ISO 22283:2020

Traditional Chinese medicine - Determination of aflatoxins in natural products by LC-FLD

This document specifies the methods for the determination of aflatoxins (AFB1, AFB2, AFG1, AFG2) in natural products using LC-FLD. It is applicable to the analysis of aflatoxins in raw materials and manufactured products, including decoction pieces derived from plants and animals.

Médecine traditionnelle chinoise — Dosage des aflatoxines dans les produits naturels par CL-DF

General Information

• Status: Published
• Publication Date: 04-Aug-2020
• ICS: 11.120.10 - Medicaments
• Technical Committee: ISO/TC 249 - Traditional Chinese medicine
• Drafting Committee: ISO/TC 249/WG 2 - Quality and safety of manufactured TCM products
• Current Stage: 9060 - Close of review
• Completion Date: 04-Mar-2031

Relations

• Consolidated By: ISO 21872-1:2017/Amd 1:2023 - Microbiology of the food chain - Horizontal method for the determination of Vibrio spp. - Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus - Amendment 1: Inclusion of performance testing of culture media and reagents
• Effective Date: 06-Jun-2022

Overview

ISO 22283:2020 - Traditional Chinese medicine - Determination of aflatoxins in natural products by LC‑FLD specifies a validated laboratory method to detect and quantify the four major aflatoxins AFB1, AFB2, AFG1 and AFG2 in raw materials and manufactured traditional Chinese medicine (TCM) products. The method uses liquid chromatography with fluorescence detection (LC‑FLD) following immunoaffinity column cleanup and either iodine or photochemical post‑column derivatization to improve fluorescence responses.

Key topics and technical requirements

• Scope: Applicable to plant‑ and animal‑derived decoction pieces, raw materials and finished TCM products.
• Sample preparation:
  • Grind to powder, sieve (24‑mesh), mix 15.0 g sample with 3.0 g NaCl.
  • Extract with methanol:water (70:30 v/v), homogenize (>11 000 rpm, 2 min), centrifuge (2 500 rpm, 5 min).
  • Dilute supernatant, filter (0.45 µm) and perform immunoaffinity column cleanup; elute with methanol and concentrate to 0.5 ml.
• LC‑FLD system and column:
  • Stainless‑steel column packed with octadecylsilyl (C18) silica; isocratic mobile phase methanol–acetonitrile–water.
  • Fluorescence detection with excitation λex = 360 nm (or 365 nm) and emission λem = 450 nm.
  • System suitability: resolution between adjacent peaks > 1.5.
• Derivatization:
  • Iodine derivatization: 0.05% iodine solution (prepared as specified), derivatization pump flow 0.3 ml/min, reactor at 70 °C.
  • Photochemical derivatization: UV reactor at 254 nm (mercury lamp).
• Calibration and quantification:
  • Stock standards prepared for AFB1, AFB2, AFG1, AFG2; working ranges: AFB1/AFG1 0.10–100.00 ng/ml and AFB2/AFG2 0.03–30.00 ng/ml.
  • Inject 25 µl standards and samples; quantify via calibration curves.

Applications and who uses this standard

• Quality control laboratories testing TCM raw materials and finished products for mycotoxin safety.
• Pharmaceutical manufacturers and herbal product producers implementing routine aflatoxin screening.
• Regulatory bodies and food/drug safety agencies referencing an analytical method for compliance and surveillance.
• Third‑party testing laboratories and exporters/importers needing reproducible, internationally recognized LC‑FLD methodology for TCM trade.

Related standards and references

• ISO 22283:2020 was prepared by ISO/TC 249 (Traditional Chinese medicine).
• LC‑MS/MS is noted in the standard as an alternative high‑sensitivity analytical approach for aflatoxins (see Annex A for method comparison).
• Annex C of ISO 22283 lists national/regional limits and references for aflatoxin regulatory limits - consult local regulations for permissible levels.

Keywords: ISO 22283:2020, Traditional Chinese medicine, aflatoxins, AFB1, AFB2, AFG1, AFG2, LC‑FLD, immunoaffinity column, photochemical derivatization, iodine derivatization, HPLC, mycotoxin testing.

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 22283
First edition
2020-08
Traditional Chinese medicine —
Determination of aflatoxins in natural
products by LC-FLD
Médecine traditionnelle chinoise — Dosage des aflatoxines dans les
produits naturels par CL-DF
Reference number
©
ISO 2020
© ISO 2020
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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ii © ISO 2020 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definition . 1
4 Symbols and abbreviated terms . 1
5 Reagents . 2
6 Apparatus . 2
6.1 LC-FLD . 2
6.2 Chromatographic column . 2
6.3 Glass sample . 2
6.4 Electronic balance . 2
6.5 Homogenizer . 2
6.6 Centrifuge . 2
6.7 Volumetric flask . 2
7 Sample preparation . 2
8 Test method . 3
8.1 Stock solution and working solution . 3
8.2 LC-FLD conditions . 3
8.2.1 General. 3
8.2.2 LC-FLD conditions and system suitability . 3
8.2.3 Post-column derivatization . 4
8.2.4 Quantification of aflatoxins in the test sample using calibration curves. 4
8.3 Application of test method . 4
9 Sampling and preservation . 5
9.1 Sampling . 5
9.2 Sample storage . 5
Annex A (informative) LC-FLD method. 6
Annex B (informative) Chromatogram of AFG , AFG , AFB and AFB . 8
2 1 2 1
Annex C (informative) Reference of national, regional and organizational limits of
aflatoxins in natural products . 9
Annex D (informative) Method validation .11
Bibliography .12
Foreword
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This document was prepared by Technical Committee ISO/TC 249, Traditional Chinese medicine.
Any feedback or questions on this document should be directed to the user’s national standards body. A
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iv © ISO 2020 – All rights reserved

Introduction
Aflatoxins are naturally occurring mycotoxins produced by certain fungi, which can be found in a
variety of agriculture products, contaminated foods and natural medicines, including natural products,
decoction pieces and manufactured products. At least 14 different aflatoxins, mainly produced by
Aspergillus flavus and Aspergillus parasiticus, have been reported to be produced in nature. Among
these, aflatoxin B (AFB ) is considered the most toxic. Other important aflatoxins include aflatoxin B ,
1 1 2
M , M , G , G , Q Q and aflatoxicol. AFB , AFB , AFG and AFG are produced by Aspergillus flavus and
1 2 1 2 1, 2 1 2 1 2
Aspergillus parasiticus, while AFM and AFM are formed from AFB and AFB metabolism, respectively.
1 2 1 2
It has been well established that most aflatoxins are highly toxic and carcinogenic. Humans, in
particular young children, are less tolerant to aflatoxin toxicity. There are frequent reports of detection
of toxic aflatoxins in herbal medicines. Therefore, aflatoxins, in particular AFB and the total amount
of AFB , AFB , AFG and AFG , should be tested and limited as a quality and safety control measure
1 2 1 2
for natural products. There are two main methods to detect aflatoxins in natural products: the liquid
chromatography tandem mass spectrometry (LC-MS/MS) method and the liquid chromatography
coupled with fluorescence detector (LC-FLD) method. LC-FLD is preferentially chosen due to its high
sensitivity, high accuracy and reasonable operating cost (see Annex A, Table A.1).
INTERNATIONAL STANDARD ISO 22283:2020(E)
Traditional Chinese medicine — Determination of
aflatoxins in natural products by LC-FLD
1 Scope
This document specifies the methods for the determination of aflatoxins (AFB , AFB , AFG , AFG ) in
1 2 1 2
natural products using LC-FLD.
It is applicable to the analysis of aflatoxins in raw materials and manufactured products, including
decoction pieces derived from plants and animals.
2 Normative references
There are no normative references in this document.
3 Terms and definition
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
aflatoxin
mycotoxin produced mainly by Aspergillus flavus and Aspergillus parasiticus
Note 1 to entry: At least 13 different types of aflatoxin are produced in nature, and most of these are known to be
highly toxic and carcinogenic.
Note 2 to entry: Aflatoxin B and the sum of aflatoxins B , B , G and G shall be tested and limited.
1 1 2 1 2
4 Symbols and abbreviated terms
AFB aflatoxin B
1 1
AFB aflatoxin B
2 2
AFG aflatoxin G
1 1
AFG aflatoxin G
2 2
HPLC high-performance liquid chromatography
LC-FLD liquid chromatography coupled with fluorescence detector
LC-MS/MS liquid chromatography tandem mass spectrometry
5 Reagents
The purity of the reagents used shall be checked by running a blank determination. The chromatogram
obtained from the solvents shall have a baseline without noticeable peaks that would interfere with
targeted aflatoxins.
5.1 Water, of appropriate purity (the resistivity of water shall be at least 18,2 MΩ).
5.2 Methanol, CH OH, of HPLC grade.
5.3 Acetonitrile, CH CN, of HPLC grade.
5.4 Sodium chloride, NaCl, of AR (analytical) grade.
6 Apparatus
6.1 LC-FLD
The LC-FLD apparatus consists of a solvent pump system, a sample injector, a chromatographic column
(a column temperature controller may be used), a detector and a data acquisition system (or an
integrator or a chart recorder). The mobile phase is supplied from one or several reservoirs and flows
through the column and detector at a constant flow rate. The detector shall be a fluorescence detector.
6.2 Chromatographic column
A stainless-steel column sealed with octadecylsilyl silica gel for chromatography shall be used.
6.3 Glass sample
All glassware shall be thoroughly cleaned before use. The glassware used for aflatoxin analysis shall be
placed in a specific container filled with 0,5 % to 1,0 % sodium hypochlorite solution for more than 2 h
and then washed with an adequate amount of fresh running water. Finally, all glassware shall be rinsed
with distilled water and dried before use.
6.4 Electronic balance
The electronic balance shall be accurate to a minimum of 0,01 mg.
6.5 Homogenizer
The homogenizer shall have a rotation speed of up to 15 000 r/min.
6.6 Centrifuge
The centrifuge shall have a rotation speed of up to 5 000 r/min.
6.7 Volumetric flask
Volumetric flasks with a capacity of 2,0 ml and 50,0 ml shall be used.
7 Sample preparation
1) All natural products shall be crushed into powders and screened through a 24-mesh sieve.
2 © ISO 2020 – All rights reserved

2) A mixture of 15,0 g powders and 3,0 g sodium chloride shall be added into a 75,0 ml mixed solution
of methanol and water at 70:30 volume fraction.
3) The mixture shall be homogenized at a speed of higher than 11 000 rpm for 2 min and centrifuged
at 2 500 rpm for 5 min.
4) 15 ml of supernatant shall be added to a 50,0 ml volumetric flask and diluted with water, then
shaken and filtered through a 0,45 μm filter paper.
5) About 20,0 ml of the filtrate shall be passed through the immunoaffinity column at a flow rate of
3 ml/min. The column shall be washed with 20,0 ml of water and the eluent shall be abandoned
until the air has passed through the column to extrude the water.
6) The column shall be eluted with methanol and the eluent shall be collected and concentrated to
0,5 ml by nitrogen. The concentrated eluent shall be diluted with 0,5 ml of the mixed solution of
methanol and water at 50:50 volume fraction in the HPLC vial before use.
8 Test method
8.1 Stock solution and working solution
Stock solution shall be prepared by mixing a solution of aflatoxin standards (1,0 μg/ml, 0,3 μg/ml,
1,0 μg/ml and 0,3 μg/ml of AFB , AFB , AFG and AFG , respectively). A series of working solutions shall
1 2 1 2
be prepared by diluting the stock solution to 0,10 ng/ml to 100,00 ng/ml (AFB and AFG ) and 0,03 ng/
1 1
ml to 30,00 ng/ml (AFB and AFG ), respectively, with mobile phase of methanol and acetonitrile.
2 2
8.2 LC-FLD conditions
8.2.1 General
The LC-FLD method based on two different methods of derivatization, pre- and post-column
derivatization, shall be used for the simultaneous determination of aflatoxins. Commonly, post-
column derivatization methods, such as photochemical derivatization, iodine derivatization and
electrochemically generated bromine derivatization, have been applied in many countries, regions and
organizations including Europe, China, the United States, Japan and South Korea. The LC-FLD method
based on i
...