1、October 2009DEUTSCHE NORM English price group 13No part of this standard 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!$ZB;“1553124
2、www.din.deDDIN EN ISO 22959Animal and vegetable fats and oils Determination of polycyclic aromatic hydrocarbons by on-linedetection (ISO 22959:2009)English version of DIN EN ISO 22959:2009-10Tierische und pflanzliche Fette und le Bestimmung polycyclischer aromatischer Kohlenwasserstoffe durch gekopp
3、elteDonor-Akzeptor-Komplex-Chromatographie und HPLC mit Fluoreszenzdetektion(ISO 22959:2009)Englische Fassung DIN EN ISO 22959:2009-10www.beuth.deDocument comprises 28 pagesdonor-acceptor complex chromatography and HPLC with fluorescenceDIN EN ISO 22959:2009-10 2 National foreword This standard has
4、been prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France), Subcommittee SC 11 “Animal and vegetable fats and oils” (Secretariat: BSI, United Kingdom) in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-prod
5、ucts Methods of sampling and analysis” (Secretariat: AFNOR, France). The responsible German bodies involved in its preparation were the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee) and the Deutsche Gesellschaft fr Fettwissenschaft
6、 e. V. (German Fat Research Society) in the Joint Committee Gemein-schaftsausschuss fr die Analytik von Fetten, len, Fettprodukten, verwandten Stoffen und Rohstoffen. The English version of ISO 22959:2009 contains an error in 3.2 (wrong CAS No. for naphthalene). This has been corrected in the German
7、 version of DIN EN ISO 22959, and correspondingly in the English version. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 661 DIN EN ISO 661 ISO 1042 DIN EN ISO 1042 ISO 5555 DIN EN ISO 5555 ISO 5725-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO
8、5725-2 National Annex NA (informative) Bibliography DIN EN ISO 661, Animal and vegetable fats and oils Preparation of test sample DIN EN ISO 1042, Laboratory glassware One-mark volumetric flasks DIN EN ISO 5555, Animal and vegetable fats and oils Sampling DIN ISO 5725-1, Accuracy (trueness and preci
9、sion) of measurement methods and results Part 1: General principles and definitions 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 method EUROPEAN STANDARD
10、NORME EUROPENNE EUROPISCHE NORM EN ISO 22959 May 2009 ICS 67.200.10 English Version Animal and vegetable fats and oils - Determination of polycyclic aromatic hydrocarbons by on-line donor-acceptor complex chromatography and HPLC with fluorescence detection (ISO 22959:2009) Corps gras dorigines anima
11、le et vgtale - Dtermination de la teneur en hydrocarbures aromatiques polycycliques par chromatographie de complexe donneur-accepteur et CLHP avec dtection par fluorescence (ISO 22959:2009) Tierische und pflanzliche Fette und le - Bestimmung polycyclischer aromatischer Kohlenwasserstoffe durch gekop
12、pelte Donor-Akzeptor-Komplex-Chromatographie und HPLC mit Fluoreszenzdetektion (ISO 22959:2009) This European Standard was approved 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 European Standard the sta
13、tus 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 CEN Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German).
14、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 status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republ
15、ic, 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 United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE N
16、ORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 22959:2009: EContents 2 DIN EN ISO 22959:2009-10 EN ISO 22959:2009 (E)Page Fo
17、reword .3 Introduction.4 1 Scope5 2 Normative references5 3 Terms and definitions .5 4 Principle .6 5 Reagents, materials and standards.7 6 Apparatus.9 7 Sampling and preparation of the test sample 9 8 Sample preparation.10 9 Procedure.11 9.1 HPLC analysis11 9.2 Identification of PAHs .11 10 Calcula
18、tion of individual PAHs 11 11 Method validation data12 11.1 Accuracy 12 11.2 Within-laboratory precision12 11.3 Recovery 12 11.4 Dynamic range.12 11.5 Limit of quantification.12 12 Precision 13 12.1 International collaborative trial 13 12.2 Repeatability 13 12.3 Reproducibility 13 13 Test report13 A
19、nnex A (informative) Example of the individual parts of an HPLC system 14 Annex B (informative) Example of the operating conditions of the pumps of the HPLC system 15 Annex C (informative) Example of the operating conditions of the column thermostat and the detector of the HPLC system.16 Annex D (in
20、formative) Example of the autosampler programme of the HPLC system17 Annex E (informative) Tubing connections of the HPLC system.18 Annex F (informative) Chromatogram of a standard calibration sample19 Annex G (informative) Determination precision for a sunflower oil, range 0,1 g/kg to 3,5 g/kg 20 A
21、nnex H (informative) Determination precision for an olive oil, range 0,1 g/kg to 3,5 g/kg .21 Annex I (informative) Determination precision for a soybean oil, range 0,1 g/kg to 3,5 g/kg.22 Annex J (informative) Determination precision for a coconut oil, range 0,1 g/kg to 3,5 g/kg 23 Annex K (informa
22、tive) Interlaboratory test.24 Bibliography26 Foreword 3 EN ISO 22959:2009 (E)DIN EN ISO 22959:2009-10 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 publi
23、cation of an identical text or by endorsement, at the 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 CENELE
24、C shall not be held responsible for identifying any or 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
25、, 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 The text of ISO 22959:2009 has been approved
26、by CEN as a EN ISO 22959:2009 without any modification. This document (EN ISO 22959:2009) has been prepared by Technical Committee ISO/TC 34 “Agricultural food products” in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats Introduction Polycyclic aromatic hydroca
27、rbons (PAHs) are formed during pyrolytic processes such as the incomplete combustion of organic substances 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
28、is a health concern due to their carcinogenicity. Different levels of PAHs have been observed in crude 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
29、 known methods of analysis of PAHs in edible fats and oils include complex and laborious extraction and 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 don
30、ors (-electrons) and the strong interaction of the PAHs with an electron acceptor stationary phase results 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
31、oils and fats, which can easily be applied as a routine analysis. The method consists of an LC-LC coupling 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 re
32、duces the amount of solvent used and saves considerable time. The DACC column clean-up is fast and is carried 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
33、 system can run 24 h/day. Finally, losses of volatile PAHs during solvent evaporation, for example, are 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 calibr
34、ation samples are subjected to the same treatment as the samples to be analysed. The system uses conventional HPLC instrumentation. 4 DIN EN ISO 22959:2009-10 EN ISO 22959:2009 (E)1 Scope This International Standard specifies a high performance liquid chromatographic (HPLC) procedure for the determi
35、nation 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) oil, and is possibly applicable to other oils, dependent on the determination of appropriate parameters. The lowest level of quantif
36、ication 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 concentration 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,
37、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/kg have not been validated. PAHs which can be determined by this method are: anthracene, phenanthrene, fluoranthene, pyrene, chrysen
38、e, benzoaanthracene, benzoepyrene, benzoapyrene, perylene, benzoghiperylene, anthanthrene, dibenzoa,hanthracene, coronene, indeno1,2,3-cdpyrene, benzoafluoranthene, benzobfluoranthene, benzokfluoranthene. 2 Normative references The following referenced documents are indispensable for the application
39、 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, Animal and vegetable fats and oils Preparation of test sample 3 Terms and definitions For the purposes of this d
40、ocument, the following terms and definitions apply. 3.1 polycyclic aromatic hydrocarbon PAH compound that contains two or more condensed (fused) aromatic hydrocarbon rings and whose content can be determined according to the method specified in this International Standard 5 EN ISO 22959:2009 (E)DIN
41、EN ISO 22959:2009-10 3.2 light polycyclic aromatic hydrocarbon compound with two to four condensed (fused) aromatic hydrocarbon rings EXAMPLES Compound CAS No. Compound CAS No. Compound CAS No. acenaphthene 83-32-9 benzoepyrene 192-97-2 naphthalene acenaphthylene 208-96-8 chrysene 218-01-9 phenanthr
42、ene 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 heavy polycyclic aromatic hydrocarbon compound with five and more condensed (fused) aromatic hydrocarbon rings EXAMPLES Compound CAS No. Compound CAS No. Compound
43、 CAS No. benzoapyrene (1,2-benzopyrene) 50-32-8 benzokfluoranthene 207-08-9 dibenzoa,hanthracene (1,2,5,6-dibenzoanthracene) 53-70-3 benzoafluoranthene 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-
44、0 3.4 polycyclic aromatic hydrocarbon content mass fraction of a polycyclic aromatic hydrocarbon or polycyclic aromatic hydrocarbon mixture in a matrix EXAMPLES Individual polycyclic aromatic hydrocarbon content; light polycyclic aromatic hydrocarbon content; heavy polycyclic aromatic hydrocarbon co
45、ntent. NOTE The content is expressed as a mass fraction in micrograms per kilogram. 4 Principle The PAHs in edible oils are determined by on-line coupling of donor-acceptor complex chromatography (DACC) and HPLC with fluorescence detection. The oil samples are eluted over a column with a modified st
46、ationary phase (DACC column) which will act as an electron acceptor. This column will retain the PAHs (electron donors) by - interactions. After elution of the oil, the PAHs are transferred on-line to the analytical reversed phase column. The individual PAHs are detected at different wavelengths. Th
47、e retention times of the PAHs are used to identify the individual compounds. The levels of the PAHs in the oil samples are calculated by external calibration. 6 DIN EN ISO 22959:2009-10 EN ISO 22959:2009 (E)91-20-3 5 Reagents, materials and standards WARNING The method requires harmful reagents. Res
48、pect normal laboratory safety regulations. All PAHs are suspected carcinogenic compounds. Therefore, it is essential that the preparation 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
49、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, HPLC grade, mass fraction wC3H8O 99,9 %. 5.1.4 Toluene, HPLC grade, mass fraction wC7H8 99,9 %. 5.1.5 Water, HPLC grade. 5.2 Standards.1)5.2.1 Anthracene, mass fraction
