ISO 20948:2024

Vegetable fats and oils — Determination of aflatoxins B1, B2, G1 and G2 by immunoaffinity column clean-up and high-performance liquid chromatography

This document specifies a method for the determination of the aflatoxins B1, B2, G1 and G2 in vegetable fats and oils, including peanut oil, sesame oil, olive oil, corn oil, sunflower oil, rapeseed oil and coconut oil, using immunoaffinity column clean-up and high-performance liquid chromatography with post-column derivatization. The limits of quantification for the aflatoxins B1, B2, G1 and G2, and for the sum of aflatoxins B1, B2, G1 and G2, are 1 μg/kg, 0,25 μg/kg, 0,5 μg/kg, 0,25 μg/kg and 1 μg/kg, respectively. The validation was carried out over the following concentration ranges: — aflatoxin B1 = 1 μg/kg to 20 μg/kg; — total aflatoxins = 2 μg/kg to 52 μg/kg.

Titre manque

General Information

• Status: Published
• Publication Date: 04-Nov-2024
• ICS: 67.200.10 - Animal and vegetable fats and oils
• Technical Committee: ISO/TC 34/SC 11 - Animal and vegetable fats and oils
• Drafting Committee: ISO/TC 34/SC 11 - Animal and vegetable fats and oils
• Current Stage: 6060 - International Standard published
• Start Date: 05-Nov-2024
• Due Date: 05-Nov-2024
• Completion Date: 05-Nov-2024

Standards Content (Sample)

International
Standard
ISO 20948
First edition
Vegetable fats and oils —
2024-11
Determination of aflatoxins
B , B , G and G by
1 2 1 2
immunoaffinity column clean-
up and high-performance liquid
chromatography
Reference number
© ISO 2024
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 2
6 Apparatus and equipment . 3
7 Procedure . 4
7.1 Sampling .4
7.2 Sample pre-treatment .4
7.2.1 Extraction .4
7.2.2 IAC clean-up .5
7.3 Analysis .5
7.3.1 HPLC conditions .5
7.3.2 Post-column derivatization.5
7.3.3 Calibration graph .5
7.3.4 Identification .5
7.3.5 Determination .5
8 Calculations . 6
9 Precision . 6
9.1 Results of interlaboratory test .6
9.2 Repeatability .6
9.3 Reproducibility .6
10 Test report . 7
Annex A (normative) Reference standard solutions and typical chromatogram of AFs . 8
Annex B (normative) Determination of the exact concentration of AF stock standard solutions .10
Annex C (informative) Results of collaborative trial .11
Bibliography . 19

iii
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
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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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
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This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, Animal
and vegetable fats and oils, in collaboration with AOAC INTERNATIONAL.
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
Introduction
Aflatoxins (AFs) are carcinogenic toxins that can naturally contaminate oleaginous seeds and fruits, leading
to the potential risk of the consumption of edible oils contaminated by aflatoxins. Regulatory limits for
AFs in edible oils have been established in several countries. This document specifies a method for the
determination of the aflatoxins B , B , G and G in vegetable fats and oils. The method is based on AOAC
1 2 1 2
[1]
Official Method 2013.05 and the validation has been extended to include corn oil, sunflower oil, rapeseed
oil and coconut oil.
v
International Standard ISO 20948:2024(en)
Vegetable fats and oils — Determination of aflatoxins B ,
B , G and G by immunoaffinity column clean-up and high-
2 1 2
performance liquid chromatography
1 Scope
This document specifies a method for the determination of the aflatoxins B , B , G and G in vegetable
1 2 1 2
fats and oils, including peanut oil, sesame oil, olive oil, corn oil, sunflower oil, rapeseed oil and coconut oil,
using immunoaffinity column clean-up and high-performance liquid chromatography with post-column
derivatization.
The limits of quantification for the aflatoxins B , B , G and G , and for the sum of aflatoxins B , B , G and G ,
1 2 1 2 1 2 1 2
are 1 μg/kg, 0,25 μg/kg, 0,5 μg/kg, 0,25 μg/kg and 1 μg/kg, respectively.
The validation was carried out over the following concentration ranges:
— aflatoxin B = 1 μg/kg to 20 μg/kg;
— total aflatoxins = 2 μg/kg to 52 μg/kg.
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
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 Principle
Test samples are extracted with methanol–water (a volume fraction of 55 + 45). After shaking and
centrifuging, the lower layer is filtered, diluted with water, and filtered through glass microfibre filter
paper. The filtrate is passed through an immunoaffinity column, and the toxins are eluted with methanol.
The toxins are subjected to high-performance liquid chromatography with fluorescence detector (HPLC-
FLD) analysis after post column derivatization.
WARNING — Aflatoxins are generally considered to be carcinogenic, neurotoxic and
[2]
immunosuppressive. Observe appropriate safety precautions for handling such compounds and
in particular avoid handling in dry form as their electrostatic nature can result in dispersion and
inhalation. Glassware can be decontaminated with 4 % sodium hypochlorite solution. Attention is
[3][4]
drawn to the statement made by the International Agency for Research on Cancer (WHO) .

5 Reagents
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only
distilled water or water of grade 1 in accordance with ISO 3696. Solvents shall be of quality for LC analysis.
5.1 Methanol, LC grade or equivalent.
5.2 Acetonitrile, LC grade or equivalent.
5.3 Sodium chloride (NaCl).
5.4 Potassium chloride (KCl).
5.5 Hydrochloric acid, c(HCl) = 12 mol/l.
5.6 Disodium hydrogen phosphate (Na HPO ).
2 4
5.7 Potassium dihydrogen phosphate (KH PO ).
2 4
5.8 Phosphate-buffered saline (PBS) buffer, pH 7,40.
Dissolve 8 g NaCl (5.3), 1,2 g Na HPO (5.6), 0,2 g KH PO (5.7) and 0,2 g KCl (5.4) in about 990 ml of water.
2 4 2 4
Adjust the pH to 7,4 with HCl (5.5) and make up to 1 l with water. Alternatively, a PBS solution of equivalent
properties may be prepared from commercially available PBS material.
5.9 Potassium bromide (KBr).
5.10 Nitric acid, 65 %
5.11 Extraction solvent, mix 55 volume parts of methanol (5.1) and 45 volume parts of water.
5.12 Washing solution, mix 10 volume parts of methanol (5.1) and 90 volume parts of water.
5.13 Aflatoxin (AF) standards:
©1)
— aflatoxin B (AFB , C H O , CAS Registry Number 1162-65-8), purity ≥ 98 %;
1 1 17 12 6
©
— aflatoxin B (AFB , C H O , CAS RN 7220-81-7), purity ≥ 98 %;
2 2 17 14 6
©
— aflatoxin G (AFG , C H O , CAS RN 1165-39-5), purity ≥ 98 %;
1 1 17 12 7
©
— aflatoxin G (AFG , C H O , CAS RN 7241-98-7), purity ≥ 98 %.
2 2 17 14 7
All standards shall be either certified standard solutions or in a crystalline form. Store all materials at –18 °C.
5.14 AF stock standard solutions
Prepare eac
...