BS EN ISO 16050-2011 Foodstuffs Determination of aflatoxin B1 and the total content of aflatoxins B1 B2 G1 and G2 in cereals nuts and derived products High-performance liquid chrom.pdf

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1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN ISO 16050:2011Foodstuffs Determination of aflatoxin B1, and the total content of aflatoxins B1, B2, G1 and G2 in cereals, nuts and derived products High-performance liquid

2、chromatographic method (ISO 16050:2003)BS EN ISO 16050:2011 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO 16050:2011. It is identical to ISO 16050:2003. It supersedes BS EN 12955:1999, which is withdrawn.The UK participation in its preparation was entruste

3、d to T e c h n i c a l C o m m i t t e e A W / - / 3 , F o o d a n a l y s i s - H o r i z o n t a l m e t h o d s .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 contrac

4、t. Users are responsible for its correct application. BSI 2011 ISBN 978 0 580 73140 2 ICS 67.050; 67.060; 67.080.10 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee

5、on 31 July 2011.Amendments issued since publicationDate T e x t a f f e c t e dEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 16050 July 2011 ICS 67.060; 67.080.10 Supersedes EN 12955:1999English Version Foodstuffs - Determination of aflatoxin B1, and the total content of aflatoxins B1, B2

6、, G1 and G2 in cereals, nuts and derived products - High-performance liquid chromatographic method (ISO 16050:2003) Produits alimentaires - Dosage de laflatoxine B1 et dtermination de la teneur totale en aflatoxines B1, B2, G1 et G2 dans les crales, les fruits coque et les produits drivs - Mthode pa

7、r chromatographie liquide haute performance (ISO 16050:2003) Lebensmittel - Bestimmung von Aflatoxin B1 und der Summe von Aflatoxin B1, B2, G1 und G2 in Getreiden, Nssen und verwandten Produkten - Hochleistungsflssigchromatographisches Verfahren (ISO 16050:2003) This European Standard was approved b

8、y CEN on 17 June 2011. 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-to-date lists and bibliographical references concerning such national standar

9、ds may be obtained on application to the CEN-CENELEC 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 noti

10、fied to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuani

11、a, 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 Centre: Avenue Marnix 17, B-1000 Brussels 2011 CEN

12、All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 16050:2011: EBS EN ISO 16050:2011 EN ISO 16050:2011 (E) 3 Foreword The text of ISO 16050:2003 has been prepared by Technical Committee ISO/TC 34 “Food products” of the International O

13、rganization for Standardization (ISO) and has been taken over as EN ISO 16050:2011 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, either by publication of an iden

14、tical text or by endorsement, at the latest by January 2012, and conflicting national standards shall be withdrawn at the latest by January 2012. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held

15、 responsible for identifying any or all such patent rights. This document supersedes EN 12955:1999. Originally, EN 12955:1999 “Foodstuffs - Determination of aflatoxin B1, and the sum of aflatoxins B1, B2, G1and G2in cereals, shell-fruits and derived products - High performance liquid chromatographic

16、 method with post column derivatization and immunoaffinity column clean up“ was the basis for ISO 16050. In order to avoid having two equal standards on CEN- and ISO-level on the same topic, it was decided to take over ISO 16050 as EN ISO 16050 and to withdraw EN 12955 as soon as EN ISO 16050 is pub

17、lished. 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, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

18、 Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 16050:2003 has been approved by CEN as a EN ISO 16050:2011 without any modification. BS EN

19、ISO 16050:2011Reference numberISO 16050:2003(E)ISO 2003INTERNATIONAL STANDARD ISO16050First edition2003-09-01Foodstuffs Determination of aflatoxin B1, and the total content of aflatoxins B1, B2, G1and G2in cereals, nuts and derived products High-performance liquid chromatographic method Produits ali

20、mentaires Dosage de laflatoxine B1et dtermination de la teneur totale en aflatoxines B1, B2, G1et G2dans les crales, les fruits coque et les produits drivs Mthode par chromatographie liquide haute performance BS EN ISO 16050:2011ISO 16050:2003(E) PDF disclaimer This PDF file may contain embedded typ

21、efaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infr

22、inging Adobes licensing policy. The ISO Central Secretariat accepts no liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters were opt

23、imized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below. ISO 2003 All rights reserved. Unless otherwise specified, n

24、o part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case post

25、ale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2003 All rights reservedBS EN ISO 16050:2011ISO 16050:2003(E) ISO 2003 All rights reserved iiiContents Page Foreword iv 1 Scope 1 2 Normative references . 1 3

26、 Principle . 1 4 Reagents 1 5 Apparatus 4 6 Procedure 5 6.1 General. 5 6.2 Extraction. 5 6.3 Clean-up. 6 6.4 HPLC operating conditions 6 6.5 Identification 6 6.6 Calibration graph 6 6.7 Determination 7 7 Calculation of results 7 8 Precision 8 8.1 Interlaboratory test . 8 8.2 Repeatability 8 8.3 Repr

27、oducibility 9 9 Test report . 9 Annex A (informative) Results of interlaboratory test 10 Bibliography . 12 BS EN ISO 16050:2011ISO 16050:2003(E) iv ISO 2003 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (IS

28、O 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 committee. International organizations, gove

29、rnmental 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 with the rules given in the ISO/IEC

30、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 approval by at least 75 % of the member

31、 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 16050 was prepared by Technical Committee ISO/TC 34, Food products. It is

32、 based on EN 12955:1999 elaborated by CEN/TC 275, Food analysis Horizontal methods. BS EN ISO 16050:2011INTERNATIONAL STANDARD ISO 16050:2003(E) ISO 2003 All rights reserved 1Foodstuffs Determination of aflatoxin B1, and the total content of aflatoxins B1, B2, G1and G2in cereals, nuts and derived pr

33、oducts High-performance liquid chromatographic method WARNING The use of this standard involves hazardous materials and operations. 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 appropriat

34、e safety and health practice and to determine the applicability of regulatory limitations prior to use. 1 Scope This International Standard specifies a reverse-phase high-performance liquid chromatographic method, with immunoaffinity column clean-up and post-column derivatization, for the determinat

35、ion of aflatoxins in cereals, nuts and derived products. The limit of quantification for aflatoxin B1, and for the sum of aflatoxins B1, B2, G1and G2, is 8 g/kg. The method has been validated for maize containing 24,5 g/kg, for peanut butter containing 8,4 g/kg, and for raw peanuts containing 16 g/k

36、g of total aflatoxins. It has also been shown that this method can be used for oilseed products, dried fruits and derived products. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies.

37、For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 3696:1987, Water for analytical laboratory use Specification and test methods 3 Principle The test sample is extracted with a mixture of methanol and water. The sample extract is filtered, d

38、iluted with water, and applied to an affinity column containing antibodies specific for aflatoxins B1, B2, G1and G2. The aflatoxins are isolated, purified and concentrated on the column then removed from the antibodies with methanol. The aflatoxins are quantified by reverse-phase high-performance li

39、quid chromatography (HPLC) with fluorescence detection and post-column derivatization. 4 Reagents Use only reagents recognized analytical grade, unless otherwise stated. 4.1 Water, according to grade 1 of ISO 3696:1987. 4.2 Sodium chloride. BS EN ISO 16050:2011ISO 16050:2003(E) 2 ISO 2003 All rights

40、 reserved4.3 Iodine, crystalline, or as an alternative, pyridinium hydrobromide perbromide (PBPB)1). 4.4 Aflatoxin, in crystal form or as a film ampoule. WARNING Aflatoxins are carcinogenic to human subjects. Attention is drawn to the statement made by the International Agencies for Research on Canc

41、er (WHO) (see 1, 2). Adequately protect from daylight the laboratory where the analyses are carried out. This may be achieved effectively by using ultraviolet (UV) absorbing foil on the windows in combination with subdued light (no direct sunlight), or curtains or blinds in combination with artifici

42、al light (fluorescent tubes are acceptable). 4.5 Acetonitrile, HPCL grade. 4.6 Methanol, analytical grade. 4.7 Methanol, HPLC grade. 4.8 Toluene, analytical grade. WARNING Toluene is highly flammable and harmful. Standard preparation involving this solvent shall be performed in a fume cupboard. Oper

43、ations outside the fume cupboard, such as measurement of standards by UV spectrometry, shall be performed with the standards in closed containers. 4.9 Toluene/acetonitrile mixture Mix 98 parts per volume of toluene (4.8) with 2 parts per volume of acetonitrile (4.5) (see Warning in 4.8). 4.10 Extrac

44、tion solvent Mix 7 parts per volume of methanol (4.6) with 3 parts per volume of water (4.1). Other extraction solvent mixtures which are compatible with the mobile phase may also be used if proved to be more effective or recommended by the manufacturer of the immunoaffinity (IA) column. 4.11 Mobile

45、 phase Mix 3 parts per volume of water (4.1) with 1 part per volume of acetonitrile (4.5) and 1 part per volume of methanol (4.7). Degas the solution before use. 4.12 Post-column derivatization reagent Dissolve 100 mg of iodine (4.3) in 2 ml of methanol (4.6). Add 200 ml of water (4.1), stir for 1 h

46、, then filter through a 0,45 m membrane filter (5.8). Prepare the solution the week of use and store the solution in the dark or in a brown glass bottle. Before use, stir the solution for 10 min. As an alternative, dissolve 50 mg of PBPB (4.3) in 1 000 ml of water. This solution may be used for up t

47、o 4 days if stored in a dark place at room temperature. 4.13 Aflatoxin B1, B2, G1and G2stock solutions WARNING Protect solutions containing aflatoxin from light as far as possible (keep in the dark, use aluminium foil or amber-coloured glassware). 1) CAS: 39416-48-3 (CAS = Chemical Abstract Service)

48、. BS EN ISO 16050:2011ISO 16050:2003(E) ISO 2003 All rights reserved 3Dissolve aflatoxin B1, B2, G1and G2separately in the toluene/acetonitrile mixture (4.9) to give separate solutions containing 10 g/ml. To determine the exact concentration of aflatoxin in each stock solution, record the absorption

49、 curve at a wavelength between 330 nm and 370 nm in 1 cm quartz glass cells (5.7) using a spectrometer (5.6) with a toluene/acetonitrile mixture (4.9) as reference. Calculate the aflatoxin concentration of each aflatoxin, i, in micrograms per millilitre, using Equation (1): max1000iiiAMd=(1) where Amax is the absorbance determined at the maximum of the absorption curve; Miis the molecular mass of each aflatoxin, in grams; iis the molar absorption coefficient of each aflatoxin in tol