1、December 2009DEUTSCHE NORM English price group 10No part of this standard 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!$8“1562156www.
2、din.deDDIN EN 15791Animal feeding stuffs Determination of Deoxynivalenol in animal feed High performance liquid chromatographic method with UV detectionand immunoaffinity column clean-upEnglish version of DIN EN 15791:2009-12Futtermittel Bestimmung von Deoxynivalenol in Futtermitteln Hochleistungsfl
3、ssigkeitschromatografie-(HPLC-)Verfahren mittels UV-Detektion undReinigung an einer ImmunoaffinittssuleEnglische Fassung DIN EN 15791:2009-12www.beuth.deDocument comprises 17 pagesDIN EN 15791:2009-12 National foreword This standard has been prepared by Technical Committee CEN/TC 327 “Animal feeding
4、 stuffs Methods of sampling and analysis” (Secretariat: NEN, Netherlands). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-03-03 AA Futtermit
5、tel. The DIN Standard corresponding to the International Standard referred to in this document is as follows: EN ISO 3696 DIN ISO 3696 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods 2 EUROPEAN STANDARDNORME EUROPENNEEUROP
6、ISCHE NORMEN 15791September 2009ICS 65.120English VersionProduits alimentaires - Dosage du dsoxynivalnol dans lesaliments pour animaux - Mthode de chromatographieliquide haute performance avec dtection UV et purificationsur colonne dimmuno-affinitFuttermittel - Bestimmung von Deoxynivalenol inFutter
7、m itteln - Hochleis ngsf ssigkeitschromatografie- (HPLC-)Verfahrenmittels UV-Detektion und Reinigung an einer ImmunoaffinittssuleThis European Standard was approved by CEN on 1 August 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for gi
8、ving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three official vers
9、ions (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 the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, B
10、ulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARD
11、IZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15791:2009: EAnimal feeding stuffs - Determination of Deoxyn
12、ivalenol in animal feed -High performance liquid chromatographic method with UV detection and immunoaffinity column clean-upltuEN 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 Calcu
13、lation . 9 9 Precision 10 10 Test report 10 Annex A (informative) Precision data 12 Annex B (informative) Chromatogram . 14 Bibliography . 15 DIN EN 15791:2009-12 EN 15791:2009 (E) 3 Foreword This document (EN 15791:2009) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs”, t
14、he 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 March 2010, and conflicting national standards shall be withdrawn at the latest by March 2010. Attention is
15、drawn 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 Eur
16、opean Free Trade Association. 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 a
17、nd health practices and determine 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 Repu
18、blic, Denmark, Estonia, Finland, 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. DIN EN 15791:2009-12 EN 15791:2009 (E) 4 1 Scope Th
19、is Standard is applicable to the 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 edi
20、tion 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 and test methods (ISO 3696:1987) 3 Principle Deoxynivalenol (DON) is extracted from the commodity using water
21、. The aqueous extract is then cleaned 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 dou
22、ble-distilled water or water of 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
23、Deoxynivalenol (DON), with a minimum 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 ca
24、rried out inside a fume cupboard. 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.
25、 4.5 Mobile Phase Mix 15 parts per 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.
26、 Degas this solution before use. DIN EN 15791:2009-12 EN 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.
27、1) to 5 mg of DON (4.2) for a solution 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
28、/ml. To determine the exact concentration 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 concentra
29、tion of deoxynivalenol, Don, in micrograms 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 deoxy
30、nivalenol in acetonitrile (4.1), (here: 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
31、 the dark for up to 3 months at 4C to 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 sto
32、ck solution (4.7), equivalent to 500 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,
33、into a 5 ml volumetric flask (5.11). Make up to the mark with acetonitrile (4.1). This will result in the DON working solution of 10 g/ml. DIN EN 15791:2009-12 EN 15791:2009 (E) 6 4.10 DON Calibration solutions Calibration solutions are prepared from the 10 g/ml DON working solution (4.9). For examp
34、le, add the volumes of 10 g/ml DON working 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 s
35、aturation of the detector signal, and there 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 clea
36、n-up columns The immunoaffinity (IA) column 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 instructi
37、ons). 5 Apparatus Usual laboratory equipment 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 equivale
38、nt 5.5 Mill (various screens) 5.6 Tumble mixer 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 DIN EN 15791:2009-12 EN 15791:2009 (E) 7 5.10 Filter, binder-free glass microfiber with ca. 2 m pore size 5.11 Volumet
39、ric flasks, with volumes of 2 ml, 5 ml, and 10 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-fr
40、ee, at flows appropriate for the analytical column 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, Octadecylsilan
41、e (ODS) 250 mm x 4,6 mm I.D., 3 m particle size, 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.
42、14.5 UV detector, capable of measuring at 220 nm 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 auto
43、sampler (5.14.4) but with minimum volume of 2,0 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 t
44、ruly representative and has not been damaged or 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 th
45、at has been demonstrated to give complete homogenisation 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. DIN EN 15791:2009-12 EN 15791:2009
46、 (E) 8 6.2 Extraction Weigh a 25,0 g test portion into 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
47、.7). 6.3 Immunoaffinity Column Clean-up Attach a reservoir (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
48、 pass slowly through the column by gravity at a rate 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
49、all the eluates from this stage of the clean-up procedure. 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 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
