EN 17280:2019
(Main)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
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
Standards Content (Sample)
SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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.
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
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.
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].
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.
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 r
...
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