1、BRITISH STANDARD BS EN ISO 15788-2:2004 Animal and vegetable fats and oils Determination of stigmastadienes in vegetable oils Part 2: Method using high-performance liquid chromatography (HPLC) The European Standard EN ISO 15788-2:2004 has the status of a British Standard ICS 67.200.10 BS EN ISO 1578
2、8-2:2004 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 6 December 2004 BSI 6 December 2004 ISBN 0 580 41395 0 National foreword This British Standard is the official English language version of EN ISO 15788-2:2004. It is identical with ISO
3、15788-2:2003. The UK participation in its preparation was entrusted to Technical Committee AW/307, Oilseeds, animal and vegetable fats and oils, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The Br
4、itish Standards which implement international or European publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards O
5、nline. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its 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 respon
6、sible international/European 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 cove
7、r, the EN ISO title page, the EN ISO foreword page, the ISO title page, pages ii to iv, pages 1 to 10, an inside back cover and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUR
8、OPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM ENISO157882 November2004 ICS67.200.10 Englishversion AnimalandvegetablefatsandoilsDeterminationof stigmastadienesinvegetableoilsPart2:Methodusinghigh performanceliquidchromatography(HPLC)(ISO157882:2003) CorpsgrasdoriginesanimaleetvgtaleDosagedes stigmasta
9、dinesdansleshuilesvgtalesPartie2: Mthodeparchromatographieliquidehauteperformance (CLHP)(ISO157882:2003) TierischeundpflanzlicheFetteundleBestimmungder StigmastadieneinPflanzenlenTeil2:Verfahrenmit Hochleistungsflssigchromatographie(HPLC)(ISO15788 2:2003) ThisEuropeanStandardwasapprovedbyCENon13Febr
10、uary2003. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheCentralSecretar
11、iatortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheCentralSecretariathasthesamestatusast heofficial versions. CENmembersarethenationalstandardsb
12、odiesofAustria,Belgium,Cyprus,CzechRepublic,Denmark,Estonia,Finland,France, Germany,Greece,Hungary,Iceland,Ireland,Italy,Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,Poland,Portugal, Slovakia, Slovenia,Spain,Sweden,SwitzerlandandUnitedKingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPEN
13、DENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2004CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.ENISO157882:2004:E I NESO 51-887:2002( 4E) 2 Foreword The text of ISO 15788-2:2003 has been prepared by Tech
14、nical Committee ISO/TC 34 “Agricultural food products“ of the International Organization for Standardization (ISO) and has been taken over as EN ISO 15788-2:2004 by Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-products - Methods of sampling and analysis“,
15、 the secretariat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by May 2005, and conflicting national standards shall be withdrawn at the latest by May 2005. According to
16、the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxe
17、mbourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. Endorsement notice The text of ISO 15788-2:2003 has been approved by CEN as EN ISO 15788-2:2004 without any modifications. ENISO157882:2004 Reference number ISO 15788-2:2003(E) OS
18、I 3002INTERNATIONAL STANDARD ISO 15788-2 First edition 2003-02-15 Animal and vegetable fats and oils Determination of stigmastadienes in vegetable oils Part 2: Method using high-performance liquid chromatography (HPLC) Corps gras dorigines animale et vgtale Dosage des stigmastadines dans les huiles
19、vgtales Partie 2: Mthode par chromatographie liquide haute performance (CLHP) ENISO157882:2004IS-88751 O2:(3002E) DPlcsid Fremia ihTs PDF file mya ctnoian emdebt dedyfepcaes. In ccacnadrow eitA hebods licnesilop gnic,y tihs file mirp eb yatnde iv roweb detu slahl ton ide ebtlnu deess teh typfecaes w
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22、y ttah lborp aem lertait gno it is f,dnuo plsaee ifnrom ttneC ehlar Secterirata ta teh serddaig sleb nevwo. ISO 3002 All irthgs erse.devr lnUeto sswrehise specified, on trap fo this lbupictaion maeb y cudorperro de tuilizi den yna form ro na ybm ynae,s lecetrinoc ro mceinahcla, incliduntohp gcoiypod
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24、tsser edevrENISO157882:2004IS-88751 O2:(3002E) I SO 3002 All irhgts seredevr iiiContents Page Foreword iv 1 Scope 1 2 Normative references. 1 3 Terms and definitions. 1 4 Principle. 2 5 Reagents 2 6 Apparatus. 3 7 Sampling 4 8 Preparation of test sample. 4 8.1 General. 4 8.2 External standard method
25、 4 8.3 Internal standard method. 4 9 Procedure. 5 9.1 High-pressure liquid chromatography (HPLC) 5 9.2 Identification of steradienes 5 10 Expression of results 6 10.1 External standard 6 10.2 Internal standard. 6 11 Precision 7 11.1 Interlaboratory test. 7 11.2 Repeatability 7 11.3 Reproducibility 7
26、 12 Test report 7 Annex A (informative) Results of interlaboratory test 8 Annex B (informative) Examples of chromatograms 9 Bibliography . 10 ENISO157882:2004IS-88751 O2:(3002E) iv I SO 3002 All irhgts seredevrForeword ISO (the International Organization for Standardization) is a worldwide federatio
27、n 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 established has the right to be represented on that committee.
28、 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 standardization. International Standards are drafted in accordance wi
29、th the rules given in the ISO/IEC 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 appro
30、val by at least 75 % of the member 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 15788-2 was prepared by Technical Commi
31、ttee ISO/TC 34, Food products, Subcommittee SC 11, Animal and vegetable fats and oils. ISO 15788 consists of the following parts, under the general title Animal and vegetable fats and oils Determination of stigmastadienes in vegetable oils: Part 1: Method using capillary-column gas chromatography (R
32、eference method) Part 2: Method using high-performance liquid chromatography (HPLC) ENISO157882:2004INTENRATIONAL TSANDADR IS-88751 O2:(3002E)I SO 3002 All irhgts seredevr 1Animal and vegetable fats and oils Determination of stigmastadienes in vegetable oils Part 2: Method using high-performance liq
33、uid chromatography (HPLC) 1 Scope This part of ISO 15788 specifies a method for the determination of steradienes, especially stigmastadienes. Steradienes are formed by dehydration of sterols during bleaching and also partially during steam washing and deodorization. The method is also suitable as a
34、screening method to detect the presence of refined vegetable oils in virgin oils such as virgin olive oil. NOTE ISO 15788-1 is the reference method for the determination of stigmastadienes in vegetable oils whilst this part of ISO 15788 can be used as a rapid screening method. In view of the precisi
35、on of this method (see Annex A), samples of virgin olive oils close to the limit adopted by international regulations (IOOC, EC) can be verified by the GLC method given in ISO 15788-1. 2 Normative references The following referenced documents are indispensable for the application of this document. F
36、or dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 3696:1987, Water for analytical use Specification and test method ISO 12228, Animal and vegetable fats and oils Determination of individu
37、al and total sterols content Gas chromatographic method 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 stigmastadienes content that part of the stigmastadienes separated by liquid chromatography under the conditions specified in this Interna
38、tional Standard NOTE It is expressed in milligrams per kilogram. 3.2 steradienes content that part of all the steradienes separated by liquid chromatography under the conditions specified in this International Standard NOTE It is expressed in milligrams per kilogram. ENISO157882:2004IS-88751 O2:(300
39、2E) 2 I SO 3002 All irhgts seredevr4 Principle Steradienes are separated from the major part of other lipids as non-polar fat compounds, using petroleum ether on a silica gel column. The petroleum ether eluate is concentrated then analysed by RP-18-HPLC and UV-detection at 235 nm. They are quantifie
40、d, depending on the kind of sample, using an internal or external standard. 5 Reagents WARNING Attention is drawn to the regulations which prescribe the handling of dangerous matter. Technical, organizational and personal safety measurements shall be followed. Use only reagents of recognized analyti
41、cal grade, unless otherwise stated. 5.1 Water, of at least grade 1 according to ISO 3696:1987. 5.2 Silica gel 60 for column chromatography, particle size 0,063 mm to 0,200 mm, or 0,063 mm to 0,100 mm 1) , with a water content of 2 g per 100 g. Dry the silica gel in a porcelain dish for 12 h at 160 C
42、 in a drying oven and cool to room temperature in a desiccator. For adjusting the silica gel to a water content of 2 g per 100 g, weigh (to the nearest 1 g) 98 g of the dried silica gel in a conical flask with ground glass stopper and add 2 g of water (weighed to the nearest 0,01 g). Shake vigorousl
43、y for 1 min and allow the silica gel to stand overnight in an airtight vessel. 5.3 Petroleum ether, boiling range 40 C to 60 C. 5.4 Acetonitrile, chromatography grade. 5.5 tert-Butyl methyl ether, chromatography grade. 5.6 Isooctane. 5.7 3,5-Cholestadiene 2) , with a known purity of at least 95 g pe
44、r 100 g. Check the purity of the cholestadiene standard by gas chromatography using 5-cholestane as internal standard. For this test, see the conditions for the gas chromatographic method for the determination of sterols specified in ISO 12228. The response factor of the flame ionization detector sh
45、ould be 1,0. Take into consideration the obtained concentration during the determination of the steradienes. 5.8 Stock solutions and standard solutions 5.8.1 3,5-Cholestadiene stock solution, of concentration 1 mg/ml. Weigh, to the nearest 0,1 mg, 50,0 mg of 3,5-cholestadiene into a 50 ml measuring
46、flask. Dissolve and dilute to the mark with tert-butyl methyl ether (5.5). 5.8.2 3,5-Cholestadiene standard solution for HPLC 1) For example, No. 7734 or 15101 from E. Merck, 64271 Darmstadt, Germany. 2) For example, No. C6012 from Sigma Chemie GmbH, Grnwalder Weg 30, 82041 Deisenhofen, Germany. Thi
47、s information is given for the convenience of users of this part of ISO 15788 and does not constitute an endorsement by ISO of these products. ENISO157882:2004IS-88751 O2:(3002E) I SO 3002 All irhgts seredevr 35.8.2.1 External standard solution, of concentration 10 g/ml. Pipette 100 l of the 3,5-cho
48、lestadiene stock solution (5.8.1) into a 10 ml measuring flask and fill up to the mark with acetonitrile/tert-butyl methyl ether (5.8.4). A 20 l aliquot of this solution contains 0,20 g. This is injected into the HPLC column. The concentration of the standard solution depends on the nature of the oil to be analysed. In the case of virgin oils with stigmastadienes contents of less than 0,5 mg/kg, the concentratio
