Traditional Chinese medicine -- Quality and safety of raw materials and finished products made with raw materials

This document specifies requirements for identity testing within a quality control framework for raw materials and finished products used in and as traditional Chinese medicine (TCM) and derivative forms. It is applicable to natural products used in TCM, including starting materials and finished products of herbal origin.

Médecine traditionnelle chinoise -- Qualité et sécurité des matières premières et des produits finis fabriqués à partir de matières premières

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INTERNATIONAL ISO
STANDARD 19609-2
First edition
2021-01
Traditional Chinese medicine —
Quality and safety of raw materials
and finished products made with raw
materials —
Part 2:
Identity testing of constituents of
herbal origin
Médecine traditionnelle chinoise — Qualité et sécurité des matières
premières et des produits finis fabriqués à partir de matières
premières —
Partie 2: Identification des constituants d'origine végétale
Reference number
ISO 19609-2:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO 19609-2:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 19609-2:2021(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Minimum requirements for the testing of the identity of starting materials and

finished products ................................................................................................................................................................................................. 2

5 Requirements for macroscopic, microscopic and organoleptic description tests ...........................3

5.1 General ........................................................................................................................................................................................................... 3

5.2 Macroscopic description test ...................................................................................................................................................... 4

5.2.1 Application ............................................................................................................................................................................ 4

5.2.2 Macroscopic examination ........................................................................................................................................ 4

5.2.3 Assessment ........................................................................................................................................................................... 4

5.3 Microscopic description test ....................................................................................................................................................... 4

5.3.1 Application ............................................................................................................................................................................ 4

5.3.2 Microscopic examination .......................................................................................................................................... 4

5.3.3 Assessment ........................................................................................................................................................................... 4

5.4 Organoleptic description test ..................................................................................................................................................... 5

5.4.1 Application ............................................................................................................................................................................ 5

5.4.2 Organoleptic examination ........................................................................................................................................ 5

5.4.3 Assessment ........................................................................................................................................................................... 5

6 Chromatographic identification tests with HPLC and TLC ........................................................................................ 5

6.1 General ........................................................................................................................................................................................................... 5

6.2 Sample preparation ............................................................................................................................................................................ 6

6.2.1 General...................................................................................................................................................................................... 6

6.2.2 Reagents .................................................................................................................................................................................. 6

6.2.3 Apparatus ............................................................................................................................................................................... 7

6.2.4 Sample preparation method for solids ......................................................................................................... 7

6.2.5 Sample preparation method for semi-solid materials .................................................................... 7

6.2.6 Sample preparation method for liquids ....................................................................................................... 8

6.2.7 Solid-phase extraction (SPE) ................................................................................................................................. 9

6.2.8 Test solution......................................................................................................................................................................... 9

6.3 Identification of constituents with HPLC and diode array detection ....................................................... 9

6.3.1 General...................................................................................................................................................................................... 9

6.3.2 HPLC fingerprint testing method ...................................................................................................................... 9

6.4 Identification of typical constituents with TLC ........................................................................................................11

6.4.1 Group selected identification tests with TLC .......................................................................................11

7 Chemical identification tests ................................................................................................................................................................16

Bibliography .............................................................................................................................................................................................................................17

© ISO 2021 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 19609-2:2021(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to

the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see

www .iso .org/ iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 249, Traditional Chinese medicine.

A list of all parts in the ISO 19609 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO 19609-2:2021(E)
Introduction

The ISO 19609 series consists of four parts with different content as shown in Figure 1.

Figure 1 — Overview of the ISO 19609 series

To ensure the safety and efficacy of herbal medicinal products, it is imperative to check the identity

of the raw materials and the finished products. One of the aims of the identity check is to prevent the

accidental use of falsifications. In order to ensure safety, it is also essential to establish adequate simple

testing methods.

As a general basis we can expect in a typical medicinal plant about 1 million constituents. When specific

extracts are made from this plant, the number of ingredients is reduced to about 50 000. In classical

phytochemical analysis, only one compound is usually analytically identified or quantified as a so-called

marker without consideration of the secondary substance matrix from this multicomponent mixture.

This results from the test practice for synthetic chemical monopreparations, in which the efficacy is

based only on a high-dose active substance. This practice results from the analytical quantification of

the relevant effective plasma concentration in the blood of the treated patients.

Phytopharmaceuticals are not based on only one active substance, but on a combination of synergistic

compounds (many to thousands). Classical phytotherapy worldwide does not usually use only individual

herbs but combinations of several herbs in preparations containing a correspondingly higher number of

individual compounds (multitarget theory).

The analytical methods of the pharmacopoeias use one or more marker compounds without

significance for the respective effectiveness. This cannot reflect the difficulty of synergistic substances

as mentioned previously.
© ISO 2021 – All rights reserved v
---------------------- Page: 5 ----------------------
ISO 19609-2:2021(E)

For preparations based on several raw herbal materials or extracts there are currently no test methods

[7]

available. In the Chinese Pharmacopoeia , only one or two markers are often used for such finished

products, although many more different raw materials are integrated.

Legally, the quality of these products as remedies must be estimated in an appropriate way.

For analytical quality assessment of such combinations, there are thus two basic approaches:

1) Marker-based identification test

For each herbal raw material or extract used, a test methodology that can be implemented in the

entire product must be individually redeveloped and validated for each of the associated markers.

For a combination product with six herbals, herbal parts or exudates, as well as animals and minerals,

six independent test methods for each single material based on these six marker compounds are

required for release. This approach is currently mandatory for phytopharmaceuticals in countries

[11]
which apply the European Pharmacopoeia .
2) Three-dimensional ingredient overview chromatography (fingerprint)

With this novel valid method, it is possible to record the entire visible and ultraviolet (UV-VIS)

spectroscopic detectable ingredient spectrum of a combination product (finished product) with

only one liquid chromatographic separation method [one high-performance liquid chromatography

(HPLC) run instead of x different ones]. In comparison with corresponding reference extracts of the

associated individual materials, this is a clear assignment without compulsory use of the various

expensive marker substances possible. This makes a cost-effective, reliable and fast product release

possible, without sacrificing product quality.

NOTE Experts agree that the presence of a marker compound as the only criterion for the identity of the used

material is not sufficient. Experience of recent years has shown that synthetic active principles or only defined

marker constituents were used instead of the real herb material (ephedra problem). Over a long time, high risks

resulted from the addition of a racemic mixture of ephedrine to optimize the ephedra material (with a mixture of

natural and non-natural isomers), which led in the end to a total prohibition of this material worldwide.

As a method for determining adequate identity, a non-specific HPLC fingerprint method is suitable. This

method makes it possible to ensure the identity of the material, both in terms of the retention times of

various ingredient patterns and in terms of the UV-VIS-spectra of the individual signals.

Here the question arises as to whether an individualized testing method for each herb (as mostly

required in pharmacopoeias) or a general fingerprint method over the whole range of ingredients is to

be used. The disadvantage of a method which is not optimally matched to each single herb, however, is

easily outweighed by the advantage that complex mixtures of different raw materials in the resulting

product can also be identified only with one single method. In addition, the found distribution pattern

can give further conclusions on the used extraction procedure.

A universal method must be established over the entire hydrophilic to lipophilic region to realize

efficiently the plurality of components in one analytical method, in a sufficiently secure way, with a

photodiode array detection (PDA) as well as a diode array detection (DAD). Now the achieved spectra

can become assigned to the underlying components of the individual raw materials. In exceptional

cases it might be necessary to make certain improvements for individual products which have to be

analysed (modification of one of the three-dimensional specifications: time, intensity and spectrum).

vi © ISO 2021 – All rights reserved
---------------------- Page: 6 ----------------------
INTERNATIONAL STANDARD ISO 19609-2:2021(E)
Traditional Chinese medicine — Quality and safety of
raw materials and finished products made with raw
materials —
Part 2:
Identity testing of constituents of herbal origin
1 Scope

This document specifies requirements for identity testing within a quality control framework for raw

materials and finished products used in and as traditional Chinese medicine (TCM) and derivative

forms. It is applicable to natural products used in TCM, including starting materials and finished

products of herbal origin.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 19609-1, Traditional Chinese medicine — Quality and safety of raw materials and finished products

made with raw materials — Part 1: General requirements
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
active substance
substance of physiological or pharmacological action
3.2
decoction piece

prescription medicinal processed from Chinese Materia Medica under the direction of TCM and

processing methods for Chinese medicines and derivative forms, which can be directly used in clinical

practice or the production of prepared medicines

[SOURCE: ISO 18668-1:2016, 3.3, modified — Note 1 to entry amalgamated with definition.]

3.3
finished product
commercial product intended for sale and use, including decoction pieces (3.2)
© ISO 2021 – All rights reserved 1
---------------------- Page: 7 ----------------------
ISO 19609-2:2021(E)
3.4
manufacturing

process that creates a finished product (3.2) from a starting material (3.8) in a form suitable for its

intended purpose, including packaging
3.5
monograph
detailed written study of a single specialized subject or an aspect of it
EXAMPLE Description of a herb in a pharmacopoeia.
3.6
raw material
substance going into or involved in the manufacturing (3.4) of a bulk product
[SOURCE: ISO 22716:2007, 2.28]
3.7
Rf-value

distance travelled by a given component divided by the distance travelled by the solvent front in thin

layer chromatography
3.8
starting material

material received by a manufacturer to be commercially processed, manufactured or packaged

Note 1 to entry: This includes raw materials (3.6) and other materials, for example solvents, excipients and

capsule shells.
4 Minimum requirements for the testing of the identity of starting materials and
finished products

General quality control methods shall include the following identification tests and, if applicable, an

“assay” as established in typical monographs:
a) macroscopic description test;
b) microscopic description test;
c) organoleptic description test;
d) test with HPLC;
e) test with thin layer chromatography (TLC).
The use of the methods depends on the material being tested (see Table 1).
2 © ISO 2021 – All rights reserved
---------------------- Page: 8 ----------------------
ISO 19609-2:2021(E)
Table 1 — Overview of identity testing methods for different materials
Starting material Starting material Mixture of Finished product
with monograph without starting materials
monograph
a) macroscopic According to According to Not applicable Not applicable
description test monograph scientific literature
b) microscopic According to According to According to Not applicable
description test monograph scientific literature scientific literature
c) organoleptic According to According to Not applicable Not applicable
description test monograph scientific literature
d) HPLC test According to Clause 6 Clause 6 Clause 6
monograph
or alternative
Clause 6
e) TLC test According to Clause 6 Clause 6 Clause 6
monograph or
alternative
Clause 6
5 Requirements for macroscopic, microscopic and organoleptic description tests
5.1 General
For sample collection see ISO 19609-1.
Visual and organoleptic examinations include the following three methods:
a) Macroscopic description test for identification of herbal materials.

This description consists of the form, size, colour, surface characters and texture (including cut

surface or fracture characters) of the crude materials and prepared slices.

— “Form” refers to the shape of crude materials and prepared slices. Wrinkled herbs, leaves or

flowers can be moistened, softened and spread.

— “Size” refers to the length, diameter and thickness of crude materials and prepared slices. In

general, a millimetre ruler is used for the measurement.

— “Colour” refers to the colour and glossiness of crude materials and prepared slices observed in

daylight. If the colour is described in a combination of two colours, the main colour is the latter.

— “Surface characters”, texture and cut surface of crude materials or prepared slices is described

without pretreatment.
b) Microscopic description test for identification of herbal materials.

Microscopic identification is a method where the application of a microscope is used to identify

the characters of tissues, stomata and stomata index, cells or cell contents in sections, powders,

disintegrated tissues or surface slides of prepared slices of crude materials and dosage form,

including powders of prepared slices of crude materials. Representative samples are chosen to

be identified and slides are prepared to meet the requirements of identification for each crude

material.
© ISO 2021 – All rights reserved 3
---------------------- Page: 9 ----------------------
ISO 19609-2:2021(E)

If the samples being examined are pulverised or ground (e.g. in the case of decoction pieces), the

resulting material should conform with the requirements in the monograph and ISO/TS 21310, as

well as the general criteria.
c) Organoleptic description test for identification of herbal materials.
— Odour
— Taste

NOTE Historically, the exact visual examination of the herbs was the only criterion for identification.

This is still used in addition to the described modern testing methods for starting materials. For uncut,

cut and powdered herbal materials, microscopic, macroscopic and organoleptic tests are established.

5.2 Macroscopic description test
5.2.1 Application

Macroscopic description tests are applicable for starting materials. Mixtures of starting materials and

finished products cannot be described typically by macroscopic description tests.

5.2.2 Macroscopic examination

The macroscopic examination is normally done without any apparatus for the description. The use of

a magnifying glass or binoculars is appropriate for the macroscopic examination of test samples. The

magnification shall be sufficient to allow adequate characterization of the smallest characteristics to be

classified in the test sample. The use of an appropriate lamp is recommended.
5.2.3 Assessment

The examinations shall be compared with authenticated reference data from monographs or other

reliable scientific data. If the results are identical with the reference data this part of the identity can be

confirmed.
5.3 Microscopic description test
5.3.1 Application

Microscopic description tests are applicable for starting materials. Mixtures of starting materials and

finished products cannot be described typically by microscopic description tests.

5.3.2 Microscopic examination
The sample collection shall be done in accordance with ISO 19609-1:2021, 6.2.

If the samples being examined are pulverised or ground (e.g. in the case of decoction pieces) the

resulting material should conform with the requirements in the monograph.

The microscopic examination should be done in accordance with ISO/TS 21310 and can be applied to

other materials, such as powdered herbals.
5.3.3 Assessment

The examinations shall be compared with authenticated reference data from monographs or other

reliable scientific data. If the results are identical with the reference data this part of the identity can be

confirmed.
4 © ISO 2021 – All rights reserved
---------------------- Page: 10 ----------------------
ISO 19609-2:2021(E)
5.4 Organoleptic description test
5.4.1 Application

Organoleptic description tests are applicable for starting materials. Mixtures of starting materials and

finished products cannot be described typically by an organoleptic description test.

NOTE Organoleptic tests of odour and taste of raw materials are useful, but are often unsuitable for finished

products because of the use of tasteless “dosage forms” or corresponding odourless and taste-neutralizing

additives.
5.4.2 Organoleptic examination

When examining the odour, smell directly or, if necessary, fracture, crush or rub the samples. When

necessary, moisten the sample with hot water before examination.

When examining the taste, use a small quantity of the sample directly. In some cases it can be

appropriate to macerate the sample in hot water and taste the extract.
Specific requirements of pharmacopoeias should be taken into account.
WARNING — Be cautious when tasting poisonous, hazardous or toxic materials.
5.4.3 Assessment

The examinations shall be compared with authenticated reference data from monographs or other

reliable scientific data. If the results are identical to the reference data, this part of the identity can be

confirmed.
6 Chromatographic identification tests with HPLC and TLC
6.1 General

Chromatographic identification tests shall be applied only for the determination of the identity of

starting materials and finished products based on herbal raw materials and ingredients, as well as

materials after advanced processing (e.g. boiling, steaming, roasting).

Gas chromatographic tests can be additionally applied for the detection of volatile compounds, such as

essential oils, and other components with characteristic odour, such as fatty acids, after hydrolysis of

lipids and others.

Chromatographic methods for hydrophilic to slightly lipophilic compounds are suitable for the analysis

of identity. In this case specific and non-specific separation processes shall be used.

Specific separation methods such as TLC detect, with a certain specificity, classical active component

groups in the field of herbal secondary products (e.g. alkaloids, flavonoids, saponins, essential oils and

other classes of natural compounds).

Non-specific separation methods such as HPLC are suitable to show the total spectrum of ingredients of

a herbal or a related product in its entirety and complexity.

In order to perform a chromatographic separation, the test samples (dosage forms) have to be

transferred into an analysable test solution in a connected upstream sample preparation step.

© ISO 2021 – All rights reserved 5
---------------------- Page: 11 ----------------------
ISO 19609-2:2021(E)
6.2 Sample preparation
6.2.1 General

For a classical analysis with chromatographic methods, it is necessary to dissolve the ingredients or

active principles, which shall be tested by suitable methods. Chromatographic techniques separate the

components, followed by an appropriate method of detection. This separation pattern can be used for

both determination of the identity and a possible quantification (assay) of relevant compounds.

For a sufficient analysis, sample preparation with appropriate methods shall ensure that neither solids,

particles nor emulsions are used.

Figure 2 gives an overview of the sample preparation of solids, semi-solid materials and liquids.

Less common dosage forms such as chewing gums, ear preparations, foams, suppositories, tampons,

poultices, sticks, medicinal distillates, liniments, smeared films, sprays, pessaries and others are not

included in the sample preparation procedure. For these products a suitable connected upstream

sample preparation method has to be adapted individually or newly developed and validated.

Alternatively, chloroform can be used.
Figure 2 — Sample preparation procedure
6.2.2 Reagents
6.2.2.1 Methanol gradient grade/HPLC grade (MeOH).
6 © ISO 2021 – All rights reserved
---------------------- Page: 12 ----------------------
ISO 19609-2:2021(E)
6.2.2.2 Chloroform gradient grade/HPLC grade (CHCl ).
WARNING — Work with chloroform can have critical effects on the health of staff.
6.2.2.3 Hexane gradient grade/HPLC grade.
6.2.2.4 Water gradient grade/HPLC grade.
6.2.2.5 Ethyl acetate gradient grade/HPLC grade.
6.2.2.6 Diethyl ether gradient grade/HPLC grade.
6.2.3 Apparatus
Use the usual laboratory equipment and, in particular, an ultrasonic bath.
6.2.4 Sample preparation method for solids

For the examination of solid testing materials there are two processing methods for further handling.

Materials in powder form, such as powders, aerosols, microparticles and nanoparticles, can be used

directly for extracting.

Coarse materials such as granules and dry extract particles, as well as herbs and ready dosage forms

...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 19609-2
ISO/TC 249
Traditional Chinese medicine —
Secretariat: SAC
Quality and safety of raw materials
Voting begins on:
2020-10-02 and manufacturing products made
with raw materials —
Voting terminates on:
2020-11-27
Part 2:
Identity testing of constituents of
herbal origin
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/FDIS 19609-2:2020(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. ISO 2020
---------------------- Page: 1 ----------------------
ISO/FDIS 19609-2:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/FDIS 19609-2:2020(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Minimum requirements for the testing of the identity of starting materials and

finished products ................................................................................................................................................................................................. 2

5 Requirements for macroscopic, microscopic and organoleptic description tests ...........................3

5.1 General ........................................................................................................................................................................................................... 3

5.2 Macroscopic description test ...................................................................................................................................................... 4

5.2.1 Application ............................................................................................................................................................................ 4

5.2.2 Macroscopic examination ........................................................................................................................................ 4

5.2.3 Assessment ........................................................................................................................................................................... 4

5.3 Microscopic description test ....................................................................................................................................................... 4

5.3.1 Application ............................................................................................................................................................................ 4

5.3.2 Microscopic examination .......................................................................................................................................... 4

5.3.3 Assessment ........................................................................................................................................................................... 4

5.4 Organoleptic description test ..................................................................................................................................................... 5

5.4.1 Application ............................................................................................................................................................................ 5

5.4.2 Organoleptic examination ........................................................................................................................................ 5

5.4.3 Assessment ........................................................................................................................................................................... 5

6 Chromatographic identification tests with HPLC and TLC ........................................................................................ 5

6.1 General ........................................................................................................................................................................................................... 5

6.2 Sample preparation ............................................................................................................................................................................ 6

6.2.1 General...................................................................................................................................................................................... 6

6.2.2 Reagents .................................................................................................................................................................................. 6

6.2.3 Apparatus ............................................................................................................................................................................... 7

6.2.4 Sample preparation method for solids ......................................................................................................... 7

6.2.5 Sample preparation method for semi-solid materials .................................................................... 7

6.2.6 Sample preparation method for liquids ....................................................................................................... 8

6.2.7 Solid-phase extraction (SPE) ................................................................................................................................. 9

6.2.8 Test solution......................................................................................................................................................................... 9

6.3 Identification of constituents with HPLC and diode array detection ....................................................... 9

6.3.1 General...................................................................................................................................................................................... 9

6.3.2 HPLC fingerprint testing method ...................................................................................................................... 9

6.4 Identification of typical constituents with TLC ........................................................................................................11

6.4.1 Group selected identification tests with TLC .......................................................................................11

7 Chemical identification tests ................................................................................................................................................................16

Bibliography .............................................................................................................................................................................................................................17

© ISO 2020 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO/FDIS 19609-2:2020(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to

the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see

www .iso .org/ iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 249, Traditional Chinese medicine.

A list of all parts in the ISO 19609 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2020 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/FDIS 19609-2:2020(E)
Introduction

The ISO 19609 series consists of four parts with different content as shown in Figure 1.

Figure 1 — Overview of the ISO 19609 series

To ensure the safety and efficacy of herbal medicinal products, it is imperative to check the identity

of the raw materials and the finished products. One of the aims of the identity check is to prevent the

accidental use of falsifications. In order to ensure safety, it is also essential to establish adequate simple

testing methods.

As a general basis we can expect in a typical medicinal plant about 1 million constituents. When specific

extracts are made from this plant, the number of ingredients is reduced to about 50 000. In classical

phytochemical analysis, only one compound is usually analytically identified or quantified as a so-called

marker without consideration of the secondary substance matrix from this multicomponent mixture.

This results from the test practice for synthetic chemical monopreparations, in which the efficacy is

based only on a high-dose active substance. This practice results from the analytical quantification of

the relevant effective plasma concentration in the blood of the treated patients.

Phytopharmaceuticals are not based on only one active substance, but on a combination of synergistic

compounds (many to thousands). Classical phytotherapy worldwide does not usually use only individual

herbs but combinations of several herbs in preparations containing a correspondingly higher number of

individual compounds (multitarget theory).

The analytical methods of the pharmacopoeias use one or more marker compounds without

significance for the respective effectiveness. This cannot reflect the difficulty of synergistic substances

as mentioned previously.
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For preparations based on several raw herbal materials or extracts there are currently no test methods

[5]

available. In the Chinese Pharmacopoeia , only one or two markers are often used for such finished

products, although many more different raw materials are integrated.

Legally, the quality of these products as remedies must be estimated in an appropriate way.

For analytical quality assessment of such combinations, there are thus two basic approaches:

1) Marker-based identification test

For each herbal raw material or extract used, a test methodology that can be implemented in the

entire product must be individually redeveloped and validated for each of the associated markers.

For a combination product with six herbals, herbal parts or exudates, as well as animals and minerals,

six independent test methods for each single material based on these six marker compounds are

required for release. This approach is currently mandatory for phytopharmaceuticals in countries

[9]
which apply the European Pharmacopoeia .
2) Three-dimensional ingredient overview chromatography (fingerprint)

With this novel valid method, it is possible to record the entire visible and ultraviolet (UV-VIS)

spectroscopic detectable ingredient spectrum of a combination product (finished product) with

only one liquid chromatographic separation method [one high-pressure liquid chromatography

(HPLC) run instead of x different ones]. In comparison with corresponding reference extracts of the

associated individual materials, this is a clear assignment without compulsory use of the various

expensive marker substances possible. This makes a cost-effective, reliable and fast product release

possible, without sacrificing product quality.

NOTE Experts agree that the presence of a marker compound as the only criterion for the identity of the used

material is not sufficient. Experience of recent years has shown that synthetic active principles or only defined

marker constituents were used instead of the real herb material (ephedra problem). Over a long time, high risks

resulted from the addition of a racemic mixture of ephedrine to optimize the ephedra material (with a mixture of

natural and non-natural isomers), which led in the end to a total prohibition of this material worldwide.

As a method for determining adequate identity, a non-specific HPLC fingerprint method is suitable. This

method makes it possible to ensure the identity of the material, both in terms of the retention times of

various ingredient patterns and in terms of the UV-VIS-spectra of the individual signals.

Here the question arises as to whether an individualized testing method for each herb (as mostly

required in pharmacopoeias) or a general fingerprint method over the whole range of ingredients is to

be used. The disadvantage of a method which is not optimally matched to each single herb, however, is

easily outweighed by the advantage that complex mixtures of different raw materials in the resulting

product can also be identified only with one single method. In addition, the found distribution pattern

can give further conclusions on the used extraction procedure.

A universal method must be established over the entire hydrophilic to lipophilic region to realize

efficiently the plurality of components in one analytical method, in a sufficiently secure way, with a

photodiode array detection (PDA) as well as a diode array detection (DAD). Now the achieved spectra

can become assigned to the underlying components of the individual raw materials. In exceptional

cases it might be necessary to make certain improvements for individual products which have to be

analysed (modification of one of the three-dimensional specifications: time, intensity and spectrum).

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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 19609-2:2020(E)
Traditional Chinese medicine — Quality and safety of raw
materials and manufacturing products made with raw
materials —
Part 2:
Identity testing of constituents of herbal origin
1 Scope

This document specifies requirements for identity testing within a quality control framework for raw

materials and finished products used in and as traditional Chinese medicine (TCM) and derivative

forms. It is applicable to natural products used in TCM, including starting materials and finished

products of herbal origin.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 19609-1, Traditional Chinese medicine — Quality and safety of raw materials and manufacturing

products made with raw materials — Part 1: General requirements
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
active substance
substance of physiological or pharmacological action
3.2
decoction piece

prescription medicinal processed from Chinese Materia Medica under the direction of TCM and

processing methods for Chinese medicines and derivative forms, which can be directly used in clinical

practice or the production of prepared medicines

[SOURCE: ISO 18668-1:2016, 3.3, modified — Note 1 to entry amalgamated with definition.]

3.3
finished product
commercial product intended for sale and use, including decoction pieces (3.2)
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3.4
manufacturing

process that creates a finished product (3.2) from a starting material (3.8) in a form suitable for its

intended purpose, including packaging
3.5
monograph
detailed written study of a single specialized subject or an aspect of it
EXAMPLE Description of a herb in a pharmacopoeia.
3.6
raw material
substance going into or involved in the manufacturing (3.4) of a bulk product
[SOURCE: ISO 22716:2007, 2.28]
3.7
Rf-value

distance travelled by a given component divided by the distance travelled by the solvent front in thin

layer chromatography
3.8
starting material

material received by a manufacturer to be commercially processed, manufactured or packaged

Note 1 to entry: This includes raw materials (3.6) and other materials, for example solvents, excipients and

capsule shells.
4 Minimum requirements for the testing of the identity of starting materials and
finished products

General quality control methods shall include the following identification tests and, if applicable, an

“assay” as established in typical monographs:
a) macroscopic description test;
b) microscopic description test;
c) organoleptic description test;
d) test with high-performance liquid chromatography (HPLC);
e) test with thin layer chromatography (TLC).
The use of the methods depends on the material being tested (see Table 1).
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ISO/FDIS 19609-2:2020(E)
Table 1 — Overview of identity testing methods for different materials
Starting material Starting material Mixture of Finished product
with monograph without starting materials
monograph
a) m a c r o s c op ic According According Not applicable Not applicable
description test monograph scientific literature
b) m ic r o s c op ic According According According Not applicable
description test monograph scientific literature scientific literature
c) o r g a n ol e p t ic According According Not applicable Not applicable
description test monograph scientific literature
d) HPLC test According Clause 6 Clause 6 Clause 6
monograph
or alternative
Clause 6
e) TLC test According Clause 6 Clause 6 Clause 6
monograph or
alternative
Clause 6
5 Requirements for macroscopic, microscopic and organoleptic description tests
5.1 General
For sample collection see ISO 19609-1.
Visual and organoleptic examinations include the following three methods:
a) Macroscopic description test for identification of herbal materials.

This description consists of the form, size, colour, surface characters and texture (including cut

surface or fracture characters) of the crude materials and prepared slices.

— “Form” refers to the shape of crude materials and prepared slices. Wrinkled herbs, leaves or

flowers can be moistened, softened and spread.

— “Size” refers to the length, diameter and thickness of crude materials and prepared slices. In

general, a milimetre ruler is used for the measurement.

— “Colour” refers to the colour and glossiness of crude materials and prepared slices observed in

daylight. If the colour is described in a combination of two colours, the main colour is the latter.

— “Surface characters”, texture and cut surface of crude materials or prepared slices is described

without pretreatment.
b) Microscopic description test for identification of herbal materials.

Microscopic identification is a method where the application of a microscope is used to identify

the characters of tissues, stomata and stomata index, cells or cell contents in sections, powders,

disintegrated tissues or surface slides of prepared slices of crude materials and dosage form,

including powders of prepared slices of crude materials. Representative samples are chosen to

be identified and slides are prepared to meet the requirements of identification for each crude

material.
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ISO/FDIS 19609-2:2020(E)

If the samples being examined are pulverised or ground (e.g. in the case of decoction pieces), the

resulting material should conform with the requirements in the monograph and ISO/TS 21310, as

well as the general criteria.
c) Organoleptic description test for identification of herbal materials.
— Odour
— Taste

NOTE Historically, the exact visual examination of the herbs was the only criterion for identification.

This is still used in addition to the described modern testing methods for starting materials. For uncut,

cut and powdered herbal materials, microscopic, macroscopic and organoleptic tests are established.

5.2 Macroscopic description test
5.2.1 Application

Macroscopic description tests are applicable for starting materials. Mixtures of starting materials and

finished products cannot be described typically by macroscopic description tests.

5.2.2 Macroscopic examination

The macroscopic examination is normally done without any apparatus for the description. The use of

a magnifying glass or binoculars is appropriate for the macroscopic examination of test samples. The

magnification shall be sufficient to allow adequate characterization of the smallest characters to be

classified in the test sample. The use of an appropriate lamp is recommended.
5.2.3 Assessment

The examinations shall be compared with authenticated reference data from monographs or other

reliable scientific data. If the results are identical with the reference data this part of the identity can be

confirmed.
5.3 Microscopic description test
5.3.1 Application

Microscopic description tests are applicable for starting materials. Mixtures of starting materials and

finished products cannot be described typically by microscopic description tests.

5.3.2 Microscopic examination
The sample collection shall be done in accordance with ISO 19609-1:2020, 6.2.

If the samples being examined are pulverised or ground (e.g. in the case of decoction pieces) the

resulting material should conform with the requirements in the monograph.

The microscopic examination should be done in accordance with ISO/TS 21310 and can be applied to

other materials, such as powdered herbals.
5.3.3 Assessment

The examinations shall be compared with authenticated reference data from monographs or other

reliable scientific data. If the results are identical with the reference data this part of the identity can be

confirmed.
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5.4 Organoleptic description test
5.4.1 Application

Organoleptic description tests are applicable for starting materials. Mixtures of starting materials and

finished products cannot be described typically by an organoleptic description test.

NOTE Organoleptic tests of odour and taste of raw materials are useful, but are often unsuitable for finished

products because of the use of tasteless “dosage forms” or corresponding odourless and taste-neutralizing

additives.
5.4.2 Organoleptic examination

When examining the odour, smell directly or, if necessary, fracture, crush or rub the samples. When

necessary, moisten the sample with hot water before examination.

When examining the taste, use a small quantity of the sample directly. In some cases it can be

appropriate to macerate the sample in hot water and taste the extract.
Specific requirements of pharmacopoeias should be taken into account.
WARNING — Be cautious when tasting poisonous, hazardous or toxic materials.
5.4.3 Assessment

The examinations shall be compared with authenticated reference data from monographs or other

reliable scientific data. If the results are identical to the reference data, this part of the identity can be

confirmed.
6 Chromatographic identification tests with HPLC and TLC
6.1 General

Chromatographic identification tests shall be applied only for the determination of the identity of

starting materials and finished products based on herbal raw materials and ingredients, as well as

materials after advanced processing (e.g. boiling, steaming, roasting).

Gas chromatographic tests can be additionally applied for the detection of volatile compounds, such as

essential oils, and other components with characteristic odour, such as fatty acids, after hydrolysis of

lipids and others.

Chromatographic methods for hydrophilic to slightly lipophilic compounds are suitable for the analysis

of identity. In this case specific and unspecific separation processes shall be used.

Specific separation methods such as TLC detect – with a certain specificity – classical active component

groups in the field of herbal secondary products (e.g. alkaloids, flavonoids, saponines, essential oils and

other classes of natural compounds).

Non-specific separation methods such as HPLC are suitable to show the total spectrum of ingredients of

a herbal or a related product in its entirety and complexity.

In order to perform a chromatographic separation, the test samples (dosage forms) have to be transfered

into an analysable test solution in a connected upstream sample preparation step.

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ISO/FDIS 19609-2:2020(E)
6.2 Sample preparation
6.2.1 General

For a classical analysis with chromatographic methods, it is necessary to dissolve the ingredients or

active principles, which shall be tested by suitable methods. Chromatographic techniques separate the

components, followed by an appropriate method of detection. This separation pattern can be used for

both determination of the identity and a possible quantification (assay) of relevant compounds.

For a sufficient analysis, sample preparation with appropriate methods shall ensure that neither solids,

particles nor emulsions are used.

Figure 2 gives an overview of the sample preparation of solids, semi-solid materials and liquids.

Less common dosage forms such as chewing gums, ear preparations, foams, suppositories, tampons,

poultices, sticks, medicinal distillates, liniments, smeared films, sprays, pessaries and others are not

included in the sample preparation procedure. For these products a suitable connected upstream

sample preparation method has to be adapted individually or newly developed and validated.

Alternative chloroform can be used.
Figure 2 — Sample preparation procedure
6.2.2 Reagents
6.2.2.1 Methanol gradient grade/HPLC grade (MeOH).
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ISO/FDIS 19609-2:2020(E)
6.2.2.2 Chloroform gradient grade/HPLC grade (CHCl ).
WARNING — Work with chloroform can have critical effects on the health of staff.
6.2.2.3 Hexane gradient grade/HPLC grade.
6.2.2.4 Water gradient grade/HPLC grade.
6.2.2.5 Ethylacetate gradient grade/HPLC grade.
6.2.2.6 Diethyl ether grad
...

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