BS-684-2.47-1998 ISO-15302-1998.pdf

BRITISH STANDARD BS 684-2.47: 1998 ISO 15302: 1998 Methods of analysis of fats and fatty oils Part 2: Other methods Section 2.47: Determination of benzoapyrene content by reverse-phase high-performance liquid chromatography ICS 67.200.10 Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:04 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 684-2.47:1998 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Board, was published under the authority of the Standards Board and comes into effect on 15 August 1998 © BSI 04-1999 ISBN 0 580 29946 5 National foreword This British Standard reproduces verbatim ISO 15302:1998 and implements it as the UK national standard. The UK participation in its preparation was entrusted to Technical Committee AW/11, Animal and vegetable fats and oils, which has the responsibility to: aid enquirers to understand the text; present to the responsible 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. A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document 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 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. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, the ISO title page, page ii, pages 1 to 4 and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover. Amendments issued since publication Amd. No.DateComments Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:04 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 684-2.47:1998 © BSI 04-1999i Contents Page National forewordInside front cover Forewordii 1Scope1 2Normative reference1 3Principle1 4Reagents1 5Apparatus1 6Sampling2 7Preparation of test sample2 8Procedure2 9Expression of results3 10Precision3 11Test report3 Annex A (informative) Results of interlaboratory test4 Annex B (informative) Bibliography4 Table A.1 Statistical results4 Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:04 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ii blank Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:04 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:04 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 684-2.47:1998 ii © BSI 04-1999 Foreword ISO (the International Organization for 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 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. 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. International Standard ISO 15302 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Subcommittee SC 11, Animal and vegetable fats and oils. Annex A and Annex B of this International Standard are for information only. Descriptors: Agricultural products, food products, animal fats, vegetable fats, animal oils, vegetable oils, chemical analysis, determination of content, benzopyrene, high performance liquid chromatography. Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:04 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 684-2.47:1998 © BSI 04-19991 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 from 0,1 4g/kg to 10 4g/kg. 2 Normative reference The following standard contains provisions, which through reference in this text, constitute provisions of this International Standard. At the time of publication the edition indicated was valid. All standards are subject to revision and parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent edition of the standard indicated below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 661:1989, Animal and vegetable fats and oils Preparation of test sample. 3 Principle Adsorption of a suitable amount of sample on an alumina column, followed by elution with light petroleum of any benzoapyrene present, and subsequent analysis of the eluate by HPLC using a fluorimetric detector. 4 Reagents All reagents shall be of recognized analytical grade. 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. 4.1 Water, double distilled, filtered through a membrane filter of 0,45 4m pore size; deionized water obtained by purifying demineralized water systems may also be used. 4.2 Light petroleum (boiling point range between 40 °C and 60 °C), or hexane, redistilled over potassium hydroxide pellets (4 g/l). 4.3 Acetonitrile, suitable for HPLC. 4.4 Tetrahydrofuran, suitable for HPLC. 4.5 Toluene, suitable for HPLC. 4.6 Sodium sulfate, granular, anhydrous. 4.7 Alumina, activity grade 4, prepared from neutral aluminium oxide, activity grade super 1, deactivated by the addition of 10 ml distilled water to 90 g of alumina. CAUTION THE REACTION IS EXOTHERMIC AND PRESSURE MAY BUILD UP. Shake the container for about 15 min and allow the contents to equilibriate for 24 h. Store the alumina in a closed vessel at ambient temperature. 4.8 Benzoapyrene, 99,0 % purity. CAUTION BENZOaPYRENE IS A KNOWN CARCINOGEN. CARRY OUT ALL WORK WITH IT IN A FUME HOOD, WEARING GLOVES TO MINIMIZE EXPOSURE. 4.9 Benzoapyrene solutions1) 4.9.1 Stock solution Weigh, to the nearest 0,1 mg, about 12,5 mg of benzoapyrene in a 25 ml graduated flask. Dissolve it in toluene (4.5) and fill to the mark. This solution contains about 0,5 mg/ml benzoapyrene and should be stored in the dark at 4 °C when it is stable for 6 months at least. 4.9.2 Standard solutions Prepare two standard solutions containing approximately 0,2 4g/ml and 0,01 4g/ml of benzoapyrene respectively by diluting aliquots of the stock solution (4.9.1) with acetonitrile (4.3). 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 Glass column for chromatography, 300 mm long, 15 mm internal diameter, fitted with sintered glass discs, and polytetrafluoroethylene (PTFE) tap. 5.2 Water baths (two), maintained at 35 °C ± 1 °C and 65 °C ± 1 °C. 5.3 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 determinations. 5.4 High-performance liquid chromatograph, consisting of an HPLC pump, injection valve with 10 4l sample loop, reverse-phase column, electronic integrator and chart recorder. NOTEIf an autosampler is used, the sample loop should be flushed with acetonitrile between subsequent injections. 1) A suitable reference material is available from the Commission of the European Community Bureau of Reference (BCR), rue de la Loi 200, B-1049, Brussels, Belgium. Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:04 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 684-2.47:1998 2 © BSI 04-1999 5.5 Columns for HPLC analysis 5.5.1 Reverse-phase guard column, capable of resolving benzoapyrene from co-extractives, together with appropriate precolumn e.g. stainless-steel precolumn 75 mm long, 4,6 mm internal diameter, packed with Lichrosorb RP-18 (5 4m particle size).2) 5.5.2 HPLC reverse-phase column, 250 mm long, of 4,6 mm internal diameter (stainless steel), for polycyclic aromatic hydrocarbons (PAHs) (e.g. Chromspher 5 PAH or Vydac 201 TP5).3) 5.6 Fluorescence detector, with emission wavelength 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. 5.7 Crimp-top minivials, of about 1 ml volume, with Teflon-layered septa and aluminium caps. 5.8 Hand crimper, for crimping the caps onto the vials. 5.9 Disposable pipettes 6 Sampling Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 5555. It is important the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage. 7 Preparation of test sample Prepare the test sample in accordance with ISO 661. 8 Procedure 8.1 Clean up of sample 8.1.1 Weigh, to the nearest 0,001 g, about 2 g of the oil sample into a 10 ml graduated flask. Dissolve in light petroleum (4.2) and dilute to the mark. 8.1.2 Fill the chromatography column (5.1) to half its height with light petroleum (4.2). Rapidly weigh 22 g of alumina (4.7) into a small beaker and transfer the alumina immediately to the column, promoting settling of the alumina by gently tapping the column. 8.1.3 Add anhydrous sodium sulfate (4.6) to the top of the column to form a layer about 30 mm deep. 8.1.4 Open the tap and allow the light petroleum to fall to the level of the top of the sodium sulfate layer. 8.1.5 Place a 20 ml graduated flask under the column. 8.1.6 Pipette 2,00 ml of the oil solution (8.1.1) onto the column. Rinse the column with minimal amounts of light petroleum, allowing the solvent layer to run into the sodium sulfate layer between rinsings. 8.1.7 Elute the column with light petroleum (4.2) with a flow of about 1 ml/min, discarding the first 20 ml of eluate and collecting the next 60 ml of eluate in a 100 ml round-bottomed flask. 8.1.8 Evaporate the eluate in the water bath (5.2) set at 65 °C, to about 0,5 ml to 1,0 ml, and transfer the concentrated solution into a pre-weighed (to nearest 0,1 mg) crimp-top minivial (5.5). 8.1.9 Continue the evaporation from the minivial, in the water bath (5.2) set at 35 °C under a gentle stream of nitrogen (about 25 ml/min) until nearly dry. Rinse the round-bottomed flask with about 1 ml of light petroleum and transfer the rinsing quantitatively to the minivial, continuing the evaporation under nitrogen. Repeat the rinsing and transfer to the minivial once more. 8.1.10 Continue the evaporation at 35 °C under nitrogen until dry. 8.1.11 Weigh the minivial to the nearest 0,1 mg, and calculate the mass of the residue. 8.1.12 Stopper the minivial with the Teflon-layered septum and aluminium cap and store at 4 °C. 8.2 High-performance liquid chromatography 8.2.1 Use a mixture of 88/12 (V/V) acetonitrile (4.3)/water (4.1) as elution solvent. Degas the elution solvent to remove oxygen in order to avoid fluorescence quenching. Use helium purging or an on-line vacuum degasser. 8.2.2 Elute at a flow rate of about 1 ml/min. 8.2.3 Prepare four dilutions of the standard benzoapyrene solutions (4.9.2) such that injection of 10 4l of each will give readings corresponding to 0,04 ng, 0,2 ng, 1,0 ng and 2,0 ng of benzoapyrene. From these, construct a four-point calibration curve using the peak areas from the integrator and chart recorder. 2) Lichrosorb RP-18 is an example of a product commercially available. This information is given for the convenience of users of this International Standard and does not consitute an endorsement by ISO of this product. 3) Chromspher 5 PAH and Vydac 201 TP5 are examples of products commercially available. This information is given for the convenience of users of this International Standard and does not consitute an endorsement by ISO of these products. Licensed Copy: sheffieldun sheffieldun, na, Wed Dec 06 13:38:04 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 684-2.47:1998 © BSI 04-19993 8.2.4 Tetrahydrofuran proved to be the optimal solvent for residue analysis of oils and fats following the clean-up procedure (8.1). Injection of volumes in excess of the specified 10 4l will give rise to problems. Do not store the samples in tetrahydrofuran for a prolonged period as benzoapyrene is not stable in this solvent. 8.3 Sample analysis 8.3.1 Inject 20 4l of tetrahydrofuran (4.4) into the minivial containing the cleansed residue (8.1.12). Dissolve the residue by careful swirling, avoiding contact of the solvent with the septum. With the calibration curve (8.2.3) benzoapyrene levels of 0,1 4g/kg to 10 4g/kg can be determined. For concentrations above 10 4g/kg, the residue solution (8.3.2) should be diluted further with tetrahydrofuran, or a smaller volume than 10 4l (8.3.2) should be injected. 8.3.2 Inject an accurately known volume of about 10 4 of the dissolved residue into the HPLC column and start the chromatogram running. Care should be taken to ensure that not more than 1,5 mg of residue is introduced into the column. If a larger amount of residue is present, the amount of tetrahydrofuran shall be adjusted or the clean-up step shall be repeated. 9 Expression of results Calculate the benzoapyrene content of the test sample using the following equation: where Express the results to the nearest 0,1 4g/kg. 10 Precision 10.1 Repeatability The absolute difference between two independent single test results, obtained using the same method on identical test material in the same laboratory by the same operator using the same equipment, within a short interval of time, will in not more than 5 % of cases be greater than the repeatability limit (r) given in Table A.1. 10.2 Reproducibility The absolute difference between two single test results, obtained using the same method on identical test material in different laboratories with different operators using different equipment, will in not more than 5 % of cases be greater than the reproducibility limit (R) given in Table A.1. 11 Test report The test report shall specify all information necessary for the complete identification of the sample;