1、July 2017 English price group 10No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67.200.10!%h8b“2692163www.din.deDIN
2、 EN ISO 15302Animal and vegetable fats and oils Determination of benzoapyrene Reversephase high performance liquid chromatography method (ISO 15302:2017);English version EN ISO 15302:2017,English translation of DIN EN ISO 15302:2017-07Tierische und pflanzliche Fette und le Bestimmung des Benzoapyren
3、Gehalts UmkehrphasenHochleistungsflssigchromatographisches Verfahren (ISO 15302:2017);Englische Fassung EN ISO 15302:2017,Englische bersetzung von DIN EN ISO 15302:2017-07Corps gras dorigines animale et vgtale Dtermination du benzoapyrne Mthode par chromatographie liquide haute performance polarit d
4、e phase inverse (ISO 15302:2017);Version anglaise EN ISO 15302:2017,Traduction anglaise de DIN EN ISO 15302:2017-07SupersedesDIN EN ISO 15302:201008www.beuth.deDocument comprises 15 pagesDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritati
5、ve.07.17 DIN EN ISO 15302:2017-07 2 A comma is used as the decimal marker. National foreword This document (EN ISO 15302:2017) has been prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France) in collaboration with Technical Committee CEN/TC 307 “Oil seeds, vegetable an
6、d animal fats and oils and their by-products Methods of sampling and analysis” (Secretariat: AFNOR, France). The responsible German bodies involved in its preparation were DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products) and t
7、he Deutsche Gesellschaft fr Fettwissenschaft (German Fat Research Society), Joint Working Committee NA 057-05-05 AA “Joint committee of DIN and DGF for the analysis of fats, oils and products thereof, related and primary products”. The DIN Standards corresponding to the International Standards refer
8、red to in this document are as follows: ISO 661 DIN EN ISO 661 ISO 5555 DIN EN ISO 5555 ISO 5725-2 DIN ISO 5725-2 Amendments This standard differs from DIN EN ISO 15302:2010-08 as follows: a) milk and milk products (or fat coming from milk and milk products) are now excluded from the Scope of the st
9、andard; b) Clause 3 “Terms and definitions” has been updated; c) the standard has been editorially revised and brought in line with the current rules of presentation. Previous editions DIN EN ISO 15302: 2007-04, 2010-08 National Annex NA (informative) Bibliography DIN EN ISO 661, Animal and vegetabl
10、e fats and oils Preparation of test sample DIN EN ISO 5555, Animal and vegetable fats and oils Sampling DIN ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement metho
11、d EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 15302 April 2017 ICS 67.200.10 Supersedes EN ISO 15302:2010 English Version Animal and vegetable fats and oils Determination of benzoapyrene Reverse-phase high performance liquid chromatography method (ISO 15302:2017) Corps gras dorigines an
12、imale et vgtale Dtermination du benzoapyrne Mthode par chromatographie liquide haute performance polarit de phase inverse (ISO 15302:2017) Tierische und pflanzliche Fette und le Bestimmung des Benzoapyren-Gehalts Umkehrphasen-Hochleistungs-flssigchromatographisches Verfahren (ISO 15302:2017) This Eu
13、ropean Standard was approved by CEN on 13 April 2017. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references c
14、oncerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member
15、 into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, Franc
16、e, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPIS
17、CHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 15302:2017 E EN ISO 15302:2017 (E) 2 Contents Page European foreword . 3 Foreword 4 1
18、 Scope 5 2 Normative references 5 3 Terms and definitions . 5 4 Principle . 5 5 Reagents . 5 6 Apparatus . 7 7 Sampling . 8 8 Preparation of test sample 8 9 Procedure. 8 10 Expression of results . 10 10.1 Calculation of relative response factors 10 10.2 Calculation of benzoapyrene content 11 11 Prec
19、ision 11 11.1 Repeatability 11 11.2 Reproducibility . 11 12 Test report 11 Annex A (informative) Results of interlaboratory tests 12 Bibliography . 13 DIN EN ISO 15302:2017-07 EN ISO 15302:2017 (E) 3 European foreword This document (EN ISO 15302:2017) has been prepared by Technical Committee ISO/TC
20、34 “Food products” in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-products Methods of sampling and analysis” the secretariat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by
21、publication of an identical text or by endorsement, at the latest by October 2017, and conflicting national standards shall be withdrawn at the latest by October 2017. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CEN
22、ELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN ISO 15302:2010. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Bel
23、gium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Swede
24、n, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 15302:2017 has been approved by CEN as EN ISO 15302:2017 without any modification. DIN EN ISO 15302:2017-07 ForewordISO (the International Organization for Standardization) is a worldwide federation of national standar
25、ds 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. International organi
26、zations, 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. The procedures used to develop this document and those intended for its f
27、urther maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ di
28、rectives).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. Details of any patent rights identified during the development of the document will be in t
29、he Introduction and/or on the ISO list of patent declarations received (see www .iso .org/ patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement. For an explanation on the voluntary nature of standards, the meaning of I
30、SO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www .iso .org/ iso/ foreword .html.This document was prepared by Technical
31、Committee ISO/TC 34, Food products, Subcommittee SC 11, Animal and vegetable fats and oils.This third edition cancels and replaces the second edition (ISO 15302:2007), of which it constitutes a minor revision. The scope of this document has been revised to exclude its application to milk and milk pr
32、oducts and their derivatives.4 EN ISO 15302:2017 (E) DIN EN ISO 15302:2017-07 1 ScopeThis document specifies a method for the determination of benzoapyrene in crude or refined edible oils and fats by reverse-phase high performance liquid chromatography (HPLC) using fluorimetric detection in the rang
33、e 0,1 g/kg to 50 g/kg.Milk and milk products (or fat coming from milk and milk products) are excluded from the scope of this document.2 Normative referencesThe following documents are referred to in the text in such a way that some or all of their content constitutes requirements of this document. F
34、or dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 661, Animal and vegetable fats and oils Preparation of test sample3 Terms and definitionsFor the purposes of this document, the following
35、terms and definitions apply.ISO and IEC maintain terminological databases for use in standardization at the following addresses: IEC Electropedia: available at http:/ www .electropedia. org/ ISO Online browsing platform: available at http:/ www. iso. org/ obp3.1benzoapyrene contentmass fraction of b
36、enzoapyrene in the test portion, as determined using the method specified in this documentNote 1 to entry: The content is expressed in micrograms per kilogram.4 PrincipleA test portion is dissolved in light petroleum and benzobchrysene is added as internal standard. A suitable amount of sample is ad
37、sorbed on an alumina column and eluted with light petroleum to remove any benzoapyrene present.5 ReagentsUse only reagents of recognized analytical grade, unless otherwise specified. Where analytical grade solvents other than the recommended ones are used, a full blank analysis shall be carried out
38、and the results of this blank analysis reported.5 EN ISO 15302:2017 (E) DIN EN ISO 15302:2017-07 SAFETY PRECAUTIONS Attention is drawn to regulations which specify handling procedures for dangerous substances. Users should be aware of and comply with technical, organizational, and personal safety me
39、asures.5.1 Water, double distilled, filtered through a membrane filter of pore size 0,45 m; deionized water obtained by purifying demineralized water systems may also be used.5.2 Light petroleum, (boiling point range 40 C to 60 C), or hexane, redistilled over potassium hydroxide pellets (4 g/l).5.3
40、Acetonitrile, suitable for HPLC.5.4 Tetrahydrofuran, suitable for HPLC.5.5 Acetonitrile-tetrahydrofuran mixture, prepared by mixing 90 ml acetonitrile (5.3) and 10 ml tetrahydrofuran (5.4).5.6 Toluene, suitable for HPLC.5.7 Sodium sulfate, granular, anhydrous.5.8 Alumina activity grade 4, prepared f
41、rom neutral aluminium oxide, activity grade super 11), deactivated by the addition of 10 ml water (5.1) to 90 g of alumina.Due to the differences in activity of alumina of various brands, a check is recommended to confirm that the deactivation procedure is appropriate for total benzoapyrene recovery
42、 from a reference sample.CAUTION THE DEACTIVATION REACTION IS EXOTHERMIC AND PRESSURE CAN BUILD UP.Shake the container for about 15 min and allow the contents to equilibrate for 24 h. Store the alumina in a closed vessel at ambient temperature.5.9 Benzoapyrene2), of purity 99,0 % by mass.CAUTION BEN
43、ZOaPYRENE IS A KNOWN CARCINOGEN. CARRY OUT ALL WORK WITH IT IN A FUME HOOD, WEARING GLOVES TO MINIMIZE EXPOSURE.5.9.1 Benzoapyrene stock solution in toluene, 0,5 mg/ml.Weigh, to the nearest 0,1 mg, about 12,5 mg of benzoapyrene in a 25 ml graduated flask. Dissolve it in toluene (5.6) and make up to
44、the mark with that solvent.Store the solution in the dark at 4 C where it is stable for at least 6 months.5.9.2 Benzoapyrene standard solutions.Prepare two standard solutions containing approximately 0,2 g/ml and 0,01 g/ml of benzoapyrene, respectively, by diluting aliquots of the stock solution (5.
45、9.1) with acetonitrile.1) “Aluminium oxide 90 active neutral” is an example of a suitable product available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this product. Equivalent products may be used if they can
46、 be shown to lead to the same results.2) A suitable reference material is available from the Joint Research Centre of the European Commission, Institute for Reference Materials and Measurements (IRMM). This information is given for the convenience of users of this document and does not constitute an
47、 endorsement by ISO of this product.6 EN ISO 15302:2017 (E) DIN EN ISO 15302:2017-07 5.10 Benzobchrysene3)internal standard solution in acetonitrile.Prepare a stock solution containing, to the nearest nanogram, approximately 10 ng/l. Dilute this solution by a factor of 10 in a volumetric flask to ob
48、tain an internal standard solution with a concentration of approximately 1 ng/l.NOTE This solution can also be prepared by dissolving benzobchrysene3), 99,0 % by mass, in acetonitrile.6 ApparatusUsual laboratory apparatus and, in particular, the following.6.1 Glass column for chromatography, of leng
49、th 300 mm and internal diameter 15 mm, fitted with sintered glass discs, and polytetrafluoroethylene (PTFE) tap.6.2 Water baths, one maintained at (35 2) C and another at (65 2) C.6.3 Flash evaporator, a rotary evaporator with vacuum and a water bath set at 40 C may be used. Care should be taken to prevent cross contamination. Clean the system thoroughly between dete
