1、BRITISH STANDARDBS EN ISO 14501:2007Milk and milk powder Determination of aflatoxin M1 content Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatographyICS 67.100.10g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g5
2、0g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN ISO 14501:2007This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 March 2008 BSI 2008ISBN 978 0 580 55233 5National forewordThis
3、 British Standard is the UK implementation of EN ISO 14501:2007. It supersedes BS EN ISO 14501:1999 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/5, Chemical analysis of milk and milk products.A list of organizations represented on this committee
4、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 its correct application.Compliance with a British Standard cannot confer immunity from legal obligations.Amendments issued since publicationA
5、md. No. Date CommentsEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 14501October 2007ICS 67.100.10 Supersedes EN ISO 14501:1998 English VersionMilk and milk powder - Determination of aflatoxin M1 content -Clean-up by immunoaffinity chromatography and determinationby high-performance liquid ch
6、romatography (ISO 14501:2007)Lait et lait en poudre - Dtermination de la teneur enaflatoxine M1 - Purification par chromatographiedimmunoaffinit et dtermination par chromatographie enphase liquide haute performance (ISO 14501:2007)Milch und Milchpulver - Bestimmung des Gehalts anAflatoxin M1- Reinin
7、gung durch Immunaffinitts-Chromatographie und Bestimmung mit Hochleistungs-Flssigchromatographie (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 Euro
8、peanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three official versions (English,
9、 French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cypru
10、s, 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 STANDARDIZATIONCOMIT E
11、UROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2007 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 14501:2007: EForeword This document (EN ISO 14501:2007) has been pre
12、pared by Technical Committee ISO/TC 34 “Agricultural food products“ in collaboration 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, eithe
13、r by publication of an identical text or by endorsement, at the latest by April 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
14、 of the following countries are bound to implement this European Standard: 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, S
15、lovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 14501:2007 has been approved by CEN as a EN ISO 14501:2007 without any modification. BS EN ISO 14501:2007iiiForeword ISO (the International Organization for Standardization) is a worldwide federa
16、tion of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committ
17、ee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance
18、 with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires ap
19、proval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 14501IDF 171 was prepared by Technic
20、al Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. This second edition of ISO 14501IDF 171 cancels and replaces the first edition (ISO 14501:1998), which has been technically re
21、vised. BS EN ISO 14501:2007IDF 171:2007iv Foreword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing Committees carrying out the techni
22、cal work. IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Publication as an International Stan
23、dard requires approval by at least 50 % of the IDF National Committees casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 14501IDF 1
24、71 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. All work was carried out by the Joint IDF-ISO Action Team on Organic contaminants of the Standing Com
25、mittee on Analytical methods for additives and contaminants under the aegis of its project leader, Mr. L. Srensen (DK). This edition of ISO 14501IDF 171 cancels and replaces IDF 171:1995, which has been technically revised. BS EN ISO 14501:2007IDF 171:20071Milk and milk powder Determination of aflat
26、oxin M1content Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography 1 Scope This International Standard specifies a method for the determination of aflatoxin M1content in milk and milk powder. The limit of detection is 0,08 g/kg for whole milk powder,
27、 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. Aflatoxins are carcinogenic to humans. Attention is
28、 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 aluminium foil. 3 The use of non-acid-washed glassware
29、 (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. sulfuric acid, 2 mol/l) for several hours, then
30、 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 for glassware coming into contact with carcinog
31、enic 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 aflatoxin M1content concentration or mass fraction o
32、f 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 the test portion through an immunoaffinity colu
33、mn that contains specific antibodies bound onto a solid support material. BS EN ISO 14501:2007IDF 171:20072 As the sample passes through the column, the antibodies are selectively bound with any aflatoxin M1(antigen) present and form an antibody-antigen complex. All other components of the sample ma
34、trix 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) coupled with fluorimetric detection. 4 Reagents Use only reagent
35、s 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 column shall have a maximum capacity of not less than 100 ng
36、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 to 80 ng/l when a volume of 50 ml of sample is applied). Any
37、 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 Capacity check. Dilute 1,0 ml of aflatoxin M1standard stock
38、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 the amount of aflatoxin M1eluted from the column by HPLC aft
39、er 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 working solution of 0,005 g/ml (4.4.3) to 10 ml with water. Mix we
40、ll 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 column by HPLC after preparing a suitable dilution of the fina
41、l 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. Other volumes in the same proportion may be used. Degas the s
42、olution (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 Aflatoxin M1standard solutions. 4.4.1 Aflatoxin M1standard cal
43、ibration solution. BS EN ISO 14501:2007IDF 171:20073Prepare 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 actual aflatoxin M1concentration by measurement of the absorbance at the maxim
44、um 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 nm and 370 nm. Measure the absorbance, A, at its maximum absorption wavelengt
45、h, 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, in grams per mole, of aflatoxin M1(M = 328 g/mol); d is the optical pathlengt
46、h, 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 concentration, dilute the aflatoxin M1standard calibration solution (4.4.1) wit
47、h 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 solution in a refrigerator at a temperature between 1 C and 5 C in the dark. Un
48、der 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 solution and prepare a fresh standard stock solution. 4.4.3 Aflatoxin M1stand
49、ard 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 the 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/ml, and 0,40 ng/m
