1、BRITISH STANDARD BS EN ISO 6867:2001 BS 5766-24: 2001 Animal feeding stuffs Determination of vitamin E content Method using high-performance liquid chromatography The European Standard EN ISO 6867:2000 has the status of a British Standard ICS 65.120 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMIT
2、TED BY COPYRIGHT LAWBS EN ISO 6867:2001 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 March 2001 BSI 03-2001 ISBN 0 580 36932 3 National
3、foreword This British Standard is the official English language version of EN ISO 6867:2000. It is identical with ISO 6867:2000. The UK participation in its preparation was entrusted to Technical Committee AW/10, Animal feeding stuffs, which has the responsibility to: A list of organizations represe
4、nted on this committee can be obtained on request to its secretary. Cross-references Attention is drawn to the fact that CEN and CENELEC standards normally include an annex which lists normative references to international publications with their corresponding European publications. The British Stan
5、dards which implement these international or European publications 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
6、include all the necessary 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. aid enquirers to understand the text; present to the responsible international/E
7、uropean committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor 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 EN ISO title p
8、age, the EN ISO foreword page, the ISO title page, pages ii and iii, a blank page, pages 1 to 11, the annex ZA page, an inside back cover and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date
9、 CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM ENISO6867 December2000 ICS06.012 Englishversion AnimalfeedingstuffsDeterminationofvitaminEcontent Methodusinghighperformanceliquidchromatography(ISO 6867:2000) AlimentsdesanimauxDterminationdelateneuren vitamineEMthodeparchromatographieliquideh
10、aute performance(ISO6867:2000) FuttermittelBestimmungdesGehaltsanVitaminE HochleistungsflssigchromatographischesVerfahren(ISO 6867:2000) ThisEuropeanStandardwasapprovedbyCENon1December2000. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEurope
11、an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheManagementCentreortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlanguagema
12、debytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheManagementCentrehasthesamestatusasthe official versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,CzechRepublic,Denmark,Finland,France,Germany,Greece, Iceland,Ireland,Italy,Luxembourg,Netherlands,Nor
13、way,Portugal,Spain,Sweden,SwitzerlandandUnitedKingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2000CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.ENISO
14、6867:2000EForeword ThetextoftheInternationalStandardISO6867:2000hasbeenpreparedbyTechnicalCommittee ISO/TC34“Agriculturalfoodproducts“incollaborationwithTechnicalCommitteeCEN/TC327 “AnimalfeedingstuffsMethodsofsamplingandanalysis“,thesecretariatofwhichisheldbyNEN . ThisEuropeanStandardshallbegiventh
15、estatusofanationalstandard,eitherbypublicationofan identicaltextorbyendorsement,atthelatestbyJune2001,andconflictingnationalstandardsshallbe withdrawnatthelatestbyJune2001 . AccordingtotheCEN/CENELECInternalRegulations,thenationalstandardsorganizationsofthe followingcountriesareboundtoimplementthisE
16、uropeanStandard:Austria,Belgium,Czech Republic,Denmark,Finland,France,Germany,Greece,Iceland,Ireland,Italy,Luxembourg, Netherlands,Norway,Portugal,Spain,Sweden,SwitzerlandandtheUnitedKingdom. Endorsementnotice ThetextoftheInternationalStandardISO6867:2000wasapprovedbyCENasaEuropeanStandard withoutan
17、ymodification. NOTE:NormativereferencestoInternationalStandardsarelistedinannexZA(normative). ENISO6867:2000 Reference number ISO 6867:2000(E) INTERNATIONAL STANDARD ISO 6867 First edition 2000-12-01 Animal feeding stuffs Determination of vitamin E content Method using high- performance liquid chrom
18、atography Aliments des animaux Dtermination de la teneur en vitamine E Mthode par chromatographie liquide haute performance ENISO6867:2000 ii ENISO6867:2000 ISO 7686:(0002)Eiii Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (I
19、SO 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, gov
20、ernmental 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
21、 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 % of the member bodies casting a vote. Attention is drawn to the possibility that some of t
22、he 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 6867 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal feeding stuffs. An
23、nex A of this International Standard is for information only. ENISO6867:2000 ENISO6867:2000 INTENRATIONAL TSANDADR ISO 7686:(0002)E1 Animal feeding stuffs Determination of vitamin E content Method using high-performance liquid chromatography 1 Scope This International Standard specifies a method for
24、 the determination of the vitamin E (DL- -tocopherol) content of animal feeding stuffs and pet foods using high performance liquid chromatography. 2 Normative references The following normative documents contain provisions which, through reference in this text, constitute provisions of this Internat
25、ional Standard. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of the normative documents i
26、ndicated below. For undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid International Standards. ISO 3696:1987, Water for analytical laboratory use Specifications and test methods. ISO 6498, Animal feeding
27、 stuffs Preparation of test samples. 3P r i n c i p l e A test portion of the sample is saponified with ethanolic potassium hydroxide solution and the vitamin E is extracted into light petroleum. The light petroleum is removed by evaporation and the residue is dissolved in hexane. The vitamin E conc
28、entration in the hexane extract is determined by normal-phase liquid chromatography using conditions that separate DL- -tocopherol from other tocopherols. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise stated. 4.1 Water, complying with at least grade 3 in accordance wi
29、th ISO 3696. 4.2 Potassium hydroxide solution. Dissolve 500 g of potassium hydroxide in water (4.1) and dilute to 1 litre. 4.3 Ethanol, w(C 2 H 5 OH) = 95 % (by volume), or equivalent industrial methylated spirit. 4.4 Hexane, HPLC grade. 4.5 Light petroleum, boiling range 40 Ct o6 0C; the residue on
30、 evaporation shall be less than 20 mg/l. 4.6 Vitamin E standard substance: DL- -tocopherol, minimum purity not less than 96,0 %. The purity of the standard substance should be checked spectrophotometrically (see 8.5.2). ENISO6867:2000 ISO 7686:(0002)E 2 4.7 1,4-Dioxan, HPLC grade. 4.8 Sodium sulfate
31、 (Na 2 SO 4 ), anhydrous. 4.9 Sodium ascorbate solution, =1 00g /l . 4.10 Inert gas,e .g .ni tro ge n . 4.11 Mobile phase for liquid chromatography. Mix 30 ml 1,4-dioxan (4.7) with 970 ml hexane (4.4). Filter through a membrane filter (5.5) before use. 4.12 Ethanol, w(C 2 H 5 OH) = 96 % (by volume).
32、 4.13 Methanol (CH 3 OH), HPLC grade. 5 Apparatus Using laboratory apparatus and, in particular, the following. 5.1 High-performance liquid chromatograph, consisting of the following. 5.1.1 Pump, set to deliver a constant eluent volume flow rate of 1,5 ml/min. 5.1.2 HPLC injection device. 5.1.3 Colu
33、mn, length 250 mm, internal diameter 4,6 mm, packed with a stationary phase consisting of silica. A column with at least 5 000 theoretical plates and a k value of 0,8 m, both with respect to DL- -tocopherol, has been found to be satisfactory. The particle size should not be smaller than 5 m and not
34、greater than 10 m. Other systems may be used provided that a satisfactory separation of vitamin E from other co-extractives is achieved. 5.1.4 Detector, allowing the measurement of fluorescence emitted at a wavelength of 326 nm when the column eluent is irradiated with ultraviolet light at a wavelen
35、gth of 293 nm, with integrator/recorder. 5.2 Boiling water bath. 5.3 Rotary vacuum evaporator, with water bath at 40 C. 5.4 Extraction apparatus (see Figure 1) consisting of the following: a cylinder of 1 litre capacity fitted with a ground glass neck and stopper; a ground glass joint, fitting the c
36、ylinder and equipped with an adjustable tube passing through the centre; and a side-arm. The adjustable tube should have a U-shaped lower end and a jet at the opposite end so that the upper liquid layer in the cylinder may be transferred to a separating funnel of 1 litre capacity. Other extraction e
37、quipment such as conical flasks and separating funnels may be used in place of the apparatus shown in Figure 1, provided that satisfactory recoveries of vitamin E are achieved. 5.5 Membrane filter, 0,45 m pore size, for filtration of mobile phase (4.11) and sample test solutions. ENISO6867:2000 ISO
38、7686:(0002)E3 5.6 Grinding apparatus, capable of grinding the sample so that it passes through a sieve with 1 mm apertures. 5.7 UV (or UV/Visible) spectrometer, capable of measuring absorbance at the wavelengths defined in 8.5.2, equipped with quartz cells of 10 mm path length. Key 1 Cylinder, of ca
39、pacity 1 litre, with ground-glass neck 5 Bottle, of capacity 1 litre, with ground-glass joint 2 Light petroleum layer 6 Side-arm 3 Aqueous layer + saponified feed 7 Adjustable tube 4J e t Figure 1 Example of extraction apparatus ENISO6867:2000 ISO 7686:(0002)E 4 6 Sampling It is important that the l
40、aboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is g iv e ni nI S O64 97 1 . Store the sample in such a way that deteri
41、oration and change in its composition are prevented. 7 Preparation of test samples Prepare the test sample in accordance with ISO 6498. Just prior to starting the analysis, grind a portion of the well-mixed laboratory sample so that it passes through a sieve with 1 mm apertures. Mix thoroughly. Homo
42、genize canned pet foods. Pass semi-moist pet foods through a mincer with 4-mm apertures. 8 Procedure 8.1 General Because of the sensitivity of vitamin E to UV radiation and air, perform all operations away from natural and strong fluorescent light and as rapidly as is consistent with accurate workin
43、g. Use amber glassware where possible. Complete each assay within one working day. 8.2 Saponification Weigh, to the nearest 0,1 g, approximately 50 g of the prepared sample (see clause 7) into a 1 litre conical flask. Add to the test portion 200 ml of ethanol (4.3) whilst swirling the flask to dispe
44、rse the sample. Add 2 ml of sodium ascorbate solution (4.9), mix by swirling and then add 50 ml of potassium hydroxide solution (4.2) and swirl again. Fit a reflux condenser to the flask and immerse the flask in the boiling water bath (5.2). Allow the contents of the flask to reflux for 30 min, swir
45、ling occasionally. NOTE In exceptional cases some products may require a longer saponification time. Cool the flask to room temperature under a stream of cold water. Transfer the contents of the flask into the extraction cylinder (see 5.4). 8.3 Extraction of vitamin E Rinse the saponification flask
46、with two 25 ml portions of ethanol (4.3) and transfer the rinsings to the cylinder. Repeat the rinsing of the flask with two 125 ml portions of light petroleum (4.5) and one 250 ml portion of water (4.1), each time transferring the rinsings to the cylinder. Stopper the cylinder and shake well for 1
47、min, releasing the pressure from time to time. Cool the cylinder under a stream of cold water while waiting for the two liquid phases to separate, before removing the stopper. ENISO6867:2000 ISO 7686:(0002)E5 When the layers have separated, remove the stopper, wash the sides of the stopper with a fe
48、w millilitres of light petroleum (4.5) and insert the adjustable tube (see 5.4), positioning the lower open end so that it is just above the level of the interface. By application of a slight pressure of inert gas (4.10) to the side arm tube, transfer the upper, light petroleum layer to a 1 litre separating funnel (see 5.4). Add 125 ml of light petroleum (4.5) to the cylinder, stopper and shake well for 1 min. Allow the layers to sep
