SIST EN 12393-2:2009
(Main)Foods of plant origin - Multiresidue methods for the gas chromatographic determination of pesticide residues - Part 2: Methods for extraction and cleanup
Foods of plant origin - Multiresidue methods for the gas chromatographic determination of pesticide residues - Part 2: Methods for extraction and cleanup
This European Standard specifies methods for the extraction and clean-up of food samples of plant origin for quantitative determination of pesticide residues.
Different solvents can be used for this purpose. These pesticide residues are generally associated with other co-extracted compounds which would interfere in the analysis. To purify the crude extracts to be analysed, several techniques can be used.
This standard contains the following extraction and clean-up methods that have been subjected to interlaboratory studies and /or are adopted throughout Europe:
- method L: Extraction with acetone, liquid-liquid partition with dichloromethane and clean-up on a silica gel/charcoal column [1];
- method M: Extraction with acetone and liquid-liquid partition with dichloromethane/light petroleum, if necessary clean-up on Florisil® 1) [2], [3], [4];
- method N: Extraction with acetone, liquid-liquid partition with dichloromethane or cyclohexane/ethyl acetate and clean-up with gel permeation and silica gel chromatography [5], [6];
- method P: Extraction with ethyl acetate, and if necessary, clean-up by gel permeation chromatography [7].
This European Standard specifies the details of methods L to P for the extraction and the clean-up of food samples of plant origin. Several solvents at different volumes are used for extraction. Techniques of clean-up are listed such as liquid-liquid partition, liquid chromatography on various adsorbents and gel permeation chromatography.
A table providing the couples (matrix/pesticide) which have been submitted to collaborative studies and a list of indicative applicability of the method to different pesticides are given for each method, wherever possible.
Pflanzliche Lebensmittel - Multiverfahren zur gaschromatographischen Bestimmung von Pestizidrückständen - Teil 2: Verfahren zur Extraktion und Reinigung
Diese Europäische Norm legt Verfahren für die Extraktion und Reinigung pflanzlicher Lebensmittel zur Bestimmung von Pestizidrückständen fest.
Zu diesem Zweck können verschiedene Lösemittel verwendet werden. Die Pestizidrückstände liegen im Allgemeinen gemeinsam mit mitextrahierten Begleitstoffen vor, die bei der Analyse stören können. Zur Reinigung dieser rohen Extrakte können unterschiedliche Verfahren angewendet werden.
Diese Europäische Norm enthält die folgenden Extraktions und Reinigungsverfahren, die in Ringversuchen erfolgreich geprüft worden und/oder in ganz Europa anerkannt sind:
Verfahren L: Extraktion mit Aceton, Flüssig/flüssig Verteilung mit Dichlormethan und Reinigung an einer Kieselgel/Aktivkohle Säule [1];
Verfahren M: Extraktion mit Aceton und Flüssig/flüssig Verteilung mit Dichlormethan/Petroleumbenzin, falls erforderlich, Reinigung an Florisil® ) [2], [3], [4];
Verfahren N: Extraktion mit Aceton, Flüssig/flüssig Verteilung mit Dichlormethan oder Cyclohexan/Ehtyl¬acetat und Reinigung durch Gelpermeations und Kieselgel Chromatographie [5], [6];
Verfahren P: Extraktion mit Ethylacetat und, falls erforderlich, Reinigung durch Gelpermeations-Chromatographie [7].
Diese Europäische Norm legt Einzelheiten der Verfahren L bis P zur Extraktion und Reinigung von Proben pflanzlicher Lebensmittel fest. Zur Extraktion werden mehrere Lösemittel in unterschiedlichen Mengen verwendet. Als Reinigungsverfahren werden z. B. die Flüssig/flüssig Verteilung, die Flüssigkeitschromatogra¬phie an verschiedenen Adsorptionsmitteln und die Gelpermeations Chromatographie aufgeführt.
Soweit möglich, ist in jedem Verfahren der jeweilige Anwendungsbereich beschrieben, und die Pestizid/ Matrix-Kombinationen, die in Ringversuchen untersucht wurden, sind tabellarisch aufgelistet, siehe Tabelle 3.
Aliments d'origine végétale - Méthodes multirésidus de détermination par chromatographie en phase gazeuse de résidus de pesticides - Partie 2: Méthodes d'extraction et de purification
La présente Norme européenne spécifie des méthodes pour l'extraction et la purification d'échantillons d'aliments d’origine végétale en vue du dosage des résidus de pesticides.
Différents solvants peuvent ainsi être utilisés. Ces résidus de pesticides sont généralement associés à d'autres composés co-extraits pouvant interférer avec l'analyse. Plusieurs méthodes peuvent être utilisées pour purifier les extraits bruts devant être analysés.
La présente Norme européenne contient les méthodes suivantes qui ont été soumises à des études interlaboratoires et/ou qui sont adoptées dans toute l'Europe :
méthode L : Extraction par acétone, partage liquide-liquide par dichlorométhane et purification sur colonne de silicagel/charbon actif [1] ;
méthode M : Extraction par acétone, partage liquide-liquide par dichlorométhane/éther de pétrole et purification sur colonne de Florisil® ) si nécessaire [2], [3], [4] ;
méthode N : Extraction par acétone, partage liquide-liquide par dichlorométhane ou cyclohexane/acétate d’éthyle et purification par chromatographie par perméation sur gel et au silicagel [5], [6] ;
méthode P : Extraction avec acétate d'éthyle et, si nécessaire, purification par chromatographie par perméation de gel [7].
La présente Norme européenne détaille les méthodes L à P, pour l'extraction et la purification d'échantillons d'aliments d’origine végétale. L'extraction fait appel à plusieurs solvants, à différents volumes. Les techniques de purifications telles que la séparation liquide-liquide, la chromatographie liquide sur divers adsorbants et la chromatographie sur colonne de perméation de gel sont indiquées.
Un tableau contenant les couples (matrice/pesticide) qui ont été soumis à des études collaboratives, ainsi qu'une liste indicative d'applicabilité de la méthode aux différents pesticides sont donnés pour chaque méthode, lorsque c'est possible.
Živila rastlinskega izvora - Multirezidualne metode za določevanje ostankov pesticidov s plinsko kromatografijo - 2. del: Metode za ekstrakcijo in čiščenje
General Information
Relations
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Pflanzliche Lebensmittel - Multiverfahren zur gaschromatographischen Bestimmung von Pestizidrückständen - Teil 2: Verfahren zur Extraktion und ReinigungAliments d'origine végétale - Méthodes multirésidus de détermination par chromatographie en phase gazeuse de résidus de pesticides - Partie 2: Méthodes d'extraction et de purificationFoods of plant origin - Multiresidue methods for the gas chromatographic determination of pesticide residues - Part 2: Methods for extraction and cleanup67.080.01Sadje, zelenjava in njuni proizvodi na splošnoFruits, vegetables and derived products in general67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 12393-2:2008SIST EN 12393-2:2009en,fr,de01-februar-2009SIST EN 12393-2:2009SLOVENSKI
STANDARDSIST EN 12393-2:19991DGRPHãþD
SIST EN 12393-2:2009
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 12393-2November 2008ICS 67.080.01Supersedes EN 12393-2:1998
English VersionFoods of plant origin - Multiresidue methods for the gaschromatographic determination of pesticide residues - Part 2:Methods for extraction and cleanupAliments d'origine végétale - Méthodes multirésidus dedétermination par chromatographie en phase gazeuse derésidus de pesticides - Partie 2: Méthodes d'extraction etde purificationPflanzliche Lebensmittel - Multiverfahren zurgaschromatographischen Bestimmung vonPestizidrückständen - Teil 2: Verfahren zur Extraktion undReinigungThis European Standard was approved by CEN on 13 September 2008.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN 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 translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, 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 STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2008 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 12393-2:2008: ESIST EN 12393-2:2009
EN 12393-2:2008 (E) 2 Contents Page Foreword.3 Introduction.4 1 Scope.5 2 Normative references.5 3 Principles.5 4 General: Summary of procedures.6 5 Method L: Extraction with acetone, liquid-liquid partition with dichloromethane and clean-up on a silica gel/charcoal column.7 6 Method M: Extraction with acetone and liquid-liquid partition with dichloromethane/light petroleum, if necessary clean-up on Florisil®.13 7 Method N: Extraction with acetone, liquid-liquid partition with dichloromethane or cyclohexane/ethyl acetate, clean-up with gel permeation and silica gel chromatography.16 8 Method P: Extraction with ethyl acetate, and if necessary, clean-up by gel permeation chromatography.34 Annex A (informative)
Average water content of crops and foods.37 Bibliography.38
SIST EN 12393-2:2009
EN 12393-2:2008 (E) 3 Foreword This document (EN 12393-2:2008) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by May 2009, and conflicting national standards shall be withdrawn at the latest by May 2009. 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 12393-2:1998 with following significant technical changes: a) deletion of method O: Extraction with acetonitrile, liquid-liquid partition with light petroleum and clean-up on a Florisil® column; b) addition of an alternative partition step and expansion of the pesticide spectrum in method N: Extraction with acetone, liquid-liquid partition with dichloromethane or cyclohexane/ethyl acetate, clean-up with gel permeation and silica gel chromatography. This European Standard EN 12393 “Foods of plant origin - Multiresidue methods for the gas chromatographic determination of pesticide residues” is divided in three parts: - Part 1 "General considerations" provides general considerations with regard to reagents, apparatus, gas chromatography, etc., applying to each of the analytical selected methods.
- Part 2 "Methods for extraction and clean-up" presents methods L to P for the extraction and clean-up using techniques such as liquid-liquid partition, adsorption column chromatography or gel permeation column chromatography, etc.
- Part 3 "Determination and confirmatory tests" gives some recommended techniques for the qualitative and the quantitative measurements of residues and the confirmation of the results.
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, 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 the United Kingdom. SIST EN 12393-2:2009
EN 12393-2:2008 (E) 4 Introduction This European Standard comprises a range of multi-residue methods of equal status: no single method can be identified as the prime method because, in this field, methods are continuously developing. The selected methods included in this standard have been validated and/or are widely used throughout Europe. Because these methods can be applied to the very wide range of food commodities/pesticide combinations, using different systems for determination, there are occasions when variations in equipment used, extraction, clean-up and chromatographic conditions are appropriate to improve method performance, see Clause 3. SIST EN 12393-2:2009
EN 12393-2:2008 (E) 5 1 Scope This European Standard specifies methods for the extraction and clean-up of food samples of plant origin for quantitative determination of pesticide residues.
Different solvents can be used for this purpose. These pesticide residues are generally associated with other co-extracted compounds which would interfere in the analysis. To purify the crude extracts to be analysed, several techniques can be used. This standard contains the following extraction and clean-up methods that have been subjected to interlaboratory studies and /or are adopted throughout Europe: method L: Extraction with acetone, liquid-liquid partition with dichloromethane and clean-up on a silica gel/charcoal column [1]; method M: Extraction with acetone and liquid-liquid partition with dichloromethane/light petroleum, if necessary clean-up on Florisil® 1) [2], [3], [4]; method N: Extraction with acetone, liquid-liquid partition with dichloromethane or cyclohexane/ethyl acetate and clean-up with gel permeation and silica gel chromatography [5], [6]; method P: Extraction with ethyl acetate, and if necessary, clean-up by gel permeation chromatography [7]. This European Standard specifies the details of methods L to P for the extraction and the clean-up of food samples of plant origin. Several solvents at different volumes are used for extraction. Techniques of clean-up are listed such as liquid-liquid partition, liquid chromatography on various adsorbents and gel permeation chromatography. A table providing the couples (matrix/pesticide) which have been submitted to collaborative studies and a list of indicative applicability of the method to different pesticides are given for each method, wherever possible. 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. EN 12393-1:2008, Foods of plant origin – Multiresidue methods for the gas chromatographic determination of pesticide residues – Part 1: General considerations EN 12393-3, Foods of plant origin – Multiresidue methods for the gas chromatographic determination of pesticide residues – Part 3: Determination and confirmatory tests 3 Principles As already described in the introduction, in certain occasions it is possible to improve the method performance by variations in equipment used, extraction, clean-up and chromatographic conditions. Such variations shall be always clearly documented and demonstrated to give valid results.
1) Florisil® is an example of a suitable product available commercially. This information is given for convenience of users of this standard and does not constitute an endorsement by CEN of this product. SIST EN 12393-2:2009
EN 12393-2:2008 (E) 6 The pesticide residues are extracted from the sample by the use of appropriate solvents, so as to obtain the maximum efficiency of extraction of the pesticide residues and minimum co-extracted substances which can give rise to interferences in the determination. Any interfering materials are removed from the sample extract to obtain a solution of the extracted pesticide residues in a solvent which is suitable for quantitative examination by the selected method of determination. 4 General: Summary of procedures 4.1 Extraction The extraction procedures are summarized in Table 1. Table 1 —Extraction procedures Methods Mass of samples Volume of solvent Ratio
(M S) g (V S) ml M S / V S g/ml L 100 Acetone: 200 ½
M 100 Acetone: 200 ½
N
100 a Acetone: 200 ½
P 50 Ethyl acetate: 100 ½
a Only relevant if the water content of the matrix is greater than 70 %. 4.2 Clean-up 4.2.1 Liquid-liquid partition The liquid-liquid partition procedures are summarized in Table 2. Table 2 —Liquid-liquid partition Methods Aliquot portion of extract Volume of added water Volume of solvent Ratio
(A E) ml (V W) ml (V S) ml A E / V W L 50 (= 20 %) 250 50 1/5 M 80 0 200 - a N 200 x a 100 - a a Depends on the water content of the matrix. Two techniques of liquid-liquid partition are proposed: with added water (methods L, N); no added water (method M).
4.2.2 Adsorption column chromatography Methods: L, M, N with different adsorbents: silica gel, charcoal, Florisil®, used pure or in mixture. SIST EN 12393-2:2009
EN 12393-2:2008 (E) 7 4.2.3 Gel permeation chromatography with BioBeads® S-X 32) Method N, and, if needed, method P. 5 Method L: Extraction with acetone, liquid-liquid partition with dichloromethane and clean-up on a silica gel/charcoal column 5.1 Principle The chopped test portion is homogenized in acetone and the homogenate is filtered. An aliquot portion of the filtrate is diluted with water and extracted with dichloromethane. The organic phase is concentrated and chromatographed on a column of silica gel and activated charcoal. The pesticide residues are eluted with a mixture of dichloromethane, toluene and acetone. The eluate is concentrated for examination by GC. 5.2 Reagents 5.2.1 General All reagents shall be suitable for the analysis of pesticide residues and in accordance with EN 12393-1:2008, Clause 4. 5.2.2 Acetone 5.2.3 Dichloromethane 5.2.4 n-Hexane 5.2.5 Toluene 5.2.6 Eluting mixture: dichloromethane/toluene/acetone 5+1+1 (V/V/V) 5.2.7 Sodium chloride solution, saturated 5.2.8 Sodium sulfate
Heat at 500 °C for at least 4 h, allow to cool and store in a stoppered bottle. 5.2.9 Activated charcoal 5.2.10 Silica gel 60 for column chromatography, 63 µm to 200 µm (70 mesh to 230 mesh) 5.2.11 Celite® 5453) (optional) 5.3 Apparatus Usual laboratory equipment in accordance with EN 12393-1 and, in particular, the following:
2) BioBeads® S-X 3 is an example of a suitable product available commercially. This information is given for convenience of users of this standard and does not constitute an endorsement by CEN of this product. 3) Celite® 545 is an example of a suitable product available commercially. This information is given for convenience of users of this standard and does not constitute an endorsement by CEN of this product. SIST EN 12393-2:2009
EN 12393-2:2008 (E) 8 5.3.1 High speed blender or homogenizer, with a suitable blender cup 5.3.2 Solvent evaporator, with a water bath, capable of being maintained at 40 °C 5.3.3 Chromatographic column, with a sintered glass disk and a polytetrafluoroethylene (PTFE) stopcock, 25 mm internal diameter, 400 mm long 5.4 Procedure 5.4.1 Preparation of the sample Chop the sample into small pieces and mix thoroughly. 5.4.2 Extraction Weigh 100 g of the coarsely comminuted sample into a 1 l beaker, add 200 ml of acetone and homogenize for approximately 30 s. If necessary, Celite® 545 can be used additionally as a filter aid. Rinse the homogenizer with 50 ml of acetone and reserve the washing for rinsing the beaker and the Büchner funnel later. Filter the homogenate with suction through a moistened round filter paper in a Büchner funnel. Rinse the filter cake with the 50 ml portion of acetone used earlier as washing liquid. Thoroughly shake the filtrate and measure its volume. Take exactly one-fifth of this filtrate and shake it vigorously for at least 2 min with 250 ml of water, 25 ml of sodium chloride solution (5.2.7) and 50 ml of dichloromethane in a 1 l separatory funnel. If the filtrate is not shaken sufficiently well, the recovery is possibly reduced substantially. Repeat this extraction with 50 ml of dichloromethane. Combine the dichloromethane phases, and dry on 30 g of sodium sulfate (5.2.8) for 30 min. Filter the dried extract through a filter paper. Rinse the flask and filter paper with 30 ml of dichloromethane applied in three portions. Evaporate the filtrate to approximately 2 ml, and remove the last traces of solvent by swirling the flask manually. Dissolve the residue in 10 ml of dichloromethane. 5.4.3 Column preparation Fill the chromatographic column (5.3.3) with dichloromethane to a level of 1 cm. Slurry 5 g of silica gel (5.2.10) in 15 ml of eluting mixture (5.2.6), and pour the slurry into the column. Drain off the supernatant. Next, thoroughly mix 15 g of silica gel and 1 g of activated charcoal in a 50 ml beaker, and slowly add 35 ml of eluting mixture (Caution: Generation of heat). Do not add more than 35 ml of eluting mixture, otherwise the suspension will become separated into phases, resulting in poor passage of active ingredients through the column. Add the activated charcoal-silica gel mixture onto the silica gel in the chromatographic column, by pouring it through a funnel, at first slowly and then in a gush, at the same time stirring constantly and with the column stopcock open. Use any eluate that has already passed through the column for rinsing the flask. Drain the eluting mixture to a level 2 cm above the packing, and top the column with a total of 5 g of sodium sulfate added in small portions. Next pre-wash the column with 50 ml of eluting mixture. 5.4.4 Clean-up Transfer the dichloromethane solution derived from 5.4.2 quantitatively to the prepared column, completing the transfer with a total of 5 ml of dichloromethane. Collect the liquid already flowing through the column and the subsequent eluate in a 250 ml round-bottomed flask. Elute the column with 140 ml of eluting mixture (5.2.6). Evaporate the collected eluates to approximately 30 ml. Transfer it to a 50 ml round-bottomed flask and evaporate again to approximately 2 ml. Beforehand, empty the receiver of the rotary evaporator. Do not on any account rotary-evaporate the solution to dryness. Transfer the solution to a graduated test tube and dilute with n-hexane to 5,0 ml. SIST EN 12393-2:2009
EN 12393-2:2008 (E) 9 5.5 Gas chromatography Use a gas chromatographic system suitable for determining organohalogen, organophosphorus and organonitrogen pesticide residues. 5.6 Collaborative studies Couples of matrices and pesticides which have been submitted to collaborative studies4) are presented in Table 3. Table 3 — Matrices and pesticides
carrot potato savoy cabbage spinach tomato yellow pea bromophos + +
+
bromopropylate
+ +
captan
+
chlorpropham
+
chlorpyrifos
+ +
cypermethrin
+
o, p'-DDE +
p, p'-DDE +
+
o, p'-DDT +
p, p'-DDT +
+
diazinon +
+
+ dichlofluanid +
dicofol
+ +
dieldrin + + + + + + α-endosulfan
+
β-endosulfan
+ +
endosulfan sulfate
+ +
endrin
+
ethion
+
4) For the collaborative studies, the activated charcoal and the silica gel 60, 63 µm to 200 µm (70 mesh to 230 mesh) from the Merck Company were used. This information is given for convenience of users of this standard and does not constitute an endorsement by CEN of these products. SIST EN 12393-2:2009
EN 12393-2:2008 (E) 10 Table 3 (continued)
carrot potato savoy cabbage spinach tomato yellow pea fenarimol
+
fenitrothion +
+
fenpropathrin
+
folpet
+
α-HCH
+
heptachlor epoxide +
+
iprodione
+
lindane (γ-HCH) + + + + + + malathion
+
+ mecarbam
+
parathion +
+ + +
permethrin
+
phosalone +
+
+ pirimiphos-methyl
+
+ + + procymidone
+
propham
+
quintozene
+
+ tetradifon
+
tolclofos-methyl
+
vinclozoline + +
+ +
5.7 Applicability The following pesticides can be analysed by this method: Aldrin Dieldrin Metribuzin Ametryn Dimethachlor Mevinphos Atrazine Dimethoate Naled Azinphos-ethyl Dioxathion Nitrofen Azinphos-methyl Disulfoton Paraoxon Aziprotryne Ditalimfos Parathion Bifenthrin α-Endosulfan Parathion-methyl Bromacil ß-Endosulfan Pendimethalin Bromophos Endosulfan sulfate Permethrin SIST EN 12393-2:2009
EN 12393-2:2008 (E) 11 Bromophos-ethyl Ethion Perthane
Bromopropylate Ethoprophos Phenkapton
Bupirimate Etrimfos Phorate
Captafol Fenamiphos Phosalone
Captan Fenarimol Pirimiphos-methyl Carbophenothion Fenchlorphos Procymidone
Chlorbenside Fenitrothion Profenofos
Chlorfenson Fenpropathrin Profluralin
Chlorfenvinphos Fenson Prometryn
Chlorflurenol Fensulfothion Propazine
Chlorpropham Fenthion Propham Chlorobenzilate Fenvalerate Propyzamide
Chloropropylate Fluchloralin Prothiofos Chlorpyrifos Flucythrinate Pyrazophos
Chlorpyrifos-methyl Fluorodifen Pyrethrins Chlorthal Fluvalinate Quinalphos
Chlorthiophos Folpet Quintozene
Cyanazine Fonofos Simazine
Cyanofenphos Formothion Sulfotep
Cyanophos α-HCH Tecnazene
Cyfluthrin ß-HCH Terbacil
λ-Cyhalothrin Heptachlor Terbufos
Cypermethrin Heptachlor epoxide Terbutryn
p,p'-DDD Heptenophos Tetrachlorvinphos
o,p'-DDE Iodofenphos Tetradifon
p,p'-DDE Iprodione Tetramethrin o,p'-DDT Isofenphos Tetrasul
p,p'-DDT Lindane Thionazin
SIST EN 12393-2:2009
EN 12393-2:2008 (E) 12 Deltamethrin Malaoxon Tolclofos-methyl
Desmetryn Malathion Tolylfluanid
Dialifos Mecarbam Triadimefon
Diazinon Metalaxyl Tri-allate Dichlobenil Metazachlor Triazophos
Dichlofenthion Methidathion Trichloronat
Dichlofluanid Methoprotryne Trifluralin
Dichlorvos Methoxychlor Vinclozolin Dicofol Metolachlor
Crops and foods on which the method was tested: Apples Grapes Pineapples Apricots Head cabbage Plums Aubergines Honey Potatoes Beans Kohlrabi Radishes (large and small types) Carrots Leeks Red cabbage Celeriac Lettuce Savoy cabbage Cherries Mandarin oranges Spinach Chillies Mushrooms Strawberries Chinese cabbage Oranges Sweet peppers Corn salad Parsley Tomatoes Cucumbers Peaches Witloof chicory Dandelion Pears
Endives Peas
SIST EN 12393-2:2009
EN 12393-2:2008 (E) 13 6 Method M: Extraction with acetone and liquid-liquid partition with dichloromethane/light petroleum, if necessary clean-up on Florisil® 6.1 Principle The chopped test portion is homogenized in acetone and the homogenate is filtered. An aliquot portion of the filtrate is extracted with a mixture of light petroleum and dichloromethane and then with dichloromethane. The organic phase can be injected directly without clean up into a gas chromatograph with an appropriate detector or purified on a Florisil® column. The eluates are concentrated for examination by GC. 6.2 Reagents 6.2.1 General All reagents shall be suitable for the analysis of pesticide residues and in accordance with EN 12393-1:2008, Clause 4. 6.2.2 Acetone 6.2.3 Light petroleum, boiling range 40 °C to 60 °C 6.2.4 Sodium chloride
Heat at 500 °C for at least 4 h, allow to cool, and store in a stoppered bottle. 6.2.5 Dichloromethane 6.2.6 Acetonitrile 6.2.7 Sodium sulfate Heat at 500 °C for at least 4 h, allow to cool, and store in a stoppered bottle. 6.2.8 Florisil® (Floridin or equivalent), 150 µm to 250 µm (60 mesh to 100 mesh) Activate by heating at 130 °C to 135 °C for at least 5 h, allow to cool in a desiccator and transfer to an airtight stoppered jar. The adsorbent thus treated keeps its activity only for 4 days. It can subsequently be reactivated by the same treatment. The activity of the adsorbent should be checked from time to time by eluting pesticide standard materials as described in the method. 6.2.9 Diethyl ether, peroxide-free, containing 2 % (V+V) ethanol 6.2.10 Eluting solvent A: diethyl ether/light petroleum 6+94 (V/V) 6.2.11 Eluting solvent B: diethyl ether/light petroleum 15+85 (V/V) 6.2.12 Eluting solvent C: diethyl ether/light petroleum 50+50 (V/V) 6.2.13 Eluting solvent D: dichloromethane/light petroleum 20+80 (V/V) 6.2.14 Eluting solvent E: dichloromethane/light petroleum/acetonitrile 50+49,65+0,35 (V/V/V) SIST EN 12393-2:2009
EN 12393-2:2008 (E) 14 6.2.15 Eluting solvent F: dichloromethane/light petroleum/acetonitrile 50+48,5+1,5 (V/V/V) 6.3 Apparatus
Usual laboratory equipment in accordance with EN 12393-1 and, in particular, the following: 6.3.1 High speed blender or homogenizer, with a suitable blender cup 6.3.2 Chromatographic column, with a PTFE stopcock, 22 mm internal diameter, 300 mm long 6.3.3 Solvent evaporator, Kuderna Danish or equivalent 6.4 Procedure 6.4.1 Preparation of the sample Chop finely the test sample and mix carefully to obtain homogeneous test portions. If the water content of the sample is less than 30 %, adjust it to about 80 % by adding water. 6.4.2 Extraction and partition Weigh 100 g (m) of the prepared sample into the blender cup (6.3.1) and add 200 ml of acetone. Blend at high speed for 3 min. Transfer the mixture to a Büchner funnel containing a filter paper moistened with acetone, filter under suction into the Büchner flask and measure the volume of the filtrate. Pour 80 ml of filtrate in a 1 l separating funnel with 100 ml of dichloromethane and 100 ml of light petroleum (6.2.3). Shake for 3 min and leave to separate layers. Transfer the lower aqueous layer to a second 1 l separating funnel. Dry upper organic layer from the first separatory funnel by passing through 3 cm of sodium sulfate (6.2.7) supported on washed glass wool in 10 cm funnel collecting in a round-bottomed flask. Add 7 g of sodium chloride to the aqueous phase and shake for 30 s until sodium chloride (6.2.4) is dissolved. Add 100 ml of dichloromethane and shake for 3 min. Let the layers separate. Transfer the aqueous phase to a third separating funnel and dry the organic phase again on the same sodium sulfate. Add to the third separating funnel 100 ml of dichloromethane and shake for 3 min, separate and discard the aqueous phase and dry the dichloromethane phase on the same layer of sodium sulfate. Wash the sodium sulfate with 50 ml of dichloromethane and concentrate all organic phases to 2 ml. Add 100 ml of light petroleum and again concentrate to 2 ml and again until all dichloromethane disappears. Add 20 ml of acetone and reconcentrate to 2 ml (Vfinal). This concentrate may be injected directly into a gas chromatograph equipped with HECD (Hall detector), NPD or FPD (method M). In some cases, a clean-up is recommended for determination by ECD: methods M1 or M2. For purification, the sample extract is concentrated to 1 ml of acetone (instead of 2 ml) and diluted to a volume of 10 ml with light petroleum. 6.4.3 Clean-up 6.4.3.1 Method M1 Place a plug of cotton wool in the bottom of the chromatographic column (6.3.2) and fill with light petroleum (6.2.3) on 20 cm. Pour 20 g of Florisil® (6.2.8) and tap along the walls of the column to settle the adsorbent. Cover the top of the adsorbent with 1 cm to 2 cm of sodium sulfate (6.2.7). Wash the adsorbent with approximately 30 ml of light petroleum. Place the evaporator flask under the column to receive the eluate. Transfer the extract for purification as described in 6.4.2, to the column, allowing it to pass through at a rate of not more than 5 ml/min. Rinse the container with two 5 ml portions of light petroleum, pouring the rinsings onto SIST EN 12393-2:2009
EN 12393-2:2008 (E) 15 the column, rinse the walls of the chromatographic column with additional small portions of light petroleum and elute at 5 ml/min with 200 ml eluting solvent A (6.2.10). Elute further with 200 ml of eluting solvent B (6.2.11) into a separate receiver and finally with 200 ml of eluant C (6.2.12). Concentrate each eluate to a suitable definite volume (Vfinal) for examination by GC. 6.4.3.2 Method M2 Place a plug of cotton wool in the bottom of the chromatographic column (6.3.2) and fill with light petroleum (6.2.3) on 20 cm. Pour 20 g of Florisil® (6.2.8) and tap along the walls of the column to settle the adsorbent. Cover the top of the adsorbent with 1 cm to 2 cm of sodium sulfate (6.2.7).
Wash the adsorbent with approximately 30 ml of light petroleum. Place the evaporator flask under the column to receive the eluate. Transfer the extract for purification as described in 6.4.2 to the column, allowing it to pass through at a rate of not more than 5 ml/min. Rinse the container with two 5 ml portions of light petroleum, pouring the rinsings onto the column, rinse the walls of the chromatographic column with additional small portions of light petroleum and elute at 5 ml/min with 200 ml of eluting solvent D (6.2.13). Elute further with 200 ml of eluting solvent E (6.2.14) into a separate receiver and finally with 200 ml of eluant F (6.2.15). Concentrate each eluate to a suitable definite volume (Vfinal) for examination by GC. 6.5 Gas chromatography and calculations Use a gas chromatographic system suitable for determining organohalogen, organophosphorus and organo-nitrogen pesticide residues. The equivalent sample weight in milligrams per microlitre of final extract solution is calculated according to Equation (1): ρ = final
10
20080VmWw×−+δ (1) where ρ
is the sample equivalent in milligrams per microlitre; m
is the mass of the test portion, in grams (here: 100 g); W
is the amount of water present in sample, in grams; δw
is the density of water, in milligrams per microlitre; Vfinal
is the volume of final extract solution, in millilitres; 10 ml is the contraction volume. Contraction volume is taken as 10 ml for samples containing 80 g/100 g to 95 g/100 g of water when 200 ml of acetone is used for extraction. Consult reference documents on food composition for average water content. An example of average water content for some crops and vegetables is given in Table A.1. The water content of most fresh fruit and vegetables can be assumed to be 85 g/100 g. NOTE The sample amount in the final extract solution for Florisil® clean-up or GC, can be calculated exactly by measuring the total volume of the original acetone extract. SIST EN 12393-2:2009
EN 12393-2:2008 (E) 16 7 Method N: Extraction with acetone, liquid-liquid partition with dichloromethane or cyclohexane/ethyl acetate, clean-up with gel permeation and silica gel chromatography 7.1 Principle The chopped test portion is homogenized in acetone, after addition of water, depending on the natural water content of the sample, in order to ensure an acetone/water ratio of 2/1 (V+V). The homogenate is filtered. An aliquot portion of the filtrate is saturated with sodium chloride and diluted with dichloromethane, resulting in separa
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.