DIN EN 15891-2010 Foodstuffs - Determination of deoxynivalenol in cereals cereal products and cereal based foods for infants and young children - HPLC method with immunoaffinity co.pdf

1、December 2010 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).I

2、CS 67.060; 67.230!$lYX“1735453www.din.deDDIN EN 15891Foodstuffs Determination of deoxynivalenol in cereals, cereal products and cerealbased foods for infants and young children HPLC method with immunoaffinity column cleanup and UV detectionEnglish translation of DIN EN 15891:2010-12Lebensmittel Best

3、immung von Deoxynivalenol in Getreide, Getreideerzeugnissen und Suglings- undKleinkindernahrung auf Getreidebasis HPLC-Verfahren mit Reinigung an einer Immunoaffinittssule und UV-DetektionEnglische bersetzung von DIN EN 15891:2010-12Denres alimentaires Dosage du doxynivalnol dans les crales, les pro

4、duits craliers, et crales pourdjeuner en alimentation infantile Mthode par CLHP avec purification sur colonne dimmunoaffinit et dtection UVTraduction anglaise de DIN EN 15891:2010-12www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authoritative.2

5、111.10 DIN EN 15891:2010-12 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). Preliminary work was done by Working Group WG 5 “Biotoxins”. The responsible German b

6、ody involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-01-03 AA Biotoxine. Mycotoxins are extremely toxic secondary metabolites produced by fungi. Food that is grown, harvest

7、ed or stored under humid conditions can be infected by fungi whose metabolites invade the food. Because some mycotoxins are extremely toxic for humans, their reliable detection is especially important for consumer health and protection. In Germany the regulation Mykotoxin-Hchstmengenverordnung gover

8、ns the maximum allowable content of mycotoxins in food. Since 2004 this regulation has contained specifications not only for aflatoxins, but also for ochratoxin A, fumonisins, deoxynivalenol (DON), and zearalenone. National regulations have been supplemented by EU-wide regulations regarding the maxi

9、mum content of contaminants in food since 2001. The maximum content of mycotoxins in certain foods is also laid down in various other regulations. The DIN Standard corresponding to the European Standard referred to in this document is as follows: EN ISO 3696 DIN ISO 3696 National Annex NA (informati

10、ve) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods 2 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15891 September 2010 ICS 67.060; 67.230 English Version Foodstuffs - Determination of deoxynivalenol in cereals, cereal products and cereal based f

11、oods for infants and young children -HPLC method with immunoaffinity column cleanup and UV detection Denres alimentaires - Dosage du doxynivalnol dans les crales, les produits craliers, et crales pour djeuner en alimentation infantile - Mthode par CLHP avec purification sur colonne dimmunoaffinit et

12、 dtection UV Lebensmittel - Bestimmung von Deoxynivalenol in Getreide, Getreideerzeugnissen und Suglings- und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinittssule und UV-Detektion This European Standard was approved by CEN on 28 August 2010. CEN members ar

13、e 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 application to

14、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 translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the sam

15、e 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, Netherlands, Norway, Poland, Po

16、rtugal, 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 2010 CEN All rights of exploitation in any form and by any mea

17、ns reserved worldwide for CEN national Members. Ref. No. EN 15891:2010: EEN 15891:2010 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Apparatus .76 Procedure .87 HPLC analysis . 108 Calculation . 129 Precision 1210 Test report . 14Annex A (informative) Typ

18、ical chromatogram 15Annex B (informative) Precision data . 16Bibliography . 19DIN EN 15891:2010-12 EN 15891:2010 (E) 3 Foreword This document (EN 15891:2010) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This Europe

19、an 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 2011, and conflicting national standards shall be withdrawn at the latest by March 2011. Attention is drawn to the possibility that some of the elements

20、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. Annexes A and B are informative. This document has been prepared under a mandate give to CEN by the European Commission and the European Free Trade As

21、sociation. 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, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Icela

22、nd, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15891:2010-12 EN 15891:2010 (E) 4 1 Scope This European Standard specifies a method for the determination of deoxyni

23、valenol (DON) in cereals (grain and flour), cereal based foods and cereal based foods for infants and young children by high performance liquid chromatography (HPLC) with immunoaffinity cleanup and UV detection. This method has been validated in three interlaboratory studies. The first study was for

24、 the analysis of samples of wheat, rice flour, oat flour, maize, polenta, and wheat based breakfast cereal ranging from 85,4 g/kg to 1 768 g/kg, the second study was for wheat and maize ranging from 165 g/kg to 4 700 g/kg and the third study was for cereal based foods for infants and young children

25、ranging from 58 g/kg to 452 g/kg. For further information on the validation, see Clause 9 and Annex B. 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 resp

26、onsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references,

27、 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 (ISO 3696:1987) 3 Principle Deoxynivalenol is extracted from the sample usin

28、g water. The aqueous extract is cleaned up on an immunoaffinity column to remove impurities from the sample. Deoxynivalenol is then quantitatively determined by HPLC and UV detection. 4 Reagents 4.1 General Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3

29、696:1995, unless otherwise specified. Solvents shall be of quality for HPLC analysis. Commercially available solutions with equivalent properties to the reagents listed may be used. 4.2 Disodium hydrogen phosphate, anhydrous or Na2HPO412 H2O. 4.3 Potassium chloride, KCl. 4.4 Potassium dihydrogen pho

30、sphate, KH2PO4. 4.5 Sodium chloride, NaCl. 4.6 Sodium hydroxide, NaOH. 4.7 Hydrochloric acid solution, mass fraction w(HCl) = 37 % in water. DIN EN 15891:2010-12 EN 15891:2010 (E) 5 4.8 Hydrochloric acid solution, substance concentration c(HCl) = 0,1 mol/l. Dilute 8,28 ml of hydrochloric acid soluti

31、on (4.7) to 1 l with water. 4.9 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l. Dissolve 4 g of sodium hydroxide (4.6) in 1 l of water. 4.10 Phosphate buffered saline (PBS) solution, c(NaCl) = 120 mmol/l, c(KCl) = 2,7 mmol/l, c(phosphate buffer) = 10 mmol/l, pH = 7,4. Dissolve 8,0 g of sodium chlori

32、de (4.5), 1,2 g of anhydrous disodium hydrogen phosphate or 2,9 g of Na2HPO412 H2O (4.2), 0,2 g of potassium dihydrogen phosphate (4.4) and 0,2 g of potassium chloride (4.3) in 900 ml of water. After dissolution, adjust the pH to 7,4 with hydrochloric acid solution (4.8) or sodium hydroxide solution

33、 (4.9) as appropriate, then dilute to 1 l with water. Alternatively, a PBS solution with equivalent properties can be prepared from commercially available PBS material. 4.11 Acetonitrile. WARNING Acetonitrile is hazardous and samples shall be blended using an explosion proof blender which is housed

34、within a fume cupboard. After blending, samples shall be filtered inside a fume cupboard. 4.12 Polyethylene glycol (PEG), with a molecular mass of approximately 8 000 g/mol. 4.13 Methanol. 4.14 Acetic acid, with a mass fraction of 96 % or glacial acetic acid, with a mass fraction of 100 %. 4.15 Dilu

35、ent for HPLC analysis. Mix 9,5 parts per volume of methanol (4.13) with 90,5 parts per volume of water. 4.16 HPLC mobile phase. Mix 15 parts per volume of methanol (4.13) with 85 parts per volume of water. Add 0,1 parts per volume of acetic acid (4.14).The exact amount of methanol used and the use o

36、f acetic acid will depend on the HPLC column chosen for analysis and shall be adjusted if necessary. Degas this solution before use. 4.17 Wash solvent. Mix 50 parts per volume of methanol (4.13) with 50 parts per volume of water. 4.18 Immunoaffinity (IA) column. The immunoaffinity column shall conta

37、in antibodies raised against DON. The column shall have a capacity of not less than 1 000 ng of DON and shall have a recovery of not less than 80 % when 500 ng of DON are applied in 1 ml to 2 ml of water. DIN EN 15891:2010-12 EN 15891:2010 (E) 6 4.19 Deoxynivalenol, purity not less than 97 % mass fr

38、action. 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. 4.20 Deoxynivalenol stock solution 1, mass concentration 1,25 mg/ml. Add 4,0 ml of acetonitrile (4.11) to appro

39、ximately 5 mg of deoxynivalenol (4.19) to form a solution with a concentration of approximately 1,25 mg/ml. Alternatively, available commercial solutions with equivalent properties can be used. Store this solution in a freezer at approximately - 18 C. A solution stored in this way is stable for 12 m

40、onths. Confirm the concentration of the solution if it is older than six months. 4.21 Deoxynivalenol stock solution 2, 250 g/ml. Dilute 800 l of stock solution 1 (4.20) to 4 ml with acetonitrile (4.11) to form a solution with a concentration of approximately 250 g/ml. Store this solution in a freeze

41、r at approximately - 18 C. A solution stored in this way is stable for 12 months. Confirm the concentration of the solution if it is older than six months. 4.22 Deoxynivalenol standard solution A. Dilute 200 l of stock solution 2 (4.21) to 2,0 ml with acetonitrile (4.11) to form a solution with a co

42、ncentration of approximately 25 g/ml. To determine the exact mass concentration, record the absorption curve between a wavelength of 200 nm to 270 nm, e.g. in 5 nm steps; in the spectrometer (5.16) against acetonitrile as reference. Identify the wavelength for maximum absorption and calculate the ma

43、ss concentration of deoxynivalenol, DON, in micrograms per millilitre using Equation (1): bMA=100maxDON(1) where Amax is the absorption determined at the maximum of the absorption curve (here: at 220 nm); M is the molar mass, in grams per mole, of deoxynivalenol (M = 296,3 g/mol); is the molar absor

44、ption coefficient, in square metres per mole, of deoxynivalenol in acetonitrile (4.11) (here: 681 m2/mol, see 1); b is the optical path length, in centimetres, of the quartz cell. Calculate the mass concentration of the stock solution 2 (4.21), DON2, in micrograms per millilitre using Equation (2):

45、10DONDON2= (2) Store this solution in a freezer at approximately - 18 C. A solution stored in this way is stable for 12 months. Confirm the concentration of the solution if it is older than six months. NOTE Preparation of standard solutions can be carried out gravimetrically by accurately weighing t

46、he deoxynivalenol standard material and the solvent used to dissolve it. DIN EN 15891:2010-12 EN 15891:2010 (E) 7 4.23 Deoxynivalenol spiking solution, = 100 g/ml. Pipette an aliquot of the stock solution 2 (4.21) equivalent to 500 g of deoxynivalenol in a 5 ml volumetric flask. Dilute to the mark w

47、ith acetonitrile (4.11). Store this solution in a freezer at approximately - 18 C. A solution stored in this way is stable for 12 months. Confirm the concentration of the solution if it is older than six months. 4.24 Deoxynivalenol standard solution B, = 10 g/ml. Pipette 500 l of the spiking solutio

48、n (4.23) in a 5 ml volumetric flask. Dilute to the mark with acetonitrile (4.11). Store this solution in a freezer at approximately - 18 C. A solution stored in this way is stable for 12 months. Confirm the concentration of the solution if it is older than six months. 5 Apparatus 5.1 General Usual l

49、aboratory glassware and equipment and, in particular the following. 5.2 Analytical balance, capable of weighing to 0,000 1 g. 5.3 Laboratory balance, capable of weighing to 0,1 g. 5.4 High speed blender or homogenizer. 5.5 Laboratory shaker or magnetic stirrer, speed adjustable to approximately 500 min-1. 5.6 Vortex mixer, or equivalent. 5.7 Centrifuge, capable of a centrifugal force of 2 500 g. 5.8 Centrifuge tube, of 250 ml capaci