BS EN 15791-2009 Foodstuffs - Determination of deoxynivalenol in animal feed - HPLC method with immunoaffinity column clean-up《食品 动物饲料中脱氧萎镰菌醇的检测 免疫亲和柱纯化HPLC法》.pdf

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1、BS EN 15791:2009ICS 65.120NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDFoodstuffs Determination ofDeoxynivalenolin animal feed HPLC method withimmunoaffinity columnclean-upThis British Standardwas published underthe authority of theStandards Policy andStrateg

2、y Committee on 30September 2009 BSI 2009ISBN 978 0 580 61807 9Amendments/corrigenda issued since publicationDate CommentsBS EN 15791:2009National forewordThis British Standard is the UK implementation of EN 15791:2009.The UK participation in its preparation was entrusted to TechnicalCommittee AW/10,

3、 Animal feeding stuffs.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a British Standard cannot

4、confer immunityfrom legal obligations.BS EN 15791:2009EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15791September 2009ICS 65.120English VersionFoodstuffs - Determination of Deoxynivalenol in animal feed -HPLC method with immunoaffinity column clean-upProduits alimentaires - Dosage du dsoxynival

5、nol dans lesaliments pour animaux - Mthode de chromatographieliquide haute performance avec dtection UV et purificationsur colonne dimmuno-affinitFuttermittel - Bestimmung von Deoxynivalenol inFuttermitteln - HPLC-Verfahren mit Reinigung an einerImmunoaffinittssuleThis European Standard was approved

6、 by CEN on 1 August 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandar

7、ds may be obtained on application to the CEN 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 translationunder the responsibility of a CEN member into its own language and notified to th

8、e CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherla

9、nds, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any f

10、orm and by any means reservedworldwide for CEN national Members.Ref. No. EN 15791:2009: EBS EN 15791:2009EN 15791:2009 (E) 2 Contents Page Foreword . 3 1 Scope 4 2 Normative references . 4 3 Principle . 4 4 Reagents 4 5 Apparatus. 6 6 Procedure. 7 7 HPLC determination 8 8 Calculation . 9 9 Precision

11、 10 10 Test report 10 Annex A (informative) Precision data 12 Annex B (informative) Chromatogram . 14 Bibliography . 15 BS EN 15791:2009EN 15791:2009 (E) 3 Foreword This document (EN 15791:2009) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs”, the secretariat of which is

12、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 March 2010, and conflicting national standards shall be withdrawn at the latest by March 2010. Attention is drawn to the possibility th

13、at 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 Free Trade Associatio

14、n. WARNING The use of this standard can involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and det

15、ermine the applicability of regulatory limitations prior to use. 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, Cyprus, Czech Republic, Denmark, Estonia, Fin

16、land, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 15791:2009EN 15791:2009 (E) 4 1 Scope This Standard is applicable to the

17、 determination of deoxynivalenol (DON) in animal compound feed at concentrations of 150 g/kg up to at least 4 000 g/kg. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated

18、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 3696:1987) 3 Principle Deoxynivalenol (DON) is extracted from the commodity using water. The aqueous extract is then cl

19、eaned up with an immunoaffinity column to remove impurities from the sample. Subsequently DON is quantitatively determined by HPLC with UV detection. 4 Reagents During the analysis, unless otherwise stated, use only reagents of recognised analytical grade and only double-distilled water or water of

20、grade 1 as defined in EN ISO 3696. Solvents shall be of quality for HPLC analysis. 4.1 Acetonitrile WARNING Acetonitrile is hazardous and handling shall be carried out inside a fume cupboard. Appropriate safety equipment (lab coat, goggles, gloves) shall be worn. 4.2 Deoxynivalenol (DON), with a min

21、imum purity of 97 % WARNING Deoxynivalenol is highly toxic. Gloves and safety glasses shall be worn at all times and all standard and sample preparation stages shall be carried out in a fume cupboard. 4.3 Methanol WARNING Methanol is hazardous and handling shall be carried out inside a fume cupboard

22、. Appropriate safety equipment (lab coat, goggles, gloves) shall be worn. 4.4 Glacial acetic acid WARNING Glacial acetic acid is hazardous and handling shall be carried out inside a fume cupboard. Appropriate safety equipment (lab coat, goggles, gloves) shall be worn. 4.5 Mobile Phase Mix 15 parts p

23、er volume of methanol (4.3) with 84,9 parts per volume of water and 0,1 parts of glacial acetic acid (4.4). The exact amount of methanol used and whether acetic acid will have to be used depends on the HPLC column chosen for analysis and must be adjusted if necessary. Degas this solution before use.

24、 BS EN 15791:2009EN 15791:2009 (E) 5 4.6 Wash Solvent Mix 50 parts per volume of methanol (4.3) with 50 parts per volume of water. 4.7 DON stock solution 250 g Deoxynivalenol per ml of Acetonitrile. May be prepared by the following: Add 4,0 ml of acetonitrile (4.1) to 5 mg of DON (4.2) for a solutio

25、n of 1,25 mg/ml. Dilute 1 000 l of the 1,25 mg/ml solution to 5,0 ml with acetonitrile for the stock solution of 250 g/ml. Dilute 200 l of the 250 g/ml stock solution in a 2,0 ml volumetric flask (5.11) with acetonitrile to create a diluted stock solution of 25 g/ml. To determine the exact concentra

26、tion record the absorption curve of this 25 g/ml diluted stock solution with the spectrophotometer (5.15) in the range of 200 nm to 270 nm in a 1 cm quartz cell with acetonitrile (4.1) as reference. Determine the absorption at 220 nm. Calculate the mass concentration of deoxynivalenol, Don, in micro

27、grams per millilitre using equation 1: ()dMAmlgDON=100/25max(1) where: Amaxis the absorption determined at the maximum of the absorption curve (here: at 220 nm); M is the molar mass of deoxynivalenol (M = 296,3 g/mol); is the molar absorption coefficient of deoxynivalenol in acetonitrile (4.1), (her

28、e: 681 m2/mol 12,6 m2/mol 1); d is the optical path length of the quartz cell in centimetres (here: 1 cm). Calculate the exact concentration of the 250 g/ml stock solution by the following equation: ()()10/25/250 = mlgmlgDONDON (2) Stock solution may be stored in the dark for up to 3 months at 4C to

29、 8C or at least 6 months at below -18 C. NOTE Stock solution preparation can be carried out gravimetrically by accurately weighing the DON standard material and the solvent used to dissolve it. 4.8 DON spiking solution Pipette an aliquot of the calibrated DON stock solution (4.7), equivalent to 500

30、g DON, into a 5 ml volumetric flask (5.11). Make up to the mark with acetonitrile (4.1). This will result in the spiking solution of 100 g/ml. 4.9 DON working solution Pipette an aliquot of the calibrated diluted DON stock solution (4.7), equivalent to 50 g DON, into a 5 ml volumetric flask (5.11).

31、Make up to the mark with acetonitrile (4.1). This will result in the DON working solution of 10 g/ml. BS EN 15791:2009EN 15791:2009 (E) 6 4.10 DON Calibration solutions Calibration solutions are prepared from the 10 g/ml DON working solution (4.9). For example, add the volumes of 10 g/ml DON working

32、 solution (4.9) shown in the table below into 10 ml volumetric flasks (5.11). Fill the flasks up to the mark with mobile phase (4.5). Deviations are permissible as long as the lowest level is above the limit of detection, the highest level does not lead to saturation of the detector signal, and ther

33、e are at least two more levels equidistantly in between. Table 1 Preparation of standard solutions Calibration solution DON Working solution (4.9) (l) DON concentration ng/ml 1 450 450 2 375 3753 300 300 4 225 2255 150 150 6 75 754.11 DON immunoaffinity clean-up columns The immunoaffinity (IA) colum

34、n contains antibodies raised against deoxynivalenol. The column shall have a capacity of not less than 2 500 ng of DON and shall give a recovery of not less than 70% when 25 ng of DON are applied in 1 ml to-2 ml of water (depending on manufacturers instructions). 5 Apparatus Usual laboratory equipme

35、nt and in particular the following: 5.1 Analytical balance, with d=0,001g for sample weighing, with d=0,01mg for gravimetrical preparation of the DON stock solution (4.7) 5.2 Homogeniser/ High Speed Blender 5.3 Laboratory shaker 5.4 Vortex Mixer, or equivalent 5.5 Mill (various screens) 5.6 Tumble m

36、ixer 5.7 Screw cap flasks, with volumes of 250 ml and 500 ml 5.8 Funnels, of appropriate size 5.9 Filter, cellulose with ca. 30 m pore size BS EN 15791:2009EN 15791:2009 (E) 7 5.10 Filter, binder-free glass microfiber with ca. 2 m pore size 5.11 Volumetric flasks, with volumes of 2 ml, 5 ml, and 10

37、ml 5.12 Graduated pipettes, with volumes of 1 ml and 5 ml 5.13 Adjustable Pipettors or gas-tight glass syringes, with volumes of 100 l and 1 000 l 5.14 HPLC system consisting of: 5.14.1 Pump, capable at least of generating binary gradients, pulsation-free, at flows appropriate for the analytical col

38、umn 5.14.2 Analytical column Any column which allows for sufficient separation of deoxynivalenol from other interfering components is suitable. Examples are: Phenomenex ODS3-Prodigy (15 cm x 4,6 mm i.d.), 5 m particle size, 100 pore size, Octadecylsilane (ODS) 250 mm x 4,6 mm I.D., 3 m particle size

39、, 80 pore size, Octadecyl (C18) 250 mm x 4,6 mm I.D., 5 m particle size, 180 pore size 5.14.3 Pre-column (optional), appropriate for the analytical column used 5.14.4 Autosampler, capable of injecting appropriate volumes with sufficient repeatability 5.14.5 UV detector, capable of measuring at 220 n

40、m 5.14.6 Data collection system 5.15 UV spectrophotometer, for checking the concentration of the DON stock solution (4.7) 5.16 Reservoirs, of appropriate size with adaptors to fit the immunoaffinity columns 5.17 Glass vials, of appropriate size for autosampler (5.14.4) but with minimum volume of 2,0

41、 ml 5.18 Syringe filter unit, polyamide (nylon) with 0,45 m pore size 5.19 Evaporator, capable of maintaining 50C with a steady stream of air or nitrogen 6 Procedure 6.1 Sample preparation It is important that the laboratory receives a sample which is truly representative and has not been damaged or

42、 changed during transport or storage. Samples should be taken and prepared in accordance with European legislation where applicable 2. Samples should be finely ground and thoroughly mixed using a mill (5.5) and a tumble mixer (5.6) or another process that has been demonstrated to give complete homog

43、enisation before a test portion is removed for analysis. In all instances if the sample has been frozen allow it to thaw completely before sampling. Mix the sample thoroughly before removing an analytical test portion. BS EN 15791:2009EN 15791:2009 (E) 8 6.2 Extraction Weigh a 25,0 g test portion in

44、to a 250 ml or 500 ml screw cap flask (5.7). Add 200 ml of deionised water, cap and shake for 1 hour with a shaker (5.3). Prepare a funnel (5.8) with filter paper (5.9). Filtrate the extracted sample into a clean 250 ml or 500 ml screw cap flask (5.7). 6.3 Immunoaffinity Column Clean-up Attach a res

45、ervoir (5.16) to an immunoaffinity column. Add 8 ml of deionised water. Then transfer 2,0 ml of the filtered extract (see above; 0,5 ml in case of analytical results above 4 000 g/kg; see section 8) into the reservoir (5.16). Allow this solution to pass slowly through the column by gravity at a rate

46、 of 1 drop/s to 2 drops/s. When the extract has passed completely through the immunoaffinity column, pass 5 ml of deionised water through the column. Remove residual liquid by passing nitrogen or air through the column for about 5 seconds. Discard all the eluates from this stage of the clean-up proc

47、edure. Finally, place a HPLC autosampler vial (5.17) under the column and pass 0,5 ml of methanol (4.3) through the column by gravity collecting the eluate. After the last drops of methanol have passed through the column allow the methanol to remain on the column for approximately 1 minute. Then add

48、 another 1,0 ml of methanol (4.3) and continue to collect the eluate. Carefully pass nitrogen or air through the column in order to collect any residual eluate. NOTE Alternatively to the manual procedure (6.4) the immunoaffinity clean-up and elution may be performed with an automatic sample preparat

49、ion unit, provided that volumes and flow rates remain unchanged. 6.4 Preparation of the test solution for HPLC analysis Place the vial with the eluate in the evaporator (5.19) and carefully evaporate to dryness under nitrogen or air at ca. 50C. Immediately afterwards cool the HPLC vial to ambient temperature and reconstitute the residue with 0,50 ml of HPLC mobile phase (4.5). Mix well with vortex mixer (5.4) for at least 30 seconds to ensure the residue is completely re-dissolved. In case of