SIST EN ISO 15753:2007
(Main)Animal and vegetable fats and oils - Determination of polycyclic aromatic hydrocarbons (ISO 15753:2006)
Animal and vegetable fats and oils - Determination of polycyclic aromatic hydrocarbons (ISO 15753:2006)
This standard describes two methods for the determination of 15 polycyclic aromatic hydrocarbons (PAHs) in animal and vegetable fats and oils: - a general method, and - a method specific for coconut oil and short-chain vegetable oils. 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 pomace olive oil cannot be analysed using this method. The polycyclic aromatic hydrocarbons are extracted with an acetonitrile/acetone mixture followed by purification on C18 phase and then Florisil-phase bonded catridges. Determination of the content of the individual polycyclic aromatic hydrocarbons after separation is achieved by means of high- pressure liquid chromatography (HPLC) by measuring the fluorescence at various excitation and emission wavelengths.
Tierische und pflanzliche Fette und Öle - Bestimmung von polycyclischen aromatischen Kohlenwasserstoffen (ISO 15753:2006)
Diese Internationale Norm beschreibt zwei Verfahren zur Bestimmung von 15 polycyclischen aromatischen Kohlenwasserstoffen (PAKs) in tierischen und pflanzlichen Fetten und Ölen:
- ein allgemeines Verfahren und
- ein Verfahren, speziell 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 ?g/kg, mit Ausnahme von Fluoranthen und Benzo(g,h,i)perylen, deren Bestimmungsgrenze bei 0,3 ?g/kg liegt, sowie Indeno(1,2,3 c,d)pyren mit einer Bestimmungsgrenze von 1 ?g/kg.
Corps gras d'origines animale et végétale - Détermination des hydrocarbures aromatiques polycycliques (ISO 15753:2006)
L'ISO 15753:2006 décrit deux méthodes pour la détermination de 15 hydrocarbures aromatiques polycycliques (HAP) dans les corps gras d'origines animale et végétale: une méthode générale et 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.
Rastlinske in živalske maščobe in olja - Določevanje policikličnih aromatskih ogljikovodikov (ISO 15753:2006)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 15753:2007
01-marec-2007
5DVWOLQVNHLQåLYDOVNHPDãþREHLQROMD'RORþHYDQMHSROLFLNOLþQLKDURPDWVNLK
RJOMLNRYRGLNRY ,62
Animal and vegetable fats and oils - Determination of polycyclic aromatic hydrocarbons
(ISO 15753:2006)
Tierische und pflanzliche Fette und Öle - Bestimmung von polycyclischen aromatischen
Kohlenwasserstoffen (ISO 15753:2006)
Corps gras d'origines animale et végétale - Détermination des hydrocarbures
aromatiques polycycliques (ISO 15753:2006)
Ta slovenski standard je istoveten z: EN ISO 15753:2006
ICS:
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
SIST EN ISO 15753:2007 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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EUROPEAN STANDARD
EN ISO 15753
NORME EUROPÉENNE
EUROPÄISCHE NORM
September 2006
ICS 67.200.10
English Version
Animal and vegetable fats and oils - Determination of polycyclic
aromatic hydrocarbons (ISO 15753:2006)
Corps gras d'origines animale et végétale - Détermination Tierische und pflanzliche Fette und Öle - Bestimmung von
des hydrocarbures aromatiques polycycliques (ISO polycyclischen aromatischen Kohlenwasserstoffen (ISO
15753:2006) 15753:2006)
This European Standard was approved by CEN on 19 August 2006.
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 Central Secretariat 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,
Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 15753:2006: E
worldwide for CEN national Members.
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EN ISO 15753:2006 (E)
Foreword
This document (EN ISO 15753:2006) has been prepared by Technical Committee ISO/TC 34
"Agricultural 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 March 2007, and conflicting national
standards shall be withdrawn at the latest by March 2007.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary,
Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Endorsement notice
The text of ISO 15753:2006 has been approved by CEN as EN ISO 15753:2006 without any
modifications.
2
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INTERNATIONAL ISO
STANDARD 15753
First edition
2006-09-01
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 15753:2006(E)
©
ISO 2006
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ISO 15753:2006(E)
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ii © ISO 2006 – All rights reserved
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ISO 15753:2006(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) . 6
9.5 Purification on C18-bonded phase cartridge (solid/liquid extraction). 6
9.6 Purification on Florisil-bonded phase cartridge (solid/liquid extraction). 6
10 Procedure for determination of PAHs from fats and oils: Method specific for coconut oil . 7
10.1 First extraction (liquid/liquid extraction). 7
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). 8
11 High-performance liquid chromatography (HPLC) . 9
11.1 Operating conditions. 9
11.2 Detection parameters . 10
11.3 Analysis of samples and standards. 11
11.4 Confirmation of the presence of PAHs. 12
12 Expression of results. 12
13 Precision. 12
13.1 Interlaboratory test . 12
13.2 Repeatability. 13
13.3 Reproducibility. 13
14 Test report. 13
Annex A (informative) Recovery values, flow charts, chromatograms and injection sequences . 14
Annex B (informative) Results of the interlaboratory test . 19
Bibliography . 22
© ISO 2006 – All rights reserved iii
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ISO 15753:2006(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 15753 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, Animal
and vegetable fats and oils.
iv © ISO 2006 – All rights reserved
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INTERNATIONAL STANDARD ISO 15753:2006(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, and
⎯ a method specific 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 µg/kg.
2 Normative references
The following referenced documents are indispensable for the application of this document. 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 The content is given in micrograms per kilogram.
NOTE 2 In general PAHs are divided into light PAHs with two to four aromatic rings, and heavy PAHs with five or more
aromatic rings.
EXAMPLES
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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ISO 15753:2006(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 must 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 method specific 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.
This information is given for the convenience of users of this International Standard 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.
2 © ISO 2006 – All rights reserved
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ISO 15753:2006(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. To be stored at −20 °C.
Before use, allow the solution to warm up to ambient temperature for at least 1 h.
NOTE 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.
5)
6.5 Automatic evaporator , for 10 ml tube (optional), or water bath (6.6).
2) This can be obtained from, for example, Promochem.
3) This can be obtained from, for example, Varian.
This information is given for the convenience of users of this International Standard 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.
4) This can be obtained from, for example, Vydac, ref. 201TP54.
5) This can be obtained from, for example, Zymark, Zymark TurboVap LV evaporator.
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ISO 15753:2006(E)
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).
6.10 Graduated cylinders.
6.11 Microsyringe, 250 µl.
6.12 Syringe, 1 000 µl.
6.13 Graduated pipette, 5 ml.
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.
6.18 Pasteur pipettes, with cotton wool in the top part to prevent contamination.
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).
6.20 Volumetric flask, of capacity 50 ml.
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.
6) This can be obtained from, for example, Zymark, Zymark Rapid Trace.
This information is given for the convenience of users of this International Standard 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.
4 © ISO 2006 – All rights reserved
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ISO 15753:2006(E)
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 (u 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:
st
⎯ 1 day: step 1, step 2 and step 3, up to purification on C18 cartridge (see Figure A.1).
nd
⎯ 2 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.
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ISO 15753:2006(E)
9.4 Extraction (liquid/liquid extraction)
9.4.1 The flow chart of the isolation procedure is given in Figure A.1.
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 (Clause 9) is not suitable and the
method specific for coconut oil should be used (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 hexane (5.2), measured with a syringe (6.12). Close the conical tube
hermetically and store at −18 °C until further use.
9.6 Purification on Florisil-bonded phase cartridge (solid/liquid extraction)
9.6.1 Allow the extract (9.5.7) to warm up to ambient temperature for at least 1 h.
9.6.2 Cartridge conditioning: Put the cartridge (5.17) on a stand (6.19). Rinse the cartridge with 5 volumes
of 3 ml of dichloromethane (5.5) then 4 volumes of 3 ml of hexane (5.2).
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ISO 15753:2006(E)
9.6.3 Put a weighed conical tube (6.9) under the cartridge (5.17).
9.6.4 Transfer the extract (9.5.7) to the cartridge (5.17) with a Pasteur pipette (6.18).
9.6.5 Introduce 1 ml of solvent mixture 3 (5.11), using a syringe (6.12) or a graduated pipette (6.13), to the
conical tube containing the extract. Agitate it for 15 s with the vortex mixer and transfer it to the
cartridge (5.17). Rinse the tube with 2 × 2 ml of solvent mixture 3 (5.11) and transfer it onto the cartridge.
Collect the solvent eluting from the cartridge together with the elution solvent.
Pay careful attention to avoid contact between the pipette and the conical tube in order to prevent cross
contamination.
9.6.6 Elute 4 ml of solvent mixture 3 (5.11) through the cartridge (5.17). Using a syringe (6.14), inject air
into the cartridge in order to elute the remaining solvent.
9.6.7 Concentrate the solution under a stream of nitrogen (5.18), using a water bath set at 35 °C (6.6) or an
automatic evaporator (6.5), to about 1 ml (takes 10 min to 15 min). Add about 0,5 ml of toluene (5.6) (keeper)
and continue to evaporate until about 50 µl remain.
The solvent should not be removed completely.
9.6.8 The exact volume is determined by weighing the conical tube, and calculating using the density of
toluene. Add the necessary volume of solvent [MeOH/THF (5.12), or acetonitrile (5.3)], V , to make up to
add
250 µl:
m
V =−250
add
d
where
m is the sample mass, in milligrams;
3
d is the density of toluene (0,866 9 kg/m ).
9.6.9 Transfer the sample to a microvial (6.16) placed in a vial (6.15).
10 Procedure for determination of PAHs from fats and oils: Method specific for
coconut oil
10.1 First extraction (liquid/liquid extraction)
10.1.1 The flow chart of the isolation procedure is given in Figure A.2.
10.1.2 Weigh, to the
...
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