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 der 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 müssen die Verdünnungsschritte berücksichtigt werden [6].
Das Verfahren kann auch auf Getreide und Getreideerzeugnisse auf der Basis von 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 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

General Information

Status
Published
Public Enquiry End Date
19-Oct-2020
Publication Date
16-Jan-2023
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
06-Jan-2023
Due Date
13-Mar-2023
Completion Date
17-Jan-2023

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Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN 16923:2023
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
SIST EN 16923:2023 en,fr,de

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

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SIST EN 16923:2023
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SIST EN 16923:2023
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.
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SIST EN 16923:2023
EN 16923:2022 (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 ............................................................................................................................................................. 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

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SIST EN 16923:2023
EN 16923:2022 (E)
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.
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SIST EN 16923:2023
EN 16923:2022 (E)
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.

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SIST EN 16923:2023
EN 16923:2022 (E)
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 be of quality for HPLC analysis, unless otherwise specified.

---------------------- Page: 7 ----------------------
SIST EN 16923:2023
EN 16923:2022 (E)
5.1 Acetonitrile, HPLC grade.
5.2 Methanol, HPLC grade.
5.3 Solvent mixture.
Mix 20 parts of acetonitrile (5.1) and 80 parts of water (20+80, v+v).
5.4 Extraction mixture.
Mix 84 parts of acetonitrile (5.1) and 16 parts of water (84+16, v+v).
5.5 Eluent for LC-MS/MS.

Examples of eluents suitable for LC-MS/MS systems are given in Annex B. Filter the solution through a

membrane filter (6.18).
5.6 Nitrogen, purity of at least 99,9 %.

5.7 Activated charcoal for column chromatography (particle size: 63 µm to 200 µm).

5.8 Aluminium oxide (neutral, for liquid chromatography).

5.9 Finely ground/pulverized diatomaceous earth (diatomite, kieselgur), e.g. Celite® 545 .

5.10 Siliconization reagent, e.g. Surfasil™ (optional).
5.11 Cyclohexane, analytical quality, (optional).
5.12 Preparation of the diluted siliconization reagent, (optional).
Add e.g. 50 ml of a siliconization reagent (5.10) to 950 ml cyclohexane (5.11).
5.13 Formic acid, HPLC quality.

5.14 Ammonia solution, substance concentration c(NH ) = 13,4 mol/l or mass concentration

ρ(NH ) = 250 g/l.
5.15 Ammonium acetate (CH CO NH ), LC-MS/MS quality.
3 2 4

5.16 Anti-clogging material, such as washed sea sand, glass beads, or polyethylene beads, (optional).

5.17 Stock solution of T-2 toxin, mass concentration ρ = 100 μg/ml, in acetonitrile.

T-2-toxin, e.g. crystalline or as a film, purity greater than 98 % mass fraction, or as certified standard

solution.
5.18 Stock solution of HT-2 toxin, ρ = 100 μg/ml, in acetonitrile.

Celite® 545 is a trade name of a product commercially available from various suppliers. This information is given for

the convenience of users of this document 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.

Surfasil™ is a trade name of a product commercially available from various suppliers. This information is given for the

convenience of users of this document 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.
---------------------- Page: 8 ----------------------
SIST EN 16923:2023
EN 16923:2022 (E)

HT-2 toxin, e.g. crystalline or as a film, purity greater than 98 % mass fraction, or as certified standard

solution.

5.19 Internal standard solution of [ C ]-T-2 toxin, ρ = 25 μg/ml, in acetonitrile.

Stock solution of T-2 toxin, mass concentration ρ = 25 μg/ml, in acetonitrile, taking into account the

certified purity and/or concentration.

Other suitable isotopic labelled standards of T-2 toxin than the [ C ]-T-2 toxin may be used.

5.20 Internal standard solution of [ C ]-HT-2 toxin, ρ = 25 μg/ml, in acetonitrile.

Stock solution of HT-2 toxin, ρ = 25 μg/ml, in acetonitrile, taking into account the certified purity and/or

concentration.

Other suitable isotopic labelled standards of HT-2 toxin than the [ C ]-HT-2 toxin may be used.

5.21 Mixed standard solution, ρ = 500 ng/ml.

Pipette 25 µl of each T-2 toxin and HT-2 toxin stock solution (5.17 and 5.18), respectively, into a 5 ml

volumetric flask, and dilute up to the mark with solvent mixture (5.3).
This solution can be stored at −18 °C for 12 months.
5.22 Mixed internal standard solution, ρ = 1000 ng/ml.

Dilute 200 µl of internal standard solution of [ C ]-T-2 toxin (5.19) and 200 µl of internal standard

solution of [ C ]-HT-2 toxin (5.20) with solvent mixture (5.3) in a 5 ml volumetric flask.

This solution can be stored at –18 °C for 6 months.
5.23 Calibration solutions.

For the calibration of the measuring system, prepare calibration solutions within a range from 5 ng/ml

to 100 ng/ml.
Prepare e.g. the following calibration solutions as outlined in Table 1:
Table 1 — Examples of suitable calibration solutions
Mixed
Mass Mixed
Mass internal Solvent
Calibration concentration standard
concentration standard mixture
solution per isotope solution
per analyte solution (5.3)
labelled analyte (5.21)
(5.22)
ng/ml ng/ml µl µl µl
IS-Blank 0 50 – 50 950
1 5 50 10 50 940
2 10 50 20 50 930
3 20 50 40 50 910
4 40 50 80 50 870
5 60 50 120 50 830
6 80 50 160 50 790
7 100 50 200 50 750
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SIST EN 16923:2023
EN 16923:2022 (E)
6 Apparatus and equipment
Usual laboratory apparatus and, in particular, the following.
6.1 Laboratory balance, accuracy of 0,01 g.
6.2 Analytical balance, accuracy of 0,1 mg.
6.3 Ultrasonic bath.
6.4 Laboratory shaker for test tubes.

6.5 Manual dispensers, microlitre syringes or microlitre pipettes for 10 µl to 5 ml.

6.6 Dispenser, suitable for 20 ml.
6.7 250 ml-Erlenmeyer flasks with stoppers, or 250 ml-centrifuge tubes.

6.8 Syringe filters (0,45 µm), or centrifugal filters (e.g. Durapore® PVDF (0,45 µm), or Millipore

Ultrafree-MC® 0,5 ml), fitting with centrifuge for reaction vessels, e.g. Eppendorf® vessels.

6.9 Folded filter, pore size 4 µm to 7 µm, diameter 100 mm.
6.10 Laboratory centrifuge.

6.11 Cartridges (6 ml), made from polypropylene (PP) and corresponding frits from polyethylene (PE),

, 6 ml, 500 mg.
or commercially available SPE columns, e.g. CHROMABOND® Carbon/Alox/Celite®
6.12 SPE vacuum/elution station.
6.13 Laboratory shaker, e.g. overhead shaker.
6.14 Laboratory blender, e.g. Ultra Turrax® .
6.15 Test tubes, suitable for a volume up to 10,0 ml.
6.16 Siliconized test tubes (optional).

After thorough cleaning of the test tubes (6.15), fill up to the top with the diluted siliconization reagent

(5.12) and allow them to stand for 1 min. Then, pouring out the reagent solution, make sure to collect it

for repeated usage. Afterwards rinse the tubes with cyclohexane (5.11) and acetonitrile (5.1) or methanol

(5.2) successively in this order. The rinsing solutions may be used again. Finally rinse the tubes twice

with double-distilled water and allow them to dry.

WARNING — Surfasil™, being a chloride silane solvent, readily reacts with water by forming hydrochloric

acid vapour. Therefore, never rinse tubes with water directly after derivatization.

Tubes that are not siliconized, such as those made from polypropylene, may be used, if formally proved

suitable.

Durapore® PVDF, Millipore Ultrafree-MC®, Ultra Turrax ®, TurboVap®LV Zymark and Surfasil™ are trade names of

products commercially available from various suppliers. Eppendorf® vessel is an example of a product commercially

available from Eppendorf, Chromabond® is the trade name of a product, commercially available from by Macherey-Nagel.

This information is given for the convenience of users of this document 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.

---------------------- Page: 10 ----------------------
SIST EN 16923:2023
EN 16923:2022 (E)
6.17 Concentration evaporator workstation, e.g. TurboVap® Zymark , or similar.
6.18 Membrane filters for aqueous solutions (pore size 0,45 µm).
6.19 LC-MS/MS system with the following components:
6.19.1 HPLC pump.
6.19.2 Injection system.

6.19.3 HPLC column, e.g. octadecylsilane (ODS), that ensures base line separation to distinguish peaks

of the T-2 toxin and HT-2 toxin from all other signals, 150 mm length, 2,00 mm inner diameter, particle

size 5 µm, suitable reversed-phase pre-column.
Columns of different dimensions may also be used.
6.19.4 Column thermostat.
6.19.5 Tandem mass spectrometer (MS/MS).
6.19.6 Data evaluation system.
7 Procedure
7.1 Preparation of the test sample

Grind and homogenize the sample to particle sizes less than 1 mm before analysis.

7.2 Preparation of the solid phase column

Mix 42 g of activated charcoal (5.7) with 30 g of neutral Al O (5.8) and 18 g of Celite 545 (5.9) in a glass

2 3

vessel (500 ml) and homogenize with a shaker (6.13) for 1 h (ratio 7:5:3 activated charcoal/neutral

Al O /Celite 545; m/m/m). Place the homogenized mixture, 0,5 g respectively, in empty 6 ml cartridges

2 3
provided with three PE frits (2 frits below, and one on top for covering).

Alternatively, commercially available SPE-columns may be used. For this reason, clean up procedure shall

be checked for recovery and shall be optimized if necessary [7].
7.3 Extraction of T-2 toxin and HT-2 toxin

Weigh 25,0 g of the homogenized and finely ground sample (7.1) with an accuracy of 0,1 g into a 250 ml

beaker/Erlenmeyer flask, or into a 250 ml centrifuge tube (6.7), add 100 ml of the extraction mixture

(5.4) and close the vessel. Shake the mixture with a shaker (6.13) for approximately 1 h at room

temperature.

Alternatively, use a laboratory blender (6.14) for extraction. In this case, homogenize the mixture for

3 min at a great speed.

After extraction, pass slightly more than 10 ml extract through a folded filter (6.9) into a glass vessel.

Centrifuge this portion at 2 500 × g at room temperature for 10 min. Remove 10 ml of the upper solution

of the centrifugate.

If highly swelling food matrices are analysed, increase the water content in the extraction medium up to

200 % or alternatively reduce the weight of the sample amount down to 50 % of the described amount.

To prevent clogging of the swelling material, add the same amount of e.g. sea sand (5.16) as the sample

weight.
Take volume and/or weight adjustments into account in the final calculation.
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7.4 Clean-up by solid phase filtration

Plug the prepared column containing 0,5 g of activated charcoal/Al O /Celite (7.2) on the SPE station

2 3

(6.12), and place a test tube (6.15) beneath to collect the eluate. Pass 5,0 ml of the extract (7.3) through

the SPE-column and collect the eluate. Apply a low vacuum in order to obtain an elution speed of 1 drop

to 2 drops per s. Rinse the cartridge two times with 5 ml of extraction mixture (5.4), and collect the

eluates also in the same test tube.

Add 25 µl of mixed internal standard solution (5.22) to the combined eluates, and evaporate to dryness

with nitrogen (5.6) using a concentration evaporator workstation (at 45 °C for 30 min, and 70 kPa gas

pressure).

Re-dissolve the residue in 500 µl of solvent mixture (5.3) by shaking turbulently (6.4) for 60 s, and, if

necessary, apply an ultrasonic bath (6.3) for 5 min at room temperature. The solution should be filtered

through syringe filters (6.8) or centrifugal filters (0,45 µm, 0,5 ml) at minimum 10 000 g in order to

discard any micro-particles to give the injection solution.

If necessary, the process of sample purification may be interrupted without analyte loss by storing the

sample extracts (evaporated to dryness) at 4 °C for several days, or at −18 °C for two weeks.

In case the residue level exceeds the calibration range, the residue should be dissolved in more than

500 µl of solvent mixture.
7.5 LC-MS/MS-analysis

Depending on the LC-MS/MS system, inject e.g. 10 µl to 25 µl of the injection solution derived from 7.4.

Set up a suitable measuring system by weighing in analytes and optimizing the separation and detection

parameters. Annex A lists some example chromatograms in Figure A.1 to Figure A.4, and Annex B lists

some suitable parameters.

The adopted measuring systems shall meet with the following requirements as in Table 2:

Table 2 — Prerequisites for precursor ions and product ions
Minimum-Signal-to-Noise Ratio Minimum-Signal-to-Noise Ratio
Analyte
for the Quantification Trace for the Confirmation Trace
T-2 toxin 50:1 at 250 pg 30:1 at 250 pg
HT-2 toxin 10:1 at 250 pg 5:1 at 250 pg
[ C ]-T-2 toxin 50:1 at 250 pg 30:1 at 250 pg
[ C ]-HT-2 toxin 10:1 at 250 pg 5:1 at 250 pg

250 pg is the calculated absolute mass amount of the analyte injected on the column. Signal is

peak height, noise is base line noise as observed in the extracted ion chromatogram.

7.6 Identification

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

sample test solution. Identify the analyte on the basis of at least two mass transitions. In addition, the

retention times (peaks in both mass traces) and the area ratio of the two peaks shall match that of the

standard substance [8].

Identify the analyte on the basis of at least two mass transitions. In addition, the retention times (peaks

in both mass traces) and the area ratio of the two peaks shall match that of the standard substance.

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8 Calculation

For the quantification of T-2 toxin and HT-2 toxin, the corresponding isotope labelled standard is used as

internal standard. Carry out a multi-point calibration with a standard solution, for example according to

Table 1 in 5.23.

For the linear regression plot the peak area ratio from each analyte against the corresponding

concentration ratio. The mass concentration of each toxin in the injection solution is derived from linear

regression in ng/ml, see Formula (1).
A ρ
a a
ab+ (1)
Cal Cal
A ρ
IS IS
where
A is the peak area of the analyte in the calibration solution (5.23);
A is the peak area of the internal standard in the calibration solution (5.23);

a is the slope of the calibration graph determined by the calibration solutions (5.23);

Cal

ρ is the mass concentrations of the analytes in the calibration solutions (5.23), in ng/ml;

ρ is the internal standard concentration in the calibration solution (5.23), in ng/ml, usually

50 ng/ml;

b is the axis intercept of the calibration determined by the calibration solutions (5.23).

Cal

Calculate the mass concentration of the analyte in the sample test solution ρ , in ng/ml using

Formula (2):
Cal
ρρ= (2)
a IS
Cal
where
is the response of the analyte in the sample test solution;
is the response of the internal standard in the sample test solution;

is the mass concentration of the internal standard in the sample test solution of 50 ng/ml.

Calculate the mass fraction of the analyte, w in µg/kg using Formula (3):
ρ ××VV
a ex im
(3)
mV×
s Aliquot
where
is the volume of extraction solution, in ml, here 100 ml;

is the volume of solvent mixture used to re-dissolve the evaporated sample, in ml, here

0,5 ml;
is the mass of the test portion, in g, here 25 g;
is the sample extract aliquot, in ml, here 5 ml.
Aliquot
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SIST
...

SLOVENSKI STANDARD
oSIST prEN 16923:2020
01-oktober-2020

Živila - Določevanje toksinov T-2 in HT-2 v žitu in žitnih proizvodih za dojenčke in

majhne otroke z 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 LC-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 CL-SM/SM après purification par

SPE
Ta slovenski standard je istoveten z: prEN 16923
ICS:
67.060 Žita, stročnice in proizvodi iz Cereals, pulses and derived
njih products
67.230 Predpakirana in pripravljena Prepackaged and prepared
hrana foods
oSIST prEN 16923:2020 en,fr,de

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

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oSIST prEN 16923:2020
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oSIST prEN 16923:2020
DRAFT
EUROPEAN STANDARD
prEN 16923
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2020
ICS 67.060; 67.230 Will supersede 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 CL-SM/SM après Kleinkindernahrung auf Getreidebasis mit LC-MS/MS

purification par SPE nach SPE Reinigung

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

CEN/TC 275.

If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations

which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other

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

Management Centre has the same status as the official versions.

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

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

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

United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without

notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16923:2020 E

worldwide for CEN national Members.
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oSIST prEN 16923:2020
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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 ........................................................................................................................... 7

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

8 Calculation ...................................................................................................................................................... 10

9 Precision .......................................................................................................................................................... 12

10 Test report ...................................................................................................................................................... 13

Annex A (informative) Example chromatograms (API 4000™) ................................................................ 14

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

Annex C (informative) Precision data ................................................................................................................ 25

Bibliography ................................................................................................................................................................. 27

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oSIST prEN 16923:2020
prEN 16923:2020 (E)
European foreword

This document (prEN 16923:2020) 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 will supersede EN 16923:2017.
Alterations to the version of 2017 are as follows:

— The second elution step in the solid phase extraction in 7.4 is more clearly described.

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 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.

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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 http://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 be of quality for HPLC analysis, unless otherwise specified.

5.1 Acetonitrile, HPLC grade.
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5.2 Methanol, HPLC grade.
5.3 Solvent mixture.
Mix 20 parts of acetonitrile (5.1) and 80 parts of water (20+80, v+v).
5.4 Extraction mixture.
Mix 84 parts of acetonitrile (5.1) and 16 parts of water (84+16, v+v).
5.5 Eluent for LC-MS/MS.

Examples of eluents suitable for LC-MS/MS systems are given in Annex B. Filter the solution through a

membrane filter (6.18).
5.6 Nitrogen, purity of at least 99,9 %.

5.7 Activated charcoal for column chromatography (particle size: 63 µm to 200 µm).

5.8 Aluminium oxide (neutral, for liquid chromatography).

5.9 Finely ground/pulverized diatomaceous earth (diatomite, kieselgur), e.g. Celite® 545.

5.10 Siliconization reagent, e.g. Surfasil™ (optional).
5.11 Cyclohexane, analytical quality, (optional).
5.12 Preparation of the diluted siliconization reagent, (optional).
Add e.g. 50 ml of a siliconization reagent (5.10) to 950 ml cyclohexane (5.11).
5.13 Formic acid, HPLC quality.

5.14 Ammonia solution, substance concentration c(NH ) = 13,4 mol/l or mass concentration

ρ(NH ) = 250 g/l.
5.15 Ammonium acetate (CH CO NH ), LC-MS/MS quality.
3 2 4

5.16 Anti-clogging material, such as washed sea sand, glass beads, or polyethylene beads, (optional).

5.17 Stock solution of T-2 toxin, mass concentration ρ = 100 μg/ml, in acetonitrile.

5.18 Stock solution of HT-2 toxin, ρ = 100 μg/ml, in acetonitrile.

5.19 Internal standard solution of [ C ]-T-2 toxin, ρ = 25 μg/ml, in acetonitrile.

Other suitable isotopic labelled standards of T-2 toxin than the [ C ]-T-2 toxin may be used.

5.20 Internal standard solution of [ C ]-HT-2 toxin, ρ = 25 μg/ml, in acetonitrile.

Other suitable isotopic labelled standards of HT-2 toxin than the [ C ]-HT-2 toxin may be used.

Surfasil™ is a trade name of a product commercially available from various suppliers. This information is given for the

convenience of users of this document and does not constitute an endorsement by CEN of the products named.

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

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5.21 Mixed standard solution, ρ = 500 ng/ml.

Pipette 25 µl of each T-2 toxin and HT-2 toxin stock solution (5.17 and 5.18), respectively, into a 5 ml

volumetric flask, and dilute up to the mark with solvent mixture (5.3).
This solution can be stored at −18 °C for 12 months.
5.22 Mixed internal standard solution, ρ = 1000 ng/ml

Dilute 200 µl of the internal standard solutions (5.19 and 5.20) with solvent mixture (5.3) in a 5 ml

volumetric flask.
This solution can be stored at –18 °C for 6 months.
5.23 Calibration solutions.

For the calibration of the measuring system, prepare calibration solutions within a range from 5 ng/ml

to 100 ng/ml.
Prepare e.g. the following calibration solutions as outlined in Table 1:
Table 1 — Examples of suitable calibration solutions
Mixed
Mass Mixed
Mass internal Solvent
Calibration concentration standard
concentration standard mixture
solution per isotope solution
per analyte solution (5.3)
labelled analyte (5.21)
(5.22)
ng/ml ng/ml µl µl µl
IS-Blank 0 50 – 50 950
1 5 50 10 50 940
2 10 50 20 50 930
3 20 50 40 50 910
4 40 50 80 50 870
5 60 50 120 50 830
6 80 50 160 50 790
7 100 50 200 50 750
6 Apparatus and equipment
Usual laboratory apparatus and, in particular, the following.
6.1 Laboratory balance, accuracy of 0,01 g.
6.2 Analytical balance, accuracy of 0,1 mg.
6.3 Ultrasonic bath.
6.4 Laboratory shaker for test tubes.

6.5 Manual dispensers, microlitre syringes or microlitre pipettes for 10 µl to 5 ml.

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6.6 Dispenser, suitable for 20 ml.
6.7 250 ml-Erlenmeyer flasks with stoppers, or 250 ml-centrifuge tubes.

6.8 Syringe filters (0,45 µm), or centrifugal filters (e.g. Durapore® PVDF (0,45 µm), or Millipore

Ultrafree-MC® 0,5 ml), fitting with centrifuge for reaction vessels, e.g. Eppendorf® vessels.

6.9 Folded filter, pore size 4 µm to 7 µm, diameter 100 mm.
6.10 Laboratory centrifuge.

6.11 Cartridges (6 ml), made from polypropylene (PP) and corresponding frits from polyethylene

(PE), or commercially available SPE columns, e.g. CHROMABOND® Carbon/Alox/Celite® , 6 ml,

500 mg.
6.12 SPE vacuum/elution station.
6.13 Laboratory shaker, e.g. overhead shaker.
6.14 Laboratory blender, e.g. Ultra Turrax® .
6.15 Test tubes, suitable for a volume up to 10,0 ml.
6.16 Siliconized test tubes (optional).

After thorough cleaning of the test tubes (6.15), fill up to the top with the diluted siliconization reagent

(5.12) and allow them to stand for 1 min. Then, pouring out the reagent solution, make sure to collect it

for repeated usage. Afterwards rinse the tubes with cyclohexane (5.11) and acetonitrile (5.1) or

methanol (5.2) successively in this order. The rinsing solutions may be used again. Finally rinse the

tubes twice with double-distilled water and allow them to dry.

WARNING — Surfasil™, being a chloride silane solvent, readily reacts with water by forming

hydrochloric acid vapour. Therefore, never rinse tubes with water directly after derivatization.

Tubes that are not siliconized, such as those made from polypropylene, may be used, if formally proved

suitable.
6.17 Concentration evaporator workstation, e.g. TurboVap® Zymark , or similar.
6.18 Membrane filters for aqueous solutions (pore size 0,45 µm).
6.19 LC-MS/MS system with the following components:
6.19.1 HPLC pump.
6.19.2 Injection system.

Durapore® PVDF, Millipore Ultrafree-MC®, Ultra Turrax ®, TurboVap®LV Zymark and Surfasil are trade names of

products commercially available from various suppliers. Eppendorf® vessel is an example of a product commercially

available from Eppendorf, Chromabond® is the trade name of a product, commercially available from by Macherey-

Nagel. This information is given for the convenience of users of this document and does not constitute an endorsement

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

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6.19.3 HPLC column, e.g. octadecylsilane (ODS), that ensures base line separation to distinguish peaks

of the T-2 toxin and HT-2 toxin from all other signals, 150 mm length, 2,00 mm inner diameter, particle

size 5 µm, suitable reversed-phase pre-column.
Columns of different dimensions may also be used.
6.19.4 Column thermostat.
6.19.5 Tandem mass spectrometer (MS/MS).
6.19.6 Data evaluation system.
7 Procedure
7.1 Preparation of the test sample

Grind and homogenize the sample to particle sizes less than 1 mm before analysis.

7.2 Preparation of the solid phase column

Mix 42 g of activated charcoal (5.7) with 30 g of neutral Al O (5.8) and 18 g of Celite 545 (5.9) in a glass

2 3

vessel (500 ml) and homogenize with a shaker (6.13) for 1 h (ratio 7:5:3 activated charcoal/neutral

Al O /Celite 545; m/m/m). Place the homogenized mixture, 0,5 g respectively, in empty 6 ml cartridges

2 3
provided with three PE frits (2 frits below, and one on top for covering).

Alternatively, commercially available SPE-columns may be used. For this reason, clean up procedure

shall be checked for recovery and shall be optimized if necessary. [7]
7.3 Extraction of T-2 toxin and HT-2 toxin

Weigh 25,0 g of the homogenized and finely ground sample (7.1) with an accuracy of 0,1 g into a 250 ml

beaker/Erlenmeyer flask, or into a 250 ml centrifuge tube (6.7), add 100 ml of the extraction mixture

(5.4) and close the vessel. Shake the mixture manually or with a shaker (6.13) for 1 h at room

temperature.

Alternatively, use a laboratory blender (6.14) for extraction. In this case, homogenize the mixture for

3 min at a great speed.

After extraction, pass slightly more than 10 ml extract through a folded filter (6.9) into a glass vessel.

Centrifuge this portion at 2 500 × g at room temperature for 10 min, Remove 10 ml of the upper

solution of the centrifugate.

If highly swelling food matrices are analysed, increase the water content in the extraction medium up to

200 % or alternatively reduce the weight of the sample amount down to 50 % of the described amount.

To prevent clogging of the swelling material, add the same amount of e.g. sea sand (5.16) as the sample

weight.
Take volume and/or weight adjustments into account in the final calculation.
7.4 Clean-up by solid phase filtration

Plug the prepared column containing 0,5 g of activated charcoal/Al O /Celite (7.2) on the SPE station

2 3

(6.12), and place a test tube (6.15) beneath to collect the eluate. Pass 5,0 ml of the extract (7.3) through

the SPE-column and collect the eluate. Apply a low vacuum in order to obtain an elution speed of 1 drop

to 2 drops per s. Rinse the cartridge two times with 5 ml of extraction mixture (5.4), and collect the

eluates also in the same test tube.
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Add 25 µl of mixed internal standard solution (5.22) to the combined eluates, and evaporate to dryness

with nitrogen (5.6) using a concentration evaporator workstation (at 45 °C for 30 min, and 10 psi gas

pressure).

Re-dissolve the residue in 500 µl of solvent mixture (5.3) by shaking turbulently (6.4) for 60 s, and, if

necessary, apply an ultrasonic bath (6.3) for 5 min at room tem
...

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