DIN EN 16006-2011 Animal feeding stuffs - Determination of the sum of fumonisin B and B in compound animal feed with immunoaffinity clean-up and RP-HPLC with fluorescence detection.pdf

1、October 2011 Translation by DIN-Sprachendienst.English price group 12No 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).IC

2、S 65.120!$t“1819393www.din.deDDIN EN 16006Animal feeding stuffs Determination of the sum of fumonisin and in compound animal feed with immunoaffinity clean-up and RP-HPLC with fluorescencedetection after pre- or post-column derivatisationEnglish translation of DIN EN 16006:2011-10Futtermittel Bestim

3、mung der Summe der Fumonisine und in Mischfutter durchReinigunganeiner Immunoaffinittssule und RP-HPLC mit Fluoreszenzdetektion nachVor-oderNachsulenderivatisierungEnglische bersetzung von DIN EN 16006:2011-10Aliments pour animaux Dosage de la somme des fumonisines et dans les aliments pour animaux

4、avec purification par immuno-affinit et RP-HPLC avec dtection par fluorescenceaprsdrivation pr ou post colonneTraduction anglaise de DIN EN 16006:2011-10www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authoritative.B1B2B1 B2B1 B210.1125DIN EN 16

5、006:2011-10 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs Methods of sampling and analysis” (Secretariat: NEN, Netherlands). The responsible German body involved in its preparation was the Normenaussc

6、huss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-03-03 AA Futtermittel. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association, and supports essen

7、tial requirements of EU Directive(s). The DIN Standards corresponding to the International Standards referred to in this document are as follows, whereby EN ISO Standards are only listed below if these have not been published as DIN EN ISO Standards with the same number: EN ISO 3696 DIN ISO 3696 Nat

8、ional Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods 2 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16006 August 2011 ICS 65.120 English Version Animal feeding stuffs - Determination of the sum of fumonisin B1and B2in comp

9、ound animal feed with immunoaffinity clean-up and RP-HPLC with fluorescence detection after pre- or post-column derivatisation Aliments pour animaux - Dosage de la somme des fumonisines B1et B2dans les aliments pour animaux avec purification par immuno-affinit et RP-HPLC avec dtection par fluorescen

10、ce aprs drivation pr- ou post-colonne Futtermittel - Bestimmung der Summe der Fumonisine B1und B2in Mischfutter durch Reinigung an einer Immunoaffinittssule und RP-HPLC mit Fluoreszenzdetektion nach Vor- oder Nachsulenderivatisierung This European Standard was approved by CEN on 25 June 2011. CEN me

11、mbers 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 applica

12、tion 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 Manag

13、ement 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, Netherl

14、ands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2011 CEN All rights of exploitation in

15、 any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16006:2011: E2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents and materials 45 Apparatus .96 Sample preparation 107 Measurements . 128 Determination of concentrations 159 Precision 1

16、610 Test report . 17Annex A (informative) Precision data . 18Annex B (informative) Examples of a chromatogram . 21Bibliography . 23EN 16006:2011 (E) DIN EN 16006:2011-10 3 Foreword This document (EN 16006:2011) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs”, the secretar

17、iat 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 February 2012, and conflicting national standards shall be withdrawn at the latest by February 2012. Attention is drawn

18、 to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC 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

19、 Free Trade Association. WARNING The use of this protocol can involve hazardous materials, operations and equipment. This protocol does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this protocol to establish appropriate safety and he

20、alth practices and determine the applicability of regulatory limitations prior to use. WARNING Fumonisins are toxic. Gloves and safety glasses should be worn at all times and all standard and sample preparation stages should be carried out in a fume cupboard. According to the CEN/CENELEC Internal Re

21、gulations, 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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg,

22、Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. EN 16006:2011 (E) DIN EN 16006:2011-10 4 1 Scope This European Standard is applicable to the determination of Fumonisin B1 160 g NaCl (4.5); 72 g Na2HPO4*12 H2O (4.6). Adjust

23、 to pH 7,4 with 10 mol/l HCl and make up to 2 000 ml. 4.16 PBS Ready to use Dilute 100 ml of PBS concentrate (4.15) to 1 000 ml with water (4.1), or PBS tablets, Phosphate buffered saline tablets one tablet dissolved in 200 ml of water (4.1) yields 0,01 mol/l phosphate buffer, 0,002 7 mol/l potassiu

24、m chloride and 0,137 mol/l sodium chloride, pH 7,4, at 25C (e.g. Sigma P4417). 4.17 Diluent Mix 50 parts per volume methanol (4.2) with 50 parts per volume water (4.1). 4.18 Extraction solvent Mix 50 parts per volume methanol (4.2) with 50 parts per volume of PBS (4.16). EN 16006:2011 (E) DIN EN 160

25、06:2011-10 6 4.19 Reaction buffer 4.19.1 Post-column derivatisation (0,006 mol/l OPA, 0,006 mol/l NAC, 0,384 mol/l sodium carbonate, 0,216 mol/l boric acid and 0,108 mol/l potassium sulphate): Dissolve 40,7 g sodium carbonate (4.8), 13,4 g boric acid (4.9) and 18,8 g potassium sulphate (4.10) per 1,

26、0 l of water (4.1); stir for 10 min; add 800 mg of OPA (4.12) per 1,0 l of the above solution; add 1 g of NAC (4.11) per 1,0 l of the above solution; stir for 10 min; sonicate for 15 min; stir for 10 min; sonicate again for 15 min and filter the solution through a 0,45 m nylon filter (5.17). Proper

27、dissolution of the OPA is very important! The reaction buffer should not be changed within a sequence of HPLC runs. Prepare fresh for every sequence of HPLC runs. 4.19.2 Pre-column derivatisation (0,05 mol/l OPA, 0,12 mol/l BME, 0,08 mol/L disodium tetraborate, 16,7% methanol): Dissolve 40 mg OPA (4

28、.12) in 1,0 ml methanol (4.2); mix until completely dissolved; add 5,0 ml of a 0,1 mol/l solution of disodium tetraborate decahydrate (3,8 g / 100 ml; 4.7); mix thoroughly; add 50 l of BME (4.13), and mix thoroughly. Alternatively: Phthaldialdehyde Reagent. 4.20 FB1or EN 16006:2011 (E) DIN EN 16006:

29、2011-10 Separate certified solutions of Fumonisin FB1and FB2 in appropriate solvents that will be mixed such that a stock solution containing ca. 50 g/ml of each is obtained. Calculate exact concentrations from certificates and dilutions. 7 NOTE 1 The above solutions (4.20) may also be prepared grav

30、imetrically by accurately weighing the dry substance and the solvent used to dissolve it. Accurately measuring the volume of the solvent is also allowed. NOTE 2 The above solutions may be stored for up to six months at below -18C in the dark. 4.21 FB1 add 200 ml of extraction solvent (4.18), cap, an

31、d shake vigorously by hand, so that the material disperses evenly; put on a shaker (5.4) for 120 min; choose speed such that the material is mixed well without collecting in the top of the flask; allow the extracted sample to settle after shaking; of the supernatant take 5,0 ml and dilute with PBS (

32、4.16) to a total volume of 50,0 ml and mix; prepare a filter funnel (5.10) with a glass micro fibre filter (5.9), and EN 16006:2011 (E) DIN EN 16006:2011-10 filter the diluted supernatant of the extracted sample into a new flask (5.5). 11 The diluted filtered extract may be stored at 4C to 10C overn

33、ight. In case of a highly contaminated material above 10 000 g/kg (see Clause 8), take 10,0 ml of the stored filtered diluted extract and dilute again with PBS (4.16) to a total volume of 50,0 ml and mix. 6.3 Clean up Take one IAC (immunoaffinity column; 4.23) per extract; attach a reservoir (5.12),

34、 do not empty storage solution from column; to the reservoir add 25,0 ml of the filtered diluted extract (6.2); open the column outlet; allow everything to pass slowly through the column (flow rate should be one drop per second to two drops per second); after the extract has passed completely, wash

35、the IAC with 10 ml of PBS (4.16); pass air through the IAC (e.g. using a properly fitted large syringe) in order to expel excess PBS; place a 5 ml volumetric flask (5.13) or a 5 ml graduated cylinder (5.6) underneath the IAC and add 5 x 500 l of methanol (4.2) to the IAC (add next aliquot only after

36、 previous has completely passed); collect all the eluate in the volumetric flask (5.13) or graduated cylinder (5.6); add 2,0 ml of water (4.1) to the IAC after all of the methanol (4.2) has passed through the column; continue to collect the eluate in the same volumetric flask or graduated cylinder,

37、and carefully pass air through the column in order to collect most of the applied water (4.1). 6.4 Test solution For pre-column derivatisation: make up the content of the volumetric flask or graduated cylinder to the 5 ml mark with water (4.1); For post-column derivatisation: add 5 l of formic acid

38、(4.14) and make up the content of the volumetric flask or graduated cylinder to the 5 ml mark with water (4.1); Mix the content of the volumetric flask or graduated cylinder and transfer an aliquot to an autosampler vial (5.11). This test solution may be stored at 4C to 10C for up to two days. 6.5 S

39、piking procedure To determine recovery spike a fumonisin-free representative compound animal feed material with FB12) aspire 40 l test solution (6.4); 3) aspire 20 l pre-column reaction buffer (4.19.2); 4) mix 20 times, and 5) inject all. The above can be done manually (adjusting the total volume wh

40、ile maintaining the relative volumes if necessary) if it is ascertained that the solution is injected within 3 minutes after mixing. It is also important that the time period between mixing and injecting is the same for all test and calibration solutions. b) Injection volume: 80 l; c) Column tempera

41、ture: 40C; d) Flow: 1,0 ml/ min; e) Fluorescence detector: Excitation : 335 nm; Emission : 440 nm (it should be checked if these are local maxima for the fluorescence detector in use); f) Mobile phase: 1) A: 0,5% formic acid (4.14) in water (4.1); 2) B: 0,5% formic acid (4.14) in methanol (4.2). g)

42、Gradient settings (HPLC dwell volume 0,8 ml) see Table 2. EN 16006:2011 (E) DIN EN 16006:2011-10 13 Table 2 Gradient settings for the pre-column derivatisation Time (min) B (%) 0 69,514 79 14,01 10017,01 10017,02 69,5 20 69,5Instruments with different dwell volume will need adjustment of the gradien

43、t to achieve the same separation as shown in Annex A. The aim should be an apparent capacity factor at elution for FB1of k 3. 7.1.3 Post-column derivatisation Instructions for self-assembled system (5.16.5): The flow path to the chromatographic column (5.16.3) is unchanged from normal operation. The

44、 outlet of the column is connected to one of the outside ports of a mixing Tee (see 5.16.5). The tubing from column to mixing Tee should be as short as possible. The other outside port of the mixing Tee is connected to the outlet of a pump (see 5.16.5) delivering the reagent flow. This connection sh

45、ould be made of a long piece of 0,005“ ID PEEK tubing (see 5.16.5) so that a sufficient back pressure is created for the reagent pump to work properly. It is of utmost importance that the reagent flow is delivered pulsation-free. A slight pulsation can be minimized be introducing a large damping vol

46、ume between the pump and the back pressure creating PEEK tubing. Large ID PEEK tubing can serve this purpose. The remaining centre port of the mixing Tee is connected through a reagent loop to the fluorescence detector. The length, and therefore the volume, of this reagent loop is a balance between

47、retaining the resolution of the chromatographic column (short) and achieving complete reaction (long). The internal diameter is of lesser importance. If chosen too small excessive back pressure will be created. Satisfying results were achieved with a 2,5 m length of 0,02“ ID PEEK tubing. Using the e

48、quipment outlined in 5.16, the following conditions have shown to produce satisfying results: a) Injection volume: 50 l b) Column temperature: 45C c) Flow : 1,2 ml/ min (mobile phase); 0,45 ml/min (post-column reagent (4.19.1) d) Fluorescence detector: Excitation : 335 nm; Emission : 440 nm (it shou

49、ld be checked if these are local maxima for the fluorescence detector in use). e) Mobile phase: EN 16006:2011 (E) DIN EN 16006:2011-10 14 1) A: 0,1% formic acid (4.14) in water (4.1); 2) B: 0,1% formic acid (4.14) in acetonitrile (4.3). Gradient settings: see Table 3. Table 3 Gradient settings for the post column-derivatisation Time (min) B (%) 0 3413 34 13,01 9516 9516,01 34 19 34This separation is