the label. 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 Ratio of antigen to total protein

The virus concentration of each master and working seed lot Determine the hepatitis A virus antigen content by a suitable
is determined to monitor consistency of production. immunochemical method (2. 7.1). Determine the total protein
Extraneous agents by a validated method. The ratio of hepatitis A virus antigen
The working seed lot complies with the requirements for content to total protein content is within the limits approved
seed lots for virus vaccines (2. 6. 16). for the particular product.
PROPAGATION AND HARVEST OF HEPATITIS A Bovine serum albumin
VIRUS Maximum 50 ng per single human dose if foetal bovine
All processing of the cell bank and subsequent cell cultures is serum is used, determined by a suitable immunochemical
done under aseptic conditions in an area where no other cells method (2. 7. 1). Where appropriate in view of the
are handled. Animal serum (but not human serum) may be manufacturing process, other suitable protein markers may
used in the cell culture media. Serum and trypsin used in the be used to demonstrate effective purification.
preparation of cell suspensions and media are shown to be Residual chemicals
free from extraneous agents. The cell culture media may If chemical substances are used during the purification
contain a pH indicator such as phenol red and antibiotics at process, tests for these substances are carried out on the
the lowest effective concentration. Not less than 500 mL of purified harvest (or on the inactivated harvest), unless
the cell cultures employed for vaccine production is set aside validation of the process has demonstrated total clearance.
as uninfected cell cultures (control cells). Multiple harvests The concentration must not exceed the limits approved for
from the same production cell culture may be pooled and the particular product.
considered as a single harvest. INACTIVATION AND INACTIVATED HARVEST OF
Only a single harvest that complies with the following HEPATITIS A VIRUS
requirements may be used in the preparation of the vaccine. Several purified harvests may be pooled before inactivation.
When the determination of the ratio of virus concentration to In order to avoid interference with the inactivation process,
antigen content has been carried out on a suitable number of virus aggregation must be prevented or aggregates must be
single harvests to demonstrate consistency, it may removed immediately before and/or during the inactivation
subsequently be omitted as a routine test. process. The virus suspension is inactivated by a validated
Identification method; the method shall have been shown to be consistently
The test for antigen content also serves to identify the single capable of inactivating hepatitis A virus without destroying
harvest. the antigenic and immunogenic activity; for each inactivation
procedure, an inactivation curve is plotted representing
residual live virus concentration measured on at least
The single harvest complies with the test for sterility (2. 6. 1),
3 occasions (for example, on days 0, 1 and 2 of the
carried out using 10 mL for each medium.
inactivation process). If formaldehyde is used for inactivation,
Mycoplasmas (2. 6. 7) the presence of excess free formaldehyde is verified at the
The single harvest complies with the test for mycoplasmas. end of the inactivation process.
Control cells Only an inactivated harvest that complies with the following
The control cells of the production cell culture comply with a requirements may be used in the preparation of the final bulk
test for identity and the requirements for extraneous agents vaccine.
(2.6.16).
Inactivation
Antigen content Carry out an amplification test for residual infectious
Determine the hepatitis A antigen content by a suitable hepatitis A virus by inoculating a quantity of the inactivated
immunochemical method (2. 7.1) to monitor production harvest equivalent to 5 per cent of the batch or, if the harvest
consistency; the content is within the limits approved for the contains the equivalent of 30 000 doses or more, not less
particular product. than 1500 doses of vaccine into cell cultures of the same type
Ratio of virus concentration to antigen content as those used for production of the vaccine; incubate for a
The consistency of the ratio of the concentration of infectious total of not less than 70 days making not fewer than 1
virus, as determined by a suitable cell culture method, to passage of cells within that period. At the end of the
antigen content is established by validation on a suitable incubation period, carry out a test of suitable sensitivity for
number of single harvests. residual infectious virus. No evidence of hepatitis A virus
multiplication is found in the samples taken at the end of the
PURIFICATION AND PURIFIED HARVEST OF
inactivation process. Use infective virus inocula concurrently
HEPATITIS A VIRUS
as positive controls to demonstrate cellular susceptibility and
The harvest, which may be a pool of several single harvests,
absence of interference.
is purified by validated methods. If continuous cell lines are
used for production, the purification process shall have been Sterility (2. 6. 1)
shown to reduce consistently the level of host-cell DNA. The inactivated viral harvest complies with the test for
Only a purified harvest that complies with the following sterility, carried out using 10 mL for each medium.
requirements may be used in the preparation of the Bacterial endotoxins (2. 6.14)
inactivated harvest. Less than 2 IU of endotoxin in the equivalent of a single
Virus concentration human dose.
The concentration of infective virus in the purified harvest is Antigen content
determined by a suitable cell culture method to monitor Determine the hepatitis A virus antigen content by a suitable
production consistency and as a starting point for monitoring immunochemical method (2. 7. 1).
the inactivation curve. Residual chemicals
See under Purification and purified harvest.
IV- 7 42 Vaccines 2023
PREPARA.TION OF INACTIVATED INFLUENZA Sterility (2. 6.1)

VIRUS Carry out the test for sterility, using 10 mL for each
The production of influenza viruses is based on a seed-lot medium.
system. Working seed lots represent not more than Viral inactivation
15 passages from the approved reassorted virus or the Inoculate 0.2 mL of the harvest into the allantoic cavity of
approved virus isolate. The final production represents 1 each of 10 fertilised eggs and incubate at 33-37 °C for
passage from the working seed lot. The strain of influenza 3 days. The test is not valid unless at least 8 of the
virus to be used is approved by the competent authority. 10 embryos survive. Harvest 0.5 mL of the allantoic fluid
SUBSTRATE FOR PROPAGATION OF INFLUENZA from each surviving embryo and pool the fluids. Inoculate
VIRUS 0 .2 mL of the pooled fluid into a further 10 fertilised eggs
Influenza virus seed to be used in the production of vaccine and incubate at 33-37 °C for 3 days. The test is not valid
is propagated in fertilised eggs from chicken flocks free from unless at least 8 of the 10 embryos survive. Harvest about
specified pathogens (5.2.2) or in suitable cell cultures (5.2.4), 0.1 mL of the allantoic fluid from each surviving embryo and
such as chick-embryo fibroblasts or chick kidney cells examine each individual harvest by a haemagglutination test.
obtained from chicken flocks free from specified pathogens If haemagglutination is found for any of the fluids, carry out
(5.2.2). For production, the virus is grown in the allantoic for that fluid a further passage in eggs and test for
cavity of fertilised hens' eggs from healthy flocks. haemagglutination; no haemagglutination occurs.
SEED LOTS OF INFLUENZA VIRUS Ovalbumin
The haemagglutinin and neuraminidase antigens of each seed Maximum 1 µg of ovalbumin in the equivalent of 1 human
lot are identified as originating from the correct strain of dose, determined by a suitable technique using a suitable
influenza virus by suitable methods. reference preparation of ovalbumin.
Only a working virus seed lot that complies with the Antimicrobial preservative
following requirements may be used in the preparation of the Where applicable, determine the amount of antimicrobial
monovalent pooled harvest. preservative by a suitable chemical method. The content is
Bacterial and fungal contamination not less than 85 per cent and not greater than 115 per cent
Carry out the test for sterility (2. 6.1), using 10 mL for each of the intended amount.
medium. Residual chemicals
Mycoplasmas (2. 6. 7) Tests are carried out on the monovalent pooled harvest for
Carry out the test for mycoplasmas, using 10 mL. the chemicals used for inactivation, the limits being approved
by the competent authority.
PROPAGATION AND HARVEST OF INFLUENZA
VIRUS PREPARATION OF VIROSOMES
An antimicrobial agent may be added to the inoculum. After Inactivated influenza virions are solubilised using a suitable
incubation at a controlled temperature, the allantoic fluids detergent and are purified by high-speed centrifugation in
are harvested and combined to form the monovalent pooled order to obtain supernatants containing mainly influenza
harvest. An antimicrobial agent may be added at the time of antigens. After the addition of suitable phospholipids,
harvest. At no stage in the production is penicillin or virosomes are formed by removal of the detergent either by
streptomycin used. adsorption chromatography or another suitable technique
POOLED HARVEST OF INFLUENZA VIRUS Only virosomes that comply with the following requirements
To limit the possibility of contamination, inactivation is may be used in the preparation of the final bulk vaccine.
initiated as soon as possible after preparation. The virus is Haemagglutinin content
inactivated by a method that has been demonstrated on Determine the content of haemagglutinin antigen by an
3 consecutive batches to be consistently effective for the immunodiffusion test (2. 7.1), by comparison with a
manufacturer. The inactivation process shall have been haemagglutinin antigen reference preparation or with an
shown to be capable of inactivating the influenza virus antigen preparation calibrated against it.
without destroying antigenicity of haemagglutinin. Phospholipids
The inactivation process shall also have been shown to be The content and identity of the phospholipids are determined
capable of inactivating avian leucosis viruses and by suitable immunochemical or physico-chemical methods.
mycoplasmas. If the monovalent pooled harvest is stored
after inactivation, it is held at a temperature of 5 ± 3 °C. Ratio of phospholipid to haemagglutinin
If formaldehyde solution is used, the concentration does not
The ratio of phospholipid content to haemagglutinin content
exceed 0.2 g/L of CH 2 0 at any time during inactivation; is within the limits approved for the particular product.
if betapropiolactone is used, the concentration does not Residual chemicals
exceed 0.1 per cent V/V at any time during inactivation. Tests are carried out for the chemicals used during the
Only a pooled harvest that complies with the following process. The concentration of each residual chemical is
requirements may be used in the preparation of the within the limits approved for the particular product.
vrrosomes. FINAL BULK VACCINE
Haemagglutinin antigen The bulk vaccine is prepared by adding virosomes to
Determine the content of haemagglutinin antigen by an inactivated hepatitis A viruses to yield an approved
immunodiffusion test (2. 7.1), by comparison with a hepatitis A antigen:haemagglutinin ratio. Several bulks may
haemagglutinin antigen reference preparation or with an be pooled, and approved stabilisers and antimicrobial
antigen preparation calibrated against it. Carry out the test at preservatives may be added.
20-25 °C. Only a final bulk vaccine that complies with the following
2023 Vaccines IV- 7 43
Protein content method. The amount is not less than the minimum amount
The amount of protein is determined using a suitable shown to be effective and is not greater than 115 per cent of
technique, the limits being approved by the competent that stated on the label.
authorithy. Sterility (2. 6.1)
Phospholipids The vaccine complies with the test for sterility.
The content and identity of the phospholipids are determined Bacterial endotoxins (2. 6.14')
by suitable immunochemical or physico-chemical methods. Less than 2 IU of endotoxin per human dose.
The amount of phospholipids complies with the limits
approved for the particular product. ASSAY
The vaccine complies with the assay of hepatitis A vaccine
Haemagglutinin content
(2.7.14, Method A).
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2. 7.1). The amount of haemagglutinin LABELLING
must not exceed the limits approved for the particular The label states:
product. - the biological origin of the cells used for the preparation
Hepatitis A antigen content of the vaccine;
Determine the hepatitis A antigen content by a suitable - that the carrier contains influenza proteins prepared in
immunochemical method. The amount of antigen must not eggs;
exceed the limits approved for the particular product. - that the vaccine is to be shaken before use;
Ratio of hepatitis A antigen to haemagglutinin _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
The ratio of hepatitis A antigen content to haemagglutinin
content is within the limits approved for the particular
product.
Ovalbumin Hepatitis A (Inactivated Adsorbed)
Maximum 1 µg of ovalbumin per human dose, determined and Typhoid Polysaccharide
by a suitable technique using a suitable reference preparation
of ovalbumin. Vaccine
Virosome size (Ph. Bur. monograph 2597)
The size distribution of the virosome-hepatitis A virus The label may state 'HepA/fyphoid'.
mixture is within the limits approved for the particular
product. PhE~ - - - - - - - - - - - - - - - - - - - - - ~
Sterility (2. 6.1) DEFINITION

The final bulk vaccine complies with the test for sterility, Hepatitis A (inactivated, adsorbed) and typhoid
carried out using 10 mL for each medium. polysaccharide vaccine is a suspension consisting of a suitable
strain of hepatitis A virus, grown in cell cultures and
inactivated by a validated method, and of purified Vi capsular
polysaccharide obtained from Salmonella typhi Ty 2 strain or
some other suitable strain that has the capacity to produce
method. The amount is not less than 85 per cent and not
Vi polysaccharide.
greater than 115 per cent of the intended amount.
The hepatitis A antigen is adsorbed on a mineral carrier,
Residual chemicals
such as aluminium hydroxide, and the Vi capsular
If chemical substances are used during the formulation
polysaccharide consists of partly 3-O-acetylated
process, tests for these substances are carried out, the limits
repeated units of 2-acetylamino-2-deoxy-o-
being approved by the competent authorithy.
galactopyranuronic acid with cr-(1--.4) linkages.
FINAL LOT The product is presented either as a liquid mixture
The final bulk vaccine is distributed aseptically into sterile containing the hepatitis A component and the typhoid
containers. The containers are then closed so as to avoid Vi polysaccharide component or as 2 separate liquids, one
contamination. containing the hepatitis A component and the other the
Only a final lot that complies with each of the requirements typhoid Vi polysaccharide component, which are mixed
given below under Identification, Tests and Assay may be together immediately before use.
(where applicable) and antimicrobial preservative content PRODUCTION
(where applicable) have been carried out on the final bulk GENERAL PROVISIONS
vaccine with satisfactory results, these tests may be omitted The 2 components are prepared as described in the
on the final lot. If the assay is carried out in viva, provided it monographs Hepatitis A vaccine (inactivated, adsorbed) (1107)
has been carried out with satisfactory results on the final bulk and Typhoid polysaccharide vaccine (1160) and comply with
vaccine, it may be omitted on the final lot. the requirements prescribed therein.
IDENTIFICATION Reference preparation The hepatitis A reference preparation
is part of a representative batch shown to be at least as
The assay (2. 7.14') serves also to identify the vaccine.
immunogenic in animals as a batch that, in clinical studies in
TESTS young healthy adults, produced not less than 95 per cent
Free formaldehyde (2. 4.18) seroconversion, corresponding to a level of neutralising
Maximum 0.2 g/L. antibody accepted to be protective, after a full-course primary
Antimicrobial preservative immunisation. An antibody level not less than 20 mIU/mL
Where applicable, determine the amount of antimicrobial determined by enzyme-linked immunosorbent assay is
preservative by a suitable chemical or physico-chemical recognised as being protective.
IV-7 44 Vaccines 2023
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 liq_uid 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 µg 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 (2.5.13)
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. 6.1) Sterility (2. 6.1)
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
satisfactory results on the final bulk containing the 0-Acetyl groups (2.5.19)
hepatitis A component, it may be omitted on the final lot. 0.085 µmol (± 25 per cent) per dose (25 µg of
polysaccharide).
CHARACTERS
If the vaccine is presented as 2 separate liq_uids, test A is carried ASSAY
out using the hepatitis A component and test B is carried out using Hepatitis A component
the typhoid Vi polysaccharide component. Test C is carried out if The vaccine complies with the assay of hepatitis A vaccine
the vaccine is presented as a liq_uid mixture of both components or (2.7.14).
immediately after mixing both components if the vaccine is Typhoid Vi polysaccharide component
presented as 2 separate liq_uids. Determine Vi polysaccharide by a suitable immunochemical
A. Whitish, cloudy suspension. method (2. 7. 1), using a reference purified polysaccharide.
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 liq_uids, identificatwn not more than 120 per cent.
test A is carried out using the hepatitis A component and LABELLING
identificatwn test B is carried out using the typhoid Vi The label states:
polysaccharide component. If the vaccine is presented as a liq_uid - 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
A. The assay (2.7.14) serves also to identify the vaccine. dose (25 µg);
B. Typhoid Vi polysaccharide is identified by a suitable - the total quantity of polysaccharide in the container;
immunochemical method (2. 7.1) using specific antibodies. - the type of cells used for production of the vaccine;
- the name and amount of the adsorbent used;
TESTS
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
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
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 The assay (2. 7.15) 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. Bur. monograph 1526) Aluminium (2.5.13)
The label may state 'HepA/HepB'.
PhE~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
adsorbent.
DEFINITION Free formaldehyde (2. 4.18)
Hepatitis A (inactivated) and hepatitis B (rDNA) vaccine Maximum 0.2 g/L.
(adsorbed) is a suspension consisting of a suitable strain of Antimicrobial preservative
hepatitis A virus, grown in cell cultures and inactivated by a Where applicable, determine the amount of antimicrobial
validated method, and of hepatitis B surface antigen preservative by a suitable chemical or physico-chemical
(HBsAg), a component protein of hepatitis B virus obtained method. The amount is not less than the minimum amount
by recombinant DNA technology; the antigens are adsorbed shown to be effective and is not greater than 115 per cent of
on a mineral carrier, such as aluminium hydroxide or that stated on the label.
hydrated aluminium phosphate.
Sterility (2. 6.1)
PRODUCTION The vaccine complies with the test for sterility.
GENERAL PROVISIONS
The two components are prepared as described in the
Less than 2 IU per human dose.
monographs on Hepatitis A vaccine (inactivated,
adsorbed) (1107) and Hepatitis B vaccine (rDNA) (1056) and ASSAY
comply with the requirements prescribed therein. Hepatitis A component
Reference preparation The reference preparation is part of a The vaccine complies with the assay of hepatitis A vaccine
representative batch shown to be at least as immunogenic in (2. 7.14).
animals as a batch that, in clinical studies in young healthy Hepatitis B component
adults, produced not less than 95 per cent seroconversion, The vaccine complies with the assay of hepatitis B vaccine
corresponding to a level of neutralising antibody recognised (rDNA) (2.7.15).
to be protective, after a full-course primary immunisation.
LABELLING
For hepatitis A, an antibody level not less than 20 mIU/mL
The label states:
determined by enzyme-linked immunosorbent assay is
- the amount of hepatitis A virus antigen and hepatitis B
recognised as being protective. For hepatitis B, an antibody
surface antigen per container;
level not less than 10 mIU/mL against HBsAg is recognised
- the type of cells used for production of the vaccine;
as being protective.
- the name and amount of the adsorbent used;
FINAL BULK VACCINE - that the vaccine must be shaken before use;
The final bulk vaccine is prepared from one or more - that the vaccine must not be frozen.
inactivated harvests of hepatitis A virus and one or more _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ph Eur
batches of purified antigen.
Antimicrobial preservative Hepatitis B Vaccine (rDNA)
preservative by a suitable chemical or physico-chemical (Ph. Bur. monograph 1056)
method. The amount is not less than 85 per cent and not
The label may state 'HepB'.
greater than 115 per cent of the intended amount.
Ph Eur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Sterility (2. 6.1)
The final bulk vaccine complies with the test for sterility, DEFINITION
carried out using 10 mL for each medium. Hepatitis B vaccine (rDNA) is a preparation of hepatitis B
FINAL LOT surface antigen (HBsAg), a component protein of hepatitis B
Only a final lot that complies with each of the requirements virus; the antigen may be adsorbed on a mineral carrier such
given below under Identification, Tests and Assay may be as aluminium hydroxide or hydrated aluminium phosphate.
released for use. Provided that the tests for free formaldehyde The vaccine may also contain the adjuvant 3-O-desacyl-4'-
(where applicable) and antimicrobial preservative content monophosphoryl lipid A. The antigen is obtained by
(where applicable) have been carried out on the final bulk recombinant DNA technology.
vaccine with satisfactory results, they may be omitted on the PRODUCTION
final lot. If the assay of the hepatitis A and/or the hepatitis B GENERAL PROVISIONS
component is carried out in viva, then provided it has been The vaccine shall have been shown to induce specific,
carried out with satisfactory results on the final bulk vaccine, protective antibodies in man. The production method shall
it may be omitted on the final lot. have been shown to yield consistently vaccines that comply
IDENTIFICATION with the requirements for immunogenicity and safety.
Hepatitis A component Hepatitis B vaccine (rDNA) is produced by the expression of
The assay (2. 7.14) serves also to identify the vaccine. the viral gene coding for HBsAg in yeast (Saccharomyces
cerevisiae) or mammalian cells (Chinese hamster ovary
(CHO) cells or other suitable cell lines), purification of the detect a potential contaminant at a concentration of
resulting HBsAg and the rendering of this antigen into an 1 per cent of total protein. Not less than 95 per cent of the
immunogenic preparation. The suitability and safety of the total protein consists of hepatitis B surface antigen.
cells are approved by the competent authority. Composition
The vaccine may contain the product of the S gene (major The content of proteins, lipids, nucleic acids and
protein), a combination of the S gene and pre-S2 gene carbohydrates is determined.
products (middle protein) or a combination of the S gene, Host-cell- and vector-derived DNA
the pre-S2 gene and pre-SI gene products (large protein). If mammalian cells are used for production, not more than
Reference preparation Part of a representative batch shown to 10 pg of DNA in the quantity of purified antigen equivalent
be at least as immunogenic in animals as a batch that, in to a single human dose of vaccine.
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. 6.1)
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
structure of the antigen is established. The morphological Additional tests on the purified antigen may be required
characteristics of the antigen particles are established by depending on the production method used: for example, a
electron microscopy. The mean buoyant density of the test for residual animal serum where mammalian cells are
antigen particles is determined by a physico-chemical used for production or tests for residual chemicals used
method, such as gradient centrifugation. The antigenic during extraction and purification.
epitopes are characterised. The protein fraction of the antigen ADSORBED 3-O-DESACYL-4' -MONOPHOSPHORYL
is characterised in terms of the primary structure (for LIPIDABULK
example, by determination of the amino-acid composition, by If 3-O-desacyl-4 '-monophosphoryl lipid A is included in the
partial amino-acid sequence analysis and by peptide vaccine it complies with the monograph 3-0-desacyl-4' -
mapping). monophosphoryl lipid A (2537). Where 3-0-desacyl-4'-
CULTURE AND HARVEST monophosphoryl lipid A liquid bulk is adsorbed prior to
Identity, microbial purity, plasmid retention and consistency inclusion in the vaccine, the adsorbed 3-O-desacyl-4'-
of yield are determined at suitable production stages. monophosphoryl lipid A bulk complies with the following
If mammalian cells are used, tests for extraneous agents and requirements.
mycoplasmas are performed in accordance with general Degree of adsorption of 3-0-desacyl-4 ' -
chapter 2. 6.16. Tests for extraneous agents in viral vaccines for monophosphoryl lipid A
human use, but using 200 mL of harvest in the test in cell The content of non-adsorbed 3-O-desacyl-4 ' -
culture for other extraneous agents. monophosphoryl lipid A in the adsorbed 3-O-desacyl-4 ' -
PURIFIED ANTIGEN monophosphoryl lipid A bulk is determined by a suitable
Only a purified antigen that complies with the following method, for example gas chromatographic quantification of
requirements may be used in the preparation of the final bulk the 3-0-desacyl-4'-monophosphoryl lipid A (2537) fatty acids in
vaccine. the supernatant, evaporated to dryness, after 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. 6.1)
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 FINAL BULK VACCINE
subtype ad or an in-house reference, by a suitable An antimicrobial preservative, a mineral carrier, such as
immunochemical method (2. 7.1) such as radio-immunoassay aluminium hydroxide or hydrated aluminium phosphate, and
(RIA), enzyme-linked immunosorbent assay (ELISA), the adjuvant 3-O-desacyl-4'-monophosphoryl lipid A may be
immunoblot (preferably using a monoclonal antibody included in the formulation of the final bulk.
directed against a protective epitope) or single radial Only a final bulk vaccine that complies with the following
diffusion. The antigen/protein ratio is within the limits requirements may be used in the preparation of the final lot.
approved for the specific product.
The molecular weight of the major band revealed following Where applicable, determine the amount of antimicrobial
sodium dodecyl sulfate polyacrylamide gel electrophoresis preservative by a suitable chemical or physico-chemical
(SDS-PAGE) performed under reducing conditions method. The amount is not less than 85 per cent and not
corresponds to the value expected from the known nucleic greater than 115 per cent of the intended amount.
acid and polypeptide sequences and possible glycosylation.
Sterility (2. 6.1)
Antigenic purity The final bulk vaccine complies with the test, carried out
The purity of the antigen is determined by comparison with using 10 mL for each medium.
a reference preparation using liquid chromatography or other
FINAL LOT
suitable methods such as SDS-PAGE with staining by acid
Only a final lot that complies with each of the requirements
blue 92 and silver. A suitable method is sensitive enough to
given below under Identification, Tests and Assay may be
(where applicable) and antimicrobial preservative content
Inactivated Influenza Vaccine
(where applicable) have been carried out on the final bulk (Whole Virion)
vaccine with satisfactory results, they may be omitted on the
(Influenza Vaccine (Whole Virion, Inactivated),
final lot. If the assay is carried out in vivo, then provided it
vaccine, it may be omitted on the final lot. The label may state 'Flu'.
Degree of adsorption When Inactivated Influenza Vaccine or Influenza Vaccine is
The degree of adsorption of the antigen and, where prescribed or demanded and the form is not stated,
applicable, 3-O-desacyl-4'-monophosphoryl lipid A is Inactivated Influenza Vaccine (Whole Virion), Inactivated
assessed. Influenza Vaccine (Split Virion) or Inactivated Influenza
Vaccine (Surface Antigen) may be dispensed or supplied.
IDENTIFICATION
The assay or, where applicable, the electrophoretic profile, Ph Eur - - - - - - - - - - - - - - - - - - - - ~
serves also to identify the vaccine. In addition, where DEFINITION

applicable, the test for 3-O-desacyl-4 1-monophosphoryl lipid Influenza vaccine (whole virion, inactivated) is a sterile,
A content also serves to identify the 3-O-desacyl-4 1- aqueous suspension of a strain or strains of influenza virus,
monophosphoryl lipid A-containing vaccine. type A or B, or a mixture of strains of the 2 types grown
TESTS individually in fertilised hens' eggs and inactivated in such a
Aluminium (2.5.13) manner that their antigenic properties are retained.
Maximum 1.25 mg per single human dose, if aluminium The stated amount of haemagglutinin antigen for each strain
hydroxide or hydrated aluminium phosphate is used as the present in the vaccine is 15 µg per dose, unless clinical
adsorbent. evidence supports the use of a different amount.
The vaccine is a slightly opalescent liquid.
3-0-Desacyl-4'-monophosphoryl lipid A
Minimum 80 per cent and maximum 120 per cent of the PRODUCTION
intended amount. CHOICE OF VACCINE STRAIN
Where applicable, determine the content of 3-O-desacyl-4'- The World Health Organization (WHO) 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
Elaboration of a European Pharmacopoeia. It is now
Where applicable, determine the content of antimicrobial
common practice to use reassorted strains giving high yields
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 µg of 3-O-desacyl-4'-monophosphoryl
VIRUS SEED LOT
lipid A.
The production of vaccine is based on a seed-lot system
ASSAY established by subculture of the candidate vaccine virus
The vaccine complies with the assay of hepatitis B vaccine (CVV). This CW is the approved virus isolate or reassorted
(rDNA) (2. 7.15). virus supplied by WHO designated laboratories, or
established by vaccine manufacturers. Working seed lots
LABELLING
represent not more than 15 passages from the CW.
The label states:
The final vaccine represents 1 passage from the working seed
- the amount of HBsAg per container;
lot. The haemagglutinin and neuraminidase antigens of each
- the type of cells used for production of the vaccine;
seed lot are identified as originating from the correct strain of
- the name and amount of the adjuvant and/or adsorbent
influenza virus by suitable methods.
used;
- that the vaccine must be shaken before use; Only a working virus seed lot that complies with the
- that the vaccine must not be frozen. following requirements may be used in the preparation of the
monovalent pooled harvest.
- - - - - - - - - - - - - - - - - - - - - PhEur
Carry out the test for sterility (2. 6.1), using 10 mL for each
medium.
Mycoplasmas (2. 6. 7)
Carry out the test for mycoplasmas, using 10 mL.
IV- 7 48 Vaccines 2023
VIRUS PROPAGATION AND HARVEST FINAL LOT

An antimicrobial agent may be added to the inoculum. After The final bulk vaccine is distributed aseptically into sterile,
incubation at a controlled temperature, the allantoic fluids tamper-evident containers. The containers are closed so as to
are harvested and combined to form a monovalent pooled prevent contamination.
harvest. An antimicrobial agent may be added at the time of Only a final lot that is satisfactory with respect to each of the
harvest. At no stage in the production is penicillin or requirements given below under Tests and Assay may be
streptomycin used. released for use. Provided that the test for residual infectious
MONO VALENT POOLED HARVEST virus has been performed with satisfactory results on each
To limit the possibility of contamination, inactivation is monovalent pooled harvest and that the tests for free
initiated as soon as possible after preparation. The virus is formaldehyde, ovalbumin and total protein have been
inactivated by a method that has been demonstrated on performed with satisfactory results on the final bulk vaccine,
3 consecutive batches to be consistently effective for the they may be omitted on the final lot.
manufacturer. The inactivation process shall have been IDENTIFICATION
shown to be capable of inactivating the influenza virus The assay serves to confirm the antigenic specificity of the
without destroying its antigenicity; the process should cause vaccine.
minimum alteration of the haemagglutinin and
neuraminidase antigens. The inactivation process shall also TESTS
have been shown to be capable of inactivating avian leucosis Residual infectious virus
viruses and mycoplasmas. If the monovalent pooled harvest is Inoculate 0.2 mL of the vaccine into the allantoic cavity of
stored after inactivation, it is held at 5 ± 3 °C. each of 10 fertilised eggs and incubate at 33-37 °C for
If formaldehyde solution is used, the concentration does not 3 days. The test is not valid unless at least 8 of the
exceed 0.2 g/L of CH 2 0 at any time during inactivation; 10 embryos survive. Harvest 0.5 mL of the allantoic fluid
if betapropiolactone is used, the concentration does not from each surviving embryo and pool the fluids. Inoculate
exceed 0.1 per cent VIV at any time during inactivation. 0 .2 mL of the pooled fluid into a further 10 fertilised eggs
Before or after the inactivation process, the monovalent and incubate at 33-37 °C for 3 days. The test is not valid
pooled harvest is concentrated and purified by high-speed unless at least 8 of the 10 embryos survive. Harvest about
centrifugation or other suitable method. 0 .1 mL of the allantoic fluid from each surviving embryo and
examine each individual harvest for live virus by a
Only a monovalent pooled harvest that complies with the
haemagglutination test. If haemagglutination is found for any
following requirements may be used in the preparation of the
of the fluids, carry out for that fluid a further passage in eggs
final bulk vaccine.
and test for haemagglutination; no haemagglutination occurs.
Haemagglutinin antigen
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2. 7.1), by comparison with a
haemagglutinin antigen reference preparation or with an
antigen preparation calibrated against it 1 • Carry out the test
at 20-25 °C.
label.
Neuraminidase antigen
The presence and type of neuraminidase antigen are
Maximum 0.2 g/L, where applicable.
confirmed by suitable enzymatic or immunological methods
on the first 3 monovalent pooled harvests from each working Ovalbumin
seed lot. Not more than the quantity stated on the label and in any
case not more than 1 µg per human dose, determined by a
Sterility (2. 6.1)
suitable immunochemical method (2. 7.1) using a suitable
Carry out the test for sterility, using 10 mL for each
reference preparation of ovalbumin.
medium.
Total protein
Residual infectious virus
Not more than 6 times the total haemagglutinin content of
Carry out the test described below under Tests.
the vaccine as determined in the assay, but in any case, not
FINAL BULK VACCINE more than 100 µg of protein per virus strain per human dose
Appropriate quantities of the monovalent pooled harvests are and not more than a total of 300 µg of protein per human
blended to make the final bulk vaccine. dose.
Only a final bulk vaccine that complies with the following Sterility (2. 6.1)
requirements may be used in the preparation of the final lot. It complies with the test for sterility.
Antimicrobial preservative Bacterial endotoxins (2.6.14)
Where applicable, determine the amount of antimicrobial Less than 100 IU per human dose.
not less than 85 per cent and not greater than 115 per cent ASSAY
of the intended amount. Determine the content of haemagglutinin antigen by an
immunodiffusion test (2. 7.1), by comparison with a
Sterility (2. 6.1)
haemagglutinin antigen reference preparation or with an
antigen preparation calibrated against it 1 • Carry out the test
at 20-25 °C. The confidence limits (P = 0.95) are not less
estimated haemagglutinin antigen content. The lower
1 Reference haemagglutinin antigens are available from the National
confidence limit (P = 0.95) is not less than 80 per cent of the
Institute for Biological Standards and Control, Blanche Lane, South
amount stated on the label for each strain.
Mimms, Potters Bar, Hertfordshire EN6 3QG, Great Britain.
LABELLING SUBSTRATE FOR VIRUS PROPAGATION

The label states: Influenza virus seed and all vaccine batches are propagated in
- that the vaccine has been prepared on eggs; fertilised eggs from chicken flocks free from specified
- the strain or strains of influenza virus used to prepare the pathogens (SPF) (5.2.2).
vaccine; VIRUS SEED LOTS
- the method of inactivation; The production of vaccine is based on a seed-lot system.
- the haemagglutinin antigen content, in micrograms per The attenuated donor virus strains and the wild-type virus
virus strain per dose; strains used for the production of the attenuated master seed
- the maximum amount of ovalbumin; lots are identified by historical records that include
- the season during which the vaccine is intended to information on their origins and the tests used in their
protect. characterisations.
- - - - - - - - - - - - - - - - - - - - - - Ph Eur 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
Influenza Vaccine (Live, Nasal) of attenuated master virus seed lots. This assay is omitted if
reverse genetics method (e.g. plasmid rescue) is used.
(Ph. Bur. monograph 2772) The production of the attenuated master virus seed lot must
PhEur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 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 must express the
the requirements for immunogenicity, safety and stability. haemagglutinin and neuraminidase from the wild type virus
CHOICE OF VACCINE STRAIN strain and other proteins from the attenuated donor virus
The W odd Health Organization reviews the world 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 reassortment methods or reverse genetics remaining genes with those of the attenuated donor strain;
(e.g. plasmid rescue). The wild type virus strains used for the - genetic stability by sequencing, cold adapted and
production of the attenuated vaccine virus seed lots must temperature sensitive phenotypes determination and
have been approved by the competent authority. attenuation test upon several passages in the substrate.
The complete history of production of the attenuated vaccine Only an attenuated master virus seed lot that complies with
virus strain including description of the derivation of the the following requirements may be used in the preparation of
seeds from the attenuated donor virus strain(s) and the the harvest.
WHO recommended wild virus strain(s) shall be approved by Identification
the competent authority. For each attenuated master virus seed lot, the haemagglutinin
During development studies and whenever a new HA and neuraminidase antigens are identified using suitable
subtype of influenza A virus (i.e. non-HI, non-H3 subtype) methods.
or a new influenza B virus type differing from the currently Cold adapted and temperature sensitive phenotype
circulating genetic lineages is included in the vaccine, the For each attenuated master virus seed lot a test is carried out
neurovirulence of the master virus seed lots is assessed using in cell cultures to demonstrate the cold adapted and
suitable animal models (e.g. in mice) with the attenuated temperature sensitive phenotypes of the seed lot.
donor virus strain as a comparator. The new strain shall not The attenuated master virus seed lot complies with the test:
be more neurovirulent than the comparator. - For the cold adaptation, if the loss of virus titre between
Genotypic and phenotypic characterisations of attenuated the incubation at + 25 °C and + 33 °C is not more than
donor virus strain(s) are undertaken using techniques for 2.0 log10 of infectious units as expressed in fluorescent
identification of attenuation markers and nucleotide focus units (FFU).
sequences.
- For the temperature sensitivity, if the loss of virus titre Microbiological contamination
between the incubation at+ 33 °C and +37 °C (for The bioburden test using a membrane filtration is carried out
strains B) or 39 °C (for strains A) is not less than on each single harvest or on each monovalent pooled harvest
2.0 log 10 of infectious units as expressed in FFU. to determine the total viable aerobic count and to verify the
Attenuation absence of yeast and mould using selective media. The total
For each attenuated master virus seed lot, an in vivo viable aerobic count is within the limit approved by the
attenuation test is carried out on ferrets. The conditions of competent authority. Verification of absence of Vibrio,
the test such as inoculation dose and observation period are Shigella and Salmonella is carried out using supplementary
established in validation studies. The attenuation test is specific validated techniques approved by the competent
performed by intranasal inoculation of ferrets, free from authority.
antibodies against influenza virus, with the attenuated master MONO VALENT BULK
virus seed lot. The animals are monitored for a defined Monovalent bulks are prepared by pooling a number of
number of days post-inoculation for signs of influenza-like satisfactory single harvests or monovalent pooled harvests of
illness, including nasal discharge, frequent sneezing, severe the same virus type. The monovalent bulk is concentrated
lethargy, or fever. and purified by high-speed centrifugation or another suitable
At the conclusion of the monitoring period, animals are method then filtered through a bacteria retentive filter.
euthanized. Nasal turbinate and lung tissues are collected Only a monovalent bulk that complies with the following
and analysed for the presence of infectious virus using a requirements may be used in the preparation of the final bulk
suitable infectivity assay. vaccine.
For a master virus seed lot to be identified as attenuated, the Identification
virus must be detected in samples of nasal turbinate tissues Each monovalent bulk is identified as influenza virus of the
and samples from lung tissues from individual animals, and given type using suitable haemagglutinin type specific assay.
must demonstrate that the virus growth is restricted or shows Virus concentration
no virus replication. In addition, there are no signs of The virus concentration of each monovalent bulk is
influenza-like illness in the inoculated animals. determined by titration using a suitable validated in vitro
Virus concentration assay (e.g. fluorescent focus assay).
The virus concentration of each attenuated master virus seed Cold adapted and temperature sensitive phenotype
lot is determined by titration in cell cultures using a suitable Each monovalent bulk complies with the test as described
validated in vitro cell-based assay (e.g. fluorescent focus under Virus seed lots.
assay) to monitor the consistency of production.
Attenuation test
Extraneous agents (2. 6.16) The attenuation test is performed by intranasal inoculation of
Each attenuated master virus seed lot complies with the ferrets, free from antibodies against influenza virus, with each
requirements for virus seed lots. monovalent bulk test sample as described under Virus seed
Avian leucosis viruses lots.
For each attenuated master virus seed lot, the absence of If sufficient consistency data are available, and approved by
avian leucosis viruses is verified using a suitable method. the competent authority, only the first 3 monovalent bulks
PROPAGATION AND HARVEST following the introduction of a new attenuated master virus
All processing of the fertilised eggs is done under aseptic seed lot are tested on ferrets.
conditions in an area where no other infectious agents or Wherever possible, in accordance with the provisions of the
cells are handled at the same time. After inoculation and European Convention for the Protection of Vertebrate
incubation at a controlled temperature, only eggs containing Animals used for Experimental and Other Scientific
living and typical chick embryos are harvested. Purposes, manufacturers are encouraged to develop validated
The percentage of rejected eggs is recorded. After in vitro alternative methods to the animal test for monovalent
homogenisation and clarification by centrifugation, the bulks using appropriate tools such as molecular methods or
clarified allantoic fluid is tested as described below and kept other suitable methods for determination of viral attenuation
at -70 °C or colder until further processing. No human markers.
protein is added to the virus suspension at any stage during

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British

Incorporating the requeriments of the 10th edition of the

See General Notices

Effective date: 1 January 2023

London: The Stationery Office

THE DEPARTMENT OF HEALTH AND SOCIAL CARE

THE DEPARTMENT OF HEALTH (NI)

© Crown Copyright 2022

First Published 2022

CONTENTS OF THE GENERAL NOTICES

Generai Notices of the European Pharmacopoeia Immw1og10ounns, 1mmunos~ra anu v accmes

Weights and Measures

Me --CH 3 Bus -CH(CH 1 )CH 2 CH,,

Manufacture of Formulated Preparations

Freshly and Recently Prepared

Descriptive term Approximate volume of solvent in millilitres

Assays and Tests

Biological Assays and Tests

In all cases fiducial limits of error are based on a probability of 95 % (P = 0. 95).

Reference Substances and Reference Preparations

Action and Use

Crude Drugs; Traditional Herbal and Complementary Medicines

1.5.3 MONOGRAPHS ON MEDICINAL PRODUCTS CONTAINING CHEMICALLY

1.1.1.1 Quality systems

1.1.1.2 Conventional terms

Competent authority The national, supranational or international body or organisation vested

1.1.1.3 References to regulatory documents

1.1.2 Compliance with the Pb. Eur.

1. 1.2.2 Demonstration of compliance with the Ph. Eur.

1.1.2.3 Demonstration of suitability of monographs

1.1.2.4 Validation and implementation of Pb. Eur. analytical procedures

such cases, validation of tht> prncNinrt>s ls thP rt>.;;:pcm~lhlllty nf thP- 11'-Pr,

1.1.2.5 Alternative analytical procedures

1. 1.2.6 Pharmacopoeia! harmonisation

1.2.5 Drying and ignition to constant mass

1.2. 7 Reagents and solvents

1.2.8 Expression of content

1.2.9 Caution statements

1.3.1 Materials for containers and containers

1.4 GENERAL MONOGRAPHS AND GENERAL MONOGRAPHS ON DOSAGE

1.5 INDIVIDUAL MONOGRAPHS

1.5.1.2 Relative atomic and molecular masses, formulae

1.5.1.3 CAS registry number

1.5.1.6 Potential adulteration

1.5.1.9 Tests and assays

1.5.1.13 Functionality-related characteristics of excipients

1.5.2 MONOGRAPHS ON HERBAL DRUGS

1.5.3 MONOGRAPHS ON MEDICINAL PRODUCTS CONTAINING CHEMICALLY

1.5.3.1 Related substances

1.7. ABBREVIATIONS AND SYMBOLS

A Absorbance mp Melting puilll

Abbreviations used in the monographs on immunoglobulins, immunosera

IP Institut Pasteur, Collection Nationale de NCTC National Collection of Type Cultures

Wave number t' one per metre 1/m m-1

Wavelength 2 micrometre µm 10- 6 m

Area A,S square metre m2 m2

Volume V cubic metre m3 m3 1 mL = 1 cm3 = 10- 6 m'

Frequency V hertz Hz s-1

Velocity, speed V metre per m/s m-s- 1

Force F ne""'ton N m-kg·s 2 1 dyne= 1 g-cm·s 2 ::: 10 5 N

Pressure, stress p pascal Pa m- 1-kg-s 2

1 atm = 101 325 Pa= 101.325 kPa

Dynamic 1/ pascal second Pa-s m- 1 -kg-s 1

Kinematic V square metre m2/s m2,s-1 Pa-s•m 3-kg- 1 1 St= 1 cm 2 •s- 1