EN 17641:2022

Foodstuffs - Multimethod for the determination of aflatoxins, deoxynivalenol, fumonisins, ochratoxin A, T-2 toxin, HT-2 toxin and zearalenone by LC-MS/MS

This document describes a method using isotopically labelled standards for the quantitative determination of aflatoxins B1, B2, G1, G2 and M1 (AFB1, AFB2, AFG1, AFG2 and AFM1), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEN), T-2 and HT-2 toxins (T-2 and HT-2) and fumonisins B1 and B2 (FB1 and FB2) in foods by liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS).
A specific immunoaffinity column (IAC) clean-up is needed for aflatoxins (AFs) and OTA in food intended for infants and young children (e.g. infant cereals, milk-based powders), in spices, in dried fruits and in nuts.
The method has been validated through an intercollaborative study on different commodity groups: cereals and cereal-based products including food for infant and young children, nuts, spices, dried fruits and milk powder. The measuring range of each mycotoxin in these naturally contaminated and/or spiked food samples were:
- AFB1: 0,085 7 µg/kg - 11,4 µg/kg;
- AFB2: 0,079 2 µg/kg - 12,5 µg/kg;
- AFG1: 0,062 8 µg/kg - 20,9 µg/kg;
- AFG2: 0,052 0 µg/kg - 15,0 µg/kg;
- AFM1: 0,034 2 µg/kg - 0,110 µg/kg;
- OTA: 0,448 µg/kg - 17,2 µg/kg;
- DON: 45,2 µg/kg - 743 µg/kg;
- ZEN: 9,57 µg/kg - 131 µg/kg;
- T-2: 10,3 µg/kg - 57,9 µg/kg;
- HT-2: 9,50 µg/kg - 81,8 µg/kg;
- FB1: 31,1 µg/kg - 4 260 µg/kg;
- FB2: 44,2 µg/kg - 1 300 µg/kg.
The measuring ranges of the method for each mycotoxin/matrix combination are given in Table 8.

Lebensmittel - Multiverfahren für die Bestimmung von Aflatoxinen, Deoxynivalenol, Fumonisinen, Ochratoxin A, T-2-Toxin, HT-2-Toxin und Zearalenon mittels LC-MS/MS

Dieses Dokument beschreibt ein Verfahren, das isotopenmarkierte Standards verwendet für die quantitative Bestimmung der Aflatoxine B1, B2, G1, G2 und M1 (AFB1, AFB2, AFG1, AFG2 und AFM1), Ochratoxin A (OTA), Deoxynivalenol (DON), Zearalenon (ZEN), T 2 und HT 2-Toxine (T 2 und HT 2) und Fumonisine B1 und B2 (FB1 und FB2) in Lebensmitteln durch Flüssigchromatographie (LC, en.: liquid chromatography), gekoppelt mit Tandem-Massenspektrometrie (MS/MS).
Für Aflatoxine (AF) und OTA in für Säuglinge und Kleinkinder bestimmter Nahrung (z. B. Zerealien für Kleinkinder, Pulver auf Milchbasis), in Gewürzen, in Trockenfrüchten und in Nüssen ist eine spezifische Immunaffinitätssäulen (IAC, en.: immunoaffinity column)-Reinigung erforderlich.
Das Verfahren wurde durch eine Ringversuchstudie zu verschiedenen Lebensmittelgruppen validiert: Getreide und Produkte auf Getreidebasis einschließlich Nahrung für Säuglinge und Kleinkinder, Nüsse, Gewürze, Trockenfrüchte und Milchpulver. Die Messbereiche der einzelnen Mykotoxine in diesen natürlich kontaminierten und/oder aufgestockten Lebensmittelproben waren:
—   AFB1:   0,085 7 µg/kg – 11,4 µg/kg;
—   AFB2:   0,079 2 µg/kg – 12,5 µg/kg;
—   AFG1:   0,062 8 µg/kg – 20,9 µg/kg;
—   AFG2:   0,052 0 µg/kg – 15,0 µg/kg;
—   AFM1:   0,034 2 µg/kg – 0,110 µg/kg;
—   OTA:   0,448 µg/kg – 17,2 µg/kg;
—   DON:   45,2 µg/kg – 743 µg/kg;
—   ZEN:   9,57 µg/kg – 131 µg/kg;
—   T 2:   10,3 µg/kg – 57,9 µg/kg;
—   HT 2:   9,50 µg/kg – 81,8 µg/kg;
—   FB1:   31,1 µg/kg – 4 260 µg/kg;
—   FB2:   44,2 µg/kg – 1 300 µg/kg.
Die Messbereiche der Verfahren für jede Mykotoxin-/Matrix-Kombination sind in Tabelle 8 angegeben.

Produits alimentaires - Multiméthode de détermination de la teneur en aflatoxines, déoxynivalénol, fumonisines, ochratoxine A, toxine T-2, toxine HT-2 et zéaralénone par CL-SM/SM

Le présent document décrit une méthode utilisant des étalons marqués aux isotopes pour la détermination quantitative des teneurs en aflatoxines B1, B2, G1, G2 et M1 (AFB1, AFB2, AFG1, AFG2 et AFM1), ochratoxine A (OTA), déoxynivalénol (DON), zéaralénone (ZEN), toxines T-2 et HT-2 (T-2 et HT 2) et fumonisines B1 et B2 (FB1 et FB2) dans les aliments par chromatographie liquide (CL) couplée à une spectrométrie de masse en tandem (SM/SM).
Une purification spécifique sur colonne d’immuno-affinité (CIA) est nécessaire pour les aflatoxines (AF) et l’OTA présentes dans les aliments pour nourrissons et enfants en bas âge (par exemple, céréales infantiles, lait en poudre), les épices, les fruits secs et les fruits à coques.
La méthode a été validée lors d’un essai interlaboratoires portant sur différents groupes de denrées : céréales et produits à base de céréales, y compris aliments pour nourrissons et enfants en bas âge, fruits à coques, épices, fruits secs et lait en poudre. Les gammes de mesure de chaque mycotoxine dans ces échantillons d’aliments naturellement contaminés et/ou dopés étaient les suivantes :
- AFB1 : 0,085 7 µg/kg - 11,4 µg/kg ;
- AFB2 : 0,079 2 µg/kg - 12,5 µg/kg ;
- AFG1 : 0,062 8 µg/kg - 20,9 µg/kg ;
- AFG2 : 0,052 0 µg/kg - 15,0 µg/kg ;
- AFM1 : 0,034 2 µg/kg - 0 110 µg/kg ;
- OTA : 0,448 µg/kg - 17,2 µg/kg ;
- DON : 45,2 µg/kg - 743 µg/kg ;
- ZEN : 9,57 µg/kg - 131 µg/kg ;
- T-2 : 10,3 µg/kg - 57,9 µg/kg ;
- HT-2 : 9,50 µg/kg - 81,8 µg/kg ;
- FB1 : 31,1 µg/kg - 4 260 µg/kg ;
- FB2 : 44,2 µg/kg - 1 300 µg/kg.
Les gammes de mesure de la méthode pour chaque combinaison mycotoxine/matrice sont indiquées dans le Tableau 8.

Živila - Hkratna metoda za določevanje aflatoksina, deoksinivalenola, fumonizinov, ohratoksina A, toksinov T-2 in HT-2 ter zearalenona z LC-MS/MS

Ta dokument opisuje metodo redčenja izotopov za kvantitativno določanje aflatoksinov B1, B2, G1, G2 in M1 (AFB1, AFB2, AFG1, AFG2 in AFM1), okratoksina A (OTA), deoksinivalenola (DON), zearalenona (ZEN), toksinov T-2 in HT-2 (T-2 in HT-2) ter fumonizinov B1 in B2 (FB1 in FB2) v živilih z visokozmogljivo tekočinsko kromatografijo (LC) v povezavi s tandemsko masno spektrometrijo (MS/MS).
Za aflatoksine (AF) in OTA v hrani za dojenčke (npr. kosmiči za dojenčke, praški na osnovi mleka), v začimbah, suhem sadju in oreščkih je potrebno posebno čiščenje imunoafinitetne kolone (IAC).
Metoda je bila potrjena z medstrokovno študijo o različnih skupinah živil: žita in proizvodi na osnovi žit, vključno s hrano za dojenčke in majhne otroke, oreščki, začimbami, suhim sadjem in mlekom v prahu. Območja koncentracij vsakega mikotoksina v teh naravno kontaminiranih vzorcih hrane in/ali vzorcih hrane z dodatki so bila:
–   aflatoksin B1:   od 0,0857 µg/kg do 11,4 µg/kg;
–   aflatoksin B2:   od 0,0792 µg/kg do 12,5 µg/kg;
–   aflatoksin G1:   od 0,0628 µg/kg do 20,9 µg/kg;
–   aflatoksin G2:   od 0,0520 µg/kg do 15,0 µg/kg;
–   aflatoksin M1:   od 0,0342 µg/kg do 0,110 µg/kg;
–   ohratoksin A:   od 0,448 µg/kg do 17,2 µg/kg;
–   deoksinivalenol:   od 45,2 µg/kg do 743 µg/kg;
–   zearalenon:   od 9,57 µg/kg do 131 µg/kg;
–   toksin T-2:      od 10,3 µg/kg do 57,9 µg/kg;
–   toksin HT-2:   od 9,50 µg/kg do 81,8 µg/kg;
–   fumonizin B1:   od 31,1 µg/kg do 4262 µg/kg;
–   fumonizin B2:   od 44,2 µg/kg do 1299 µg/kg.
Merilna območja metode za vsako kombinacijo mikotoksin/matrika so navedena v preglednici 7.

General Information

• Status: Published
• Publication Date: 06-Sep-2022
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 5 - Biotoxins
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 07-Sep-2022
• Due Date: 21-Jul-2022
• Completion Date: 07-Sep-2022
• Ref Project: SIST EN 17641:2022 - Foodstuffs - Multimethod for the determination of aflatoxins, deoxynivalenol, fumonisins, ochratoxin A, T-2 toxin, HT-2 toxin and zearalenone by LC-MS/MS

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN 17641:2022
01-november-2022

Živila - Hkratna metoda za določevanje aflatoksina, deoksinivalenola, fumonizinov,

ohratoksina A, toksinov T-2 in HT-2 ter zearalenona z LC-MS/MS

Foodstuffs - Multimethod for the determination of aflatoxins, deoxynivalenol, fumonisins,

ochratoxin A, T-2 toxin, HT-2 toxin and zearalenone by LC-MS/MS
Lebensmittel - Multimethode für die Bestimmung von Aflatoxinen, Deoxynivalenol,
Fumonisinen, Ochratoxin A, T-2-Toxin, HT-2-Toxin und Zearalenon mittels LC-MS/MS

Produits alimentaires - Multiméthode de détermination de la teneur en aflatoxines,

déoxynivalénol, fumonisines, ochratoxine A, toxine T-2, toxine HT-2 et zéaralénone par

CL-SM/SM
Ta slovenski standard je istoveten z: EN 17641:2022
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN 17641:2022 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 17641:2022
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SIST EN 17641:2022
EN 17641
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2022
EUROPÄISCHE NORM
ICS 67.050
English Version
Foodstuffs - Multimethod for the determination of
aflatoxins, deoxynivalenol, fumonisins, ochratoxin A, T-2
toxin, HT-2 toxin and zearalenone by LC-MS/MS

Produits alimentaires - Multiméthode de Lebensmittel - Multimethode für die Bestimmung von

détermination de la teneur en aflatoxines, Aflatoxinen, Deoxynivalenol, Fumonisinen, Ochratoxin

déoxynivalénol, fumonisines, ochratoxine A, toxine T-2, A, T-2-Toxin, HT-2-Toxin und Zearalenon mittels LC-

toxine HT-2 et zéaralénone par CL-SM/SM MS/MS
This European Standard was approved by CEN on 24 July 2022.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17641:2022 E

worldwide for CEN national Members.
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SIST EN 17641:2022
EN 17641:2022 (E)
Contents Page

European foreword ............................................................................................................................................. 3

Introduction .......................................................................................................................................................... 4

1 Scope .......................................................................................................................................................... 5

2 Normative references .......................................................................................................................... 5

3 Terms and definitions ......................................................................................................................... 5

4 Principles ................................................................................................................................................. 6

5 Reagents ................................................................................................................................................... 6

6 Apparatus and equipment ...............................................................................................................12

7 Procedure...............................................................................................................................................14

7.1 Preparation of the laboratory sample .........................................................................................14

7.2 Test portions weighing .....................................................................................................................14

7.2.1 Cereals, cereal-based products ......................................................................................................14

7.2.2 Milk powders, nuts, spices, dried fruits ......................................................................................14

7.3 Spiking with ISTD solutions ............................................................................................................14

7.4 Extraction ...............................................................................................................................................14

7.4.1 General procedure ..............................................................................................................................14

7.4.2 Without IAC clean-up .........................................................................................................................15

7.4.3 With IAC clean-up ................................................................................................................................15

8 LC-MS/MS analysis ..............................................................................................................................16

8.1 Operating conditions .........................................................................................................................16

8.2 Injection sequence ..............................................................................................................................16

8.3 Data treatment .....................................................................................................................................17

9 Calculations ...........................................................................................................................................17

9.1 Identification and confirmation.....................................................................................................17

9.2 Calibration curve and measuring range .....................................................................................17

9.3 Calculations ...........................................................................................................................................18

10 Precision .................................................................................................................................................19

10.1 General ....................................................................................................................................................19

10.2 Repeatability .........................................................................................................................................19

10.3 Reproducibility ....................................................................................................................................19

11 Test report .............................................................................................................................................22

Annex A (informative) Precision data.........................................................................................................23

Annex B (informative) Overview of the sample preparation .............................................................39

Annex C (informative) Example conditions for suitable LC-MS/MS systems ................................40

Bibliography ........................................................................................................................................................49

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SIST EN 17641:2022
EN 17641:2022 (E)
European foreword

This document (EN 17641:2022) has been prepared by Technical Committee CEN/TC 275 “Food

analysis - Horizontal methods”, the secretariat of which is held by DIN.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by March 2023, and conflicting national standards shall be

withdrawn at the latest by March 2023.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

Any feedback and questions on this document should be directed to the users’ national standards body.

A complete listing of these bodies can be found on the CEN website.

According to the CEN-CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,

Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,

Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North

Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the United

Kingdom.
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SIST EN 17641:2022
EN 17641:2022 (E)
Introduction

Mycotoxins are fungal metabolites that may occur in various foodstuffs such as cereals, nuts, spices, fruits,

oil seeds, or coffee. Mycotoxins can be produced before harvest in the crop and even after harvest if

climate conditions are favourable for further fungal growth. Milk can be contaminated as well by

Aflatoxin M , the major metabolite of Aflatoxin B , when cows are fed with Aflatoxin B contaminated

1 1 1

feed. To protect consumer health, maximum levels for mycotoxins in foodstuffs have been established in

a broad range of food commodities including those intended for infants and young children consumption.

WARNING 1 — Suitable precaution and protection measures need to be taken when carrying out

working steps with harmful chemicals. The latest version of the hazardous substances ordinance,

Regulation (EC) No 1907/2006 [1], should be taken into account as well as appropriate national

statements.

WARNING 2 — The use of this document can involve hazardous materials, operations and

equipment. This document does not purport to address all the safety problems associated with its

use. It is the responsibility of the user of this document to establish appropriate safety and health

practices and determine the applicability of regulatory limitations prior to use.

WARNING 3 — Aflatoxins are known to have carcinogenic effects and to be both acutely and

chronically toxic. Aflatoxins B , B , G , G and M are classified as carcinogenic to humans

1 2 1 2 1

(Group 1) by the International Agency for Cancer Research (IARC). Fumonisin B , fumonisin B

1 2

and ochratoxin A have been classified as possibly carcinogenic to humans (Group 2B) and

zearalenone, deoxynivalenol and T-2 as not classifiable as to their carcinogenicity to humans

(Group 3) [2].
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SIST EN 17641:2022
EN 17641:2022 (E)
1 Scope

This document describes a method using isotopically labelled standards for the quantitative

determination of aflatoxins B , B , G , G and M (AFB1, AFB2, AFG1, AFG2 and AFM1), ochratoxin A

1 2 1 2 1

(OTA), deoxynivalenol (DON), zearalenone (ZEN), T-2 and HT-2 toxins (T-2 and HT-2) and fumonisins B

and B (FB1 and FB2) in foods by liquid chromatography (LC) coupled with tandem mass spectrometry

(MS/MS).

A specific immunoaffinity column (IAC) clean-up is needed for aflatoxins (AFs) and OTA in food intended

for infants and young children (e.g. infant cereals, milk-based powders), in spices, in dried fruits and in

nuts.

The method has been validated through an intercollaborative study on different commodity groups:

cereals and cereal-based products including food for infant and young children, nuts, spices, dried fruits

and milk powder. The measuring range of each mycotoxin in these naturally contaminated and/or spiked

food samples were:
— AFB1: 0,085 7 µg/kg – 11,4 µg/kg;
— AFB2: 0,079 2 µg/kg – 12,5 µg/kg;
— AFG1: 0,062 8 µg/kg – 20,9 µg/kg;
— AFG2: 0,052 0 µg/kg – 15,0 µg/kg;
— AFM1: 0,034 2 µg/kg – 0,110 µg/kg;
— OTA: 0,448 µg/kg – 17,2 µg/kg;
— DON: 45,2 µg/kg – 743 µg/kg;
— ZEN: 9,57 µg/kg – 131 µg/kg;
— T-2: 10,3 µg/kg – 57,9 µg/kg;
— HT-2: 9,50 µg/kg – 81,8 µg/kg;
— FB1: 31,1 µg/kg – 4 260 µg/kg;
— FB2: 44,2 µg/kg – 1 300 µg/kg.

The measuring ranges of the method for each mycotoxin/matrix combination are given in Table 8.

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696)

3 Terms and definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— IEC Electropedia: available at https://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
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SIST EN 17641:2022
EN 17641:2022 (E)
4 Principles

The mycotoxins are extracted from the test portion with water and acidified acetonitrile and a liquid-

liquid partition is initiated after addition of magnesium sulphate and sodium chloride salts. The resulting

acetonitrile supernatant is then defatted with hexane. Depending on the mycotoxin/matrix combination,

the sample extract is then submitted to two different protocols (a general scheme is given in Annex B):

— without IAC clean-up: generic procedure for the determination of all mycotoxins in cereals and cereal-

based products. An aliquot of the acetonitrile supernatant is evaporated to dryness, then

reconstituted in a methanol-water solution and subsequently analysed by LC-MS/MS;

— with IAC clean-up: specific procedure for AFs and OTA determination in food intended for infants and

young children (e.g. infant cereals, milk powder) for sensitivity purpose and in spices, nuts and dried

fruits to prevent matrix effects in the mass spectrometer instrument. An aliquot of the acetonitrile

supernatant is first diluted in a phosphate buffered saline (PBS) solution and the resulting mixture

is then applied onto an IAC containing antibodies specific to AFs and OTA. The IAC is washed with

water and the mycotoxins are eluted with methanol. The eluate is evaporated to dryness,

reconstituted in a methanol-water solution, and subsequently analysed by LC-MS/MS.

Quantification is performed by the isotopic dilution approach using C isotopically labelled mycotoxins

as internal standards (ISTDs).
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 LC-MS grade for LC-MS analysis, unless otherwise

specified. Commercially available solutions with equivalent properties to those listed may be used.

WARNING 4 — Decontamination procedures for laboratory wastes of AFs were developed by the

IARC [3], [4].
5.1 Nitrogen compressed gas, purity equivalent to 99,99 % or better.
5.2 Water, HPLC grade.
5.3 Water, LC-MS grade.
5.4 Methanol (MeOH), analytical grade.
5.5 Methanol (MeOH), LC-MS grade.
5.6 Acetonitrile (MeCN), analytical grade.
5.7 Formic acid, analytical grade.
5.8 Ammonium formate, LC-MS grade.
5.9 Acetic acid glacial, analytical grade.
5.10 n-Hexane, analytical grade.
5.11 Magnesium sulphate (MgSO ) anhydrous, analytical grade.
5.12 Sodium chloride (NaCl), analytical grade.
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SIST EN 17641:2022
EN 17641:2022 (E)
5.13 Partitioning salts mixture, MgSO -NaCl (4 + 1, m + m).

Weigh 4,0 g ± 0,01 g of MgSO (5.11) and 1,00 g ± 0,01 g of NaCl (5.12) into a 15 ml polypropylene tube.

Alternatively, a ready-to-use partitioning salt mixture may be supplied by commercial sources.

5.14 Potassium chloride, (KCl) analytical grade.
5.15 Potassium dihydrogen phosphate, (KH PO ) analytical grade.
2 4
5.16 Disodium hydrogen orthophosphate, (Na HPO ) analytical grade.
2 4
5.17 Sodium hydroxide, (NaOH) analytical grade.
5.18 Sodium hydroxide solution, (NaOH) molar concentration c = 0,1 mol/l.
Dissolve 0,4 g of sodium hydroxide (5.17) in 100 ml water (5.2).
5.19 Hydrochloric acid solution, (HCl) analytical grade, w(HCl) = 37 %.
5.20 Hydrochloric acid diluted solution, (HCl) c = 0,1 mol/l.

Add 8,4 ml of hydrochloric acid solution (5.19) into a 1 l volumetric flask and fill-up to the mark with

water (5.2).
5.21 Phosphate buffered saline (PBS) solution, pH 7,3 ± 0,2.

Weigh 0,20 g of KCl (5.14), 0,20 g of KH PO (5.15), 1,15 g of Na HPO (5.16) and 8,00 g of NaCl (5.12) to

2 4 2 4

the nearest 0,01 g and transfer into a 1 l volumetric flask. Dissolve with approximately 900 ml of water

(5.2).

After dissolution adjust the pH to 7,3 ± 0,2 with either HCl solution (5.20) or NaOH solution (5.18), then

fill up to the mark with water (5.2).

Alternatively, a PBS solution with equivalent properties may be prepared from commercially available

PBS material.
5.22 Extraction solution, acetic acid (87 mmol/l) in acetonitrile.

Mix 500 ml of acetonitrile (5.6) and 5 ml of acetic acid glacial (5.9) into a 1 l volumetric flask. Fill-up to

the mark with acetonitrile (5.6) and mix well. This solution can be used for 3 months if stored at room

temperature.
5.23 Diluting solution, methanol-water (15 + 85, V + V).

Mix 15 ml of methanol (5.4) with 85 ml of water (5.2) into a 100 ml volumetric flask. This solution can be

used for 3 months if stored at room temperature.

5.24 Mycotoxins analytical standard, e.g. crystalline, as a film or as a certified standard solution.

5.24.1 AFB1.
5.24.2 AFB2.
5.24.3 AFG1.
5.24.4 AFG2.
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SIST EN 17641:2022
EN 17641:2022 (E)
5.24.5 AFM1.
5.24.6 OTA.
5.24.7 DON.
5.24.8 ZEN.
5.24.9 T-2.
5.24.10 HT-2.
5.24.11 FB1.
5.24.12 FB2.
5.25 Mycotoxin stock standard solutions.

The individual stock standard solutions are either prepared by dissolving pure substance in an

appropriate solvent or prepared from dried-down films and subsequently reconstituted according to the

certificate of each individual standard or purchased as ready-to-use solutions (5.24).

The mycotoxins covered in this document dissolve well in acetonitrile, with the exception of fumonisins

for which acetonitrile/water solution (50 + 50, V + V) is recommended for the preparation.

5.26 Mycotoxin working standard solutions.

Prepare the working standard solutions as described hereafter by combining the appropriate volumes of

each individual stock standard solutions (5.25), using the appropriate pipettes (6.1) and the mentioned

solvents. These solutions are used to build the calibration curve (5.29) and for the preparation of positive

control samples (7.6).

5.26.1 AFs working standard solution 1, AFB1, AFB2, AFG1 and AFG2, each at mass concentration

ρ = 0,1 µg/ml in acetonitrile.

5.26.2 AFs working standard solution 2, AFB1, AFB2, AFG1 and AFG2, each at ρ = 0,01 µg/ml in

acetonitrile.
5.26.3 AFM1 working standard solution 1, ρ = 0,1 µg/ml in acetonitrile.
5.26.4 AFM1 working standard solution 2, ρ = 0,01 µg/ml in acetonitrile.

5.26.5 [DON, T-2, HT-2, ZEN]- working standard solution, in acetonitrile at mass concentrations given

in Table 1.
Table 1 — [DON, T-2, HT-2, ZEN]- working standard solution
Compound Mass concentration
µg/ml
DON 5,0
T-2 2,5
HT-2 2,5
ZEN 2,0
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5.26.6 Fumonisins (FBs) working standard solution, FB1 and FB2, each at ρ = 5,0 µg/ml in

acetonitrile-water solution (50 + 50, V + V).

5.26.7 OTA working standard solution, ρ = 0,1 µg/ml in methanol-water solution (15 + 85, V + V).

5.27 C-isotopically labelled mycotoxin analytical standards, as a certified standard solution.

13 13 13

5.27.1 C-isotopically labelled AFB1( C-AFB1), e.g. ( C )-AFB1, ρ = 0,5 µg/ml in acetonitrile.

13 13 13

5.27.2 C-isotopically labelled AFB2 ( C-AFB2), e.g. ( C )-AFB2, ρ = 0,5 µg/ml in acetonitrile.

13 13 13

5.27.3 C-isotopically labelled AFG1 ( C-AFG1), e.g. ( C )-AFG1, ρ = 0,5 µg/ml in acetonitrile.

13 13 13

5.27.4 C-isotopically labelled AFG2 ( C-AFG2), e.g. ( C )-AFG2, ρ = 0,5 µg/ml in acetonitrile.

13 13 13

5.27.5 C-isotopically labelled AFM1 ( C-AFM1), e.g. ( C )-AFM1, ρ = 0,5 µg/ml in acetonitrile.

13 13 13

5.27.6 C-isotopically labelled OTA ( C-OTA), e.g. ( C )-OTA, ρ = 10 µg/ml in acetonitrile.

13 13 13

5.27.7 C-isotopically labelled DON ( C-DON), e.g. ( C )-DON, ρ = 25 µg/ml in acetonitrile.

13 13 13

5.27.8 C-isotopically labelled ZEN ( C-ZEN), e.g. ( C )-ZEN, ρ = 25 µg/ml in acetonitrile.

13 13 13

5.27.9 C-isotopically labelled T-2 ( C-T2), e.g. ( C )-T-2, ρ = 25 µg/ml in acetonitrile.

13 13 13
5.27.10 C-isotopically labelled HT-2 ( C-HT-2), e.g. ( C )-HT-2, ρ = 25 µg/ml in
acetonitrile.
13 13 13

5.27.11 C-isotopically labelled FB1 ( C-FB1), e.g. ( C )-FB1, ρ = 25 µg/ml in acetonitrile-

water solution (50 + 50, V + V).
13 13 13

5.27.12 C-isotopically labelled FB2 ( C-FB2), e.g. ( C )-FB2, ρ = 10 µg/ml in acetonitrile-

water solution (50 + 50, V + V).
5.28 ISTD working standard solutions.

Prepare the ISTD solutions as described hereafter by combining the appropriate volumes of individual

isotopically labelled mycotoxin solutions (5.27), using the appropriate pipettes (6.1) and the mentioned

solvent. These solutions are used to build the calibration curve (5.29) and to spike each test portion

before extraction (7.3).
13 13 13 13 13
5.28.1 C-AFs solution, C-AFB1, C-AFB2, C-AFG1, C-AFG2, each at ρ = 0,1 µg/ml in
acetonitrile.
5.28.2 C-AFM1 solution, ρ = 0,1 µg/ml in acetonitrile.

5.28.3 C-[DON, T-2, HT-2, ZEN] solution, in acetonitrile at concentrations given in Table 2.

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Table 2 — C-[DON, T-2, HT-2, ZEN] ISTD solution
Compound Mass concentration
µg/ml
5,0
C-DON
2,5
C-T-2
2,5
C-HT-2
2,0
C-ZEN
13 13 13

5.28.4 C-FBs solution, C-FB1 and C-FB2, each at ρ = 10 µg/ml in acetonitrile-water solution

(50 + 50, V + V).

5.28.5 C-OTA solution, ρ = 0,1 µg/ml in methanol-water solution (15 + 85, V + V).

5.29 Standard solutions for external calibration curve.

Prepare the standard solutions for calibration into nine separate 15 ml polypropylene tubes, as described

in Table 3 for example. Evaporate to dryness under a stream of nitrogen at 40 °C then continue to prepare

the standard solutions for calibration as described in Table 4 for example.

Sonicate the calibrants CAL 0 to CAL 8 for about 1 min. Transfer these solutions into glass vials and store

them at approximately −20 °C protected from light for up to 3 months.

Mass concentration of each mycotoxin in each calibrant solution is given in Table 5.

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Table 3 — Example of pipetting scheme for the preparation of the calibration solutions before

the evaporation step
a a
Mycotoxin CAL 0 CAL 1 CAL 2 CAL 3 CAL 4 CAL 5 CAL 6
CAL 7 CAL 8
µl µl µl µl µl µl µl µl µl
AFs (5.26.1) 0 - - - 20 40 160 320 640
AFs (5.26.2) - 5 20 40 - - - - -
AFM1 (5.26.3) 0 - - - 20 40 160 320 640
AFM1 (5.26.4) - 5 20 40 - - - - -
[DON, T-2, HT-2, ZEN]
0 5 10 20 40 80 160 320 640
(5.26.5)
FBs (5.26.6) 0 5 10 20 40 80 160 320 640
C-AFs (5.28.1) 20 20 20 20 20 20 20 20 20
20 20 20 20 20 20 20 20 20
C-AFM1(5.28.2)
C-[DON, T-2, HT-2,
20 20 20 20 20 20 20 20 20
ZEN] (5.28.3)
C-FBs (5.28.4) 20 20 20 20 20 20 20 20 20

The calibration range can be extended for quantification of highly contaminated samples (9.2). Typically, CAL 7 and

CAL 8 can be prepared as described in Table 3 to extend the range by a factor of 2 (CAL 7) and a factor of 4 (CAL 8).

NOTE Robustness of the method is not affected as long as the same ISTD solutions are used for both preparing calibration

standard solutions and spiking test portions (7.3).

Table 4 — Example of pipetting scheme for the preparation of the calibration solutions following

the evaporation step
a a
Compound CAL 0 CAL 1 CAL 2 CAL 3 CAL 4 CAL 5 CAL 6
CAL 8
CAL 7
µl µl µl µl µl µl µl µl µl
OTA (5.26.7) 0 2,5 5 10 20 40 160 320 640
20 20 20 20 20 20 20 20 20
C-OTA (5.28.5)
MeOH-H O (15 + 85,
1 980 1978 1 975 1 970 1 960 1 940 1 820 1 660 1 340
V + V) (5.23)

The calibration range can be extended for quantification of highly contaminated samples (9.2). Typically, CAL 7 and

CAL 8 can be prepared as described in Table 3 to extend the range by a factor of 2 (CAL 7) and a factor of 4 (CAL 8).

NOTE 1 Robustness of the method is not affected as long as the same ISTD solutions are used for both preparing calibration

standard solutions and spiking test portions (7.3).

NOTE 2 OTA solutions are added after the evaporation step to avoid unpredictable OTA losses upon evaporation.

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Table 5 — Example mass concentrations of mycotoxins and ISTD in calibration solutions

Mycotoxin CAL 0 CAL 1 CAL 2 CAL 3 CAL 4 CAL 5 CAL 6 CAL 7 CAL 8
ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml
AFs 0 0,025 0,1 0,2 1 2 8 16 32
AFM1 0 0,025 0,1 0,2 1 2 8 16 32
DON 0 12,5 25 50 100 200 400 800 1600
T-2 and HT-2 0 6,25 12,5 25 50 100 200 400 800
ZEN 0 5 10 20 40 80 160 320 640
FBs 0 12,5 25 50 100 200 400 800 1600
OTA 0 0,125 0,25 0,5 1 2 8 16 32
1 1 1 1 1 1 1 1 1
C-AFs
1 1 1 1 1 1 1 1 1
C-AFM1
50 50 50 50 50 50 50 50 50
C-DON
13 13
25 25 25 25 25 25 25 25 25
C-T-2 & C-HT-2
20 20 20 20 20 20 20 20 20
C-ZEN
100 100 100 100 100 100 100 100 100
C-FBs
1 1 1 1 1 1 1 1 1
C-OTA
6 Apparatus and equipment

Glassware and equipment (graduated cylinders, glass funnels, beakers, pipettes, etc.) and, in particular,

the following.
6.1 Pipettes, suited for organic solvent in the range 1 μl to 1 ml.
6.2 Conical polypropylene screw cap centrifuge tube, 50 ml with cap.
6.3 Conical polypropylene screw cap centrifuge tube, 15 ml with cap.
6.4 Polypropylene microcentrifuge tube, 1,5 ml.
6.5 HPLC glass vial, 1,5 ml with screw cap.

6.6 Adjustable mechanical vertical or horizontal shaker, capable to shake at 300 min .

6.7 Laboratory shaker.
6.8 Ultrasonic water bath.
6.9 Laboratory balance, accuracy of 0,01 g.
6.10 Analytical balance, accuracy of 0,1 mg.
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6.11 Centrifuge, with rotors adapted for polypropylene tubes of 15 ml and 50 ml volume, capable of

generating a relative centrifugal force of 4 000 g.

6.12 Centrifuge, with rotors adapted for polypropylene tubes of 1,5 ml volume, capable of generating a

relative centrifugal force of 8 500 g.
6.13 Sample concentrator, with temperature control and nitrogen gas supply.
6.14 Vacuum manifold for SPE clean-up, with taps.
6.15 Polypropylene reservoirs (approx. 25 ml), adapted for SPE columns.
6.16 Disposable syringe, 5 ml.
6.17 Plastic Pasteur pipette, non-sterile, 7 ml.
6.18 IAC for AFB1, AFB2, AFG1, AFG2 and OTA.

The IAC contains antibodies raised against AFB1, AFB2, AFG1, AFG2 and OTA with a capacity greater than

100 ng .
6.19 IAC for AFM1.

The IAC contains antibodies raised against AFM1 with a capacity greater than 100 ng.

Alternatively, an IAC containing antibodies raised against AFB1, AFB2, AFG1, AFG2 with a cross-reactivity

to AFM1 might also suitable .
6.20 LC-MS/MS system, with the following components:

6.20.1 LC pump, capable of delivering a binary gradient at flow rates appropriate for the analytical

column in use with sufficient accuracy.
6.20.2 Degasser, optional, for degassing LC mobile phases.

6.20.3 Injection system, capable of injecting an appropriate volume of injection solution with sufficient

accuracy.

6.20.4 LC column, capable to retain the first eluting analyte at least twice the retention time

corresponding to the void volume of the column. Examples of suitable columns and gradients are given

in Annex C.

6.20.5 LC pre-column, optional, with the same stationary phase material as the LC column (6.20.4).

6.20.6 Column oven, capable to maintain a constant temperature.

6.20.7 Tandem mass spectrometer (e.g. triple quadrupole or quadrupole linear ion trap), equipped

with an electrospray ionization (ESI) interface and operated in multiple r
...

SLOVENSKI STANDARD
oSIST prEN 17641:2021
01-marec-2021
Živila - Večelementna metoda za določevanje aflatoksina, deoksinivalenola,
fumonizinov, ohratoksina A, toksinov T-2 in HT-2 ter zearalenona z LC-MS/MS

Foodstuffs - Multimethod for the determination of aflatoxins, deoxynivalenol, fumonisins,

ochratoxin A, T-2 toxin, HT-2 toxin and zearalenone by LC-MS/MS
Lebensmittel — Multimethode für die Bestimmung von Aflatoxinen, Deoxynivalenol,
Fumonisinen, Ochratoxin A, T 2 Toxin, HT 2 Toxin und Zearalenon mittels LC MS/MS

Produits alimentaires — Multiméthode de détermination de la teneur en aflatoxines,

déoxynivalénol, fumonisines, ochratoxine A, toxine T-2, toxine HT-2 et zéaralénone par

CL-SM/SM
Ta slovenski standard je istoveten z: prEN 17641
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
oSIST prEN 17641:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 17641:2021
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oSIST prEN 17641:2021
DRAFT
EUROPEAN STANDARD
prEN 17641
NORME EUROPÉENNE
EUROPÄISCHE NORM
January 2021
ICS 67.050
English Version
Foodstuffs - Multimethod for the determination of
aflatoxins, deoxynivalenol, fumonisins, ochratoxin A, T-2
toxin, HT-2 toxin and zearalenone by LC-MS/MS

Produits alimentaires - Multiméthode de Lebensmittel - Multimethode für die Bestimmung von

détermination de la teneur en aflatoxines, Aflatoxinen, Deoxynivalenol, Fumonisinen, Ochratoxin

déoxynivalénol, fumonisines, ochratoxine A, toxine T-2, A, T 2 Toxin, HT 2 Toxin und Zearalenon mittels LC

toxine HT-2 et zéaralénone par CL-SM/SM MS/MS

This 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, 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.

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

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17641:2021 E

worldwide for CEN national Members.
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oSIST prEN 17641:2021
prEN 17641:2021 (E)
Contents Page

European foreword ...................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Principles ........................................................................................................................................................... 6

5 Reagents ............................................................................................................................................................. 6

6 Apparatus and equipment ......................................................................................................................... 12

7 Procedure ........................................................................................................................................................ 13

8 LC-MS/MS analysis ........................................................................................................................................ 16

9 Calculations ..................................................................................................................................................... 17

10 Precision .......................................................................................................................................................... 18

11 Test report ....................................................................................................................................................... 22

Annex A (informative) Precision data .................................................................................................................. 23

Annex B (informative) Overview of the sample preparation ....................................................................... 39

Annex C (informative) Example conditions for suitable LC-MS/MS systems ......................................... 40

C.1 General.............................................................................................................................................................. 40

C.2 LC system settings examples..................................................................................................................... 40

C.3 MS system settings examples.................................................................................................................... 43

Bibliography ................................................................................................................................................................. 48

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European foreword

This document (prEN 17641:2021) 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.
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oSIST prEN 17641:2021
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Introduction

Mycotoxins are fungal metabolites that may occur in various foodstuffs such as cereals, nuts, spices,

fruits, oil seeds, or coffee. Mycotoxins can be produced before harvest in the crop and even after harvest

if climate conditions are favourable for further fungal growth. Milk can be contaminated as well by

Aflatoxin M , the major metabolite of Aflatoxin B , when cows are fed with Aflatoxin B contaminated

1 1 1

feed. To protect consumer health, maximum levels for mycotoxins in foodstuffs have been established

in a broad range of food commodities including those intended for infants and young children

consumption.

WARNING 1 — Suitable precaution and protection measures need to be taken when carrying out

working steps with harmful chemicals. The latest version of the hazardous substances

ordinance, Regulation (EC) No 1907/2006 [1], should be taken into account as well as

appropriate national statements.

WARNING 2 — The use of this document can involve hazardous materials, operations and

equipment. This document does not purport to address all the safety problems associated with

its use. It is the responsibility of the user of this document to establish appropriate safety and

health practices and determine the applicability of regulatory limitations prior to use.

WARNING 3 — Aflatoxins are known to have carcinogenic effects and to be both acutely and

chronically toxic. Aflatoxins B , B , G , G and M are classified as carcinogenic to humans (Group

1 2 1 2 1

1) by the International Agency for Cancer Research (IARC). Fumonisin B , fumonisin B and

1 2

ochratoxin A have been classified as possibly carcinogenic to humans (Group 2B) and

zearalenone, deoxynivalenol and T-2 as not classifiable as to their carcinogenicity to humans

(Group 3) [2].
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1 Scope

This document describes an isotope dilution method for the quantitative determination of aflatoxins B ,

B , G , G and M (AFB1, AFB2, AFG1, AFG2 and AFM1), ochratoxin A (OTA), deoxynivalenol (DON),

2 1 2 1

zearalenone (ZEN), T-2 and HT-2 toxins (T-2 and HT-2) and fumonisins B and B (FB1 and FB2) in

1 2

foods by liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS).

A specific immunoaffinity column (IAC) clean-up is needed for aflatoxins (AFs) and OTA in infant foods

(e.g. infant cereals, milk-based powders), in spices, in dried fruits and in nuts.

The method has been validated through an intercollaborative study on different commodity groups:

cereals and cereal-based products including food for infant and young children, nuts, spices, dried fruits

and milk powder. The ranges of concentrations of each mycotoxin in these naturally contaminated

and/or spiked food samples were:
— aflatoxin B : 0,0857 µg/kg – 11,4 µg/kg;
— aflatoxin B : 0,0792 µg/kg – 12,5 µg/kg;
— aflatoxin G : 0,0628 µg/kg – 20,9 µg/kg;
— aflatoxin G : 0,0520 µg/kg – 15,0 µg/kg;
— aflatoxin M : 0,0342 µg/kg – 0,110 µg/kg;
— ochratoxin A: 0,448 µg/kg – 17,2 µg/kg;
— deoxynivalenol: 45,2 µg/kg – 743 µg/kg;
— zearalenone: 9,57 µg/kg – 131 µg/kg;
— T-2 toxin: 10,3 µg/kg – 57,9 µg/kg;
— HT-2 toxin: 9,50 µg/kg – 81,8 µg/kg;
— fumonisin B : 31,1 µg/kg – 4 262 µg/kg;
— fumonisin B : 44,2 µg/kg – 1 299 µg/kg.

The measuring ranges of the method for each mycotoxin/matrix combination are given in Table 7.

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

EN ISO 3696, Water for analytical laboratory use — Specification and test methods (ISO 3696)

3 Terms and definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
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4 Principles

The mycotoxins are extracted from the test portion with water and acidified acetonitrile and a liquid-

liquid partition is initiated after addition of magnesium sulphate and sodium chloride salts. The

resulting acetonitrile supernatant is then defatted with hexane. Depending on the mycotoxin/matrix

combination, the sample extract is then submitted to two different protocols (a general scheme is given

in Annex B):

— without IAC clean-up: generic procedure for the determination of all mycotoxins in cereals and

cereal-based products. An aliquot of the acetonitrile supernatant is evaporated to dryness, then

reconstituted in a methanol-water solution and subsequently analysed by LC-MS/MS;

— with IAC clean-up: specific procedure for AFs and OTA determination in infant foods (e.g. infant

cereals, milk powder) for sensitivity purpose and in spices, nuts and dried fruits to prevent matrix

effects in the mass spectrometer instrument. An aliquot of the acetonitrile supernatant is first

diluted in a phosphate buffered saline (PBS) solution and the whole extract is then applied onto an

IAC containing antibodies specific to AFs and OTA. The IAC is washed with water and the

mycotoxins are eluted with methanol. The eluate is evaporated to dryness, reconstituted in a

methanol-water solution, and subsequently analysed by LC-MS/MS.

Quantification is performed by the isotopic dilution approach using C isotopically labelled mycotoxins

as internal standards (ISTDs).
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 LC-MS grade for LC-MS analysis, unless otherwise

specified. Commercially available solutions with equivalent properties to those listed may be used.

WARNING 1 — Decontamination procedures for laboratory wastes of aflatoxins were developed by the

IARC [3, 4].
5.1 Nitrogen compressed gas, purity equivalent to 99,99 % or better.
5.2 Water, HPLC grade.
5.3 Water, LC-MS grade.
5.4 Methanol (MeOH), analytical grade.
5.5 Methanol (MeOH), LC-MS grade.
5.6 Acetonitrile (MeCN), analytical grade.
5.7 Formic acid, analytical grade.
5.8 Ammonium formate, LC-MS grade.
5.9 Acetic acid glacial, analytical grade.
5.10 n-Hexane, analytical grade.
5.11 Magnesium sulphate (MgSO ) anhydrous, analytical grade.
5.12 Sodium chloride (NaCl), analytical grade.
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5.13 Partitioning salts mixture, MgSO -NaCl (4 + 1, w + w).

Weigh 4,0 g ± 0,2 g of MgSO (5.11) and 1,00 g ± 0,05 g of NaCl (5.12) into a 15 ml polypropylene tube.

Alternatively, a ready-to-use partitioning salt mixture can be supplied by commercial sources.

5.14 Potassium chloride, (KCl) analytical grade.
5.15 Potassium dihydrogen phosphate, (KH PO ) analytical grade.
2 4
5.16 Disodium hydrogen orthophosphate, (Na HPO ) analytical grade.
2 4
5.17 Sodium hydroxide, (NaOH) analytical grade.
5.18 Sodium hydroxide solution, (NaOH) molar concentration c = 0,1 mol/l.
Dissolve 0,4 g of sodium hydroxide (5.17) in 100 ml water (5.2).
5.19 Hydrochloric acid solution, (HCl) analytical grade, φ(HCl) = 37 %.
5.20 Hydrochloric acid diluted solution, c = 0,1 mol/l.

Add 8,4 ml of hydrochloric acid solution (5.19) into a 1-l volumetric flask and complete to volume with

water (5.2).
5.21 Phosphate buffered saline (PBS) solution, pH 7,3 ± 0,2

Weigh 0,20 g of KCl (5.14), 0,20 g of KH PO (5.15), 1,15 g of Na HPO (5.16) and 8,00 g of NaCl (5.12) to

2 4 2 4

the nearest 0,01 g and transfer into a 1-l volumetric flask. Dissolve in water (5.2) and add 900 ml of

water (5.2).

After dissolution adjust the pH to 7,3 ± 0,2 with either HCl solution (5.20) or NaOH solution (5.18), then

fill up to the mark with water (5.2).

Alternatively, a PBS solution with equivalent properties may be prepared from commercially available

PBS material.
5.22 Extraction solution, acetic acid 5 ml/l in acetonitrile.

Mix 500 ml of acetonitrile (5.6) and 5 ml of acetic acid glacial (5.9) into a 1-l volumetric flask. Complete

to volume with acetonitrile (5.6) and mix well. This solution can be used for 3 months if stored at room

temperature.
5.23 Diluting solution, methanol-water (15 + 85, V + V).

Mix 15 ml of methanol (5.4) with 85 ml of water (5.2) into a 100-ml volumetric flask. This solution can

be used for 3 months if stored at room temperature.

5.24 Mycotoxins analytical standard, e.g. crystalline, as a film or as a certified standard solution.

5.24.1 Aflatoxin B (AFB1).
5.24.2 Aflatoxin B (AFB2).
5.24.3 Aflatoxin G (AFG1).
5.24.4 Aflatoxin G (AFG2).
5.24.5 Aflatoxin M (AFM1).
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5.24.6 Ochratoxin A (OTA).
5.24.7 Deoxynivalenol (DON).
5.24.8 Zearalenone (ZEN).
5.24.9 T-2 Toxin (T-2).
5.24.10 HT-2 Toxin (HT-2).
5.24.11 Fumonisin B (FB1).
5.24.12 Fumonisin B (FB2).
5.25 Mycotoxin stock standard solutions.

The individual stock standard solutions are either prepared by dissolving neat (solid) standards in an

appropriate solvent or prepared from dried-down films and subsequently reconstituted according to

the certificate of each individual standard or purchased as ready-to-use solutions (5.24).

The mycotoxins covered in this document dissolve well in acetonitrile, with the exception of fumonisins

for which acetonitrile/water solution (50 + 50, V + V) is recommended for the preparation of individual

stock solutions.
5.26 Mycotoxin working standard solutions.

Prepare the working standard solutions as described hereafter by combining the appropriate volumes

of each individual stock standard solutions (5.25), using the appropriate pipets (6.1) and the mentioned

solvent. These solutions are used to build the calibration curve (5.29) and for the preparation of

positive control samples (7.6).

5.26.1 Aflatoxins (AFs) composite working standard solution, AFB1, AFB2, AFG1 and AFG2, each

at mass concentration ρ = 0,1 µg/ml in acetonitrile.

5.26.2 Aflatoxins (AFs) composite working standard solution, AFB1, AFB2, AFG1 and AFG2, each

at ρ = 0,01 µg/ml in acetonitrile.
5.26.3 AFM1 working standard solution, ρ = 0,1 µg/ml in acetonitrile.
5.26.4 AFM1 working standard solution, ρ = 0,01 µg/ml in acetonitrile.

5.26.5 [DON, T-2, HT-2, ZEN]-composite working standard solution, in acetonitrile at mass

concentrations given in Table 1.
Table 1 — [DON, T-2, HT-2, ZEN]-composite working standard solution
Compound Mass concentration
µg/ml
DON 5,0
T-2 2,5
HT-2 2,5
ZEN 2,0
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5.26.6 Fumonisins (FBs) composite working standard solution, FB1 and FB2, each at

ρ = 5,0 µg/ml in acetonitrile-water solution (50 + 50, V + V).

5.26.7 OTA working standard solution, ρ = 0,1 µg/ml in methanol-water solution (15 + 85, V + V).

5.27 C-isotopically labelled mycotoxin analytical standards, as a certified standard solution.

13 13 13

5.27.1 C-isotopically labelled aflatoxin B ( C-AFB1), e.g. ( C )-aflatoxin B , ρ = 0,5 µg/ml in

1 17 1
acetonitrile.
13 13 13

5.27.2 C-isotopically labelled aflatoxin B ( C-AFB2), e.g. ( C )-aflatoxin B , ρ = 0,5 µg/ml in

2 17 2
acetonitrile.
13 13 13

5.27.3 C-isotopically labelled aflatoxin G1 ( C-AFG1), e.g. ( C17)-aflatoxin G1, ρ = 0,5 µg/ml in

acetonitrile.
13 13 13

5.27.4 C-isotopically labelled aflatoxin G ( C-AFG2), e.g. ( C )-aflatoxin G , ρ = 0,5 µg/ml in

2 17 2
acetonitrile.
13 13 13

5.27.5 C-isotopically labelled aflatoxin M ( C-AFM1), e.g. ( C )-aflatoxin M , ρ = 0,5 µg/ml in

1 17 1
acetonitrile.
13 13 13

5.27.6 C-isotopically labelled ochratoxin A ( C-OTA), e.g. ( C )-ochratoxin A, ρ = 10 µg/ml in

acetonitrile.
13 13 13

5.27.7 C-isotopically labelled deoxynivalenol ( C-DON), e.g. ( C )-deoxynivalenol, ρ = 25 µg/ml

in acetonitrile.
13 13 13

5.27.8 C-isotopically labelled zearalenone ( C-ZEN), e.g. ( C18)-zearalenone, ρ = 25 µg/ml in

acetonitrile.
13 13 13

5.27.9 C-isotopically labelled T-2 toxin ( C-T2), e.g. ( C )-T-2 toxin, ρ = 25 µg/ml in acetonitrile.

13 13 13

5.27.10 C-isotopically labelled HT-2 toxin ( C-HT2), e.g. ( C )-HT-2 toxin, ρ = 25 µg/ml in

acetonitrile.
13 13 13

5.27.11 C-isotopically labelled fumonisin B ( C-FB1), e.g. ( C )-fumonisin B , ρ = 25 µg/ml in

1 34 1
acetonitrile-water solution (50 + 50, V + V).
13 13 13

5.27.12 C-isotopically labelled fumonisin B ( C-FB2), e.g. ( C )-fumonisin B , ρ = 10 µg/ml in

2 34 2
acetonitrile-water solution (50 + 50, V + V).
5.28 Internal standard (ISTD) solutions.

Prepare the ISTD solutions as described hereafter by combining the appropriate volumes of individual

isotopically labelled mycotoxin solutions, using the appropriate pipets (6.1) and the mentioned solvent.

These solutions are used to build the calibration curve (5.29) and to spike each test portion before

extraction (7.3).
13 13 13 13 13 13

5.28.1 C-Aflatoxins ( C-AFs) internal standard solution, C-AFB1, C-AFB2, C-AFG1, C-

AFG2, each at ρ = 0,1 µg/ml in acetonitrile.
5.28.2 C-AFM1 internal standard solution, ρ = 0,1 µg/ml in acetonitrile.
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5.28.3 C-[DON, T-2, HT-2, ZEN] internal standard solution, in acetonitrile at concentrations given

in Table 2.
Table 2 — C-[DON, T-2, HT-2, ZEN] internal standard solution
Compound Mass concentration
µg/ml
C-DON 5,0
C-T-2 2,5
C-HT-2 2,5
C-ZEN 2,0
13 13 13 13

5.28.4 C-Fumonisins ( C-FBs) internal standard solution, C-FB1 and C-FB2, each at

ρ = 10 µg/ml in acetonitrile-water solution (50 + 50, V + V).

5.28.5 C-OTA internal standard solution, ρ = 0,1 µg/ml in methanol-water solution (15 + 85, V + V).

5.29 Standard solutions for external calibration curve.

Into nine separate 15 ml polypropylene tubes, prepare the standard solutions for calibration as

described in Table 3a for example.

Evaporate to dryness under a stream of nitrogen at 40 °C then continue to prepare the standard

solutions for calibration as described in Table 3b for example.

Sonicate the calibrants CAL 0 to CAL 8 for about 1 min. Transfer these solutions into glass vials and

store them at −20 °C protected from light for up to 3 months.
Concentration of each mycotoxin in each calibrant solution is given in Table 4.

Table 3a — Example pipetting scheme for the preparation of the calibration solutions before the

evaporation step
a a
Mycotoxin CAL 0 CAL 1 CAL 2 CAL 3 CAL 4 CAL 5 CAL 6 CAL 7 CAL 8
µl µl µl µl µl µl µl µl µl
AFs (5.26.1) 0 - - - 20 40 160 320 640
AFs (5.26.2) - 5 20 40 - - - - -
AFM1 (5.26.3) 0 - - - 20 40 160 320 640
AFM1 (5.26.4) - 5 20 40 - - - - -
[DON, T-2, HT-2,
0 5 10 20 40 80 160 320 640
ZEN] (5.26.5)
FBs (5.26.6) 0 5 10 20 40 80 160 320 640
C-AFs (5.28.1) 20 20 20 20 20 20 20 20 20
C-AFM1(5.28.2) 20 20 20 20 20 20 20 20 20
C-[DON, T-2, HT-2,
20 20 20 20 20 20 20 20 20
ZEN] (5.28.3)
C-FBs (5.28.4) 20 20 20 20 20 20 20 20 20

The calibration range can be extended for quantification of highly contaminated samples (9.2). Typically, CAL 7 and

CAL 8 can be prepared as described in Table 3a to extend the range by a factor of 2 (CAL 7) and a factor of 4 (CAL 8).

NOTE Robustness of the method is not affected as long as the same ISTD solutions are used for both preparing

calibration standard solutions and spiking test portions (7.3).
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Table 3b — Example pipetting scheme for the preparation of the calibration solutions following

the evaporation step
Compound CAL 0 CAL 1 CAL 2 CAL 3 CAL 4 CAL 5 CAL 6 CAL 7 CAL 8
a a
µl µl µl µl µl µl µl µl µl
OTA (5.26.7) 0 2,5 5 10 20 40 160 320 640
C-OTA (5.28.5) 20 20 20 20 20 20 20 20 20
MeOH-
H O (15 + 85, 1 980 1978 1 975 1 970 1 960 1 940 1 820 1 660 1 340
V + V) (5.23)

The calibration range can be extended for quantification of highly contaminated samples (9.2). Typically,

CAL 7 and CAL 8 can be prepared as described in Table 3a to extend the range by a factor of 2 (CAL 7) and a

factor of 4 (CAL 8).

NOTE 1 Robustness of the method is not affected as long as the same ISTD solutions are used for both preparing

calibration standard solutions and spiking test portions (7.3).

NOTE 2 OTA solutions are added after the evaporation step to avoid unpredictable OTA losses upon

evaporation.

Table 4 — Example mass concentrations of mycotoxins and related ISTD in calibration solutions

Mycotoxin CAL 0 CAL 1 CAL 2 CAL 3 CAL 4 CAL 5 CAL 6 CAL 7 CAL 8
ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml
AFs 0 0,025 0,1 0,2 1 2 8 16 32
AFM1 0 0,025 0,1 0,2 1 2 8 16 32
DON 0 12,5 25 50 100 200 400 800 1600
T-2 and HT-2 0 6,25 12,5 25 50 100 200 400 800
ZEN 0 5 10 20 40 80 160 320 640
FBs 0 12,5 25 50 100 200 400 800 1600
OTA 0 0,125 0,25 0,5 1 2 8 16 32
C-AFs 1 1 1 1 1 1 1 1 1
C-AFM1 1 1 1 1 1 1 1 1 1
C-DON 50 50 50 50 50 50 50 50 50
13 13
C-T-2 & C-HT- 25 25 25 25 25 25 25 25 25
C-ZEN 20 20 20 20 20 20 20 20 20
C-FBs 100 100 100 100 100 100 100 100 100
C-OTA 1 1 1 1 1 1 1 1 1
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6 Apparatus and equipment

Glassware and equipment (graduated cylinders, glass funnels, beakers, pipette, etc.) and, in particular,

the following.
6.1 Pipettes, suited for organic solvent in the range 1 μl to 1 ml.
6.2 Conical polypropylene screw cap centrifuge tube, 50 ml with caps.
6.3 Conical polypropylene screw cap centrifuge tube, 15 ml with caps.
6.4 Polypropylene microcentrifuge tube, 1,5 ml.
6.5 HPLC glass vial, 1,5 ml with screw cap.

6.6 Adjustable mechanical vertical or horizontal shaker, capable to shake at 300 r/min.

6.7 Laboratory shaker.
6.8 Ultrasonic water bath.
6.9 Laboratory balance, accuracy: 0,01 g.
6.10 Analytical balance, accuracy: 0,000 1 g.

6.11 Centrifuge, with rotors adapted for polypropylene tubes of 15 ml and 50 ml volume, capable of

generating a relative centrifugal force of 4 000 g.

6.12 Centrifuge, with rotors adapted for polypropylene tubes of 15 ml volume, capable of generating a

relative centrifugal force of 8 400 g.
6.13 Sample concentrator, with temperature control and nitrogen gas supply.
6.14 Vacuum manifold for SPE clean-up, with taps.
6.15 Polypropylene reservoirs (approx. 25 ml), adapted for SPE columns.
6.16 Disposable syringe, 5 ml.
6.17 Plastic Pasteur pipette, non-sterile, 7 ml.
6.18 Immunoaffinity columns (IAC) for AFB1, AFB2, AFG1, AFG2 and OTA.

The IAC contains antibodies raised against AFB , AFB , AFG , AFG and OTA with a capacity greater than

1 2 1 2
100 ng .
6.19 Immunoaffinity columns for AFM1

The IAC contains antibodies raised against aflatoxin M with a capacity greater than 100 ng.

Alternatively, an IAC containing antibodies raised against AFB1, AFB2, AFG1, AFG2 with a cross-

reactivity to AFM1 might be also suitable .
1 ®

AFLAOCHRA PREP column from R-biopharm, is an example of a suitable product available commercially. This

information is given for the convenience of users of this document 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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6.20 LC-MS/MS system, with the following components:

6.20.1 LC pump, capable of delivering a binary gradient at flow rates appropriate for the analytical

column in use with sufficient accuracy.
6.20.2 Degasser, optional, for degassing LC mobile phases.

6.20.3 Injection system, capable of injecting an appropriate volume of injection solution with

sufficient accuracy.

6.20.4 LC column, capable to retain the first eluting analyte at at least twice the retention time

corresponding to the void volume of the column. Examples of suitable columns and gradients are given

in Annex C.

6.20.5 LC pre-column, optional, with the same stationary phase material as the LC column (6.20.4).

6.20.6 Column oven, capable to maintain a constant temperature.

6.20.7 Triple stage mass spectrometer (e.g. triple quadrupole or quadrupole linear ion trap),

equipped with an electrospray ionization (ESI) interface and operated in multiple reaction monitoring

(MRM) mode. Any ionization mode (typically negative or positive) giving sufficient yield may be

employed.
6.20.8 Computer-based instrument control and data evaluation system.
7 Procedure
7.1 Preparation of the laboratory sample

Samples shall be stored in air-tight containers, protected from light and thoroughly mixed before

analysis. Finely grind and/or extensively homogenize the laboratory samples.
7.2 Test portions weighing
7.2.1 Cereals, cereals-based products

Weigh a test portion of 5,00 g of the homogeneous laboratory sample to the nearest 0,05 g into a 50 ml

polypropylene tube (6.2).
7.2.2 Milk powders, nuts, spices, dried fruits

Weigh a test portion of 2,00 g of the homogeneous laboratory sample to the nearest 0,02 g into a 50 ml

polypropylene tube (6.2).
7.3 Spiking with internal standard (ISTD) solutions

Spike each test portion with 50 µl of ISTD solutions (5.28) as shown in Table 5. The choice of the ISTD to

be spiked depends on the final purpose of the analysis, i.e. the mycotoxin(s) to be monitored.

NOTE Robustness of the method is not affected as long as the same ISTD solutions are used for both

preparing calibration standards (5.29) and spiking test portions.
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Table 5 — Spiking of ISTD solutions
ISTD solutions Spiking Amount of each
level ISTD added on test
portion
µl ng
C-AFs, ρ = 0,1 µg/ml (5.28.1) 50 5
C-AFM1, ρ = 0,1 µg/ml (5.28.2) 50 5
13 a
C-[DON, T-2, HT-2, ZEN] (5.28.3) 50 250, 125, 125, 100
C-FBs mix, ρ = 10 µg/ml (5.28.
...