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1、 Reference numbers ISO 14674:2005(E) IDF 190:2005(E) ISO and IDF 2005 INTERNATIONAL STANDARD ISO 14674 IDF 190 First edition 2005-05-01 Milk and milk powder Determination of aflatoxin M1 content Clean-up by immunoaffinity chromatography and determination by thin-layer chromatography Lait et lait en
2、poudre Dtermination de la teneur en aflatoxin M1 Purification par chromatographie dimmunoaffinit et dtermination par chromatographie sur couche mince Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for
3、 Resale, 04/20/2007 03:27:49 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14674:2005(E) IDF 190:2005(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited
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6、e by ISO member bodies and IDF national committees. In the unlikely event that a problem relating to it is found, please inform the ISO Central Secretariat at the address given below. ISO and IDF 2005 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or u
7、tilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO or IDF at the respective address below. ISO copyright office International Dairy Federation Case postale 56 CH-1211 Geneva 20 Diamant Building Boulevard
8、Auguste Reyers 80 B-1030 Brussels Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88 Fax + 41 22 749 09 47 Fax + 32 2 733 04 13 E-mail copyrightiso.org E-mail infofil-idf.org Web www.iso.org Web www.fil-idf.org Published in Switzerland ii ISO and IDF 2005 All rights reserved Copyright International Organi
9、zation for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 03:27:49 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14674:2005(E) IDF 190:2005(E) ISO and IDF 2005 All rights reserved iii Co
10、ntents Page Forewordiv 1 Scope1 2 Terms and definitions.1 3 Principle.1 4 Reagents2 5 Apparatus.3 6 Sampling4 7 Preparation of test samples.5 7.1 Milk or liquid milk products .5 7.2 Milk powders .5 7.3 Immunoaffinity clean-up.5 8 Procedure.6 8.1 Unidirectional TLC6 8.2 Bidirectional TLC 6 9 Calculat
11、ion and expression of results7 9.1 Calculation.7 9.2 Expression of results8 10 Precision8 10.1 Interlaboratory test .8 10.2 Repeatability8 10.3 Reproducibility8 11 Test report9 Annex A (informative) Results of interlaboratory test10 Bibliography .11 Copyright International Organization for Standardi
12、zation Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 03:27:49 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14674:2005(E) IDF 190:2005(E) iv ISO and IDF 2005 All rights reserved Foreword ISO (the Inter
13、national Organization for Standardization) is a worldwide federation 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
14、been established has the right to be represented on that committee. 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 st
15、andardization. International Standards are drafted in accordance 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
16、for voting. Publication as an International Standard requires approval 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
17、all such patent rights. ISO 14674IDF 190 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately by AOA
18、C International. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 03:27:49 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14674:2005(E) IDF 190:2005
19、(E) ISO and IDF 2005 All rights reserved v 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 techn
20、ical work. IDF collaborates with ISO and AOAC International 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
21、as an International Standard requires approval by at least 50 % of the 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 ri
22、ghts. ISO 14674IDF 190 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International. A
23、ll work was carried out by the Joint ISO/IDF/AOAC Action Team, Organic contaminants, of the Standing Committee on Analytical methods for additives and contaminants, under the aegis of its project leader, Mrs S Dragacci (FR). Copyright International Organization for Standardization Provided by IHS un
24、der license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 03:27:49 MDTNo reproduction or networking permitted without license from IHS -,-,- Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical St
25、andards 1/9972545001 Not for Resale, 04/20/2007 03:27:49 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARD ISO 14674:2005(E) IDF 190:2005(E) ISO and IDF 2005 All rights reserved 1 Milk and milk powder Determination of aflatoxin M1 content Clean-up by im
26、munoaffinity chromatography and determination by thin-layer chromatography WARNING The method described in this International Standard requires the use of aflatoxin M1 solutions. Aflatoxins are carcinogenic to human subjects. Attention is drawn to the statement made by the International Agency for R
27、esearch on Cancer (WHO). 5 Aflatoxins are subject to light degradation. Adequately protect analytical work from daylight and keep aflatoxin standard solutions protected from light, for example by using amber vials or aluminium foil. The use of non-acid-washed glassware (e.g. vials, tubes, flasks) fo
28、r aqueous aflatoxin solutions can cause a loss of aflatoxin. Take special care with new glassware. Before use, soak the new glassware in diluted acid (e.g. 2 mol/l sulfuric acid) for several hours, then rinse extensively with distilled water to remove all traces of acid. Check to ensure that the pH
29、is in the range of 6 to 8 by using a pH-paper. Use the decontamination procedure for laboratory wastes developed and validated by the International Agency for Research on Cancer (WHO). 5 1 Scope This International Standard specifies a method for the determination of the aflatoxin M1 (AFM1) content o
30、f milk and milk powder by a method including a clean-up step using immunoaffinity chromatography followed by a thin-layer chromatography (IAC-TLC). The method is applicable to raw milk, low fat or skimmed liquid milk and milk powder. The lowest quantity of AFM1 that can commonly be determined is 2 n
31、g, which corresponds to a limit of quantification close to 0,10 g/l for liquid milk or dissolved milk powder (for a spot of 20 l). 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 aflatoxin M1 content mass fraction of substances determined by
32、the method specified in this International Standard NOTE The aflatoxin M1 content is expressed as micrograms per litre for liquid milk products, and as micrograms per kilogram for milk powder. 3 Principle Aflatoxin M1 (AFM1) is extracted by passing the test portion through an immunoaffinity column.
33、The column contains specific antibodies bound onto a solid support material. As the sample passes through the column, the antibodies selectively bind with any AFM1 (antigen) present and form an antibody-antigen complex. All Copyright International Organization for Standardization Provided by IHS und
34、er license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 03:27:49 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14674:2005(E) IDF 190:2005(E) 2 ISO and IDF 2005 All rights reserved other components of the sample matrix are washed o
35、ff the column with water. Then the AFM1 is eluted from the column with methanol and acetonitrile. After concentration of the eluate, the amount of AFM1 is determined by one-dimensional thin-layer chromatography. In the case of interference, two-dimensional thin- layer chromatography is carried out t
36、o separate the AFM1 from its impurities. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or water of equivalent purity. 4.1 Pure solvents. WARNING Some of the pure solvents (e.g. chloroform, acetonitrile, toluene and metha
37、nol) are toxic. Take all necessary precautions where needed. 4.1.1 Chloroform (CHCl3). 4.1.2 Acetonitrile (CH3CN). 4.1.3 Diethyl ether (C2H5)2O. 4.1.4 Methanol (CH3OH). 4.1.5 Toluene (C6H5CH3). 4.1.6 Acetone (CH3.COCH3), optional. 4.1.7 Isopropanol (CH3CHOHCH3), optional. 4.2 Acetonitrile/methanol s
38、olution, of volume ratio 3:2. Add 30 ml of acetonitrile (4.1.2) to 20 ml of methanol (4.1.4) and mix. 4.3 Toluene/acetonitrile solution, of volume ratio 9:1. Add 9 ml of toluene (4.1.5) to 1 ml of acetonitrile (4.1.2) and mix. Use this solution to resuspend the AFM1 standard solutions (4.5) and the
39、evaporated eluate before the TLC analysis. 4.4 TLC development solvents. 4.4.1 Unidirectional TLC solution. Prepare a 100 ml unidirectional TLC solution by adding 4 ml of methanol (4.1.4) and 1 ml of water to 95 ml of diethyl ether (4.1.3) and mix well (volume ratio 95:4:1). 4.4.2 Bidirectional TLC
40、solution, optional. Prepare a 100 ml bidirectional TLC solution by adding 10 ml of acetone (4.1.6) and 3 ml of isopropanol (4.1.7) to 87 ml of chloroform (4.1.1) and mix well (volume ratio 87:10:3). 4.5 Aflatoxin M1 standard solution. 4.5.1 AFM1 standard stock solution. Prepare an AFM1 standard stoc
41、k solution with a nominal concentration of 10 g/ml chloroform (4.1.1); i.e. by resuspending a lyophilized film of 10 g of AFM1 to 1 ml of chloroform. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for
42、 Resale, 04/20/2007 03:27:49 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14674:2005(E) IDF 190:2005(E) ISO and IDF 2005 All rights reserved 3 In accordance with the AOAC protocol 6, determine the concentration of the AFM1 standard stock solution by measuring its abs
43、orbance at the wavelength of maximum absorption and use a calibrated spectrometer to record the absorbance of the standard stock solution against chloroform (4.1.1), used as blank, at between = 200 nm and = 400 nm. Check the purity of the AFM1 by recording the spectrum between 200 nm and 400 nm. Mea
44、sure the absorbance (A) at the wavelength for maximum absorption (max), i.e. close to 365 nm. Calculate the mass concentration, c, expressed in micrograms per millilitre, by using the following equation: 100/cAM= where A is the numerical value of the absorbance measured at max; M is the numerical va
45、lue of the molar mass of the AFM1, in grams per mole (M = 328 g/mol); is the numerical value of the absorption coefficient of AFM1 in chloroform, in square metres per mole ( = 1 995 m2mol1). Keep the AFM1 standard stock solution in a well-stoppered amber-coloured vial protected from light. Store the
46、 standard solution at below 0 C. Under these conditions, the AFM1 standard stock solution is stable for about one year. 4.5.2 AFM1 standard working solution. 4.5.2.1 Working solution A. Use a volumetric pipette or a Hamilton-like microsyringe (5.2) to transfer 50 l of AFM1 standard stock solution (4
47、.5.1) into a vial. Evaporate the solution to dryness. Resuspend the dried solution with 500 l of toluene/acetonitrile solution (4.3) to obtain an AFM1 standard working solution with concentration of 1 g/ml (working solution A). Use solution A to spot onto TLC plates for test samples with a high cont
48、amination level or when the determination level is close to 0,50 g/l. 4.5.2.2 Working solution B. Transfer 100 l of solution A to a vial. Add 900 l of toluene/acetonitrile solution (4.3) to obtain an AFM1 standard working solution with concentration of 0,1 g/ml (working solution B). Use solution B to spot onto TLC plates for test samples with a low contamination level or when the determination level is close to 0,10 g/l. 5 Apparatus Usual laborat