oSIST prEN 17641:2021

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 an isotope dilution method 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 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 B1:   0,0857 µg/kg - 11,4 µg/kg;
-   aflatoxin B2:   0,0792 µg/kg - 12,5 µg/kg;
-   aflatoxin G1:   0,0628 µg/kg - 20,9 µg/kg;
-   aflatoxin G2:   0,0520 µg/kg - 15,0 µg/kg;
-   aflatoxin M1:   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 B1:   31,1 µg/kg - 4 262 µg/kg;
-   fumonisin B2:   44,2 µg/kg - 1 299 µg/kg.
The measuring ranges of the method for each mycotoxin/matrix combination are given in Table 7.

Lebensmittel — Multimethode 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 der Isotopenverdünnung 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), gekoppelt mit Tandem-Massen-spektrometrie (MS/MS).
Für Aflatoxine (AF) und OTA in Säuglings- und Kleinkindernahrung (z. B. Zerealien für Kleinkinder, Pulver auf Milchbasis), in Gewürzen, in Trockenfrüchten und in Nüssen ist eine spezifische Immunaffinitätssäulen (IAC)-Reinigung erforderlich.
Das Verfahren wurde durch eine gemeinschaftliche Studie zu verschiedenen Bereichen validiert: Getreide und Produkte auf Getreidebasis einschließlich Nahrung für Säuglinge und Kleinkinder, Nüsse, Gewürze, Trockenfrüchte und Milchpulver. Die Konzentrationsbereiche der einzelnen Mykotoxine in diesen natürlich kontaminierten und/oder aufgestockten Lebensmittelproben waren:
-   Aflatoxin B1:   0,085 7 µg/kg - 11,4 µg/kg;
-   Aflatoxin B2:   0,079 2 µg/kg - 12,5 µg/kg;
-   Aflatoxin G1:   0,062 8 µg/kg - 20,9 µg/kg;
-   Aflatoxin G2:   0,052 0 µg/kg - 15,0 µg/kg;
-   Aflatoxin M1:   0,034 2 µg/kg - 0,110 µg/kg;
-   Ochratoxin A:   0,448 µg/kg - 17,2 µg/kg;
-   Deoxynivalenol:   45,2 µg/kg - 743 µg/kg;
-   Zearalenon:   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 B1:   31,1 µg/kg - 4 262 µg/kg;
-   Fumonisin B2:   44,2 µg/kg - 1 299 µg/kg.
Die Messbereiche der Verfahren für jede Mykotoxin-/Matrix-Kombination sind in Tabelle 7 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 de dilution isotopique 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) coupée 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 (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 concentrations de chaque mycotoxine dans ces échantillons d’aliments naturellement contaminés et/ou dopés étaient les suivantes :
-   aflatoxine B1 :   0,0857 µg/kg - 11,4 µg/kg ;
-   aflatoxine B2 :   0,0792 µg/kg - 12,5 µg/kg ;
-   aflatoxine G1 :   0,0628 µg/kg - 20,9 µg/kg ;
-   aflatoxine G2 :   0,0520 µg/kg - 15,0 µg/kg ;
-   aflatoxine M1 :   0,0342 µg/kg - 0,110 µg/kg ;
-   ochratoxine A :   0,448 µg/kg - 17,2 µg/kg ;
-   déoxynivalénol :   45,2 µg/kg - 743 µg/kg ;
-   zéaralénone :   9,57 µg/kg - 131 µg/kg ;
-   toxine T-2 :   10,3 µg/kg - 57,9 µg/kg ;
-   toxine HT-2 :   9,50 µg/kg - 81,8 µg/kg ;
-   fumonisine B1 :   31,1 µg/kg - 4 262 µg/kg ;
-   fumonisine B2 :   44,2 µg/kg - 1 299 µg/kg.
Les gammes de mesure de la méthode pour chaque combinaison mycotoxine/matrice sont indiquées dans le Tableau 7.

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

General Information

• Status: Not Published
• Public Enquiry End Date: 24-Feb-2021
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 4020 - Public enquire (PE) (Adopted Project)
• Start Date: 01-Feb-2021
• Due Date: 21-Jun-2021
• Completion Date: 22-Mar-2021
• Ref Project: prEN 17641 - 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
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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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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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.
...