1、BRITISH STANDARD BS5766-16: 1999 ISO14718: 1998 Methods for analysis of animal feeding stuffs Part16: Determination of aflatoxin B 1content of mixed feeding stuffs (high-performance liquid chromatographic method) ICS65.120BS5766-16:1999 This BritishStandard, having been prepared under the directiono
2、f the Consumer Products and Services Sector Committee, was published underthe authority of the Standards Committee and comes into effect on 15 May1999 BSI02-2000 ISBN 0 580 32126 6 National foreword This BritishStandard reproduces verbatim ISO14718:1998 and implements it as the UK national standard.
3、 The UK participation in its preparation was entrusted to Technical Committee AW/10, Animal feeding stuffs, which has the responsibility to: aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change,
4、 and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The BritishStandards which implement international or Europe
5、an publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necess
6、ary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pagesi andii, theISO ti
7、tle page, pages ii to iv, pages1 to12 and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover. Amendments issued since publication Amd. No. Date CommentsBS5766-16:1999 BSI
8、02-2000 i Contents Page National foreword Inside front cover Foreword iii Text of ISO14718 1ii blankBS5766-16:1999 ii BSI 02-2000 Contents Page Foreword iii 1 Scope 1 2 Normative reference 1 3 Principle 1 4 Reagents and materials 1 5 Apparatus 3 6 Sampling 5 7 Preparation of the test sample 5 8 Proc
9、edure 5 8.1 General 5 8.2 Determination of the absorption spectrum of the aflatoxin B 1standard solution 6 8.3 Extraction 6 8.4 Clean-up 6 8.4.1 Florisil purification 6 8.4.2 C 18purification 6 8.5 High-performance liquid chromatography 7 8.5.1 General 7 8.5.2 HPLC pump settings 7 8.5.3 Post-column
10、pump settings for HPLC with iodine derivatization 7 8.5.4 Fluorescence detector 7 8.5.5 Injector 7 8.5.6 Check of chromatographic separation 7 8.5.7 Check of the stability of the system 7 8.5.8 Check of linearity 7 8.5.9 Injection of sample extracts 7 9 Calculation of results 8 9.1 Calculation of th
11、e aflatoxin B 1content of the aflatoxin B 1standard solution 8 9.2 Calculation of the mass of aflatoxin B 1in the injected reference calibration solution 8 9.3 Calculation of the mass of aflatoxin B 1in the test solution 8 9.4 Calculation of the aflatoxin B 1content of the sample 8 10 Accuracy 8 10.
12、1 Systematic error 8 10.2 Precision 9 10.2.1 Interlaboratory test 9 10.2.2 Repeatability 9 10.2.3 Reproducibility 9 11 Test report 9 Annex A (informative) Results of interlaboratory test 10 Annex B (informative) Equivalence of iodine and bromine derivatization 10 Figure 1 Diagrammatic representation
13、 of the HPLC system for derivatization with iodine 4 Figure 2 Diagrammatic representation of the HPLC system for derivatization with bromine 4 Table A.1 Statistical results of the interlaboratory test of the method applying derivatization with iodine 10 Table A.2 Summary of the published statistical
14、 results of the interlaboratory test 10 Table B.1 Statistical results of interlaboratory tests of the equivalence of iodine and bromine derivatization 11BS5766-16:1999 BSI 02-2000 iii Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bo
15、dies (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 committee. International organizatio
16、ns, 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 with the rules given in the
17、ISO/IEC Directives, Part3. 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 least75% of the member bodies casting a vote. International Standard ISO14718 was prepared by
18、Technical Committee ISO/TC34, Agricultural food products, Subcommittee SC10, Animal feeding stuffs. Annex A and Annex B of this International Standard are for information only. Descriptors: Agricultural products, food, animal feeding products, mixtures, chemical analysis, determination of content, a
19、flatoxins, high performance liquid chromatography.iv blankBS5766-16:1999 BSI 02-2000 1 1 Scope This International Standard specifies a high-performance liquid chromatographic (HPLC) method for the determination of aflatoxin B 1content of animal feeding stuffs including those containing citrus pulp.
20、The lower limit of determination is14g/kg. NOTE 1This International Standard may be applicable for the determination of the aflatoxin B 1content of a number of raw materials and straight feeding stuffs such as corn gluten, groundnut, palm kernel, copra, citrus pulp, tapioca, soya bean, rice bran, po
21、llard, rape seed, niger seed and cotton seed (seereferences1 and2). These materials were, however, not included in the collaborative testing of the method. NOTE 2This International Standard may also be applicable for the determination of the content of the sum of the aflatoxins B 1 , B 2 , G 1 , and
22、 G 2 . However, the method has not been validated for this parameter by collaborative testing. 2 Normative reference The following normative document contains provisions which, through reference in this text, constitute provisions of this International Standard. For dated references, subseqent amend
23、ments to, or revisions of, this publication do not apply. However, parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent edition of the normative document indicated below. For undated references, the latest edition of the
24、 normative document referred to applies. Members of IEC and ISO editions maintain registers of currently valid International Standards. ISO6498:1998, Animal feeding stuffs Preparation of test sample. 3 Principle The sample is extracted with chloroform. The extract is filtered and an aliquot portion
25、is purified on a Florisil 1)cartridge and a C 18cartridge. Thefinal separation and determination is achieved by high-performance liquid chromatography (HPLC) using a reverse-phase C 18column, followed by post-column derivatization with iodine or bromine, and fluorescence detection. 4 Reagents and ma
26、terials Use only reagents of recognized analytical grade. 4.1 Water, demineralized or deionized, with resistivity of at least10M7cm, or water of at least equivalent purity. 4.2 Concentrated sulfuric acid, c(H 2 SO 4 )=18mol/l, (H 2 SO 4 )=1,84g/ml. 4.3 Sulfuric acid, c(H 2 SO 4 )=2mol/l. Carefully a
27、dd105ml of concentrated sulfuric acid(4.2) to895ml of water and mix well. Avoid excessive heating of the solution. 4.4 Control sample Prepare a control sample of about2kg of compound feed with an aflatoxin B 1content of about54g/kg by combining samples of previous determinations with an aflatoxin B
28、1content of about54g/kg. Mix thoroughly. The aflatoxin B 1content of the control sample should be determined five times by two analysts following the procedure described in clause8. From the results the mean aflatoxin B 1content, the standard deviation and the coefficient of variation should be calc
29、ulated. 4.5 Acid-washed Celite545, or product of equivalent quality 2) . 4.6 Florisil Sep-Pak style cartridge, Waters No.51960, or product of equivalent quality 3) . 4.7 C 18Sep-Pak style cartridge, Waters No.51910, or product of equivalent quality 3) . 4.8 Acetone 4.9 Methanol 4.10 Acetonitrile 4.1
30、1 Chloroform, stabilized with ethanol (mass fraction0,5% to1,0%). WARNING: Chloroform is a toxic substance. Avoid inhalation of and exposure to chloroform. Work in a fumehood when handling the solvent and solutions thereof. The adsorption characteristics of the Florisil cartridge(4.6) may change if
31、stabilizers other than ethanol are used. When chloroform as described is not available, the adsorption characteristics should be verified in accordance with clause8. 1) Florisil is the trade-name of a commercially available product. This information is given for the convenience of users of this Inte
32、rnational Standard and does not constitute an endorsement by ISO of this product. Equivalent products may be used if they can be shown to lead to the same results. 2) Celite is the trade-name of a commercially available product. 3) Florisil is the trade-name of a commercially available product. Flor
33、isil Sep-Pak style cartridge article number51960 and C 18Sep-Pak style catridge article number51910, from Waters Associates (Milwaukee, USA), are examples of suitable products available commercially. This information is given for the convenience of users of this International Standard and does not c
34、onstitute an endorsement by ISO of these products. Equivalent products may be used if they can be shown to lead to the same results.BS5766-16:1999 2 BSI 02-2000 4.12 Mixture of acetone and water, 98+2 (byvolume). Combine980ml of acetone(4.8) and20ml of water(4.1). Mix well. 4.13 Mixture of acetone a
35、nd water, 15+85 (by volume). Combine150ml of acetone(4.8) and850ml of water(4.1). Mix well. 4.14 Mixture of acetone and water,5+95 (by volume). Combine50ml of acetone(4.8) and950ml of water(4.1). Mix well. 4.15 Mixture of methanol and water, 20+80 (byvolume). Combine200ml of methanol(4.9) and800ml o
36、f water(4.1). Mix well. 4.16 Concentrated nitric acid, c(HNO 3 )=14mol/l, (HNO 3 )=1,40g/ml, for HPLC with bromine derivatization. 4.17 Potassium bromide (KBr), for HPLC with bromine derivatization. 4.18 Mobile phase for HPLC. 4.18.1 Mobile phase for HPLC with iodine derivatization. Combine120ml of
37、acetonitrile(4.10),210ml of methanol(4.9) and390ml of water(4.1) and mix. Filter the eluent through a0,454m PTFE membrane filter using the solvent filtration system(5.1) and degas for10min in the ultrasonic bath(5.2) before use. NOTEThe composition of the mobile phase solvent may need adjustment dep
38、ending on the characteristics of the HPLC column used. 4.18.2 Mobile phase for HPLC with bromine derivatization Combine400ml of acetonitrile(4.10),700ml of methanol(4.9) and1300ml of water(4.1) and mix. Add to the mixture286mg of potassium bromide(4.17) and1524l of concentrated nitric acid(4.16). Mi
39、x well and degas with a stream of inert gas for15min. 4.19 Saturated iodine solution for HPLC with iodine derivatization Add2g of iodine to400ml of water. Mix for at least90min and filter through a0,454m PTFE membrane filter(see5.1). Prepare the solution fresh on the day of use. Protect the saturate
40、d solution from light to prevent photodegradation. 4.20 Sodium hypochlorite solution (householdquality), (active chlorine)=100g/l. 4.21 Sodium hypochlorite solution, volume fraction1%. Dilute10ml of sodium hypochlorite solution(4.20) with990ml of water-acetone mixture(4.14). 4.22 Inert gas, e.g.nitr
41、ogen. 4.23 Aflatoxin B 1standard material (C 17 H 12 O 6 ),2,3,6a!,9a!-tetrahydro-4- methoxycyclopentacfuro3,2:4,5-furo 2,3-h1benzopyran-1,11-dione; Chemical Abstracts Service Registry (CAS) number1162-65-8. WARNINGS 1 Mycotoxins are extremely toxic substances. Perform all manipulations in a designa
42、ted fume cupboard. Take special precautions when toxins are in a dry form because of their electrostatic nature and resulting tendency to disperse in working areas. 2 Aflatoxins are sensitive to UV radiation. Therefore, conduct all operations in the absence of sunlight or artificial white light. Pro
43、vide sufficient, but not excessive, illumination with tungsten filament lamps. Lowenergy lamps and fluorescent tubes may be used, but the use of amber glassware (vials,volumetric flasks) is recommended. 3 Glassware that has been in contact with solutions of aflatoxin B 1has to be soaked overnight in
44、 a hypochlorite solution(4.21), before cleaning, in order to remove traces of aflatoxin B 1 . 4.24 Aflatoxin B 1standard solution, (aflatoxinB 1 ). 104g/ml. Transfer the content of an ampoule containing aflatoxin B 1 (4.23) to a flask and dissolve in chloroform(4.11). Transfer the solution to a conv
45、enient size volumetric flask and dilute to the mark with chloroform so as to obtain a solution with an aflatoxin B 1content of about104g/ml. Mix. Transfer the solution to amber vials or an airtight screw-cap bottle and store in a cool place(4 C) in the dark, well sealed and wrapped in aluminium foil
46、. 4.25 Aflatoxin B 1stock standard solution. Transfer quantitatively2,5ml of the aflatoxin B 1standard solution(4.24) to a50ml volumetric flask and dilute to the mark with chloroform(4.11). Transfer the solution to amber vials or an airtight screw-cap bottle and store in a cool place(4 C) in the dar
47、k, well sealed and wrapped in aluminium foil. 4.26 Aflatoxin B 1calibration solutions for HPLCBS5766-16:1999 BSI 02-2000 3 4.26.1 Calibration solution I, (aflatoxin B 1 ). 4ng/ml. Allow the volumetric flask with stock standard solution(4.25) to reach room temperature in the aluminium foil (a few hou
48、rs). Transfer4004l of the stock standard solution (equivalent to about200ng of aflatoxin B 1 ) to an acid-washed50ml volumetric flask, and evaporate the solution to dryness in a stream of inert gas(4.22). Dissolve the residue in20ml of the water-acetone mixture(4.13). Dilute to the mark with the wat
49、er-acetone mixture and mix well. 4.26.2 Calibration solution II, (aflatoxinB 1 ). 3ng/ml. Transfer quantitatively7,5ml of the calibration solution I(4.26.1) to an acid-washed10ml volumetric flask. Dilute to the mark with the water-acetone mixture(4.13) and mix well. 4.26.3 Reference calibration solution, (aflatoxinB 1 ). 2ng/ml. Transfer quantitatively25ml of the calibration solution I(4.26.1) to an acid-washed50ml volumetric flask. Dilute to the mark with the water-acetone mixture(4.13) and mix well. This solution is used
