1、BS EN 16877:2016Animal feeding stuffs: Methodsof sampling and analysis Determination of T-2 and HT-2toxins, Deoxynivalenol andZearalenone, in feed materialsand compound feed by LC-MSBSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS EN 16877:2016 BRITISH STANDARDNati
2、onal forewordThis British Standard is the UK implementation of EN 16877:2016. The UK participation in its preparation was entrusted to TechnicalCommittee AW/10, Animal feeding stuffs.A list of organizations represented on this committee can be obtained on request to its secretary.This publication do
3、es not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 89779 5 ICS 65.120; 71.040.50 Compliance with a British Standard cannot confer immunit
4、y from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 December 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS EN 16877:2016EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16877 Nov
5、ember 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 Zearalenone, in feed materials and compound feed by LC-MS Aliments des animaux - Mthodes dchantillonnage et danalyse - Dosage par CL-SM
6、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 Untersuchungsverfahren - Bestimmung von T-2- und HT-2-Toxinen, Deoxynivalenol und Zearalenon in Einzelfuttermitteln und Mischfuttermitteln mittel
7、s 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 conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliogra
8、phical 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 responsibil
9、ity of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic
10、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 United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISAT
11、ION 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 worldwide for CEN national Members. Ref. No. EN 16877:2016 E BS EN 16877:2016EN 16877:2016 (E) 2 Contents Page European forewo
12、rd . 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 Spiking procedure 10 7 Measurements . 11 7.1 General . 11 7.2 LC conditions 11 7.3 MS conditions 11 7.4 Batch c
13、omposition . 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 Precision 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
14、-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) Precision data . 15 Annex B (informative) Examples . 20 B.1 Example 1 20 B.1.1 General . 20 B.1.2 LC conditions
15、 20 B.1.3 MS conditions 21 BS EN 16877:2016EN 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.3.2 LC conditions 24 B.3.3 MS conditions . 24 Annex C (informative) Examples of chromatograms according to the sett
16、ings of the examples in Annex B 26 Bibliography . 31 BS EN 16877:2016EN 16877:2016 (E) 4 European foreword This document (EN 16877:2016) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs - Methods of sampling and analysis”, the secretariat of which is held by NEN. This Europ
17、ean 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 standards shall be withdrawn at the latest by May 2017. Attention is drawn to the possibility that some of the elements of
18、this document may be the subject of patent rights. CEN shall not be held responsible for identifying any or all such patent rights. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. According to the CEN-CENELEC Internal R
19、egulations, 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland
20、, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16877:2016EN 16877:2016 (E) 5 Introduction WARNING The method described in this standard implies the use of reagents tha
21、t 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 measures for the health and safety protection of the personnel prior to use of the standard and to ensure that regulatory and le
22、gal requirements are complied with. BS EN 16877:2016EN 16877:2016 (E) 6 1 Scope This method of analysis is applicable to the determination of HT-2 toxin (HT2) in the tested 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
23、 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 extend beyond the tested ranges. It is the responsibility of the laboratory to prove that the limit of quantitation (LOQ) for HT-2
24、 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 document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, t
25、he latest edition of the referenced document (including any amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987) 3 Principle Finely ground and homogeneous test material is suspended in water. After addition of ethyl acetate the s
26、ample 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 the extraction tube. The organic phase is transferred to a clean vial for possible storage. An aliquote of the organic phase is mi
27、xed with stable-isotope labelled analogues of the analytes and evaporated to dryness in deactivated glass vials. After reconstitution of the dry extract with organic mobile phase modifier and water, and thorough mixing, the analytes are quantified with a Liquid Chromatography-Mass Spectrometry (LC-M
28、S) 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 associated safety problems. It is the responsibility of the user of this standard to take appropriate measures for the health and s
29、afety 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 (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.).
30、 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). 4.10 HT-2 toxin (HT2). BS EN 16877:2016EN 16877:2016 (E) 7 4.11 T-2 toxin (T2). 4.12 Zearalenone (ZON). 4.13 13C15-Deoxynivalenol (
31、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 containing Deoxynivalenol (4.9), HT-2 toxin (4.10), T-2 toxin (4.11), and Zearalenone (4.12) in neat acetonitrile (4.7) at relevant concentrat
32、ions. 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 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 coll
33、aborative 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 deactivated 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 th
34、e 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 the working solution is applicable to the calibration range of the different compounds. Only prepare enough volume for one full calibrat
35、ion. 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 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)
36、. 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) in neat acetonitrile (4.7) at the same concentrations as the respective native compounds in the Multitoxin stock solution (4.17). NOTE Th
37、is 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 solution (4.18) such that six equidistant calibration levels across the calibration range result. Proceed as described in 6.3, “Test so
38、lution”. 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 number of levels may be adjusted to local needs and requirements. BS EN 16877:2016EN 16877:2016 (E) 8 Table 1 Example calibration solutio
39、ns 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 6,4 490 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 fort
40、ification of the tested mycotoxins which is sufficiently stable. 5 Apparatus 5.1 Mill: Single mill or multiple mills capable of comminuting test materials to particle 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)
41、, 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 Spray 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 arb
42、itrary 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 for the convenience of users of this International Standard and their mentioning does not constitute an endorsement by CEN of the products n
43、amed. 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 Ultima PT with ESI interface the following settings have shown to satisfy the performance requirements and provide overall acceptable results (s
44、ee Figure C.3 and Figure C.4 for chromatograms). BS EN 16877:2016EN 16877:2016 (E) 22 B.2.2 LC conditions Dwell volume: the original static mixer was replaced 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; Colum
45、n 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 % 0 92 8 0,67 50 50 8 33 67 8,01 5 95 9,5 5 95 9,51 92 8 11,5 92 8
46、 B.2.3 MS conditions The run is divided in to four segments around the four analyte peaks. The ion transitions in “selected reaction monitoring” mode as in Table B.4 are measured. BS EN 16877:2016EN 16877:2016 (E) 23 Table B.4 Ion transitions for Example 2 Item Segment 1 Segment 2 Segment 3 Segment
47、4 Run time min 0 4,0 4,0 6,2 6,2 7,2 7,2 11,5 Analyte DON + 13C15-DON HT2 + 13C22-HT2 T2 + 13C24-T2 ZON + 13C18-ZON Adduct Protonated Sodium Sodium Deprotonated Transitions (Collision Energy eV) 297- 231 (18), 297- 249 (18), 312- 263 (18), 312- 276 (18) 447- 285 (21), 447- 345 (18), 469- 300 (17), 4
48、69- 362 (17) 489- 245 (24), 489- 327 (21), 513- 260 (20), 513- 344 (19) 317- 131 (18), 317- 175 (18), 335- 185 (18), 335- 290 (18) Cone voltage V 50 85 80 60 Polarity Pos Pos Pos Neg Spray voltage V 2,500 2,500 2,500 2,500 Desolvation temperature C 350 350 350 350 Desolvation Gas Flow L/h 700 700 70
49、0 700 Cone Gas Flow L/h 100 100 100 100 Source temperature C 120 120 120 120 Any tradenames, trademarks, product names and/or suppliers named above are only named 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.3 Example 3 B.3.1 General With a LC-MS system consisting of an Agilent 1200 SL HPLC and an Applied Biosy
