1、July 2010 Translation by DIN-Sprachendienst.English price group 11No 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).ICS 6
2、7.060; 67.230!$i“1705757www.din.deDDIN EN 15850Foodstuffs Determination of zearalenone in maize based baby food, barley flour,maize flour, polenta, wheat flour and cereal based foods for infants andyoung children HPLC method with immunoaffinity column cleanup and fluorescencedetectionEnglish transla
3、tion of DIN EN 15850:2010-07Lebensmittel Bestimmung von Zearalenon in Suglingsnahrung auf Maisbasis, Gerstenmehl, Maismehl,Maisgrie, Weizenmehl und Lebensmittel auf Getreidebasis fr Suglinge undKleinkinder HPLC-Verfahren mit Reinigung an einer Immunoaffinittssule und FluoreszenzdetektionEnglische be
4、rsetzung von DIN EN 15850:2010-07Produits alimentaires Dosage de la zaralnone dans la farine dorge, de mas et de bl, la polenta et lesproduits pour nourrissons et jeunes enfants base de crales Mthode par chromatographie liquide haute performance avec purification sur colonnedimmunoaffinit et dtectio
5、n par fluorescenceTraduction anglaise de DIN EN 15850:2010-07www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authoritative.2007.10 DIN EN 15850:2010-07 A comma is used as the decimal marker. National foreword This standard has been prepared by T
6、echnical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). Preliminary work was done by Working Group WG 5 “Biotoxins”. The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultu
7、ral Products Standards Committee), Working Committee NA 057-01-03 AA Biotoxine. Mycotoxins are extremely toxic secondary metabolites produced by fungi. Food that is grown, harvested or stored under humid conditions can be infected by fungi whose metabolites invade the food. Because some mycotoxins a
8、re extremely toxic for humans, their reliable detection is especially important for consumer health and protection. In Germany the regulation Mykotoxin-Hchstmengenverordnung governs the maximum allowable content of mycotoxins in food. Since 2004 this regulation has contained specifications not only
9、for aflatoxins, but also for ochratoxin A, fumonisins, deoxynivalenol (DON), and zearalenone. National regulations have been supplemented by EU-wide regulations regarding the maximum content of contaminants in food since 2001. The maximum content of mycotoxins in certain foods is also laid down in v
10、arious other regulations. 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 STANDAR
11、D NORME EUROPENNE EUROPISCHE NORM EN 15850 April 2010 ICS 67.060 English Version Foodstuffs - Determination of zearalenone in maize based baby food, barley flour, maize flour, polenta, wheat flour and cereal based foods for infants and young children - HPLC method with immunoaffinity column cleanup
12、and fluorescence detection Produits alimentaires - Dosage de la zaralnone dans la farine dorge, de mas et de bl, la polenta et les produits pour nourrissons et jeunes enfants base de crales - Mthode par chromatographie liquide haute performance avec purification sur colonne dimmunoaffinit et dtectio
13、n par fluorescence Lebensmittel - Bestimmung von Zearalenon in Suglingsnahrung auf Maisbasis, Gerstenmehl, Maismehl, Maisgrie, Weizenmehl und Lebensmittel auf Getreidebasis fr Suglinge und Kleinkinder - HPLC-Verfahren mit Reinigung an einer Immunoaffinittssule und Fluoreszenzdetektion This European
14、Standard was approved by CEN on 27 February 2010. 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 bibliographical references conce
15、rning such national standards 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 translation under the responsibility of a CEN member into its ow
16、n language and notified to the CEN 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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvi
17、a, Lithuania, Luxembourg, Malta, Netherlands, 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 Brussel
18、s 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15850:2010: EEN 15850:2010 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Apparatus .76 Procedure .97 HPLC analysis . 108 Calculatio
19、n . 129 Precision 1210 Test report . 13Annex A (informative) Typical chromatograms 15Annex B (informative) Precision data . 16Bibliography . 18DIN EN 15850:2010-07 EN 15850:2010 (E) 3 Foreword This document (EN 15850:2010) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizont
20、al methods”, the secretariat of which is held by DIN. 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 October 2010, and conflicting national standards shall be withdrawn at the latest by October 2
21、010. Attention is 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 Comm
22、ission and the European 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 ap
23、propriate safety and 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,
24、Croatia, 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 the United Kingdom. DIN EN 15850:2010-07 EN
25、15850:2010 (E) 4 1 Scope This European Standard specifies a method for the determination of zearalenone in maize based baby food, barley flour, maize flour, polenta, wheat flour and cereal based foods for infants and young children by high performance liquid chromatography (HPLC) with immunoaffinity
26、 cleanup and fluorescence detection. This method has been validated in two interlaboratory studies. The first study was for the analysis of samples of maize based baby food, barley flour, maize flour, polenta and wheat flour ranging from 10 g/kg to 335 g/kg, and the second study was for samples of c
27、ereal based foods for infants and young children ranging from 9 g/kg to 44 g/kg. Further information on validation, see Clause 9 and Annex B. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited
28、 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 A test portion is extracted with aqueous acetonitrile or methanol acco
29、rding to the products analyzed. The extract is then diluted with phosphate buffered saline (PBS) to give an aqueous extract that is applied to an immunoaffinity column containing antibodies specific for zearalenone. Zearalenone is purified and concentrated on the column and removed from the antibodi
30、es using acetonitrile or methanol as eluent. Zearalenone is quantified by reverse-phase high performance liquid chromatography (RP-HPLC) with fluorescence detection. 4 Reagents 4.1 General Use only reagents of recognised analytical grade and water complying with grade 1 of EN ISO 3696:1995, unless o
31、therwise specified. Solvents shall be of quality for HPLC analysis, unless otherwise specified. Commercially available solutions with equivalent properties to those listed may be used. 4.2 Disodium hydrogen phosphate, Na2HPO4anhydrous or Na2HPO412 H2O. 4.3 Potassium chloride (KCl). 4.4 Potassium dih
32、ydrogen phosphate, KH2PO4. 4.5 Sodium chloride (NaCl). 4.6 Sodium hydroxide (NaOH). 4.7 Hydrochloric acid solution, mass fraction w(HCl) = 37 % in water. 4.8 Hydrochloric acid solution, substance concentration c(HCl) = 0,1 mol/l. Dilute 8,28 ml of hydrochloric acid solution (4.7) to 1 l with water.
33、DIN EN 15850:2010-07 EN 15850:2010 (E) 5 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
34、sodium chloride (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 hydro
35、xide solution (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 whi
36、ch is housed within a fume cupboard. After blending, samples shall be filtered inside a fume cupboard. 4.12 Methanol, HPLC grade. 4.13 Methanol, technical grade. 4.14 Extraction solvent A. Mix 75 parts per volume of acetonitrile (4.11) with 25 parts per volume of water. 4.15 Injection solvent A for
37、HPLC analysis. Mix four parts per volume of acetonitrile (4.11) with six parts per volume of water. 4.16 HPLC mobile phase A. Mix 53 parts per volume of acetonitrile (4.11) with 47 parts per volume of water. Filter and degas the HPLC mobile phase before use. 4.17 Extraction solvent B. Mix 75 parts p
38、er volume of methanol (4.13) with 25 parts per volume of water. 4.18 Washing solvent. Mix 15 parts per volume of methanol (4.12) with 85 parts per volume of PBS (4.10). 4.19 Injection solvent B for HPLC analysis. Mix five parts per volume of methanol (4.12) with five parts per volume of water. 4.20
39、HPLC mobile phase B. Mix 75 parts per volume of methanol (4.12) with 25 parts per volume of water. Filter and degas the HPLC mobile phase before use. DIN EN 15850:2010-07 EN 15850:2010 (E) 6 4.21 Immunoaffinity column. The immunoaffinity column shall contain antibodies raised against zearalenone. Th
40、e column shall have a capacity of not less than 1 500 ng of zearalenone and shall give a recovery of not less than 80 % when 75 ng of zearalenone is applied in 10 ml of a mixture of 15 parts per volume of methanol and 85 parts per volume of PBS. 4.22 Zearalenone, in crystal form or as a film in ampo
41、ules, purity not less than 98 % mass fraction or in form of commercially available Zearalenone solution. WARNING Zearalenone is an oestrogenic compound and should be treated with extreme caution. Gloves and safety glasses shall be worn at all times and all standard and sample preparation stages shal
42、l be carried out in a fume cupboard. 4.23 Zearalenone stock solution, c 200 g/ml. Add 4,0 ml of acetonitrile (4.11) to 5 mg of zearalenone (4.22) to form a solution with a mass concentration of approximately 1,25 mg/ml. Dilute 800 l of this solution to 5 ml with acetonitrile (4.11) to form a stock s
43、olution with a concentration of approximately 200 g/ml. Store this solution in a freezer at - 18 C to - 20 C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for 12 months. Confirm the mass concentration of the solution if it is older than six months.
44、4.24 Zearalenone spiking solution, c 10 g/ml. Dilute 250 l of stock solution (4.23) with 4,75 ml of acetonitrile (4.11) to form a solution with a mass concentration of approximately 10 g/ml. To determine the exact concentration, record the absorption curve of this solution between 200 nm to 300 nm i
45、n a 1 cm quartz cell in the spectrometer (5.25) with acetonitrile (4.11) as reference. Identify the wavelength for maximum absorption ( is approximately 274 nm). Calculate the mass concentration of zearalenone, zon, in micrograms per millilitre using Equation (1): bMA=100maxzon(1) where Amax is the
46、absorption determined at the maximum of the absorption curve (274 nm); M is the molar mass, in grams per mole, of zearalenone (M = 318,4 g/mol); is the molar absorption coefficient, in square metres per mole of zearalenone in acetonitrile (4.11) ( 1 262 m2/mol, see 1); b is the optical path length,
47、in centimetres, of the quartz cell. Store this solution in a freezer at - 18 C to - 20 C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for 12 months. Confirm the mass concentration of the solution if it is older than six months. 4.25 Zearalenone sta
48、ndard solution A, = 2 g/ml, for maize based baby food, barley flour, maize flour, polenta and wheat flour. Transfer an aliquot of the spiking solution (4.24) equivalent to 10 g of zearalenone into either a vial (5.9) or a calibrated volumetric flask (5.10). Add acetonitrile (4.11) to make the total
49、volume up to 5 ml. DIN EN 15850:2010-07 EN 15850:2010 (E) 7 Store this solution in a freezer at - 18 C to - 20 C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for 12 months. Confirm the mass concentration of the solution if it is older than six months. 4.26 Zearalenone standard solution B, = 0,4 g/ml, for cereal based foods for infants and young children. Transfer an aliquot of the spi
