DIN EN 16877-2017 Animal feeding stuffs - Methods of sampling and analysis - Determination of T-2 and HT-2 toxins Deoxynivalenol and Zearalenone in feed materials and compound feed.pdf

1、March 2017 English price group 15No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 65.120; 71.040.50!%bVV“2635151www.

2、din.deDIN EN 16877Animal feeding stuffs Methods of sampling and analysis Determination of T2 and HT2 toxins, Deoxynivalenol and Zearalenone, in feed materials and compound feed by LCMS;English version EN 16877:2016,English translation of DIN EN 16877:2017-03Futtermittel Probenahme und Untersuchungsv

3、erfahren Bestimmung von T2 und HT2Toxinen, Deoxynivalenol und Zearalenon in Einzelfuttermitteln und Mischfuttermitteln mittels LCMS;Englische Fassung EN 16877:2016,Englische bersetzung von DIN EN 16877:2017-03Aliments des animaux Mthodes dchantillonnage et danalyse Dosage par CLSM des toxines T2 et

4、HT2, du doxynivalnol et de la zaralnone dans les matires premires pour aliments et les aliments composs;Version anglaise EN 16877:2016,Traduction anglaise de DIN EN 16877:2017-03www.beuth.deDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authorit

5、ative.Document comprises 33 pages 02.17 2 A comma is used as the decimal marker. National foreword This document (EN 16877:2016) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs - Methods of sampling and analysis” (Secretariat: NEN, Netherlands), Working Group WG 5 “Natural

6、 toxins” (Secretariat: NEN, Netherlands), with the active participation of German experts. The responsible German body involved in its preparation was DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products), Working Committee NA 057-

7、03-03 AA Futtermittel (Spiegelausschuss zu CEN/TC 327). The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 5725-2 DIN ISO 5725-2 ISO 5725-5 DIN ISO 5725-5 National Annex NA (informative) Bibliography DIN ISO 5725-2, Accuracy (trueness and

8、precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method DIN ISO 5725-5, Accuracy (trueness and precision) of measurement methods and results Part 5: Alternative methods for the determination of the

9、 precision of a standard measurement method DIN EN 16877:2017-03 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16877 November 2016 ICS 65.120; 71.040.50 English Version Animal feeding stuffs: Methods of sampling and analysis - Determination of T-2 and HT-2 toxins, Deoxynivalenol and Zearaleno

10、ne, in feed materials and compound feed by LC-MS Aliments des animaux - Mthodes dchantillonnage et danalyse - Dosage par CL-SM des toxines T-2 et HT-2, du doxynivalnol et de la zaralnone dans les matires premires pour aliments et les aliments composs Futtermittel - Probenahme- und Untersuchungsverfa

11、hren - Bestimmung von T-2- und HT-2-Toxinen, Deoxynivalenol und Zearalenon in Einzelfuttermitteln und Mischfuttermitteln mittels LC-MS This European Standard was approved by CEN on 26 September 2016. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the condit

12、ions 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

13、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 standa

14、rds 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, Slovakia, Slov

15、enia, 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, B-1000 Brussels 2016 CEN All rights of exploitation in any form and by any means reserved

16、 worldwide for CEN national Members. Ref. No. EN 16877:2016 E EN 16877:2016 (E) 2 Contents Page European foreword . 4 Introduction 5 1 Scope 6 2 Normative references 6 3 Principle . 6 4 Reagents . 6 5 Apparatus . 8 6 Procedures. 9 6.1 Sample preparation 9 6.2 Extraction . 9 6.3 Test solution 10 6.4

17、Spiking procedure 10 7 Measurements . 11 7.1 General . 11 7.2 LC conditions 11 7.3 MS conditions 11 7.4 Batch composition . 11 7.5 Peak identification . 11 7.6 Determination of DON, HT2, T2, and ZON in calibration or and test solutions . 11 7.7 Calibration 11 8 Determination of mass fraction 12 9 Pr

18、ecision 13 9.1 Interlaboratory study . 13 9.2 Repeatability 13 9.2.1 General . 13 9.2.2 HT-2 toxin . 13 9.2.3 T-2 toxin 13 9.2.4 DON 13 9.2.5 ZON 13 9.3 Reproducibility . 13 9.3.1 General . 13 9.3.2 HT-2 toxin . 14 9.3.3 T-2 toxin 14 9.3.4 DON 14 9.3.5 ZON 14 10 Test report 14 Annex A (informative)

19、Precision data . 15 Annex B (informative) Examples . 20 B.1 Example 1 20 B.1.1 General . 20 B.1.2 LC conditions 20 B.1.3 MS conditions 21 DIN EN 16877:2017-03 EN 16877:2016 (E) 3 B.2 Example 2 21 B.2.1 General . 21 B.2.2 LC conditions 22 B.2.3 MS conditions . 22 B.3 Example 3 23 B.3.1 General . 23 B

20、.3.2 LC conditions 24 B.3.3 MS conditions . 24 Annex C (informative) Examples of chromatograms according to the settings of the examples in Annex B 26 Bibliography . 31 DIN EN 16877:2017-03 EN 16877:2016 (E) 4 European foreword This document (EN 16877:2016) has been prepared by Technical Committee C

21、EN/TC 327 “Animal feeding stuffs - Methods of sampling and analysis”, the secretariat of which is held by NEN. 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 2017, and conflicting national st

22、andards shall be withdrawn at the latest by May 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

23、 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 Repub

24、lic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. DIN E

25、N 16877:2017-03 EN 16877:2016 (E) 5 Introduction WARNING The method described in this standard implies the use of reagents that pose a hazard to health. The standard does not claim to address all associated safety problems. It is the responsibility of the user of this standard to take appropriate me

26、asures for the health and safety protection of the personnel prior to use of the standard and to ensure that regulatory and legal requirements are complied with. DIN EN 16877:2017-03 EN 16877:2016 (E) 6 1 Scope This method of analysis is applicable to the determination of HT-2 toxin (HT2) in the tes

27、ted range of 22 g/kg to 178 g/kg, T-2 toxin (T2) in the tested range of 7 g/kg to 50 g/kg, Deoxynivalenol (DON) in the tested range of 88 g/kg to 559 g/kg, and Zearalenone (ZON) in the tested range of 14 g/kg to 430 g/kg in cereals and cereal-based compound animal feed. The actual working ranges may

28、 extend beyond the tested ranges. It is the responsibility of the laboratory to prove that the limit of quantitation (LOQ) for HT-2 and T-2 toxin is 10 g/kg, for DON 100 g/kg, and for ZON 20g/kg. 2 Normative references The following documents, in whole or in part, are normatively referenced in this

29、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 3696:1995, Water for analytical laboratory use - Specification and test methods (

30、ISO 3696:1987) 3 Principle Finely ground and homogeneous test material is suspended in water. After addition of ethyl acetate the sample is agitated. Then sodium sulphate is added to facilitate phase separation and after a delay the sample is centrifuged to pellet particulate matter at the bottom of

31、 the extraction tube. The organic phase is transferred to a clean vial for possible storage. An aliquote of the organic phase is mixed with stable-isotope labelled analogues of the analytes and evaporated to dryness in deactivated glass vials. After reconstitution of the dry extract with organic mob

32、ile phase modifier and water, and thorough mixing, the analytes are quantified with a Liquid Chromatography-Mass Spectrometry (LC-MS) system. 4 Reagents WARNING The method described in this standard implies the use of reagents that pose a hazard to health. The standard does not claim to address all

33、associated safety problems. It is the responsibility of the user of this standard to take appropriate measures for the health and safety protection of the personnel prior to use of the standard and to ensure that regulatory and legal requirements are complied with. 4.1 Water (deionized). 4.2 Water (

34、LC-MS grade, double-distilled or water of grade 1 as defined in EN ISO 3696:1995). 4.3 Methanol (LC-MS grade). 4.4 Methanol (p.a.). 4.5 Ethyl acetate (p.a.). 4.6 Formic acid (98-100 %, LC-MS grade). 4.7 Acetonitrile (LC-MS grade). 4.8 Sodium sulfate, anhydrous, granulated. 4.9 Deoxynivalenol (DON).

35、4.10 HT-2 toxin (HT2). DIN EN 16877:2017-03 EN 16877:2016 (E) 7 4.11 T-2 toxin (T2). 4.12 Zearalenone (ZON). 4.13 13C15-Deoxynivalenol (13C15-DON). 4.14 13C22-HT-2 toxin (13C22-HT2). 4.15 13C24-T-2 toxin (13C24-T2). 4.16 13C18-Zearalenone (13C18-ZON). 4.17 Multitoxin stock solution: A mixture contai

36、ning Deoxynivalenol (4.9), HT-2 toxin (4.10), T-2 toxin (4.11), and Zearalenone (4.12) in neat acetonitrile (4.7) at relevant concentrations. When preparing this solution the certified purities of the mycotoxin reference materials need to be properly accounted for. In any case the purities shall be

37、95 %. NOTE 1 3,2 g/ml DON, 0,5 g/ml HT-2 toxin, 0,3 g/ml T-2 toxin, and 0,3 g/ml ZON in neat acetonitrile have been used during the collaborative study. This solution is stable for three months in the dark at 28 C. To compare a new stock solution against an old one add 25 l of each into separate dea

38、ctivated vials (5.6) and proceed as described in “Test solution” (6.3). NOTE 2 If 6.4“Spiking procedure” is executed at least 6 ml of the stock solution are needed. 4.18 Multitoxin working solution: Dilute Multitoxin stock solution (4.17) with Methanol (4.3) such that the resulting concentration in

39、the working solution is applicable to the calibration range of the different compounds. Only prepare enough volume for one full calibration. NOTE Adding 188 l of the Multitoxin stock solution described in 4.17, Note 1 to a 3 ml volumetric flask and making up to the mark with methanol will result in

40、a solution containing 0,2 g/ml DON, 0,031 g/ml HT-2 toxin, 0,019 g/ml T-2 toxin, and 0,019 g/ml ZON in methanol/acetonitrile (94/6, v/v). 4.19 Multi internal standard (ISTD) stock solution: A mixture containing 13C15-DON (4.13), 13C22-HT-2 toxin (4.14), 13C24-T-2 toxin (4.15), and 13C18-ZON (4.16) i

41、n neat acetonitrile (4.7) at the same concentrations as the respective native compounds in the Multitoxin stock solution (4.17). NOTE This solution is stable for three months in the dark at (28) C. 4.20 Calibration: To six deactivated glass vials (5.6) add different volumes of the Multitoxin working

42、 solution (4.18) such that six equidistant calibration levels across the calibration range result. Proceed as described in 6.3, “Test solution”. Table 1 below shows example calibration levels using the solution described in the Note to 4.18 above. Once it has been shown that there is linearity the n

43、umber of levels may be adjusted to local needs and requirements. DIN EN 16877:2017-03 EN 16877:2016 (E) 8 Table 1 Example calibration solutions Volume of Multitoxin working solution (4.18.) Total mass of analyte per vial l ng DON HT-2 T-2 ZON 25 5 0,78 0,48 0,48 180 36 5,6 3,4 3,4 335 67 10 6,4 6,4

44、490 98 15 9,3 9,3 645 129 20 12 12 800 160 25 15 15 4.21 Quality control material: An appropriate material with natural contamination or fortification of the tested mycotoxins which is sufficiently stable. 5 Apparatus 5.1 Mill: Single mill or multiple mills capable of comminuting test materials to p

45、article sizes of 231 (16), 297- 249 (13), 312- 263 (9), 312- 276 (9) 447- 285 (22), 447- 345 (20), 469- 300 (19), 469- 362 (18) 489- 245 (30), 489- 327 (25), 513- 260 (26), 513- 344 (23) 317- 131 (25), 317- 175 (22), 335- 185 (26), 335- 290 (21) Tube Lens V 80 110 140 80 Polarity Pos Pos Pos Neg Spr

46、ay Voltage V 2800 2800 2400 2000 Vaporizer temperature C 350 350 350 350 Sheath Gas Pressure arbitrary units 30 30 30 30 Aux Gas Pressure arbitrary units 10 10 10 10 Transfer Capillary temperature C 320 320 320 320 Any tradenames, trademarks, product names and/or suppliers named above are only named

47、 for the convenience of users of this International Standard and their mentioning does not constitute an endorsement by CEN of the products named. Equivalent products may lead to the same results. B.2 Example 2 B.2.1 General With a LC-MS system consisting of a HP1100 HPLC and a Micromass Quattro Ult

48、ima PT with ESI interface the following settings have shown to satisfy the performance requirements and provide overall acceptable results (see Figure C.3 and Figure C.4 for chromatograms). DIN EN 16877:2017-03 EN 16877:2016 (E) 22 B.2.2 LC conditions Dwell volume: the original static mixer was repl

49、aced by a low-volume peek mixing Tee; Injection volume: 5 l; Column Supelco Ascentis Express C18, 75 2,1 mm, particle size 2,7 m fused-core; Column temperature: 40 C; Flow rate: 0,3 ml/min; Mobile phase A: 0,1 % formic acid (4.6.) in water (4.2); Mobile phase B: 0,1 % formic acid (4.6.) in methanol (4.3). Table B.3 Gradient settings Run time min Mobile phase A % Mobile phase B %