ISO 22283:2020
(Main)Traditional Chinese medicine — Determination of aflatoxins in natural products by LC-FLD
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
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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
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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.
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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 u
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