EN 17641:2022
(Main)Foodstuffs - Multimethod for the determination of aflatoxins, deoxynivalenol, fumonisins, ochratoxin A, T-2 toxin, HT-2 toxin and zearalenone by LC-MS/MS
Foodstuffs - Multimethod for the determination of aflatoxins, deoxynivalenol, fumonisins, ochratoxin A, T-2 toxin, HT-2 toxin and zearalenone by LC-MS/MS
This document describes a method using isotopically labelled standards for the quantitative determination of aflatoxins B1, B2, G1, G2 and M1 (AFB1, AFB2, AFG1, AFG2 and AFM1), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEN), T-2 and HT-2 toxins (T-2 and HT-2) and fumonisins B1 and B2 (FB1 and FB2) in foods by liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS).
A specific immunoaffinity column (IAC) clean-up is needed for aflatoxins (AFs) and OTA in food intended for infants and young children (e.g. infant cereals, milk-based powders), in spices, in dried fruits and in nuts.
The method has been validated through an intercollaborative study on different commodity groups: cereals and cereal-based products including food for infant and young children, nuts, spices, dried fruits and milk powder. The measuring range of each mycotoxin in these naturally contaminated and/or spiked food samples were:
- AFB1: 0,085 7 µg/kg - 11,4 µg/kg;
- AFB2: 0,079 2 µg/kg - 12,5 µg/kg;
- AFG1: 0,062 8 µg/kg - 20,9 µg/kg;
- AFG2: 0,052 0 µg/kg - 15,0 µg/kg;
- AFM1: 0,034 2 µg/kg - 0,110 µg/kg;
- OTA: 0,448 µg/kg - 17,2 µg/kg;
- DON: 45,2 µg/kg - 743 µg/kg;
- ZEN: 9,57 µg/kg - 131 µg/kg;
- T-2: 10,3 µg/kg - 57,9 µg/kg;
- HT-2: 9,50 µg/kg - 81,8 µg/kg;
- FB1: 31,1 µg/kg - 4 260 µg/kg;
- FB2: 44,2 µg/kg - 1 300 µg/kg.
The measuring ranges of the method for each mycotoxin/matrix combination are given in Table 8.
Lebensmittel - Multiverfahren für die Bestimmung von Aflatoxinen, Deoxynivalenol, Fumonisinen, Ochratoxin A, T-2-Toxin, HT-2-Toxin und Zearalenon mittels LC-MS/MS
Dieses Dokument beschreibt ein Verfahren, das isotopenmarkierte Standards verwendet für die quantitative Bestimmung der Aflatoxine B1, B2, G1, G2 und M1 (AFB1, AFB2, AFG1, AFG2 und AFM1), Ochratoxin A (OTA), Deoxynivalenol (DON), Zearalenon (ZEN), T 2 und HT 2-Toxine (T 2 und HT 2) und Fumonisine B1 und B2 (FB1 und FB2) in Lebensmitteln durch Flüssigchromatographie (LC, en.: liquid chromatography), gekoppelt mit Tandem-Massenspektrometrie (MS/MS).
Für Aflatoxine (AF) und OTA in für Säuglinge und Kleinkinder bestimmter Nahrung (z. B. Zerealien für Kleinkinder, Pulver auf Milchbasis), in Gewürzen, in Trockenfrüchten und in Nüssen ist eine spezifische Immunaffinitätssäulen (IAC, en.: immunoaffinity column)-Reinigung erforderlich.
Das Verfahren wurde durch eine Ringversuchstudie zu verschiedenen Lebensmittelgruppen validiert: Getreide und Produkte auf Getreidebasis einschließlich Nahrung für Säuglinge und Kleinkinder, Nüsse, Gewürze, Trockenfrüchte und Milchpulver. Die Messbereiche der einzelnen Mykotoxine in diesen natürlich kontaminierten und/oder aufgestockten Lebensmittelproben waren:
— AFB1: 0,085 7 µg/kg – 11,4 µg/kg;
— AFB2: 0,079 2 µg/kg – 12,5 µg/kg;
— AFG1: 0,062 8 µg/kg – 20,9 µg/kg;
— AFG2: 0,052 0 µg/kg – 15,0 µg/kg;
— AFM1: 0,034 2 µg/kg – 0,110 µg/kg;
— OTA: 0,448 µg/kg – 17,2 µg/kg;
— DON: 45,2 µg/kg – 743 µg/kg;
— ZEN: 9,57 µg/kg – 131 µg/kg;
— T 2: 10,3 µg/kg – 57,9 µg/kg;
— HT 2: 9,50 µg/kg – 81,8 µg/kg;
— FB1: 31,1 µg/kg – 4 260 µg/kg;
— FB2: 44,2 µg/kg – 1 300 µg/kg.
Die Messbereiche der Verfahren für jede Mykotoxin-/Matrix-Kombination sind in Tabelle 8 angegeben.
Produits alimentaires - Multiméthode de détermination de la teneur en aflatoxines, déoxynivalénol, fumonisines, ochratoxine A, toxine T-2, toxine HT-2 et zéaralénone par CL-SM/SM
Le présent document décrit une méthode utilisant des étalons marqués aux isotopes pour la détermination quantitative des teneurs en aflatoxines B1, B2, G1, G2 et M1 (AFB1, AFB2, AFG1, AFG2 et AFM1), ochratoxine A (OTA), déoxynivalénol (DON), zéaralénone (ZEN), toxines T-2 et HT-2 (T-2 et HT 2) et fumonisines B1 et B2 (FB1 et FB2) dans les aliments par chromatographie liquide (CL) couplée à une spectrométrie de masse en tandem (SM/SM).
Une purification spécifique sur colonne d’immuno-affinité (CIA) est nécessaire pour les aflatoxines (AF) et l’OTA présentes dans les aliments pour nourrissons et enfants en bas âge (par exemple, céréales infantiles, lait en poudre), les épices, les fruits secs et les fruits à coques.
La méthode a été validée lors d’un essai interlaboratoires portant sur différents groupes de denrées : céréales et produits à base de céréales, y compris aliments pour nourrissons et enfants en bas âge, fruits à coques, épices, fruits secs et lait en poudre. Les gammes de mesure de chaque mycotoxine dans ces échantillons d’aliments naturellement contaminés et/ou dopés étaient les suivantes :
- AFB1 : 0,085 7 µg/kg - 11,4 µg/kg ;
- AFB2 : 0,079 2 µg/kg - 12,5 µg/kg ;
- AFG1 : 0,062 8 µg/kg - 20,9 µg/kg ;
- AFG2 : 0,052 0 µg/kg - 15,0 µg/kg ;
- AFM1 : 0,034 2 µg/kg - 0 110 µg/kg ;
- OTA : 0,448 µg/kg - 17,2 µg/kg ;
- DON : 45,2 µg/kg - 743 µg/kg ;
- ZEN : 9,57 µg/kg - 131 µg/kg ;
- T-2 : 10,3 µg/kg - 57,9 µg/kg ;
- HT-2 : 9,50 µg/kg - 81,8 µg/kg ;
- FB1 : 31,1 µg/kg - 4 260 µg/kg ;
- FB2 : 44,2 µg/kg - 1 300 µg/kg.
Les gammes de mesure de la méthode pour chaque combinaison mycotoxine/matrice sont indiquées dans le Tableau 8.
Živila - Hkratna metoda za določevanje aflatoksina, deoksinivalenola, fumonizinov, ohratoksina A, toksinov T-2 in HT-2 ter zearalenona z LC-MS/MS
Ta dokument opisuje metodo redčenja izotopov za kvantitativno določanje aflatoksinov B1, B2, G1, G2 in M1 (AFB1, AFB2, AFG1, AFG2 in AFM1), okratoksina A (OTA), deoksinivalenola (DON), zearalenona (ZEN), toksinov T-2 in HT-2 (T-2 in HT-2) ter fumonizinov B1 in B2 (FB1 in FB2) v živilih z visokozmogljivo tekočinsko kromatografijo (LC) v povezavi s tandemsko masno spektrometrijo (MS/MS).
Za aflatoksine (AF) in OTA v hrani za dojenčke (npr. kosmiči za dojenčke, praški na osnovi mleka), v začimbah, suhem sadju in oreščkih je potrebno posebno čiščenje imunoafinitetne kolone (IAC).
Metoda je bila potrjena z medstrokovno študijo o različnih skupinah živil: žita in proizvodi na osnovi žit, vključno s hrano za dojenčke in majhne otroke, oreščki, začimbami, suhim sadjem in mlekom v prahu. Območja koncentracij vsakega mikotoksina v teh naravno kontaminiranih vzorcih hrane in/ali vzorcih hrane z dodatki so bila:
– aflatoksin B1: od 0,0857 µg/kg do 11,4 µg/kg;
– aflatoksin B2: od 0,0792 µg/kg do 12,5 µg/kg;
– aflatoksin G1: od 0,0628 µg/kg do 20,9 µg/kg;
– aflatoksin G2: od 0,0520 µg/kg do 15,0 µg/kg;
– aflatoksin M1: od 0,0342 µg/kg do 0,110 µg/kg;
– ohratoksin A: od 0,448 µg/kg do 17,2 µg/kg;
– deoksinivalenol: od 45,2 µg/kg do 743 µg/kg;
– zearalenon: od 9,57 µg/kg do 131 µg/kg;
– toksin T-2: od 10,3 µg/kg do 57,9 µg/kg;
– toksin HT-2: od 9,50 µg/kg do 81,8 µg/kg;
– fumonizin B1: od 31,1 µg/kg do 4262 µg/kg;
– fumonizin B2: od 44,2 µg/kg do 1299 µg/kg.
Merilna območja metode za vsako kombinacijo mikotoksin/matrika so navedena v preglednici 7.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-november-2022
Živila - Hkratna metoda za določevanje aflatoksina, deoksinivalenola, fumonizinov,
ohratoksina A, toksinov T-2 in HT-2 ter zearalenona z LC-MS/MS
Foodstuffs - Multimethod for the determination of aflatoxins, deoxynivalenol, fumonisins,
ochratoxin A, T-2 toxin, HT-2 toxin and zearalenone by LC-MS/MS
Lebensmittel - Multimethode für die Bestimmung von Aflatoxinen, Deoxynivalenol,
Fumonisinen, Ochratoxin A, T-2-Toxin, HT-2-Toxin und Zearalenon mittels LC-MS/MS
Produits alimentaires - Multiméthode de détermination de la teneur en aflatoxines,
déoxynivalénol, fumonisines, ochratoxine A, toxine T-2, toxine HT-2 et zéaralénone par
CL-SM/SM
Ta slovenski standard je istoveten z: EN 17641:2022
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 17641
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2022
EUROPÄISCHE NORM
ICS 67.050
English Version
Foodstuffs - Multimethod for the determination of
aflatoxins, deoxynivalenol, fumonisins, ochratoxin A, T-2
toxin, HT-2 toxin and zearalenone by LC-MS/MS
Produits alimentaires - Multiméthode de Lebensmittel - Multimethode für die Bestimmung von
détermination de la teneur en aflatoxines, Aflatoxinen, Deoxynivalenol, Fumonisinen, Ochratoxin
déoxynivalénol, fumonisines, ochratoxine A, toxine T-2, A, T-2-Toxin, HT-2-Toxin und Zearalenon mittels LC-
toxine HT-2 et zéaralénone par CL-SM/SM MS/MS
This European Standard was approved by CEN on 24 July 2022.
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, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17641:2022 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principles . 6
5 Reagents . 6
6 Apparatus and equipment .12
7 Procedure.14
7.1 Preparation of the laboratory sample .14
7.2 Test portions weighing .14
7.2.1 Cereals, cereal-based products .14
7.2.2 Milk powders, nuts, spices, dried fruits .14
7.3 Spiking with ISTD solutions .14
7.4 Extraction .14
7.4.1 General procedure .14
7.4.2 Without IAC clean-up .15
7.4.3 With IAC clean-up .15
8 LC-MS/MS analysis .16
8.1 Operating conditions .16
8.2 Injection sequence .16
8.3 Data treatment .17
9 Calculations .17
9.1 Identification and confirmation.17
9.2 Calibration curve and measuring range .17
9.3 Calculations .18
10 Precision .19
10.1 General .19
10.2 Repeatability .19
10.3 Reproducibility .19
11 Test report .22
Annex A (informative) Precision data.23
Annex B (informative) Overview of the sample preparation .39
Annex C (informative) Example conditions for suitable LC-MS/MS systems .40
Bibliography .49
European foreword
This document (EN 17641:2022) has been prepared 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 March 2023, and conflicting national standards shall be
withdrawn at the latest by March 2023.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations 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, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the United
Kingdom.
Introduction
Mycotoxins are fungal metabolites that may occur in various foodstuffs such as cereals, nuts, spices, fruits,
oil seeds, or coffee. Mycotoxins can be produced before harvest in the crop and even after harvest if
climate conditions are favourable for further fungal growth. Milk can be contaminated as well by
Aflatoxin M , the major metabolite of Aflatoxin B , when cows are fed with Aflatoxin B contaminated
1 1 1
feed. To protect consumer health, maximum levels for mycotoxins in foodstuffs have been established in
a broad range of food commodities including those intended for infants and young children consumption.
WARNING 1 — Suitable precaution and protection measures need to be taken when carrying out
working steps with harmful chemicals. The latest version of the hazardous substances ordinance,
Regulation (EC) No 1907/2006 [1], should be taken into account as well as appropriate national
statements.
WARNING 2 — The use of this document can involve hazardous materials, operations and
equipment. This document does not purport to address all the safety problems associated with its
use. It is the responsibility of the user of this document to establish appropriate safety and health
practices and determine the applicability of regulatory limitations prior to use.
WARNING 3 — Aflatoxins are known to have carcinogenic effects and to be both acutely and
chronically toxic. Aflatoxins B , B , G , G and M are classified as carcinogenic to humans
1 2 1 2 1
(Group 1) by the International Agency for Cancer Research (IARC). Fumonisin B , fumonisin B
1 2
and ochratoxin A have been classified as possibly carcinogenic to humans (Group 2B) and
zearalenone, deoxynivalenol and T-2 as not classifiable as to their carcinogenicity to humans
(Group 3) [2].
1 Scope
This document describes a method using isotopically labelled standards for the quantitative
determination of aflatoxins B , B , G , G and M (AFB1, AFB2, AFG1, AFG2 and AFM1), ochratoxin A
1 2 1 2 1
(OTA), deoxynivalenol (DON), zearalenone (ZEN), T-2 and HT-2 toxins (T-2 and HT-2) and fumonisins B
and B (FB1 and FB2) in foods by liquid chromatography (LC) coupled with tandem mass spectrometry
(MS/MS).
A specific immunoaffinity column (IAC) clean-up is needed for aflatoxins (AFs) and OTA in food intended
for infants and young children (e.g. infant cereals, milk-based powders), in spices, in dried fruits and in
nuts.
The method has been validated through an intercollaborative study on different commodity groups:
cereals and cereal-based products including food for infant and young children, nuts, spices, dried fruits
and milk powder. The measuring range of each mycotoxin in these naturally contaminated and/or spiked
food samples were:
— AFB1: 0,085 7 µg/kg – 11,4 µg/kg;
— AFB2: 0,079 2 µg/kg – 12,5 µg/kg;
— AFG1: 0,062 8 µg/kg – 20,9 µg/kg;
— AFG2: 0,052 0 µg/kg – 15,0 µg/kg;
— AFM1: 0,034 2 µg/kg – 0,110 µg/kg;
— OTA: 0,448 µg/kg – 17,2 µg/kg;
— DON: 45,2 µg/kg – 743 µg/kg;
— ZEN: 9,57 µg/kg – 131 µg/kg;
— T-2: 10,3 µg/kg – 57,9 µg/kg;
— HT-2: 9,50 µg/kg – 81,8 µg/kg;
— FB1: 31,1 µg/kg – 4 260 µg/kg;
— FB2: 44,2 µg/kg – 1 300 µg/kg.
The measuring ranges of the method for each mycotoxin/matrix combination are given in Table 8.
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.
EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696)
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at https://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
4 Principles
The mycotoxins are extracted from the test portion with water and acidified acetonitrile and a liquid-
liquid partition is initiated after addition of magnesium sulphate and sodium chloride salts. The resulting
acetonitrile supernatant is then defatted with hexane. Depending on the mycotoxin/matrix combination,
the sample extract is then submitted to two different protocols (a general scheme is given in Annex B):
— without IAC clean-up: generic procedure for the determination of all mycotoxins in cereals and cereal-
based products. An aliquot of the ace
...
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