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 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

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

La présente Norme européenne décrit une méthode de dépistage permettant de doser l’aflatoxine B1, le déoxynivalénol, la fumonisine B1 et B2, l’ochratoxine A, les toxines HT-2 et T-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 d’évaluer la conformité du produit alimentaire aux limites réglementaires ou de déterminer si un certain niveau prédéfini (la concentration cible du dépistage, CCD) est dépassé 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 le seuil établi est dépassé et que l’échantillon peut contenir une ou plusieurs mycotoxines à un niveau supérieur à la CCD.
Pour une identification complète et une quantification exacte, une deuxième méthode d’analyse quantitative de confirmation est requise (ne fait pas partie du domaine d’application de la présente Norme).
La méthode est appropriée pour différents types de produits alimentaires et a été validée pour des matrices représentatives provenant de quatre groupes de denrées :
-   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 ;
-   haute teneur en eau : jus de raisin.
Pendant la validation, des seuils ont été établis 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
Public Enquiry End Date
19-Oct-2018
Publication Date
06-Nov-2019
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
23-Oct-2019
Due Date
28-Dec-2019
Completion Date
07-Nov-2019

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SLOVENSKI STANDARD
SIST EN 17279:2019
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
SIST EN 17279:2019 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 17279:2019

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SIST EN 17279:2019


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.

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SIST EN 17279:2019
EN 17279:2019 (E)
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
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

2

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SIST EN 17279:2019
EN 17279:2019 (E)
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.
3

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SIST EN 17279:2019
EN 17279:2019 (E)
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.
4

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SIST EN 17279:2019
EN 17279:2019 (E)
1 Scope
This document specifies a screening method for the determination of aflatoxin B , deoxynivalenol,
1
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;
1
— deoxynivalenol: 250 µg/kg to 865 µg/kg;
— fumonisin B : 200 µg/kg to 790 µg/kg;
1
— fumonisin B : 110 µg/kg to 230 µg/kg;
2
— 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)
5

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SIST EN 17279:2019
EN 17279:2019 (E)
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.
4
5.6 Aflatoxin B (AB1) e.g. crystalline, as a film or as certified standard solution.
1
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.
1
5.9 Fumonisin B (FB2) e.g. crystalline, as a film or as certified standard solution.
2
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.
1
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.
1
6

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EN 17279:2019 (E)
13 13
5.17 C Fumonisin B ( C-FB2) e.g. solution ρ = 25 mg/l, in acetonitrile/water.
2
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 = 0,25 by default), in g/ml;
STC is the screening target concentration (mass fraction) in the sample, in µg/kg.
EXAMPLE For a mycotoxin with an STC of 1 000 µg/kg in the sample, the mass concentration of this
mycotoxin in the mixed mycotoxin stock solution is 5 000 ng/ml.
5.24 Mixed stock solution
Prepare a mixed stock solution containing all individual mycotoxins at a mass concentration calculated
according to Formula (1), using the appropriate pipets (6.6) and a mixture of acetonitrile and water
(80+20, v+v). This solution can be used for six months if stored in the dark at 4°C.
This mixed stock solution may be used for the preparation of positive control samples (7.4).
5.25 Mixed internal standard (ISTD) solution (isotopically labelled mycotoxins).
Isotopically labelled mycotoxins are generally available as certified standard solutions. Prepare a mixed
ISTD solution in a mixture of acetonitrile and water (80+20, v+v), containing all isotopically labelled
mycotoxins at a mass concentration calculated according to Formula (1).
EXAMPLE For a mycotoxin with an STC of 10 µg/kg in the sample, the mass concentration of the
corresponding isotopic label in the mixed ISTD solution is 50 ng/ml.
This solution can be used for six months if stored in the dark at 4 °C.
This solution is used as internal calibrant and is to be added to the mixed standard solution (5.26) and
each of the sample extracts (7.3).
7

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SIST EN 17279:2019
EN 17279:2019 (E)
5.26 Mixed standard solution.
Prepare a mixed standard solution by combining 1 part per volume of the mixed mycotoxin stock
solution (5.24), 1 part per volume of the mixed ISTD solution (5.25), 8 parts per volume of extraction
solution (5.22), and 10 parts per volume of water (5.2). A volume of typically 400 µl is freshly prepared
for each batch of analysis.
EXAMPLE Preparation in vial:
20 µl mixed stock solution (5.24);
20 µl mixed ISTD solution (5.25);
160 µl extraction solution (5.22);
200 µl water (5.2).
The mixed standard solution is used to check correct measurement of the mycotoxins and their isotopic
labelled analogues (7.5.4).
6 Apparatus and equipment
Usual laboratory glassware and equipment, in particular, the following:
6.1 Conical polypropylene screw cap centrifuge tubes, 50 ml with caps.
6.2 Analytical balance, accuracy: 0,01 mg.
6.3 Laboratory balance, accuracy: 0,01 g.
6.4 Adjustable mechanical vertical or horizontal shaker or rotary tumbling machine.
6.5 Laboratory shaker.
6.6 Pipets, adjustable, e.g. 10 µl to 100 µl and 100 µl to 1000 µl, suited for organic solvents (e.g.
positive displacement pipets), with appropriate tips.
6.7 Centrifuge, capable of generating a relative centrifugal force of 3 000 g.
6.8 Vials, 1,5 ml to 2 ml, made of glass or polypropylene with screw cap.
6.9 Syringe filter or centrifuge filter, 0,20 µm to 0,45 µm, made of nylon or polytetrafluoroethylene
(PTFE).
6.10 Auto sampler vials, of appropriate size for the auto sampler in use, e.g. glass with insert vials,
filter vials (PTFE, 0,45 μm), with crimp cap or equivalent.
6.11 LC-MS/MS system, with the following components:
6.11.1 LC pump, capable of delivering a binary gradient at flow rates appropriate for the analytical
column in use with sufficient accuracy.
6.11.2 Injection system, capable of injecting an appropriate volume of injection solution with
sufficient accuracy.
6.11.3 LC column, capable of retaining the target mycotoxins, preferably with a retention factor of at
least two.
8

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6.11.4 Column oven, capable of maintaining a constant temperature.
6.11.5 Tandem mass spectrometer (MS/MS), capable of ionization of the mycotoxins (either
resulting in positive or negative ions), performing Multiple Reaction Monitoring (MRM), and with a
sufficiently wide dynamic range.
NOTE Instruments capable of alternating measurement of positive and negative ions (pos/neg switching) are
beneficial because these can cover all target analytes within one run.
6.11.6 Data evaluation system.
7 Procedure
7.1 Preparation of the test sample
Finely grind the laboratory sample and homogenize it.
7.2 Extraction
The amount of homogenized test sample examined is 5 g. For samples homogenized by slurry mixing,
the amount of slurry corresponding to 5 g of the original sample is used.
Weigh the test portion indicated in Table 1 to the nearest 0,01 g into a 50 ml centrifuge tube (6.1).
Add water (5.1) and acidified acetonitrile (5.22) to the sample as indicated in Table 1. Close the tube,
and shake thoroughly by hand. Make sure dry samples are suspended into the liquid.
Table 1 — Sample set-up
Sample Test Added Extraction MgSO
4
portion water solution
e
(5.5)
d
(5.22)
 g ml ml g
a
5 10 10 5
Sample with moisture content < 15 %
b 10 5 10 5
Slurried sample 1+1
b 12,5 2,5 10 5
Slurried sample 1+1,5
b 15 0 10 5
Slurried sample 1+2
b 20 0 10 7,5
Slurried sample 1+3
c 5 5 10 5
Samples with moisture content > 85 %
a
e.g. dry milled cereals.
b
x + y means: x g sample with y ml of water.
c
most fresh fruits/vegetables, liquids .
d
ratio of water and extraction solvent is 1+1, v+v, except for 'Slurried sample 1+3' (1+0,67, v+v).
e
the amount of magnesium sulfate is 0,5 g per ml of total water (from sample and added) in the
extraction tube.
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Place the tubes in a mechanical shaker (6.4) and shake for 30 min.
Open the tube, add the amount of magnesium sulfate (5.5) indicated in Table 1 to the tube, close the
tube, immediately shake for approximately 5 s to avoid formation of lumps of magnesium sulfate. Shake
the tube vigorously for approximately 30 s by hand or in a mechanical shaker (6.5).
Centrifuge the tube at approximately 3 000 g for at least 5 min to aid settlement of particulate matter
and phase separation.
NOTE After phase partitioning, the volume of the acetonitrile phase (upper layer) is approximately 10,7 ml
and contains approximately 17 % of water [4].
7.3 Preparation of the sample test solution
Using the appropriate pipets, add 10 parts per volume of extract into a vial (6.8 or 6.10), furthermore
add 1 part per volume of mixed ISTD solution (5.25) and 9 parts per volume of water (5.1). Mix using a
shaker (6.5) for at least 5 s.
NOTE 1 Volumes of 200 μl of extract, 20 μl of mixed ISTD solution and 180 μl of water have shown to work
well.
The final extract can be turbid. Turbid extracts can be injected without adverse effects on the analysis.
In case precipitates occur, these shall be removed by centrifugation or by filtration, either using a filter
vial (6.10), centrifuge filter, or a syringe filter (6.9). In the latter case, the total volume of 400 µl
indicated above might be too small.
NOTE 2 The final extract composition is acidic acetonitrile and water (approximately 1+1, v+v). A lower
content of organic solvent can result in solubility limitations of higher concentrations of less water soluble
mycotoxins such as zearalenone.
7.4 Preparation of control samples
With each batch of samples, one negative control and one positive control is used.
The negative control is either a sample free of the target mycotoxins (non-detectable or < 10 % of the
STC), or, if not available, a reagent blank.
To create a reagent blank, perform extraction (7.2) and subsequent steps without adding the sample
test portion.
The positive control is a sample free of the target mycotoxins (non-detectable or < 10 % of the STC)
which is spiked with the mycotoxins at the STC. Alternatively, a reference material known to contain the
target mycotoxins at a concentration close to the STC is used.
For preparation of a positive control sample: spike a sample free of the target mycotoxins by adding
1,0 ml of mixed stock solution (5.24) to a test sample free of the target mycotoxins.
7.5 LC-MS/MS analysis
7.5.1 General
The LC-MS/MS system, injection volume, mobile phase composition and gradient, acquisition
conditions, and data processing parameters shall be such that the targeted mycotoxins are detected
at ≤ 25 % of the [cut-off value × STC], with sufficient selectivity to obtain an acceptable low false suspect
rate.
Examples of LC-MS/MS settings are given in Annex A.
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7.5.2 LC conditions
LC conditions shall be fit-for-purpose, i.e. such that the requirements indicated in 7.5.1 and in 6.11 are
met, but preferably also resulting in short run times. For the latter the use of relatively short columns
(e.g. 50 mm) is beneficial.
The injection volume is a compromise between peak shape of the early eluting mycotoxins and
sensitivity. If instrument sensitivity allows, small injection volumes (e.g. 2 µl) are recommended.
7.5.3 MS conditions
The measurement of one transition (the most favourable one in terms of signal-to-noise ratio (S/N))
suffices. Measurement of more transitions is optional. The most favourable transition can differ for
different instruments and eluents used in LC (e.g. protonated molecules or sodium/ammonium adducts
+ -
in ESI , deprotonated molecules or acetate/formate adducts in ESI ). Ideally, positive and negative ions
are detected in one run if the instrument is capable to do so and sufficient sensitivity is achieved using
the same eluent. Alternatively, two injections are required, with or without using different eluents.
The method relies on internal calibration of each mycotoxin against its isotopically labelled internal
standard in the same extract. For this it is essential that the corresponding transitions are used (same
13
adduct, same fragment, only differing in m/z due to number of C-atoms).
13
In Annex B the m/z values of corresponding pairs of native and C-labelled precursors and product
ions are given for a number of commonly used transitions for the mycotoxins in LC-MS/MS.
Furthermore, all analyte specific MS parameters, including the collision energy, shall be the same for the
two corresponding transitions.
7.5.4 Injection sequence
Start a batch of measurements with a solvent injection (extraction solution (5.22) and water (1+1, v+v))
to check non-contamination of the system.
Inject the mixed standard solution (5.26) to verify correct detection of all mycotoxins and their isotopic
labelled analogues, followed by a solvent injection to check for possible carry-over. Then inject the
negative control, the positive control and the sample extracts. End the sequence with another injection
of the positive control.
7.5.5 Determination of the mycotoxins in the test solutions
Process the data using the appropriate integration software. Peak areas are used for all subsequent
calculations. Check peak assignment and integration for the measured transitions and adjust if needed.
The peak areas of the mycotoxins and their corresponding isotopically labelled internal standards in the
mixed standard solution (5.26) should not differ more than 30 % when the corresponding transitions
and the same MS settings are used [1].
8 Calculation
8.1 Calculation of relative response
Calculate the response of the mycotoxins in controls and in the samples, RR , relative to their
sample
isotopically labelled internal standards according to Formula (2):
 
A
sample
  (2)
RR =
sample
 
AIS(STC)
 
where
11

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A is the peak area of the mycotoxin in the sample;
sample
A is the peak area of the isotopically labelled internal standard at the
IS(STC)
STC in the sample.
8.2 Classification of the samples
The samples are classified as negative or suspect based on the RR and the cut-off value at the
sample
STC as established during the interlaboratory study. The cut-off data are given in Annex C.
The sample is negative when: RR < cut-off value at the STC
sample
The sample is suspect when: RR ≥ cut-off value at the STC
sample
Results for the samples are valid when the positive controls are suspect and the negative controls are
negative.
9 Test report
The test report should comply with EN ISO/IEC 17025 and shall contain at least the following data:
a) all information necessary for the identification of the sample (kind of sample, origin of sample,
designation);
b) a reference to this European Standard;
c) the date and type of sampling procedure (if known);
d) the date of receipt;
e) the date of test;
f) the test results and the units in which they have been expressed;
g) any particular points observed in the course of the test;
h) any operations not specified in the method or regarded as optional, which might have affected the
results.
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Annex A
(informative)

Example conditions for suitable LC-MS/MS systems
A.1 System settings example 1
A.1.1 LC conditions
UHPLC system: ®,
1
Acquity UPLC (Waters)
Column: ®, 1
50 × 2,1 mm, 1,8 µm HSS T3 (Waters)
Injection volume: 2 µl
Column temperature: 40°C
Flow rate: 0,40 ml/min
Eluent A: Water, containing 0,1 % formic acid and 300 mg/l (4,8 mmol/l) ammonium
formate
Eluent B: Methanol, containing 0,1 % formic acid and 300 mg/l (4,8 mmol/l)
ammonium formate
Table A.1 — Eluent composition
Time Eluent A Eluent B
min % %
0,0 100 0
1,0 100 0
1,5 50 50
3,5 0 100
4,5 0 100
5,0 100 0
6,0 100 0

1)
Acquity UPLC is a trade name of a product supplied by Waters. This information is given for the convenience of users of this
European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if
they can be shown to lead to the same results.
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A.1.2 MS conditions
®, 2
MS system: 6500 Qtrap (AB Sciex)
Acquisition is performed by alternating measurement of positive and negative ions.
Generic settings:

Experiment 1 Experiment 2
+ -
 ESI ESI
Scan type MRM MRM
Scheduled MRM No No
Polarity positive negative
Resolution Q1 unit unit
Resolution Q3 unit unit
Intensity threshold 0 cycles per s 0 cycles per s
Settling time 30 ms 30 ms
MR pause 5 ms 5 ms
MCA No No
Step Size 0 Da 0 Da
Curtain gas (CUR) 40 40
Collision gas (CAD) medium medium
Temperature (TEM) 400 400
Ion Source Gas 1 (GS1) 50 50
Ion Source Gas 2 (GS2) 50 50
IonSpray Voltage (IS) 4000 −4000
Entrance Potential (EP) 10 −10
Dwell time for all transitions 5 ms 5 ms

2 ®
6500 Qtrap is a trade name of a product supplied by AB Sciex. This info
...

SLOVENSKI STANDARD
oSIST prEN 17279:2018
01-oktober-2018
äLYLOD9HþHOHPHQWQDPHWRGD]DSUHJOHGRKUDWRNVLQD$DIODWRNVLQD%
GHRNVLQLYDOHQROD]HDUDOHRQDWHUIXPRQL]LQRY%LQ%YåLYLOLKUD]HQYKUDQL]D
GRMHQþNHLQPDMKQHRWURNHV+3/&0606
Foodstuffs - Multimethod for the screening of ochratoxin A, aflatoxin B1, deoxynivalenol,
zearalenone and fumonisin B1 and B2 in foodstuffs, excluding foods for infants and
young children, by HPLC-MS/MS
Lebensmittel - Multiverfahren mit LC-MS/MS zum Screening auf Ochratoxin A, Aflatoxin
B1, Deoxynivalenol, Zearalenon, T2-HT-2-Toxin und Fumonisin B1 und B2 in
Lebensmitteln außer Lebensmittel für Säuglinge und Kleinkinder
Produits alimentaires - Multiméthode de dépistage de l'ochratoxine A, de l'aflatoxine B1,
du déoxynivalénol, de la zéaralénone et de la fumonisine B1 et B2 dans les produits
alimentaires, à l'exception des aliments pour nourrissons et jeunes enfants, par CLHP-
SM/SM
Ta slovenski standard je istoveten z: prEN 17279
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
oSIST prEN 17279:2018 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 17279:2018


DRAFT
EUROPEAN STANDARD
prEN 17279
NORME EUROPÉENNE

EUROPÄISCHE NORM

August 2018
ICS 67.050
English Version

Foodstuffs - Multimethod for the screening of ochratoxin
A, aflatoxin B1, deoxynivalenol, zearalenone and fumonisin
B1 and B2 in foodstuffs, excluding foods for infants and
young children, by HPLC-MS/MS
Produits alimentaires - Multiméthode de dépistage de Lebensmittel - Multiverfahren mit LC-MS/MS zum
l'ochratoxine A, de l'aflatoxine B1, du déoxynivalénol, Screening auf Ochratoxin A, Aflatoxin B1,
de la zéaralénone et de la fumonisine B1 et B2 dans les Deoxynivalenol, Zearalenon, T2-HT-2-Toxin und
produits alimentaires, à l'exception des aliments pour Fumonisin B1 und B2 in Lebensmitteln außer
nourrissons et jeunes enfants, par CLHP-SM/SM Lebensmittel für Säuglinge und Kleinkinder
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 275.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.


EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17279:2018 E
worldwide for CEN national Members.

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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
7.1 Preparation of control samples . 9
7.2 Extraction of mycotoxin from the samples . 9
7.3 Test solution. 10
7.4 LC-MS/MS analysis . 11
8 Calculation . 12
9 Test report . 12
Annex A (informative) Precision data . 13
Annex B (informative) Example conditions for suitable LC-MS/MS systems . 22
13
Annex C (informative) Corresponding pairs of native and C-labelled precursors and
product ions for a number of commonly used transitions . 26
Bibliography . 29
2

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European foreword
This document (prEN 17279:2018) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
3

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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 B1, deoxynivalenol, fumonisin B1 and B2, ochratoxin A, HT-2 and
T-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 (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.
4

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1 Scope
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.
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 3696:1995, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987)
5

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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 http://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 high performance liquid
chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS). The relative response of
each mycotoxin to its isotopic label, added to the final extract at the screening target concentration
(STC), is tested against an established cut-off level.
5 Reagents
Use only reagents of recognized analytical grade and water complying with grade 1 of
EN ISO 3696:1995, 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 3696:1995.
5.3 Acetonitrile, p.a.
5.4 Acetic acid, purity greater than 98 % (w/w).
5.5 Magnesium sulfate (MgSO ) - anhydrous, p.a.
4
5.6 Aflatoxin B1 (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 B1 (FB1) e.g. crystalline, as a film or as certified standard solution.
5.9 Fumonisin B2 (FB2) e.g. crystalline, as a film or as certified standard solution.
5.10 HT-2 toxin (HT2) 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 (T2) e.g. crystalline, as a film or as certified standard solution.
5.13 Zearalenone (ZEN) e.g. crystalline, as a film or as certified standard solution.
13
5.14 C Aflatoxin B1 (U-AB1) e.g. solution ρ = 0,5 mg/l, in acetonitrile.
17
13
5.15 C Deoxynivalenol (U-DON) e.g. solution ρ = 25 mg/l, in acetonitrile.
15
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13
5.16 C Fumonisin B1 (U-FB1) e.g. solution ρ = 25 mg/l, in acetonitrile/water.
34
13
5.17 C Fumonisin B2 (U-FB2) e.g. solution ρ = 25 mg/l, in acetonitrile/water.
34
13
5.18 C HT-2 toxin (U-HT2) e.g. solution ρ = 25 mg/l, in acetonitrile.
22
13
5.19 C Ochratoxin A (U-OTA) e.g. solution ρ = 10 mg/l, in acetonitrile.
20
13
5.20 C T-2 toxin (U-T2) e.g. solution ρ = 25 mg/l, in acetonitrile.
24
13
5.21 C Zearalenone (U-ZEN) e.g. solution ρ = 25 mg/l, in acetonitrile.
18
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 is stable for six months if stored at room temperature.
5.23 Mixed mycotoxin stock solution.
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 standard
dissolve well in acetonitrile, with the exception of fumonisins for which acetonitrile/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):
c = (20/D) × w (1)
STC
where
D is the dilution factor of the sample in the final extract (D = 4 by default);
w is the screening target concentration (mass fraction) in the sample, in µg/kg.
STC
EXAMPLE For a mycotoxin with an STC of 1 000 µg/kg in the sample, the mass concentration of this
mycotoxin in the mixed mycotoxin stock solution is 5 000 ng/ml.
Prepare a mixed mycotoxin stock solution by combining the appropriate volumes of individual
mycotoxin solutions, using the appropriate pipets (6.6) and acetonitrile/water (80+20, v+v). This
solution is stable for six months if stored at 4 °C.
This mixed mycotoxin stock solution may be used for the preparation of positive control samples (7.3).
5.24 Mixed internal standard (ISTD) intermediate solution (isotopically labelled mycotoxins).
Isotopically labelled mycotoxins are generally available as certified standard solutions. Prepare a mixed
internal standard solution in acetonitrile/water (80+20, v+v), containing all isotopically labelled
mycotoxins at a mass concentration calculated according to Formula (1).
EXAMPLE For a mycotoxin with an STC of 10 µg/kg in the sample, the mass concentration of the
corresponding isotopic label in the internal standard solution is 50 ng/ml.
This solution is stable for six months if stored in the dark at 4 °C.
This solution is used as calibrant and is to be added to each of the sample extracts (7.3).
7

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5.25 Mixed mycotoxin solvent standard solution.
Prepare a mixed mycotoxin solvent standard by combining 1 part per volume of the mixed mycotoxin
stock solution (5.23), 1 part per volume of the internal standard solution (5.24), 8 parts per volume of
acetonitrile containing 1 % acetic acid (5.22), and 10 parts per volume of water (5.2). A volume of
typically 400 µl is freshly prepared for each batch of analysis.
EXAMPLE Preparation in vial:
160 µl acetonitrile containing 1 % acetic acid (5.22);
20 µl mixed mycotoxin stock solution (5.23);
20 µl mixed internal standard intermediate solution (5.24);
200 µl water (5.2).
The mixed mycotoxin solvent standard solution is used to check correct measurement of the
mycotoxins and their isotopic labels (7.4.4).
6 Apparatus and equipment
Usual laboratory glassware and equipment, in particular, the following:
6.1 Conical polypropylene screw cap centrifuge tubes, 50 ml with caps.
6.2 Analytical balance, accuracy: 0,01 mg.
6.3 Laboratory balance, accuracy: 0,01 g.
6.4 Adjustable mechanical vertical or horizontal shaker or rotary tumbling machine.
6.5 Laboratory shaker.
6.6 Pipets, adjustable, e.g. 10 µl to 100 µl and 100 µl to 1000 µl, suited for organic solvents (e.g.
positive displacement pipets),, with appropriate tips.
6.7 Centrifuge, capable of generating a relative centrifugal force of 3 000 g.
6.8 Vials, 1,5 ml to 2 ml, made of glass vials or polypropylene with screw cap.
6.9 Syringe filter or centrifuge filter, 0,20 µm to 0,45 µm, made of nylon or polytetrafluoroethylene
(PTFE).
6.10 Auto sampler vials, of appropriate size for the auto sampler in use, e.g. glass with insert vials,
filter vials (polytetrafluoroethylene (PTFE) 0,45 μm), with crimp cap or equivalent.
6.11 LC-MS/MS system, with the following components:
6.11.1 LC pump, capable of delivering a binary gradient at flow rates appropriate for the analytical
column in use with sufficient accuracy.
6.11.2 Injection system, capable of injecting an appropriate volume of injection solution with
sufficient accuracy, and cross-contamination below 0,1 %.
6.11.3 LC column, capable of retaining the target mycotoxins, preferably with a retention factor of at
least two.
8

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6.11.4 Column oven, capable of maintaining a constant temperature.
6.11.5 Tandem mass spectrometer (MS/MS), capable of ionization of the mycotoxins (either
resulting in positive or negative ions), performing Selected Reaction Monitoring (SRM), and with a
sufficiently wide dynamic range.
NOTE Instruments capable of alternating measurement of positive and negative ions (pos/neg switching) are
beneficial because these can cover all target analytes within one run.
6.11.6 Data evaluation system
7 Procedure
7.1 Preparation of control samples
With each batch of samples, one negative control and one positive control is used.
The negative control is either a sample free of the target mycotoxins (non-detectable or < 10 % of the
STC), or, if not available, a reagent blank.
The positive control is a sample free of the target mycotoxins (non-detectable or < 10 % of the STC)
which is spiked with the mycotoxins at STC. Alternatively, a reference material known to contain the
target mycotoxins at a level close to the STC is used.
For preparation of a positive control sample: spike a sample free of the target mycotoxins by adding
1,0 ml of mixed mycotoxin solution (5.23).
7.2 Extraction of mycotoxin from the samples
Finely grind the laboratory sample and homogenize it.
The amount of homogenized test sample examined is 5 g. For samples homogenized by slurry mixing,
the amount of slurry corresponding to 5 g of the original sample is used.
Weigh the test portion indicated in Table 1, to the nearest 0,01 g (6.3), into a 50 ml centrifuge tube
(6.1).
Add water (5.1) and acidified acetonitrile (5.22) to the sample as indicated in Table 1. Close the tube,
and shake thoroughly by hand. Make sure dry samples are suspended into the liquid.
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Table 1 — Composition
Sample Test Added Extraction MgSO
4
portion water solvent e
(5.5)
d
(5.22)
 g ml ml g
a
Sample with moisture content < 15 % 5 10 10 5
b
10 5 10 5
Slurried sample 1+1
b
12,5 2,5 10 5
Slurried sample 1+1,5
b
15 0 10 5
Slurried sample 1+2
b
20 0 10 7,5
Slurried sample 1+3
c
5 5 10 5
Samples with moisture content > 85 %
a
e.g. dry milled cereals.
b
x + y means: x g sample slurried with y ml of water.
c
most fresh fruits/vegetables, liquids .
d
the ratio of water/extraction solvent is 1+1, v+v, except for slurry 1+3 (1+0,67, v+v).
e
the amount of magnesium sulfate is 0,5 g per ml of total water (from sample + added) in the
extraction tube.
Place the tubes in a mechanical shaker (6.4) and shake for 30 min.
Open the tube, add the amount of magnesium sulfate (5.5) indicated in Table 1 to the tube, close the
tube, immediately shake for approximately 5 s to avoid formation of lumps of magnesium sulfate. Shake
the tube vigorously for approximately 30 s by hand or in a mechanical shaker (6.5).
Centrifuge the tube at approximately 3 000 g (6.7) for at least 5 min to aid settlement of particulate
matter and phase separation.
NOTE After phase partitioning, the volume of the acetonitrile phase (upper layer) is approximately 10,7 ml
and contains approximately 17 % water [5].
7.3 Test solution
Using the appropriate pipets, add10 parts per volume of extract into a vial (6.8 or 6.10), furthermore
add 1 part per volume of mixed internal standard solution (5.24) and 9 parts per volume of water (5.1).
Mix using a shaker (6.5) for at least 5 s.
NOTE 1 Volumes of 200 μl of extract, 20 μl of internal standard solution (5.24), and 180 μl of water have shown
to work well.
The final extract can be turbid. Turbid extracts can be injected without adverse effects on the analysis.
In case precipitates occur, these shall be removed by centrifugation or by filtration, either using a filter
vial (6.10), centrifuge filter, or a syringe filter (6.9). In the latter case, the total volume of 400 µl
indicated above might be too small.
NOTE 2 The final extract composition is acidic acetonitrile/water (approximately 1+1, v+v). A lower content of
organic solvent can result in solubility limitations of higher levels of less water soluble mycotoxins such as
zearalenone.
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7.4 LC-MS/MS analysis
7.4.1 General
The LC-MS system, injection volume, mobile phase composition and gradient, acquisition conditions,
and data processing parameters shall be such that the targeted mycotoxins are detected at ≤ 25 % of the
[cut-off value × w ], with sufficient selectivity to obtain an acceptable low false suspect rate.
STC
Examples of LC-MS/MS settings are given in Annex B.
7.4.2 LC conditions
LC conditions shall be fit-for-purpose, i.e. such that the requirements indicated in 7.4.1 and in 6.11.4 are
met, but preferably also resulting in short run times. For the latter the use of relatively short columns
(e.g. 50 mm) is beneficial.
The injection volume is a compromise between peak shape of the early eluting mycotoxins and
sensitivity. If instrument sensitivity allows, small injection volumes (e.g. 2 µl) are recommended.
7.4.3 MS conditions
The measurement of one transition (the most favourable one in terms of S/N) suffices. Measurement of
more transitions is optional. The most favourable transition can differ for different instruments and
+
eluents used in LC (e.g. protonated molecules or sodium/ammonium adducts in ESI , deprotonated
-
molecules or acetate/formate adducts in ESI ). Ideally, positive and negative ions are detected in one
run if the instrument is capable to do so and sufficient sensitivity is achieved using the same eluent.
Alternatively, two injections are required, with or without using different eluents.
The method relies on internal calibration of each mycotoxin against its isotopically labelled internal
standard in the same extract. For this it is essential that the corresponding transitions are used. The
precursor ion and product ion used for the native and the labelled mycotoxin are the same (same
13
adduct, same fragment, only differing in m/z due to number of C-atoms).
13
In Annex C the m/z values of corresponding pairs of native and C-labelled precursors and product
ions are given for a number of commonly used transitions for the mycotoxins in LC-MS/MS.
Furthermore, all analyte specific MS parameters, including the collision energy, shall be the same for the
two corresponding transitions.
7.4.4 Injection sequence
Start a batch of measurements with a solvent injection (extraction solution (5.22)/water, 1+1 v+v) to
check non-contamination of the system.
Inject the mixed mycotoxin solvent standard solution (5.25) to verify correct detection of all mycotoxins
and their labels, followed by solvent to check for possible carry-over. Then inject the negative control,
the positive control and the sample extracts. End the sequence with another injection of the positive
control.
The peak areas of the mycotoxins and their corresponding isotopically labelled internal standards in
mix solvent standard (5.25) shall not differ more than 20 % when the corresponding transitions and the
same MS settings are used.
7.4.5 Determination of the mycotoxins in the test solutions
Process the data using the appropriate integration software. Peak areas are used for all subsequent
calculations. Check peak assignment and integration for the measured transitions and adjust if needed.
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8 Calculation
Calculate the response of the mycotoxins in controls and in the samples, RR , relative to their
Sample
isotopically labelled internal standards according to Formula (2):
 
A
sample
 
RR = (2)
sample
 
AIS(STC)
 
where
A is the peak area of the mycotoxin in the sample;
sample
A is the peak area of the isotopically labelled internal standard at STC.
IS(STC)
The sample is negative when: RR < cut-off value of the STC.
sample
The sample is suspect when: RR ≥ cut-off value of the STC.
sample
Results for the samples are valid when the positive controls are suspect and the negative control are
negative.
Calculate the cut-off value at STC according to Formula (3):
Cut-off value at STC = RR – t-value × SD (3)
STC 0,05 STC
where
RR is the mean relative response of samples containing mycotoxins at STC;
STC
t-value is the one tailed t-value for a rate of false-negative results of 5 % (1,7 for n = 20, see
0,05
[3]);
SD is the standard deviation obtained for the relative response of samples containing
STC
mycotoxins at STC, as obtained during the interlaboratory test.
Cut-off values at STC and other levels, and the relative standard deviations, (RSD in %) obtained during
R
the interlaboratory test are included in Annex A.
9 Test report
The test report should comply with EN ISO/IEC 17025 and shall contain at least the following data:
a) all information necessary for the identification of the sample (kind of sample, origin of sample,
designation);
b) a reference to this European Standard;
c) the date and type of sampling procedure (if known);
d) the date of receipt;
e) the date of test;
f) the test results and the units in which they have been expressed;
g) any particular points observed in the course of the test;
h) any operations not specified in the method or regarded as optional, which might have affected the
results.
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Annex A
(informative)

Precision data
The method was developed by RIKILT – Wageningen University and Research in collaboration with
Eurofins|WEJ-Contaminants, and was tested in 2016 in an interlaboratory study with 14 participants,
using blanks, spiked and naturally contaminated samples of cereals/cereal products, figs, peanut, and
grape juice, based on Commission Regulation (EU) 519/2014EU [3], and the AOAC Guidelines for
collaborative study procedures [6] within the frame of CEN Mandate M/520.
The data given in Table A.1 to Table A.8 were obtained in this interlaboratory test.

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Table A.1 — Precision data and cut-off values for Aflatoxin B1
Corn
Cere
-
Parameters Wheat al Wheat flour Figs Peanuts Grape juice
flake
mix
s
a b a a b a a b a a b a a b
Mass fraction in µg/kg
nd 2,0 5,1 nd 2,0 3,0 nd 2,0 0,5 nd 2,0 4,9 nd 2,0
Number of participating laboratories 13 13 13 13 13 13 13 13 13 13 13 13 13 13
Number of laboratories delivering compliant
13 13 13 13 13 13 13 13 13 13 13 13 13 13
results
Number of accepted results 26 26 26 26 26 26 26 26 18 26 22 24 26 26
Mean RR for fortifications at STC = 2 µg/kg  0,955   0,908   0,967   0,914   0,939
Mean RR samples (blank or contaminated) 0,000  2,55 0,007  1,45 0,012  0,247 0,000  2,11 0,004
Relative reproducibility standard deviation RSD  18 26  27 34  24 17
 25 20 16
R
RR cut-off value at mass fraction specified above  0,542 0,655  0,636 0,539  0,520 0,409  0,548 0,619  0,680
Percentage of false negatives at STC (RR < cut-off  0,0 0,0  3,8   4,5 0,0
 3,8 0,0 3,8
c
va
...

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