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
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
09-Oct-2019
Completion Date
09-Oct-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

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

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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.
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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.

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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,

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)

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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.

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.
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SIST EN 17279:2019
EN 17279:2019 (E)
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 = 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).
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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.
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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
portion water solution
(5.5)
(5.22)
g ml ml g
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 %
e.g. dry milled cereals.
x + y means: x g sample with y ml of water.
most fresh fruits/vegetables, liquids .

ratio of water and extraction solvent is 1+1, v+v, except for 'Slurried sample 1+3' (1+0,67, v+v).

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

adduct, same fragment, only differing in m/z due to number of C-atoms).

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):
 
sample
  (2)
RR =
sample
 
AIS(STC)
 
where
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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: ®,
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

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
...

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