SIST EN ISO 15753:2016
(Main)Animal and vegetable fats and oils - Determination of polycyclic aromatic hydrocarbons (ISO 15753:2016)
Animal and vegetable fats and oils - Determination of polycyclic aromatic hydrocarbons (ISO 15753:2016)
This International Standard describes two methods for the determination of 15 polycyclic aromatic
hydrocarbons (PAHs) in animal and vegetable fats and oils:
— a general method;
— a specific method for coconut oil and vegetable oils with short-chain fatty acids.
These methods are not quantitative for the very volatile compounds such as naphthalene, acenaphthene
and fluorene. Due to interferences provided by the matrix itself, palm oil and olive pomace oil cannot be
analysed using this method.
The quantification limit is 0,2 μg/kg for almost all compounds analysed, except for fluoranthene and
benzo(g,h,i)perylene, where the quantification limit is 0,3 μg/kg, and indeno(1,2,3-c,d)pyrene, where
the quantification limit is 1,0 μg/kg.
NOTE The results for olive pomace oil in Annex B show that this method is not applicable to this type of oil.
The precision data determined are very poor.
Milk and milk products (or fat coming from milk and milk products) are excluded from the scope of this
International Standard.
Tierische und pflanzliche Fette und Öle - Bestimmung von polycyclischen aromatischen Kohlenwasserstoffen (ISO 15753:2016)
Diese Internationale Norm beschreibt zwei Verfahren zur Bestimmung von 15 polycyclischen aromatischen Kohlenwasserstoffen (PAKs) in tierischen und pflanzlichen Fetten und Ölen:
— ein allgemeines Verfahren;
— ein spezielles Verfahren für Kokosfett und Pflanzenöle mit kurzkettigen Fettsäuren.
Diese Verfahren sind bei leicht flüchtigen Verbindungen, wie z. B. Naphthalin, Acenaphthen und Fluoren nicht quantitativ. Aufgrund von Störungen, die durch die Matrix selbst bedingt sind, können Palmöl und Oliven¬tresteröl nicht mit diesem Verfahren analysiert werden.
Die Bestimmungsgrenze beträgt bei fast allen analysierten Verbindungen 0,2 μg/kg, mit Ausnahme von Fluoranthen und Benzo(g,h,i)perylen, deren Bestimmungsgrenze bei 0,3 μg/kg liegt, sowie Indeno(1,2,3 c,d)pyren mit einer Bestimmungsgrenze von 1,0 μg/kg.
ANMERKUNG Die Ergebnisse für Oliventresteröl in Anhang B zeigen, dass dieses Verfahren nicht für diese Ölart anwendbar ist. Die ermittelten Präzisionsdaten sind sehr schlecht.
Milch und Milcherzeugnisse (oder Fett aus Milch und Milcherzeugnissen) sind vom Anwendungsbereich dieser Internationalen Norm ausgenommen.
Corps gras d'origines animale et végétale - Détermination des hydrocarbures aromatiques polycycliques (ISO 15753:2016)
ISO 15753:2016 décrit deux méthodes pour la détermination de 15 hydrocarbures aromatiques polycycliques (HAP) dans les corps gras d'origines animale et végétale:
? une méthode générale;
? une méthode spécifique pour l'huile de coprah et les huiles végétales avec acides gras à chaînes courtes.
Ces méthodes ne sont pas quantitatives pour les composés très volatils tels que le naphtalène, l'acénaphtène et le fluorène. En raison des interférences causées par la matrice elle-même, l'huile de palme et l'huile de grignons d'olive ne peuvent pas être analysées par cette méthode.
La limite de quantification est de 0,2 µg/kg pour la majorité des composés analysés, à l'exception du fluoranthène et du benzo(g,h,i)pérylène, dont la limite de quantification est de 0,3 µg/kg, et de l'indéno(1,2,3-c,d)pyrène, dont la limite de quantification est de 1,0 µg/kg.
NOTE Les résultats pour l'huile de grignons d'olive dans l'Annexe B montrent que cette méthode n'est pas applicable à ce type d'huile. Les données de fidélité déterminées sont très médiocres.
Le lait et les produits laitiers (ou la graisse provenant du lait et des produits laitiers) sont exclus du domaine d'application de la présente Norme internationale.
Rastlinske in živalske maščobe in olja - Določevanje policikličnih aromatskih ogljikovodikov (ISO 15753:2016)
Ta mednarodni standard opisuje dve metodi za določevanje 15 policikličnih aromatskih
ogljikovodikov (PAH) v živalskih in rastlinskih maščobah in oljih:
– splošno metodo;
– posebno metodo za kokosovo olje in rastlinska olja z maščobnimi kislinami s kratko verigo.
Ti metodi nista kvantitativni za zelo hlapljive spojine, kot so naftalen, acenaften in fluoren. Zaradi motenj, ki jih povzroča sama matrika, s to metodo ni mogoče analizirati palmovega olja in olja iz oljčnih tropin.
Meja kvantifikacije je 0,2 μg/kg pri skoraj vseh analiziranih spojinah, razen fluorantena in benzo(g,h,i)perilena, kjer je meja kvantifikacije 0,3 μg/kg, ter indeno(1,2,3-c,d)pirena, kjer je meja kvantifikacije 1,0 μg/kg.
OPOMBA: Rezultati za olja iz oljčnih tropin iz dodatka B kažejo, da se ta metoda ne uporablja za to vrsto olja. Pridobljeni podatki o natančnosti so zelo slabi.
Mleko in mlečni izdelki (ali maščoba iz mleka in mlečnih izdelkov) niso zajeti v tem mednarodnem standardu.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 15753:2016
01-junij-2016
1DGRPHãþD
SIST EN ISO 15753:2007
SIST EN ISO 15753:2007/A1:2011
5DVWOLQVNHLQåLYDOVNHPDãþREHLQROMD'RORþHYDQMHSROLFLNOLþQLKDURPDWVNLK
RJOMLNRYRGLNRY ,62
Animal and vegetable fats and oils - Determination of polycyclic aromatic hydrocarbons
(ISO 15753:2016)
Tierische und pflanzliche Fette und Öle - Bestimmung von polycyclischen aromatischen
Kohlenwasserstoffen (ISO 15753:2016)
Corps gras d'origines animale et végétale - Détermination des hydrocarbures
aromatiques polycycliques (ISO 15753:2016)
Ta slovenski standard je istoveten z: EN ISO 15753:2016
ICS:
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
SIST EN ISO 15753:2016 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 15753:2016
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SIST EN ISO 15753:2016
EN ISO 15753
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2016
EUROPÄISCHE NORM
ICS 67.200.10 Supersedes EN ISO 15753:2006
English Version
Animal and vegetable fats and oils - Determination of
polycyclic aromatic hydrocarbons (ISO 15753:2016)
Corps gras d'origines animale et végétale - Tierische und pflanzliche Fette und Öle - Bestimmung
Détermination des hydrocarbures aromatiques von polycyclischen aromatischen Kohlenwasserstoffen
polycycliques (ISO 15753:2016) (ISO 15753:2016)
This European Standard was approved by CEN on 11 March 2016.
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
© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 15753:2016 E
worldwide for CEN national Members.
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SIST EN ISO 15753:2016
EN ISO 15753:2016 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 15753:2016
EN ISO 15753:2016 (E)
European foreword
This document (EN ISO 15753:2016) has been prepared by Technical Committee ISO/TC 34 “Food
products” in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats
and oils and their by-products - Methods of sampling and analysis” the secretariat of which is held by
AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by October 2016, and conflicting national standards shall
be withdrawn at the latest by October 2016.
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 supersedes EN ISO 15753:2006.
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.
Endorsement notice
The text of ISO 15753:2016 has been approved by CEN as EN ISO 15753:2016 without any modification.
3
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SIST EN ISO 15753:2016
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SIST EN ISO 15753:2016
INTERNATIONAL ISO
STANDARD 15753
Second edition
2016-04-01
Animal and vegetable fats and oils —
Determination of polycyclic aromatic
hydrocarbons
Corps gras d’origines animale et végétale — Détermination des
hydrocarbures aromatiques polycycliques
Reference number
ISO 15753:2016(E)
©
ISO 2016
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SIST EN ISO 15753:2016
ISO 15753:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
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SIST EN ISO 15753:2016
ISO 15753:2016(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents and materials . 2
6 Apparatus . 3
7 Sampling . 4
8 Sample preparation . 5
9 Procedure for determination of PAHs from fats and oils: General method .5
9.1 Preliminary remarks . 5
9.2 Blank . 5
9.3 Determination of recovery values (without matrix) . 5
9.4 Extraction (liquid/liquid extraction) . 5
9.5 Purification on C18-bonded phase cartridge (solid/liquid extraction). 6
9.6 Purification on Florisil-bonded phase cartridge (solid/liquid extraction). 7
10 Procedure for determination of PAHs from fats and oils: Method specific for coconut oil .8
10.1 First extraction (liquid/liquid extraction) . 8
10.2 Second extraction (liquid/liquid extraction) . 8
10.3 Purification on C18-bonded phase cartridge (solid/liquid extraction). 8
10.4 Purification on Florisil-bonded phase cartridge (solid/liquid extraction). 9
11 High-performance liquid chromatography (HPLC) .10
11.1 Operating conditions .10
11.2 Detection parameters .10
11.3 Analysis of samples and standards .12
11.4 Confirmation of the presence of PAHs .12
12 Expression of results .12
13 Precision .13
13.1 Interlaboratory test.13
13.2 Repeatability .13
13.3 Reproducibility .13
14 Test report .13
Annex A (informative) Recovery values, flow charts, chromatograms and injection sequences .15
Annex B (informative) Results of the interlaboratory test .20
Bibliography .23
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SIST EN ISO 15753:2016
ISO 15753:2016(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 11, Animal
and vegetable fats and oils.
This second edition cancels and replaces the first edition (ISO 15753:2006), of which it constitutes a
minor revision. It also incorporates Amendment ISO 15753:2006/Amd.1:2011. A non-applicability
statement for milk and milk products has been added to the Scope.
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SIST EN ISO 15753:2016
INTERNATIONAL STANDARD ISO 15753:2016(E)
Animal and vegetable fats and oils — Determination of
polycyclic aromatic hydrocarbons
1 Scope
This International Standard describes two methods for the determination of 15 polycyclic aromatic
hydrocarbons (PAHs) in animal and vegetable fats and oils:
— a general method;
— a specific method for coconut oil and vegetable oils with short-chain fatty acids.
These methods are not quantitative for the very volatile compounds such as naphthalene, acenaphthene
and fluorene. Due to interferences provided by the matrix itself, palm oil and olive pomace oil cannot be
analysed using this method.
The quantification limit is 0,2 µg/kg for almost all compounds analysed, except for fluoranthene and
benzo(g,h,i)perylene, where the quantification limit is 0,3 µg/kg, and indeno(1,2,3-c,d)pyrene, where
the quantification limit is 1,0 µg/kg.
NOTE The results for olive pomace oil in Annex B show that this method is not applicable to this type of oil.
The precision data determined are very poor.
Milk and milk products (or fat coming from milk and milk products) are excluded from the scope of this
International Standard.
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.
ISO 661, Animal and vegetable fats and oils — Preparation of test sample
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
polycyclic aromatic hydrocarbon
PAH
compound that contains two or more condensed (fused) aromatic hydrocarbon rings and the content of
which can be determined according to the method specified in this International Standard
Note 1 to entry: The content is given in micrograms per kilogram.
Note 2 to entry: In general, PAHs are divided into light PAHs with two to four aromatic rings, and heavy PAHs
with five or more aromatic rings.
EXAMPLE Light PAHs include:
naphthalene (CAS RN [91-20-3]), acenaphthene (CAS RN [83-32-9]), acenaphthylene (CAS RN [208-96-8]),
fluorene (CAS RN [86-73-7]), anthracene (CAS RN [120-12-7]), phenanthrene (CAS RN [85-01-8]), fluoranthene
(CAS RN [206-44-0]), chrysene (CAS RN [218-01-9]), benz(a)anthracene (CAS RN [56-55-3]), pyrene
(CAS RN [129-00-0]).
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SIST EN ISO 15753:2016
ISO 15753:2016(E)
Heavy PAHs include:
benzo(a)pyrene (CAS RN [50-32-8]), benzo(b)fluoranthene (CAS RN [205-99-2]), benzo(k)fluoranthene
(CAS RN [207-08-9]), benzo(g,h,i)perylene (CAS RN [191-24-2]), dibenz(a,h)anthracene (CAS RN [53-70-3]),
indeno(1,2,3-c,d)pyrene (CAS RN [193-39-5]).
4 Principle
The polycyclic aromatic hydrocarbons are extracted with an acetonitrile/acetone mixture followed by
purification on C18 reversed-phase and then Florisil bonded-phase cartridges. Determination of the
content of the individual polycyclic aromatic hydrocarbons after separation is achieved by means of
high-pressure liquid chromatography (HPLC) and by measuring the fluorescence at various excitation
and emission wavelengths.
5 Reagents and materials
WARNING — Attention is drawn to the regulations governing the handling of dangerous matter.
Technical, organizational and personal safety measures should be followed.
Use only reagents of recognized analytical grade unless otherwise stated.
Check the quality of solvents before use by concentrating the solvent about 1 000 times by evaporation
and analysing the concentrate by HPLC (300 ml to 300 µl). The chromatogram shall be free from peaks
in the elution area of PAHs.
1)
5.1 Methanol, “ultra resi-analysed” grade.
1)
5.2 Hexane, HPLC grade.
1)
5.3 Acetonitrile, HPLC grade.
1)
5.4 Acetone, HPLC grade.
1)
5.5 Dichloromethane, HPLC grade.
1)
5.6 Toluene, HPLC grade.
1)
5.7 Water, HPLC grade.
1)
5.8 Tetrahydrofuran, HPLC grade.
5.9 Solvent mixture 1: acetonitrile/acetone (volume fraction 60 %/40 %).
Quantity used per sample: 41 ml for general method, 36 ml for specific method for coconut oil.
5.10 Solvent mixture 2: acetonitrile/acetone (volume fraction 80 %/20 %).
Quantity used per sample: 2 × 11 ml for method specific for coconut oil.
5.11 Solvent mixture 3: hexane/dichloromethane (volume fraction 75 %/25 %).
Quantity used per sample: 7 ml for general method, 2 × 7 ml for method specific for coconut oil.
1) These can be obtained from, for example, Baker. The information given in the footnotes is for the convenience of
users of this document and does not constitute an endorsement by ISO of these products. Equivalent products may
be used if they can be shown to lead to the same results.
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SIST EN ISO 15753:2016
ISO 15753:2016(E)
5.12 Mixture of tetrahydrofuran/methanol (volume fraction 50 %/50 %).
2)
5.13 Standard solution with 16 certified EPA Priority PAHs in toluene, at a concentration of
100 µg/ml (100 mg/l): naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene,
fluoranthene, pyrene, benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene,
benzo(a)pyrene, dibenz(a,h)anthracene, benzo(g,h,i)perylene, indeno(1,2,3-c,d)pyrene.
NOTE 1 This is stored at -20 °C.
Before use, allow the solution to warm up to ambient temperature for at least 1 h.
NOTE 2 Acenaphthylene is not fluorescent and, thus, it cannot be determined by these methods.
5.14 Stock standard solution, 200 ng/ml (200 µg/l).
Add 100 µl of standard solution (5.13) with a 250 µl syringe (6.11) to a 50 ml volumetric flask (6.20) and
dilute to the mark with acetonitrile.
5.15 Working standard solution, 50 ng/ml (50 µg/l).
Add 250 µl of stock standard solution (5.14) with a 250 µl syringe (6.11) to 750 µl of THF/methanol
mixture (5.12) or acetonitrile (5.3).
3)
5.16 C18 bonded-phase cartridges, 2 g phase, 12 ml capacity.
3)
5.17 Florisil bonded-phase cartridges, 500 mg phase, 3 ml capacity.
5.18 Stream of nitrogen, pressure regulated at 34,5 kPa (5 psi, about 1,5 l/min).
6 Apparatus
Usual laboratory apparatus and, in particular, the following.
The use of disposable glass tubes is acceptable. The general use of glass is necessary as plastics can
contain PAHs.
−1
6.1 Centrifuge, capable of attaining at least 4 000 min , suitable for 100 ml and 10 ml tubes.
6.2 HPLC system with binary gradient elution, with solvent reservoir of 1 l capacity, a mobile
phase liner filter, pump, autosampler, column temperature regulation set at 25 °C, fluorescence detector
programmable over time for various excitation and emission wavelengths, and computer-assisted
acquisition and data treatment.
4)
6.3 C18 reversed-phase column, 250 mm in length, 4,6 mm internal diameter, 5 µm particles,
suitable for PAH analysis.
6.4 Vortex mixer.
2) This can be obtained from, for example, Promochem.
3) This can be obtained from, for example, Varian.
4) This can be obtained from, for example, Vydac, ref. 201TP54.
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ISO 15753:2016(E)
5)
6.5 Automatic evaporator, for 10 ml tube (optional), or water bath (6.6).
Recommended operating conditions:
— temperature of water bath 35 °C;
— nitrogen pressure 34,5 kPa.
6.6 Water bath, regulated at 35 °C.
6.7 Balance, with readability of 0,1 mg.
6.8 Centrifuge tubes, of 100 ml capacity (one per sample).
6.9 Conical centrifuge tubes, of 11 ml capacity (three per sample), with PTFE septa and closed top
screw caps (one per sample).
[6]
6.10 Graduated measuring cylinders, ISO 4788 , class A.
6.11 Microsyringe, 250 µl.
6.12 Syringe, 1 000 µl.
[4]
6.13 Graduated pipette, capacity 5 ml, ISO 835 , class A.
6.14 Syringe, 5 ml, equipped with an adapter cap for SPE cartridges.
6.15 Vials for autosampler.
6.16 Microvials, of 250 µl capacity, adapted for HPLC system.
6.17 Ultrasonic bath, with water temperature not higher than 40 °C.
[7]
6.18 Pasteur pipettes, with cotton wool in the top part to prevent contamination, ISO 7712 .
6)
6.19 Device composed of stand and pincers, to hold SPE cartridges or, if available, an automatic SPE
work station.
NOTE Depending on the SPE sample processing station used, the proposed extraction methods may require
slight adaptations (times, pressure, volumes).
[5]
6.20 One-mark volumetric flask, capacity 50 ml, ISO 1042 , class A.
7 Sampling
A representative sample should have been sent to the laboratory. It should not have been damaged or
changed during transport or storage.
Sampling is not part of the method specified in this International Standard. A recommended sampling
method is given in ISO 5555.
5) This can be obtained from, for example, Zymark, Zymark TurboVap LV evaporator.
6) This can be obtained from, for example, Zymark, Zymark Rapid Trace.
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8 Sample preparation
Prepare the test sample in accordance with the method given in ISO 661. Before sampling, the liquid
samples shall be at room temperature and homogenized by magnetic agitation.
Sample the solid matrix by melting the entire sample or by melting and homogenizing several core
samples.
9 Procedure for determination of PAHs from fats and oils: General method
9.1 Preliminary remarks
In order to obtain repeatable results, the ambient temperature of the laboratory shall be regulated
(≤20 °C). This is a very important condition for the extraction of PAHs from coconut oil (or vegetable
oils containing short-chain fatty acids). These oils contain fatty acids with short and long chains; when
the ambient temperature is higher than 20 °C, the solubility of short-chain fatty acids increases.
Before use, rinse the whole vessel three times with hexane (5.2).
Each sequence of samples shall include a blank (9.2), and a standard solution extracted under the same
conditions as the sample in order to calculate the recovery values of the extraction (9.3). The recovery
values shall be within the range 70 % to 110 %. The mean recovery values are given in Table A.1.
For a quantitative analysis, two test portions shall be extracted and analysed separately, the final result
being the mean value of the results of these two subsamples.
It is not possible to complete the entire analysis within a single day. Sample extracts shall be stored
overnight under deep-freeze conditions of at least -18 °C:
— 1st day: step 1, step 2 and step 3, up to purification on C18 cartridge (see Figure A.1);
— 2nd day: step 3, purification on Florisil cartridge and preparation of HPLC system for sample
analysis (see Figure A.1);
— following night and day(s): analysis of the samples (see Table A.2).
9.2 Blank
To ensure the absence of contamination of solvents and cartridges, the purification procedure
(according to 9.5, 9.6 and Clause 11) shall first be carried out on a blank sample (sample with solvent
mixture but with the oil omitted). The chromatogram obtained shall be free from the compounds of
interest. If the chromatogram contains interferences, the source of interferences shall be determined
and eliminated. Blank values cannot be used to correct sample values as blank values are generally not
homogenous (repeatability).
9.3 Determination of recovery values (without matrix)
In order to verify the extraction efficiency of cartridges, carry out a test with a standard solution. Spike
1 750 µl of solvent mixture 1 (5.9) with 250 µl of working standard solution (5.15) with a 250 µl syringe
(6.11). Transfer to a C18 cartridge and treat as described in 9.5, 9.6 and Clause 11.
WARNING — When removing solvents under a stream of nitrogen (see 9.5.6), do not evaporate to
dryness but leave about 50 µl in the vial, otherwise, volatile PAHs will be lost.
9.4 Extraction (liquid/liquid extraction)
9.4.1 The flow chart of the isolation procedure is given in Figure A.1.
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9.4.2 Weigh, to the nearest 1 mg, about 2,5 g of the sample into a 100 ml centrifuge tube (6.8).
Add 10 ml of solvent mixture 1 (5.9).
9.4.3 Agitate the centrifuge tube for 30 s with the vortex mixer (half speed), and then put the tube in
an ultrasonic bath (6.17) for 5 min.
−1
9.4.4 Centrifuge for 5 min at 4 000 min .
9.4.5 Carefully remove the top layer with a Pasteur pipette (6.18) and transfer it to a weighed conical
tube (6.9).
9.4.6 Evaporate the solvent from the conical tube for 30 min to 40 min, under a stream of nitrogen
(5.18), using either a water bath at 35 °C (6.6) or an automatic evaporator (6.5).
9.4.7 Repeat the extraction twice with a further 10 ml of solvent mixture 1 (5.9).
Concentrate the extracts in the same conical tube under a stream of nitrogen (5.18) using water bath set
at 35 °C (6.6) or using an automatic evaporator (6.5). The fat residue should be about 200 mg to 800 mg.
If the fat residue mass is higher than 800 mg, then the general method (see Clause 9) is not suitable and
the method specific for coconut oil should be used (see Clause 10).
9.5 Purification on C18-bonded phase cartridge (solid/liquid extraction)
9.5.1 Cartridge conditioning: Put the cartridge (5.16) on a stand (6.19).
Rinse the cartridge with 2 volumes of 12 ml of methanol (5.1) then 2 volumes of 12 ml of acetonitrile (5.3).
Allow the solvent to flow through under atmospheric pressure.
9.5.2 Put a weighed conical tube (6.9) under the cartridge (5.16).
9.5.3 With a syringe (6.12) or a graduated pipette (6.13), introduce 2 ml of solvent mixture 1 (5.9) into
the conical tube containing residual fat material (9.4.6).
Agitate the tube with the vortex mixer (6.4) for 15 s. Centrifuge for 30 s. Transfer the top layer to the
cartridge (5.16) with a Pasteur pipette (6.18). Repeat the operation twice (2 ml of solvent mixture 1,
mixing, centrifuging and transferring onto the cartridge). Collect the solvent eluting from the cartridge
together with the elution solvent.
9.5.4 Add 5 ml of solvent mixture 1 (5.9) to the top of the cartridge (5.16) and allow the elution to
proceed under atmospheric pressure.
9.5.5 Using a syringe (6.14), inject air into the cartridge in order to elute the remaining solvent and any
PAHs which could be retained in the phase.
9.5.6 Remove solvents under a stream of nitrogen (5.18) using a water bath set at 35 °C (6.6) or an
automatic evaporator (6.5).
The fat residue should be not more than 50 mg.
9.5.7 Dilute the residue in 1 ml of hex
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