1、ICS 67.050Licensed Copy: Wang Bin, ISO/EXCHANGE CHINA STANDARDS, 05/03/2008 07:29, Uncontrolled Copy, (c) BSIg49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3
2、g47g36g58aflatoxin B1and the sum of aflatoxin B1, B2, G1and G2in hazelnuts, peanuts, pistachios, figs, and paprika powder High performance liquid chromatographic method with post-column derivatisation and immunoaffinity column cleanupThe European Standard EN 14123:2007 has the status of a British St
3、andardFoodstuffs Determination of BRITISH STANDARDBS EN 14123:2007BS EN 14123:2007Licensed Copy: Wang Bin, ISO/EXCHANGE CHINA STANDARDS, 05/03/2008 07:29, Uncontrolled Copy, (c) BSIThis British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 January 20
4、08 BSI 2008ISBN 978 0 580 58514 2Amendments/corrigenda issued since publicationDate Commentscontract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunity from legal obligations.National forewordThis British Standard is the UK implementation of
5、EN 14123:2007. It supersedes BS EN 14123:2003 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Food analysis Horizontal methods.A list of organizations represented on this committee can be obtained on request to its secretary.This publication do
6、es not purport to include all the necessary provisions of a EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 14123 December 2007 ICS 67.050 Supersedes EN 14123:2003 English Version Foodstuffs - Determination of aflatoxin B1and the sum of aflatoxin B1, B2, G1and G2in hazelnuts, peanuts, pistachio
7、s, figs, and paprika powder - High performance liquid chromatographic method with post-column derivatisation and immunoaffinity column cleanup Produits alimentaires - Dosage de laflatoxine B1et de la somme des aflatoxines B1, B2, G1et G2dans les noisettes, les cacahutes, les pistaches, les figues et
8、 le paprika en poudre - Mthode par purification sur colonne dimmuno-affinit suivie dune chromatographie liquide haute performance avec drivation post-colonne Lebensmittel - Bestimmung von Aflatoxin B1und der Summe von Aflatoxin B1, B2, G1und G2in Haselnssen, Erdnssen, Pistazien, Feigen und Paprikapu
9、lver - Hochleistungsflssigchromatographisches Verfahren mit Immunaffinittssulen-Reinigung und Nachsulenderivatisierung This European Standard was approved by CEN on 12 November 2007. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving t
10、his 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. This European Standard exists in three official version
11、s (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 are the national standards bodies of Austria, Belgium, B
12、ulgaria, 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 United Kingdom. EUROPEAN COMMITTEE FOR STAND
13、ARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2007 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 14123:2007: ELicensed Copy: Wang Bin, ISO/EXCHANGE
14、 CHINA STANDARDS, 05/03/2008 07:29, Uncontrolled Copy, (c) BSIEN 14123:2007 (E) 2 Contents Page Foreword3 1 Scope 3 2 Normative references 3 3 Principle3 4 Reagents.4 5 Apparatus .7 6 Procedures .9 7 Precision.13 8 Test report 16 Annex A (informative) Typical chromatograms .18 Annex B (informative)
15、Precision data24 Bibliography 29 Licensed Copy: Wang Bin, ISO/EXCHANGE CHINA STANDARDS, 05/03/2008 07:29, Uncontrolled Copy, (c) BSIEN 14123:2007 (E) 3 Foreword This document (EN 14123:2007) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of
16、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 June 2008, and conflicting national standards shall be withdrawn at the latest by June 2008. This document supersedes EN 14123
17、:2003 with the following changes: a) Validation data on hazelnut are included. 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 o
18、f this standard to establish appropriate 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 Standar
19、d: Austria, Belgium, Bulgaria, 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. 1
20、Scope This European Standard is applicable to the determination of aflatoxins B1, B2, G1and G2in hazelnuts, figs, pistachios, peanuts and paprika powder. The limit of quantification of the method is 0,8 ng/g for each aflatoxin or better (value derived from in-house and collaborative study), dependin
21、g on the equipment used. 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 amendments) applies. E
22、N ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987) 3 Principle A test portion is either extracted with a solvent solution (methanol/water) or the solvent solution plus hexane (or cyclohexane). The sample extract is filtered, diluted with phosphate buffer
23、ed saline (PBS) and applied to an immunoaffinity column (IAC) containing antibodies specific to aflatoxins B1, B2, G1and G2. The aflatoxins are eluted from the immunoaffinity column with methanol. Aflatoxins are quantified by reverse-phase high performance liquid chromatography (RP-HPLC) with post-c
24、olumn derivatization (PCD) involving bromination followed by fluorescence detection. The PCD is achieved with either electrochemically generated bromine or with pyridinium hydrobromide perbromide (PBPB). Licensed Copy: Wang Bin, ISO/EXCHANGE CHINA STANDARDS, 05/03/2008 07:29, Uncontrolled Copy, (c)
25、BSIEN 14123:2007 (E) 4 4 Reagents 4.1 General Use only reagents of recognized analytical grade and water complying with grade 3 of EN ISO 3696, unless otherwise specified. 4.2 Water, complying with grade 1 of EN ISO 3696 4.3 Phosphate buffered saline (PBS), pH = 7,4 Dissolve 0,20 g of potassium chlo
26、ride, 0,20 g of potassium dihydrogen phosphate, 1,16 g of disodium hydrogen orthophosphate (or 2,92 g of hydrogenphosphate12 H20) and 8,00 g of sodium chloride in 0,9 l of water. After dissolution, adjust the pH to 7,4 with HCl (0,1 mol/l) or NaOH (0,1 mol/l) as appropriate. Dilute to 1 l with water
27、. Commercially available phosphate buffered saline tablets with equivalent properties may be used. 4.4 Sodium chloride (NaCl) 4.5 Pyridinium hydrobromide perbromide (PBPB), CAS: 39416-48-3 4.6 Potassium bromide (KBr) 4.7 Acetonitrile, HPLC grade 4.8 Methanol, HPLC grade 4.9 Methanol, p.a. grade 4.10
28、 Toluene 4.11 Extraction solvent mixture of methanol and water Mix 8 parts per volume of methanol (4.9) with 2 parts per volume of water. 4.12 n-Hexane, cyclohexane, p.a. grade 4.13 Nitric acid, c(HNO3) = 4 mol/l Dilute 28 ml of nitric acid (volume fraction is 65 %), or 26 ml of nitric acid (volume
29、fraction is 70 %) with water to a final volume of 100 ml. 4.14 Immunoaffinity column The affinity column contains antibodies raised against aflatoxins B1, B2, G1and G2. The column shall have a maximum capacity of not less than 100 ng of aflatoxin B1and shall give a recovery of not less than 80 % for
30、 aflatoxins B1, B2, G1and not less than 60 % for aflatoxin G2when applied as an aqueous standard solution (10 % of methanol) containing 5 ng of each toxin. The maximum solvent concentration of solutions that can be applied on the column shall not exceed 12 % of methanol. Licensed Copy: Wang Bin, ISO
31、/EXCHANGE CHINA STANDARDS, 05/03/2008 07:29, Uncontrolled Copy, (c) BSIEN 14123:2007 (E) 5 4.15 HPLC mobile phase solvent (A), for use with PBPB Mix 6 parts per volume of water (4.2) with 2 parts per volume of acetonitrile (4.7) and 3 parts per volume of methanol (4.8). Degas the solution before use
32、. The mobile phase shall be free of particles and should be filtered prior use. 4.16 HPLC mobile phase solvent (B), for use with electrochemically generated bromine Mix 6 parts per volume of water (4.2) with 2 parts per volume of acetonitrile (4.7) and 3 parts per volume of methanol (4.8). Add 120 m
33、g of potassium bromide (4.6) and 350 l of nitric acid (4.13) per litre of mobile phase. Degas the solution before use. 4.17 Post-column reagent Dissolve 50 mg of PBPB (4.5) in 1 l of water. The solution may be used up to four days if stored in a dark place at room temperature. 4.18 Mixture of toluen
34、e and acetonitrile Mix 98 parts per volume of toluene (4.10) with 2 parts per volume of acetonitrile (4.7). 4.19 Aflatoxins, either in form of crystals or film in ampoules or in form of commercially available aflatoxin solutions WARNING 1 Decontamination procedures for laboratory wastes of aflatoxin
35、s were developed by the International Agency for Research on Cancer (IARC) 1, 2. WARNING 2 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 foil on the wind
36、ows 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 glassware) and store
37、 at the temperature recommended by the manufacturer (e.g. -18 C). 4.20 Aflatoxins stock solution Dissolve aflatoxin B1, B2, G1and G2separately in the mixture of toluene and acetonitrile (4.18) to give separate solutions with a concentration of 10 g/ml for each aflatoxin. Wrap the flasks tightly in a
38、luminium foil and store them at less than 4 C. To determine the exact concentration of aflatoxins in each stock solution, record the absorption curve between a wavelength of 330 nm and 370 nm in 1 cm quartz glass cells in a spectrometer with the mixture of toluene and acetonitrile (4.18) in the refe
39、rence cell. Calculate the mass concentration of each aflatoxin, i, in micrograms per millilitre, using Equation (1): bMAiii=100max (1) where: Amaxis the absorbance determined at the maximum of the absorption curve; Miis the molar mass of each aflatoxin, in grams per mol; Licensed Copy: Wang Bin, ISO
40、/EXCHANGE CHINA STANDARDS, 05/03/2008 07:29, Uncontrolled Copy, (c) BSIEN 14123:2007 (E) 6 iis the molar absorption coefficient of each aflatoxin in toluene and acetonitrile (4.18), in square metres per mol; b is the optical path length of the cell, in centimetres. Miand iof aflatoxins B1, B2, G1and
41、 G2are given in Table 1. Table 1 Molar mass and molar absorption coefficient of aflatoxins B1, B2, G1and G2(In mixture of toluene and acetonitrile (4.18) Aflatoxin Mig/mol im2/mol B1312 1930 B2314 2040 G1328 1660 G2330 1790 4.21 Mixed aflatoxins stock solution Prepare a mixed aflatoxins stock soluti
42、on containing 1000 ng/ml of aflatoxin B1and G1, 200 ng/ml of aflatoxin B2and G2in the toluene and acetonitrile mixture (4.18) by appropriate dilution of aflatoxins (B1, B2, G1and G2) stock solutions (4.20). NOTE A commercial total aflatoxins standard solution which is ready to use in a vial containi
43、ng 1000 ng/ml of total aflatoxin may be used as an alternative. 4.22 Diluted mixed aflatoxins stock solution Prepare a diluted mixed aflatoxins stock solution containing 100 ng/ml of aflatoxin B1and G1, 20 ng/ml of aflatoxin B2and G2in the toluene and acetonitrile mixture (4.18) by pipetting exactly
44、 1,0 ml of the mixed aflatoxins stock solution (4.21) into a 10 ml calibrated volumetric flask (5.10), fill to the mark with the toluene and acetonitrile mixture (4.18) and mix well. Wrap the flask tightly in aluminium foil and store it at less than 4 C or in a freezer. Before use, do not open the f
45、lask until the contents have reached room temperature to avoid incorporation of water by condensation. 4.23 Mixed aflatoxins calibration solutions Use the diluted mixed aflatoxins stock solution containing 100 ng/ml of aflatoxin B1and G1, 20 ng/ml of aflatoxin B2and G2(see 4.22) for pipetting the vo
46、lumes as given in Table 2 into a set of 10 ml volumetric flasks (5.10). Evaporate the toluene/acetonitrile solution just to dryness under a stream of nitrogen at room temperature. To each flask, add 4 ml of methanol, let aflatoxins dissolve, dilute to 10 ml with water, and shake well. Methanol and w
47、ater are subject to volume contraction when mixed, so adjust the volume again to the given volume. Licensed Copy: Wang Bin, ISO/EXCHANGE CHINA STANDARDS, 05/03/2008 07:29, Uncontrolled Copy, (c) BSIEN 14123:2007 (E) 7 Table 2 Preparation of mixed aflatoxins calibration solutions Calibration solution
48、 Mass concentration of calibration solution ng/ml Taken from diluted stock solution (4.22) l B1B2G1G21 40 0,400 0,080 0,400 0,080 2 120 1,200 0,240 1,200 0,240 3 200 2,000 0,400 2,000 0,400 4 280 2,800 0,560 2,800 0,560 5 360 3,600 0,720 3,600 0,720 4.24 Spiking solution Prepare a spiking solution b
49、y pipetting 2 ml of the mixed aflatoxins stock solution (containing 1000 ng/ml of aflatoxin B1and G1, 200 ng/ml of aflatoxin B2and G2, see 4.21) into a 10 ml calibrated volumetric flask. Evaporate the toluene/acetonitrile solution just to dryness under a stream of nitrogen at room temperature. Dilute to the mark with methanol and shake well. The concentration of this spiking solution is 200 ng/ml of aflatoxin B1and G1, and 40 ng/ml of aflatoxin B2and G2. Wrap the flask tightly in aluminium foil and store it at less than 4 C. Befo
