SIST 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 describes 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, T-2) and zearalenone (ZEA) in cereals and cereal products by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) after cleanup by solid phase extraction (SPE).
The method has been validated with both contaminated and spiked samples of wheat, wheat flour, and wheat crackers.
Validation levels for NIV ranged from 27,7μg/kg to 377,8 μg/kg.
Validation levels for DON ranged from 233,9μg/kg to 2420,0 μg/kg.
Validation levels for 3-acetyl-DON ranged from 18,5μg/kg to 136,5 μg/kg.
Validation levels for 15-acetyl-DON ranged from 11,4μg/kg to 141,8 μg/kg.
Validation levels for HT-2 ranged from 6,6 μg/kg to 133,8 μg/kg.
Validation levels for T-2 ranged from 2,1 μg/kg to 37,6 μg/kg.
Validation levels for ZEA ranged from 31,6μg/kg to 229,7 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 beschreibt 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, T 2) und Zearalenon (ZEA) in Getreide und Getreideerzeugnissen mit Hochleistungsflüssigchromatographie (HPLC) und Tandem-Massenspektrometrie (MS/MS) nach Reinigung durch Festphasenextraktion (SPE).
Das Verfahren wurde an kontaminierten und aufgestockten Proben (Weizen, Weizenmehl und Weizen-cracker) validiert.
NIV ist von 27,7 μg/kg bis 377,8 μg/kg validiert worden.
DON ist von 233,9 μg/kg bis 2 420,0 μg/kg validiert worden.
3 Acetyl-DON ist von 18,5 μg/kg bis 136,5 μg/kg validiert worden.
15 Acetyl-DON ist von 11,4 μg/kg bis 141,8 μg/kg validiert worden.
HT 2-Toxin ist von 6,6 μg/kg bis 133,8 μg/kg validiert worden.
T 2-Toxin ist von 2,1 μg/kg bis 37,6 μg/kg validiert worden.
ZEA ist von 31,6 μg/kg bis 229,7 μg/kg validiert worden.
Laborerfahrungen haben gezeigt, dass dieses Verfahren auch auf Gerste und Hafermehl und Reiscracker 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
La présente Norme européenne 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, T-2) ainsi que de la zéaralénone (ZEA) 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 contaminés et des échantillons dopés de blé, de farine de blé et de biscuits salés au blé.
Les niveaux de validation pour le NIV se situaient entre 27,7 µg/kg et 377,8 µg/kg.
Les niveaux de validation pour le DON se situaient entre 233,9 µg/kg et 2 420,0 µg/kg.
Les niveaux de validation pour le 3-acétyl-DON se situaient entre 18,5 µg/kg et 136,5 µg/kg.
Les niveaux de validation pour le 15-acétyl-DON se situaient entre 11,4 µg/kg et 141,8 µg/kg.
Les niveaux de validation pour la HT-2 se situaient entre 6,6 µg/kg et 133,8 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 ZEA se situaient entre 31,6 µg/kg et 229,7 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
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
2
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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.
3
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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].
4
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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.
5
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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)
15
ρ = 25 μg/ml, in acetonitrile.
13
5.17 Deoxynivalenol isotopically labelled internal standard ( C-DON) e.g. deoxynivalenol
13
C15-labelled (fully) ρ = 25 μg/ml, in acetonitrile.
13
5.18 3-Acetyl-DON isotopically labelled internal standard ( C-3-AcDON) e.g. 3-acetyl-DON
13
C -labelled (fully) ρ = 25 μg/ml, in acetonitrile.
17
13 13
5.19 HT-2 toxin isotopically labelled internal standard ( C-HT-2) e.g. HT-2 toxin C -labelled
22
(fully) ρ = 25 μg/ml, in acetonitrile.
1
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.
6
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13 13
5.20 T-2 toxin isotopically labelled internal standard ( C-T-2) e.g. T-2 toxin C -labelled (fully)
24
ρ = 25 μg/ml, in acetonitrile.
13 13
5.21 Zearalenone isotopically labelled internal standard ( C-ZEN) e.g. zearalenone C -labelled
18
(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
1
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
7
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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.
8
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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
3
(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
4
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
1
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.
9
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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.
13
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.
10
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8 Calculation
Calculate the mass fraction of each mycotoxin, w, in microgram per kilogram, of the sample according to
Formula (1):
V
Rb 1000
1
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;
1
V is the injection volume of the reconstituted sample extract, in millilitre;
2
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×
4
m = (2)
SPE
V
3
where
m is the mass of the extracted test portion, here: 10 g;
V is the volume of the extraction mixture, here: 50 ml;
3
V is the volume of filtered extract dried before clean-up, here: 5 ml.
4
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.
11
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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
12
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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
13
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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
...
SLOVENSKI STANDARD
oSIST prEN 17280:2018
01-oktober-2018
äLYLOD'RORþHYDQMH]HDUDOHQRQDLQWULKRWHFHQDYNOMXþQR]GHRNVLQLYDOHQRORP
'21 LQQMHJRYLPLDFHWLOLUDQLPLGHULYDWL DFHWLO'21LQDFHWLO'21
QLYDOHQRORP 1,9 LQWRNVLQRPD7LQ+7YåLWXLQåLWQLKSURL]YRGLK]/&0606
Foodstuffs - Determination of zearalenone and trichothecenes including deoxynivalenol
(DON) and its acetylated derivatives (3-acetyl- DON and 15-acetyl-DON), nivalenol (NIV)
and T-2 and HT-2 toxin in cereals and cereal products by LC-MS/MS
Lebensmittel - Bestimmung von Zearalenon und Trichothecenen einschließlich
Deoxynivalenol (DON) und den acetylierten Derivaten (3-Acetyl-DON und 15-Acetyl-
DON), Nivalenol (NIV), 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: prEN 17280:2018
ICS:
67.060 äLWDVWURþQLFHLQSURL]YRGLL] Cereals, pulses and derived
QMLK products
oSIST prEN 17280:2018 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 17280:2018
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oSIST prEN 17280:2018
DRAFT
EUROPEAN STANDARD
prEN 17280
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2018
ICS 67.060
English Version
Foodstuffs - Determination of zearalenone and
trichothecenes including deoxynivalenol (DON) and its
acetylated derivatives (3-acetyl- DON and 15-acetyl-DON),
nivalenol (NIV) and T-2 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 (DON)
ses dérivés acétylés (3-acétyl-DON et 15-acétyl-DON), und den acetylierten Derivaten (3-Acetyl-DON und 15-
du nivalénol (NIV) et des toxines T-2 et HT-2 dans les Acetyl-DON), Nivalenol (NIV), T-2- und HT-2-Toxin in
céréales et les produits céréaliers par CL-SM/SM Getreide und Getreideerzeugnissen mit LC-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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, 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
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17280:2018:2018 E
worldwide for CEN national Members.
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prEN 17280:2018 (E)
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 6
5 Reagents . 7
6 Apparatus and equipment . 9
7 Procedure. 10
7.1 Preparation of the test sample . 10
7.2 Extraction of mycotoxin from the sample . 10
7.3 Solid phase extraction clean up . 10
7.4 Preparation of the sample test solution . 10
7.5 Spiking procedure . 10
7.6 LC-MS/MS analysis . 11
7.6.1 General . 11
7.6.2 Batch composition and analytical sequence . 11
7.7 Identification . 11
7.8 Calibration . 11
8 Calculation . 12
9 Precision . 12
9.1 General . 12
9.2 Repeatability . 12
9.3 Reproducibility . 12
10 Test report . 16
Annex A (informative) Typical chromatograms . 17
A.1 Example chromatogram (API SCIEX 5000™) . 17
A.2 Example chromatogram (Thermo Q-Exactive™). 18
Annex B (informative) Example conditions for suitable LC-MS/MS systems . 19
B.1 System Settings for API SCIEX 5000™ . 19
B.1.1 Settings for chromatography . 19
B.1.2 Detector parameters . 19
B.2 System settings for Thermo Q-Exactive™ . 21
B.2.1 Settings for chromatography . 21
B.2.2 Detector Parameters . 22
2
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Annex C (informative) Additional examples for separation between 3-AcDON and 15-
AcDON . 24
TM
C.1 Settings for Accucore C18 (Thermo Scientific) . 24
®
C.2 Settings for Kinetex C18 (Phenomenex) . 24
®
C.3 Settings for Kinetex PFP (Phenomenex) . 25
Annex D (informative) Precision data . 26
Table D.8 —Precision data for 3-acetyl deoxynivalenol in wheat flour . 31
Bibliography . 41
3
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European foreword
This document (prEN 17280:2018) 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.
This document has been prepared under a standardization request given to CEN by the European
Commission and the European Free Trade Association.
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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 maize, wheat, 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].
5
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1 Scope
This document describes 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, T-2) and
zearalenone (ZEA) in cereals and cereal products by high performance liquid chromatography (HPLC)
coupled with tandem mass spectrometry (MS/MS) after cleanup by solid phase extraction (SPE).
The method has been validated with both contaminated and spiked samples of wheat, wheat flour, and
wheat crackers.
Validation levels for NIV ranged from 27,7μg/kg to 377,8 μg/kg.
Validation levels for DON ranged from 233,9μg/kg to 2420,0 μg/kg.
Validation levels for 3-acetyl-DON ranged from 18,5μg/kg to 136,5 μg/kg.
Validation levels for 15-acetyl-DON ranged from 11,4μg/kg to 141,8 μg/kg.
Validation levels for HT-2 ranged from 6,6 μg/kg to 133,8 μg/kg.
Validation levels for T-2 ranged from 2,1 μg/kg to 37,6 μg/kg.
Validation levels for ZEA ranged from 31,6μg/kg to 229,7 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 http://www.iso.org/obp
4 Principle
Trichothecenes and zearalenone are extracted from the commodities with a mixture of acetonitrile-
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 quantified by reversed phase high
performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS).
6
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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
≥
®
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 (ZEA) 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)
15
ρ = 25 μg/ml, in acetonitrile.
13 13
5.17 Deoxynivalenol isotopically labelled internal standard ( C DON) e.g. deoxynivalenol C-
15
labelled (fully) ρ = 25 μg/ml, in acetonitrile.
13 13
5.18 3-Acetyl-DON isotopically labelled internal standard ( C 3-AcDON) e.g. 3-acetyl-DON C-
17
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
22
(fully) ρ = 25 μg/ml, in acetonitrile.
13 13
5.20 T-2 toxin isotopically labelled internal standard ( C24T-2) e.g. T-2 toxin C-labelled (fully)
ρ = 25 μg/ml, in acetonitrile.
13 13
5.21 ZEA toxin isotopically labelled internal standard ( C ZEA) e.g. zearalenone C-labelled
18
(fully) ρ = 25 μg/ml, in acetonitrile.
7
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5.22 Mixed mycotoxin stock solutions.
Prepare a mixed mycotoxin stock solution in acetonitrile (5.4), containing: NIV (5.9), DON (5.10), 3-
AcDON(5.11), 15-AcDON (5.12), HT-2 (5.13), T-2 (5.14),and ZEA (5.15) to be used for spiking purposes
(7.5).
5.23 Mixed mycotoxin working solution.
Prepare a mixed mycotoxin working solution in acetonitrile (5.4) containing: 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; ZEA (5.15), 0,5 µg/ml. This solution is used for calibration purposes
(5.25).
5.24 Mixed internal standard (ISTD) working solution.
Isotopically labelled mycotoxins are generally available as certified standard solutions in acetonitrile.
Prepare mixed ISTD working solution by mixing the commercial individual ISTD solutions to obtain a
13 13 13
mixture containing C NIV (5.16), 1,25 µg/ml, C DON (5.17), 6,25 µg/ml, C 3-AcDON (5.18),
15 15 17
13 13 13
0,75 µg/ml, C HT-2 (5.19), 0,25, µg/ml, C T-2 (5.20), 0,25, µg/ml, and C ZEA (5.21), 0,5 µg/ml in
22 24 18
acetonitrile (5.4).
5.25 Calibration solutions.
Add different volumes of the mixed mycotoxin working solution (5.23) to six autosampler vials (6.11)
as listed in Table 1 to obtain six calibration levels across the calibration range. Proceed as described in
7.4.
Table 1 — Example of suitable calibration solutions
Mass concentration of calibration solutions (µg/ml)
Calibrati Mixed NIV DON 3-AcDON 15- HT-2 T-2 ZEA
on mycotoxin AcDON
solution working
solution
µl µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml
1 25 0,078 0,391 0,047 0,047 0,016 0,016 0,031
2 50 0,156 0,781 0,094 0,094 0,031 0,031 0,063
3 100 0,313 1,563 0,188 0,188 0,063 0,063 0,125
4 200 0,625 3,125 0,375 0,375 0,125 0,125 0,250
5 400 1,250 6,250 0,750 0,750 0,250 0,250 0,500
6 600 1,875 9,375 1,125 1,125 0,375 0,375 0,750
Mass concentration of isotopically labelled analyte (µg/ml) in all
calibration solutions
0,316 1,563 0,188 0,188 0,063 0,063 0,125
Once it has been shown that there is linearity, the number of levels may be adjusted accordingly to local needs and
requirements.
5.26 HPLC injection solvent.
The composition of HPLC injection solvent depends on the applied LC conditions (see 7.1). Examples of
eluents suitable for LC-MS/MS systems are given in Annex B.
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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,for solvent extraction 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 Calibrated microlitre syringe(s) or microlitre pipette(s), 10 µl to 1000 µl.
6.9 Glass 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, and cross-contamination below 0,1 %.
6.14.3 LC column, capable of separating the seven analytes and ensuring a baseline resolution of
mycotoxins monitored in positive from those monitored in negative ion mode. The minimum acceptable
retention time for the analytes under examination shall be at least twice the retention time
corresponding to the void volume of the column.
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 analyzers or Parallel Reaction Monitoring (PRM) in case of MS/high resolution mass
spectrometry (HRMS) analyzers.
Any ionization source providing sufficient yield may be employed.
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7 Procedure
7.1 Preparation of the test sample
Finely grind the laboratory sample and homogenize it.
7.2 Extraction of mycotoxin from the sample
Weigh 10,0 g, to the nearest 0,1 g (6.2), of the test sample into a 100 ml conical flask (6.5). Add 50 ml 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 (6.7) of the filtered extract into a 10 ml glass tube (6.9) and evaporate to dryness
under a stream of air or nitrogen (5.1) at approximately 40 °C.
Redissolve the residue by adding first 100 μl (6.8) of methanol (5.5) and shake for approximately 1 min.
Then add 900 μl (6.8) 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.
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 working solution (5.24) to the SPE eluate and/or the relevant volumes of
mixed mycotoxin working solution (5.23) as listed in Table 1.
Evaporate to dryness the SPE eluate and/or the mixed mycotoxin working solution volumes 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 of HPLC injection solvent (5.26) and mix thoroughly for
at least 10 s.
Filter the solution through a syringe or centrifuge filter (6.12).
Transfer the solution into autosampler vials (6.11).
7.5 Spiking procedure
For the determination of the recovery, carry out a spiking procedure using the mixed mycotoxin stock
solutions (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
solvent is added, and distribute the solution evenly over the materials. Evaporate the spiking solvent at
room temperature.
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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 to obtain an acceptably low false suspect rate.
Annex A illustrates some example chromatograms, and Annex B gives some suitable parameters.
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 Batch composition and analytical 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 highest calibration solution followed by a reagent blank to
check for possible carry over.
Subsequently inject the sample test solutions. At the end of the batch, re-inject the calibration series.
For larger batches of samples, inject calibration solution number 3 after every approximately 10
samples.
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 area ratio of the two
peaks shall match that of the standard substance.
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.
13
Divide the peak area of 15-AcDON by the peak area of C 3-AcDON.
17
Prepare a calibration curve for each of the seven analytes (NIV, DON, 3-AcDON, 15-AcDON, HT-2, T-2,
and ZEA) by plotting the peak area ratios of each analyte calculated in the calibration solutions against
the corresponding amount (µg) of analyte injected on column. Estimate slope and possible intercept of
each of the seven calibration curves by using linear regression.
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8 Calculation
Calculate the mass fraction of each mycotoxin, w, in microgram per kilogram of the sample according to
Formula (1):
R b V 1
1
w= − ×−
aa V m
2 SPE
(1)
where
R is the peak area ratio of the relevant analyte and the corresponding internal standard in the
sample test solution;
a is the slope of the linear function from calibration data (7.8);
b is the intercept of the linear function from calibration data (7.8);
V is the volume of the reconstituted extract after cleanup (here: 0,4 ml);
1
V is the volume of the reconstituted extract injected into the LC system;
2
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×
4
m =
SPE
V
3
(2)
where
m is the mass of the extracted test portion (here: 10 g);
V is the volume of the extraction solvent (here: 50 ml);
3
V is the volume of filtered extract dried before clean-up (here: 5 ml).
4
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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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 1
...
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