《BS-ISO-6870-2002.pdf》由会员分享，可在线阅读，更多相关《BS-ISO-6870-2002.pdf（16页珍藏版）》请在三一文库上搜索。
1、BRITISH STANDARD BS ISO 6870:2002 Animal feeding stuffs Qualitative determination of zearalenone ICS 65.120 ? Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:29:26 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 6870:2002 This British Standard was published under the authority of the Sta
2、ndards Policy and Strategy Committee on 1 May 2003 BS 1 May 2003 ISBN 0 580 39870 6 National foreword This British Standard reproduces verbatim ISO 6870:2002 and implements it as the UK national standard. The UK participation in its preparation was entrusted to Technical Committee AW/10, Animal feed
3、ing stuffs, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international publications referred to in this document may be found in the BSI Catalogue under the s
4、ection entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application
5、. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor
6、related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii and iii, a blank page, pages 1 to 8, an inside back cover and a back cover. The BSI copyright date displayed in t
7、his document indicates when the document was last issued. Amendments issued since publication Amd. No. DateComments Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:29:26 GMT+00:00 2006, Uncontrolled Copy, (c) BSI INTERNATIONAL STANDARD ISO 6870 Second edition 2002-04-15 Reference number IS
8、O 6870:2002(E) Animal feeding stuffs Qualitative determination of zearalenone Aliments des animaux Dosage qualitatif de la zaralnone BS ISO 6870:2002 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:29:26 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ISO :0786(2002)E ii FDP dicslaimre This FDP
9、 fam elitnoc yt deddebme niayfep.seca In roccatiw ecnadA hycilop gnisnecil sebod, thf sim eliarp eb ytnieiv ro dewb detu sllah ton eb tidesselnu de tt ehyfepseca hwich rae meil era deddebct desnetsni dna olat no delmoc ehtupre epfrromign tide ehti.gn In odaolnwding htis life, rapticca setpe trehiet
10、neh serilibisnopty fo ton fnirisebodA gnign ilop gnisnecilcy. TI ehSO tneCrceS larteraita catpecon s illibayti in ihts aera. ebodA si t armedarak fo dAS ebotsysme Inroctarop.de teDailfo s tfos ehtware prtcudos t desuo crtaet ehFDP si ff eb nac elit ni dnuoG ehlarene Info talerive to tf eh;eli teh PD
11、F-rctaeirap nomateers weer tpof dezimior rptnii.gn vErec yaeeb sah ert nakt neo rusnet etah tf ehi elis tiusbaf elob esu ry ISO merebm seidob. In the kilnuley evtne ttah a borpmel talerign to ti f si,dnuo saelpfni erom teh tneCS larterceratai ta thserdda es igvne olebw. ISO 2002 Ar llieser sthgrvsse
12、lnU .de torehceps esiwfi,dei rap ont iht fos bupliactim noarper eb yro decudo tuzilina ni dey from b roy nasnaem y, tcelernahcem ro cinoicla, inclidung tohppocoying m dnaicrfomli, tiwtuoh permini noiss wrtif gnirmo ietI rehSO ta tserdda eheb slow or SIsO memreb ydob in toc ehtnury fo tr ehtseuqeer.
13、ISO copyrithg fofiec tsop esaCela 65 G 1121-HCeneva 02 T.le + 22 14 10 947 11 Fxa + 14 47 2290 9 74 E-liam cpoyrigthsio.ch Web wwwsi.oc.h BS ISO 6870:2002 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:29:26 GMT+00:00 2006, Uncontrolled Copy, (c) BSI iii Foreword ISO (the International Or
14、ganization 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 been establ
15、ished 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 standardizati
16、on. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. 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 %
17、of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. International Standard ISO 6870 was prepared by Te
18、chnical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal feeding stuffs. This second edition cancels and replaces the first edition (ISO 6870:1985), of which it constitutes a minor revision. The title has been changed to stress that the method is only qualitative and the scope now stat
19、es that the method is for screening purposes only. The temperature range in 3.8.2 has been corrected to 0 C to 5 C. Annex A of this International Standard is for information only. BS ISO 6870:2002 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:29:26 GMT+00:00 2006, Uncontrolled Copy, (c)
20、BSI Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:29:26 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ANRETNIITOTS LANDNADRAISO 7862002:0)E( 1 Animal feeding stuffs Qualitative determination of zearalenone 1Scope This International Standard specifies a qualitative method for the determinati
21、on of zearalenone in animal feeding stuffs and, in particular, in maize. This method is for screening purposes only. The limit of determination of zearalenone is approximately . NOTEAlthough sorghum gives interfering fluorescent spots identical to those of zearalenone, the method is still applicable
22、 to this feed because the values are different after development of the chromatogram in the second direction. These spots are not developed by the specified confirmation technique. 2Principle A test portion is extracted with a mixture of acetonitrile and potassium chloride solution, then filtered, a
23、nd an aliquot portion is defatted with isooctane, followed by purification in a mixture of acetonitrile, water and lead acetate in the presence of diatomaceous earth. After filtration, an aliquot portion is extracted with chloroform which is subsequently evaporated. The dry extract is dissolved in a
24、 mixture of benzene and acetonitrile. Two-dimensional thin-layer chromatography is performed on an aliquot portion of this solution. The zearalenone content is determined by visual measurement or by measurement of the intensity of fluorescence of the spot under UV light by comparison with known quan
25、tities of zearalenone applied to the same plate. The identity of the zearalenone is confirmed using bis-diazotized benzidine reagent. 3Reagents Use only reagents of recognized analytical grade and distilled or demineralized water or water of equivalent purity. 3.1Acetonitrile. 3.2Isooctane. 3.3Chlor
26、oform. WARNING Chloroform is a toxic substance. Avoid inhalation of and exposure to chloroform. Work in a fumehood when handling the solvent and solutions thereof. 3.4Benzene/acetonitrile, mixture, by volume. WARNING Benzene is toxic by inhalation and contact with skin and is highly flammable. 3.5De
27、veloping solvents. 3.5.1Toluene/ethyl acetate/formic acid, mixture, by volume. 3.5.2Chlorofom/ethanol, mixture, by volume. 3.6Potassium chloride, solution. 50g/kg Rf 98+2 6+3+1 95+5 40 g/l BS ISO 6870:2002 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:29:26 GMT+00:00 2006, Uncontrolled C
28、opy, (c) BSI 2 3.7Lead acetate solution, prepared as follows. Weigh of lead acetate into a one-mark volumetric flask, add of acetic acid, dilute to the mark with water and mix. 3.8Bis-diazotized benzidine reagent, prepared as follows. WARNING Benzidine is a carcinogen, and is toxic by inhalation, co
29、ntact with the skin and ingestion. 3.8.1Preparation of benzidine solution Place of benzidine in a flask containing of water and of hydrochloric acid and make up to volume with water. Keep this solution protected from light in a brown glass bottle. 3.8.2Preparation of the reagent Cool equal volumes o
30、f the benzidine solution (3.8.1) and of a sodium nitrite solution to between and . Thoroughly mix the two solutions. The solution obtained is dark purple and turbid. Leave to attain room temperature (yellow colour) before use. Prepare this reagent just before use. 3.9Diatomaceous earth (Celite 545),
31、 hydrochloric acid washed. 3.10Nitrogen. 3.11Zearalenone, standard solution of concentration , in benzene. Determine the absorption spectrum of the solution between and by means of a spectrometer, using silica optical cells and using benzene as reference. Record the maximum absorbance, , which is cl
32、ose to . Calculate the zearalenone concentration, in micrograms per millilitre, of the solution, by means of the formula where 318is the molar mass of zearalenone; 6 060is the molar extinction coefficient. 4Apparatus Usual laboratory equipment and, in particular, the following. 4.1Grinder, suitable
33、for preparing a product to pass completely through a sieve of aperture size . 4.2Shaker, capable of producing about 100 oscillations per minute. 4.3Filter papers, medium grade (a rapid grade filter paper gives a turbid solution; a slow grade filter paper will become clogged). 200 g1 000 ml3 ml 5 g/l
34、 0,5 g100 ml20 ml1,5 ml 100 g/l0 C 5 C 10g/ml 300 nm330 nm 10 mmA 317 nm 318 A 1 000 6 060 1 mm BS ISO 6870:2002 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:29:26 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 3 4.4Rotary evaporator with round bottom flask. 4.5Apparatus for thin-layer chro
35、matography, i.e. apparatus required for the preparation of the plates (4.6) and application of spots (capillary pipettes or microsyringes), a developing tank, and apparatus for spraying the reagent (3.8) on the plates. 4.6Glass plates for thin-layer chromatography, of dimensions , prepared as follow
36、s (the quantities indicated are sufficient for the preparation of five plates). Weigh of silica gel G-HR into a conical flask, add of water, stopper and mix thoroughly for . Spread the slurry over the plates in such a way that a uniform layer of thickness is obtained. Allow to dry in air and store t
37、he plates in a desiccator. Activate the plates before use by placing them in an oven, maintained at for . Commercially available prepared plates may be used if the results obtained are comparable to the results obtained with plates prepared as specified in the previous paragraph. 4.7Short wavelength
38、 UV lamp (wavelength ). The intensity of irradiation shall be such as to clearly distinguish a spot of of zearalenone on thin-layer plate when the lamp is placed at a distance of from the plate. WARNING In view of the danger of UV light to the eyes, eye protection shall be worn. 4.8Test tubes, of ca
39、pacity , with a polyethylene stopper. 4.9Fluorodensitometer (optional, but desirable). 4.10Water bath, capable of being maintained at . 4.11Conical flask, of capacity , with a ground glass stopper. 4.12Separating funnels, of capacity . 4.13Measuring cylinders, of capacities and . 4.14Pipettes, of ca
40、pacities and . 4.15Microsyringes. 5Sampling Take the laboratory sample of the product to be tested in accordance with the International Standard appropriate to the product concerned, unless sampling for the determination of zearalenone is excluded from its field of application. If an appropriate Int
41、ernational Standard does not exist, the parties concerned shall reach agreement on this subject, taking into account the characteristics of the product to be sampled. 6Procedure 6.1Preparation of test sample Grind the sample so that it passes completely through a sieve of aperture size . Mix thoroug
42、hly. 6.2Test portion Weigh, to the nearest , of the test sample into the conical flask (4.11). 200 mm200 mm 30 g60 ml1 min 0,25 mm 110 C 3 C 1 h 253 nm 25 ng 100 mm 10 ml 60 C 1 C 500 ml 250 ml 100 ml250 ml 50 ml100 ml 1 mm 0,01 g 50 g500 ml BS ISO 6870:2002 Licensed Copy: sheffieldun sheffieldun, n
43、a, Mon Nov 27 03:29:26 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 4 6.3Extraction Add of the acetonitrile (3.1), and of the potassium chloride solution (3.6) carefully measured from a measuring cylinder (4.13). Stopper the flask, mix and shake for with the shaker (4.2). Filter through a filter paper
44、 (4.3). Transfer, by means of a pipette (4.14), of the filtrate to a separating funnel (4.12) and defat by carrying out two successive extractions each time with of the isooctane (3.2). Collect the acetonitrile phase in the round-bottom flask of the rotary evaporator (4.4) and evaporate to dryness u
45、nder reduced pressure. 6.4Purification Add to the residue obtained, of the acetonitrile (3.1), of water and of the lead acetate solution (3.7), carefully measured from a measuring cylinder (4.13). Mix and allow to separate for in the water bath (4.10) maintained at . A precipitate is formed. Add of
46、the diatomaceous earth (3.9) and filter through a filter paper (4.3). Transfer by means of a pipette (4.14), of the filtrate to a separating funnel (4.12) and carry out three successive extractions, each time with of the chloroform (3.3). Dry the chloroform fractions over sodium sulfate. Collect the
47、 chloroform fraction in the round-bottom flask of the rotary evaporator (4.4) and evaporate almost to dryness under reduced pressure. Transfer the residue quantitatively to the test tube (4.8) by rinsing with chloroform, then evaporate to dryness under nitrogen (3.10) on the water bath (4.10). Cauti
48、ously add, using a microsyringe, of the benzene/acetonitrile mixture (3.4) and stopper the tube tightly. 6.5Two-dimensional thin-layer chromatography 6.5.1Application of solutions (see Figure 1) Draw on a plate (4.6) two straight lines parallel to adjacent sides (at and , respectively, from the edge
49、s) to mark the limit of migration of the solvent fronts. Apply the following solutions to the plate by means of microsyringes: at point A, of the purified extract (6.4); at point B, of the standard solution (3.11); at point C, of the standard solution (3.11); at point D, of the standard solution (3.11); at point E, of the standard solution (3.11). Dry under a stream of air or of nitrogen. The spots obtained should have, at most, a diameter of about . 6.5.2Development (se