1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN ISO 15302:2010Animal and vegetable fatsand oils Determination ofbenzoapyrene Reverse-phase high performance liquidchromatography method (ISO15302:2007)BS EN ISO 15302:2010
2、BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO15302:2010. It is identical to ISO 15302:2007.The UK participation in its preparation was entrusted to TechnicalCommittee AW/307, Oil seeds, animal and vegetable fats and oilsand their by products.A list of orga
3、nizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2010ISBN 978 0 580 69271 0ICS 67.200.10Compliance with a British Standard c
4、annot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 30 September 2010.Amendments issued since publicationDate Text affectedEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 15302 April 2010 ICS 67
5、.200.10 Supersedes EN ISO 15302:2007English Version Animal and vegetable fats and oils - Determination of benzoapyrene - Reverse-phase high performance liquid chromatography method (ISO 15302:2007) Corps gras dorigines animale et vgtale - Dtermination du benzoapyrne - Mthode par chromatographie liqu
6、ide haute performance polarit de phase inverse (ISO 15302:2007) Tierische und pflanzliche Fette und le - Bestimmung des Benzoapyren-Gehalts - Umkehrphasen-Hochleistungsflssigchromatographisches Verfahren (ISO 15302:2007) This European Standard was approved by CEN on 18 March 2010. CEN members are bo
7、und 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 obtained on application to the
8、CEN 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 Management Centre has the same st
9、atus as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portug
10、al, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2010 CEN All rights of exploitation in any form and by any means r
11、eserved worldwide for CEN national Members. Ref. No. EN ISO 15302:2010: EBS EN ISO 15302:2010EN ISO 15302:2010 (E) 3 Foreword The text of ISO 15302:2007 has been prepared by Technical Committee ISO/TC 34 “Food products” of the International Organization for Standardization (ISO) and has been taken o
12、ver as EN ISO 15302:2010 by 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 publication
13、 of an identical text or by endorsement, at the latest by October 2010, and conflicting national standards shall be withdrawn at the latest by October 2010. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall
14、not be held responsible for identifying any or all such patent rights. This document supersedes EN ISO 15302:2007. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulga
15、ria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice
16、The text of ISO 15302:2007 has been approved by CEN as a EN ISO 15302:2010 without any modification. BS EN ISO 15302:2010ISO 15302:2007(E) ISO 2007 All rights reserved iiiForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO mem
17、ber 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, governmen
18、tal 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 Direc
19、tives, 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 approval by at least 75 % of the member bodi
20、es 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 15302 was prepared by Technical Committee ISO/TC 34, Food products, Subcommitt
21、ee SC 11, Animal and vegetable fats and oils. This second edition cancels and replaces the first edition (ISO 15302:1998). BS EN ISO 15302:2010BS EN ISO 15302:2010INTERNATIONAL STANDARD ISO 15302:2007(E) ISO 2007 All rights reserved 1Animal and vegetable fats and oils Determination of benzoapyrene R
22、everse-phase high performance liquid chromatography method 1 Scope This International Standard 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 range 0,
23、1 g/kg to 50 g/kg. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 661
24、, Animal and vegetable fats and oils Preparation of test sample 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 benzoapyrene content mass fraction of benzoapyrene in the test portion, as determined using the method specified in this Internati
25、onal Standard NOTE The content is expressed in micrograms per kilogram. 4 Principle A 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 benzoapyren
26、e present. 5 Reagents Use 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 and the results of this blank analysis reported. SAFETY PRECAUTIONS Attention is dr
27、awn to regulations which specify handling procedures for dangerous substances. Users should be aware of and comply with technical, organizational, and personal safety measures. BS EN ISO 15302:2010ISO 15302:2007(E) 2 ISO 2007 All rights reserved5.1 Water, double distilled, filtered through a membran
28、e 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 Acetonitrile, suitable for HPLC. 5.4 Tetrahydrofuran, suitabl
29、e 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 from neutral aluminium oxide, activity grade super 11), d
30、eactivated 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 REAC
31、TION IS EXOTHERMIC AND PRESSURE MAY 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 BENZOaPYRENE IS A KNOWN CARCINOGEN. CARRY OUT ALL WORK
32、 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 the mark with that solvent. Store the solution in
33、 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 (5.9.1) with acetonitrile. 1) “Aluminium oxide 90
34、active neutral” is an example of a suitable product available commercially. This information is given for the convenience of users of this International Standard 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 re
35、sults. 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 International Standard and does not constitute an endorsement by I
36、SO of this product. BS EN ISO 15302:2010ISO 15302:2007(E) ISO 2007 All rights reserved 35.10 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,
37、to obtain an internal standard solution with a concentration of approximately 1 ng/l. NOTE This solution may also be prepared by dissolving benzobchrysene3), 99,0 % by mass, in acetonitrile. 6 Apparatus Usual laboratory apparatus and, in particular, the following. 6.1 Glass column for chromatography
38、, of length 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 b
39、e taken to prevent cross contamination. Clean the system 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 sam
40、ple loop shall be flushed with acetonitrile between consecutive injections. 6.5 Columns for HPLC analysis 6.5.1 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
41、4,6 mm, packed with Lichrosorb RP-18 (of particle size 5 m)4). 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
42、at 406 nm (slit 10 nm) and excitation wavelength at 384 nm (slit 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 vi
43、als. 6.9 Disposable pipettes. 3) A suitable reference material is 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 International Stan
44、dard 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 International Standard and does not constitute an endorsement by ISO of this product. Equivalent products may be
45、 used if they can be shown to lead to the same results. BS EN ISO 15302:2010ISO 15302:2007(E) 4 ISO 2007 All rights reserved7 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the
46、method specified in this International Standard. A recommended sampling method is given in ISO 55551. 8 Preparation of test sample Prepare the test sample in accordance with ISO 661. 9 Procedure 9.1 Clean up of sample 9.1.1 Weigh, to the nearest 0,001 g, about 0,400 g of the fat or oil into a glass
47、beaker and dissolve in 2 ml of light petroleum (5.2). Add 20 l of the internal standard solution (5.10) by means of a microsyringe. This is equivalent to 50 g/kg when calculated on the sample mass. If a high level of benzoapyrene is expected, then add 50 l of the internal standard solution (5.10). T
48、his is equivalent to 125 g/kg when calculated on the sample mass. 9.1.2 Fill the chromatography column (6.1) to half its height with light petroleum (5.2). Rapidly weigh 22 g of alumina (5.8) into a small beaker and transfer the alumina immediately to the column, then gently tap the column to effect
49、 settling of the alumina. 9.1.3 Add anhydrous sodium sulfate (5.7) to the top of the column to form a layer about 30 mm deep. 9.1.4 Open the tap and allow the light petroleum to fall to the level of the top of the sodium sulfate layer. 9.1.5 Place a 20 ml graduated flask under the column. 9.1.6 Introduce the oil solution (9.1.1) on to the column. Rinse the column with minimal amounts of light petroleum (5.2), allowing the solvent layer to run into the sodium sulfa
