SIST-TS CEN/TS 16621:2014
(Main)Food analysis - Determination of benzo[a]pyrene, benz[a]anthracene, chrysene and benzo[b]fluoranthene in foodstuffs by high performance liquid chromatography with fluorescence detection (HPLC-FD)
Food analysis - Determination of benzo[a]pyrene, benz[a]anthracene, chrysene and benzo[b]fluoranthene in foodstuffs by high performance liquid chromatography with fluorescence detection (HPLC-FD)
CEN/TS 16621 specifies a method for the determination of benzo[a]pyrene (BaP) plus benz[a]anthracene (BaA), benzo[b]fluoranthene (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 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 μg/kg to 1,00 μg/kg and from 4,95 μg/kg to 23,53 μg/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 matrices 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 μg/kg. In addition, the method was tested in-house and shown to be applicable also for the quantification of the other 12 PAHs of the 15+1 EU priority PAHs set (benzo[c]fluorene (BcL), benzo[j]fluoranthene (BjF), benzo[k]fluoranthene (BkF), cyclopenta[cd]pyrene (CPP), dibenz[a,h]anthracene (DhA), 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 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 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 (see [1] and [2]).
Lebensmittelanalytik - Bestimmung von Benzo(a)pyren, Benz(a)anthracen, Chrysen und Benzo(b)fluoranthen in Lebensmitteln mittels Hochleistungs-Flüssigkeitschromatographie mit Fluoreszenzdetektion (HPLC-FD)
Die vorliegende Technische Spezifikation legt ein Verfahren zur Bestimmung von Benzo[a]pyren (BaP) plus Benz[a]anthracen (BaA), Benzo[b]fluoranthen (BbF) und Chrysen (CHR) in verschiedenen Lebensmittel¬matrices fest. Das Verfahren beruht auf der Reinigung mit Größenausschlusschromatographie (en: size exclusion chromatography, SEC) mit anschließender quantitativer Bestimmung mittels Hochleistungs-Flüssig-chromatographie (HPLC) mit programmierbarer Fluoreszenzdetektion. Dieses Verfahren wurde laborintern durch die Analyse aufgestockter Proben von Speiseöl, frischen Muscheln, geräuchertem Fisch, geräucherten Fleischerzeugnissen, verarbeiteten Lebensmitteln auf Getreidebasis (Getreidebeikost) für Kleinkinder, Säuglingsnahrung, Schokolade und Nahrungsergänzungsmitteln (Isoflavonen) bei Konzentrationen im Bereich von 0,25 μg/kg bis 1,00 μg/kg und von 4,95 μg/kg bis 23,53 g/kg, in Abhängigkeit vom polycyclischen aromatischen Kohlenwasserstoff (PAK) oder von der Matrix, validiert.
Das Verfahren hat sich als anwendbar auf eine Vielzahl weiterer Matrices erwiesen, wie z. B. Fleisch-erzeugnisse, frischen Fisch, Paprika, Röstkaffee, Brot, Kräuter, Frühstückszerealien, Bier, Sonnenblumenöl, Oliven und gebratene Tomaten, bei einer Bestimmungsgrenze von weniger als 0,5 µg/kg.
Zusätzlich wurde das Verfahren laborintern geprüft und erwies sich auch für die quantitative Bestimmung der anderen 12 PAK der 15 1 von der EU als prioritär eingestuften PAK (Benzo[c]fluoren (BcL), Benzo[j]fluoranthen (BjF), Benzo[k]fluoranthen (BkF), Cyclopenta[cd]pyren (CPP), Dibenz[a,h]anthracen (DhA), 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 allen vorstehend aufgeführten Matrices und bei ähnlichen Konzentrationsbereichen als anwendbar, mit Ausnahme von CPP, bei dem eine UV Detektion anzuwenden war, mit Bestimmungsgrenzen von mehr als 8 µg/kg.
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 [1] und [2].
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 liquide à haute performance avec détection de fluorescence (HPLC-FD)
La présente Spécification technique décrit une méthode de dosage du benzo[a]pyrène (BaP) ainsi que du benzo[a]anthracène (BaA), benzo[b]fluoranthène (BbF) et chrysène (CHR) dans plusieurs matrices d’aliments. La méthode repose sur la purification par chromatographie d’exclusion stérique (SEC), suivie de la quantification par chromatographie en phase liquide à haute performance (HPLC) avec détection de fluorescence programmable. Cette méthode a été validée en interne par le biais de l’analyse d’échantillons dopés d’huile d’olive alimentaire, de moules fraîches, de poisson fumé, de produits à base de viande fumée, de produits alimentaires à base de céréales pour jeunes enfants, de formules infantiles, de chocolat et de compléments alimentaires (isoflavones) à des niveaux de 0,25 μg/kg à 1,00 μg/kg et de 4,95 μg/kg à 23,53 g/kg, en fonction de l’hydrocarbure aromatique polycyclique (HAP) ou de la matrice.
La méthode s’est avérée adaptée à un grand nombre d’autres matrices telles que les produits à base de viande, le poisson frais, le paprika, le café torréfié, le pain, les herbes aromatiques, les céréales pour petit-déjeuner, la bière, l’huile de tournesol, les olives et la tomate frite, avec une limite de quantification inférieure à 0,5 µg/kg.
De plus, la méthode a été soumise à un essai en interne et s’est révélée appropriée à la quantification de 12 autres HAP sur les 15+1 HAP prioritaires de l’UE (benzo[c]fluorène (BcL), benzo[j]fluoranthène (BjF), benzo[k]fluoranthène (BkF), cyclopenta[cd]pyrène (CPP), dibenzo[a,h]anthracène (DhA), 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 toutes les matrices indiquées ci-dessus et à des niveaux similaires, hormis pour le CPP, où une détection UV a dû être utilisée avec des limites de quantification supérieures à 8 µg/kg.
Pour doser les HAP dans les corps gras alimentaires, deux autres normes 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 tekočinsko kromatografijo visoke ločljivosti s fluorescentno detekcijo (HPLC-FD)
Standard CEN/TS 16621 navaja metodo za določevanje benzo[a]pirena (BaP), benz[a]antracena (BaA), benzo[b]fluorantena (BbF) in krizena (CHR) v različnih živilskih matricah. Metoda je osnovana na kromatografiji z ločevanjem po velikosti (SEC), ki ji sledi kvantifikacija s tekočinsko kromatografijo visoke ločljivosti (HPLC) s programabilno fluorescentno detekcijo. Ta metoda je bila interno validirana prek analize primešanih vzorcev jedilnega olivnega olja, svežih klapavic, prekajene ribe, prekajenih mesnih izdelkov, procesirane hrane za otroke na osnovi žitaric, formul za dojenčke, čokolade in prehranskih nadomestil (izoflavonov) v količinah od 0,25 μg/kg do 1,00 μg/kg in od 4,95 μg/kg do 23,53 μg/kg, odvisno od policikličnega aromatskega ogljikovodika (PAH) ali matrice. Ta metoda je v skladu z lastnostmi, ki so določene za BaP, BaA, BbF in CHR v veljavni zakonodaji [3]. Ta metoda je ustrezna za številne druge matrice – mesne izdelke, sveže ribe, papriko, praženo kavo, kruh, zelišča, žitarice, pivo, sončnično olje in pečene paradižnike – z mejo kvantifikacije pod 0,5 μg/kg. Z internimi testi se je metoda izkazala kot ustrezna tudi za kvantifikacijo drugih 12 policikličnih aromatskih ogljikovodikov izmed 15 + 1 prednostnih policikličnih aromatskih ogljikovodikov, ki jih je določila EU (benzo[c]fluoren (BcL), benzo[j]fluoranten (BjF), benzo[k]fluoranten (BkF), ciklopenta[cd]piren (CPP), dibenz[a,h]antracen (DhA), dibenzo[a,e]piren (DeP), benzo[ghi]perilen (BgP), dibenzo[a,h]piren (DhP), dibenzo[a,i]piren (DiP), dibenzo[a,l]piren (DlP), indeno[1,2,3-cd]piren (IcP), 5-metilkrizen (5MC)) v vseh zgoraj navedenih matrikah v podobnih mejah, razen za CPP, kjer je bilo treba uporabiti zaznavanje UV z mejami kvantifikacije nad 8 μg/kg. Za določevanje policikličnih aromatičnih ogljikovodikov v jedilnih maščobah in oljih sta na voljo dva dodatna standarda – EN ISO 22959 in EN ISO 15753 (glej [1] in [2]).
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN/TS 16621:2014
01-julij-2014
$QDOL]HåLYLO'RORþHYDQMHEHQ]R>D@SLUHQDEHQ]>D@DQWUDFHQDNUL]HQDLQEHQ]R>E@
IOXRUDQWHQDYåLYLOLKVWHNRþLQVNRNURPDWRJUDILMRYLVRNHORþOMLYRVWLVIOXRUHVFHQWQR
GHWHNFLMR +3/&)'
Food analysis - Determination of benzo[a]pyrene, benz[a]anthracene, chrysene and
benzo[b]fluoranthene in foodstuffs by high performance liquid chromatography with
fluorescence detection (HPLC-FD)
Lebensmittelanalytik - Bestimmung von Benzo(a)pyren, Benz(a)anthracen, Chrysen und
Benzo(b)fluoranthen in Lebensmitteln mittels Hochleistungs-Flüssigkeitschromatographie
mit Fluoreszenzdetektion (HPLC-FD)
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 liquide à haute performance avec détection de fluorescence (HPLC-FD)
Ta slovenski standard je istoveten z: CEN/TS 16621:2014
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST-TS CEN/TS 16621:2014 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
SIST-TS CEN/TS 16621:2014
---------------------- Page: 2 ----------------------
SIST-TS CEN/TS 16621:2014
TECHNICAL SPECIFICATION
CEN/TS 16621
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
April 2014
ICS 67.050
English Version
Food analysis - Determination of benzo[a]pyrene,
benz[a]anthracene, chrysene and benzo[b]fluoranthene in
foodstuffs by high performance liquid chromatography with
fluorescence detection (HPLC-FD)
Analyse des produits alimentaires - Dosage du Lebensmittelanalytik - Bestimmung von Benzo[a]pyren,
benzo(a)pyrène, benzo(a)anthracène, chrysène et Benz[a]anthracen, Chrysen und Benzo[b]fluoranthen in
benzo(b)fluoranthène dans les denrées alimentaires par Lebensmitteln mittels Hochleistungs-
chromatographie en phase liquide à haute performance Flüssigkeitschromatographie mit Fluoreszenzdetektion
avec détection de fluorescence (HPLC-FD) (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 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 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, 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
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 16621:2014 E
worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST-TS CEN/TS 16621:2014
CEN/TS 16621:2014 (E)
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 (informative) 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
2
---------------------- Page: 4 ----------------------
SIST-TS CEN/TS 16621:2014
CEN/TS 16621:2014 (E)
Foreword
This document (CEN/TS 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 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 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, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
3
---------------------- Page: 5 ----------------------
SIST-TS CEN/TS 16621:2014
CEN/TS 16621:2014 (E)
1 Scope
This Technical Specification specifies a method for the determination of benzo[a]pyrene (BaP) plus
benz[a]anthracene (BaA), benzo[b]fluoranthene (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 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 μg/kg to 1,00 μg/kg and from 4,95 μg/kg to 23,53 µg/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 matrices 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 µg/kg.
In addition, the method was tested in-house and shown to be applicable also for the quantification of the other
12 PAHs of the 15+1 EU priority PAHs set (benzo[c]fluorene (BcL), benzo[j]fluoranthene (BjF),
benzo[k]fluoranthene (BkF), cyclopenta[cd]pyrene (CPP), dibenz[a,h]anthracene (DhA), 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 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 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 (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 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. 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 otherwise 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 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.
4
---------------------- Page: 6 ----------------------
SIST-TS CEN/TS 16621:2014
CEN/TS 16621:2014 (E)
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 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 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 compressed 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 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 Polycyclic aromatic hydrocarbons.
4.17.1 Benzo[a]pyrene (BaP).
4.17.2 Chrysene (CHR).
4.17.3 Benzo[b]fluoranthene (BbF).
5
---------------------- Page: 7 ----------------------
SIST-TS CEN/TS 16621:2014
CEN/TS 16621:2014 (E)
4.17.4 Benz[a]anthracene (BaA).
4.17.5 Benzo[k]fluoranthene (BkF).
4.17.6 Dibenzo[a,h]anthracene (DhA).
4.17.7 Benzo[g,h,i]perylene (BgP).
4.17.8 Indeno[1,2,3-cd]pyrene (IcP).
4.17.9 Benzo[c]fluorene (BcL).
4.17.10 Cyclopenta[c,d]pyrene (CPP).
4.17.11 5 – Methylchrysene (5MC).
4.17.12 Benzo[j]fluoranthene (BjF).
4.17.13 Dibenzo[a,l]pyrene (DlP).
4.17.14 Dibenzo[a,e]pyrene (DeP).
4.17.15 Dibenzo[a,i]pyrene (DiP).
4.17.16 Dibenzo[a,h]pyrene (DahP).
4.17.17 15+1 EU priority PAHs standard solution containing 10 μg/ml each, in appropriate organic
solvent, preferably acetonitrile.
4.18 PAH4 standard solution
Prepare a standard solution of 10 μg/ml PAH4 in acetonitrile by weighing carefully the proper amounts of the 4
PAHs (BaP, BaA, BbF and CHR) individually in acetonitrile. 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 µl of the 10 μg/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.
4.20 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 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 µl of the 10 μg/ml 15+1 PAHs
standard solution (4.17.17) into a calibrated 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
applied.
6
---------------------- Page: 8 ----------------------
SIST-TS CEN/TS 16621:2014
CEN/TS 16621:2014 (E)
In case of commercial availability of 15+1 EU priority PAHs standard solution containing 10 μg/ml each, in
acetonitrile, the 15+1 PAHs stock solution can be prepared directly by diluting 500 µl of that solution up to
10 ml with acetonitrile.
4.22 15+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 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, 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 nearest 0,1 g.
5.7 Glass syringe, of 5 ml capacity.
5.8 Microsyringes, of 250 μl, 500 μl and 1 000 μl capacity.
5.9 Calibrated volumetric flasks, of 5 ml and 10 ml capacity.
5.10 Displacement pipettes, of 200 μl, with appropriate tips.
5.11 Glass vials, approximately 1,8 ml capacity and crimp caps.
5.12 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 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 µm.
5.13.4 UV detector, capable to provide λ = 254 nm.
5.13.5 Fraction collector.
5.13.6 Recorder, integrator or computer based data processing system.
5.13.7 Calibration
7
---------------------- Page: 9 ----------------------
SIST-TS CEN/TS 16621:2014
CEN/TS 16621:2014 (E)
Connect the columns to the system and calibrate the SEC cleanup columns following the instructions of the
manufacturer. The calibration solution requires the following compounds: corn oil, methoxychlor, perylene and
sulfur. The system calibration will determine the time spent from the injection of the sample to the elution of
PAHs by using perylene as indicator. The fraction or fractions containing the PAHs can be selected by
programming the fractions collector.
NOTE Other SEC columns can be used provided that the SEC system, after calibration, is able to include the PAHs
in the selected fraction or fractions, with acceptable recovery rates and free of disturbing interferences.
5.14 HPLC apparatus, comprising the following:
5.14.1 Injection system, suitable for 100 µl injection volume.
5.14.2 Mobile phase pump (gradient), capable of pumping 1 ml/min pulse free.
5.14.3 Programmable fluorescence detector.
5.14.4 Computer based data processing system.
5.14.5 Analytical reverse-phase HPLC specific for PAHs, separating column, C18, base deactivated
octadecyl silane (ODS) (recommended 4,6 mm x 250 mm column with 5 µm particle size), and a suitable
corresponding reverse phase guard column.
5.14.6 Degasser, (optional).
5.14.7 Column oven, capable to operate at 28 °C ± 1 °C.
5.14.8 UV detector, capable to provide λ = 223 nm.
5.15 PTFE filters, 0,20 µm or 0,45 µm.
5.16 Glass tubes, of 10 ml capacity, suitable for the evaporator (5.4) and the fraction collector (5.13.5).
5.17 Glass tubes, of 10 ml capacity, capped.
5.18 Grinding mill.
6 Procedure
6.1 Sample preparation
In general, only the edible parts of foodstuffs will be analysed. The edible parts of shellfish shall be thoroughly
washed with water and dried by slightly pressing with filter papers. The edible portions to be analysed shall be
ground and well mixed. They can be stored in closed glass or aluminium containers under frozen conditions
up to the moment of their analysis.
6.2 Extraction
Weigh, to the nearest 0,1 g, a 25 g test portion of prepared sample into a 250 ml-round bottomed glass tube,
(5.3). Add 100 ml of dichloromethane (4.5), (V , see Clause 8). Blend for 3 min with a high speed blender
1
(5.1).
Filter an aliquot of the lower organic layer through a prefolded paper filter. The filtered extract shall be clear,
otherwise repeat this step with some anhydrous sodium sulphate (4.16) in the filter. Filter an aliquot of the
filtered extract through a 0,20 μm or 0,45 μm PTFE filter (5.15), using a 5 ml glass syringe (5.7) and purify by
SEC.
8
---------------------- Page: 10 ----------------------
SIST-TS CEN/TS 16621:2014
CEN/TS 16621:2014 (E)
In the case of samples of edible oils, the extraction step is replaced by just a dispersion of the sample in
dichloromethane as follows: Pour a 5 ml portion of oil in a previously tared glass tube (5.17) and take note of
the sample weight to the nearest 0,1 g. Add 5 ml of dichloromethane (4.5), cap the tube and mix once by
inversion. After that, mix by stirring one min in vortex mixer (5.1) and filter an aliquot of the mixture through a
0,20 µm or 0,45 µm PTFE filter (5.15
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.