EN 16923:2022
(Main)Foodstuffs - Determination of T-2 toxin and HT-2 toxin in cereals and cereal products for infants and young children by SPE clean up and HPLC-MS/MS
Foodstuffs - Determination of T-2 toxin and HT-2 toxin in cereals and cereal products for infants and young children by SPE clean up and HPLC-MS/MS
This document describes a method for the determination of T-2 toxin and HT-2 toxin in cereals and cereal-based products, e.g. oats, intended for nutrition of infants and young children by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) after cleanup by solid phase extraction (SPE) [5].
The method has been validated for HT-2 toxin in oat flour at levels of 9,3 µg/kg and 28,1 µg/kg, oat flakes at levels of 16,5 µg/kg and 21,4 µg/kg, and breakfast cereals (containing oat flakes) at a level of 8,1 µg/kg and for T-2 toxin in oat flour at levels of 4,4 µg/kg and 8,3 µg/kg, oat flakes at levels of 4,9 µg/kg and 6,6 µg/kg and breakfast cereals (containing oat flakes) at a level of 3,5 µg/kg.
Laboratory experiences [6] have shown that the method is also applicable to highly swelling materials (dry cereal-based porridges and modified starches), but these were not examined in the method validation study. Details are outlined in 7.3.
The method can also be applied to oat-by-products at higher levels of T-2- and HT-2 toxin. In this case, the dilution steps need to be considered [6].
The method can also be applied to cereals and cereal products for infants and young children based on e.g. wheat, barley and rice. In this case, the method needs to be in-house-validated for each material. At the time of the interlaboratory study, planned range was 10 µg/kg to 100 µg/kg, and it is known from the pre-study that the method works well in the whole range, although final validation was only done in the range from 3,5 µg/kg to 28,1 µg/kg.
Lebensmittel - Bestimmung von T-2-Toxin und HT-2-Toxin in Getreide und Säuglings- und Kleinkindernahrung auf Getreidebasis mit HPLC-MS/MS nach SPE-Reinigung
Dieses Dokument beschreibt ein Verfahren zur Bestimmung von T-2-Toxin und HT-2-Toxin durch Hochleistungsflüssigchromatographie (HPLC, en: high performance liquid chromatography) in Kopplung mit Tandem Massenspektrometrie (MS/MS) nach Festphasenreinigung (SPE, en: solid phase extraction) [5] in Getreide und getreidebasierten Produkten, z. B. Haferflocken, die für die Ernährung von Säuglingen und Kleinkindern vorgesehen sind.
Das Verfahren wurde für HT-2 Toxin in Hafermehl bei Konzentrationen von 9,3 µg/kg und 28,1 µg/kg, in Haferflocken bei Konzentrationen von 16,5 µg/kg und 21,4 µg/kg und bei Frühstückscerealien (die Haferflocken enthalten) bei einer Konzentration von 8,1 µg/kg sowie für T-2-Toxin in Hafermehl bei Konzentrationen von 4,4 µg/kg und 8,3 µg/kg, in Haferflocken bei Konzentrationen von 4,9 µg/kg und 6,6 µg/kg und bei Frühstückscerealien (die Haferflocken enthalten) bei einer Konzentration von 3,5 µg/kg validiert.
Laborerfahrungen [6] haben gezeigt, dass das Verfahren auch für stark aufquellende Materialien (auf trockenem Getreide basierendem Porridge und modifizierte Stärken) anwendbar ist, jedoch wurden diese in der Validierungsuntersuchung des Verfahrens nicht überprüft. Einzelheiten hierzu sind in 7.3 enthalten.
Das Verfahren kann ebenso bei Hafer-Nebenprodukten bei höheren Konzentrationen von T-2-Toxin und HT-2-Toxin angewendet werden. In diesem Fall ist es erforderlich die Verdünnungsschritte zu berücksichtigen [6].
Das Verfahren kann auch auf Getreide und Getreideerzeugnisse auf der Basis von z. B. Weizen, Gerste und Reis für Säuglinge und Kleinkinder angewendet werden. In diesem Falle ist es erforderlich, das Verfahren im Labor für jedes Material zu validieren (In house Validation). Zum Zeitpunkt des Ringversuches war ein Bereich von 10 µg/kg bis 100 µg/kg zur Validierung vorgesehen, und die Vorstudien haben gezeigt, dass das Verfahren diesen gesamten Bereich gut abdeckt, obwohl das Verfahren letztendlich nur für den Bereich von 3,5 µg/kg bis 28,1 µg/kg validiert wurde.
Produits alimentaires - Dosage des toxines T-2 et HT-2 dans les céréales et les produits céréaliers pour nourrissons et enfants en bas âge par purification par SPE et CLHP-SM/SM
Le présent document décrit une méthode de dosage des toxines T-2 et HT-2 dans les céréales et les produits céréaliers, par exemple l’avoine, destinés à l’alimentation des nourrissons et des enfants en bas âge par chromatographie liquide à haute performance (CLHP) couplée à une spectrométrie de masse en tandem (SM/SM) après purification par extraction en phase solide (SPE) [5].
La méthode a été validée pour la toxine HT-2 présente dans la farine d’avoine à des niveaux de 9,3 µg/kg et 28,1 µg/kg, dans les flocons d’avoine à des niveaux de 16,5 µg/kg et 21,4 µg/kg et dans les céréales pour petit-déjeuner (contenant des flocons d’avoine) à un niveau de 8,1 µg/kg, et pour la toxine T-2 présente dans la farine d’avoine à des niveaux de 4,4 µg/kg et 8,3 µg/kg, dans les flocons d’avoine à des niveaux de 4,9 µg/kg et 6,6 µg/kg et dans les céréales pour petit-déjeuner (contenant des flocons d’avoine) à un niveau de 3,5 µg/kg.
Les expériences menées en laboratoire [6] ont démontré que la méthode est également applicable aux matériaux qui gonflent beaucoup (bouillies à base de céréales sèches et amidons modifiés), mais ces matériaux n’ont pas été examinés lors de l’étude de validation de la méthode. Le paragraphe 7.3 fournit plus d’informations.
La méthode peut également être appliquée aux sous-produits de l’avoine à des teneurs en toxines T-2 et HT-2 plus élevées. Dans ce cas, les étapes de dilution doivent être prises en compte [6].
La méthode peut également être appliquée aux céréales et aux produits céréaliers pour nourrissons et enfants en bas âge à base par exemple de blé, d’orge et de riz. Dans ce cas, la méthode doit être validée en interne pour chaque matrice. Lors de l’étude interlaboratoires, la gamme prévue allait de 10 µg/kg à 100 µg/kg et il est connu, grâce à l’étude préalable, que la méthode fonctionne bien sur toute l’étendue de la gamme, bien que la validation finale ait seulement été faite pour la gamme de concentrations de 3,5 µg/kg à 28,1 µg/kg.
Živila - Določevanje toksinov T-2 in HT-2 v žitu in žitnih proizvodih za dojenčke in majhne otroke s HPLC-MS/MS po čiščenju s SPE
Ta dokument opisuje metodo za določanje toksinov T-2 in HT-2 v žitu in žitnih proizvodih za dojenčke oziroma majhne otroke s tekočinsko kromatografijo visoke ločljivosti (HPLC) v kombinaciji s tandemsko masno spektrometrijo (MS/MS) po čiščenju z ekstrakcijo na trdni fazi (SPE) [5].
Metoda je potrjena za toksin HT-2 v ovseni moki pri koncentracijah 9,3 μg/kg in 28,1 μg/kg, ovsenih kosmičih pri koncentracijah 16,5 μg/kg in 21,4 μg/kg ter žitih za zajtrk (ki vsebujejo ovsene kosmiče) pri koncentracijah 8,1 μg/kg, ter za toksin T-2 v ovseni moki pri koncentracijah 4,4 μg/kg in 8,3 μg/kg, ovsenih kosmičih pri koncentracijah 4,9 μg/kg in 6,6 μg/kg ter žitih za zajtrk (ki vsebujejo ovsene kosmiče) pri koncentraciji 3,5 μg/kg.
Laboratorijske izkušnje [6] kažejo, da se metoda uporablja tudi za materiale, ki močno nabreknejo (kaše iz suhih žit in modificiranih škrobov), vendar ti materiali niso bili raziskani v študiji validativnosti metode. Podrobnosti so opredeljene v točki 7.3.
Metoda se lahko uporabi tudi za stranske proizvode iz ovsa z višjimi ravnmi toksinov T-2 in HT-2. V tem primeru je treba upoštevati korake za redčenje [6].
Metodo je mogoče uporabiti tudi pri žitih in žitnih izdelkih za dojenčke oziroma majhne otroke na osnovi npr. pšenice, ječmena in riža. V tem primeru je treba metodo interno validirati za vsak izdelek. V medlaboratorijski raziskavi je bilo načrtovano območje od 10 µg/kg do 100 µg/kg, iz predhodne raziskave pa je znano, da metoda dobro deluje v celotnem območju, čeprav je bila končna validacija opravljena le v območju od 3,5 µg/kg do 28,1 µg/kg.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-februar-2023
Nadomešča:
SIST EN 16923:2017
Živila - Določevanje toksinov T-2 in HT-2 v žitu in žitnih proizvodih za dojenčke in
majhne otroke s HPLC-MS/MS po čiščenju s SPE
Foodstuffs - Determination of T-2 toxin and HT-2 toxin in cereals and cereal products for
infants and young children by SPE clean up and HPLC-MS/MS
Lebensmittel - Bestimmung von T-2-Toxin und HT-2-Toxin in Getreide und Säuglings-
und Kleinkindernahrung auf Getreidebasis mit HPLC-MS/MS nach SPE-Reinigung
Produits alimentaires - Dosage des toxines T-2 et HT-2 dans les céréales et les produits
céréaliers pour nourrissons et enfants en bas âge par SPE et CLHP-SM/SM
Ta slovenski standard je istoveten z: EN 16923:2022
ICS:
67.060 Žita, stročnice in proizvodi iz Cereals, pulses and derived
njih products
67.230 Predpakirana in pripravljena Prepackaged and prepared
hrana foods
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 16923
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2022
EUROPÄISCHE NORM
ICS 67.060; 67.230 Supersedes EN 16923:2017
English Version
Foodstuffs - Determination of T-2 toxin and HT-2 toxin in
cereals and cereal products for infants and young children
by SPE clean up and HPLC-MS/MS
Produits alimentaires - Dosage des toxines T-2 et HT-2 Lebensmittel - Bestimmung von T 2 Toxin und HT 2
dans les céréales et les produits céréaliers pour Toxin in Getreide und Säuglings- und
nourrissons et enfants en bas âge par purification par Kleinkindernahrung auf Getreidebasis mit HPLC
SPE et CLHP-SM/SM MS/MS nach SPE-Reinigung
This European Standard was approved by CEN on 9 October 2022.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 16923:2022 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 . 5
6 Apparatus and equipment . 8
7 Procedure. 9
7.1 Preparation of the test sample . 9
7.2 Preparation of the solid phase column . 9
7.3 Extraction of T-2 toxin and HT-2 toxin . 9
7.4 Clean-up by solid phase filtration . 10
7.5 LC-MS/MS-analysis . 10
7.6 Identification . 10
8 Calculation . 11
9 Precision . 12
9.1 General . 12
9.2 Repeatability . 12
9.3 Reproducibility . 12
10 Test report . 13
Annex A (informative) Example chromatograms (API 4000™) .
Annex B (informative) Example conditions for suitable LC-MS/MS systems . 18
B.1 System settings for SCIEX API 4000™ and SCIEX API 4000™ QTrap . 18
B.1.1 Settings for chromatography . 18
B.1.2 Detector parameters . 18
B.2 System settings for SCIEX API 2000 . 20
B.2.1 Settings for chromatography . 20
B.2.2 Detector parameters . 21
B.3 System settings for SCIEX API 3000™ . 22
B.3.1 Settings for chromatography . 22
B.3.2 Detector parameters . 22
B.4 System settings for Micromass Quattro LC . 23
B.4.1 Settings for chromatography . 23
B.4.2 Detector parameters . 24
Annex C (informative) Precision data . 25
Bibliography . 27
European foreword
This document (EN 16923:2022) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2023, and conflicting national standards shall be
withdrawn at the latest by May 2023.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 16923:2017.
In comparison with the previous edition, the following technical modifications have been made:
— the second elution step in the solid phase extraction in 7.4 is more clearly described.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the United
Kingdom.
Introduction
The mycotoxin T-2 toxin and its metabolite HT-2 toxin belong to the group of trichothecenes which are
produced by various Fusarium species. Cereals like maize, wheat, barley, oats and rye 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 latest version of the hazardous substances ordinance, Regulation (EC)
No 1907/2006 [3], should be taken into account as well as appropriate national statements, e.g. such as
in [4].
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 — T-2 toxin and its metabolite HT-2 toxin are known to have carcinogenic effects.
1 Scope
This document describes a method for the determination of T-2 toxin and HT-2 toxin in cereals and
cereal-based products, e.g. oats, intended for nutrition of infants and young children by high performance
liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) after cleanup by solid
phase extraction (SPE) [5].
The method has been validated for HT-2 toxin in oat flour at levels of 9,3 µg/kg and 28,1 µg/kg, oat flakes
at levels of 16,5 µg/kg and 21,4 µg/kg, and breakfast cereals (containing oat flakes) at a level of 8,1 µg/kg
and for T-2 toxin in oat flour at levels of 4,4 µg/kg and 8,3 µg/kg, oat flakes at levels of 4,9 µg/kg and
6,6 µg/kg and breakfast cereals (containing oat flakes) at a level of 3,5 µg/kg.
Laboratory experiences [6] have shown that the method is also applicable to highly swelling materials
(dry cereal-based porridges and modified starches), but these were not examined in the method
validation study. Details are outlined in 7.3.
The method can also be applied to oat-by-products at higher levels of T-2- and HT-2 toxin. In this case,
the dilution steps need to be considered [6].
The method can also be applied to cereals and cereal products for infants and young children based on
e.g. wheat, barley and rice. In this case, the method needs to be in-house-validated for each material. At
the time of the interlaboratory study, planned range was 10 µg/kg to 100 µg/kg, and it is known from the
pre-study that the method works well in the whole range, although final validation was only done in the
range from 3,5 µg/kg to 28,1 µg/kg.
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:
— ISO Online browsing platform: available at https://www.iso.org/obp/ui
— IEC Electropedia: available at https://www.electropedia.org/
4 Principle
T-2 toxin and HT-2 toxin are extracted with acetonitrile-water mixture and by shaking manually or with
a laboratory blender. A solid phase extraction column or a pass through column is used to clean up and
concentrate the filtered and diluted extract, see also [7]. The toxins are determined by HPLC coupled with
tandem mass spectrometry.
5 Reagents
Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696,
unless otherwise specified. Solvents shall
...
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