1、BRITISH STANDARD BS 5766-7: 1988 ISO 6651:1987 Methods for Analysis of animal feeding stuffs Part 7: Determination of aflatoxin B 1 ISO title: Animal feeding stuffs Determination of aflatoxin B 1content UDC 636.085:543:615.918:582.282.123.4BS5766-7:1988 This British Standard, having been prepared un
2、der the directionof the Food and Agriculture Standards Committee,was published underthe authority of the BoardofBSI and comes into effect on 30December1988 BSI 12-1999 First published April 1984 First revision December 1988 The following BSI references relate to the work on this standard: Committee
3、reference FAC/14 Draft (ref. 87/54980) announced in BSI News December 1987 ISBN 0 580 16807 7 Committees responsible for this British Standard The preparation of this British Standard was entrusted by the Food and Agriculture Standards Committee (FAC/-) to Technical Committee FAC/14 upon which the f
4、ollowing bodies were represented: Association of Public Analysts Department of Trade and Industry (Laboratory of the Government Chemist) Grain and Feed Trade Association Limited Ministry of Agriculture, Fisheries and Food National Farmers Union Overseas Development Administration (Tropical Developme
5、nt and Research Institute) Pet Food Manufacturers Association Royal Association of British Dairy Farmers United Kingdom Agricultural Supply Trade Association Ltd. Amendments issued since publication Amd. No. Date of issue CommentsBS5766-7:1988 BSI 12-1999 i Contents Page Committees responsible Insid
6、e front cover National foreword ii 1 Scope 1 2 Field of application 1 3 Reference 1 4 Principle 1 5 Reagents 1 6 Apparatus 3 7 Sampling 3 8 Procedure 4 9 Expression of results 7 10 Test report 8 Figure 1 Application of solutions 9 Figure 2 Interpretation of the chromatogram 10 Figure 3 Confirmation
7、test 11 Publication referred to Inside back coverBS5766-7:1988 ii BSI 12-1999 National foreword This Part of BS 5766 has been prepared under the direction of the Food and Agriculture Standards Committee and is identical with ISO6651:1987 “Animal feeding stuffs Determination of aflatoxin B 1content”,
8、 published by the International Organization for Standardization (ISO). It is a revision of BS5766-7:1984, which is withdrawn and from which it differs in that a factor in5.14.1 a) has been corrected. In the United Kingdom, animal feeding stuffs are controlled by legislation. The method in this Part
9、 of BS5766 is technically equivalent to the method given in the Feeding Stuffs (Sampling and analysis) Regulations 1982. Terminology and conventions. The text of the International Standard has been approved as suitable for publication as a British Standard without deviation. Some terminology and cer
10、tain conventions are not identical with those used in British Standards; attention is drawn especially to the following. The comma has been used as a decimal marker. In British Standards it is current practice to use a full point on the baseline as the decimal marker. In British Standards it is curr
11、ent practice to use the symbol “L” for litre (and in its submultiples) rather than “l” and to use the spelling “sulphur”, etc. instead of “sulfur”, etc. Wherever the words “International Standard” appear, referring to this standard, they should be read as “Part of BS5766”. Cross-references. The Tech
12、nical Committee has reviewed the provisions of ISO6498:1983, referred to in8.1.2, and has decided that they are acceptable for use in conjunction with this standard. The United Kingdom did not approve ISO6498 but, in the present context, test samples prepared in accordance with ISO6498 or BS5766 “Me
13、thods for analysis of animal feeding stuffs” Part 10 “Preparation of test samples” are equally acceptable. A related British Standard for ISO565:1983, referred to in the footnote to6.2, is BS410:1986 “Specification for test sieves”, the relevant requirements of which are technically equivalent. Addi
14、tional information. Where the words “simple” and “mixed” are used in relation to feeding stuffs, they should be read as “straight” and “compound” respectively, as these are the terms commonly used in the United Kingdom. Water complying with grade3 of BS3978 “Specification for water for laboratory us
15、e” is suitable for reagents (seeclause5). Care should be taken to ensure that the chloroform (see5.1) is stabilized with the specified proportion of ethanol as some suppliers use higher proportions. It is recommended that ready-to-use TLC plates (see6.7) be used. The requirement in 8.8 may be constr
16、ued as meaning that the mean of the two values determined is to be reported as the result. With reference to the information in9.2, the following is considered to be a more useful form of expression. Repeatability. The difference between the values of two determinations, carried out on the same test
17、 sample either simultaneously or in rapid succession by the same analyst will not exceed30% (relative value) of their mean, otherwise the determinations should be repeated. Reproducibility. The difference between two single and independent values obtained by two analysts working in different laborat
18、ories on parts of the same test sample will not exceed80% (relative value) of the mean of the values of the two laboratories on more than one occasion in20.BS5766-7:1988 BSI 12-1999 iii A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standa
19、rds 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, pages i to iv, pages1 to 12, an inside back cover and a back cover. This sta
20、ndard 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.iv blankBS5766-7:1988 BSI 12-1999 1 1 Scope This International Standard specifies two methods for the determination of the aflatoxin B 1conten
21、t of animal feeding stuffs. 2 Field of application 2.1 Method A is applicable to the following simple feeding stuffs: oilseeds and oilseed residues, and in particular ground-nut, copra, linseed, soya, sesame, babassu palm; manioc meal; maize germ expeller; cereals and cereal products; pea meal; pota
22、to pulp and flour. In the presence of substances interfering with the determination by method A, it is recommended that the determination be carried out in accordance with method B. 2.2 Method B is applicable to mixed feeding stuffs and to simple feeding stuffs not mentioned in2.1. This method is no
23、t applicable to feeding stuffs containing citrus pulp. 2.3 The lower limit of detection of aflatoxin B 1is0,01mg/kg. 3 Reference ISO 6498, Animal feeding stuffs Preparation of test samples. 4 Principle Extraction of the test portion with chloroform, filtration, and purification of an aliquot portion
24、 on a silica gel column. Evaporation of the eluate and dissolution of the residue in a specified volume of chloroform or mixture of benzene and acetonitrile. Thin-layer chromatography, one-dimensional for method A and two-dimensional for method B, of an aliquot portion of this solution. Determinatio
25、n of the aflatoxin B 1content, either visually or by fluorodensitometry, by examination of the chromatogram under ultraviolet light and comparison with known quantities of standard aflatoxin B 1applied to the same plate as the test portion extract. Confirmation of the identity of aflatoxin B 1by for
26、mation of the hemiacetal derivative. 5 Reagents All reagents shall be of recognized analytical quality. The water used shall be distilled water or water of at least equivalent purity. 5.1 Chloroform, stabilized with0,5 to1,0% of96%(V/V) ethanol. 5.2 n-Hexane 5.3 Diethyl ether, anhydrous, free from p
27、eroxides. 5.4 Benzene/acetonitrile, (98 + 2) mixture. Mix 98 volumes of benzene with 2 volumes of acetonitrile. 5.5 Chloroform/methanol, (97 + 3) mixture. Mix 97 volumes of chloroform with 3 volumes of methanol.BS5766-7:1988 2 BSI 12-1999 5.6 Developing solvents 1) 5.6.1 Chloroform/acetone, (90 + 10
28、) mixture. Mix 90 volumes of chloroform with 10 volumes of acetone, in an unsaturated tank. 5.6.2 Diethyl ether/methanol/water, (96+3+1) mixture. Mix 96 volumes of diethyl ether, 3 volumes of methanol and 1 volume of water, in an unsaturated tank. 5.6.3 Diethyl ether/methanol/water, (94+4,5+1,5) mix
29、ture. Mix 94 volumes of diethyl ether with4,5 volumes of methanol and1,5 volumes of water, in a saturated tank. 5.6.4 Chloroform/methanol, (94 + 6) mixture. Mix 94 volumes of chloroform with 6 volumes of methanol, in a saturated tank. 5.6.5 Chloroform/methanol, (97 + 3) mixture. Mix 97 volumes of ch
30、loroform with 3 volumes of methanol, in a saturated tank. 5.7 Silica gel, for column chromatography, of particle size0,05to0,20mm. 5.8 Silica gel, G-HR or equivalent, for thin-layer chromatography. 5.9 Diatomaceous earth (Hyflosupercel), acid-washed. 5.10 Sodium sulfate, anhydrous granules. 5.11 Tri
31、fluoroacetic acid 5.12 Inert gas, for example nitrogen. 5.13 Sulfuric acid, 50% (V/V) solution. 5.14 Aflatoxin B 1 , standard solution containing about0,14g of aflatoxin B 1per millilitre, in the chloroform(5.1) or in the benzene/acetonitrile mixture (5.4). WARNING Aflatoxins are highly carcinogenic
32、 and must be handled with great care. Prepare and check the solution as follows. 5.14.1 Preparation of stock solution and determination of concentration Prepare a solution of aflatoxin B 1in the chloroform(5.1) or the benzene/acetonitrile mixture(5.4) such that the concentration is between8 and104g/
33、ml. Determine the absorption spectrum between330and370nm by means of the spectrophotometer(6.9). Measure the absorbance (A) at363nm in the case of the chloroform solution, or at348nm in the case of the benzene/acetonitrile mixture solution. Calculate the concentration of aflatoxin B 1 , in microgram
34、s per millilitre of solution, from the formulae: a) for the chloroform solution: b) for the solution in the benzene/acetonitrile mixture: 5.14.2 Dilution Dilute the stock solution (5.14.1), as appropriate, away from daylight, to obtain a standard solution with a concentration of aflatoxin B 1of abou
35、t0,1g/ml. If kept in a refrigerator at 4 C, this solution is stable for 2 weeks. 1) The solvents should be used in covered tanks. When saturated tanks are specified, this is achieved by lining the tanks with absorbent paper and allowing the interiors to become saturated with solvent vapour.BS5766-7:
36、1988 BSI 12-1999 3 5.14.3 Testing of chromatographic purity of the solution Onto a plate (6.7), apply a spot of54I of the standard aflatoxin B 1solution of concentration8 to104g/ml (5.14.1). Develop the chromatogram as indicated in8.5.1. Under ultraviolet light, the chromatogram shall show only one
37、spot and no fluorescence shall be perceptible in the original deposition zone. 5.15 Aflatoxin B 1and B 2(see the warning in5.14), solutions for qualitative testing, containing about0,14g of aflatoxin B 1and B 2per millilitre, in the chloroform (5.1) or in the benzene/acetonitrile mixture (5.4). Thes
38、e concentrations are given as a guide. They shall be adjusted so as to obtain the same intensity of fluorescence for both aflatoxins (see8.5.1). 6 Apparatus Usual laboratory equipment, and in particular: 6.1 Grinder/mixer 6.2 Sieve, of aperture size1,0mm. 2) 6.3 Shaking apparatus or magnetic stirrer
39、 6.4 Chromatographic tubes, made of glass (internal diameter22mm, length300mm), with a polytetrafluoroethylene tap and a250ml reservoir, plugged at the bottom end with cotton or glass wool. 6.5 Rotary vacuum evaporator, with a 500 ml round-bottomed flask. 6.6 Apparatus for thin-layer chromatography
40、(TLC), i.e.that necessary for the preparation of the plates(6.7) and application of spots (capillary pipettes or microsyringes), a developing tank, and spraying apparatus for applying the sulfuric acid(5.13) to the plates. 6.7 Glass TLC plates, 200mm 200mm, prepared as follows (the quantities indica
41、ted are sufficient to cover five plates). Place30g of the silica gel (5.8) in a conical flask, add60ml of water, stopper and shake for1min. Spread the suspension on the plates so as to obtain a uniform layer0,25mm thick. Leave to dry in the air and then store in a desiccator containing silica gel. A
42、t the time of use, activate the plates by keeping them in an oven at110 C for1h. Ready-to-use plates are suitable if they give results similar to those obtained with the plates prepared as indicated above. 6.8 Long-wavelength (360 nm) ultraviolet lamp The intensity of irradiation shall make it possi
43、ble for a spot of 1,0ng of aflatoxin B 1to be clearly distinguished on a TLC plate at a distance of10cm from the lamp. WARNING Ultraviolet light is dangerous to the eyes. Protective goggles shall be worn. 6.9 Spectrophotometer, suitable for making measurements in the ultraviolet region of the spectr
44、um. 6.10 Fluorodensitometer (optional). 6.11 Fluted filter paper 6.12 Graduated tube, of capacity 10,0ml, with a polyethylene stopper. 6.13 Conical flask, of capacity 500ml, with a ground glass stopper. 6.14 Pipette, of capacity 50 ml. 6.15 Balance 7 Sampling Take the laboratory sample from the mate
45、rial to be sampled in accordance with the International Standard for the material concerned unless sampling for the determination of aflatoxin is excluded from its field of application. If no appropriate International Standard exists, agreement shall be reached between the parties concerned, taking
46、into account the characteristics of the material being sampled. 2) See ISO 565, Test sieves Woven metal wire cloth, perforated plate and electroformed sheet Nominal sizes of openings.BS5766-7:1988 4 BSI 12-1999 8 Procedure 8.1 Preparation of the test sample 8.1.1 If the sample contains more than5% o
47、f fat, it shall be defatted with light petroleum before grinding. In such cases, the analytical results shall be expressed in terms of the mass of the non-defatted sample. 8.1.2 Grind the laboratory sample so that it completely passes through the sieve (6.2). Mix thoroughly. (SeeISO6498.) 8.2 Test p
48、ortion Weigh, to the nearest0,01g, 50g of the prepared test sample into the conical flask (6.13). 8.3 Extraction Add to the test portion (8.2) 25g of the diatomaceous earth (5.9), 25ml of water, and250ml of the chloroform (5.1) accurately measured from a measuring cylinder. Stopper the flask, and sh
49、ake or stir for30min using the apparatus (6.3). Filter through the fluted filter paper (6.11), taking care to discard the first10ml of the filtrate, and subsequently collect at least50ml of the filtrate. 8.4 Column clean-up 8.4.1 Preparation of the column Fill two-thirds of the chromatographic tube (6.4) with the chloroform (5.1) and add5g of the sodium sulfate(5.10). Check that the upper surface of the sodium sulfate layer is flat, then add10g, in small portions, of the silica gel (5.7). Stir carefully after each addition to eliminate
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