SIST EN 16619:2015
(Main)Food analysis - Determination of benzo[a]pyrene, benz[a]anthracene, chrysene and benzo[b]fluoranthene in foodstuffs by gas chromatography mass spectrometry (GC-MS)
Food analysis - Determination of benzo[a]pyrene, benz[a]anthracene, chrysene and benzo[b]fluoranthene in foodstuffs by gas chromatography mass spectrometry (GC-MS)
This European Standard specifies a method for the determination of 4 of the 15+1 EU priority polycyclic aromatic hydrocarbons (PAHs), identified as target PAHs. They are benz[a]anthracene (BaA), benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF) and chrysene (CHR). The method allows their quantification in the presence of the other 12 EU priority PAHs (benzo[j]fluoranthene (BjF), cyclopenta[cd]pyrene (CPP), benzo[k]fluoranthene (BkF), dibenz[a,h]anthracene (DhA), benzo[c]fluorene (BcL), dibenzo[a,e]pyrene (DeP), benzo[ghi]perylene (BgP), dibenzo[a,h]pyrene (DhP), dibenzo[a,i]pyrene (DiP), dibenzo[a,l]pyrene (DlP), indeno[1,2,3-cd]pyrene (IcP), 5-methylchrysene (5MC)) in extruded wheat flour, smoked fish, dry infant formula, 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 pressurised liquid extraction (PLE). Soxhlet extraction may be applied 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) No 836/2011 (see [1]).
The method has been validated in an interlaboratory study via the analysis of both naturally contaminated and spiked samples, ranging from 0,5 µg/kg to 11,9 µg/kg. However, linearity of the instrument response was proven for the concentration range 0,5 µg/kg to 20 µg/kg.
For the determination of PAHs in edible fats and oils, two other CEN standards are also available, EN ISO 22959 and EN ISO 15753, for more information see [2] and [3].
Lebensmittelanalytik - Bestimmung von Benzo[a]pyren, Benz[a]anthracen, Chrysen und Benzo[b]fluoranthen in Lebensmitteln mit Gaschromatographie und Massenspektrometrie (GC-MS)
Die vorliegende Europäische Norm legt ein Verfahren zur Bestimmung von 4 der 15 1 von der EU als prioritär eingestuften polyzyklischen aromatischen Kohlenwasserstoffe (PAK) fest, die als Ziel PAK bezeichnet werden. Diese Verbindungen sind Benz[a]anthracen (BaA), Benzo[a]pyren (BaP), Benzo[b]fluoranthen (BbF) und Chrysen (CHR). Das Verfahren ermöglicht deren quantitative Bestimmung in Gegenwart der anderen 12 von der EU als prioritär eingestuften PAK (Benzo[j]fluoranthen (BjF), Cyclopenta[cd]pyren (CPP), Benzo[k]fluoranthen (BkF), Dibenz[a,h]anthracen (DhA), Benzo[c]fluoren (BcL), Dibenzo[a,e]pyren (DeP), Benzo[ghi]perylen (BgP), Dibenzo[a,h]pyren (DhP), Dibenzo[a,i]pyren (DiP), Dibenzo[a,l]pyren (DlP), Indeno[1,2,3 cd]pyren (IcP), 5 Methylchrysen (5MC)) in extrudiertem Weizenmehl, geräuchertem Fisch, pulverförmigem Säuglingsnahrungsmittel, Wurstbrät, gefriergetrockneten Muscheln, Speiseöl und Weizen¬mehl mittels Gaschromatographie-Massenspektrometrie (GC MS). Die Extraktion von PAK aus Feststoff¬proben erfolgt durch beschleunigte Lösemittelextraktion (PLE). Als Alternative zur PLE kann die Soxhlet-Extraktion angewendet werden. Die Aufreinigung der Probe erfolgt unter Anwendung der folgenden Techniken in der angegebenen Reihenfolge: Größenausschlusschromatographie (en: size exclusion chromatography, SEC) und Festphasenextraktion (en: solid phase extraction, SPE).
Dieses Verfahren entspricht den in der Verordnung (EU) Nr. 836/2011 der Kommission (siehe [1]) festgelegten Leistungskenngrößen.
Das Verfahren wurde in einem Ringversuch durch die Analyse sowohl natürlich kontaminierter als auch aufgestockter Proben im Bereich von 0,5 µg/kg bis 11,9 µg/kg validiert. Die Linearität des Ansprechverhaltens des Gerätes wurde jedoch für den Konzentrationsbereich von 0,5 µg/kg bis 20 µg/kg nachgewiesen.
Zur Bestimmung von PAK in Speisefetten und ölen stehen auch zwei andere CEN Normen zur Verfügung, EN ISO 22959 und EN ISO 15753, hinsichtlich weiterführender Informationen siehe [2] und [3].
Analyse des produits alimentaires - Dosage du benzo(a)pyrène, benzo(a)anthracène, chrysène et benzo(b)fluoranthène dans les denrées alimentaires par chromatographie en phase gazeuse couplée à la spectrométrie de masse (CG-SM)
La présente Norme européenne spécifie une méthode de dosage de 4 des 15+1 hydrocarbures aromatiques polycycliques (HAP) prioritaires de l'UE, identifiés comme HAP cibles : le benzo[a]anthracène (BaA), le benzo[a]pyrène (BaP), le benzo[b]fluoranthène (BbF) et le chrysène (CHR). La méthode permet de quantifier la présence des 12 autres HAP prioritaires de l'UE (benzo[j]fluoranthène (BjF), cyclopenta[cd]pyrène (CPP), benzo[k]fluoranthène (BkF), dibenzo[a,h]anthracène (DhA), benzo[c]fluorène (BcL), dibenzo[a,e]pyrène (DeP), benzo[ghi]pérylène (BgP), dibenzo[a,h]pyrène (DhP), dibenzo[a,i]pyrène (DiP), dibenzo[a,l]pyrène (DlP), indéno[1,2,3-cd]pyrène (IcP), 5-méthylchrysène (5MC)) dans la farine de blé extrudée, le poisson fumé, les laits infantiles en poudre, la chair à saucisse, les moules lyophilisées, les huiles alimentaires et la farine de blé, par chromatographie en phase gazeuse couplée à la spectrométrie de masse (CG-SM). Pour les échantillons solides, l’extraction est effectuée par extraction sous fluide pressurisé (PLE) ou Soxhlet. La purification de l'échantillon est effectuée en appliquant les techniques suivantes dans l'ordre indiqué : chromatographie d'exclusion stérique (SEC) et extraction en phase solide (SPE).
Cette méthode est conforme aux caractéristiques de performance spécifiées dans le Règlement (UE) n° 836/2011 de la Commission (voir [1]).
La méthode a été validée lors d'un essai interlaboratoires impliquant l’analyse d’échantillons naturellement contaminés et d’échantillons enrichis, sur un intervalle de concentration de 0,5 µg/kg à 11,9 µg/kg. Toutefois, la linéarité de la réponse de l'instrument a été démontrée pour la gamme de concentrations de 0,5 µg/kg à 20 µg/kg.
Pour doser les HAP dans les corps gras alimentaires, deux autres normes CEN sont également disponibles, l'EN ISO 22959 et l'EN ISO 15753, pour plus d'informations voir [1] et [2].
Analize živil - Določevanje benzo(a)pirena, benz(a)antracena, krizena in benzo(b)fluorantena v živilih s plinsko kromatografijo z masno spektrometrijo (GS-MS)
General Information
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittelanalytik - Bestimmung von Benzo[a]pyren, Benz[a]anthracen, Chrysen und Benzo[b]fluoranthen in Lebensmitteln mit Gaschromatographie und Massenspektrometrie (GC-MS)Analyse des produits alimentaires
- Dosage du benzo(a)pyrène, benzo(a)anthracène, chrysène et benzo(b)fluoranthène dans les denrées alimentaires par chromatographie en phase gazeuse couplée à la spectrométrie de masse (CG-SM)Food analysis - Determination of benzo[a]pyrene, benz[a]anthracene, chrysene and benzo[b]fluoranthene in foodstuffs by gas chromatography mass spectrometry (GC-MS)67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 16619:2015SIST EN 16619:2015en,fr,de01-julij-2015SIST EN 16619:2015SLOVENSKI
STANDARD
SIST EN 16619:2015
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16619
April 2015 ICS 67.050 English Version
Food analysis - Determination of benzo[a]pyrene, benz[a]anthracene, chrysene and benzo[b]fluoranthene in foodstuffs by gas chromatography mass spectrometry (GC-MS) Analyse des produits alimentaires
- Dosage du benzo(a)pyrène, benzo(a)anthracène, chrysène et benzo(b)fluoranthène dans les denrées alimentaires par chromatographie en phase gazeuse couplée à la spectrométrie de masse (CG-SM)
Lebensmittelanalytik - Bestimmung von Benzo[a]pyren, Benz[a]anthracen, Chrysen und Benzo[b]fluoranthen 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 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 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 versions.
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, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16619:2015 ESIST EN 16619:2015
EN 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 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
SIST EN 16619:2015
EN 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 text 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 responsible 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 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, the national 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, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 16619:2015
EN 16619:2015 (E) 4 1 Scope This European Standard specifies a method for the determination of 4 of the 16 EU priority polycyclic aromatic hydrocarbons (PAHs), identified as target PAHs. They are benz[a]anthracene (BaA), benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF) and chrysene (CHR). The method allows their quantification in the presence of the other 12 EU priority PAHs (benzo[j]fluoranthene (BjF), cyclopenta[cd]pyrene (CPP), benzo[k]fluoranthene (BkF), dibenz[a,h]anthracene (DhA), benzo[c]fluorene (BcL), dibenzo[a,e]pyrene (DeP), benzo[ghi]perylene (BgP), dibenzo[a,h]pyrene (DhP), dibenzo[a,i]pyrene (DiP), dibenzo[a,l]pyrene (DlP), indeno[1,2,3-cd]pyrene (IcP), 5-methylchrysene (5MC)) in extruded wheat flour, smoked fish, dry infant formula, 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 collaborative 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) No 836/2011 (see [1]). In particular the specifications for the limit of detection (LOD) and of the limit of quantification (LOQ) (0,30 µg/kg and 0,90 µg/kg respectively) were met. The method has been validated in an interlaboratory study via the analysis of both naturally contaminated and spiked samples, ranging from 0,5 µg/kg to 11,9 µg/kg. However, linearity of the instrument response was proven for the concentration range 0,5 µg/kg to 20 µg/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] 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 any 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 the 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 a small volume, filtered and purified by SEC, using a mixture of ethyl acetate and cyclohexane as eluent. After SEC, 200
SEC fraction. The SEC fraction is evaporated to about 200 µl, and cleaned up by SPE on silica, using cyclohexane as eluent. The cleaned up sample extract is evaporated again to about 200 µl. 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 column 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. SIST EN 16619:2015
EN 16619:2015 (E) 5 4 Reagents 4.1 General Use 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 commercial 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) are 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=ρ (1) where
density (in g/ml); m measured 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 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 chemical waste according to 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 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 oxide) granular, 90
size. 4.6 Sand, 50 mesh to 70 mesh particle size. 4.7 n-Hexane. SIST EN 16619:2015
EN 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 (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 columns 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/Eurachem 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 list 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, benzo[j]fluoranthene, and benzo[k]fluoranthene are potentially interfering with the target analytes and are therefore used for evaluation of selectivity. SIST EN 16619:2015
EN 16619:2015 (E) 7 Table 1 — Names and structures of the native PAHs Namea CAS number Structure Name a CAS number Structure Benz[a]anthracene (BaA) 56–55–3
Figure 1 Benzo[b]fluoranthene (BbF) 205–99–2
Figure 2 Benzo[a]pyrene (BaP) 50–32–8
Figure 3 Chrysene
(CHR) 218–01–9
Figure 4 Triphenylene (TRP) 217–59–4
Figure 5 Benzo[j]fluoranthene (BjF) 205–82–3
Figure 6 Benzo[k]fluoranthene (BkF) 207–08–9
Figure 7
a The acronym is given in parenthesis, the target analytes are given in bold. 4.16 Stable isotope 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 trial contained the stable isotope labelled PAHs at a level of about 100 µg/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 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 benz[a]anthracene-13C6, 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. SIST EN 16619:2015
EN 16619:2015 (E) 8 Table 2 — Names and structures of 13C labelled PAHs Name CAS number Structure Benz[a]anthracene-13C6 not available
and
Figure 8 Benzo[a]pyrene-13C4 not available
Figure 9 Benzo[b]fluoranthene-13C6 not available
Figure 10 Chrysene-13C6 not 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 native PAH and the stable isotope labelled analogue applied for its quantification. Table 3 — Correspondence stable isotope labelled PAHs and native PAHs Stable isotope labelled PAHs Native PAH a Benz[a]anthracene 13C6 Benz[a]anthracene (BaA) Benzo[a]pyrene 13C4 Benzo[a]pyrene (BaP) Benzo[b]fluoranthene 13C6 Benzo[b]fluoranthene (BbF) Chrysene 13C6 Chrysene (CHR) a The acronym is given in parenthesis. 4.17 9-fluorobenzo[k]fluoranthene (FBkF), as injection standard, neat or in form of a commercially available solution 9-fluorobenzo[k]fluoranthene (FBkF) is used as injection standard and added to the sample extract prior to injection into the GC-MS. Both neat material and commercially available solutions may be used. The concentration of a commercial solution is preferably 100 µg/kg in toluene. Table 4 — Name and structure of the injection standard Name a CAS number Structure 9-fluorobenzo[k]fluoranthene (FBkF) 113600–15–0
Figure 12 a The acronym is given in parenthesis. SIST EN 16619:2015
EN 16619:2015 (E) 9 5 Standard preparation 5.1 General All standard solutions are preferably prepared gravimetrically. The tare masses of all recipients and the masses after each preparation step are recorded and used for calculation of the standard concentrations. Volumetric preparation of standard solutions may be applied as well, provided that the used volumetric glassware complies with EN ISO 1042:1999. Standard solutions may be prepared from neat materials, from commercial single substance solutions, or from commercial mixes. However, compatibility of the solvents of commercial solutions with toluene shall be taken into account. Preferably, a commercial mixed PAH standard solution should be used for the preparation of intermediate standard solution (5.7) and calibration solutions (5.8). The concentration of this solution shall be in the same order of magnitude as the mixed PAH stock solution (5.4). 5.2 Injection standard solution Prepare a solution of FBkF (4.17) in toluene (4.10) with a concentration of approximately 400 ng/g. The conversion of mass-per-mass units (ng/g) to mass-per-volume units (ng/ml) is done via the density equation (Formula (1)). For toluene a density value of 0,866 9 g/ml is applied. This solution will be used for the spiking of the sample extract before measurement by GC-MS (see 7.9) to assess the recovery of stable isotope labelled PAHs (see 10.2). Store this solution in the dark and at a temperature below 10 °C. A solution stored in this way is stable for at least six months. If longer stability is proven, the solution can still be applied. 5.3 PAH stock solutions
A solution in toluene (4.10) with a concentration in the range from 50 µg/g to 150 µg/g shall be prepared for each of the native PAHs listed in Table 1 in case neat reference materials are applied for the preparation of calibrants. These PAH stock solutions are prepared by weighing from 1 mg to 5 mg of each neat substance into glass weighing cylinders (6.1) using the microbalance (6.4). The weighing cylinder (6.1) is transferred with tweezers into a 100 ml amber glass volumetric flask (6.2). About 40 ml of toluene is added and weighed with an analytical balance (6.5). Other amber glass recipients may be applied, provided that solvent evaporation is minimized during standard preparation. Each flask shall be sonicated for a couple of minutes to achieve complete dissolution of the native PAHs in the solvent. Once the solutions are homogeneous, they are transferred for storage into 40 ml amber glass vials (6.3). These solutions will be used for the preparation of mixed PAH stock solution (5.4) and, finally, of calibration solutions (see 5.8). Store these solutions in the dark and at a temperature below 10 °C. A solution stored in this way is stable for at least 12 months. If longer stability is proven, the solution can still be applied. 5.4 Mixed PAH stock solutions Prepare, from the PAH stock solutions (5.3), a solution in toluene (4.10) with a concentration of approximately 2 µg/g. For this purpose, both the PAH stock solutions (5.3) and the toluene (4.10) are weighed using an analytical balance (6.5). This solution will be used for the preparation of the intermediate standard solutions (5.7) and, finally, of the calibration solutions (see 5.8). SIST EN 16619:2015
EN 16619:2015 (E) 10 Store this solution in the dark, at a temperature below 10 °C. A solution stored in this way was proven to be stable for at least 12 months. If longer stability is proven, the solution can still be applied. 5.5 Labelled PAH stock solution Prepare with the individual solutions of stable isotope labelled PAHs (4.16) listed in Table 2, a solution in toluene (4.10) with a concentration of approximately 700 ng/g. Use an analytical balance (6.5) for this purpose. This solution will be used for the preparation of the process solution (5.6). Store this solution in the dark and at a temperature below 10 °C. A solution stored in this way is stable for at least 12 months. If longer stability is proven, the solution can still be applied. 5.6 Process solution Prepare, from the Labelled PAH stock solution (5.5), a solution in toluene (4.10) with a concentration of approximately 150 ng/g. This concentration is obtained by adding 4,5 ml of the labelled PAH stock solution (5.5) to 16 ml of toluene (4.10). The exact amounts of the two components are determined gravimetrically with an analytical balance (6.5). This solution will be used for spiking of the test portion (see 7.2). Store this solution in the dark and at a temperature below 10 °C. A solution stored in this way is stable for at least six months. If longer stability is proven, the solution can still be applied. 5.7 Intermediate solutions Prepare the intermediate solutions which will be used for calibration from the mixed PAH stock solution (5.4) by dilution in toluene (4.10). These solutions will be used for the preparation of the calibration solutions (see 5.8). The concentrations of PAHs in these solution shall be approximately two times the concentrations of PAHs in the calibration solutions (see 5.8), hence in the range from 10 ng/g to 250 ng/g. The required amounts of mixed PAH stock solution (5.4) are listed in Table 5. They are pipetted into a 100 ml amber volumetric flask (6.2) and made up to volume with toluene (4.10). The standard concentrations are calculated from gravimetrical data, which are recorded at each preparation step. Use an analytical balance (6.5) for weighing. SIST EN 16619:2015
EN 16619:2015 (E) 11 Table 5 — Nominal volumes of the mixed PAH stock solution (5.4) to be pipetted in order to prepare 100 ml of each of the listed concentration levels of the intermediate solutions Intermediate solution Nominal volume of mixed PAH stock solution (5.4) ml Nominal PAH a concentration in the intermediate solution ng/g b IS 1 0,5 10 IS 2 1,0 20 IS 3 2,5 50 IS 4 4,0 80 IS 5 5,5 110 IS 6 7,0 140 IS 7 8,5 170 IS 8 10,0 200 IS 9 12,5 250 a The concentration level refers to the individual target PAHs. b The given concentrations are indicative. The real concentrations shall be determined from gravimetrical data. Store these solutions in the dark and at a temperature below 10 °C. A solution stored in this way is stable for at least 12 months. If longer stability is proven, the solution can still be applied. 5.8 Calibration solutions Prepare the calibration solutions from the intermediate solutions (5.7), the process solution (5.6), and the injection standard solution (5.2). Record the tare weight of the applied GC autosampler vials. Pipette directly in the GC autosampler vials (6.21.4), approximately 500 µl of the intermediate solutions (IS 1 to IS 9) (5.7), approximately 400 µl of the process solution (5.6), and approximately 100 µl of the injection standard solution (5.2). Weigh all solutions with an analytical balance (6.5). Calculate the concentrations from the gravimetrical data. The concentration levels of calibration solutions are summarized in Table 6. Table 6 — Indicative compositions of the PAH calibration solutions Calibration solution PAHa concentration in the calibration solution ng/g b Corresponding to PAH a concentration in the sample
Stable isotope labelled PAH concentration in the calibration solution ng/g b Concentration of FBkF in the calibration solution
ng/gb CS 1 5 0,5 60,0 40,0 CS 2 10 1,0 60,0 40,0 CS 3 25 2,5 60,0 40,0 CS 4 40 4,0 60,0 40,0 CS 5 55 5,5 60,0 40,0 CS 6 70 7,0 60,0 40,0 CS 7 85 8,5 60,0 40,0 CS 8 100 10,0 60,0 40,0 CS 9 125 12,5 60,0 40,0 a The concentration level refers to the 4 target PAHs. b The final concentration shall be adjusted accordingly with the weighing and the exact concentration of the PAH in the mixed PAH stock solution (5.4). SIST EN 16619:2015
EN 16619:2015 (E) 12 5.9 Standard solution for assessment of chromatographic selectivity Prepare a standard solution in toluene containing all PAHs listed in Table 1 (including triphenylene, benzo[j]fluoranthene, and benzo[k]fluoranthene) at a concentration from 10 ng/g to 100 ng/g. This solution is used only for the characterization of the chromatographic separation (see 10.3). 6 Apparatus WARNING — All glassware shall be meticulously cleaned (except disposable glassware). The glassware is first thoroughly washed with laboratory detergent and hot water. All glassware used for the preparation and storage of standards (e.g. glass weighing cylinders (6.1) and amber volumetric flasks (6.2)) is rinsed before use with toluene (4.10) and dried in the fume hood under ambient conditions. Glassware used for other purposes (e.g. PLE solvent collection bottles (6.8.6)) is rinsed before use with cyclohexane and acetone (4.8) and dried either in the fume hood or in a drying cabinet. Usual laboratory glassware and equipment and, in particular, the following: 6.1 Glass cylinders, for weighing of neat PAHs, approximately of 1 ml volume, preferably made from amber glass. Such vials are not commercialised for this particular purpose. However, several suppliers offer neckless, flat bottom glass vials with an outer diameter of 8 mm and a length of about 30 mm to 40 mm, which are suitable for the weighing of small amounts of solid substance. 6.2 Amber glass volumetric flasks, of various volumes (5 ml to 100 ml), according to EN ISO 1042:1999. 6.3 Amber glass vials, 40 ml, with PTFE layered screw caps. 6.4 Micro-balance, accuracy to the nearest 0,000 001 g. 6.5 Analytical balance, accuracy to the nearest 0,000 01 g. 6.6 Laboratory balance, accuracy to the nearest 0,01 g. 6.7 Porcelain mortar and pestle, capacity of the mortar shall be at least 200 ml. 6.8 Pressurized liquid extraction (PLE) apparatus1) comprising the following: 6.8.1 PLE cells, of 33 ml volume.
6.8.2 Cellulose filters, 30 mm diameter. 6.8.3 Sample carousel. 6.8.4 Degasser. 6.8.5 Extraction chamber.
1) Dionex ASE® 300 is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of these products. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 16619:2015
EN 16619:2015 (E) 13 6.8.6 Solvent collection bottles, compatible with the PLE apparatus, capable of collecting about 50 ml extract, with light protection. 6.8.7 Pressure control device, for the supply and release of the pressurizing gas (4.3) in the extraction cell. 6.8.8 Temperature control device. 6.8.9 Instrument control and data processing system. 6.9 Soxhlet apparatus (alternatively to PLE apparatus) The apparatus comprises a heating mantle, a round bottom flask of 250 ml, a Soxhlet extractor of 85 ml capacity, a suitable cellulose thimble, a suitable condenser, and a cryostat or other cooling device. 6.10 Evaporation apparatus An evaporator capable of evaporation under controlled temperature and vacuum condition, or an alternative concentration workstation shall be used for the evaporation of extracts. The evaporation apparatus shall be equipped with, either round bottom flasks or glass tubes, of appropriate volumes: approximately 250 ml for the evaporation of PLE extracts (approximately 100 ml), and 100 ml for evaporation of the collected eluent of SEC (approximately 50 ml). 6.11 Glass Pasteur capillary pipettes, 230 mm length. 6.12 Glass test tubes, 10 ml capacity. 6.13 Glass syringe, luer tip, 10 ml capacity. 6.14 Polytetrafluoroethylene (PTFE) membrane filter, diameter of 25 mm and 5 µm pore size. 6.15 Size Exclusion Chromatography (SEC) apparatus, comprising the following: 6.15.1 Liquid pump, supplying a flow rate of 4,0 ml/min. 6.15.2 Evaporating device (optional). 6.15.3 Sample carousel, for vials of 10 ml capacity (optional). 6.15.4 Amber sample collection vials, about 5 ml capacity. 6.15.5 Manual or automated injection system, capable of injecting 5 ml. 6.15.6 SEC column 2), with the following characteristics: 50 g of styrene-
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