1、Animal and vegetable fats and oils Determination of benzoapyrene Reverse-phase high performance liquid chromatography methodBS EN ISO 15302:2017BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06National forewordThis British Standard is the UK implementation of EN ISO 1
2、5302:2017. It supersedes BS EN ISO 15302:2010, which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/307, Oilseeds, animal and vegetable fats and oils and their by-products.A list of organizations represented on this committee can be obtained on request t
3、o its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2017 Published by BSI Standards Limited 2017ISBN 978 0 580 96369 8ICS 67.200.10Compliance with a British Stand
4、ard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 September 2017.Amendments/corrigendaissuedsincepublicationDate Text affectedBRITISH STANDARDBS EN ISO 15302:2017EUROPEAN STANDARDNORME EUROPE
5、NNEEUROPISCHE NORMEN ISO 15302April 2017ICS 67.200.10 Supersedes EN ISO 15302:2010EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGCEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN Ref. No. EN ISO 15302:2017: EAll rights of exp
6、loitation in any form and by any means reserved worldwide for CEN national MembersAnimal and vegetable fats and oils - Determination of benzoapyrene - Reverse-phase high performance liquid chromatography method (ISO 15302:2017)Corps gras dorigines animale et vgtale - Dtermination du benzoapyrne - Mt
7、hode par chromatographie liquide haute performance polarit de phase inverse (ISO 15302:2017)Tierische und pflanzliche Fette und le - Bestimmung des Benzoapyren-Gehalts - Umkehrphasen-Hochleistungsflssigchromatographisches Verfahren (ISO 15302:2017)This European Standard was approved by CEN on 13 Apr
8、il 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 concerning such national standards may be obtai
9、ned 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 into its own language and notified to the CEN-
10、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, France, Germany, Greece, Hungary, Iceland, Ireland, I
11、taly, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.English VersionEN ISO 15302:2017 (E)European forewordThis document (EN ISO 15302:2017) has been prepared by Technical Committe
12、e ISO/TC 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,
13、either by 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 a
14、nd/or CENELEC 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: Aust
15、ria, Belgium, 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, Spai
16、n, Sweden, Switzerland, Turkey and the United Kingdom.Endorsement noticeThe text of ISO 15302:2017 has been approved by CEN as EN ISO 15302:2017 without any modification.iiBS EN ISO 15302:2017ISO 15302:2017(E)Foreword iv1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 15 Rea
17、gents 16 Apparatus . 36.5 Columns for HPLC analysis . 37 Sampling 48 Preparation of test sample . 49 Procedure. 49.1 Clean up of sample . 49.2 High performance liquid chromatography . 59.3 Sample analysis 510 Expression of results 610.1 Calculation of relative response factors 610.2 Calculation of b
18、enzoapyrene content 711 Precision . 711.1 Repeatability . 711.2 Reproducibility . 712 Test report . 7Annex A (informative) Results of interlaboratory tests 8Bibliography 9 ISO 2017 All rights reserved iiiContents PageBS EN ISO 15302:2017ISO 15302:2017(E)ForewordISO (the International Organization fo
19、r 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 established has the
20、 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 standardization. The proce
21、dures used to develop this document and those intended for its further 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 editoria
22、l rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).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 i
23、dentified during the development of the document will be in the 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 explanat
24、ion on the voluntary nature of standards, the meaning of ISO 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/f
25、oreword.html.This document was prepared by Technical 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
26、revised to exclude its application to milk and milk products and their derivatives.iv ISO 2017 All rights reservedBS EN ISO 15302:2017Animal and vegetable fats and oils Determination of benzoapyrene Reverse-phase high performance liquid chromatography method1 ScopeThis document specifies a method fo
27、r 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 range 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
28、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. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced docume
29、nt (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 terms and definitions apply.ISO and IEC maintain terminological databases for use in standardization at the following a
30、ddresses: IEC Electropedia: available at http:/www.electropedia.org/ ISO Online browsing platform: available at http:/www.iso.org/obp 3.1benzoapyrene contentmass fraction of benzoapyrene in the test portion, as determined using the method specified in this documentNote 1 to entry: The content is exp
31、ressed 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 adsorbed on an alumina column and eluted with light petroleum to remove any benzoapyrene present.5 ReagentsUse only reagents of
32、 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 and the results of this blank analysis reported.INTERNATIONAL STANDARD ISO 15302:2017(E) ISO 2017 All rights reserved 1BS EN
33、ISO 15302:2017ISO 15302:2017(E)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 measures.5.1 Water, double distilled, filtered through a membrane fi
34、lter 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 Acetonitrile, suitable for HPLC.5.4 Tetrahydrofuran, suitable for
35、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 from neutral aluminium oxide, activity grade super 11), deactivated
36、 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 from a reference sample.CAUTION THE DEACTIVATION REACTION IS EXOT
37、HERMIC 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 Benzoapyrene 2), of purity 99,0 % by mass.CAUTION BENZOaPYRENE IS A KNOWN CARCINOGEN. CARRY OUT ALL WORK WITH IT IN A
38、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 the mark with that solvent.Store the solution in the dark at 4 C
39、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 (5.9.1) with acetonitrile.1) “Aluminium oxide 90 active neutral” is
40、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 be shown to lead to the same results.2) A suitable reference mat
41、erial 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 endorsement by ISO of this product.2 ISO 2017 All rights reserve
42、dBS EN ISO 15302:2017ISO 15302:2017(E)5.10 Benzobchrysene 3)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 obtain an internal standard solution with a conc
43、entration 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 length 300 mm and internal diameter 15 mm, fitted
44、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
45、thoroughly between determinations.6.4 High performance liquid chromatograph, consisting of an HPLC pump, injection valve with 50 l sample loop, reverse-phase column, electronic integrator and chart recorder.If an autosampler is used, the sample loop shall be flushed with acetonitrile between consecu
46、tive injections.6.5 Columns for HPLC analysis6.5.1 Reverse-phase guard column, capable of resolving benzoapyrene from co-extractives, together with appropriate precolumn e.g. stainless-steel precolumn of length 75 mm and internal diameter 4,6 mm, packed with Lichrosorb RP-18 (of particle size 5 m)4)
47、.6.5.2 HPLC reverse-phase column, of length 250 mm and internal diameter 4,6 mm (stainless steel), for polycyclic aromatic hydrocarbons (PAHs) (e.g. Chromspher 5 PAH or Vydac 201 TP5)4).6.6 Fluorescence detector, with emission wavelength at 406 nm (slit 10 nm) and excitation wavelength at 384 nm (sl
48、it 10 nm). The detector shall be capable of the required performance to carry out the analysis.6.7 Crimp-top minivials, of about 1 ml volume, with PTFE-layered septa and aluminium caps.6.8 Hand crimper, for crimping the caps onto the vials.6.9 Disposable pipettes.3) A suitable reference material is
49、available from the Joint Research Centre of the European Commission, Institute for Reference Materials and Measurements (IRMM) or Dr. Ehrenstorfer GmbH. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this product.4) Examples of suitable products available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this produ
