1、BS EN 15850:2010ICS 67.060NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDFoodstuffs Determination ofzearalenone in maizebased baby food,barley flour, maizeflour, polenta, wheatflour and cereal basedfoods for infantsand young children HPLC methodwith immunoaffin
2、itycolumn cleanup andfluorescence detectionThis British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 May2010 BSI 2010ISBN 978 0 580 64288 3Amendments/corrigenda issued since publicationDate CommentsBS EN 15850:2010National forewordThis British Stand
3、ard is the UK implementation of EN 15850:2010. The UK participation in its preparation was entrusted to TechnicalCommittee AW/-/3, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to
4、include all the necessary provisionsof a contract. Users are responsible for its correct application. Compliance with a British Standard cannot confer immunityfrom legal obligations.BS EN 15850:2010EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15850 April 2010 ICS 67.060 English Version Foods
5、tuffs - 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 and fluorescence detection Produits alimentaires - Dosage de la zaralnone dans la fari
6、ne 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 dtection par fluorescence Lebensmittel - Bestimmung von Zearalenon in Suglingsnahrung auf Mai
7、sbasis, Gerstenmehl, Maismehl, Maisgrie, Weizenmehl und Lebensmittel auf Getreidebasis fr Suglinge und Kleinkinder - HPLC-Verfahren mit Reinigung an einer Immunoaffinittssule und Fluoreszenzdetektion This European Standard was approved by CEN on 27 February 2010. CEN members are bound to comply with
8、 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 the CEN Management Cen
9、tre 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 same status as the offici
10、al 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, Portugal, Romania, Slova
11、kia, 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 means reserved worldwide
12、for CEN national Members. Ref. No. EN 15850:2010: EBS EN 15850:2010EN 15850:2010 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Apparatus .76 Procedure .97 HPLC analysis . 108 Calculation . 129 Precision 1210 Test report . 13Annex A (informative) Typical c
13、hromatograms 15Annex B (informative) Precision data . 16Bibliography . 18BS EN 15850:2010EN 15850:2010 (E) 3 Foreword This document (EN 15850:2010) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standar
14、d 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 2010. Attention is drawn to the possibility that some of the elements of thi
15、s 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 Association. WARNING The use of this sta
16、ndard 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 determine the applicability of re
17、gulatory 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, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany
18、, 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 15850:2010EN 15850:2010 (E) 4 1 Scope This European Standard specifies a method for the deter
19、mination 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 cleanup and fluorescence detection. This method has been validated in two inter
20、laboratory 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 cereal based foods for infants and young children ranging from 9 g/kg to 44 g/kg.
21、 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 applies. For undated references, the latest edition of the referenced document
22、(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 according to the products analyzed. The extract is then diluted with phosphate buffe
23、red 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 antibodies using acetonitrile or methanol as eluent. Zearalenone is quantified by revers
24、e-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 otherwise specified. Solvents shall be of quality for HPLC analysis, unless other
25、wise 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 dihydrogen phosphate, KH2PO4. 4.5 Sodium chloride (NaCl). 4.6 Sodium hydroxide (NaO
26、H). 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. BS EN 15850:2010EN 15850:2010 (E) 5 4.9 Sodium hydroxide solution, c(NaOH) = 0,1
27、 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 chloride (4.5), 1,2 g of anhydrous disodium hydrogen phosphate or 2,9 g of Na2
28、HPO412 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 (4.9) as appropriate, then dilute to 1 l with water. Alternatively, a P
29、BS 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 within a fume cupboard. After blending, samples shall be filtered inside
30、 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 HPLC analysis. Mix four parts per volume of acetonitrile (4.11) with six parts per vo
31、lume 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 per volume of methanol (4.13) with 25 parts per volume of water. 4.18 Washing solvent.
32、 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 HPLC mobile phase B. Mix 75 parts per volume of methanol (4.12) with 25 parts per vol
33、ume of water. Filter and degas the HPLC mobile phase before use. BS EN 15850:2010EN 15850:2010 (E) 6 4.21 Immunoaffinity column. The immunoaffinity column shall contain antibodies raised against zearalenone. The column shall have a capacity of not less than 1 500 ng of zearalenone and shall give a r
34、ecovery 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 ampoules, purity not less than 98 % mass fraction or in form of commercially available Zearale
35、none 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 shall be carried out in a fume cupboard. 4.23 Zearalenone stock solution, c 200 g/ml. Add 4,0
36、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 solution with a concentration of approximately 200 g/ml. Store this solution in a freezer a
37、t - 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. 4.24 Zearalenone spiking solution, c 10 g/ml. Dilute 250 l of stock solution (4.23) with 4
38、,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 in a 1 cm quartz cell in the spectrometer (5.25) with acetonitrile (4.11) as reference. Ide
39、ntify 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 absorption determined at the maximum of the absorption curve (274 nm); M is the molar mass
40、, 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, in centimetres, of the quartz cell. Store this solution in a freezer at - 18 C to - 20 C.
41、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 standard solution A, = 2 g/ml, for maize based baby food, barley flour, maize flour, polenta
42、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 volume up to 5 ml. BS EN 15850:2010EN 15850:2010 (E) 7 Store this solution in a freezer at
43、 - 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 yo
44、ung children. Transfer an aliquot of the spiking solution (4.24) equivalent to 2 g of zearalenone into either a vial (5.9) or a calibrated volumetric flask (5.10). Add acetonitrile (4.11) to make the total volume up to 5 ml. Store this solution in a freezer at - 18 C to - 20 C. Allow to reach room t
45、emperature 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. 5 Apparatus 5.1 General Usual laboratory glassware and equipment and, in particular, the following. 5.2 High speed blender or homog
46、enizer. 5.3 Analytical balance, capable of weighing to 0,000 1 g. 5.4 Laboratory balance, capable of weighing to 0,1 g. 5.5 Adjustable vertical or horizontal shaker. 5.6 Vortex mixer, or equivalent. 5.7 Mills, various screens. 5.8 Tumble mixer. 5.9 Glass vials, of various sizes 5.10 Volumetric flask
47、s, of 3 ml or 5 ml and 10 ml capacity. 5.11 Beaker, of 250 ml capacity. 5.12 Conical flask, with screw cap or glass stopper of 100 ml, 250 ml and 500 ml capacity. 5.13 Filter paper, e.g., qualitative, strong, fast flow, 24 cm diameter, 30 m pore size, prefolded or equivalent. 5.14 Glass microfibre f
48、ilter, e.g. 1,6 m retention size or equivalent. 5.15 Pipettes, of e.g. 25 l to 250 l, 1 ml, 5 ml and 10 ml capacity. 5.16 Displacement micropipettes or syringes, gas tight of e.g. 100 l, 500 l, 1 000 l capacity. BS EN 15850:2010EN 15850:2010 (E) 8 5.17 Vacuum manifold or automated system, to accommo
49、date immunoaffinity columns. 5.18 Reservoirs, of 50 ml to 75 ml capacity, and attachments for immunoaffinity columns. 5.19 Plastic syringes, 5 ml. 5.20 Vacuum pump, capable of for example pulling a vacuum of 1 kPa and or pumping 18 l/min. 5.21 Solvent vacuum filtration system, fitted with 47 mm glass microfibre filter. 5.22 Disposable syringe filter unit, nylon with a pore size of 0,45 m. Prior to usage, verify that no zearalenone losses occur during filtra
