1、BRITISH STANDARDBS EN 14123:2007Foodstuffs Determination of aflatoxin 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 St
2、andard EN 14123:2007 has the status of a British StandardICS 67.050g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN 14123:2007This British Stan
3、dard was published under the authority of the Standards Policy and Strategy Committee on 31 January 2008 BSI 2008ISBN 978 0 580 58514 2National forewordThis British Standard is the UK implementation of EN 14123:2007. It supersedes BS EN 14123:2003 which is withdrawn.The UK participation in its prepa
4、ration 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 does not purport to include all the necessary provisions of a contract. Users are responsible for it
5、s correct application.Compliance with a British Standard cannot confer immunity from legal obligations.Amendments/corrigenda issued since publicationDate CommentsEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 14123 December 2007 ICS 67.050 Supersedes EN 14123:2003 English Version Foodstuffs -
6、Determination of aflatoxin 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 cleanup Produits alimentaires - Dosage de laflatoxine B1et de l
7、a somme des aflatoxines B1, B2, G1et G2dans les noisettes, les cacahutes, les pistaches, les figues et 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
8、B1und der Summe von Aflatoxin B1, B2, G1und G2in Haselnssen, Erdnssen, Pistazien, Feigen und Paprikapulver - Hochleistungsflssigchromatographisches Verfahren mit Immunaffinittssulen-Reinigung und Nachsulenderivatisierung This European Standard was approved by CEN on 12 November 2007. CEN members are
9、 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
10、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
11、 status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Ro
12、mania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION 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 reser
13、ved worldwide for CEN national Members. Ref. No. EN 14123:2007: EEN 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 (informativ
14、e) Precision data24 Bibliography 29 EN 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 which is held by DIN. This European Standard shall be given the status of a national standard, e
15、ither 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 14123:2003 with the following changes: a) Validation data on hazelnut are included. WARNING The use o
16、f 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 applicabi
17、lity 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, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, German
18、y, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. 1 Scope This European Standard is applicable to the determination of aflatoxins B1, B2, G1and G2in
19、 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), depending on the equipment used. 2 Normative references The following referenced documents are indispens
20、able 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. EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987)
21、 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 buffered saline (PBS) and applied to an immunoaffinity column (IAC) containing antibodies specific to
22、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-column derivatization (PCD) involving bromination followed by fluorescence detection. The PCD is
23、achieved with either electrochemically generated bromine or with pyridinium hydrobromide perbromide (PBPB). EN 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 wi
24、th grade 1 of EN ISO 3696 4.3 Phosphate buffered saline (PBS), pH = 7,4 Dissolve 0,20 g of potassium chloride, 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 dissolutio
25、n, 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. 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 Po
26、tassium bromide (KBr) 4.7 Acetonitrile, HPLC grade 4.8 Methanol, HPLC grade 4.9 Methanol, p.a. grade 4.10 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,
27、c(HNO3) = 4 mol/l Dilute 28 ml of nitric acid (volume fraction is 65 %), or 26 ml of nitric acid (volume 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 ma
28、ximum capacity of not less than 100 ng of aflatoxin B1and shall give a recovery of not less than 80 % for 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 so
29、lutions that can be applied on the column shall not exceed 12 % of methanol. EN 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 befo
30、re use. 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
31、 120 mg 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
32、toluene 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 afl
33、atoxins 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 th
34、e 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 glassware) and
35、 store 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 tightl
36、y in aluminium 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 th
37、e reference 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; EN 14123:2007 (E) 6 ii
38、s 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 G2are given in Table 1. Table 1 Molar mass and molar absorption coefficient of aflatoxins B1
39、, 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 solution containing 1000 ng/ml of aflatoxin B1and G1, 200 ng/ml of aflatoxin B2and G2in the toluene
40、 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 containing 1000 ng/ml of total aflatoxin may be used as an alternative. 4.22 Diluted mixed aflatoxins
41、 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 1,0 ml of the mixed aflatoxins stock solution (4.21) into a 10 ml calibrated volumetric flas
42、k (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 flask until the contents have reached room temperature to avoid incorporation of water by cond
43、ensation. 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 volumes as given in Table 2 into a set of 10 ml volumetric flasks (5.10). Evaporate the toluene
44、/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 water are subject to volume contraction when mixed, so adjust the volume again to the given vo
45、lume. EN 14123:2007 (E) 7 Table 2 Preparation of mixed aflatoxins calibration solutions Calibration solution 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
46、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 by 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. Evapo
47、rate 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 alumini
48、um foil and store it at less than 4 C. Before use, do not open the flask until the contents have reached room temperature to avoid incorporation of water by condensation. 5 Apparatus 5.1 General All glassware coming into contact with aqueous solutions of aflatoxins shall be washed with acid solution
49、 before use. Many laboratory washing machines do this as part of the washing program. Otherwise soak such laboratory glassware in sulfuric acid (2 mol/l) for several hours (e.g. 15 h overnight), then rinse well (e.g. 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 may cause losses of aflatoxins. In practice, the treatment is necessary for round bottomed flasks, volumetric flasks, measuring cylinders, vials or t
