Foodstuffs - Determination of zearalenone and trichothecenes including deoxynivalenol and its acetylated derivatives (3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol), nivalenol T-2 toxin and HT-2 toxin in cereals and cereal products by LC-MS/MS

This document specifies a procedure for the determination of nivalenol (NIV), deoxynivalenol (DON) and its acetyl derivatives (3-acetyl-DON and 15-acetyl-DON), HT-2 and T-2 toxins (HT-2 and T-2) and zearalenone (ZEN) in cereals and cereal products by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) after clean-up by solid phase extraction (SPE).
The method has been validated with samples of wheat, wheat flour, and wheat crackers. The wheat and the wheat flour was prepared from a mixture of wheat and fungi infected wheat kernels. The wheat crackers were baked from wheat flour and water spiked with the target mycotoxins.
Validation levels for NIV ranged from 27,7 μg/kg to 378 μg/kg.
Validation levels for DON ranged from 234 μg/kg to 2420 μg/kg.
Validation levels for 3-acetyl-DON ranged from 18,5 μg/kg to 137 μg/kg.
Validation levels for 15-acetyl-DON ranged from 11,4 μg/kg to 142 μg/kg.
Validation levels for HT-2 ranged from 6,6 μg/kg to 134 μg/kg.
Validation levels for T-2 ranged from 2,1 μg/kg to 37,6 μg/kg.
Validation levels for ZEN ranged from 31,6 μg/kg to 230 μg/kg.
Laboratory experiences have shown that this method is also applicable to barley and oat flour, and rye based crackers [5], however, this has not been validated in a collaborative study.

Lebensmittel - Bestimmung von Zearalenon und Trichothecenen einschließlich Deoxynivalenol und den acetylierten Derivaten (3-Acetyl-Deoxynivalenol und 15-Acetyl-Deoxynivalenol, Nivalenol sowie T-2- und HT-2-Toxin in Getreide und Getreideerzeugnissen mit LC-MS/MS

Dieses Dokument legt ein Verfahren für die Bestimmung von Nivalenol (NIV), Deoxynivalenol (DON) und den Acetylderivaten (3 Acetyl DON und 15 Acetyl DON), HT 2- und T 2 Toxin (HT 2 und T 2) und Zearalenon (ZEN) in Getreide und Getreideerzeugnissen mit Hochleistungsflüssigchromatographie (HPLC) und Tandem Massenspektrometrie (MS/MS) nach Reinigung durch Festphasenextraktion (SPE) fest.
Das Verfahren wurde an Weizen, Weizenmehl und Weizencracker validiert. Der Weizen und das Weizenmehl wurden aus einer Mischung von Weizen und mit Pilzen befallenen Weizenkörnern hergestellt. Die Weizen¬cracker wurden aus Weizenmehl und Wasser, das mit den entsprechenden Mykotoxinen angereichert war, gebacken.
NIV ist von 27,7 μg/kg bis 378 μg/kg validiert worden.
DON ist von 234 μg/kg bis 2 420 μg/kg validiert worden.
3 Acetyl DON ist von 18,5 μg/kg bis 137 μg/kg validiert worden.
15 Acetyl DON ist von 11,4 μg/kg bis 142 μg/kg validiert worden.
HT 2 Toxin ist von 6,6 μg/kg bis 134 μg/kg validiert worden.
T 2 Toxin ist von 2,1 μg/kg bis 37,6 μg/kg validiert worden.
ZEN ist von 31,6 μg/kg bis 230 μg/kg validiert worden.
Laborerfahrungen haben gezeigt, dass dieses Verfahren auch auf Gerste und Hafermehl und auf Roggen basierende Cracker anwendbar ist [5], dies wurde jedoch nicht im Ringversuch validiert.

Produits alimentaires - Dosage de la zéaralénone et des trichothécènes y compris du déoxynivalénol (DON) et ses dérivés acétylés (3-acétyl-DON et 15-acétyl-DON), du nivalénol (NIV) et des toxines T-2 et HT-2 dans les céréales et les produits céréaliers par CL-SM/SM

Le présent document décrit un mode opératoire de dosage du nivalénol (NIV), du déoxynivalénol (DON) et de ses dérivés acétylés (3-acétyl-DON et 15-acétyl-DON), des toxines HT-2 et T 2 (HT-2 et T-2) ainsi que de la zéaralénone (ZEN) dans les céréales et les produits céréaliers par chromatographie liquide haute performance (CLHP) couplée à une spectrométrie de masse en tandem (SM/SM) après purification par extraction en phase solide (SPE).
La méthode a été validée avec des échantillons de blé, de farine de blé et de biscuits salés au blé. Le blé et la farine de blé ont été préparés à partir d’un mélange de blé et de grains de blé infectés par des champignons. Les biscuits salés au blé ont été confectionnés à partir de farine de blé et d’eau supplémentée avec les mycotoxines d’intérêt.
Les niveaux de validation pour le NIV se situaient entre 27,7 μg/kg et 378 μg/kg.
Les niveaux de validation pour le DON se situaient entre 234 μg/kg et 2 420 μg/kg.
Les niveaux de validation pour le 3-acétyl-DON se situaient entre 18,5 μg/kg et 137 μg/kg.
Les niveaux de validation pour le 15-acétyl-DON se situaient entre 11,4 μg/kg et 142 μg/kg.
Les niveaux de validation pour la HT-2 se situaient entre 6,6 μg/kg et 134 μg/kg.
Les niveaux de validation pour la T-2 se situaient entre 2,1 μg/kg et 37,6 μg/kg.
Les niveaux de validation pour la ZEN se situaient entre 31,6 μg/kg et 230 μg/kg.
Les expériences en laboratoire ont montré que cette méthode est également applicable à la farine d’orge et d’avoine, ainsi qu’aux biscuits salés à base de seigle [5]. Toutefois, elle n’a pas été validée lors d’une étude comparative.

Živila - Določevanje zearalenona in trihotecena, vključno z deoksinivalenolom in njegovimi acetiliranimi derivati (3-acetil-deoksinivalenol in 15-acetil-deoksinivalenol), nivalenolom toksinov T-2 in HT-2, v žitu in žitnih proizvodih z LC-MS/MS

Ta dokument opisuje postopek določanja nivalenola (NIV), deoksinivalenola (DON) in njegovih acetilnih derivatov (3-acetil-DON in 15-acetil-DON), toksinov HT-2 in T-2 (HT-2, T-2) ter zearalenona (ZEA) v žitu in žitnih proizvodih z visokozmogljivo tekočinsko kromatografijo (HPLC) skupaj s tandemsko masno spektrometrijo (MS/MS) po čiščenju z ekstrakcijo na trdni fazi (SPE).
Metoda je bila potrjena s kontaminiranimi in primešanimi vzorci pšenice, pšenične moke in pšeničnih krekerjev.
Ravni validacije za NIV so se gibale od 27,7 μg/kg do 377,8 μg/kg.
Ravni validacije za DON so se gibale od 233,9 μg/kg do 2420,0 μg/kg.
Ravni validacije za 3-acetil-DON so se gibale od 18,5 μg/kg do 136,5 μg/kg.
Ravni validacije za 15-acetil-DON so se gibale od 11,4 μg/kg do 141,8 μg/kg.
Ravni validacije za HT-2 so se gibale od 6,6 μg/kg do 133,8 μg/kg.
Ravni validacije za T-2 so se gibale od 2,1 μg/kg do 37,6 μg/kg.
Ravni validacije za ZEA so se gibale od 31,6 μg/kg do 229,7 μg/kg.
Izkušnje v laboratoriju so pokazale, da je ta metoda uporabna tudi za ječmenovo in ovseno moko ter ržene krekerje [5], vendar to ni bilo potrjeno v medlaboratorijski raziskavi.

General Information

Status
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Publication Date
30-Jan-2020
Withdrawal Date
29-Apr-2020
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
09-Oct-2019
Due Date
02-Nov-2019
Completion Date
09-Oct-2019

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SLOVENSKI STANDARD
SIST EN 17280:2019
01-december-2019
Živila - Določevanje zearalenona in trihotecena, vključno z deoksinivalenolom in
njegovimi acetiliranimi derivati (3-acetil-deoksinivalenol in 15-acetil-

deoksinivalenol), nivalenolom toksinov T-2 in HT-2, v žitu in žitnih proizvodih z LC-

MS/MS

Foodstuffs - Determination of zearalenone and trichothecenes including deoxynivalenol

and its acetylated derivatives (3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol),

nivalenol T-2 toxin and HT-2 toxin in cereals and cereal products by LC-MS/MS
Lebensmittel - Bestimmung von Zearalenon und Trichothecenen einschließlich

Deoxynivalenol und den acetylierten Derivaten (3-Acetyl-Deoxynivalenol und 15-Acetyl-

Deoxynivalenol, Nivalenol sowie T-2- und HT-2-Toxin in Getreide und
Getreideerzeugnissen mit LC-MS/MS

Produits alimentaires - Dosage de la zéaralénone et des trichothécènes y compris du

déoxynivalénol (DON) et ses dérivés acétylés (3-acétyl-DON et 15-acétyl-DON), du

nivalénol (NIV) et des toxines T-2 et HT-2 dans les céréales et les produits céréaliers par

CL-SM/SM
Ta slovenski standard je istoveten z: EN 17280:2019
ICS:
67.060 Žita, stročnice in proizvodi iz Cereals, pulses and derived
njih products
SIST EN 17280:2019 en,fr,de

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

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SIST EN 17280:2019
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SIST EN 17280:2019
EN 17280
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2019
EUROPÄISCHE NORM
ICS 67.060
English Version
Foodstuffs - Determination of zearalenone and
trichothecenes including deoxynivalenol and its acetylated
derivatives (3-acetyl-deoxynivalenol and 15-acetyl-
deoxynivalenol), nivalenol T-2 toxin and HT-2 toxin in
cereals and cereal products by LC-MS/MS

Produits alimentaires - Dosage de la zéaralénone et des Lebensmittel - Bestimmung von Zearalenon und

trichothécènes y compris du déoxynivalénol (DON) et Trichothecenen einschließlich Deoxynivalenol und den

ses dérivés acétylés (3-acétyl-DON et 15-acétyl-DON), acetylierten Derivaten (3-Acetyl-Deoxynivalenol und

du nivalénol (NIV) et des toxines T-2 et HT-2 dans les 15-Acetyl-Deoxynivalenol, Nivalenol sowie T-2- und

céréales et les produits céréaliers par CL-SM/SM HT-2-Toxin in Getreide und Getreideerzeugnissen mit

LC-MS/MS
This European Standard was approved by CEN on 5 August 2019.

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

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

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

member.

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

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

Centre has the same status as the official versions.

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

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

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

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

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17280:2019 E

worldwide for CEN national Members.
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SIST EN 17280:2019
EN 17280:2019 (E)
Contents Page

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

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

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

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

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

4 Principle ............................................................................................................................................................. 5

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

6 Apparatus and equipment ........................................................................................................................... 8

7 Procedure........................................................................................................................................................... 9

8 Calculation ...................................................................................................................................................... 11

9 Precision .......................................................................................................................................................... 11

10 Test report ...................................................................................................................................................... 15

Annex A (informative) Typical chromatograms ............................................................................................ 16

Annex B (informative) Example conditions for suitable LC-MS/MS systems ...................................... 18

Annex C (informative) Additional examples for separation between 3-AcDON and 15-

AcDON ............................................................................................................................................................... 24

Annex D (informative) Precision data ............................................................................................................... 27

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

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SIST EN 17280:2019
EN 17280:2019 (E)
European foreword

This document (EN 17280:2019) has been prepared by Technical Committee CEN/TC 275 “Food

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

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

identical text or by endorsement, at the latest by April 2020, and conflicting national standards shall be

withdrawn at the latest by April 2020.

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

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

This document has been prepared under a mandate given to CEN by the European Commission and the

European Free Trade Association.

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

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

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

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

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
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SIST EN 17280:2019
EN 17280:2019 (E)
Introduction

The mycotoxins nivalenol, deoxynivalenol, and its acetyl derivatives (3-acetyl deoxynivalenol, 15-acetyl

deoxynivalenol), T-2 toxin and its metabolite HT-2 toxin, and zearalenone are produced by various

Fusarium species. Cereals like wheat, maize, barley, oats, rye and relevant derived products are most

likely to be affected.

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

working steps with harmful chemicals. The hazardous substances ordinance, Regulation (EC)

No 1907/2006 [3], 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 — Fusarium toxins (zearalenone, deoxynivalenol, T-2 and HT-2 toxins) have been

implicated as the causative agents in a variety of animal diseases, such as pulmonary oedema,

infertility, diarrhoea, vomiting, anorexia, leukopenia, immunosuppression, skin and

gastrointestinal irritation, hemorraging, etc., and have been associated to some human diseases.

The IARC has defined zearalenone, deoxynivalenol and T-2 as not classifiable as to their

carcinogenicity to humans (Group 3) [4].
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SIST EN 17280:2019
EN 17280:2019 (E)
1 Scope

This document specifies a procedure for the determination of nivalenol (NIV), deoxynivalenol (DON)

and its acetyl derivatives (3-acetyl-DON and 15-acetyl-DON), HT-2 and T-2 toxins (HT-2 and T-2) and

zearalenone (ZEN) in cereals and cereal products by high performance liquid chromatography (HPLC)

coupled with tandem mass spectrometry (MS/MS) after clean-up by solid phase extraction (SPE).

The method has been validated with samples of wheat, wheat flour, and wheat crackers. The wheat and

the wheat flour were prepared from a mixture of wheat and fungi infected wheat kernels. The wheat

crackers were baked from wheat flour and water spiked with the target mycotoxins.

Validation levels for NIV ranged from 27,7 μg/kg to 378 μg/kg.
Validation levels for DON ranged from 234 μg/kg to 2420 μg/kg.
Validation levels for 3-acetyl-DON ranged from 18,5 μg/kg to 137 μg/kg.
Validation levels for 15-acetyl-DON ranged from 11,4 μg/kg to 142 μg/kg.
Validation levels for HT-2 ranged from 6,6 μg/kg to 134 μg/kg.
Validation levels for T-2 ranged from 2,1 μg/kg to 37,6 μg/kg.
Validation levels for ZEN ranged from 31,6 μg/kg to 230 μg/kg.

Laboratory experiences have shown that this method is also applicable to barley and oat flour, and rye

based crackers [5], however, this has not been validated in a collaborative study.

2 Normative references

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

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

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

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

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

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

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

Trichothecenes and zearalenone are extracted from the homogenized sample material with a mixture of

acetonitrile and water. The extract is filtered and evaporated to dryness. The residue is dissolved with a

mixture of methanol and water and applied to a polymeric solid phase extraction column. The

mycotoxins are purified and concentrated on the column then released using methanol as eluent.

Isotopically labelled mycotoxins are added to the column eluate before evaporating it to dryness. After

reconstitution of the dry extract with the injection solvent, the mycotoxins are detected by reversed

phase HPLC-MS/MS.
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SIST EN 17280:2019
EN 17280:2019 (E)
5 Reagents

Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696,

unless otherwise specified.
5.1 Nitrogen or oil-free compressed air.
5.2 Water, deionized.
5.3 Water, HPLC quality.
5.4 Acetonitrile, HPLC quality.
5.5 Methanol, HPLC quality.
5.6 Ammonium acetate, for mass spectrometry, c(CH COONH ) ≥ 99,0 %.
3 4
5.7 Extraction mixture.

Mix 84 parts per volume of acetonitrile (5.4) and 16 parts per volume of water (5.2).

5.8 Solid phase extraction (SPE) columns, containing 60 mg of a balanced hydrophilic/lipophilic

® 1

polymer able to retain both polar and non-polar compounds (Waters Oasis HLB is suitable).

5.9 Nivalenol (NIV) e.g. crystalline, as a film or as certified standard solution.

5.10 Deoxynivalenol (DON) e.g. crystalline, as a film or as certified standard solution.

5.11 3-Acetyl-DON (3-AcDON) e.g. crystalline, as a film or as certified standard solution.

5.12 15-Acetyl-DON (15-AcDON) e.g. crystalline, as a film or as certified standard solution.

5.13 HT-2 toxin (HT-2) e.g. crystalline, as a film or as certified standard solution.

5.14 T-2 toxin (T-2) e.g. crystalline, as a film or as certified standard solution.

5.15 Zearalenone (ZEN) e.g. crystalline, as a film or as certified standard solution.

13 13

5.16 Nivalenol isotopically labelled internal standard ( C-NIV) e.g. nivalenol C -labelled (fully)

ρ = 25 μg/ml, in acetonitrile.

5.17 Deoxynivalenol isotopically labelled internal standard ( C-DON) e.g. deoxynivalenol

C15-labelled (fully) ρ = 25 μg/ml, in acetonitrile.

5.18 3-Acetyl-DON isotopically labelled internal standard ( C-3-AcDON) e.g. 3-acetyl-DON

C -labelled (fully) ρ = 25 μg/ml, in acetonitrile.
13 13

5.19 HT-2 toxin isotopically labelled internal standard ( C-HT-2) e.g. HT-2 toxin C -labelled

(fully) ρ = 25 μg/ml, in acetonitrile.

Oasis HLB column is an example available commercially from Waters. This information is given for the

convenience of users of this European Standard and does not constitute an endorsement by CEN of the products

named. Equivalent products may be used if they can be shown to lead to the same results.

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SIST EN 17280:2019
EN 17280:2019 (E)
13 13

5.20 T-2 toxin isotopically labelled internal standard ( C-T-2) e.g. T-2 toxin C -labelled (fully)

ρ = 25 μg/ml, in acetonitrile.
13 13

5.21 Zearalenone isotopically labelled internal standard ( C-ZEN) e.g. zearalenone C -labelled

(fully) ρ = 25 μg/ml, in acetonitrile.
5.22 Mixed stock solution.

Prepare a mixed mycotoxin stock solution in acetonitrile (5.4), containing e.g.: NIV (5.9) 12,5 µg/ml;

DON (5.10) 62,5 µg/ml; 3-AcDON (5.11) 7,5 µg/ml; 15-AcDON (5.12) 7,5 µg/ml; HT-2 (5.13) 2,5 µg/ml;

T-2 (5.14) 2,5 µg/ml; ZEN (5.15) 5,0 µg/ml. This solution can be used for spiking purposes (7.5).

5.23 Mixed standard solution.

Prepare a mixed standard solution in acetonitrile (5.4) containing e.g.: NIV (5.9), 1,25 µg/ml; DON

(5.10), 6,25 µg/ml; 3-AcDON (5.11), 0,75 µg/ml; 15-AcDON (5.12), 0,75 µg/ml; HT-2 (5.13), 0,25 µg/ml;

T-2 (5.14), 0,25 µg/ml; ZEN (5.15), 0,5 µg/ml. This solution is used for calibration purposes (5.25).

5.24 Mixed internal standard (ISTD) solution.

Isotopically labelled mycotoxins are generally available as certified standard solutions in acetonitrile.

Prepare a mixed ISTD solution by mixing the commercial individual ISTD solutions to obtain a mixture

13 13 13

containing e.g. C-NIV (5.16), 1,25 µg/ml; C-DON (5.17), 6,25 µg/ml; C-3-AcDON (5.18), 0,75 µg/ml;

13 13 13

C-HT-2 (5.19), 0,25 µg/ml; C-T-2 (5.20), 0,25 µg/ml; C-ZEN (5.21), 0,5 µg/ml in acetonitrile (5.4).

5.25 Calibration solutions.

Add different volumes of the mixed standard solution (5.23) and the mixed ISTD solution (5.24) to six

autosampler vials (6.11) e.g. as listed in Table 1 to obtain six calibration levels across the calibration

range. Evaporate to dryness in an evaporator (6.13) under a stream of air or nitrogen (5.1) at

approximately 40 °C.

Re-dissolve the dried residue by adding 400 µl (V ) of HPLC injection solvent (5.26) and mix thoroughly

for at least 10 s.
Table 1 — Example of suitable calibration solutions
Mass concentration of calibration solutions
Calibration Mixed Mixed NIV DON 3- 15- HT-2 T-2 ZEN
solution standard ISTD AcDON AcDON
solution solution
µl µl µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml
1 25 100 0,078 0,391 0,047 0,047 0,016 0,016 0,031
2 50 100 0,156 0,781 0,094 0,094 0,031 0,031 0,063
3 100 100 0,313 1,563 0,188 0,188 0,063 0,063 0,125
4 200 100 0,625 3,125 0,375 0,375 0,125 0,125 0,250
5 400 100 1,250 6,250 0,750 0,750 0,250 0,250 0,500
6 600 100 1,875 9,375 1,125 1,125 0,375 0,375 0,750

Mass concentration of isotopically labelled analytes (µg/ml) in all calibration solutions

0,313 1,563 0,188 0,188 0,063 0,063 0,125
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SIST EN 17280:2019
EN 17280:2019 (E)
5.26 HPLC injection solvent.

The composition of HPLC injection solvent depends on the applied LC conditions. Examples of eluents

suitable for LC-MS/MS systems are given in Annex B.
6 Apparatus and equipment
Usual laboratory glassware and equipment, in particular, the following.
6.1 Analytical balance, accuracy of 0,01 mg.
6.2 Laboratory balance, accuracy of 0,01 g.

6.3 Adjustable mechanical vertical or horizontal shaker, with suitable 100 ml flasks.

6.4 Paper filter, pore size 20 μm to 25 μm.
6.5 Conical flasks, with screw top or glass stopper.
6.6 Vacuum manifold to accommodate solid phase extraction columns.
6.7 Single marked pipettes, 5 ml capacity.
6.8 Microlitre syringe(s) or microlitre pipette(s), 10 µl to 1000 µl.
6.9 Tubes of 10 ml with caps.
6.10 4 ml vials with caps.
6.11 Autosampler vials with caps.
6.12 Disposable filter unit, with pore size of 0,2 µm, regenerated cellulose.
6.13 Concentration evaporator workstation.
6.14 LC-MS/MS system, with the following components:

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

column in use with sufficient accuracy.

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

sufficient accuracy.

6.14.3 LC column, capable of retaining the target mycotoxins, preferably with a retention factor of at

least two. The two isomers 15-AcDON and 3-AcDON cannot be distinguished individually by specific

Multiple Reaction Monitoring (MRM) transitions. Therefore the LC column and gradient shall be able to

separate both compounds. Examples of suitable LC columns and gradients are reported in Annex B.

6.14.4 Column oven, capable of maintaining a constant temperature.

6.14.5 Tandem mass spectrometer (MS/MS), capable of ionization of the mycotoxins (either

resulting in positive or negative ions), performing Selected Reaction Monitoring (SRM) in case of

MS/MS analysers or Parallel Reaction Monitoring (PRM) in case of MS/high resolution mass

spectrometry (HRMS) analysers.
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SIST EN 17280:2019
EN 17280:2019 (E)
Any ionization source providing sufficient yield may be employed.
6.14.6 Data evaluation system.
7 Procedure
7.1 Preparation of the test sample
Finely grind the laboratory sample and homogenize it.
7.2 Extraction

Weigh 10,0 g to the nearest 0,1 g into a 100 ml conical flask (6.5). Add 50 ml (V ) of extraction mixture

(5.7) and shake vigorously for 60 min with a shaker (6.3).
Filter through a paper filter (6.4).

Pipette a 5 ml aliquot (V ) of the filtered extract into a 10 ml tube (6.9) and evaporate to dryness under

a stream of air or nitrogen (5.1) at approximately 40 °C.

Re-dissolve the residue by adding first 100 μl of methanol (5.5) and shake for approximately 1 min.

Then add 900 μl of water (5.3) and shake again for approximately 1 min.
7.3 Solid phase extraction clean up
Connect the SPE column (5.8) to the vacuum manifold (6.6).

Activate and condition the SPE column (5.8) by passing through 2 ml of methanol (5.5), then 2 ml of

water (5.3).

Pass the whole volume (1 ml) of reconstituted extract at a flow rate of about one drop per second

through the column and discard the eluate. Dry the column by applying a gentle air flow.

Wash the column with 1 ml of water (5.3) and discard the eluate. Dry the column.

Elute the mycotoxins with 1 ml of methanol (5.5). Collect the eluate in a 4 ml vial (6.10). Pass air

through the column to completely recover the eluate.
7.4 Preparation of the sample test solution
Add 100 µl of the mixed ISTD solution (5.24) to the SPE eluate.

Evaporate the SPE eluate to dryness in an evaporator (6.13) under a stream of air or nitrogen (5.1) at

approximately 40 °C.

Re-dissolve the dried residue by adding 400 µl (V ) of HPLC injection solvent (5.26) and mix thoroughly

for at least 10 s.

Filter the re-dissolved residue through a syringe filter or centrifuge filter (6.12).

Transfer the sample test solution into autosampler vials (6.11).
7.5 Spiking procedure

For the determination of the recovery, carry out a spiking procedure using the mixed stock solution

(5.22). The spiking level shall be within the calibration range and preferably shall correspond to the

middle concentration of the calibration curve. Take care that no more than 1 ml of the spiking solution

is added, and distribute the solution evenly over the sample materials. Evaporate the spiked solution at

room temperature.
Alternatively, a certified reference material can be applied.
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SIST EN 17280:2019
EN 17280:2019 (E)
7.6 LC-MS/MS analysis
7.6.1 General

Optimize analytical parameters (selection of the ionization mode, selection of the masses of precursor

and product ions, optimization of cone voltages and collision energies) by infusion and injection of

standard solutions of the analytes.

A combination of analytical column, mobile phase composition, gradient settings and injection volume

shall be such that it allows obtaining acceptable separation and reliable results at the required levels,

with sufficient selectivity.

Annex A illustrates some example chromatograms, and Annex B gives some suitable parameters.

Chromatographic separation of 3-AcDON and 15-AcDON is necessary to provide individual data on the

two isomers. Some additional examples of LC columns and relevant operating conditions allowing the

separation between 3-AcDON and 15-AcDON are given in Annex C.
7.6.2 Injection sequence

Always start a batch of measurements by injecting a LC injection solvent (5.26) to prove non-

contamination of the system.

Then inject the calibration range from the least concentrated to the most concentrated.

Then inject a solvent blank to check for possible carry over.
Subsequently inject the sample test solutions.

At the end of the batch, inject the low-end point, to ensure that the end-of-series sensitivity is the same

as at the beginning, and to ensure that there has been no change in retention times.

7.7 Identification

Identify each mycotoxin by comparing retention times of calibration solution with that of the sample

test solution. Identify the analyte on the basis of at least two mass transitions. The retention time and

the ion ratio of the two peaks shall match that of the standard substance. Confirm the identity of the

sample peaks using the retention time ± 0,2 min of the mean observed for the calibration standards [6].

Ion ratios, defined as the response of the peak with the lower area/response of the peak with the higher

area, shall match (± 30 % of average) the calibration standards from the same sequence [6].

7.8 Calibration

For each injection calculate the ratio of the peak area of each analyte to the peak area of the respective

labelled analogue. These peak area ratios are used in all subsequent calculations.

Divide the peak area of 15-AcDON by the peak area of C-3-AcDON.

Prepare a calibration curve for each of the seven analytes (NIV, DON, 3-AcDON, 15-AcDON, HT-2, T-2,

and ZEN) by plotting the peak area ratios of each analyte calculated in the calibration solutions (Y-axis)

against the corresponding amount (μg) of analyte injected on column (X-axis). Estimate slope and

intercept of each of the seven calibration curves by using linear regression.
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SIST EN 17280:2019
EN 17280:2019 (E)
8 Calculation

Calculate the mass fraction of each mycotoxin, w, in microgram per kilogram, of the sample according to

Formula (1):
Rb 1000
w= −× × (1)
aa V m
2 SPE
where

R is the peak area ratio of the relevant analyte and the corresponding internal standard in the

sample test solution;
a -1
is the slope of the calibration curve from calibration data (7.8), in µg ;
b is the intercept of the calibration curve from calibration data (7.8);
V is the volume of the reconstituted extract after clean-up, here: 0,4 ml;
V is the injection volume of the reconstituted sample extract, in millilitre;
1000 is a conversion factor;
m is the sample equivalent weight purified on SPE column, here: 1 g.
SPE
Calculate the sample equivalent weight (m ) according to Formula (2):
SPE
mV×
m = (2)
SPE
where
m is the mass of the extracted test portion, here: 10 g;
V is the volume of the extraction mixture, here: 50 ml;
V is the volume of filtered extract dried before clean-up, here: 5 ml.
9 Precision
9.1 General

Details of the interlaboratory test of the precision of the method are summarized in Annex D. The values

derived from the interlaboratory test may not be applicable to analyte concentration ranges and

matrices other than given in Annex D.
9.2 Repeatability

The absolute difference between two single test results found on identical test material by one operator

using the same apparatus within the shortest feasible time interval will exceed the repeatability limit r

in Tables 2 to Table 8 in not more than 5 % of the cases.
9.3 Reproducibility

The absolute difference between two single test results found on identical test material reported by two

laboratories will exceed the reproducibility limit R in Tables 2 to Table 8 in not more than 5 % of the

cases.
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SIST EN 17280:2019
EN 17280:2019 (E)
Table 2 — Validation data for nivalenol
Sample x̅ r R
µg/kg µg/kg µg/kg
wheat 194,1 31,76 91,81
wheat 87,12 81,12 85,02
wheat 186,9 57,12 21,06
wheat 377,8 151,2 268,6
wheat flour 116,0 19,79 59,80
wheat flour 52,89 50,46 49,26
wheat flour 107,7 12,63 28,41
wheat flour 214,8 21,26 58,50
wheat crackers 70,71 16,39 40,33
wheat crackers 27,69 11,62 24,65
wheat crackers 64,87 11,27 53,26
wheat crackers 123,8 20,14 75,89
Table 3 — Validation data for deoxynivalenol
Sample x̅ r R
µg/kg µg/kg µg/kg
wheat 1212 124,8 322,5
wheat 635,4 53,91 263,0
wheat 1201 355,7 366,4
wheat 2420 146,1 435,3
wheat flour 692,5 97,12 188,3
wheat flour 351,0 65,68 313,9
wheat flour 613,0 64,50 244,7
wheat flour 1234 114,2 354,4
wheat crackers 448,7 34,98 235,4
wheat crackers 233,9 92,80 158,2
wheat crackers 487,6 68,32 204,5
wheat crackers 1004 374,1 389,1
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SIST EN 17280:2019
EN 17280:2019 (E)
Table 4 — Validation data for 3-acetyl deoxynivalenol
Sample x̅ r R
µg/kg µg/kg µg/kg
wheat 136,5 21,74 30,56
wheat 49,10 4,074 37,27
wheat 83,68 21,99 18,40
wheat 67,68 12,14 14,64
wheat flour 67,01 16,07 75,53
wheat flour 30,22 5,800 27,09
wheat flour 58,95 16,57 52,23
wheat flour 96,22 14,32 26,37
wheat crackers 44,96 7,433 38,08
wheat crackers 18,47 8,603 13,24
wheat crackers 26,22 4,924 14,89
wheat crackers 49,96 16,21 28,30
Table 5 — Validation data for 15-acetyl deoxynivalenol
Sample x̅ r R
µg/kg µg/kg µg/kg
wheat 141,84 33,87 138,28
wheat 35,64 5,788 44,38
wheat 41,48 10,75 28,79
wheat 30,14 6,023 32,26
wheat flour 68,25 11,40 59,06
wheat flour 12,87 3,744 12,22
wheat flour 24,38 9,390 28,63
wheat flour 28,64 7,720 29,14
wheat crackers 46,24 11,14 47,57
wheat crackers 11,41 8,049 14,36
wheat crackers 16,42 4,751 15,53
wheat crackers 32,34 5,374 30,35
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SIST EN 17280:2019
EN 17280:2019 (E)
Table 6 — Validation data for HT-2 toxin
Sample x̅ r R
µg/kg µg/kg µg/kg
wheat 46,40 9,544 42,51
wheat 17,33 3,209 12,53
wheat 36,29 31,25 35,92
wheat 133,8 38,34 100,7
wheat flour 21,60 3,660 5,981
wheat flour 6,63 4,813 7,110
wheat flour 14,18 3,530 11,64
wheat flour 32,97 8,489 24,71
wheat crackers 10,73 1,427 7,924
wheat crackers 7,988 3,133 6,835
wheat crackers 19,59 8,357 11,30
wheat crackers 38,02 11,22 32,62
Table 7 — Validation data for T-2 toxin
Sample x̅ r R
µg/kg µg/kg µg/kg
wheat 45,89 15,55 35,82
wheat 27,50 8,670 24,92
wheat 47,79 44,93 55,16
wheat 18,01 5,930 17,72
wheat flour 20,85 3,691 20,58
wheat flour 11,53 2,347 10,51
wheat flour 26,85 4,770 6,392
wheat flour 37,57 7,590 38,14
wheat c
...

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