EN ISO 16050:2011

Foodstuffs - Determination of aflatoxin B1, and the total content of aflatoxins B1, B2, G1 and G2 in cereals, nuts and derived products - High-performance liquid chromatographic method (ISO 16050:2003)

ISO 16050:2003 specifies a reverse-phase high-performance liquid chromatographic method, with immunoaffinity column clean-up and post-column derivatization, for the determination of aflatoxins in cereals, nuts and derived products. The limit of quantification for aflatoxin B1, and for the sum of aflatoxins B1, B2, G1 and G2, is 8 micrograms per kilogram.
The method has been validated for maize containing 24,5 micrograms per kilogram, for peanut butter containing 8,4 micrograms per kilogram, and for raw peanuts containing 16 micrograms per kilogram of total aflatoxins. It has also been shown that this method can be used for oilseed products, dried fruits and derived products.

Lebensmittel - Bestimmung von Aflatoxin B1 und der Summe von Aflatoxin B1, B2, G1 und G2 in Getreiden, Nüssen und verwandten Produkten - Hochleistungsflüssigchromatographisches Verfahren (ISO 16050:2003)

Diese Internationale Norm legt ein Verfahren zur Bestimmung von Aflatoxinen in Getreiden, Nüssen und verwandten Produkten fest, wobei die Umkehrphasen Hochleistungsflüssigchromatographie mit Reinigung an einer Immunoaffinitätssäule und Nachsäulenderivatisierung angewandt wird. Die Bestimmungsgrenze für Aflatoxin B1 und für die Summe von Aflatoxin B1, B2, G1 und G2 beträgt 8 µg/kg.
Das Verfahren wurde für Mais mit 24,5 μg/kg, Erdnusscreme mit 8,4 μg/kg und unbehandelte Erdnüsse mit 16 μg/kg Gesamtaflatoxingehalt validiert. Außerdem wurde gezeigt, dass dieses Verfahren auf Ölsaaten, Trockenobst und verwandte Produkte angewendet werden kann.

Produits alimentaires - Dosage de l'aflatoxine B1 et détermination de la teneur totale en aflatoxines B1, B2, G1 et G2 dans les céréales, les fruits à coque et les produits dérivés - Méthode par chromatographie liquide à haute performance (ISO 16050:2003)

L'ISO 16050:2003 spécifie une méthode par chromatographie liquide à haute performance en phase inversée, avec purification sur colonne d'immunoaffinité et dérivation post-colonne, pour le dosage des aflatoxines dans les céréales, les fruits à coque et les produits dérivés. La limite de quantification de l'aflatoxine B1 et de la somme des aflatoxines B1, B2, G1 et G2 est de 8 µg/kg.
Cette méthode a été validée sur du maïs, du beurre d'arachide et des arachides brutes ayant respectivement une teneur en aflatoxines totales de 24,5 µg/kg, 8,4 µg/kg et 16 µg/kg. Il a également été démontré que la présente méthode peut être utilisée pour les produits oléagineux, les fruits secs et les produits dérivés.

Živila - Določevanje aflatoksina B1 in vsote aflatoksinov B1, B2, G1 in G2 v žitih in oreščkih ter njihovih proizvodih - Metoda tekočinske kromatografije visoke ločljivosti (ISO 16050:2003)

Ta mednarodni standard opredeljuje metodo tekočinske kromatografije visoke ločljivosti z reverzno fazo, z imunoafinitetnim kolonskim čiščenjem in derivacijo za kolono, za določevanje aflatoksinov v žitih in oreščkih ter njihovih proizvodih. Meja kvantifikacije za aflatoksin B1 in za vsoto aflatoksinov B1, B2, G1 in G2, je 8 μg/kg. Metoda je bila potrjena za koruzo, ki vsebuje 24,5 μg/kg, za arašidovo maslo, ki vsebuje 8,4 μg/kg, in za surove arašide, ki vsebujejo 16 μg/kg celotnih aflatoksinov. Prav tako je bilo prikazano, da se ta metoda lahko uporablja za proizvode iz oljnic in sušeno sadje ter njihove proizvode.

General Information

• Status: Published
• Publication Date: 12-Jul-2011
• Withdrawal Date: 30-Jan-2012
• ICS: 67.060 - Cereals, pulses and derived products
• ICS: 67.080.10 - Fruits and derived products
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 5 - Biotoxins
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 13-Jul-2011
• Due Date: 29-May-2012
• Completion Date: 13-Jul-2011
• Ref Project: SIST EN ISO 16050:2011 - Foodstuffs - Determination of aflatoxin B1, and the total content of aflatoxins B1, B2, G1 and G2 in cereals, nuts and derived products - High-performance liquid chromatographic method (ISO 16050:2003)

Relations

• Replaces: EN 12955:1999 - Foodstuffs - Determination of aflatoxin B1, and the sum of aflatoxins B1, B2, G1 and G2 in cereals, shell-fruits and derived products - High performance liquid chromatographic method with post column derivatization and immunoaffinity column clean up
• Effective Date: 16-Jul-2011

Overview

EN ISO 16050:2011 (identical to ISO 16050:2003) specifies a validated analytical procedure for the determination of aflatoxin B1 and the total content of aflatoxins B1, B2, G1 and G2 in cereals, nuts and derived products. The method uses reverse‑phase high‑performance liquid chromatography (HPLC) with immunoaffinity column (IA) clean‑up and post‑column derivatization plus fluorescence detection. The standard sets a limit of quantification (LOQ) of 8 µg/kg for aflatoxin B1 and for the sum of B1+B2+G1+G2 and includes validated performance data for specific matrices.

Key topics and technical requirements

• Analytical principle: extraction with methanol/water, IA column affinity purification, elution and quantification by reverse‑phase HPLC with fluorescence detection after post‑column derivatization.
• Clean‑up: immunoaffinity columns specific to aflatoxins B1, B2, G1, G2.
• Derivatization reagents: iodine solution or pyridinium hydrobromide perbromide (PBPB) as post‑column oxidizing reagents (document lists preparation details).
• Typical reagents and solvents: methanol/water extraction (7:3 v/v), mobile phase (water:acetonitrile:methanol 3:1:1), toluene/acetonitrile mixture for stock standards (as described in the standard).
• Quantification and calibration: use of aflatoxin standard solutions, UV spectrometric determination of stock concentrations, and calibration graphs for HPLC response.
• Performance and validation: LOQ = 8 µg/kg; method validated for maize (total aflatoxins 24.5 µg/kg), peanut butter (8.4 µg/kg), raw peanuts (~16 µg/kg). Method applicability extended to oilseed products, dried fruits and derived products.
• Safety and handling: aflatoxins are carcinogenic - the standard highlights light protection, fume hood use for solvents (toluene), and safe laboratory practice.

Applications

• Regulatory compliance testing for aflatoxin limits in cereals, nuts and related food products.
• Food industry quality control: incoming raw material screening, product release testing and supplier verification.
• Import/export surveillance: testing consignments for aflatoxin contamination to meet trade and sanitary requirements.
• Analytical laboratories and research performing quantitative aflatoxin analysis or method validation.

Who should use this standard

• Accredited food testing laboratories and quality assurance teams in cereal, nut and oilseed processing.
• Public health and food safety authorities, customs laboratories, and contract testing providers.
• Method developers and researchers working on mycotoxin analysis.

Related standards

• Supersedes EN 12955:1999 (withdrawn).
• References ISO 3696 (water for analytical use) and other ISO/CEN food analysis guidance.

Keywords: EN ISO 16050:2011, ISO 16050, aflatoxin B1, total aflatoxins, HPLC method, immunoaffinity column, post‑column derivatization, cereals, nuts, food safety testing.

Standards Content (Sample)

SLOVENSKI STANDARD
01-oktober-2011
1DGRPHãþD
SIST EN 12955:2000
äLYLOD'RORþHYDQMHDIODWRNVLQD%LQYVRWHDIODWRNVLQRY%%*LQ*YåLWLKLQ
RUHãþNLKWHUQMLKRYLKSURL]YRGLK0HWRGDWHNRþLQVNHNURPDWRJUDILMHYLVRNH
ORþOMLYRVWL,62
Foodstuffs - Determination of aflatoxin B1, and the total content of aflatoxins B1, B2, G1
and G2 in cereals, nuts and derived products - High-performance liquid chromatographic
method (ISO 16050:2003)
Lebensmittel - Bestimmung von Aflatoxin B1 und der Summe von Aflatoxin B1, B2, G1
und G2 in Getreiden, Nüssen und verwandten Produkten -
Hochleistungsflüssigchromatographisches Verfahren (ISO 16050:2003)
Produits alimentaires - Dosage de l'aflatoxine B1 et détermination de la teneur totale en
aflatoxines B1, B2, G1 et G2 dans les céréales, les fruits à coque et les produits dérivés
- Méthode par chromatographie liquide à haute performance (ISO 16050:2003)
Ta slovenski standard je istoveten z: EN ISO 16050:2011
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
67.060 äLWDVWURþQLFHLQSURL]YRGLL] Cereals, pulses and derived
QMLK products
67.080.10 Sadje in sadni proizvodi Fruits and derived products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 16050
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2011
ICS 67.060; 67.080.10 Supersedes EN 12955:1999
English Version
Foodstuffs - Determination of aflatoxin B1, and the total content
of aflatoxins B1, B2, G1 and G2 in cereals, nuts and derived
products - High-performance liquid chromatographic method
(ISO 16050:2003)
Produits alimentaires - Dosage de l'aflatoxine B1 et Lebensmittel - Bestimmung von Aflatoxin B1 und der
détermination de la teneur totale en aflatoxines B1, B2, G1 Summe von Aflatoxin B1, B2, G1 und G2 in Getreiden,
et G2 dans les céréales, les fruits à coque et les produits Nüssen und verwandten Produkten -
dérivés - Méthode par chromatographie liquide à haute Hochleistungsflüssigchromatographisches Verfahren (ISO
performance (ISO 16050:2003) 16050:2003)
This European Standard was approved by CEN on 17 June 2011.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same
status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2011 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16050:2011: E
worldwide for CEN national Members.

Contents Page
Foreword .3

Foreword
The text of ISO 16050:2003 has been prepared by Technical Committee ISO/TC 34 “Food products” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 16050:2011 by
Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the secretariat of which is held by
DIN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by January 2012, and conflicting national standards shall be withdrawn at
the latest by January 2012.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 12955:1999.
Originally, EN 12955:1999 "Foodstuffs - Determination of aflatoxin B , and the sum of aflatoxins B B , G
1 1, 2 1
and G in cereals, shell-fruits and derived products - High performance liquid chromatographic method with
post column derivatization and immunoaffinity column clean up" was the basis for ISO 16050. In order to
avoid having two equal standards on CEN- and ISO-level on the same topic, it was decided to take over
ISO 16050 as EN ISO 16050 and to withdraw EN 12955 as soon as EN ISO 16050 is published.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 16050:2003 has been approved by CEN as a EN ISO 16050:2011 without any modification.

INTERNATIONAL ISO
STANDARD 16050
First edition
2003-09-01
Foodstuffs — Determination of aflatoxin
B , and the total content of aflatoxins B ,
1 1
B , G and G in cereals, nuts and
2 1 2
derived products — High-performance
liquid chromatographic method
Produits alimentaires — Dosage de l'aflatoxine B et détermination de
la teneur totale en aflatoxines B , B , G et G dans les céréales, les
1 2 1 2
fruits à coque et les produits dérivés — Méthode par chromatographie
liquide à haute performance
Reference number
ISO 16050:2003(E)
©
ISO 2003
ISO 16050:2003(E)
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ii © ISO 2003 — All rights reserved

ISO 16050:2003(E)
Contents Page
Foreword. iv
1 Scope. 1
2 Normative references. 1
3 Principle. 1
4 Reagents. 1
5 Apparatus. 4
6 Procedure. 5
6.1 General. 5
6.2 Extraction. 5
6.3 Clean-up. 6
6.4 HPLC operating conditions. 6
6.5 Identification. 6
6.6 Calibration graph. 6
6.7 Determination. 7
7 Calculation of results. 7
8 Precision. 8
8.1 Interlaboratory test. 8
8.2 Repeatability. 8
8.3 Reproducibility. 9
9 Test report. 9
Annex A (informative) Results of interlaboratory test . 10
Bibliography . 12

ISO 16050:2003(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 16050 was prepared by Technical Committee ISO/TC 34, Food products. It is based on EN 12955:1999
elaborated by CEN/TC 275, Food analysis — Horizontal methods.

iv © ISO 2003 — All rights reserved

INTERNATIONAL STANDARD ISO 16050:2003(E)

Foodstuffs — Determination of aflatoxin B , and the total
content of aflatoxins B , B , G and G in cereals, nuts and
1 2 1 2
derived products — High-performance liquid chromatographic
method
WARNING — The use of this standard involves hazardous materials and operations. This standard
does not purport to address all the safety problems associated with its use. It is the responsibility of
the user of this standard to establish appropriate safety and health practice and to determine the
applicability of regulatory limitations prior to use.
1 Scope
This International Standard specifies a reverse-phase high-performance liquid chromatographic method, with
immunoaffinity column clean-up and post-column derivatization, for the determination of aflatoxins in cereals,
nuts and derived products. The limit of quantification for aflatoxin B , and for the sum of aflatoxins B , B , G
1 1 2 1
and G , is 8 µg/kg.
The method has been validated for maize containing 24,5 µg/kg, for peanut butter containing 8,4 µg/kg, and
for raw peanuts containing 16 µg/kg of total aflatoxins. It has also been shown that this method can be used
for oilseed products, dried fruits and derived products.
2 Normative references
The following referenced documents are indispensable for the application 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 3696:1987, Water for analytical laboratory use — Specification and test methods
3 Principle
The test sample is extracted with a mixture of methanol and water. The sample extract is filtered, diluted with
water, and applied to an affinity column containing antibodies specific for aflatoxins B , B , G and G . The
1 2 1 2
aflatoxins are isolated, purified and concentrated on the column then removed from the antibodies with
methanol. The aflatoxins are quantified by reverse-phase high-performance liquid chromatography (HPLC)
with fluorescence detection and post-column derivatization.
4 Reagents
Use only reagents recognized analytical grade, unless otherwise stated.
4.1 Water, according to grade 1 of ISO 3696:1987.
4.2 Sodium chloride.
ISO 16050:2003(E)
1)
4.3 Iodine, crystalline, or as an alternative, pyridinium hydrobromide perbromide (PBPB) .
4.4 Aflatoxin, in crystal form or as a film ampoule.
WARNING — Aflatoxins are carcinogenic to human subjects. Attention is drawn to the statement
made by the International Agencies for Research on Cancer (WHO) (see [1], [2]).
Adequately protect from daylight the laboratory where the analyses are carried out. This may be
achieved effectively by using ultraviolet (UV) absorbing foil on the windows in combination with
subdued light (no direct sunlight), or curtains or blinds in combination with artificial light (fluorescent
tubes are acceptable).
4.5 Acetonitrile, HPCL grade.
4.6 Methanol, analytical grade.
4.7 Methanol, HPLC grade.
4.8 Toluene, analytical grade.
WARNING — Toluene is highly flammable and harmful. Standard preparation involving this solvent
shall be performed in a fume cupboard. Operations outside the fume cupboard, such as measurement
of standards by UV spectrometry, shall be performed with the standards in closed containers.
4.9 Toluene/acetonitrile mixture
Mix 98 parts per volume of toluene (4.8) with 2 parts per volume of acetonitrile (4.5) (see Warning in 4.8).
4.10 Extraction solvent
Mix 7 parts per volume of methanol (4.6) with 3 parts per volume of water (4.1).
Other extraction solvent mixtures which are compatible with the mobile phase may also be used if proved to
be more effective or recommended by the manufacturer of the immunoaffinity (IA) column.
4.11 Mobile phase
Mix 3 parts per volume of water (4.1) with 1 part per volume of acetonitrile (4.5) and 1 part per volume of
methanol (4.7). Degas the solution before use.
4.12 Post-column derivatization reagent
Dissolve 100 mg of iodine (4.3) in 2 ml of methanol (4.6). Add 200 ml of water (4.1), stir for 1 h, then filter
through a 0,45 µm membrane filter (5.8). Prepare the solution the week of use and store the solution in the
dark or in a brown glass bottle. Before use, stir the solution for 10 min.
As an alternative, dissolve 50 mg of PBPB (4.3) in 1 000 ml of water. This solution may be used for up to
4 days if stored in a dark place at room temperature.
4.13 Aflatoxin B , B , G and G stock solutions
1 2 1 2
WARNING — Protect solutions containing aflatoxin from light as far as possible (keep in the dark, use
aluminium foil or amber-coloured glassware).

1) CAS: 39416-48-3 (CAS = Chemical Abstract Service).
2 © ISO 2003 — All rights reserved

ISO 16050:2003(E)
Dissolve aflatoxin B , B , G and G separately in the toluene/acetonitrile mixture (4.9) to give separate
1 2 1 2
solutions containing 10 µg/ml.
To determine the exact concentration of aflatoxin in each stock solution, record the absorption curve at a
wavelength between 330 nm and 370 nm in 1 cm quartz glass cells (5.7) using a spectrometer (5.6) with a
toluene/acetonitrile mixture (4.9) as reference. Calculate the aflatoxin concentration of each aflatoxin, ρ , in
i
micrograms per millilitre, using Equation (1):
AM××1000
max i
ρ = (1)
i
ε ×d
i
where
A is the absorbance determined at the maximum of the absorption curve;

max
M is the molecular mass of each aflatoxin, in grams;
i
ε is the molar absorption coefficient of each aflatoxin in toluene/acetonitrile;
i
NOTE This value is determined in a solution that contains c = 1 mol/l of aflatoxin and in a cell with the optical
pathlength d = 1 cm. The molar absorption coefficient (ε) is usually given without a unit of measurement, but
−1 −1
from the equation A = ε × c × d, the following unit can be derived for it: l◊mol ◊cm .
d is the optical pathlength of the cell, in centimetres.
M and ε are given in Table 1.
i i
Table 1 — Molecular mass and molar absorption coefficient of aflatoxins B , B , G and G
1 2 1 2
Aflatoxin M ε
i i
B 312 19 300
B 314 20 400
G 328 16 600
G 330 17 900
NOTE A mixture of toluene and acetonitrile (98 + 2) is used as
solvent.
4.14 Stock solution of mixed aflatoxins
Prepare a stock solution containing 500 ng/ml of aflatoxin B , 125 ng/ml of aflatoxin B , 250 ng/ml of
1 2
aflatoxin G and 125 ng/ml of aflatoxin G in toluene/acetonitrile (4.9). If the solution has to be stored, weigh
1 2
the flask before storage. Wrap the flask tightly in aluminium foil and store it at approximately 4 °C. Immediately
before use, reweigh the flask and record any change in mass after storage.
NOTE Normal exposure to UV light during absorbance measurement results in no observable conversion to
photoproducts.
4.15 Standard solution of mixed aflatoxins
Transfer each quantity, as specified in Table 2, o
...