1、BS EN 15851:2010ICS 67.060NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDFoodstuffs Determination ofaflatoxin B1 in cerealbased foods for infantsand young children HPLC methodwith immunoaffinitycolumn cleanup andfluorescence detectionThis British Standard was p
2、ublished under the authority of the Standards Policy and Strategy Committee on 31 May2010 BSI 2010ISBN 978 0 580 64289 0Amendments/corrigenda issued since publicationDate CommentsBS EN 15851:2010National forewordThis British Standard is the UK implementation of EN 15851:2010. The UK participation in
3、 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 include all the necessary provisionsof a contract. Users are responsib
4、le for its correct application. Compliance with a British Standard cannot confer immunityfrom legal obligations.BS EN 15851:2010EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15851 April 2010 ICS 67.060 English Version Foodstuffs - Determination of aflatoxin B1in cereal based foods for infants
5、 and young children - HPLC method with immunoaffinity 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 dimm
6、unoaffinit et dtection par fluorescence Lebensmittel - 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
7、 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 t
8、he 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 same
9、 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, Por
10、tugal, 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 mean
11、s reserved worldwide for CEN national Members. Ref. No. EN 15851:2010: EBS EN 15851:2010EN 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 (info
12、rmative) Typical chromatogram 13Annex B (informative) Precision data . 14Bibliography . 15BS EN 15851:2010EN 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
13、 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 2010. Attention is drawn to the possibility that some of th
14、e 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 European Free Trade Association. WARNING Th
15、e 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 and health practices and determine the ap
16、plicability 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, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland
17、, 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. BS EN 15851:2010EN 15851:2010 (E) 4 1 Scope This European Standard specifies a met
18、hod for the determination of aflatoxin B1in baby food 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,
19、07 g/kg to 0,18 g/kg. For further information on the 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 editio
20、n 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 a mixture of methanol and water. The extract is filtered, diluted with phosphate buffer
21、ed saline (PBS) to a specified solvent concentration, 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 hi
22、gh performance liquid chromatography (RP-HPLC) with 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
23、General Use only reagents of recognized 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 otherwise specified. Commercially available solutions with equivalent properties to those listed may be us
24、ed. WARNING Dispose of waste solvents according to 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 hydr
25、ogen phosphate, Na2HPO4anhydrous or Na2HPO412 H2O. 4.5 Potassium bromide. 4.6 Potassium chloride. 4.7 Potassium dihydrogen phosphate, KH2PO4. 4.8 Sodium chloride. BS EN 15851:2010EN 15851:2010 (E) 5 4.9 Sodium hydroxide. 4.10 Hydrochloric acid solution, mass fraction w(HCl) = 37 % in water. 4.11 Hyd
26、rochloric acid solution, substance concentration 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(Na
27、Cl) = 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 (4.8), 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
28、water. After dissolution, adjust the pH to 7,4 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
29、hydrobromide perbromide (PBPB), CAS: 39416-48-3. 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. 4.1
30、7 Methanol, technical grade. 4.18 Toluene. 4.19 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 aceto
31、nitrile (4.15) and three parts per volume of methanol (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
32、of methanol (4.16). Add 120 mg of potassium bromide (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 solutio
33、n may be used up to four days if stored in a dark place at room temperature. BS EN 15851:2010EN 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 colum
34、n shall contain antibodies raised against 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 pe
35、r volume of water. 4.26 Aflatoxin B1, in 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 effective
36、ly by using ultraviolet (UV) absorbing foil 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, us
37、e aluminium foil or amber-coloured glassware). 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
38、absorption curve between 330 nm and 370 nm 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
39、 Equation (1): bMA=100maxafl(1) where Amax 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
40、toluene and acetonitrile (4.24) (1 930 m2/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 th
41、e concentration of the solution if it is 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 an
42、d acetonitrile (4.24). This solution contains 5,00 ng/ml aflatoxin B1. BS EN 15851:2010EN 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 w
43、ater by condensation. A solution stored 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
44、 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 aflatoxin B1. Wrap the flask tightly in aluminium foil and store it at less than 4 C. Before use, do
45、not remove the aluminium foil until the contents have reached room temperature to avoid incorporation of water by condensation. A solution stored in this way is stable for at least three months. 5 Apparatus WARNING All glassware coming into contact with aqueous solutions of aflatoxins shall be washe
46、d with acid solution before use. Many laboratory washing machines do this as part of the washing programme. Otherwise soak laboratory glassware coming into contact with aqueous solutions of aflatoxins in sulfuric acid (c = 2 mol/l) for several hours (e.g. 15 h overnight), then rinse well (e.g. at le
47、ast three times) with water to remove all traces of acid. Check the absence of acid with pH paper. This treatment is necessary, because the use of non-acid washed glassware can cause losses of aflatoxins. In practice, the treatment is necessary for round bottomed flasks, volumetric flasks, measuring
48、 cylinders, vials or tubes used for calibration solutions and final extracts (particularly autosampler vials), and Pasteur pipettes, if these are used to transfer calibration solutions or extracts. Usual laboratory glassware and equipment and, in particular, the following. 5.1 Analytical balance, ca
49、pable of weighing to 0,000 1 g. 5.2 Laboratory balance, capable of weighing to 0,01 g. 5.3 Adjustable vertical or horizontal shaker. 5.4 Filter paper, e.g. 24 cm diameter, prefolded. 5.5 Conical flask, with screw top or glass stopper of 500 ml capacity. 5.6 Glass microfibre filter, retention size 1,6 m or smaller. 5.7 Reservoir, of 75 ml capacity with luer tip connector and attachments for immunoaffinity column (IAC). 5.8 Hand pump, 20 ml syringe with luer lock or rubber stopp
