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!$izO“1708744www.din.deDDIN EN 15851Foodstuffs Determination of aflatoxin B1 in cereal based foods for infantsand young children HPLC method with immunoaffinity column cleanup and fluorescencedetectionEnglish translation of DIN EN 15851:2010-07Lebensmittel Bestimmung von Aflatoxin B1 in
3、 Suglings- und Kleinkindernahrung aufGetreidebasis HPLC-Verfahren mit Reinigung an einer Immunoaffinittssule und FluoreszenzdetektionEnglische bersetzung von DIN EN 15851:2010-07Produits alimentaires Dosage de laflatoxine B1 dans les produits pour nourrissons et jeunes enfants base de crales Mthode
4、de chromatographie liquide haute performance avec purification sur colonnedimmunoaffinit et dtection par fluorescenceTraduction anglaise de DIN EN 15851:2010-07www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authoritative.1707.10 DIN EN 15851:20
5、10-07 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 body involved in its pr
6、eparation 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, harvested or stored under hum
7、id 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 governs the maximum allowab
8、le 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 maximum content of contami
9、nants 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 International Standard referred to in this document is as follows: EN ISO 3696 DIN ISO 3696 National Annex NA (informative) Bibliography
10、DIN ISO 3696, Water for analytical laboratory use Specification and test methods 2 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15851 April 2010 ICS 67.060 English Version Foodstuffs - Determination of aflatoxin B1in cereal based foods for infants and young children - HPLC method with immuno
11、affinity column cleanup and fluorescence detection Produits alimentaires - Dosage de laflatoxine B1dans les produits pour nourrissons et jeunes enfants base de crales - Mthode de chromatographie liquide haute performance avec purification sur colonne dimmunoaffinit et dtection par fluorescence Leben
12、smittel - Bestimmung von Aflatoxin B1in Suglings- und Kleinkindernahrung auf Getreidebasis - 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 the CEN/CENELEC Interna
13、l 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 Centre or to any CEN member
14、. 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 official versions. CEN members
15、 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, Slovakia, Slovenia, Spain, Sw
16、eden, 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 for CEN national Members
17、. Ref. No. EN 15851:2010: EEN 15851:2010 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Apparatus .76 Procedure .87 HPLC analysis 98 Calculation . 119 Precision 1110 Test report . 12Annex A (informative) Typical chromatogram 13Annex B (informative) Precisi
18、on data . 14Bibliography . 15DIN EN 15851:2010-07 EN 15851:2010 (E) 3 Foreword This document (EN 15851:2010) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the status of a nation
19、al 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 this document may be the subject of patent
20、 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 standard can involve hazardous materials,
21、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 regulatory limitations prior to use. Acco
22、rding 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, Iceland, Ireland, It
23、aly, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15851:2010-07 EN 15851:2010 (E) 4 1 Scope This European Standard specifies a method for the determination of aflatoxin B1in baby fo
24、od by high performance liquid chromatography (HPLC) with immunoaffinity cleanup and fluorescence detection. This method has been validated in an interlaboratory study via the analysis of both naturally contaminated and spiked samples ranging from 0,07 g/kg to 0,18 g/kg. For further information on th
25、e 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 (including any amendme
26、nts) 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 a mixture of methanol and water. The extract is filtered, diluted with phosphate buffered saline (PBS) to a specified solvent concentratio
27、n, and applied to an immunoaffinity column containing antibodies specific to aflatoxin B1. Aflatoxin B1is purified and concentrated on the column and removed from the antibodies using methanol as eluent. Aflatoxin B1is quantified by reverse-phase high performance liquid chromatography (RP-HPLC) with
28、 post column derivatization (PCD) involving bromination followed by fluorescence detection. The post column derivatization is achieved with either electrochemically generated bromine or with pyridinium hydrobromide perbromide (PBPB). 4 Reagents 4.1 General Use only reagents of recognized analytical
29、grade and water complying with grade 1 of EN ISO 3696:1995, unless otherwise specified. Solvents shall be of quality for HPLC analysis, unless otherwise specified. Commercially available solutions with equivalent properties to those listed may be used. WARNING Dispose of waste solvents according to
30、applicable environmental rules and regulations. Decontamination procedures for laboratory wastes have been reported by the International Agency for Research on Cancer (IARC), see 4. 4.2 Helium purified compressed gas. 4.3 Nitrogen. 4.4 Disodium hydrogen phosphate, Na2HPO4anhydrous or Na2HPO412 H2O.
31、4.5 Potassium bromide. 4.6 Potassium chloride. 4.7 Potassium dihydrogen phosphate, KH2PO4. 4.8 Sodium chloride. DIN EN 15851:2010-07 EN 15851:2010 (E) 5 4.9 Sodium hydroxide. 4.10 Hydrochloric acid solution, mass fraction w(HCl) = 37 % in water. 4.11 Hydrochloric acid solution, substance concentrati
32、on c(HCl) = 0,1 mol/l. Dilute 8,28 ml of hydrochloric acid solution (4.10) to 1 l with water. 4.12 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l. Dissolve 4 g of sodium hydroxide (4.9) in 1 l of water. 4.13 Phosphate buffered saline (PBS) solution, c(NaCl) = 120 mmol/l, c(KCl) = 2,7 mmol/l, c(phosp
33、hate buffer) = 10 mmol/l, pH = 7,4. Dissolve 8,0 g of sodium chloride (4.8), 1,2 g of anhydrous disodium hydrogen phosphate or 2,9 g of Na2HPO412 H2O (4.2), 0,2 g of potassium dihydrogen phosphate (4.7) and 0,2 g of potassium chloride (4.6) in 900 ml of water. After dissolution, adjust the pH to 7,4
34、 with hydrochloric acid solution (4.11) or sodium hydroxide solution (4.12) as appropriate, then dilute to 1 l with water. Alternatively, a PBS solution with equivalent properties can be prepared from commercially available PBS material. 4.14 Pyridinium hydrobromide perbromide (PBPB), CAS: 39416-48-
35、3. 4.15 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 a fume cupboard. 4.16 Methanol, HPLC grade. 4.17 Methanol, technical grade. 4.18 Toluene. 4.1
36、9 Extraction solvent. Mix eight parts per volume of methanol (4.17) with two parts per volume of water. 4.20 Nitric acid, c(HNO3) = 4 mol/l. 4.21 HPLC mobile phase A, for use with PBPB. Mix six parts per volume of water with two parts per volume of acetonitrile (4.15) and three parts per volume of m
37、ethanol (4.16). Degas mobile phase A with for example helium (4.2). 4.22 HPLC mobile phase B, for use with electrochemically generated bromine. Mix six parts per volume of water with two parts per volume of acetonitrile (4.15) and three parts per volume of methanol (4.16). Add 120 mg of potassium br
38、omide (4.5) and 350 l of nitric acid (4.20) per litre of mobile phase. Degas mobile phase B with for example helium (4.2). 4.23 Post-column reagent. Dissolve 50 mg of PBPB (4.14) in 1 l of water. To be used with mobile phase solvent A (4.21). The solution may be used up to four days if stored in a d
39、ark place at room temperature. DIN EN 15851:2010-07 EN 15851:2010 (E) 6 4.24 Mixture of toluene and acetonitrile. Mix nine parts per volume of toluene (4.18) with one part per volume of acetonitrile (4.15). 4.25 Immunoaffinity column. The immunoaffinity column shall contain antibodies raised against
40、 aflatoxin B1. The column shall have a capacity of not less than 100 ng of aflatoxin B1and shall give a recovery of not less than 80 % when 5 ng of aflatoxin B1are applied as a standard solution in a mixture of ten parts per volume of methanol and 90 parts per volume of water. 4.26 Aflatoxin B1, in
41、crystal form or as a film in ampoules or in form of commercially available aflatoxin B1solution. WARNING Aflatoxins are subject to light degradation. Protect the laboratory, where the analyses are done, adequately from daylight. This can be achieved effectively by using ultraviolet (UV) absorbing fo
42、il on the windows in combination with subdued light (no direct sunlight) or curtains or blinds in combination with artificial light (fluorescent tubes are acceptable). Protect aflatoxin containing solutions from light as much as possible (keep in the dark, use aluminium foil or amber-coloured glassw
43、are). 4.27 Aflatoxin B1stock solution, c 10 g/ml. Prepare a solution of aflatoxin B1in the mixture of toluene and acetonitrile (4.24) to give a solution with a mass concentration of approximately 10 g/ml. To determine the exact mass concentration, record the absorption curve between 330 nm and 370 n
44、m in 1 cm quartz cells in a spectrometer (5.14) with the mixture of toluene and acetonitrile (4.24) as reference. Identify the wavelength for maximum absorption (between 330 nm and 370 nm). Calculate the mass concentration of aflatoxin B1, afl, in g/ml, using Equation (1): bMA=100maxafl(1) where Ama
45、x is the absorption determined at the maximum of the absorption curve (between 330 nm and 370 nm); M is the molar mass, in g/mol, of aflatoxin B1(M = 312 g/mol); is the molar absorption coefficient, in square metres per mole, of aflatoxin B1in the mixture of toluene and acetonitrile (4.24) (1 930 m2
46、/mol, see 5); b is the optical path length, in centimetres, of the quartz cell. Store this solution in a freezer at approximately - 18 C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for 12 months. Confirm the concentration of the solution if it is
47、older than 12 months. 4.28 Aflatoxin B1standard solution, = 5,00 ng/ml. Pipette a volume of aflatoxin B1stock solution (4.27) containing exactly 1,00 g aflatoxin B1into a 200 ml calibrated volumetric flask and dilute to the mark with the mixture of toluene and acetonitrile (4.24). This solution cont
48、ains 5,00 ng/ml aflatoxin B1. DIN EN 15851:2010-07 EN 15851:2010 (E) 7 Wrap the flask tightly in aluminium foil and store it at less than 4 C. Before use, do not remove the aluminium foil until the contents have reached room temperature to avoid incorporation of water by condensation. A solution sto
49、red in this way is stable for at least four weeks. 4.29 Aflatoxin B1spiking solution, = 2 g/ml. Pipette a volume of aflatoxin B1stock solution (4.27) containing exactly 20 g aflatoxin B1into a 10 ml calibrated volumetric flask. Evaporate the mixture of toluene and acetonitrile solution just to dryness under a stream of nitrogen at room temperature. Make up to volume with methanol (4.16) and shake well. The concentration of this spiking solution is 2 g/ml for
