1、January 2008DEUTSCHE NORM English price group 10No part of this standard 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 67.100.10!$K_M“1406042
2、www.din.deDDIN EN ISO 14501Milk and milk powder Determination of aflatoxin M1 content Clean-up by immunoaffinity chromatography and determination byhigh-performance liquid chromatography (ISO 14501:2007)English version of DIN EN ISO 14501:2008-01Milch und Milchpulver Bestimmung des Gehalts an Aflato
3、xin M1 Reinigung durch Immunaffinitts-Chromatographie und Bestimmung mitHochleistungs-Flssigchromatographie (ISO 14501:2007)Englische Fassung DIN EN ISO 14501:2008-01SupersedesDIN EN ISO 14501:1999-01www.beuth.deDocument comprises 14 pagesDIN EN ISO 14501:2008-01 2 National foreword This standard fa
4、lls in the domain of Technical Committee CEN/TC 302 “Milk and milk products Methods of sampling and analysis” (Secretariat: NEN, the Netherlands). It is based on International Standard ISO 22160:2007 which was prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France), Su
5、bcommittee SC 5 “Milk and milk products” (Secretariat: NEN, the Netherlands). Based on the results of parallel voting, ISO 14501:2007 has been adopted as European Standard. The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und land-wirtschaftliche Produkte
6、(Food and Agricultural Products Standards Committee), Technical Committee Milch und Milchprodukte Probenahme- und Untersuchungsverfahren. The DIN Standards corresponding to the International Standard referred to in the bibliography of the EN are as follows: ISO 707 DIN EN ISO 707 ISO 5725-1 DIN ISO
7、5725-1 ISO 5725-2 DIN ISO 5725-2 Amendments This standard differs from DIN EN ISO 14501:1999-01 as follows: a) The test is now carried out using acetonitrile instead of chloroform. b) The results of the interlaboratory trial have been revised (see Annex A). c) The standard has been editorially revis
8、ed to take account of the new style rules for standards. Previous editions DIN EN ISO 14501: 1999-01 National Annex NA (informative) Bibliography DIN EN ISO 707, Milk and milk products Guidance on sampling DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: G
9、eneral principles and definitions DIN ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 14501 Octob
10、er 2007 ICS 67.100.10 Supersedes EN ISO 14501:1998English Version Milk and milk powder - Determination of aflatoxin M1 content - Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography (ISO 14501:2007) Lait et lait en poudre - Dtermination de la teneur e
11、n aflatoxine M1 - Purification par chromatographie dimmunoaffinit et dtermination par chromatographie en phase liquide haute performance (ISO 14501:2007) Milch und Milchpulver - Bestimmung des Gehalts an Aflatoxin M1- Reiningung durch Immunaffinitts-Chromatographie und Bestimmung mit Hochleistungs-F
12、lssigchromatographie (ISO 14501:2007) This European Standard was approved by CEN on 13 October 2007. CEN members are 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
13、-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 versions (English, French, German). A version in any other language made by translation u
14、nder 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, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece
15、, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management
16、 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 ISO 14501:2007: EForeword This document (EN ISO 14501:2007) has been prepared by Technical Committee ISO/TC 34 “Food products” in col
17、laboration with Technical Committee CEN/TC 302 “Milk and milk products Methods of sampling and analysis” the secretariat of which is held by NEN. 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 Ap
18、ril 2008, and conflicting national standards shall be withdrawn at the latest by April 2008. This document supersedes EN ISO 14501:1998. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: A
19、ustria, 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. Endors
20、ement notice The text of ISO 14501:2007 has been approved by CEN as a EN ISO 14501:2007 without any modification. 2 EN ISO 14501:2007 (E) DIN EN ISO 14501:2008-01 1 Scope This International Standard specifies a method for the determination of aflatoxin M1content in milk and milk powder. The limit of
21、 detection is 0,08 g/kg for whole milk powder, i.e. 0,008 g/l for reconstituted liquid milk. The method is also applicable to low fat milk, skimmed milk, low fat milk powder, and skimmed milk powder. CAUTION 1 The method described in this protocol requires the use of solutions of aflatoxin M1. Aflat
22、oxins are carcinogenic to humans. Attention is drawn to the statement made by the International Agency for Research on Cancer 4, 5. 2 Protect the laboratory in which the analyses are performed adequately from daylight and keep aflatoxin standard solutions protected from light, e.g. by using aluminiu
23、m foil. 3 The use of non-acid-washed glassware (e.g. tubes, vials, flasks, beakers, syringes) for aqueous aflatoxin solutions may cause loss of aflatoxin. Moreover, brand new laboratory glassware, before coming into contact with aqueous solutions of aflatoxin, should be soaked in dilute acid (e.g. s
24、ulfuric acid, 2 mol/l) for several hours, then rinsed well with distilled water to remove all traces of acid (check to ensure pH is in the range 6 to 8). 4 Use decontamination procedures for laboratory wastes such as solid compounds, solutions in organic solvents, aqueous solutions and spills, and f
25、or glassware coming into contact with carcinogenic materials. Suitable decontamination procedures have been developed and validated by the International Agency for Research on Cancer 4, 5. 2 Terms and definitions For the purposes of this document the following terms and definitions apply. 2.1 aflato
26、xin M1content concentration or mass fraction of substances determined by the procedure specified in this International Standard. NOTE The aflatoxin M1concentration is expressed in micrograms per litre and the mass fraction in micrograms per kilogram. 3 Principle Aflatoxin M1is extracted by passing t
27、he test portion through an immunoaffinity column that contains specific antibodies bound onto a solid support material. 3 EN ISO 14501:2007 (E) DIN EN ISO 14501:2008-01 As the sample passes through the column, the antibodies are selectively bound with any aflatoxin M1(antigen) present and form an an
28、tibody-antigen complex. All other components of the sample matrix are washed off the column with water. Then aflatoxin M1is eluted from the column and the eluate is collected. The amount of aflatoxin M1present in this eluate is determined by means of high-performance liquid chromatography (HPLC) cou
29、pled with fluorimetric detection. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and only distilled or demineralized water or water of equivalent purity. 4.1 Immunoaffinity column. The immunoaffinity column shall contain antibodies against aflatoxin M1. The
30、column shall have a maximum capacity of not less than 100 ng of aflatoxin M1(which corresponds to 2 g/l when a volume of 50 ml of a test portion is applied). It shall give a recovery of not less than 80 % for aflatoxin M1when a standard solution containing 4 ng of toxin is applied (which corresponds
31、 to 80 ng/l when a volume of 50 ml of sample is applied). Any immunoaffinity column meeting the performance specifications mentioned above can be used. The performance of the columns shall be checked regularly and at least once for every batch of columns (see the procedure in 4.1.1 and 4.1.2). 4.1.1
32、 Capacity check. Dilute 1,0 ml of aflatoxin M1standard stock solution (4.4.2) to 50 ml with water. Mix well and apply the whole volume to the immunoaffinity column carefully following the recommendations given by the manufacturer for the use of columns. Wash the column and elute the toxin. Determine
33、 the amount of aflatoxin M1eluted from the column by HPLC after preparing a suitable dilution of the final eluate. Calculate the capacity for the aflatoxin. Compare the result with the requirements given in 4.1. 4.1.2 Recovery check. Use a pipette (5.4) to dilute 0,8 ml of aflatoxin M1standard worki
34、ng solution of 0,005 g/ml (4.4.3) to 10 ml with water. Mix well and apply the whole volume to the immunoaffinity column carefully following the recommendations given by the manufacturer for the use of columns. Wash the column and elute the toxin. Determine the amount of aflatoxin M1eluted from the c
35、olumn by HPLC after preparing a suitable dilution of the final eluate. Calculate the recovery for the aflatoxin. Compare the result with the requirements given in 4.1. 4.2 Acetonitrile, pure, HPLC grade. 4.2.1 Acetonitrile solution, 25 %. Add 250 ml of acetonitrile (4.2) to 750 ml of water and mix.
36、Other volumes in the same proportion may be used. Degas the solution (eluent) before using it. 4.2.2 Acetonitrile solution, 10 %. Add 100 ml of acetonitrile (4.2) to 900 ml of water and mix. Other volumes in the same proportion may be used. Degas the solution before using it. 4.3 Nitrogen gas. 4.4 A
37、flatoxin M1standard solutions. 4.4.1 Aflatoxin M1standard calibration solution. 4 EN ISO 14501:2007 (E) DIN EN ISO 14501:2008-01 Prepare an aflatoxin M1standard calibration solution by dissolving aflatoxin M1(C17H12O7) in acetonitrile (4.2) to give a nominal concentration of 10 g/ml. Determine the a
38、ctual aflatoxin M1concentration by measurement of the absorbance at the maximum absorption wavelength of the solution as follows. Use the spectrophotometer (5.14) to record the absorbance of the aflatoxin M1standard calibration solution against acetonitrile (4.2) as blank at wavelengths between 330
39、nm and 370 nm. Measure the absorbance, A, at its maximum absorption wavelength, max, which is close to 350 nm. Calculate the concentration, c1, expressed in micrograms per millilitre, by using Equation (1): 1100cAMd =(1) where: A is the numerical value of the absorbance at max; M is the molar mass,
40、in grams per mole, of aflatoxin M1(M = 328 g/mol); d is the optical pathlength, in centimetres (d = 1 cm); is the numerical value of the absorption coefficient, in square metres per mole, of the toxin in acetonitrile ( = 1 985 m2mol1). 4.4.2 Aflatoxin M1standard stock solution. After checking its co
41、ncentration, dilute the aflatoxin M1standard calibration solution (4.4.1) with acetonitrile (4.2) to an aflatoxin M1standard stock solution of 0,1 g/ml. The standard stock solution shall be well stoppered and wrapped in aluminium foil to protect it from light. Store the aflatoxin M1standard stock so
42、lution in a refrigerator at a temperature between 1 C and 5 C in the dark. Under these conditions the stock solution is stable for at least two months. If the standard stock solution is more than two months old, determine the aflatoxin M1concentration before use. If there is any change, discard the
43、solution and prepare a fresh standard stock solution. 4.4.3 Aflatoxin M1standard working solutions. Before preparing the aflatoxin M1standard working solutions, allow the standard stock solution (4.4.2) to attain ambient temperature. Prepare the standard working solutions on the day of use. Dilute t
44、he aflatoxin M1standard stock solution (4.4.2) with the 10 % acetonitrile solution (4.2.2) to an aflatoxin M1concentration of 0,005 g/ml. Remove aliquots of the diluted standard stock solution to prepare a series of standard working solutions containing, for example, 0,05 ng/ml, 0,10 ng/ml, 0,20 ng/
45、ml, and 0,40 ng/ml of aflatoxin M1by diluting with the 10 % acetonitrile solution (4.2.2). Other final dilutions may be chosen, depending on the injection loop volume. 5 Apparatus Usual laboratory equipment and, in particular, the following. 5.1 Disposable syringes, of capacities 10 ml and 50 ml. 5
46、EN ISO 14501:2007 (E) DIN EN ISO 14501:2008-01 5.2 Vacuum system i.e. Bchner flask, Vac-Elut system 1)or peristaltic pump. 5.3 Centrifuge, capable of producing a radial acceleration of at least 2 000g. 5.4 Pipettes, of capacities 1,0 ml, 2,0 ml and 50,0 ml or suitable autopipette. 5.5 Glass beakers,
47、 of capacity 250 ml. 5.6 One-mark volumetric flask, of capacity 100 ml. 5.7 Water baths, capable of operating at 30 C 2 C, at between 35 C and 37 C and 50 C 5 C. 5.8 Filter paper, Whatman No. 4 1)or equivalent. 5.9 Graduated conical glass tubes, with ground glass neck and stopper of capacities 5 ml,
48、 10 ml and 20 ml. 5.10 HPLC apparatus, equipped with a pulse-free pump, capable of producing a constant volume flow rate of about 1 ml/min, and an injector system, with a fixed or variable injection volume loop, capable of injecting volumes of 20 l to 500 l. 5.11 Reversed phase HPLC analytical colum
49、n, with 3 m or 5 m octadecyl silica packing and a guard column filled with reverse phase material. 5.12 Fluorescence detector, capable of providing about 365 nm excitation and 435 nm emission wavelengths and of detecting (signal to noise ratio: 5) aflatoxin M1when 0,02 ng is injected under appropriate chromatographic conditions. 5.13 Strip chart recorder, with a printer or plotter, or electronic integrator or computer-based data processing system. 5.14 Spectrophotometer, capable of measuring at wavelengths from
