EN 15741:2020

Animal feeding stuffs: Methods of sampling and analysis - Determination of OCPs and PCBs by GC-MS

This document specifies a gas chromatographic mass spectrometric (GC/MS) method for the determination of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in animal feeding stuffs and oil.
The method is applicable to animal feeding stuffs consisting of less than 20 % by mass and oil/fatty samples containing residues of one or more of the following OCPs and PCBs and some of their isomers and degradation products:
   aldrin;
   dieldrin;
   chlordane, as the sum of chlordane isomers and oxychlordane;
   dichlorodiphenyltrichloroethane (DDT), as the sum of isomers op'-DDT, pp'-DDT, pp'-TDE (pp'-DDD), and pp'-DDE;
   endosulfan, as the sum of α-/β-isomers and endosulfan-sulphate;
   endrin, as the sum of endrin and delta-keto-endrin;
   heptachlor, as the sum of heptachlor and heptachlor epoxide;
   hexachlorobenzene (HCB);
   hexachlorocyclohexane isomers α-HCH (α-BHC), β-HCH (β-BHC), γ-HCH (γ-BHC or lindane);
   photo heptachlor;
   cis- and trans-nonachlor;
   non dioxin-like PCBs (ndl-PCBs), as the sum of PCB 28, 52, 101, 138, 153 and 180.
The method has been fully validated by a collaborative trial for the substances and corresponding ranges (ng/g) noted in Table 1.
The method has not been fully validated for oxychlordane, endrin ketone, cis- and trans-nonachlor and photo heptachlor in all matrices.
The method is not applicable to chlorocamphene (toxaphene), a complex mixture of polychlorinated camphenes. Chlorocamphene has a very distinctive chromatographic profile and is easily recognisable by GC/ECD. Positive identification of the toxaphene isomers can be performed by negative chemical ionisation mass spectrometry (NCI-MS), electron impact tandem mass spectrometry (EI MS × MS) or electron impact high resolution mass spectrometry (EI-HRMS), which is not within the scope of this method.
A limit of quantification (LOQ) for the mentioned organochlorine pesticides of 5 ng/g should normally be obtained. However, 10 ng/g applies for heptachlor aldrin, endrin, dieldrin, and endosulfan (α-, β- and sulphate). For the ndl-PCBs an LOQ of 0,5 to 1,0 ng/g should be obtained. The LOQs mentioned apply to the individual compounds (i.e. not the sum of two or more compounds). Individual laboratories are responsible for ensuring that the equipment that they used will achieve these LOQs. On customers' demand the standard may be applied to solely the analysis of PCBs or OCPs.

Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von OCP und PCB mittels GC-MS

Dieses Dokument legt ein gaschromatographisches massenspektrometisches Verfahren (GC MS) für die Bestimmung von Organochlorpestiziden (OCP) und polychlorierten Biphenylen (PCB) in Mischfuttermitteln und Einzelfuttermitteln (Öle und Fette, Fischmehl) fest.
Das Verfahren ist auf Mischfuttermittel mit einem Massenanteil von weniger als 20 % und für öl /fetthaltige Proben anwendbar, die Rückstände von einem oder mehreren der folgenden OCP und PCB und einige von deren Isomeren und Abbauprodukten enthalten:
—   Aldrin;
—   Dieldrin;
—   Chlordan, als die Summe von Chlordanisomeren und Oxychlordan;
—   Dichlordiphenyltrichlorethan (DDT), als die Summe der Isomere von o,p’ DDT, p,p ’DDT, p,p’ TDE (p,p’ DDD) und p,p’ DDE;
—   Endosulfan, als die Summe von α /β Isomeren und Endosulfan Sulfat;
—   Endrin, als die Summe von Endrin und delta Ketoendrin;
—   Heptachlor, als die Summe von Heptachlor und Heptachlorepoxid;
—   Hexachlorbenzol (HCB);
—   Hexachlorcyclohexan Isomere α HCH (α BHC), β HCH (β BHC), γ HCH (γ BHC oder Lindan);
—   Photoheptachlor;
—   cis  und trans Nonachlor;
—   nicht dioxinähnliche PCB (ndl PCB), als Summe von PCB 28, 52, 101, 138, 153 und 180.
Das Verfahren wurde vollständig durch einen Ringversuch für die in Tabelle 1 aufgeführten Substanzen und entsprechenden Bereiche (µg/kg) validiert.
Das Verfahren wurde für Oxychlordan, Endrin-Keton, cis   und trans Nonachlor und Photoheptachlor in allen Matrices nicht vollständig validiert.
Für die Analyt/Matrix Kombinationen, bei denen die Validierungsdaten als unzureichend angesehen wurden, können die mit diesem Verfahren erzielten Ergebnisse nur als Screening-Ergebnisse betrachtet werden, es sei denn, das Laboratorium führt eine interne Validierung durch, um nachzuweisen, dass zufriedenstellende Ergebnisse erzielt werden können.
Das Verfahren ist nicht anwendbar auf Chlorcamphen (Toxaphen), eine komplexe Mischung von poly-chlorierten Camphenen. Chlorcamphen hat ein sehr ausgeprägtes chromatographisches Profil und ist leicht erkennbar durch GC-ECD. Eine positive Identifizierung der Toxaphen Isomere kann durch eine negative chemische Ionisationsmassen¬spektrometrie (NCI MS), eine Elektronenstoß Tandem-Massenspektrometrie (EI MS × MS) oder eine hochauflösende Elektronenstoß Massenspektrometrie (EI HRMS) durchgeführt werden, die nicht in den Anwendungsbereich dieses Verfahrens fällt.
In der Regel sollte für die genannten Organochlorpestizide eine Bestimmungsgrenze (LOQ) von 6 µg/kg bis 29 µg/kg erreicht werden (siehe Tabelle 1). Für die ndl PCB sollte eine Bestimmungsgrenze von 0,5 µg/kg bis 1,0 µg/kg erreicht werden. Die genannten Bestimmungsgrenzen gelten für die einzelnen Verbindungen (d. h. nicht die Summe von zwei oder mehr Verbindungen). Die einzelnen Laboratorien sind dafür verantwortlich sicherzustellen, dass die von ihnen verwendeten Geräte diese Bestimmungsgrenzen erreichen. Auf Kundenwunsch darf die Norm nur auf die Analyse von PCB oder OCP angewendet werden.

Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Détermination des pesticides organochlorés (POC) et des polychlorobiphényles (PCB) par GC/MS

Le présent document décrit une méthode de chromatographie en phase gazeuse couplée à la spectrométrie de masse (GC-MS) pour la détermination des pesticides organochlorés (POC) et des polychlorobiphényles (PCB) dans les aliments composés et les matières premières alimentaires pour animaux (huiles et graisses, farine de poisson).
La méthode est applicable aux aliments composés constitués de moins de 20 % d’eau en masse et aux échantillons d’huile/de graisses contenant des résidus d’un ou de plusieurs des POC et PCB suivants et de certains de leurs isomères et produits de dégradation :
—   aldrine ;
—   dieldrine ;
—   chlordane, correspondant à la somme des isomères de chlordane et d’oxychlordane ;
—   dichlorodiphényltrichloréthane (DDT), correspondant à la somme des isomères de o,p’-DDT, p,p’ DDT, p,p’-TDE (p,p’-DDD) et p,p’-DDE ;
—   endosulfan, correspondant à la somme des isomères α-/β et de l’endosulfan sulfate ;
—   endrine, correspondant à la somme de l’endrine et du delta-keto-endrine ;
—   heptachlore, correspondant à la somme de l’heptachlore et de l’heptachlore époxyde ;
—   hexachlorobenzène (HCB) ;
—   isomères des hexachlorocyclohexanes α-HCH (α-BHC), β-HCH (β-BHC), γ-HCH (γ-BHC ou lindane) ;
—   photoheptachlore ;
—   cis-nonachlore et trans-nonachlore ;
—   PCB qui ne sont pas de type dioxine (NDL-PCB), correspondant à la somme des PCB 28, 52, 101, 138, 153 et 180.
La méthode a été intégralement validée par un essai interlaboratoires au regard des substances et des plages de concentrations correspondantes (ng/g) notées dans le Tableau 1.
La méthode n’a pas été intégralement validée pour l’oxychlordane, l’endrine-cétone, le cis-nonachlore, le trans-nonachlore et le photoheptachlore dans toutes les matrices.
Pour les combinaisons matrice/analyte pour lesquelles les données de validation ont été jugées insuffisantes, les résultats obtenus avec cette méthode ne peuvent être considérés que comme des résultats de dépistage, à moins que le laboratoire ne procède à une validation interne pour montrer que des résultats satisfaisants peuvent être obtenus.
La méthode ne s’applique pas au camphéchlore (toxaphène), mélange complexe de camphènes polychlorés. Le camphéchlore possède un profil chromatographique très particulier et est facilement identifiable par chromatographie en phase gazeuse avec détecteur à capture d’électrons (GC/ECD). L’identification positive des isomères du toxaphène peut être effectuée par spectrométrie de masse par ionisation chimique négative (NCI-MS), spectrométrie de masse en tandem par impact d’électrons (EI MS/MS) ou par spectrométrie de masse haute résolution par impact d’électrons (EI-HRMS), qui ne relèvent pas du domaine d’application de la présente méthode.
Il convient généralement d’obtenir une limite de quantification (LQ) des pesticides organochlorés mentionnés de 6 µg/kg à 29 µg/kg (voir le Tableau 1). Pour les NDL-PCB, il convient d’obtenir une LQ de 0,5 µg/kg à 1,0 µg/kg. Les LQ mentionnées s’appliquent aux composés individuels (c’est-à-dire non applicables à la somme de deux composés ou plus). Il incombe à chaque laboratoire de garantir que les appareils qu’ils utilisent atteignent ces LQ. Sur demande des clients, la norme peut être appliquée uniquement à l’analyse des PCB ou des POC.

Krma: metode vzorčenja in analize - Določevanje OCP in PCB z GC/MS

General Information

• Status: Published
• Publication Date: 10-Mar-2020
• Withdrawal Date: 29-Sep-2020
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
• Drafting Committee: CEN/TC 327/WG 1 - Minerals and Contaminants
• Current Stage: 9060 - Closure of 2 Year Review Enquiry - Review Enquiry
• Start Date: 02-Sep-2025
• Completion Date: 02-Sep-2025
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Mandate: M/522 - Mandate for standardisation addressed to CEN for methods of analysis in the field of animal nutrition - part II
• Ref Project: SIST EN 15741:2020 - Animal feeding stuffs: Methods of sampling and analysis - Determination of OCPs and PCBs by GC/MS

Relations

• Replaces: EN 15741:2009 - Animal feeding stuffs - Determination of OC-pesticides and PCB’s by GC/MS
• Effective Date: 15-Jul-2015

Standards Content (Sample)

SLOVENSKI STANDARD
01-maj-2020
Nadomešča:
SIST EN 15741:2009
Krma: metode vzorčenja in analize - Določevanje OCP in PCB z GC/MS
Animal feeding stuffs: Methods of sampling and analysis - Determination of OCPs and
PCBs by GC/MS
Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von OCP und
PCB mittels GC-MS
Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Détermination des
pesticides organochlorés (POC) et des polychlorobiphényles (PCB) par GC/MS
Ta slovenski standard je istoveten z: EN 15741:2020
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 15741
EUROPEAN STANDARD
NORME EUROPÉENNE
March 2020
EUROPÄISCHE NORM
ICS 65.120 Supersedes EN 15741:2009
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of OCPs and PCBs by GC/MS
Aliments des animaux : Méthodes d'échantillonnage et Futtermittel: Probenahme- und
d'analyse - Détermination des pesticides organochlorés Untersuchungsverfahren - Bestimmung von OCP und
(POC) et des polychlorobiphényles (PCB) par GC/MS PCB mittels GC/MS
This European Standard was approved by CEN on 6 January 2020.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, 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: Rue de la Science 23, B-1040 Brussels
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 15741:2020 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 7
3 Terms and definitions . 7
4 Principle . 7
5 Reagents and materials . 8
6 Apparatus . 14
7 Sampling . 18
8 Preparation of test sample . 19
9 Procedure. 19
9.1 General . 19
9.2 Test portions of animal feed stuff and oil samples . 19
9.3 Extraction of the feed test portion . 20
9.4 Clean-up . 20
9.5 Gas chromatography . 21
10 Calculation and expression of results . 21
10.1 General . 21
10.2 Calibration criteria . 22
10.3 Identification and confirmation. 22
10.4 Calculation . 22
10.5 Recovery . 24
11 Precision . 25
11.1 Interlaboratory test . 25
11.2 Repeatability and precision within participating laboratories . 25
11.3 Reproducibility and precision between participating laboratories . 25
12 Test report . 25
13 Important considerations to this method . 25
13.1 Alternative extraction techniques . 25
13.2 Different amounts of silica . 25
13.3 Different clean-up step . 25
13.4 Different internal standard . 25
13.5 Optimal GC-MS (MS) settings . 26
Annex A (informative) Description of PTV injection system (in case this is available) . 27
Annex B (informative) Collaborative trial. 28
Bibilography . 48
European foreword
This document (EN 15741:2020) has been prepared by Technical Committee CEN/TC 327 “Animal
feeding stuffs: Methods of sampling and analysis”, the secretariat of which is held by NEN.
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 September 2020, and conflicting national standards shall
be withdrawn at the latest by September 2020.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 15741:2009.
In comparison with the previous edition, the following technical modification has been made:
Regarding non dioxin-like PCBs (ndl-PCBs), this document contains two approaches that can be followed.
Method 1 concerns the original extraction and clean-up methods of the previous edition of this standard,
but combined with more sensitive detection approaches. In method 2, the extraction and clean-up
methods have been modified in order to increase the test portion. The detection of method 2 concerns
the original detection method of the previous edition of this standard.
This document has been prepared under a standardization request given to CEN by the European
Commission and the European Free Trade Association.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United
Kingdom
Introduction
WARNING — The use of this document can involve hazardous materials, operations and equipment. This
standard does not purport to address all the safety problems associated with its use. It is the
responsibility of the user of this European Standard to establish appropriate safety and health practices
and determine the applicability of regulatory limitations prior to use.
1 Scope
This document specifies a gas chromatographic mass spectrometric (GC-MS) method for the
determination of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in compound
feeds and feed materials (oils and fats, fish meal).
The method is applicable to compound feeds consisting of less than 20 % water by mass and oil/fatty
samples containing residues of one or more of the following OCPs and PCBs and some of their isomers
and degradation products:
— aldrin;
— dieldrin;
— chlordane, as the sum of chlordane isomers and oxychlordane;
— dichlorodiphenyltrichloroethane (DDT), as the sum of isomers o,p'-DDT, p,p'-DDT, p,p'-TDE (p,p'-
DDD), and p,p'-DDE;
— endosulfan, as the sum of α-/β-isomers and endosulfan-sulphate;
— endrin, as the sum of endrin and delta-keto-endrin;
— heptachlor, as the sum of heptachlor and heptachlor epoxide;
— hexachlorobenzene (HCB);
— hexachlorocyclohexane isomers α-HCH (α-BHC), β-HCH (β-BHC), γ-HCH (γ-BHC or lindane);
— photo heptachlor;
— cis- and trans-nonachlor;
— non dioxin-like PCBs (ndl-PCBs), as the sum of PCB 28, 52, 101, 138, 153 and 180.
The method has been fully validated by a collaborative trial for the substances and corresponding ranges
(µg/kg) noted in Table 1.
Table 1 — Residue compound and range of (µg/kg) collaborative trial
Compound Range (µg/kg)
all ndl-PCBs 0,7 to 39
aldrin 10 to 34
dieldrin 12 to 97
endrin 13 to 88
a a
cis-chlordane 16 to 24
trans-chlordane 7 to 25
b b
p,p’-DDT 19 to 200
o,p’-DDT 8 to 87
c c
pp’-TDE 9 to 103
pp'-DDE 21 to 264
α-endosulfan 15 to 165
β-endosulfan 26 to 331
d d
endosulfan sulphate 29 to 61
heptachlor 15 to 365
heptachlor epoxide 15 to 382
e e
HCB 8 to 170
α-HCH 21 to 247
β-HCH 6 to 84
f f
γ-HCH 17 to 186
NOTE  The following information is to be taken into consideration:
a)  Cis-chlordane has not been fully validated for chicken feed, pig feed and fish oil.
b)  p,p’-DDT has not been fully validated for pig feed and vegetable oil.
c)  pp’-TDE has not been fully validated for pig feed and fish meal.
d)  Endosulfan sulphate has not been fully validated for pig feed and vegetable oil.
e)  HCB has not been fully validated for fish oil.
f)  γ-HCH has not been fully validated for fish oil.
The method has not been fully validated for oxychlordane, endrin ketone, cis- and trans-nonachlor and
photo heptachlor in all matrices.
For those matrix-analyte combinations where the validation data were regarded insufficient, the results
obtained with this method can only be regarded as screening results, unless the laboratory performs an
in-house validation to show that satisfactory results can be obtained.
The method is not applicable to chlorocamphene (toxaphene), a complex mixture of polychlorinated
camphenes. Chlorocamphene has a very distinctive chromatographic profile and is easily recognizable by
GC/ECD. Positive identification of the toxaphene isomers can be performed by negative chemical
ionization mass spectrometry (NCI-MS), electron impact tandem mass spectrometry (EI MS × MS) or
electron impact high resolution mass spectrometry (EI-HRMS), which is not within the scope of this
method.
A limit of quantification (LOQ) for the mentioned organochlorine pesticides of 6 to 29 µg/kg should
normally be obtained (see Table 1). For the ndl-PCBs an LOQ of 0,5 to 1,0 µg/kg should be obtained. The
LOQs mentioned apply to the individual compounds (i.e. not the sum of two or more compounds).
Individual laboratories are responsible for ensuring that the equipment that they used will achieve these
LOQs. On customers' demand the standard may be applied to solely the analysis of PCBs or OCPs.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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.
EN ISO 6498, Animal feeding stuffs – Guidelines for sample preparation (ISO 6498)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
3.1
limit of detection
smallest measured content, from which it is possible to deduce the presence of the analyte with
reasonable statistical certainty
Note 1 to entry: The limit of detection is numerically equal to 3 times the standard deviation of the mean of blank
determinations (n > 10).
3.2
limit of quantification
lowest content of the analyte which can be measured with reasonable statistical certainty
Note 1 to entry: If both accuracy and precision are constant over a concentration range around the limit of
detection, then the limit of quantification is numerically equal to 6 times the standard deviation of the mean of blank
determinations (n > 10).
3.3
feed additives
substances that are added to animal nutrition
Note 1 to entry: see Regulation (EC) No.1831/2003 of the European Parliament and of the Council on additives
for use in animal nutrition for more information.
4 Principle
4.1 General
In order to check for the presence of organochlorine pesticides (OCPs), a test portion of animal feeding
stuff is fortified with internal standard ( C -PCB mix) and extracted with ethyl acetate. The extract is
concentrated and subsequently purified by:
— gel permeation chromatography (GPC), with a mixture of cyclohexane/ethyl acetate as eluting
solvent;
— chromatography on partially deactivated silica gel.
The collected fraction containing the compounds of interest is concentrated and re-dissolved in a solution
containing another internal standard (PCB 209) as a reference standard. After concentration an aliquot
of the extract is injected into a GC-MS using a splitless injector. In case more sensitivity is necessary or
less volume reduction is wanted, injection of a larger volume by means of a Programmed Temperature
Vaporizer (PTV) injector is possible. An example is described in Annex B.
For ndl-PCBs, two approaches can be followed.
4.2 Ndl-PCBs; method 1
A test portion of animal feeding stuff is fortified with internal standard ( C -PCB mix), and is extracted
with ethyl acetate. The extract is concentrated and subsequently purified by:
— gel permeation chromatography, with a mixture of cyclohexane /ethyl acetate as eluting solvent;
— chromatography on partially deactivated silica gel.
The collected fraction containing the compounds of interest is concentrated and re-dissolved in a solution
containing another internal standard (PCB 209) as a reference standard. After concentration an aliquot
of the extract is injected into a GC-MS-MS or GC-HRMS using a splitless injector. As an alternative,
injection by means of a PTV injector is possible. An example is described in Annex B.
4.3 Ndl-PCBs; method 2
A test portion of animal feeding stuff is fortified with internal standard ( C -PCB mix), and is extracted
with ethyl acetate. The extract is concentrated and subsequently purified by:
— concentrated sulfuric acid;
— gel permeation chromatography (GPC), with a mixture of cyclohexane/ethyl acetate as eluting
solvent;
— chromatography on partially deactivated silica gel.
The collected fraction containing the compounds of interest is concentrated and re-dissolved in a solution
containing another internal standard (PCB 209) as a reference standard. After concentration an aliquot
of the extract is injected into a GC-MS, using a splitless injector. As an alternative, injection by means of a
PTV injector is possible. An example is described in Annex B.
5 Reagents and materials
5.1 General
Use only reagents of recognized analytical grade and with a purity suitable for OC and PCB residue
analysis. Check the purity of the reagents by performing a blank test under the same conditions as used
in the method. The chromatogram should not show any interfering impurity at the retention time of
compounds of interest.
5.2 Chemicals
5.2.1 Ethyl acetate
5.2.2 Cyclohexane
5.2.3 Ethyl acetate/Cyclohexane = 1+1 parts by volume
Mix 500 ml of ethyl acetate (5.2.1) with 500 ml of cyclohexane (5.2.2) and mix thoroughly. Store at room
temperature in a tightly closed glass bottle.
5.2.4 Hexane
5.2.5 Decane
5.2.6 Hexane/Decane = 95+5 part by volume
Mix 950 ml of hexane (5.2.4) with 50 ml of decane (5.2.5) and mix thoroughly. Store at room temperature
in a tightly closed glass bottle.
5.2.7 Iso-octane
5.2.8 Toluene
5.2.9 Silica gel, deactivated with 3,5 % water
Heat silica gel 60 (63µm to 200µm = 70 mesh to 230 mesh), at 130 °C for at least 5 h, allow to cool in a
desiccator and store in a tightly stopped container in the desiccator. Add 3,5 ml water dropwise from a
burette, with a continuous swirling, to 96,5 g dried silica gel in a 300 ml Erlenmeyer flask with a ground
joint. Immediately stopper the flask with a ground stopper and shake vigorously for 5 min until all lumps
have disappeared. Next, shake for 2 h on a mechanical shaker, then store in a tightly stoppered container.
Deactivated silica gel is tenable during approximately 2 weeks if carefully stored.
5.2.10 Hexane/toluene = 3+7 parts by volume
Mix 30 ml of n-hexane (5.2.4) with 70 ml of toluene (5.2.8) and mix thoroughly. Store at room
temperature in a tightly closed glass bottle.
5.2.11 Concentrated H SO
2 4
5.2.12 Internal standard (PCB 209)
5.2.12.1 PCB 209 stock solution 1, 100 µg/ml
Weigh 10 mg (±0,01 mg) of PCB 209 (5.2.12) in a brown medicine glass bottle of 100 ml and add iso-
octane (5.2.7) to achieve a concentration of 100 μg/ml. Store the solution in a refrigerator at 4°C (±3°C).
The solution is tenable under these conditions during at least 5 years if the weight of the solution is
carefully controlled. Alternatively, use a commercially available standard solution of 100 µg/ml.
5.2.12.2 PCB 209 stock solution 2, 10,0 µg/ml
Dilute 10,0 ml of PCB 209 Stock solution 1 (5.2.12.1) to 100,0 ml with hexane (5.2.4). Store the solution
in a refrigerator at 4°C (±3°C). The solution is tenable under these conditions during at least 5 years if the
weight of the solution is carefully controlled.
5.2.12.3 PCB 209 working solution, concentration 1 000 ng/ml
Dilute 10 ml of PCB 209 Stock solution 2 (5.2.12.2) to 100,0 ml with hexane (5.2.4). Store the solution in
a refrigerator at 4 °C (±3 °C). The solution is tenable under these conditions during at least 5 years if the
weight of the solution is carefully controlled.
5.3 Internal standards ( C mass labelled PCBs)
5.3.1 Internal standards ( C mass labelled PCBs), 1 000 ng/ml
13 13
C PCB 28 (2,4,4’ trichlorobiphenyl, C ); CAS Number: 208263-76-7;
12 12
13 13
C PCB 52 (2,2’,5,5’ tetrachlorobiphenyl, C ); CAS Number: 208263-80-3;
12 12
13 13
C PCB 101 (2,2’,4,5,5’ pentachlorobiphenyl, C ); CAS Number: 104130-39-4;
12 12
13 13
C PCB 138 (2,2’,3’,4,4’,5 hexachlorobiphenyl, C ); CAS Number: 208263-66-5;
12 12
13 13
C PCB 153 (2,2’,4,4’,5,5’ hexachlorobiphenyl, C ); CAS Number: 185376-58-3;
12 12
13 13
C PCB 180 (2,2’,3,4,4’,5,5’ heptachlorobiphenyl, C ); CAS Number: not available.
12 12
Alternatively, use a certified mixture at a concentration of 1 000 ng/ml.
5.3.2 Internal standards ( C mass labelled PCBs), 100 ng/ml
Dilute 1,0 ml of internal standards ( C mass labelled PCBs) (5.3.1) to 10,0 ml with hexane (5.2.4). Store
the solution in a refrigerator at 4 °C (±3 °C). The solution is tenable under these conditions during at least
5 years if the weight is carefully controlled.
5.4 PCB congeners stock standard solution
5.4.1 PCB congeners stock standard solution, 10 µg/ml
PCB 28 (2,4,4’ trichlorobiphenyl); CAS Number: 7012-37-5;
PCB 52 (2,2’,5,5’ tetrachlorobiphenyl); CAS Number: 35693-99-3;
PCB 101 (2,2’,4,5,5’ pentachlorobiphenyl); CAS Number: 37680-73-2;
PCB 138 (2,2’,3’,4,4’,5 hexachlorobiphenyl); CAS Number: 35065-28-2;
PCB 153 (2,2’,4,4’,5,5’ hexachlorobiphenyl); CAS Number: 35065-27-1;
PCB 180 (2,2’,3,4,4’,5,5’ heptachlorobiphenyl); CAS Number: 35065-29-3.
Alternatively, use a certified mixture at a concentration of 10 µg/ml.
5.4.2 PCB congeners working standard solution, 2,0 µg/ml
Dilute 2,0 ml of PCB congeners stock standard solution (5.4.1) to 10,0 ml with hexane (5.2.4). Store the
solution in a refrigerator at 4 °C (±3 °C). The solution is tenable under these conditions during at least
5 years if the weight is carefully controlled.
5.4.3 PCB congeners work standard solution, 0,2 µg/ml
Dilute 1,0 ml of PCB congeners stock standard solution (5.4.2) to 10,0 ml with hexane (5.2.4). Store the
solution in a refrigerator at 4 °C (±3 °C). The solution is tenable under these conditions during at least
5 years if the weight is carefully controlled.
5.5 OC-pesticide reference standards, as follows
Each with a purity of not less than 99 %:
Aldrin:
(1R,4S,4aS,5S,8R,8aR)-1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-1,4:5,8-
dimethanonaphthalene; CAS Number: 309-00-2.
Dieldrin:
(1R,4S,4aS,5R,6R,7S,8S,8aR)-1,2,3,4,10,10-hexachloro-1,4,4a,5,6,7,8,8a-octahydro-6,7epoxy-1,4:5,8-
dimethanonaphthalene; CAS Number: 60-57-1.
Chlordane, α isomer:
1,2,4,5,6,7,8,8-octachloro-2,3,3a,4,7,7a-hexahydro-4,7-ethano-1H-indene; α isomer; CAS Number: 5103-
71-9.
Chlordane, β isomer:
1,2,4,5,6,7,8,8-octachloro-2,3,3a,4,7,7a-hexahydro-4,7-ethano-1H-indene; β isomer; CAS Number: 5103-
74-2.
Oxychlordane :
4,7-Methanoindan, 1,2,4,5,6,7,8,8-octachloro-2,3-epoxy-3a,4,7,7a-tetrahydro-, exo, endo-; CAS Number:
27304-13-8.
o,p'-DDT :
1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane; CAS Number: 789-02-6;
p,p'-DDT :
1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane; CAS Number: 50-29-3.
pp'-TDE :
(pp'-DDD) 1,1-dichloro-2,2-bis(4-chlorophenyl) ethane; CAS Number: 72-54-8.
pp'-DDE :
1,1-dichloro-2,2-bis(4-chlorophenyl) ethylene; CAS Number: 72-55-9.
Endosulfan, α stereoisomer:
6,9-Methano-2,4,3-benzodioxathiepin, 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-, 3-oxide,
(3α,5aβ, 6α,9α,9aβ); CAS Number: 959-98-8.
Endosulfan, β stereoisomer :
6,9-Methano-2,4,3-benzodioxathiepin, 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-, 3-oxide,
(3α,5aα,6β,9β,9aα); CAS Number: 33213-65-9.
Endosulfan-sulphate;
6,9-Methano-2,4,3-benzodioxathiepin, 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-, 3,3-dioxide;
CAS Number: 1031-07-8.
Endrin
(1R,4S,4aS,5S,6S,7R,8R,8aR)-1,2,3,4,10,10-hexachloro-1,4,4a,5,6,7,8,8a-octahydro-6,7-epoxy-1,4:5,8-dd;
CAS Number: 72-20-8.
Delta-keto endrin
2,5,7-Metheno-3H-cyclopenta[a]pentalen-3-one,3b,4,5,6,6,6a-hexachlorodecahydro-, 2α,
3aβ,3bβ,4β,5β,6aβ,7α,7aβ,8R*; CAS Number 53494-70-5.
Heptachlor
1,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-4,7-methanoindene; CAS Number: 76-44-8.
β-Heptachlor epoxide
1,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-4,7-methanoindene(exo); CAS Number: 1024-57-3.
HCB
hexachlorobenzene; CAS Number: 118-74-1.
α-HCH (α-BHC)
α-1,2,3,4,5,6-hexachlorocyclohexane; CAS Number: 319-84-6.
β-HCH (β-BHC)
β-1,2,3,4,5,6-hexachlorocyclohexane; CAS Number: 319-85-7.
γ-HCH (γ-BHC; lindane)
γ-1,2,3,4,5,6-hexachlorocyclohexane; CAS Number: 58-89-9.
Nonachlor, cis;
CAS Number 5103-73-1.
Nonachlor, trans;
CAS Number 39765-80-5.
Photo heptachlor;
CAS Number 33442-83-0.
Alternatively, use a certified mixture at a precise or certified concentration (approximately 10 µg/ml).
5.5.1 Pesticide stock solution 1, 100 µg/ml
Weigh 10 mg (±0,01 mg) of each individual pesticide (5.5) in separate brown medicine glass bottles of
100 ml and add iso-octane (5.2.7) to achieve a concentration of 100 μg/ml. Store the solutions in a
refrigerator at 4 °C (±3 °C). The solutions are tenable under these conditions during at least 5 years if the
weight is carefully controlled. Alternatively, use a commercially available standard solution with a precise
or certified concentration of approximately 100 µg/ml.
NOTE Dissolve ß-HCH in 10 ml toluene (5.2.8) to achieve complete solvability and dilute further with iso-
octane (5.2.7) to achieve a concentration of 100 μg/ml
5.5.2 Pesticide stock solution 2, 2,5 µg/ml
Pipet 2,5 ml of each individual pesticide stock solutions 1 (5.5.1) into a 100,0 ml graduated flask and
dilute with hexane (5.2.4) to 100,0 ml. Store the solution in a refrigerator at 4 °C (±3 °C). The solution is
tenable under these conditions during at least 5 years if the weight is carefully controlled.
5.5.3 Pesticide working solution, 0,5 µg/ml
Pipet 2,0 ml of pesticide stock solution 2 (5.5.2) into a 10,0 ml graduated flask and dilute with hexane
(5.2.4) to 10,0 ml. Store the solution in a refrigerator at 4 °C (±3 °C). The solution is tenable under these
conditions during at least 5 years if the weight is carefully controlled.
5.6 Calibration solutions
Prepare calibration mixtures according to Table 2 in a final volume of 1,0 ml of an alkane solvent (5.2.6
or 5.2.7) and store them at 4 °C (±3 °C).
Table 2 — Calibration mixtures
C -PCB PCB 209
PCB 0,2 μg/ml OC 0,5 μg/ml OC 5,0 μg/ml
Level 1 000 ng/ml Solvent
mix 100 ng/ml
(5.4.3) (5.5.3) (5.5.2)
(5.2.12.3)
(5.3.2)
μl ng/ml μl ng/ml μl ng/ml μl ng/ml µl ng/ml µl
1 0 0 0 0 0 n.a. 100 10 50 50 850
2 5 1 20 10 0 n.a. 100 10 50 50 825
3 10 2 50 25 0 n.a. 100 10 50 50 790
4 25 5 250 125 0 n.a. 100 10 50 50 575
5 100 20 0 n.a. 100 500 100 10 50 50 650
6 250 50 0 n.a. 250 1 250 100 10 50 50 350
5.7 Glass vial, 100 ml, including PTFE-lined screwcaps
5.8 Glass wool
Heated at 160 °C – 200 °C during at least 24 h.
5.9 Sodium sulphate, anhydrous
Heated at 160 °C – 200 °C during at least 24 h.
5.10 Helium gas, purity 5,0 or higher.
5.11 Nitrogen gas, purity 5,0 or higher.
5.12 GC sampler vial, 2 ml.
5.13 Glass graduated evaporation tubes, 50 ml
5.14 Chromatographic tubes, glass or PTFE
Chromatographic tube with solvent reservoir.
5.15 Autosampler vial, with limited volume insert
5.16 Glass tubes, approximately 50 ml
5.17 Glass tubes, approximately 4 ml
5.18 Anhydrous sodium sulphate
6 Apparatus
6.1 General
Usual laboratory glassware and equipment and, in particular, the following:
NOTE All technical descriptions below are examples of possible system setups and parameters that are scaled
or adopted to the user's equipment.
6.2 Analytical balance, accuracy 0,01 mg
6.3 Analytical balance, accuracy 10 mg
6.4 Mechanical shaker
6.5 GPC clean-up system
Equilibrate the GPC-system (6.5) under the recommended operating conditions and check the GPC
column performance as subscribed in EPA method 3640 [1].
WARNING — In case the recovery of β-HCH and γ-chlordane in the GPC control with a standard solution
is too low, the start of the collection time of the OC/PCB fraction is too late. In case the recovery of HCB is
too low, the end time of the OC/PCB fraction is too early.
6.5.1 HPLC-pump
The HPLC pump shall be capable of maintaining a flow-rate of 1,0 ml per min of the solvent mixture
specified under 5.2.3.
6.5.2 Automated injection system
The automated injection system shall be capable of performing a series of unattended injections of a
volume of 500 μl out of 2 ml GC sampler vial (5.12).
6.5.3 GPC-column
The GPC-column shall be capable of performing a separation as specified by criteria in EPA Method 3640
[1].
EXAMPLE A column with length of 45 cm, and 10 mm internal diameter, packed with stationary phase Bio
Beads SX-3 can be used for this purpose. In this example, the OCP/PCB containing fraction elutes between 16 min –
26 min. Other GPC columns with different dimensions and stationary phases may perform equally, but the
performance should be tested before use.
6.5.4 Fraction collector
6.6 Evaporation system, equipped with 50 ml graduated glass tubes and nitrogen gas
6.7 Evaporation block, equipped with heater and nitrogen gas
The evaporation block shall be able to contain 4 ml glass tubes (5.17).
6.8 Gas chromatograph-mass spectrometer (single quadrupole)
6.8.1 General
The gas chromatograph shall be capable of working with capillary columns. The use of a capillary column
coated with a mid-range polarity stationary phase (dimensions 30 m × 0,25 mm, film thickness 0,25 µm)
is recommended. The column flow is kept constant at 1,3 ml per min.
The temperature program starts at an initial temperature of 80 °C where it is kept for 3 min. After this
the temperature is ramped with 5 °C per min to a final temperature of 280 °C where it is kept for 10 min.
Finally, the GC is cooled to 80 °C.
6.8.2 Mass-spectrometer – SIM mode operation
The mass-spectrometer shall be capable of monitoring in full scan mode or selected ion monitoring (SIM),
and should be tuned according to the manufacturer's description.
The selected mass fragments are shown in Table 3 (PCBs) and Table 4 (OCs). At least one quantifier ion
and two qualifier ions have to be measured. Depending on the retention time of the components, SIM
windows are composed, preferably with less than 20 fragments. The selection of other quantifier or
qualifier masses is allowed but the suitability for identification and quantification should be
demonstrated.
Table 3 — Selected ions for PCBs
Fragment Fragment Fragment
Compound m/z m/z m/z
Quantifier Qualifier 1 Qualifier 2
PCB 28 256 258 186
PCB 52 292 290 220
PCB 101 326 324 254
PCB 153 360 358 290
PCB 138 360 358 290
PCB 180 394 396 324
C -PCB 28 268 270
C -PCB 52 304 302
C -PCB 101 338 336
C -PCB 153
372 370
C -PCB 138 372 370
C -PCB 180 406 408
PCB 209 498 500
Table 4— Selected ions for OC pesticides
Fragment Fragment Fragment
Compound m/z m/z m/z
Quantifier Qualifier 1 Qualifier 2
aldrin 263 265 293
dieldrin 263 277 237
α-chlordane (cis) 373 375 237
γ-chlordane (trans) 373 375 237
oxychlordane 387 389 185
o,p'-DDT 235 237 165
p,p'-DDT 235 237 165
p,p'-TDE 235 237 165
p,p'-DDE 246 248 318
α-endosulfan 339 341 272
β-endosulfan 339 341 272
endosulfan-sulphate 387 389 272
endrin 263 265 281
endrin-ketone 317 319 281
heptachlor 272 274 237
heptachlor epoxide 353 355 263
HCB 284 286 249
α-HCH 181 183 219
β-HCH 181 183 219
γ-HCH 181 183 219
cis-nonachlor 409 407 237
trans-nonachlor 409 407 237
photo heptachlor 337 339 266
6.9 Gas chromatograph-mass spectrometer (triple quadrupole)
6.9.1 General
The gas chromatograph shall be capable of working with capillary columns. The use of a capillary column
coated with a mid-range polarity stationary phase (dimensions 30 m × 0,25 mm, film thickness 0,25 µm)
is recommended. The column flow is kept constant at 1,3 ml per min.
The temperature program starts at an initial temperature of 80 °C where it is kept for 3 min. After this
the temperature is ramped with 5 °C per min to a final temperature of 280 °C where it is kept for 10 min.
Finally, the GC is cooled to 80 °C.
6.9.2 Mass-spectrometer
The mass-spectrometer shall be capable of Multiple Reaction Monitoring (MRM), and should be tuned
according to the manufacturer's description.
6.9.2.1 MRM mode operation
The selected mass fragments are shown in Table 5 and Table 6. At least one quantifier transition and one
qualifier transition shall be measured. Depending on the retention time of the components, MRM

windows are composed. Collision energy should be optimized for the used instrument, the shown values
are indicative. In case a MRM transition is disturbed by interferences in certain matrices, the MS shall be
optimized for an alternative transition that does not suffer from interferences.
Table 5 — MRM Transitions for PCBs
Quantifier Qualifier Collision
Dwell
energy
Compound Transition Transition
ms
V
m/z m/z
PCB 28 256 → 186 258 → 186 75 25
PCB 52 290 → 220 292 → 222 75 25
PCB 101 324 → 254 326 → 256 75 25
PCB 153 360 → 290 358 → 288 75 25
PCB 138 360 → 290 358 → 288 75 25
PCB 180 394 → 324 396 → 326 75 25
C -PCB 28 268 → 198 270 → 198 75
C -PCB 52
302 → 232 304 → 234 75
13 25
C -PCB 101 336 → 266 338 → 268 75
13 25
C -PCB 153 372 → 302 370 → 300 75
13 25
C -PCB 138 372 → 302 370 → 300 75
C -PCB 180
406 → 336 408 → 338 75
PCB 209 498 → 428  25 25
Table 6 — MRM Transitions for OC Pesticides
Quantifier Qualifier Collision
Dwell
Energy
Compound Transition Transition
ms
V
m/z m/z
aldrin 263 → 228 263 → 193 75 20
dieldrin 263 → 193 263 → 228 75 35
α-chlordane (cis) 373 → 266 373 → 301 75 28
γ-chlordane (trans) 373 → 266 373 → 301 75 28
oxychlordane 185 → 121 185 → 149 75 15
o,p'-DDT 235 → 165 235 → 199 75 30
p,p'-DDT 235 → 165 235 → 199 75 30
p,p'-TDE 235 → 165 235 → 199 75 30
p,p'-DDE 246 → 176 318 → 248 75 28
α-endosulfan 241 → 206 195 → 160 75 15
β-endosulfan 195 → 159 241 → 206 75 10
endosulfan sulphate 272 → 237 274 → 239 75 14
endrin 281 → 245 263 → 193 75 11
endrin ketone 317 → 101 317 → 245 75 20
heptachlor 272 → 237 274 → 239 75 18
heptachlor epoxide 353 → 263 353 → 289 75 12
HCB 284 → 249 249 → 214 75 16
α-HCH 181 → 145 219 → 183 75 17
β-HCH 181 → 145 219 → 183 75 17
γ-HCH 181 → 145 219 → 183 75 17
cis-nonachlor 407 → 300 407 → 263 75 25
trans-nonachlor 373 → 266 373 → 301 75 25
photo heptachlor 339 → 266 301 → 266 75 25
6.9.2.2 Full scan mode operation
The mass-range is scanned from 50 u to 550 u at 3 scans per second. The scan rate may vary, but there
need to be at least 10 scans per peak.
7 Sampling
The sample should be truly representative and shall not have been damaged or changed during prior
transportation or storage. Sampling is not part of the method specified in this document. A recommended
sampling method is given in EN ISO 6497 [2].
8 Preparation of test sample
Prepare the test sample in accordance with EN ISO 6498. Dry or low moisture products such as cereals
and cereal products, mixed feeds, and hay should be ground carefully so that it passes completely through
a sieve with 1 mm apertures. Mix thoroughly.
9 Procedure
9.1 General
Analyse in each series the following samples:
1) Procedural blank;
2) Blank animal feed (n = 1) and/or blank oil (n = 1);
3) Blank animal feed spiked (n = 2) and/or blank oil spiked (n = 2);
4) All samples.
NOTE Any blank feed sample proven to be blank in a previous run can be used for quality control.
9.2 Test portions of animal feed stuff and oil samples
9.2.1 Test portion of animal feed
9.2.1.1 OCs and ndl-PCBs (method 1)
Weigh 8,0 g (±0,10 g) of the prepared test sample into a 100 ml glass vial. Fortify the sample with 160 µl
13 13
C -PCB mix work solution (5.3.2) (2 µg/kg feed). Mix the test sample and C -PCB mix solution
12 12
and incur for at least 1 h.
9.2.1.2 ndl-PCBs (method 2)
Weigh 40,0 g (±0,10 g) of the prepared test sample into a 100 ml glass vial. Fortify the sample with 160 µl
13 13
C -PCB mix work solution (5.3.2) (0,4 µg/kg feed). Mix the test sample and C -PCB mix solution
12 12
and incur for at least 1 h.
If fat content is higher than 10 % by weight, the amount of sample shall be proportionally less.
9.2.2 Test portion of oils
9.2.2.1 OCs and for ndl-PCBs (method 1)
Weigh 1,0 g (±0,10 g) of the oil sample into a glass tube of 50 ml (5.16). Fortify the sample with 200 µl
C -PCB mix Work solution (5.3.2) (20 µg/kg oil). Add 3,8 ml ethyl acetate/cyclohexane (1:1 v/v)
mixture (5.2.3) and vortex for 5 min.
9.2.2.2 ndl-PCBs (method 2)
Weigh 5,0 g (±0,10 g) of the oil sample into a glass tube of 50 ml (5.16). Fortify the sample with 200 µl
C -PCB mix Work solution (5.3.2) (4 µg/kg oil). Add 19,8 ml hexane (5.2.4) and vortex for 5 min. Add
20 ml concentrated H SO and place in an ultrasonic bath for at least 1 h. After separation of the phases
2 4
take the upper layer. Extract the remaining H SO phase with another 5 ml hexane. Collect the hexane
2 4
phases and evaporate (40 °C, N ) to a final volume of < 1,0 ml. Make the volume up to 5,0 ml using
cyclohexane/ethyl acetate (5.2.3).
9.3 Extraction of the feed test portion
9.3.1 Organochlorine pesticides and for ndl-PCBs (method 1)
Add 50 ml ethyl acetate (5.2) to the sample test portion, close the vial with a PTFE-lined screwcap and
shake it during minimal 18 h using a mechanical shaker (6.4). Filter the extract over anhydrous sodium
sulphate (5.18) (approximately 10 g in a glass funnel with some glass wool (5.8)). Alternatively the
extract can be stirred with sodium sulphate and subsequent it can be filtered over a paper filter. Measure
25 ml filtrate, bring into a graduated tube (5.16) and evaporate (40 °C, N ) to a final volume of < 1,0 ml.
Make the volume up to 2,0 ml using cyclohexane/ethyl acetate (5.2.3).
9.3.2 ndl-PCBs (method 2)
Add 250 ml ethyl acetate (5.2) to the sample test portion, close the vial with a PTFE-lined screwcap and
shake it during minimal 18 h using a mechanical shaker (6.4). Filter the extract over anhydrous sodium
sulphate (5.18) (approximately 10 gr in a glass funnel with some glass wool (5.8)). Measure 125 ml
filtrate, bring into a graduated tube (5.16) and evaporate (40 °C, N ) to a final volume of < 1,0 ml. Add
50 ml hexane and 10 ml concentrated H SO and place in an ultrasonic bath for at least 1 h. After
2 4
separation of the phases take the upper layer. Extract the remaining H SO phase with another 25 ml
2 4
hexane. Collect the hexane phases and evaporate (40 °C, N ) to a final volume of < 1,0 ml. Make the
volume up to 2,0 ml using cyclohexane/ethyl acetate (5.2.3).
9.4 Clean-up
9.4.1 Gel permeation chromatography clean-up
9.4.2 Preparation GPC system
Equilibrate the GPC-system under the recommended operating conditions (6.5) and check the GPC
column performance as subscribed in EPA method 3640 [1].
9.4.3 Purification
Inject 0,5 ml (≡ 1 or 5 g feed or 0,1 or 0,5 g oil) from the prepared samples (9.2.2 or 9.3) into the GPC-
system. Collect the fraction eluting containing the compounds of interest directly into 50 ml evaporation
tubes (5.16).The collected GPC fractions are concentrated in an evaporation system (40 °C, N , (6.6)) to
a volume of approximately 0,5 ml. Transfer the extract into a 4 ml glass tube (5.17). Rinse the evaporation
tube with 1 ml of hexane (5.2.4), and combine the solvent with the first fraction into the 4 ml glass tube.
9.4.4 Column chromatography on partially deactivated silica
Pack the chromatographic tube (5.14) in the following order: glass wool plug (5.8), 4,0 g of deactivated
silica gel (5.2.9), 5 mm to 10 mm layer of sodium sulphate (5.18), glass wool plug (5.8). Before use, rinse
the column with 10 ml of n-hexane (5.2.4) and discard the eluate. As soon as the hexane has drained to
the top of the silica gel, pipette the n-hexane solution derived from the evaporation of the sample solution
(9.4.3) on to the pre-washed silica gel column. Elute 6 times with 3 ml eluent (n-hexane: toluene) (5.2.10).
Collect eluate and concentrate in an evaporation system (40 °C, N , (6.6)) to a volume of approximately
0,5 ml. Transfer the extract into a 4 ml glass tube (5.17), and add 10 µl decane (5.2.5). Rinse the
evaporation tube (5.17) with 2 ml of hexane (5.2.4) and combine the solvent with the first fraction into
the 4 ml glass tube (5.17). Evaporate the solvent gently in an evaporation block (40 °C, N , (6.6)) until
only the decane remains. Add 10 µl PCB 209 work solution (5.2.12.3) and 180 µl hexane (5.2.4) to the
residue, and mix thoroughly. Finally, transfer the extract into the autosampler vial, using a limited volume
insert (5.15). Low recoveries for endosulfan (α, β) and/or dieldrin can be due to an insignificant elution.
If so the number of eluting cycles shall be increased.
NOTE 1 The final concentration is 5 or 25 g feed per ml or 0,5 or 2,5 g oil per ml.
...