1、BS EN ISO22959:2009ICS 67.200.10NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDAnimal and vegetablefats and oils Determination ofpolycyclic aromatichydrocarbons byon-line donor-acceptor complexchromatographyand HPLC withfluorescence detection(ISO 22959:2009)Thi
2、s British Standardwas published under theauthority of the StandardsPolicy and StrategyCommittee on 30 June2009. BSI 2009ISBN 978 0 580 58293 6Amendments/corrigenda issued since publicationDate CommentsBS EN ISO 22959:2009National forewordThis British Standard is the UK implementation of EN ISO 22959
3、:2009.The UK participation in its preparation was entrusted to TechnicalCommittee AW/307, Oil seeds, animal and vegetable fats and oils andtheir by products.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include a
4、ll the necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 22959May 2009ICS 67.200.10English VersionAnimal and vegetable fats and oil
5、s - Determination of polycyclicaromatic hydrocarbons by on-line donor-acceptor complexchromatography and HPLC with fluorescence detection (ISO22959:2009)Corps gras dorigines animale et vgtale - Dterminationde la teneur en hydrocarbures aromatiques polycycliquespar chromatographie de complexe donneur
6、-accepteur etCLHP avec dtection par fluorescence (ISO 22959:2009)Tierische und pflanzliche Fette und le - Bestimmungpolycyclischer aromatischer Kohlenwasserstoffe durchgekoppelte Donor-Akzeptor-Komplex-Chromatographie undHPLC mit Fluoreszenzdetektion (ISO 22959:2009)This European Standard was approv
7、ed by CEN on 1 May 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandard
8、s may be obtained on application to the 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 translationunder the responsibility of a CEN member into its own language and notified to the
9、 CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlan
10、ds, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any fo
11、rm and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 22959:2009: EBS EN ISO 22959:2009EN ISO 22959:2009 (E) 3 Foreword This document (EN ISO 22959:2009) has been prepared by Technical Committee ISO/TC 34 “Agricultural food products“ in collaboration with Technical Committee
12、 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 of an identical text or by endorsement, at the
13、 latest by November 2009, and conflicting national standards shall be withdrawn at the latest by November 2009. 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 not be held responsible for identifying any o
14、r all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary,
15、 Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 22959:2009 has been approved by CEN as a EN ISO 22959:2009 without any modificatio
16、n. BS EN ISO 22959:2009ISO 22959:2009(E) ISO 2009 All rights reserved iiiContents Page Foreword iv Introduction v 1 Scope . 1 2 Normative references . 1 3 Terms and definitions. 1 4 Principle. 2 5 Reagents, materials and standards 3 6 Apparatus 5 7 Sampling and preparation of the test sample 5 8 Sam
17、ple preparation 6 9 Procedure 7 9.1 HPLC analysis . 7 9.2 Identification of PAHs. 7 10 Calculation of individual PAHs 7 11 Method validation data. 8 11.1 Accuracy 8 11.2 Within-laboratory precision . 8 11.3 Recovery 8 11.4 Dynamic range 8 11.5 Limit of quantification 8 12 Precision 9 12.1 Internatio
18、nal collaborative trial . 9 12.2 Repeatability 9 12.3 Reproducibility 9 13 Test report . 9 Annex A (informative) Example of the individual parts of an HPLC system 10 Annex B (informative) Example of the operating conditions of the pumps of the HPLC system 11 Annex C (informative) Example of the oper
19、ating conditions of the column thermostat and the detector of the HPLC system 12 Annex D (informative) Example of the autosampler programme of the HPLC system. 13 Annex E (informative) Tubing connections of the HPLC system 14 Annex F (informative) Chromatogram of a standard calibration sample . 15 A
20、nnex G (informative) Determination precision for a sunflower oil, range 0,1 g/kg to 3,5 g/kg 16 Annex H (informative) Determination precision for an olive oil, range 0,1 g/kg to 3,5 g/kg. 17 Annex I (informative) Determination precision for a soybean oil, range 0,1 g/kg to 3,5 g/kg 18 Annex J (infor
21、mative) Determination precision for a coconut oil, range 0,1 g/kg to 3,5 g/kg 19 Annex K (informative) Interlaboratory test 20 Bibliography . 22 BS EN ISO 22959:2009ISO 22959:2009(E) iv ISO 2009 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide feder
22、ation 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 commit
23、tee. 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 accordanc
24、e with 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 a
25、pproval 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 22959 was prepared by Technical Com
26、mittee ISO/TC 34, Food products, Subcommittee SC 11, Animal and vegetable fats and oils. BS EN ISO 22959:2009ISO 22959:2009(E) ISO 2009 All rights reserved vIntroduction Polycyclic aromatic hydrocarbons (PAHs) are formed during pyrolytic processes such as the incomplete combustion of organic substan
27、ces or have a petrogenic origin (mineral oils). Edible fats and oils may be contaminated by environmental pollution and/or processing steps prior to refining. The presence of PAHs in fats and oils is a health concern due to their carcinogenicity. Different levels of PAHs have been observed in crude
28、edible oils. Refining of the oils (deodorization, bleaching, charcoal treatment) under the appropriate conditions reduces the content of the individual PAHs to the microgram per kilogram level. The known methods of analysis of PAHs in edible fats and oils include complex and laborious extraction and
29、 clean-up procedures to isolate the low levels of PAHs present. With the donor-acceptor complex-chromatography (DACC) technique, PAHs can be extracted from different matrices. PAHs are electron donors (-electrons) and the strong interaction of the PAHs with an electron acceptor stationary phase resu
30、lts in retention of the PAHs and elution of (the bulk of) the other components of the oil. This International Standard specifies an automated on-line method for the determination of PAHs in edible oils and fats, which can easily be applied as a routine analysis. The method consists of an LC-LC coupl
31、ing of a clean-up DACC column to an analytical column for the separation. PAHs are quantified by fluorescence detection. Compared to older techniques, this automated on-line method significantly reduces the amount of solvent used and saves considerable time. The DACC column clean-up is fast and is c
32、arried out during the HPLC run of the previous sample. The total analysis time for one sample is approximately 90 min, compared with the traditional methods which require 8 h to 10 h. Moreover, the system can run 24 h/day. Finally, losses of volatile PAHs during solvent evaporation, for example, are
33、 eliminated. The quantification limits of 0,1 g/kg of the individual PAHs have been retained with the DACC method, which automatically corrects for possibly incomplete recoveries because the calibration samples are subjected to the same treatment as the samples to be analysed. The system uses conven
34、tional HPLC instrumentation. BS EN ISO 22959:2009BS EN ISO 22959:2009INTERNATIONAL STANDARD ISO 22959:2009(E) ISO 2009 All rights reserved 1Animal and vegetable fats and oils Determination of polycyclic aromatic hydrocarbons by on-line donor-acceptor complex chromatography and HPLC with fluorescence
35、 detection 1 Scope This International Standard specifies a high performance liquid chromatographic (HPLC) procedure for the determination of polycyclic aromatic hydrocarbons (PAHs) in edible fats and oils. The method has been validated for coconut (CN), olive (OV), sunflower (SF), and soybean (BO) o
36、il, and is possibly applicable to other oils, dependent on the determination of appropriate parameters. The lowest level of quantification for the PAHs is 0,1 g/kg. The lowest possible amount of each PAH which can be distinguished from the baseline noise has not been determined. The validated concen
37、tration range of the method is 0,1 g/kg to 3,5 g/kg for each individual PAH. For samples containing (light) PAH contents 3,5 g/kg, dilution to bring the contents into the validated range is possible. It is also possible to adjust the range of the calibration curves. However, ranges exceeding 3,5 g/k
38、g have not been validated. PAHs which can be determined by this method are: anthracene, phenanthrene, fluoranthene, pyrene, chrysene, benzoaanthracene, benzoepyrene, benzoapyrene, perylene, benzoghiperylene, anthanthrene, dibenzoa,hanthracene, coronene, indeno1,2,3-cdpyrene, benzoafluoranthene, benz
39、obfluoranthene, benzokfluoranthene. 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)
40、 applies. ISO 661, 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 polycyclic aromatic hydrocarbon PAH compound that contains two or more condensed (fused) aromatic hydrocarbon rin
41、gs and whose content can be determined according to the method specified in this International Standard BS EN ISO 22959:2009ISO 22959:2009(E) 2 ISO 2009 All rights reserved3.2 light polycyclic aromatic hydrocarbon compound with two to four condensed (fused) aromatic hydrocarbon rings EXAMPLES Compou
42、nd CAS No. Compound CAS No. Compound CAS No. acenaphthene 83-32-9 benzoepyrene 192-97-2 naphthalene 50-32-8 acenaphthylene 208-96-8 chrysene 218-01-9 phenanthrene 85-01-8 anthracene 120-12-7 fluoranthene 206-44-0 pyrene 129-00-0 benzoaanthracene (1,2-benzoanthracene) 56-55-3 fluorene 86-73-7 3.3 hea
43、vy polycyclic aromatic hydrocarbon compound with five and more condensed (fused) aromatic hydrocarbon rings EXAMPLES Compound CAS No. Compound CAS No. Compound CAS No. benzoapyrene (1,2-benzopyrene) 50-32-8 benzokfluoranthene 207-08-9 dibenzoa,hanthracene (1,2,5,6-dibenzoanthracene) 53-70-3 benzoafl
44、uoranthene 203-33-8 benzoghiperylene (1,12-benzoperylene) 191-24-2 indeno1,2,3-cdpyrene 193-39-5 benzobfluoranthene 205-99-2 coronene 191-07-1 perylene 198-55-0 3.4 polycyclic aromatic hydrocarbon content mass fraction of a polycyclic aromatic hydrocarbon or polycyclic aromatic hydrocarbon mixture i
45、n a matrix EXAMPLES Individual polycyclic aromatic hydrocarbon content; light polycyclic aromatic hydrocarbon content; heavy polycyclic aromatic hydrocarbon content. NOTE The content is expressed as a mass fraction in micrograms per kilogram. 4 Principle The PAHs in edible oils are determined by on-
46、line coupling of donor-acceptor complex chromatography (DACC) and HPLC with fluorescence detection. The oil samples are eluted over a column with a modified stationary phase (DACC column) which will act as an electron acceptor. This column will retain the PAHs (electron donors) by - interactions. Af
47、ter elution of the oil, the PAHs are transferred on-line to the analytical reversed phase column. The individual PAHs are detected at different wavelengths. The retention times of the PAHs are used to identify the individual compounds. The levels of the PAHs in the oil samples are calculated by exte
48、rnal calibration. BS EN ISO 22959:2009ISO 22959:2009(E) ISO 2009 All rights reserved 35 Reagents, materials and standards WARNING The method requires harmful reagents. Respect normal laboratory safety regulations. All PAHs are suspected carcinogenic compounds. Therefore, it is essential that the pre
49、paration of the stock solutions, the standard dilutions and the samples of the calibration curve (5.3) are performed by preference in a class-2 laboratory. Furthermore, a laboratory coat and safety gloves are essential for these steps. Contaminated tissues and gloves shall be collected in a plastic bag and removed after sealing the bag. 5.1 Reagents. 5.1.1 Acetonitrile, HPLC grade, mass fraction wC2H3N 99,9 %. 5.1.2 Ethyl acetate, HPLC grade, mass fraction wC4H8O2 99,8 %. 5.1.3 2-Propanol, H
