EN 17279:2019

Foodstuffs - Multimethod for the screening of aflatoxin B1, deoxynivalenol, fumonisin B1 and B2, ochratoxin A, T-2 toxin, HT-2 toxin and zearalenone in foodstuffs, excluding foods for infants and young children, by LC-MS/MS

This document describes a screening method for the determination of aflatoxin B1, deoxynivalenol, fumonisin B1 and B2, ochratoxin A, HT-2 and T-2 toxins, and zearalenone in foodstuffs by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS).
The aim of the screening method is to test compliance of foodstuff with regulatory limits or to determine whether a certain pre-defined level (the screening target concentration, STC) is exceeded or not. The result of the screening is either "negative" or "suspect". "Negative" (screen negative) means that the targeted mycotoxins are not detected or potentially present but below the STC. "Suspect" (screen positive) means that the established cut-off level is exceeded and the sample can contain one or more mycotoxins at a level higher than the STC.
For full identification and accurate quantification a second confirmatory quantitative analysis method is required which is outside the scope of this document.
The method is suitable for various types of foodstuff and has been validated for representative matrices from four commodity groups:
-   high starch and/or protein content and low water and fat content: wheat, cereal mixture, wheat flour and cornflakes;
-   high oil content: peanuts;
-   high sugar low water content: figs;
-   high water content: grape juice.
During validation, cut-off levels were established for the following screening target concentrations:
-   aflatoxin B1: 2 µg/kg to 5 µg/kg;
-   deoxynivalenol: 250 µg/kg to 865 µg/kg;
-   fumonisin B1: 200 µg/kg to 790 µg/kg;
-   fumonisin B2: 110 µg/kg to 230 µg/kg;
-   ochratoxin A: 4 µg/kg to 9 µg/kg;
-   T-2 toxin: 25 µg/kg;
-   HT-2 toxin: 25 µg/kg to 50 µg/kg;
-   zearalenone: 30 µg/kg to 100 µg/kg.

Lebensmittel - Multiverfahren mit LC-MS/MS zum Screening auf Aflatoxin B1, Deoxynivalenol, Fumonisin B1 und B2, Ochratoxin A, T2-Toxin, HT-2-Toxin und Zearalenon in Lebensmitteln außer Lebensmittel für Säuglinge und Kleinkinder

Diese Europäische Norm beschreibt ein Screeninverfahren für die Bestimmung von Aflatoxin B1, Deoxynivalenol, Fumonisin B1 und B2, Ochratoxin A, HT 2 und T 2 Toxin und Zearalenon in Lebensmitteln mit Hochleistungsflüssigchromatographie (en: high performance liquid chromatography, HPLC) gekoppelt mit Tandem-Massenspektrometrie (MS/MS).
Das Ziel dieses Screeningverfahrens ist zu prüfen, ob das Lebensmitteln den gesetzlichen Vorgaben entspricht oder ob ein vordefiniertes Level (die "Screening-Zielkonzentration", en: screening target concentration, STC) überschritten ist oder nicht. Das Ergebnis dieses Screenings ist entweder "negativ" oder "verdächtig". "Negativ" (Screen-negativ) bedeutet, dass die interessierenden Mykotoxine nicht nachgewiesen oder möglicherweise vorhanden sind aber unter der STC liegen. "Verdächtig" (Screen-positiv) bedeutet, dass der "festgelegte cut-off-Wert" überschritten ist und dass die Probe möglicherweise ein oder mehrere Mykotoxine in Gehalten über der STC enthält.
Zur vollständigen Identifizierung und exakten Quantifizierung ist ein weiteres Verfahren erforderlich, was nicht durch diese Norm abgedeckt ist, und zwar ein quantitatives Bestätigungsverfahren.
Dieses Verfahren ist für verschiedene Lebensmittelarten geeignet und wurde repräsentative Matrices aus vier Bereichen validiert:
-   Hoher Stärke- und/oder Proteingehalt und niedriger Wasser- und Fettgehalt: Weizen, Getreide-mischung, Weizenmehl und Cornflakes;
-   hoher Fettgehalt: Erdnüsse;
-   hoher Zucker- und niedriger Wassergehalt: Feigen;
-   hoher Wassergehalt: Traubensaft.
Wärend der Validierung wurden für die folgenden Screening-Zielkonzentrationen die folgenden "cut-off"-Werte ermittelt:
-   Aflatoxin B1: 2 µg/kg bis 5 µg/kg;
-   Deoxynivalenol: 250 µg/kg bis 865 µg/kg;
-   Fumonisin B1: 200 µg/kg bis 790 µg/kg;
-   Fumonisin B2: 110 µg/kg bis 230 µg/kg;
-   Ochratoxin A: 4 µg/kg bis 9 µg/kg;
-   T 2 toxin: 25 µg/kg;
-   HT 2 toxin: 25 µg/kg bis 50 µg/kg;
-   Zearalenon: 30 µg/kg bis 100 µg/kg.

Produits alimentaires - Multiméthode de dépistage de l'aflatoxine B1, du déoxynivalénol, de la fumonisine B1 et B2, de l'ochratoxine A, de la toxine T-2, de la toxine HT-2 et de la zéaralénone dans les produits alimentaires, à l'exception des aliments pour nourrissons et jeunes enfants, par CLHP-SM/SM

Le présent document décrit une méthode de dépistage permettant de doser l’aflatoxine B1, le déoxynivalénol, la fumonisine B1 et B2, l’ochratoxine A, la toxine T-2 et la toxine HT-2, ainsi que la zéaralénone dans les produits alimentaires par chromatographie liquide haute performance (CLHP) couplée à une spectrométrie de masse en tandem (SM/SM).
Le but de la méthode de dépistage est de déterminer si une certaine concentration prédéfinie (la concentration cible du dépistage, CCD) est dépassée ou non. Le résultat du dépistage est soit « négatif » soit « suspect ». « Négatif » (dépistage négatif) signifie que les mycotoxines ciblées ne sont pas détectées ou potentiellement présentes mais en-dessous de la CCD. « Suspect » (dépistage positif) signifie que la valeur seuil établie est dépassée et que l’échantillon peut contenir une ou plusieurs mycotoxines à une concentration supérieure à la CCD.
Pour une identification complète et une quantification exacte, une méthode d’analyse quantitative de confirmation est requise (ne fait pas partie du domaine d’application du présent document).
La méthode est appropriée pour différents types de produits alimentaires et a été validée pour des matrices représentatives provenant de quatre groupes de denrées (voir données détaillées à l’Annexe C) :
—   haute teneur en amidon et/ou en protéine et faible teneur en eau et en matière grasse : blé, mélange de céréales, farine de blé et flocons de maïs ;
—   haute teneur en huile : arachides ;
—   haute teneur en sucre et faible teneur en eau : figues sèches ;
—   haute teneur en eau : jus de raisin.
Pendant la validation, des valeurs seuils ont été établies pour les concentrations cibles du dépistage suivantes :
—   aflatoxine B1 : de 2 µg/kg à 5 µg/kg ;
—   déoxynivalénol : de 250 µg/kg à 865 µg/kg ;
—   fumonisine B1 : de 200 µg/kg à 790 µg/kg ;
—   fumonisine B2 : de 110 µg/kg à 230 µg/kg ;
—   ochratoxine A : de 4 µg/kg à 9 µg/kg ;
—   toxine T-2 : 25 µg/kg ;
—   toxine HT-2 : de 25 µg/kg à 50 µg/kg ;
—   zéaralénone : de 30 µg/kg à 100 µg/kg.

Živila - Multirezidualna presejalna metoda za ugotavljanje prisotnosti aflatoksina B1, deoksinivalenola, fumonizinov B1 in B2, ohratoksina A, toksina T-2 in HT-2 ter zearalenona v živilih, razen v hrani za dojenčke in majhne otroke, s HPLC-MS/MS

Ta dokument opisuje presejalno metodo za določanje aflatoksina B1, eoksinivalenola, fumonizinov B1 in B2, ohratoksina A, toksina T-2 in HT-2 ter zearalenona v živilih z visokozmogljivo tekočinsko kromatografijo (HPLC) v povezavi s tandemsko masno spektrometrijo (MS/MS).
Namen presejalne metode je preskusiti skladnost živila z regulativnimi omejitvami oziroma ugotoviti, ali je določena vnaprej določena raven (presejalna ciljna koncentracija, STC) presežena. Rezultat presejalne metode je »negativen« ali »sumljiv«. »Negativen« rezultat (presejalno negativen) pomeni, da ciljni mikotoksini niso zaznani ali so potencialno prisotni, vendar so pod presejalno ciljno koncentracijo. »Sumljiv« rezultat (presejalno pozitiven) pomeni, da je ugotovljena mejna vrednost presežena in vzorec utegne vsebovati enega ali več mikotoksinov na ravni, višji od presejalne ciljne koncentracije.
Za popolno identifikacijo in natančno količinsko določitev je potrebna druga potrditvena metoda kvantitativne analize, ki ne spada na področje uporabe tega dokumenta.
Metoda je primerna za različne vrste živil in je potrjena za reprezentativne zmesi iz štirih proizvodnih skupin:
– živila z visoko vsebnostjo škroba in/ali beljakovin ter nizko vsebnostjo vode in maščob: pšenica, žitne mešanice, pšenična moka in koruzni kosmiči;
– živila z visoko vsebnostjo olja: arašidi;
– živila z visoko vsebnostjo sladkorja in nizko vsebnostjo vode: fige;
– živila z visoko vsebnostjo vode: grozdni sok.
Med validacijo so bile določene mejne vrednosti za naslednje presejalne ciljne koncentracije:
– aflatoksin B1: 2 µg/kg do 5 µg/kg,
– deoksinivalenol: 250 µg/kg do 865 µg/kg,
– fumonizin B1: 200 µg/kg do 790 µg/kg,
– fumonizin B2: 110 µg/kg do 230 µg/kg,
– ohratoksin A: 4 µg/kg do 9 µg/kg,
– toksin T-2: 25 µg/kg,
– toksin HT-2: 25 µg/kg do 50 µg/kg,
– zearalenon: 30 µg/kg do 100 µg/kg.

General Information

• Status: Published
• Publication Date: 08-Oct-2019
• Withdrawal Date: 29-Apr-2020
• ICS: 67.050 - General methods of tests and analysis for food 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: 09-Oct-2019
• Due Date: 02-Nov-2019
• Completion Date: 09-Oct-2019
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Mandate: M/520 - Mandate for standardisation addressed to CEN for methods of analysis for mycotoxins in food
• Ref Project: SIST EN 17279:2019 - Foodstuffs - Multimethod for the screening of aflatoxin B1, deoxynivalenol, fumonisin B1 and B2, ochratoxin A, T-2 toxin, HT-2 toxin and zearalenone in foodstuffs, excluding foods for infants and young children, by LC-MS/MS

Standards Content (Sample)

SLOVENSKI STANDARD
01-december-2019
Živila - Multirezidualna presejalna metoda za ugotavljanje prisotnosti aflatoksina
B1, deoksinivalenola, fumonizinov B1 in B2, ohratoksina A, toksina T-2 in HT-2 ter
zearalenona v živilih, razen v hrani za dojenčke in majhne otroke, s HPLC-MS/MS
Foodstuffs - Multimethod for the screening of aflatoxin B1, deoxynivalenol, fumonisin B1
and B2, ochratoxin A, T-2 toxin, HT-2 toxin and zearalenone in foodstuffs, excluding
foods for infants and young children, by LC-MS/MS
Lebensmittel - Multiverfahren mit HPLC-MS/MS zum Screening auf Aflatoxin B1,
Deoxynivalenol, Fumonisin B1 und B2, Ochratoxin A, T2-HT-2-Toxin und Zearalenon in
Lebensmitteln außer Lebensmittel für Säuglinge und Kleinkinder
Produits alimentaires - Multiméthode de dépistage de l'aflatoxine B1, du déoxynivalénol,
de la fumonisine B1 et B2, de l'ochratoxine A, de la toxine T-2, de la toxine HT-2 et de la
zéaralénone dans les produits alimentaires, à l'exception des aliments pour nourrissons
et jeunes enfants, par CLHP-SM/SM
Ta slovenski standard je istoveten z: EN 17279:2019
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 17279
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2019
EUROPÄISCHE NORM
ICS 67.050
English Version
Foodstuffs - Multimethod for the screening of aflatoxin B1,
deoxynivalenol, fumonisin B1 and B2, ochratoxin A, T-2
toxin, HT-2 toxin and zearalenone in foodstuffs, excluding
foods for infants and young children, by LC-MS/MS
Produits alimentaires - Multiméthode de dépistage de Lebensmittel - Multiverfahren mit HPLC-MS/MS zum
l'aflatoxine B1, du déoxynivalénol, de la fumonisine B1 Screening auf Aflatoxin B1, Deoxynivalenol, Fumonisin
et B2, de l'ochratoxine A, de la toxine T-2, de la toxine B1 und B2, Ochratoxin A, T2-HT-2-Toxin und
HT-2 et de la zéaralénone dans les produits Zearalenon in Lebensmitteln außer Lebensmittel für
alimentaires, à l'exception des aliments pour Säuglinge und Kleinkinder
nourrissons et jeunes enfants, par CLHP-SM/SM
This European Standard was approved by CEN on 5 August 2019.

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, Turkey 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
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17279:2019 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 6
4 Principle . 6
5 Reagents . 6
6 Apparatus and equipment . 8
7 Procedure . 9
8 Calculation . 11
9 Test report . 12
Annex A (informative) Example conditions for suitable LC-MS/MS systems . 13
Annex B (informative) Corresponding pairs of native and C-labelled precursors and
product ions for a number of commonly used transitions . 18
Annex C (informative) Precision data . 21
Bibliography . 30

European foreword
This document (EN 17279:2019) 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 April 2020, and conflicting national standards shall be
withdrawn at the latest by April 2020.
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.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
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, Turkey and the
United Kingdom.
Introduction
Mycotoxins are fungal metabolites that may occur in various foodstuffs. Cereals and cereal products,
peanuts, dried fruits and relevant derived products are most likely to be affected by the mycotoxins
covered by this document (aflatoxin B , deoxynivalenol, fumonisin B and B , ochratoxin A, HT-2 and T-
1 1 2
2 toxins, and zearalenone).
WARNING 1 — Suitable precaution and protection measures need to be taken when carrying out
working steps with harmful chemicals. The hazardous substances ordinance Regulation (EU)
1907/2006 [2] 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.
1 Scope
This document specifies a screening method for the determination of aflatoxin B , deoxynivalenol,
fumonisin B and B , ochratoxin A, T-2 toxin, HT-2 toxin, and zearalenone in foodstuffs by high
1 2
performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS).
The aim of the screening method is to determine whether a certain pre-defined concentration (the
screening target concentration, STC) is exceeded or not. The result of the screening is either “negative”
or “suspect”. “Negative” (screen negative) means that the targeted mycotoxins are not detected or
potentially present but below the STC. “Suspect” (screen positive) means that the established cut-off
value is exceeded and the sample can contain one or more mycotoxins at a concentration higher than
the STC.
For full identification and accurate quantification a confirmatory quantitative analysis method is
required which is outside the scope of this document.
The method is suitable for various types of foodstuff and has been validated for representative matrices
from four commodity groups (see detailed data in Annex C):
— high starch and/or protein content and low water and fat content: wheat, cereal mixture, wheat
flour and cornflakes;
— high oil content: peanuts;
— high sugar and low water content: dried figs;
— high water content: grape juice.
During validation, cut-off values were established for the following screening target concentrations:
— aflatoxin B : 2 µg/kg to 5 µg/kg;
— deoxynivalenol: 250 µg/kg to 865 µg/kg;
— fumonisin B : 200 µg/kg to 790 µg/kg;
— fumonisin B : 110 µg/kg to 230 µg/kg;
— ochratoxin A: 4 µg/kg to 9 µg/kg;
— T-2 toxin: 25 µg/kg;
— HT-2 toxin: 25 µg/kg to 50 µg/kg;
— zearalenone: 30 µg/kg to 100 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
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 http://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
4 Principle
The mycotoxins are extracted from the homogenized sample material, after addition of water, by
shaking with acidified acetonitrile. After a salt-induced phase partitioning and centrifugation, the
acetonitrile extract is diluted with water, optionally filtered, and analysed by HPLC coupled with
MS/MS. The relative response of each mycotoxin to its isotopic labelled analogue added to the final
extract at the screening target concentration (STC), is tested against an established cut-off value.
5 Reagents
Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696,
unless otherwise specified. Solvents shall be of quality for LC analysis, unless otherwise specified.
5.1 Water, deionised.
5.2 Water, LC-MS grade, double distilled or water of grade 1 as defined in EN ISO 3696.
5.3 Acetonitrile, pro analysis (p.a.).
5.4 Acetic acid, purity greater than 98 % (m/m).
5.5 Magnesium sulfate (MgSO ) - anhydrous, p.a.
5.6 Aflatoxin B (AB1) e.g. crystalline, as a film or as certified standard solution.
5.7 Deoxynivalenol (DON) e.g. crystalline, as a film or as certified standard solution.
5.8 Fumonisin B (FB1) e.g. crystalline, as a film or as certified standard solution.
5.9 Fumonisin B (FB2) e.g. crystalline, as a film or as certified standard solution.
5.10 HT-2 toxin (HT-2) e.g. crystalline, as a film or as certified standard solution.
5.11 Ochratoxin A (OTA) e.g. crystalline, as a film or as certified standard solution.
5.12 T-2 toxin (T-2) e.g. crystalline, as a film or as certified standard solution.
5.13 Zearalenone (ZEA) e.g. crystalline, as a film or as certified standard solution.
13 13
5.14 C Aflatoxin B ( C-AB1) e.g. solution ρ = 0,5 mg/l, in acetonitrile.
13 13
5.15 C Deoxynivalenol ( C-DON) e.g. solution ρ = 25 mg/l, in acetonitrile.
13 13
5.16 C Fumonisin B ( C -FB1) e.g. solution ρ = 25 mg/l, in acetonitrile/water.
13 13
5.17 C Fumonisin B ( C-FB2) e.g. solution ρ = 25 mg/l, in acetonitrile/water.
13 13
5.18 C HT-2 toxin ( C-HT2) e.g. solution ρ = 25 mg/l, in acetonitrile.
13 13
5.19 C Ochratoxin A ( C-OTA) e.g. solution ρ = 10 mg/l, in acetonitrile.
13 13
5.20 C T-2 toxin ( C-T2) e.g. solution ρ = 25 mg/l, in acetonitrile.
13 13
5.21 C Zearalenone ( C-ZEA) e.g. solution ρ = 25 mg/l, in acetonitrile.
5.22 Extraction solution, acetonitrile containing 1 % of acetic acid.
Add 1 part per volume of acetic acid (5.4) to 99 parts per volume of acetonitrile (5.3) and mix. This
solution can be used for six months if stored at room temperature.
5.23 Individual stock solutions
The individual solutions are either prepared by dissolving neat (solid) standards in an appropriate
solvent, or from individual stock solutions purchased as such. The mycotoxins covered in this European
Standard dissolve well in acetonitrile, with the exception of fumonisins for which a mixture of
acetonitrile and water (50+50, v+v) is recommended for preparing individual stock solutions.
Calculate for each of the individual mycotoxins the mass concentration, ρ, in ng/ml according to
Formula (1):
ρ = 20 × D × STC (1)
where
D is the dilution factor (g sample per ml final extract)(D
...