1、BSI Standards PublicationBS EN 16619:2015Food analysis Determinationof benzoapyrene,benzaanthracene, chryseneand benzobfluoranthenein foodstuffs by gaschromatography massspectrometry (GC-MS)BS EN 16619:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 16619:2
2、015.The UK participation in its preparation was entrusted to TechnicalCommittee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a co
3、ntract. Users are responsible for its correctapplication. The British Standards Institution 2015. Published by BSI StandardsLimited 2015ISBN 978 0 580 80856 2ICS 67.050Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the author
4、ity of theStandards Policy and Strategy Committee on 30 April 2015.Amendments issued since publicationDate Text affectedBS EN 16619:2015EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16619 April 2015 ICS 67.050 English Version Food analysis - Determination of benzoapyrene, benzaanthracene, chr
5、ysene and benzobfluoranthene in foodstuffs by gas chromatography mass spectrometry (GC-MS)Analyse des produits alimentaires - Dosage du benzo(a)pyrne, benzo(a)anthracne, chrysne et benzo(b)fluoranthne dans les denres alimentaires par chromatographie en phase gazeuse couple la spectromtrie de masse (
6、CG-SM) Lebensmittelanalytik - Bestimmung von Benzoapyren, Benzaanthracen, Chrysen und Benzobfluoranthen in Lebensmitteln mit Gaschromatographie und Massenspektrometrie (GC-MS) This European Standard was approved by CEN on 7 February 2015. CEN members are bound to comply with the CEN/CENELEC Internal
7、 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 CEN-CENELEC Management Centre or to any CEN
8、 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-CENELEC Management Centre has the same status as the official versio
9、ns. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Po
10、land, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2015 CEN All rights of exploitatio
11、n in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16619:2015 EBS EN 16619:2015EN 16619:2015 (E) 2 Contents Page Foreword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents .5 5 Standard preparation 9 6 Apparatus 12 7 Procedure 15 8 GC-MS analysis . 17
12、 9 Calculation and reporting 22 10 Quality control . 22 11 Precision data . 24 12 Test report . 27 Annex A (informative) Typical chromatograms 28 Annex B (informative) Precision data . 30 Annex C (informative) Precision data from single laboratory validation . 35 Bibliography . 37 BS EN 16619:2015EN
13、 16619:2015 (E) 3 Foreword This document (EN 16619:2015) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical t
14、ext or by endorsement, at the latest by October 2015 and conflicting national standards shall be withdrawn at the latest by October 2015. 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 respons
15、ible 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 purp
16、ort 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, the na
17、tional standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, L
18、ithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16619:2015EN 16619:2015 (E) 4 1 Scope This European Standard specifies a method for the determination of 4 of the 16 EU priority polycycli
19、c aromatic hydrocarbons (PAHs), identified as target PAHs. They are benzaanthracene (BaA), benzoapyrene (BaP), benzobfluoranthene (BbF) and chrysene (CHR). The method allows their quantification in the presence of the other 12 EU priority PAHs (benzojfluoranthene (BjF), cyclopentacdpyrene (CPP), ben
20、zokfluoranthene (BkF), dibenza,hanthracene (DhA), benzocfluorene (BcL), dibenzoa,epyrene (DeP), benzoghiperylene (BgP), dibenzoa,hpyrene (DhP), dibenzoa,ipyrene (DiP), dibenzoa,lpyrene (DlP), indeno1,2,3-cdpyrene (IcP), 5-methylchrysene (5MC) in extruded wheat flour, smoked fish, dry infant formula,
21、 sausage meat, freeze-dried mussels, edible oil and wheat flour, by gas-chromatography mass-spectrometry (GC-MS). The extraction of PAHs from solid samples is performed by pressurized liquid extraction (PLE). Soxhlet extraction was applied by some participants in the method validation study by colla
22、borative trial as alternative to PLE. The sample cleanup is performed by applying the following techniques in the reported sequence: size exclusion chromatography (SEC), and solid phase extraction (SPE). This method complies with the performance characteristics specified in Commission Regulation (EU
23、) No 836/2011 (see 1). In particular the specifications for the limit of detection (LOD) and of the limit of quantification (LOQ) (0,30 g/kg and 0,90 g/kg respectively) were met. The method has been validated in an interlaboratory study via the analysis of both naturally contaminated and spiked samp
24、les, ranging from 0,5 g/kg to 11,9 g/kg. However, linearity of the instrument response was proven for the concentration range 0,5 g/kg to 20 g/kg. For the determination of PAHs in edible fats and oils, two other standards are also available, EN ISO 22959 and EN ISO 15753, for more information see 2
25、and 3. 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 document (including an
26、y amendments) applies. EN ISO 1042:1999, Laboratory glassware - One-mark volumetric flasks (ISO 1042:1998) EN ISO 3696:1995, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987) 3 Principle The sample is homogenized. A test portion is mixed with desiccant, sand and th
27、e stable isotope labelled internal standard solution. It is then extracted with n-hexane or cyclohexane by pressurized liquid extraction, or alternatively by Soxhlet extraction. If applicable, co-extracted water is separated from the organic phase of the extract. The organic extract is evaporated to
28、 a small volume, filtered and purified by SEC, using a mixture of ethyl acetate and cyclohexane as eluent. After SEC, 200 l of toluene are added as a keeper to the collected SEC fraction. The SEC fraction is evaporated to about 200 l, and cleaned up by SPE on silica, using cyclohexane as eluent. The
29、 cleaned up sample extract is evaporated again to about 200 l. Finally, an injection standard solution is added to the sample prior to measurement by GC-MS. The injection is performed with a PTV, or split/splitless injection port. The chromatographic separation is obtained on a mid-polar capillary c
30、olumn with high selectivity for PAHs. The analytes are ionised by electron ionization (EI) at 70 eV. The target PAHs are recorded in Single Ion Monitoring (SIM) mode, and quantified by comparison with the stable isotope labelled analogues. BS EN 16619:2015EN 16619:2015 (E) 5 4 Reagents 4.1 General U
31、se only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696:1995, unless otherwise specified. All reagents and standard solutions shall be stored according to the specifications given by the supplier. The specifications given in this procedure for opened commercia
32、l solutions or for in-house prepared solutions aim to minimize solvent evaporation and to protect the analytes (PAHs) from degradation. Standard solutions are preferably prepared gravimetrically. Depending on the handled amount of substance a micro-balance (6.4) and/or an analytical balance (6.5) ar
33、e used for the preparation of solutions of both native and stable isotope labelled PAHs. All concentrations are expressed as mass per mass. If necessary, the concentrations expressed as mass per volume could be obtained applying the density equation (Formula (1). vm= (1) where density (in g/ml); m m
34、easured mass of the substance (in g); v volume of the solution (in ml). The density of toluene at 20 C is 0,8669 g/ml. Comprehensive information on the density of solvents at various temperatures is given in 4. All solutions and substances are used at room temperature. WARNING 1 Some PAHs are consid
35、ered 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 chemical waste according to
36、applicable environmental rules and regulations. 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 Some precaution is needed when using plastics as polypropylene or PTFE because the analytes may be absorbed
37、onto these materials. 4.2 Helium purified compressed gas (purity equivalent to 99,995 % or better). 4.3 Nitrogen purified compressed gas (purity equivalent to 99,995 % or better). 4.4 Disodium sulfate, (Na2SO4), anhydrous, granular. 4.5 Poly(acrylic acid), partial sodium salt-graft-poly(ethylene oxi
38、de) granular, 90 m to 850 m particle size. 4.6 Sand, 50 mesh to 70 mesh particle size. 4.7 n-Hexane. BS EN 16619:2015EN 16619:2015 (E) 6 4.8 Acetone. 4.9 Cyclohexane. 4.10 Toluene. 4.11 Ethyl acetate. 4.12 SEC eluent Mix 1 part per volume of cyclohexane (4.9) with 1 part per volume of ethyl acetate
39、(4.11). 4.13 SPE column For the solid phase extraction cleanup, a silica SPE column is used. Commercial cartridges of 500 mg 4 ml or self-filled cartridges of the same size containing 500 mg activated silica are used. The surface area of the silica should be around 500 m2/g. NOTE Commercial SPE colu
40、mns made of polypropylene were used in the method validation study by collaborative trial. 4.14 Reference material for quality control A certified reference material, or any other suitable quality control material (e.g left over proficiency test material) may be applied for this purpose. The CITAC/E
41、urachem Guide to Quality in Analytical Chemistry may be consulted for guidance, see 5. Analyse this material with every sample batch and use it to control the method performances along time (see 10.4). 4.15 Native reference substances - commercially available neat material or solutions of PAHs The l
42、ist of native substances analysed with this method is provided in Table 1. The target analytes are given in bold font. Commercially available, preferably certified, standard solutions are preferred due to the higher level of safety in handling. Triphenylene, benzojfluoranthene, and benzokfluoranthen
43、e are potentially interfering with the target analytes and are therefore used for evaluation of selectivity. BS EN 16619:2015EN 16619:2015 (E) 7 Table 1 Names and structures of the native PAHs NameaCAS number Structure Name aCAS number Structure Benzaanthracene (BaA) 56553 Figure 1 Benzobfluoranthen
44、e (BbF) 205992 Figure 2 Benzoapyrene (BaP) 50328 Figure 3 Chrysene (CHR) 218019 Figure 4 Triphenylene (TRP) 217594 Figure 5 Benzojfluoranthene (BjF) 205823 Figure 6 Benzokfluoranthene (BkF) 207089 Figure 7 a The acronym is given in parenthesis, the target analytes are given in bold. 4.16 Stable isot
45、ope labelled reference standards (in the form of commercially available stable isotope labelled PAH solutions) The stable isotope labelled analogues, applied for the quantification of the target PAHs are listed in Table 2. The commercial solutions used in the method validation study by collaborative
46、 trial contained the stable isotope labelled PAHs at a level of about 100 g/kg in nonane. Preference is given to 13C labelled analogues as their chemical properties best match those of the native analytes. However, alternatively to 13C labelled substances, deuterated analogues of the target analytes
47、 may be applied. The concentration levels of these solutions should be similar to the levels specified for the 13C labelled PAH solutions. NOTE 1 Highly deuterated PAHs are separated on the specified GC-column at least partially from their native analogues. NOTE 2 Both forms of benzaanthracene-13C6,
48、 which are displayed in Table 2, are equally suitable for the purpose of this standard. The 13C labelled reference material might be even supplied as a mixture, which was the case in the method validation study by collaborative trial. BS EN 16619:2015EN 16619:2015 (E) 8 Table 2 Names and structures
49、of 13C labelled PAHs Name CAS number Structure Benzaanthracene-13C6not available and Figure 8 Benzoapyrene-13C4not available Figure 9 Benzobfluoranthene-13C6not available Figure 10 Chrysene-13C6not available Figure 11 The recovery of the method is calculated based on the stable isotope labelled standards (see 10.2). Their physical-chemical properties are considered equivalent to the native PAHs. Table 3 indicates the correspondence between each nat
