1、BSI Standards Publication PD CEN/TS 16621:2014 Food analysis Determination of benzoapyrene, benzaanthracene, chrysene and benzobfluoranthene in foodstuffs by high performance liquid chromatography with fluorescence detection (HPLC- FD)PD CEN/TS 16621:2014 PUBLISHED DOCUMENT National foreword This Pu
2、blished Document is the UK implementation of CEN/TS 16621:2014. The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods. A list of organizations represented on this committee can be obtained on request to its secretary. This publication
3、 does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2014. Published by BSI Standards Limited 2014 ISBN 978 0 580 80857 9 ICS 67.050 Compliance with a British Standard cannot confer immunity from
4、 legal obligations. This Published Document was published under the authority of the Standards Policy and Strategy Committee on 30 April 2014. Amendments issued since publication Date Text affectedPD CEN/TS 16621:2014TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 1662
5、1 April 2014 ICS 67.050 English Version Food analysis - Determination of benzoapyrene, benzaanthracene, chrysene and benzobfluoranthene in foodstuffs by high performance liquid chromatography with fluorescence detection (HPLC-FD) Analyse des produits alimentaires - Dosage du benzo(a)pyrne, benzo(a)a
6、nthracne, chrysne et benzo(b)fluoranthne dans les denres alimentaires par chromatographie en phase liquide haute performance avec dtection de fluorescence (HPLC-FD) Lebensmittelanalytik - Bestimmung von Benzoapyren, Benzaanthracen, Chrysen und Benzobfluoranthen in Lebensmitteln mittels Hochleistungs
7、- Flssigkeitschromatographie mit Fluoreszenzdetektion (HPLC-FD) This Technical Specification (CEN/TS) was approved by CEN on 21 October 2013 for provisional application. The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
8、 submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard. CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available promptly at national level in an appropriate form. It is
9、permissible to keep conflicting national standards in force (in parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, E
10、stonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR S
11、TANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2014 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN/TS 16621:2014 EPD CEN/TS 16621:2014
12、 CEN/TS 16621:2014 (E) 2 Contents Page Foreword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents .4 5 Apparatus .7 6 Procedure .8 7 HPLC analysis 9 8 Calculation . 12 9 Recovery 12 10 Test report . 12 11 Precision data . 13 Annex A (informative) Typical chromatograms 14 Annex B (informa
13、tive) In-house validation data for the PAH4 in different matrices . 16 Annex C (informative) In-house performance data with a mixture of cyclohexane and ethyl acetate as alternative extraction solvent . 21 Bibliography . 23 PD CEN/TS 16621:2014 CEN/TS 16621:2014 (E) 3 Foreword This document (CEN/TS
14、16621:2014) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. 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 re
15、sponsible for identifying any or all such patent rights. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association WARNING The use of this document can involve hazardous materials, operations and equipment. This document does not
16、purport to address all the safety problems associated with its use. It is the responsibility of the user of this document to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. According to the CEN-CENELEC Internal Regulations, th
17、e national standards organizations of the following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy,
18、Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. PD CEN/TS 16621:2014 CEN/TS 16621:2014 (E) 4 1 Scope This Technical Specification specifies a method for the determination of benzoapyr
19、ene (BaP) plus benzaanthracene (BaA), benzobfluoranthene (BbF) and chrysene (CHR) in several food matrices. The method is based on size exclusion chromatography (SEC) cleanup, followed by quantification with high performance liquid chromatography (HPLC) with programmable fluorescence detection. This
20、 method has been in-house validated via the analysis of spiked samples of edible olive oil, fresh mussels, smoked fish, smoked meat products, processed cereal-based foods for young children, infant formulae, chocolate and food supplements (isoflavones) at levels ranging from 0,25 g/kg to 1,00 g/kg a
21、nd from 4,95 g/kg to 23,53 g/kg, depending on the Polycyclic Aromatic Hydrocarbon (PAH) or the matrix. This method complies with the performance characteristics specified for BaP, BaA, BbF and CHR in current legislation 3. The method has been shown to be applicable to a variety of additional matrice
22、s as meat products, fresh fish, paprika, roasted coffee, bread, herbs, breakfast cereals, beer, sunflower oil, olives and fried tomato, with a limit of quantification below 0,5 g/kg. In addition, the method was tested in-house and shown to be applicable also for the quantification of the other 12 PA
23、Hs of the 15+1 EU priority PAHs set (benzocfluorene (BcL), benzojfluoranthene (BjF), benzokfluoranthene (BkF), cyclopentacdpyrene (CPP), dibenza,hanthracene (DhA), dibenzoa,epyrene (DeP), benzoghiperylene (BgP), dibenzoa,hpyrene (DhP), dibenzoa,ipyrene (DiP), dibenzoa,lpyrene (DlP), indeno1,2,3-cdpy
24、rene (IcP), 5-methylchrysene (5MC) in all matrices listed above and at similar level ranges, except for CPP, where a UV detection had to be used with limits of quantification above 8 g/kg. For the determination of PAHs in edible fats and oils, two other standards are also available, EN ISO 22959 and
25、 EN ISO 15753 (see 1 and 2). 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced
26、document (including any amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987) 3 Principle The PAHs are extracted from solid matrices with dichloromethane. In case of edible oils, the samples are simply dispersed in dichloromethane
27、. Aliquots of crude extracts in dichloromethane are purified by SEC. The final extracts are analysed by HPLC under gradient conditions with programmable fluorescence detection. 4 Reagents Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696:1995, unless ot
28、herwise specified. Solvents shall be of quality for HPLC analysis. For storing and expiring dates of use of substances and commercially available solutions, supplier indications or certificates shall be followed. Refrigerated standard solutions shall reach room temperature before being used. WARNING
29、 1 Some PAHs are considered carcinogenic. Persons using this document should be familiar with normal laboratory practices. It is the responsibility of the user of this document to apply practices which are in agreement with applicable occupational safety and health practices. WARNING 2 Dispose chemi
30、cal waste according to applicable environmental rules and regulations. PD CEN/TS 16621:2014 CEN/TS 16621:2014 (E) 5 WARNING 3 PAHs are degraded by UV light. Protect PAHs solutions from light (keep in the dark, use aluminium foil or amber glassware). WARNING 4 The analyst shall ensure that samples do
31、 not become contaminated during sample preparation. Containers shall be rinsed with high purity acetone or hexane before use to minimize the risk of contamination. Wherever possible, apparatus and equipment coming into contact with the sample shall be made of inert materials such as aluminium, glass
32、 or polished stainless steel. Some precaution is needed when using plastics as polypropylene or PTFE because the analytes may be adsorbed onto these materials. 4.1 Helium purified compressed gas (purity equivalent to 99,995 % or better). For solvent degassing, if needed. 4.2 Nitrogen purified compre
33、ssed gas (purity equivalent to 99,995 % or better). 4.3 Acetone. 4.4 n-Hexane. 4.5 Dichloromethane. 4.6 Acetonitrile. 4.7 Methoxychlor. 4.8 Perylene. 4.9 Sulfur. 4.10 Corn oil, commercial. 4.11 HPLC mobile phase solvent A: Water. The mobile phase solvent A should be degassed. 4.12 HPLC mobile phase
34、solvent B: Acetonitrile (4.6). The mobile phase solvent B should be degassed. 4.13 Cyclohexane. 4.14 Ethyl acetate. 4.15 Mixture of cyclohexane and ethyl acetate. Mix one part per volume of cyclohexane (4.13) with one part per volume of ethyl acetate (4.14). 4.16 Anhydrous sodium sulphate. 4.17 Poly
35、cyclic aromatic hydrocarbons. 4.17.1 Benzoapyrene (BaP). 4.17.2 Chrysene (CHR). 4.17.3 Benzobfluoranthene (BbF). PD CEN/TS 16621:2014 CEN/TS 16621:2014 (E) 6 4.17.4 Benzaanthracene (BaA). 4.17.5 Benzokfluoranthene (BkF). 4.17.6 Dibenzoa,hanthracene (DhA). 4.17.7 Benzog,h,iperylene (BgP). 4.17.8 Inde
36、no1,2,3-cdpyrene (IcP). 4.17.9 Benzocfluorene (BcL). 4.17.10 Cyclopentac,dpyrene (CPP). 4.17.11 5 Methylchrysene (5MC). 4.17.12 Benzojfluoranthene (BjF). 4.17.13 Dibenzoa,lpyrene (DlP). 4.17.14 Dibenzoa,epyrene (DeP). 4.17.15 Dibenzoa,ipyrene (DiP). 4.17.16 Dibenzoa,hpyrene (DahP). 4.17.17 15+1 EU p
37、riority PAHs standard solution containing 10 g/ml each, in appropriate organic solvent, preferably acetonitrile. 4.18 PAH4 standard solution Prepare a standard solution of 10 g/ml PAH4 in acetonitrile by weighing carefully the proper amounts of the 4 PAHs (BaP, BaA, BbF and CHR) individually in acet
38、onitrile. Store this solution under refrigeration conditions. A solution stored in this way is stable for at least 12 months. If longer stability is proven, the solution can still be applied. 4.19 PAH4 stock solution Prepare a stock solution of 500 ng/ml in acetonitrile by diluting exactly 500 l of
39、the 10 g/ml PAH4 standard solution (4.18) to 10 ml with acetonitrile (4.6) into a calibrated 10 ml volumetric flask. Store this solution under refrigeration conditions. A solution stored in this way is stable for at least 12 months. If longer stability is proven, the solution can still be applied. 4
40、.20 PAH4 working solution Prepare a working solution of 50 ng/ml in acetonitrile, by diluting 1 ml of the 500 ng/ml PAH4 stock solution in acetonitrile (4.19) up to 10 ml with acetonitrile (4.6) into a calibrated 10 ml volumetric flask. Store this solution under refrigeration conditions. A solution
41、stored in this way is stable for at least six months. If longer stability is proven, the solution can still be applied. 4.21 15+1 PAHs stock solution Prepare a stock solution of 500 ng/ml in acetonitrile by pipetting exactly 500 l of the 10 g/ml 15+1 PAHs standard solution (4.17.17) into a calibrate
42、d 10 ml volumetric flask. Take them to dryness by evaporation under nitrogen, and redissolve in 10 ml of acetonitrile (4.6). Store this solution under refrigeration conditions. A solution stored in this way is stable for at least 12 months. If longer stability is proven, the solution can still be ap
43、plied. PD CEN/TS 16621:2014 CEN/TS 16621:2014 (E) 7 In case of commercial availability of 15+1 EU priority PAHs standard solution containing 10 g/ml each, in acetonitrile, the 15+1 PAHs stock solution can be prepared directly by diluting 500 l of that solution up to 10 ml with acetonitrile. 4.22 15+
44、1 PAHs working solution Prepare a working solution of 50 ng/ml in acetonitrile, by diluting 1 ml of the 500 ng/ml 15+1 PAHs stock solution in acetonitrile (4.21) to 10 ml with acetonitrile into a calibrated 10 ml volumetric flask. Store this solution under refrigeration conditions. A solution stored
45、 in this way is stable for at least six months. If longer stability is proven, the solution may still be applied. 5 Apparatus Usual laboratory glassware and equipment and, in particular, the following: 5.1 High speed blender, for solid samples and vortex mixer for liquid samples. 5.2 Filter papers,
46、suitable for qualitative analysis, prefolded. 5.3 Round bottomed glass tubes, of 250 ml capacity, suitable for the high speed blender. 5.4 Evaporator, with water bath and flushing nitrogen capability. 5.5 Analytical balance, accuracy to the nearest 0,000 1 g. 5.6 Laboratory balance, accuracy to the
47、nearest 0,1 g. 5.7 Glass syringe, of 5 ml capacity. 5.8 Microsyringes, of 250 l, 500 l and 1 000 l capacity. 5.9 Calibrated volumetric flasks, of 5 ml and 10 ml capacity. 5.10 Displacement pipettes, of 200 l, with appropriate tips. 5.11 Glass vials, approximately 1,8 ml capacity and crimp caps. 5.12
48、 Graduated pipette, of 5 ml capacity. 5.13 Size-Exclusion Chromatography (SEC) system, comprising the following: 5.13.1 HPLC pump (isocratic), capable of pumping 5 ml/min pulse free. 5.13.2 Injection system, suitable for 1,0 ml and 0,2 ml injection volume. 5.13.3 Two SEC cleanup columns, 19 mm x 150
49、 mm and 19 mm x 300 mm, connected in series, packed with high-performance, fully porous, highly cross-linked, styrene divinylbenzene copolymer particles, 10 nm pore size with nominal particle size of 15 m. 5.13.4 UV detector, capable to provide = 254 nm. 5.13.5 Fraction collector. 5.13.6 Recorder, integrator or computer based data processing system. 5.13.7 Calibration PD CEN/TS 16621:2014 CEN/TS 16621:2014 (E) 8 Connect the columns to the system and calibrate
