EN 16923-2017 en Foodstuffs - Determination of T-2 toxin and HT-2 toxin in cereals and cereal products for infants and young children by LC-MS MS after SPE cleanup.pdf

1、Foodstuffs Determination of T-2 toxin and HT-2 toxin in cereals and cereal products for infants and young children by LC-MS/MS after SPE cleanupBS EN 16923:2017BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16923

2、May 2017 ICS 67.060; 67.230 English Version Foodstuffs - Determination of T-2 toxin and HT-2 toxin in cereals and cereal products for infants and young children by LC-MS/MS after SPE cleanup Produits alimentaires - Dosage des toxines T-2 et HT-2 dans les crales et les produits craliers pour nourriss

3、ons et enfants en bas ge par CL-SM/SM aprs purification par SPE Lebensmittel - Bestimmung von T-2-Toxin und HT-2-Toxin in Getreide und Suglings- und Kleinkindernahrung auf Getreidebasis mit LC-MS/MS nach SPE-Reinigung This European Standard was approved by CEN on 27 February 2017. CEN members are bo

4、und 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

5、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

6、 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,

7、 Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17,

8、 B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16923:2017 ENational forewordThis British Standard is the UK implementation of EN 16923:2017.The UK participation in its preparation was entrusted to Technical C

9、ommittee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The Britis

10、h Standards Institution 2017 Published by BSI Standards Limited 2017ISBN 978 0 580 90469 1ICS 67.060; 67.230Compliance with a British Standard cannot confer immunity from legal obligations. This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 J

11、une 2017.Amendments/corrigenda issued since publicationDate Text affectedBRITISH STANDARDBS EN 16923:2017EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16923 May 2017 ICS 67.060; 67.230 English Version Foodstuffs - Determination of T-2 toxin and HT-2 toxin in cereals and cereal products for in

12、fants and young children by LC-MS/MS after SPE cleanup Produits alimentaires - Dosage des toxines T-2 et HT-2 dans les crales et les produits craliers pour nourrissons et enfants en bas ge par CL-SM/SM aprs purification par SPE Lebensmittel - Bestimmung von T-2-Toxin und HT-2-Toxin in Getreide und S

13、uglings- und Kleinkindernahrung auf Getreidebasis mit LC-MS/MS nach SPE-Reinigung This European Standard was approved by CEN on 27 February 2017. 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

14、 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).

15、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, Cyp

16、rus, 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 U

17、nited Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No.

18、EN 16923:2017 EBS EN 16923:2017EN 16923:2017 (E) 2 Contents Page European foreword . 3 Introduction 4 1 Scope 5 2 Normative references 5 3 Principle . 5 4 Reagents . 5 5 Apparatus and equipment . 7 6 Procedure. 9 7 Calculation 10 8 Precision 11 9 Test report 12 Annex A (informative) Example chromato

19、grams (API 4000) 13 Annex B (informative) Example conditions for suitable LC-MS/MS systems 17 Annex C (informative) Precision data 24 Bibliography . 26 BS EN 16923:2017EN 16923:2017 (E) 3 European foreword This document (EN 16923:2017) has been prepared by Technical Committee CEN/TC 275 “Food analys

20、is - 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 November 2017, and conflicting national standards shall be withdrawn at the lates

21、t by November 2017. 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 Comm

22、ission and the European Free Trade Association. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Fo

23、rmer 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 the United Kingdom. BS EN 16923:2017EN 16923:2017 (

24、E) 4 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

25、 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 hazard

26、ous 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 pri

27、or to use. WARNING 3 T-2 toxin and its metabolite HT-2 toxin are known to have carcinogenic effects. BS EN 16923:2017EN 16923:2017 (E) 5 1 Scope This European Standard 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 nu

28、trition 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 g/kg and 28,1 g/kg, oat flakes at levels of

29、 16,5 g/kg and 21,4 g/kg, and breakfast cereals (containing oat flakes) at a level of 8,1 g/kg and for T-2 toxin in oat flour at levels of 4,4 g/kg and 8,3 g/kg, oat flakes at levels of 4,9 g/kg and 6,6 g/kg and breakfast cereals (containing oat flakes) at a level of 3,5 g/kg. Laboratory experiences

30、 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 6.3. The method can also be applied to oat-by-products at higher levels of T-2- and HT

31、-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 interl

32、aboratory study, planned range was 10 g/kg to 100 g/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 g/kg to 28,1 g/kg. 2 Normative references The following documents, in whole or in part, are normativ

33、ely referenced in this document and are indispensable for its application. 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

34、and test methods (ISO 3696) 3 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

35、 7. The toxins are determined by HPLC coupled with tandem mass spectrometry. 4 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. 4.1 Aceton

36、itrile, HPLC grade. 4.2 Methanol, HPLC grade. 4.3 Solvent mixture. Mix 20 parts of acetonitrile (4.1) and 80 parts of water (20+80, v+v). 4.4 Extraction mixture. Mix 84 parts of acetonitrile (4.1) and 16 parts of water (84+16, v+v). BS EN 16923:2017EN 16923:2017 (E) 6 4.5 Eluent for LC-MS/MS. Exampl

37、es of eluents suitable for LC-MS/MS systems are given in Annex B. Filter the solution through a membrane filter (5.18). 4.6 Nitrogen, purity of at least 99,9 %. 4.7 Activated charcoal for column chromatography (particle size: 63 m to 200 m). 4.8 Aluminium oxide (neutral, for liquid chromatography).

38、4.9 Finely ground/pulverized diatomaceous earth (diatomite, kieselgur), e.g. Celite545. 4.10 Siliconization reagent, e.g. SurfaSil1)(optional). 4.11 Cyclohexane, analytical quality, (optional). 4.12 Preparation of the diluted siliconization reagent, (optional). Add e.g. 50 ml of a siliconization rea

39、gent (4.10) to 950 ml cyclohexane (4.11). 4.13 Formic acid, HPLC quality. 4.14 Ammonia solution, substance concentration c(NH3) = 13,4 mol/l or mass concentration (NH3) = 250 g/l. 4.15 Ammonium acetate (CH3CO2NH4), LC-MS/MS quality. 4.16 Anti-clogging material, such as washed sea sand, glass beads,

40、or polyethylene beads, (optional). 4.17 Stock solution of T-2 toxin, mass concentration = 100 g/ml, in acetonitrile. 4.18 Stock solution of HT-2 toxin, = 100 g/ml, in acetonitrile. 4.19 Internal standard solution of 13C24-T-2 toxin, = 25 g/ml, in acetonitrile. Other suitable isotopic labelled standa

41、rds of T-2 toxin than the 13C24-T-2 toxin may be used. 4.20 Internal standard solution of 13C22-HT-2 toxin, = 25 g/ml, in acetonitrile. Other suitable isotopic labelled standards of HT-2 toxin than the 13C22-HT-2 toxin may be used. 4.21 Mixed standard solution, = 500 ng/ml. Pipette 25 l of each T-2

42、toxin and HT-2 toxin stock solution (4.17 and 4.18), respectively, into a 5 ml volumetric flask, and dilute up to the mark with solvent mixture (4.3). This solution can be stored at 18 C for 12 months. 1) Surfasil is a trade name of a product commercially available from various suppliers. This infor

43、mation 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. BS EN 16923:2017EN 16923:2017 (E) 7 4.22 Mixed internal standard solution, = 1

44、000 ng/ml Dilute 200 l of the internal standard solutions (4.19 and 4.20) with solvent mixture (4.3) in a 5 ml volumetric flask. This solution can be stored at 18 C for 6 months. 4.23 Calibration solutions. For the calibration of the measuring system, prepare calibration solutions within a range fro

45、m 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 Calibration solution Mass concentration per analyte Mass concentration per isotope labelled analyte Mixed standard solution (4.21) Mixed internal standa

46、rd solution (4.22) Solvent mixture (4.3) ng/ml ng/ml l l l 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 5 Apparatus and equipment Usual laboratory apparatus and, in particular, the following. 5.1

47、 Laboratory balance, accuracy of 0,01 g. 5.2 Analytical balance, accuracy of 0,1 mg. 5.3 Ultrasonic bath. 5.4 Laboratory shaker for test tubes. 5.5 Manual dispensers, microlitre syringes or microlitre pipettes for 10 l to 5 ml. 5.6 Dispenser, suitable for 20 ml. 5.7 250 ml-Erlenmeyer flasks with sto

48、ppers, or 250 ml-centrifuge tubes. BS EN 16923:2017EN 16923:2017 (E) 8 5.8 Syringe filters (0,45 m), or centrifugal filters (e.g. DuraporePVDF (0,45 m), or Millipore Ultrafree-MC0,5 ml), fitting with centrifuge for reaction vessels, e.g. Eppendorfvessels2). 5.9 Folded filter, pore size 4 m to 7 m, d

49、iameter 100 mm. 5.10 Laboratory centrifuge. 5.11 Cartridges (6 ml), made from polypropylene (PP) and corresponding frits from polyethylene (PE), or commercially available SPE columns, e.g. CHROMABONDCarbon/Alox/Celite2), 6 ml, 500 mg. 5.12 SPE vacuum/elution station. 5.13 Laboratory shaker, e.g. overhead shaker. 5.14 Laboratory blender, e.g. Ultra Turrax2). 5.15 Test tubes, suitable for a volume up to 10,0 ml. 5.16 Siliconized test tubes (optional). After thorough cleanin