ISO/FDIS 23191

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

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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

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

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-

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

(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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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