Traditional Chinese medicine -- Determination of selected Aconitum alkaloids by high-performance liquid chromatography (HPLC)

This document specifies methods for the determination of the selected Aconitum alkaloids, including aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine, yunaconitine, deacetyl-yunaconitine and crassicauline A.

Médecine traditionnelle chinoise -- Dosage d'alcaloïdes d'aconit (Aconitum) sélectionnés par chromatographie liquide à haute performance (CLHP)

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Status
Published
Publication Date
28-Jul-2020
Current Stage
5060 - Close of voting Proof returned by Secretariat
Start Date
19-Jun-2020
Completion Date
18-Jun-2020
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INTERNATIONAL ISO
STANDARD 23191
First edition
2020-07
Traditional Chinese medicine —
Determination of selected Aconitum
alkaloids by high-performance liquid
chromatography (HPLC)
Médecine traditionnelle chinoise — Dosage d'alcaloïdes d'aconit
(Aconitum) sélectionnés par chromatographie liquide à haute
performance (CLHP)
Reference number
ISO 23191:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 23191:2020(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 23191:2020(E)
Contents Page

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

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

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

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

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

4 Abbreviated terms .............................................................................................................................................................................................. 1

5 Principle ........................................................................................................................................................................................................................ 2

6 Reagents ........................................................................................................................................................................................................................ 3

7 Apparatus ..................................................................................................................................................................................................................... 3

8 Sampling ........................................................................................................................................................................................................................ 4

9 Test procedures ..................................................................................................................................................................................................... 4

9.1 General ........................................................................................................................................................................................................... 4

9.2 HPLC-MS procedure ........................................................................................................................................................................... 5

9.2.1 Reference solution .......................................................................................................................................................... 5

9.2.2 Test solution......................................................................................................................................................................... 5

9.2.3 Chromatographic conditions ................................................................................................................................. 5

9.2.4 HPLC-MS conditions ..................................................................................................................................................... 5

9.2.5 Determination .................................................................................................................................................................... 5

9.3 HPLC-DAD procedure ........................................................................................................................................................................ 5

9.3.1 Reference solution .......................................................................................................................................................... 5

9.3.2 Test solution......................................................................................................................................................................... 6

9.3.3 Chromatographic conditions ................................................................................................................................. 6

9.3.4 Determination .................................................................................................................................................................... 6

10 Test report ................................................................................................................................................................................................................... 6

Annex A (informative) Typical HPLC conditions ...................................................................................................................................... 7

Annex B (informative) Typical chromatogram of the selected Aconitum alkaloids ............................................9

Annex C (informative) Typical Aconitum herb used in traditional Chinese medicine ...................................12

Annex D (informative) Reference values of national and regional limits of Aconitum

alkaloids in Aconitum carmichaelii lateral root ................................................................................................................13

Bibliography .............................................................................................................................................................................................................................14

© ISO 2020 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 23191:2020(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 ISO/TC 249, Traditional Chinese medicine.

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 23191:2020(E)
Introduction

Aconitum is a genus of about 350 species of flowering plants belonging to the family of Ranunculaceae.

The therapeutic use of as many as 76 species of Aconitum herbs in traditional Chinese medicine have

been documented for a very long time. Among the Aconitum herbs, the most well known are processed

Aconitum carmichaelii lateral root (附子), Aconitum carmichaelii root (川乌) and Aconitum kusnezoffii

root (草乌). Of approximately 80 000 traditional Chinese medicine formulae, around 10,2 % contain

Aconitum herbs. It is one of the most frequently used groups of herbal medicines in traditional Chinese

medicine. Typical Aconitum herbs used in traditional Chinese medicine are shown in Annex C, Table C.1.

Aconitum herbs contain Aconitum alkaloids which have anti-inflammatory, analgesic and cardiotonic

activities. The Aconitum alkaloids are a double-edged sword, however. At present, international trade

in Aconitum products is restricted to a few nations due to the high natural toxicity of crude Aconitum

products. Unprocessed Aconitum is highly toxic. Expert processing is required to reduce (but not

eliminate) toxicity, and where individual national regulatory schemes do not ban the herb, it is generally

restricted to a high-risk category, such as Schedule 2 in the Hong Kong Chinese Medicine regulations.

Also, there are sporadic cases of Aconitum alkaloid poisoning due to misuse reported all over the world.

Nonetheless, the toxicity of Aconitum herbs can be reduced dramatically with proper processing (such

as repeated boiling or steaming), prolonged decoction and dose control. However, testing standards for

Aconitum alkaloids have not yet been harmonized on an international level, and regulatory authorities

in many nations do not adequately differentiate highly toxic forms from less-toxic forms (or even non-

toxic forms) of Aconitum herbs.

Six kinds of Aconitum alkaloids [aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine

(BAC), benzoylmesaconine (BMA) and benzoylhypaconine (BHA)] are commonly used as chemical

markers for quality control of aconite, determined by the high-performance liquid chromatography

[1]

(HPLC) method . The AOAC Official Method 2008.11 also requires the determination of three Aconitum

alkaloids, AC, MA and HA, in dietary supplements and raw botanical materials by LC/UV detection

[2]

with confirmation by LC/MS/MS . Nevertheless, poisoning cases are still occasionally reported. From

[3]

1989 to 2010, 140 cases of Aconitum poisoning, including one fatal case, were reported in Hong Kong .

Additionally, 17 cases were reported in Taiwan from 1990 to 1999, 2017 cases in China from 1989 to

[4]

2008 and 121 cases in Korea from 1995 to 2007 . Multiple reasons for Aconitum poisoning exist and

include overdoses, inadequate processing, Aconitum contamination in other herbs, dispensing and

management errors and hidden risk factors. In the 17 cases reported in Hong Kong, yunaconitine (YAC),

crassicauline A (CCA) and 8-deacetyl-yunaconitine (DYA) were detected instead of AC, MA and HA in the

[3,4]

urine samples of the Aconitum poisoning patients . As a result, these alkaloids are considered to be

[5]

hidden risk factors and should be covered in laboratory screenings for toxic compounds . Therefore,

a method to simultaneously determine the levels of these nine alkaloids is needed for quality control of

[6]

the herb and its products in order to ensure the safe use of these medicinal materials .

This document aims to build a systematic and practical international standard for the determination of

Aconitum alkaloids with the goal of standardizing the global market, to ensure safe and effective use in

clinics and to reduce cases of Aconitum alkaloid poisoning.

As national implementation may differ, national standards bodies are invited to modify the limit values

of selected Aconitum alkaloids in their national standards. Examples of national and regional values are

given in Annex D.
© ISO 2020 – All rights reserved v
---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 23191:2020(E)
Traditional Chinese medicine — Determination of
selected Aconitum alkaloids by high-performance liquid
chromatography (HPLC)
1 Scope

This document specifies methods for the determination of the selected Aconitum alkaloids, including

aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine,

yunaconitine, deacetyl-yunaconitine and crassicauline A.
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 3696, Water for analytical laboratory use — Specification and test methods
World Health Organization, Quality control methods for herbal materials, 2011
3 Terms and definitions
No terms and definitions are listed in this document.

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/
4 Abbreviated terms
For the purposes of this document, the following abbreviated terms apply.
AC aconitine
BAC benzoylaconine
BHA benzoylhypaconine
BMA benzoylmesaconine
CAS chemical abstracts service
CCA crassicauline A
CRS chemical reference substance
DYA 8-deacetyl-yunaconitine
ESI electrospray ionization
HA hypaconitine
© ISO 2020 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO 23191:2020(E)
HPLC high-performance liquid chromatography
JAC jesaconitine
MA mesaconitine
MRM multiple reaction monitoring
MS mass spectrometer
TS test solution
UV DAD ultraviolet diode array detector
YAC yunaconitine
5 Principle

The test solutions (TSs) are analysed by HPLC on a reverse-phase column packed with octadecylsilane

bonded silica gel, with 0,1 % formic acid as the mobile phase A, acetonitrile as the mobile phase B and

by mass spectrometer (MS) or ultraviolet diode array detector (UV DAD).

Aconitum alkaloids determinable by this method are shown in Table 1. Relevant structural formulae of

Aconitum alkaloids are given in Figure 1.
Table 1 — Aconitum alkaloids determinable by HPLC method
Molar mass
Name Molecular formula CAS no.
g/mol
AC C H NO 302-27-2 645,74
34 47 11
MA C H NO 2752-64-9 631,71
33 45 11
HA C H NO 6900-87-4 615,71
33 45 10
BAC C H NO 466-24-0 603,78
32 45 10
BMA C H NO 63238-67-5 589,68
31 43 10
BHA C H NO 63238-66-4 573,67
31 43 9
YAC C H NO 70578-24-4 659,76
35 49 11
DYA C H NO 110011-77-3 645,79
33 51 10
CCA C H NO 79592-91-9 643,76
35 49 10
2 © ISO 2020 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 23191:2020(E)
Key
AC HA MA BAC BHA BMA YAC DYA CCA
R CH CH CH CH CH CH CH CH CH CH CH CH CH CH
1 2 3 3 3 2 3 3 3 2 3 2 3 2 3
R OH H OH OH H OH OH OH H
R OCOCH OCOCH OCOCH OH OH OH OCOCH OH OCOCH
3 3 3 3 3 3
R OH OH OH OH OH OH H H OH
R H H H H H H OCH OCH OCH
5 3 3 3
Figure 1 — Structural formulae of selected Aconite alkaloids
6 Reagents

Use only reagents of recognized analytical grade and water conforming with grade 3 as specified in

ISO 3696, unless otherwise specified.
— isopropanol, C H O, AR grade;
3 8
— dichloromethane, CH Cl , AR grade;
2 2
— acetonitrile, CH CN. HPLC grade;
— ethyl acetate, C H O AR grade;
4 8 2.
— methanol, CH OH HPLC grade;
3 .
— purified water, deionized;
— ammonia TS.
7 Apparatus
Use the usual laboratory apparatus and the following:
— liquid chromatograph, fitted with electrospray ionization (ESI) MS or DAD;
© ISO 2020 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO 23191:2020(E)

— chromatographic column, of stainless steel, 2,1 mm × 100 mm, with 1,7 μm particle size packing

of octadecylsilane bonded silica gel;
— ultrasonic bath, power 300 W, frequency 40 kHz;
— one-mark pipettes, of capacity 5 ml, 25 ml and 50 ml;
— conical flasks, of capacity 100 ml, with glass stopper;
— vacuum system (e.g. Büchner flask, Vac-Elut system 1 or peristaltic pump);
— analytical balance, capable of weighing to the nearest 0,001 g;
— microporous membrane, 0,22 μm, organic phase.
8 Sampling

Sampling shall be carried out in accordance with the method described in the World Health

Organization's Quality control methods for herbal materials, General advice on sampling. Samples not less

than 250 g shall be taken from each batch randomly. Sampling of Aconitum genus shall be conducted as

follows:
a) from a batch of five containers or packaging units, take a sample from each;
b) from a batch of six to 50 units, take a sample from five;

c) from a batch of over 50 units, sample 10 %, rounding up the number of units to the nearest multiple

of 10. For example, a batch of 51 units would be sampled as for 60, i.e. take samples from six

packages;

d) from each container or package selected, take three original samples from the top, middle and

bottom of the container or package;

e) the three original samples shall then be combined into a pooled sample which shall be mixed

carefully;
f) the average sample is obt
...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 23191
ISO/TC 249
Traditional Chinese medicine —
Secretariat: SAC
Determination of selected Aconitum
Voting begins on:
alkaloids by high-performance liquid
2020­04­23
chromatography (HPLC)
Voting terminates on:
2020­06­18
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 23191:2020(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 23191:2020(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
CH­1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/FDIS 23191:2020(E)
Contents Page

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

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

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

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

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

4 Abbreviated terms .............................................................................................................................................................................................. 1

5 Principle ........................................................................................................................................................................................................................ 2

6 Reagents ........................................................................................................................................................................................................................ 3

7 Apparatus ..................................................................................................................................................................................................................... 3

8 Sampling ........................................................................................................................................................................................................................ 4

9 Test procedures ..................................................................................................................................................................................................... 4

9.1 General ........................................................................................................................................................................................................... 4

9.2 HPLC­MS procedure ........................................................................................................................................................................... 5

9.2.1 Reference solution .......................................................................................................................................................... 5

9.2.2 Test solution......................................................................................................................................................................... 5

9.2.3 Chromatographic conditions ................................................................................................................................. 5

9.2.4 HPLC­MS conditions ..................................................................................................................................................... 5

9.2.5 Determination .................................................................................................................................................................... 5

9.3 HPLC­DAD procedure ........................................................................................................................................................................ 5

9.3.1 Reference solution .......................................................................................................................................................... 5

9.3.2 Test solution......................................................................................................................................................................... 6

9.3.3 Chromatographic conditions ................................................................................................................................. 6

9.3.4 Determination .................................................................................................................................................................... 6

10 Test report ................................................................................................................................................................................................................... 6

Annex A (informative) Typical HPLC conditions ...................................................................................................................................... 7

Annex B (informative) Typical chromatogram of the selected Aconitum alkaloids ............................................9

Annex C (informative) Typical Aconitum herb used in traditional Chinese medicine ...................................13

Annex D (informative) Reference values of national and regional limits of Aconitum

alkaloids in Aconitum carmichaelii lateral root ................................................................................................................14

Bibliography .............................................................................................................................................................................................................................15

© ISO 2020 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO/FDIS 23191:2020(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 non­governmental, 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 ISO/TC 249, Traditional Chinese medicine.

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 23191:2020(E)
Introduction

Aconitum is a genus of about 350 species of flowering plants belonging to the family of Ranunculaceae.

The therapeutic use of as many as 76 species of Aconitum herbs in traditional Chinese medicine have

been documented for a very long time. Among the Aconitum herbs, the most well known are processed

Aconitum carmichaelii lateral root (附子), Aconitum carmichaelii root (川乌) and Aconitum kusnezoffii

root (草乌). Of approximately 80 000 traditional Chinese medicine formulae, around 10,2 % contain

Aconitum herbs. It is one of the most frequently used groups of herbal medicines in traditional Chinese

medicine. Typical Aconitum herbs used in traditional Chinese medicine are shown in Annex C, Table C.1.

Aconitum herbs contain Aconitum alkaloids which have anti-inflammatory, analgesic and cardiotonic

activities. The Aconitum alkaloids are a double­edged sword, however. At present, international trade

in Aconitum products is restricted to a few nations due to the high natural toxicity of crude Aconitum

products. Unprocessed Aconitum is highly toxic. Expert processing is required to reduce (but not

eliminate) toxicity, and where individual national regulatory schemes do not ban the herb, it is generally

restricted to a high-risk category, such as Schedule 2 in the Hong Kong Chinese Medicine regulations.

Also, there are sporadic cases of Aconitum alkaloid poisoning due to misuse reported all over the world.

Nonetheless, the toxicity of Aconitum herbs can be reduced dramatically with proper processing (such

as repeated boiling or steaming), prolonged decoction and dose control. However, testing standards for

Aconitum alkaloids have not yet been harmonized on an international level, and regulatory authorities

in many nations do not adequately differentiate highly toxic forms from less-toxic forms (or even non-

toxic forms) of Aconitum herbs.

Six kinds of Aconitum alkaloids [aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine

(BAC), benzoylmesaconine (BMA) and benzoylhypaconine (BHA)] are commonly used as chemical

markers for quality control of aconite, determined by the high-performance liquid chromatography

[1]

(HPLC) method . The AOAC Official Method 2008.11 also requires the determination of three Aconitum

alkaloids, AC, MA and HA, in dietary supplements and raw botanical materials by LC/UV detection

[2]

with confirmation by LC/MS/MS . Nevertheless, poisoning cases are still occasionally reported. From

[3]

1989 to 2010, 140 cases of Aconitum poisoning, including one fatal case, were reported in Hong Kong .

Additionally, 17 cases were reported in Taiwan from 1990 to 1999, 2017 cases in China from 1989 to

[4]

2008 and 121 cases in Korea from 1995 to 2007 . Multiple reasons for Aconitum poisoning exist and

include overdoses, inadequate processing, Aconitum contamination in other herbs, dispensing and

management errors and hidden risk factors. In the 17 cases reported in Hong Kong, yunaconitine (YAC),

crassicauline A (CCA) and 8-deacetyl-yunaconitine (DYA) were detected instead of AC, MA and HA in the

[3,4]

urine samples of the Aconitum poisoning patients . As a result, these alkaloids are considered to be

[5]

hidden risk factors and should be covered in laboratory screenings for toxic compounds . Therefore,

a method to simultaneously determine the levels of these nine alkaloids is needed for quality control of

[6]

the herb and its products in order to ensure the safe use of these medicinal materials .

This document aims to build a systematic and practical international standard for the determination of

Aconitum alkaloids with the goal of standardizing the global market, to ensure safe and effective use in

clinics and to reduce cases of Aconitum alkaloid poisoning.

As national implementation may differ, national standards bodies are invited to modify the limit values

of selected Aconitum alkaloids in their national standards. Examples of national and regional values are

given in Annex D.
© ISO 2020 – All rights reserved v
---------------------- Page: 5 ----------------------
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 23191:2020(E)
Traditional Chinese medicine — Determination of
selected Aconitum alkaloids by high-performance liquid
chromatography (HPLC)
1 Scope

This document specifies methods for the determination of the selected Aconitum alkaloids, including

aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine,

yunaconitine, deacetyl-yunaconitine and crassicauline A.
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 3696, Water for analytical laboratory use — Specification and test methods
World Health Organization, Quality control methods for herbal materials, 2011
3 Terms and definitions
No terms and definitions are listed in this document.

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/
4 Abbreviated terms
For the purposes of this document, the following abbreviated terms apply.
AC aconitine
BAC benzoylaconine
BHA benzoylhypaconine
BMA benzoylmesaconine
CAS chemical abstracts service
CCA crassicauline A
CRS chemical reference substance
DYA 8-deacetyl-yunaconitine
ESI electrospray ionization
HA hypaconitine
© ISO 2020 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO/FDIS 23191:2020(E)
HPLC high-performance liquid chromatography
JAC jesaconitine
MA mesaconitine
MRM multiple reaction monitoring
MS mass spectrometer
TS test solution
UV DAD ultraviolet diode array detector
YAC yunaconitine
5 Principle

The test solutions (TSs) are analysed by HPLC on a reverse-phase column packed with octadecylsilane

bonded silica gel, with 0,1 % formic acid as the mobile phase A, acetonitrile as the mobile phase B and

by mass spectrometer (MS) or ultraviolet diode array detector (UV DAD).

Aconitum alkaloids determinable by this method are shown in Table 1. Relevant structural formulae of

Aconitum alkaloids are given in Figure 1.
Table 1 — Aconitum alkaloids determinable by this method
Molar mass
Name Molecular formula CAS no.
g/mol
AC C H NO 302­27­2 645,74
34 47 11
MA C H NO 2752­64­9 631,71
33 45 11
HA C H NO 6900­87­4 615,71
33 45 10
BAC C H NO 466­24­0 603,78
32 45 10
BMA C H NO 63238­67­5 589,68
31 43 10
BHA C H NO 63238­66­4 573,67
31 43 9
YAC C H NO 70578­24­4 659,76
35 49 11
DYA C H NO 110011­77­3 645,79
33 51 10
CCA C H NO 79592­91­9 643,76
35 49 10
2 © ISO 2020 – All rights reserved
---------------------- Page: 7 ----------------------
ISO/FDIS 23191:2020(E)
Key
AC HA MA BAC BHA BMA YAC DYA CCA
R CH CH CH CH CH CH CH CH CH CH CH CH CH CH
1 2 3 3 3 2 3 3 3 2 3 2 3 2 3
R OH H OH OH H OH OH OH H
R OCOCH OCOCH OCOCH OH OH OH OCOCH OH OCOCH
3 3 3 3 3 3
R OH OH OH OH OH OH H H OH
R H H H H H H OCH OCH OCH
5 3 3 3
Figure 1 — Structural formulae of selected Aconite alkaloids
6 Reagents

Use only reagents of recognized analytical grade and water conforming with grade 3 as specified in

ISO 3696, unless otherwise specified.
— isopropanol, C H O, AR grade;
3 8
— dichloromethane, CH Cl , AR grade;
2 2
— acetonitrile, CH CN. HPLC grade;
— ethyl acetate, C H O AR grade;
4 8 2.
— methanol, CH OH HPLC grade;
3 .
— purified water, deionized;
— ammonia TS.
7 Apparatus
The usual laboratory apparatus and the following:
— liquid chromatograph, fitted with electrospray ionization (ESI) MS or DAD;
© ISO 2020 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO/FDIS 23191:2020(E)

— chromatogaphic column, of stainless steel, 2,1 mm × 100 mm, with 1,7 μm particle size packing of

octadecylsilane bonded silica gel;
— ultrasonic bath, power 300 W, frequency 40 kHz;
— one-mark pipettes, of capacity 5 ml, 25 ml and 50 ml;
— conical flasks, of capacity 100 ml, with glass stopper;
— vacuum system (e.g. Büchner flask, Vac-Elut system 1 or peristaltic pump);
— analytical balance, capable of weighing to the nearest 0,001 g;
— microporous membrane, 0,22 μm, organic phase.
8 Sampling

Sampling shall be carried out in accordance with the method described in the World Health

Organization's Quality control methods for herbal materials, General advice on sampling. Samples not less

than 250 g shall be taken from each batch randomly. Sampling of Aconitum genus shall be conducted as

follows:
a) from a batch of five containers or packaging units, take a sample from each;
b) from a batch of six to 50 units, take a sample from five;

c) from a batch of over 50 units, sample 10 %, rounding up the number of units to the nearest multiple

of 10. For example, a batch of 51 units would be sampled as for 60, i.e. take samples from six

packages;

d) from each container or package selected, take three original samples from the top, middle and

bottom of the container or package;
...

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