EN 12393-2:2013
(Main)Foods of plant origin - Multiresidue methods for the determination of pesticide residues by GC or LC-MS/MS - Part 2: Methods for extraction and clean-up
Foods of plant origin - Multiresidue methods for the determination of pesticide residues by GC or LC-MS/MS - Part 2: Methods for extraction and clean-up
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 European Standard contains the following extraction and clean-up methods that have been subjected to interlaboratory studies and/or are adopted throughout Europe:
- method M: Extraction with acetone and liquid-liquid partition with dichloromethane/light petroleum, if necessary clean-up on Florisil® ) [1], [2], [3];
- method N: Extraction with acetone, liquid-liquid partition with dichloromethane or cyclohexane/ethyl acetate and clean-up with gel permeation and silica gel chromatography [4], [5];
- method P: Extraction with ethyl acetate, and if necessary, clean-up by gel permeation chromatography [6].
This European Standard specifies the details of methods M, N and 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 Bestimmung von Pestizidrückständen mit GC oder LC-MS/MS - 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 M: Extraktion mit Aceton und Flüssig/Flüssig Verteilung mit Dichlormethan/Petroleumbenzin, falls erforderlich, Reinigung an Florisil® ) [1], [2], [3];
- Verfahren N: Extraktion mit Aceton, Flüssig/Flüssig Verteilung mit Dichlormethan oder Cyclohexan/Ehtyl-acetat und Reinigung durch Gelpermeations und Kieselgel Chromatographie [4], [5];
- Verfahren P: Extraktion mit Ethylacetat und, falls erforderlich, Reinigung durch Gelpermeations-Chromatographie [6].
Diese Europäische Norm legt Einzelheiten der Verfahren M, N und 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 getestet wurden, sind tabellarisch aufgelistet, siehe Tabelle 3.
Aliments d'origine végétale - Méthodes multirésidus de détermination de résidus de pesticides par CPG ou CL-SM/SM - 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 d'extraction et de purification suivantes qui ont été soumises à des études interlaboratoires et/ou qui sont adoptées dans toute l'Europe :
- méthode M : extraction à l'acétone, partage liquide-liquide au dichlorométhane/éther de pétrole et purification au Florisil®, le cas échéant ) [1], [2], [3] ;
- méthode N : extraction par acétone, partage liquide-liquide par dichlorométhane ou cyclohexane/acétate d'éthyle et purification par chromatographie par perméation de gel et de gel de silice [4], [5] ;
- méthode P : extraction avec l'acétate d'éthyle et, si nécessaire, purification par chromatographie par perméation de gel [6].
La présente Norme européenne détaille les méthodes M, N et 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 purification telles que le partage liquide-liquide, la chromatographie liquide sur divers adsorbants et la chromatographie par perméation de gel sont mentionnées.
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 ali tekočinsko kromatografijo z masno selektivno detekcijo (LC-MS/MS) - 2. del: Metode za ekstrakcijo in čiščenje
Ta evropski standard določa metode za ekstrakcijo in čiščenje vzorcev živil rastlinskega izvora za kvantitativno določevanje ostankov pesticidov. V ta namen se lahko uporabljajo različna topila. Ti ostanki pesticidov so običajno povezani z drugimi sočasno ekstrahiranimi spojinami, ki bi motile analizo. Za purifikacijo surovih ekstraktov za analizo se lahko uporabi več tehnik. Ta evropski standard zajema naslednje metode za ekstrakcijo in čiščenje, ki so bile predmet medlaboratorijskih študij in/ali so sprejete po vsej Evropi: – metoda M: ekstrakcija z acetonom in ločitev tekoče-tekoče z diklorometanom/lahkim bencinom, po potrebi čiščenje s Florisil® 1) [1], [2], [3]; – metoda N: ekstrakcija z acetonom, ločitev tekoče-tekoče z diklorometanom ali cikloheksanom/etil acetatom ter čiščenje z gelsko permeacijsko in silikagelsko kromatografijo [4], [5]; – metoda P: ekstrakcija z etil acetatom in po potrebi čiščenje z gelsko permeacijsko kromatografijo [6]. Ta evropski standard določa podrobnosti metod M, N in P za ekstrakcijo ter čiščenje vzorcev živil rastlinskega izvora. Za ekstrakcijo se uporablja več topil v različnih količinah. Navedene so tehnike čiščenja, kot so ločitev tekoče tekoče, tekočinska kromatografija za različne adsorbente in gelska permeacijska kromatografija. Preglednica s pari (matrika/pesticid), ki so bili predloženi za medlaboratorijske študije, in seznam indikativne uporabnosti metode za različne pesticide sta navedena za vsako metodo, kadar je to mogoče.
General Information
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Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Pflanzliche Lebensmittel - Multiverfahren zur Bestimmung von Pestizidrückständen mit GC oder LC-MS/MS - Teil 2: Verfahren zur Extraktion und ReinigungAliments d'origine végétale - Méthodes multirésidus de détermination de résidus de pesticides par CPG ou CL-SM/SM - Partie 2: Méthodes d'extraction et de purificationFoods of plant origin - Multiresidue methods for the determination of pesticide residues by GC or LC-MS/MS - Part 2: Methods for extraction and clean-up67.080.01Sadje, 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 12393-2:2013SIST EN 12393-2:2014en,fr,de01-januar-2014SIST EN 12393-2:2014SLOVENSKI
STANDARDSIST EN 12393-2:20091DGRPHãþD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 12393-2
November 2013 ICS 67.050 Supersedes EN 12393-2:2008English Version
Foods of plant origin - Multiresidue methods for the determination of pesticide residues by GC or LC-MS/MS - Part 2: Methods for extraction and clean-up
Aliments d'origine végétale - Méthodes multirésidus de détermination de résidus de pesticides par CPG ou CL-SM/SM - Partie 2: Méthodes d'extraction et de purification Pflanzliche Lebensmittel - Multiverfahren zur Bestimmung von Pestizidrückständen mit GC oder LC-MS/MS - Teil 2: Verfahren zur Extraktion und Reinigung This European Standard was approved by CEN on 21 September 2013.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12393-2:2013: ESIST EN 12393-2:2014
Average water content of crops and foods . 41 Bibliography . 42
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 European 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 12393-2:2014
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 European Standard contains the following extraction and clean-up methods that have been subjected to interlaboratory studies and/or are adopted throughout Europe: method M: Extraction with acetone and liquid-liquid partition with dichloromethane/light petroleum, if necessary clean-up on Florisil® 1) [1], [2], [3]; method N: Extraction with acetone, liquid-liquid partition with dichloromethane or cyclohexane/ethyl acetate and clean-up with gel permeation and silica gel chromatography [4], [5]; method P: Extraction with ethyl acetate, and if necessary, clean-up by gel permeation chromatography [6]. This European Standard specifies the details of methods M, N and 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 documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN 12393-1:2013, Foods of plant origin — Multiresidue methods for the determination of pesticide residues by GC or LC-MS/MS — Part 1: General considerations EN 12393-3:2013, Foods of plant origin — Multiresidue methods for the determination of pesticide residues by GC or LC-MS/MS — 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. 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.
1) Florisil®,is an example of a suitable product available commercially from U.S. Silica company. This information is given for convenience of users of this standard and does not constitute an endorsement by CEN of this product. SIST EN 12393-2:2014
g ml g/ml M 100 Acetone: 200 ½
N
100 a Acetone: 200 ½
P 10 Ethyl acetate: 20 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 Method Aliquot portion of extract Volume of added water Volume of solvent Ratio
(AE) ml (VW) ml (VS) ml AE / VW 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 (method N); no added water (method M). 4.2.2 Adsorption column chromatography Methods: M, N with different adsorbents: silica gel, charcoal, Florisil®, used pure or in mixture. 4.2.3 Gel permeation chromatography with Bio-Beads® S-X32) Method N, and, if needed, method P.
2) BioBeads® S-X3 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 12393-2:2014
Heat at 500 °C for at least 4 h, allow to cool, and store in a stoppered bottle. 5.2.5 Dichloromethane 5.2.6 Acetonitrile 5.2.7 Sodium sulfate
Heat at 500 °C for at least 4 h, allow to cool, and store in a stoppered bottle. 5.2.8 Florisil® (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 four 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. 5.2.9 Diethyl ether, peroxide-free, containing 2 % (V+V) ethanol 5.2.10 Eluting solvent A: diethyl ether/light petroleum 6+94 (V/V) 5.2.11 Eluting solvent B: diethyl ether/light petroleum 15+85 (V/V) 5.2.12 Eluting solvent C: diethyl ether/light petroleum 50+50 (V/V) 5.2.13 Eluting solvent D: dichloromethane/light petroleum 20+80 (V/V) 5.2.14 Eluting solvent E: dichloromethane/light petroleum/acetonitrile 50+49,65+0,35 (V/V/V) 5.2.15 Eluting solvent F: dichloromethane/light petroleum/acetonitrile 50+48,5+1,5 (V/V/V) SIST EN 12393-2:2014
Usual laboratory equipment in accordance with EN 12393-1 and, in particular, the following: 5.3.1 High speed blender or homogenizer, with a suitable blender cup 5.3.2 Chromatographic column, with a PTFE stopcock, 22 mm internal diameter, 300 mm long 5.3.3 Solvent evaporator, Kuderna Danish3) or equivalent 5.4 Procedure 5.4.1 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. NOTE The general water content of some crops and foods is given in Table A.1. 5.4.2 Extraction and partition Weigh 100 g (m) of the prepared sample into the blender cup (5.3.1) and add 200 ml (VEx) 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 (VR1) of filtrate in a 1 l separating funnel with 100 ml of dichloromethane and 100 ml of light petroleum (5.2.3). 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 (5.2.7) supported on washed glass wool in 10 cm funnel collecting in a round-bottomed flask. Add 7 g of sodium chloride (5.2.4) to the aqueous phase and shake for 30 s until sodium chloride (5.2.4) 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 e.g. 2 ml and again until all dichloromethane disappears. Add 20 ml of acetone and reconcentrate to 2 ml (Vend). 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. 5.4.3 Clean-up 5.4.3.1 Method M1 Place a plug of cotton wool in the bottom of the chromatographic column (5.3.2) and fill with light petroleum (5.2.3) on 20 cm. Pour 20 g of Florisil® (5.2.8) 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 (5.2.7).
3) The Kuderna-Danish evaporator is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. SIST EN 12393-2:2014
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 5.4.2 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 (5.2.13). Elute further with 200 ml of eluting solvent E (5.2.14) into a separate receiver and finally with 200 ml of eluent F (5.2.15). Concentrate each eluate to a suitable definite volume, e.g. 2 ml (Vend) for examination by GC. 5.5 Gas chromatography Use a gas chromatographic system suitable for determining organohalogen, organophosphorus and organo-nitrogen pesticide residues as described in EN 12393-3. Inject an aliquot portion (Vi) of the eluates obtained in 5.4.2, 5.4.3.1 or 5.4.3.2 into the gas chromatograph. 5.6 Calculation of residues The residue R, expressed in milligrams per kilogram, of an identified analyte is calculated from Formula (1):
Ex1waterendStAR1iSt()VfVVWFRVVmF+××××=××× (1) where VEx
is the volume of acetone added in extraction step 5.4.2; f1
is the factor considering the volume contraction from mixing acetone with the water present in the test portion (Vwater). The typical value of f1 is 0,90; Vwater is the volume of water present in the test portion. 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; Vend
is the final volume of eluate solution obtained in 5.4.2, 5.4.3.1 or 5.4.3.2; WSt
is the mass of analyte injected with standard solution; FA
is the peak area obtained from final extract; SIST EN 12393-2:2014
is the portion of volume VEx used for partition in 5.4.2; Vi
is the portion of volume Vend injected into the gas chromatograph; m
is the mass of the test portion, in grams; FSt
is the peak area obtained from WSt. 6 Method N: Extraction with acetone, liquid-liquid partition with dichloromethane or cyclohexane/ethyl acetate, clean-up with gel permeation and silica gel chromatography 6.1 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 separation of excess water. Alternatively, sodium chloride and a mixture of cyclohexane and ethyl acetate are added to the homogenate and the mixture is intensively mixed. The organic phase is concentrated and cleaned up by gel permeation chromatography (GPC) on Bio-Beads S-X3® (polystyrene gel) using a mixture of cyclohexane and ethyl acetate as eluent. The residue-containing fraction is concentrated, and analysed directly by gas chromatography using a phosphorus/nitrogen selective detector, a flame photometric detector or a mass spectrometer. After solvent exchange, the same fraction can be used for determination by LC-MS/MS. For analysis by electron capture and in some cases also by nitrogen-selective detection, a supplemental clean-up on a small silica gel column may be necessary. In this clean-up step, the pesticides are separated in several fractions thus providing additional leads for identification.
6.2 Reagents 6.2.1 General All reagents shall be suitable for the analysis of pesticide residues and in accordance with EN 12393-1:2013, Clause 4. 6.2.2 Acetone 6.2.3 Dichloromethane 6.2.4 Ethyl acetate 6.2.5 Cyclohexane 6.2.6 GPC eluting mixture: cyclohexane/ethyl acetate 1+1 (V/V) 6.2.7 n-Hexane 6.2.8 Isooctane 6.2.9 Toluene 6.2.10 Water, for chromatography 6.2.11 Methanol SIST EN 12393-2:2014
Heat at 500 °C for at least 4 h, allow to cool and store in a stoppered bottle. 6.2.19 Sodium sulfate, powder
Heat at 500 °C for at least 4 h, allow to cool and store in a stoppered bottle. 6.2.20 Salt mixture: sodium sulfate + sodium chloride 1+1 (w/w) 6.2.21 Celite® 545 4)
6.2.22 Silica gel 60 for column chromatography, 63 µm to 200 µm (70 mesh to 230 mesh), deactivated with 1,5 % water Heat the silica gel for at least 5 h at 130 °C, allow to cool in a desiccator, and store in a tightly stoppered container in the desiccator. To 98,5 g of dried silica gel in a 300 ml conical flask (with ground joint), add 1,5 ml of water dropwise from a burette with continuous swirling. Immediately stopper the flask with ground stopper, shake vigorously for 5 min until all lumps have disappeared, next shake for 2 h on a mechanical shaker, and then store in a tightly stoppered container. 6.2.23 Glass wool, extracted exhaustively with acetone 6.2.24 Cotton-wool, extracted exhaustively with acetone 6.2.25 Bio-Beads® S-X3, 38 µm to 75 µm (200 mesh to 400 mesh), 3 % crosslinked, styrene divinylbenzene beads for size limit chromatography, 2 000 mol weight limit 6.2.26 Filter paper, 6 cm and 13,5 cm diameter, fast flow rate, extracted exhaustively with acetone 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 Solvent evaporator, e.g. rotary evaporator for reducing sample extracts and concentration workstation, for preparation of final LC-MS/MS extracts, or similar devices
4) Celite 545®, (high-purity fluxcalcined diatomaceous silica, especially prepared for chromatography) 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.
6.4.1.3 Plant material with low water content Weigh out 10 g to 50 g (m) of the dry or dried matrix having a water content of x g/100 g (for example 25 g to 50 g for dried fruit and dried vegetables; 10 g to 20 g for spices and tea; 50 g for cereal grains). Then add sufficient water to adjust the total water present to 100 g6). The amount of water (W) to be added is calculated from the formula W = 100 - (m × x)/100. Blend and let stand for 10 min to 20 min. Next add 200 ml of acetone and homogenize for 3 min. 6.4.2 Partition 6.4.2.1 Partition with dichloromethane Add 10 g of Celite® 545 and again homogenize for 10 s. Filter the homogenate derived from 6.4.1.2 or 6.4.1.3 through a fast flow rate filter paper (6.2.26) in a Büchner funnel, with gentle vacuum suction, until more than 200 ml of filtrate is collected. To avoid losses of solvent due to strong vacuum, it is recommended only to use a low vacuum. Do not allow the filter cake to pull dry. Measure out 200 ml of filtrate (VR1) in a graduated cylinder, and transfer to a 500 ml separatory funnel. Add 20 g of sodium chloride (6.2.18), and shake vigorously for 3 min. Next add 100 ml of dichloromethane (6.2.3), shake for 2 min, and then let stand for approximately 10 min to allow the phases to separate. Discard the lower aqueous phase. Collect the organic phase in a flask and add approximately 25 g of sodium sulfate (6.2.19), let stand for approximately 30 min with occasional swirling, and then filter through a cotton-wool plug (6.2.24) layered with 3 cm of sodium sulfate in a funnel. Collect the filtrate in a 500 ml round-bottomed flask, and rinse separatory funnel and filter twice with 20 ml portions of ethyl acetate (6.2.4). Concentrate the
5) GPC Autoprep 1001 or 1002® are examples 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 these products. 6) 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. SIST EN 12393-2:2014
6.4.3.2 Packing gel permeation column Allow the Bio-Beads® (approximately 50 g) to swell overnight in the GPC eluting mixture (6.2.6). Then pour the suspension all at once into the column (capacity of approximately 180 ml). As soon as the gel bed has settled (free from air bubbles) to a level of approximately 32 cm, insert the plunger, lower it down to the bed level, and screw it into place. If the gel bed sinks to a still lower level after prolonged operation, the plunger shall be adjusted accordingly (observe manufacturer's instructions). 6.4.3.3 Checking elution volumes For each gel permeation column before the first use, the elution conditions shall be checked on several analytes of the lower and upper elution volume range (see Table 3), and on appropriate crude extracts. To do so, load the sample loop with crude extracts or a mixture of standard solutions, elute as described in step 6.4.3.4 and determine by means of a suitable analytical method whether the added analytes are completely recovered or whether interferences are caused by non-separated impurities. The same check shall be carried out on analytes after they have been in use for a lengthy period. NOTE It has been shown that some matrices can introduce randomized adsorption effect of certain analytes on Bio-Beads® resin and can cause false negative or false positive results. 6.4.3.4 Clean-up of crude extracts To the concentrated residue of a crude extract obtained in 6.4.2.1 or 6.4.2.2 add exactly 7,5 ml of ethyl acetate and dissolve by gentle swirling. Add approximately 5 g of salt mixture (6.2.20) for binding the remaining water, swirl again, and add exactly 7,5 ml of cyclohexane to yield a total volume of 15,0 ml (VR2). Shake for approximately 20 s, allow the salt mixture to settle and filter through a fast flow-rate filter paper, and inject 5 ml of the filtrate (VR3) into one of the sample loops of the gel permeation chromatograph. Elute the gel permeation column with the GPC eluting mixture at a flow rate of 5,0 ml/min. The instrument switches of the gel permeation chromatograph are adjusted according to the checking of the elution volumes. The settings depend on the target pesticides. Typical settings are as follows (see also Table 3): dump switch to 18 min to discard 90 ml; SIST EN 12393-2:2014
ml 1 t
2 u
3 v
4 w
5 x
3,4,5-Trimethacarb 100 to 140 0 0 5 0 0 Acephate 115 to 145 0 0 0 0 5 Acetamiprid 120 to 220 0 0 0 0 5 Aclonifen 115 to 145 0 5 0 0 0 Acrinathrin a
70 to 120 4 1 0 0 0 Alachlor 125 to 150 0 0 5 0 0 Aldrin 120 to 150 5 0 0 0 0 Ametryn 115 to 190 0 0 1 3 0 Amidithion 115 to 145 0 0 0 4 3 Anilazine b
105 to 135 0 0 5 0 0 Anthraquinone 145 to 185 0 2 4 0 0 Atrazine 110 to 135 0 0 4 3 0 Azinphos-ethyl 130 to 160 0 0 5 0 0 Azinphos-methyl 145 to 180 0 0 4 0 0 Azoxystrobin 120 to 155 0 0 0 5 0 Bendiocarb 130 to 160
Benfluralin 100 to 130 5 0 0 0 0 Bensulfuron-methyl 100 to 150
Benthiavalicarb-isopropyl a 60 to 110 0 0 0 5 0 Benzoylprop-ethyl 125 to 150 0 3 3 0 0 Bifenox 115 to 150 0 3 3 0 0 Bifenthrin a 090 to 120 0 5 0 0 0 Binapacryl 100 to 130 0 5 0 0 0 Bitertanol 100 to 130 0 0 0 4 2 Boscalid 105 to 130 0 0 5 0 0 Bromacil c
105 to 140 0 0 0 5 0 Bromophos 120 to 150 4 2 0 0 0 Bromophos-ethyl 110 to 140 5 1 0 0 0 Bromopropylate 095 to 135 0 0 3 3 1 Bromoxynil octanoate 120 to 150 0 5 1 0 0 Buprofezin a 90 to 120 0 0 4 0 0 Butocarboxim-sulfoxide 120 to 160
Butoxycarboxim 105 to 140
Camphechlor (Toxaphene) 110 to 150 5 1 0 0 0
ml 1 t 2 u 3 v 4 w 5 x Captafol d
120 to 150 0 0 5 0 0 Captan d 120 to 150 0 0 5 0 0 Carbaryl 125 to 170 0 0 5 0 0 Carbendazim 135 to 200
Carbofuran 125 to 155 0 0 5 0 0 Carbophenothion 120 to 140 0 3 0 0 0 Carbophenothion-methyl 120 to 160 0 4 0 0 0 Chinomethionat 170 to 200 0 1 4 0 0 (Quinomethionate) 170 to 200 0 1 4 0 0 Chlorbenside e
120 to 155 0 0 1 0 0 Chlorbenside sulfone 130 to 160 0 0 5 0 0 α-Chlordane 110 to 140 5 0 0 0 0 γ-Chlordane 100 to 130 5 0 0 0 0 Chlorfenapyr a
085 to 105 0 5 0 0 0 Chlorfenprop-methyl 125 to 150 0 5 0 0 0 Chlorfenson 120 to 150 1 5 0 0 0 Chlorfenvinphos 110 to 140 0 0 4 3 0 Chlorfluazuron a 80 to 110
Chloridazon 130 to 155 0 0 0 4 1 Chlormephos 115 to 145 3 3 0 0 0 Chlorobenzilate 100 to 135 0 0 4 2 1 Chloroneb 145 to 170 0 5 0 0 0 Chloropropylate 100 to 135 0 0 4 2 0 Chlorsulfuron 110 to 150
Chlorothalonil 125 to 165 0 5 0 0 0 Chlorotoluron 115 to 150 0 0 0 5 2 Chloroxuron 130 to 155 0 0 1 5 0 Chlorpropham 110 to 135 0 2 4 0 0 Chlorpyrifos 110 to 140 2 4 0 0 0 Chlorpyrifos-methyl 120 to 150 1 4 0 0 0 Chlorthal-dimethyl 135 to 160 0 5 1 0 0 Chlorthiophos 115 to 155 0 4 0 0 0 Cinosulfuron 100 to 150
Clodinafop-propargyl 100 to 125 0 0 5 0 0 SIST EN 12393-2:2014
ml 1 t 2 u 3 v 4 w 5 x Clofentezine a 85 to 190
Clomazone 115 to 145 0 0 5 0 0 Cloquintocet-1-mexyl 105 to 130 0 0 2 4 0 Coumaphos 135 to 165 0 0 5 0 0 Crotoxyphos 105 to 145 0 0 0 4 0 Crufomate 100 to 140 0 0 0 3 4 Cyanazine 110 to 135 0 0 0 4 0 Cyanofenphos 115 to 145 0 2 4 0 0 Cyanophos 115 to 150 0 0 4 0 0 Cycloate 125 to 150 0 0 5 1 0 Cyfluthrin a 090 to 120 0 5 0 0 0 λ-Cyhalothrin a 090 to 110 0 5 0 0 0 Cymoxanil f
110 to 130 0 0 0 5 0 Cypermethrin 100 to 135 0 5 0 0 0 Cyproconazole 100 to 120
Cyprodinil 105 to 135 0 0 5 0 0 o,p'-DDD 110 to 140 5 0 0 0 0 p,p'-DDD 100 to 140 5 0 0 0 0 o,p'-DDE 120 to 150 5 0 0 0 0 p,p'-DDE 120 to 150 5 0 0 0 0 o,p'-DDT 120 to 150 5 0 0 0 0 p,p'-DDT 110 to 140 5 0 0 0 0 DEF g
115 to 135 0 0 5 1 0 Deltamethrin 100 to 135 0 5 0 0 0 Demeton-S-methyl 125 to 155 0 0 0 0 0 Demeton-S-methyl sulfone 120 to 160 0 0 0 2 3 Demeton-S sulfoneh
115 to 140 0 0 0 3 3 Demeton-S sulfoxide i
140 to 170 0 0 0 0 3 N-Desethyl-pirimiphos-methyl 120 to 155 0 0 1 5 0 Desmedipham 100 to 120
Dialifos 110 to 140 0 3 3 0 0 Di-allate 120 to 150 0 4 1 0 0 Diazinon 105 to 135 0 0 5 0 0 Dichlobenil 125 to 155 1 5 0 0 0 Dichlofenthion 110 to 140 3 3 0 0 0 SIST EN 12393-2:2014
ml 1 t 2 u 3 v 4 w 5 x Dichlofluanid j
100 to 140 0 3 3 0 0 p,p'-Dichlorobenzophenone k
125 to 155 0 5 0 0 0 Dichlorvos 115 to 140 0 0 1 3 0 Diclofop-methyl 135 to 165 0 0 5 0 0 Dicloran 105 to 145 0 5 0 0 0 Dicofol l
100 to 150 2 4 0 0 0 Dicrotophos 130 to 160 0 0 0 0 5 Dieldrin 120 to 150 0 5 0 0 0 Diethofencarb 105 to 130 0 0 5 0 0 Difenoconazol 110 to 140 0 0 0 3 3 Diflubenzuron 110 to 135
Diflufenican 105 to 125 0 0 5 0 0 Dimefox 120 to 155 0 0 0 0 5 Dimethachlor 135 to 165 0 0 4 2 0 Dimethoate 120 to 150 0 0 0 3 3 Dimetilan 120 to 180
Dimoxystrobin a 85 to 160 0 0 3 3 0 Dinitramine m
105 to 130 4 1 0 0 0 Dinobuton 110 to 140 0 4 2 0 0 Dinocap 100 to 120 0 5 0 0 0 Dioxathion 110 to 140 0 3 3 1 0 Diphenylamin 130 to 160 0 5 0 0 0 Disulfoton n
115 to 150 0 2 0 0 3 Disulfoton sulfone 110 to 140 0 0 5 0 0 Disulfoton sulfoxide 120 to 150 0 0 0 0 5 Ditalimfos 120 to 150 0 0 4 1 0 Diuron 115 to 135
Edifenphos 130 to 160 0 0 4 0 0 α-Endosulfan 110 to 150 2 4 0 0 0 ß-Endosulfan 110 to 150 0 5 0 0 0 Endosulfan sulfate 100 to 140 0 5 0 0 0 Endrin 130 to 160 0 5 0 0 0 EPN 135 to 160 0 5 0 0 0 Ethiofencarb-sulfone 110 to 170
Ethion 100 to 140 0 5 0 0 0 SIST EN 12393-2:2014
ml 1 t 2 u 3 v 4 w 5 x Ethofumesate 110 to 135 0 0 3 0 0 Ethoprophos 120 to 155 0 0 4 1 0 Etrimfos 105 to 140 0 0 5 0 0 Famophos 125 to 155 0 0 5 0 0 Famoxadon 95 to 115 0 0 5
0 Fenamidone 105 to 140 0 0 4 1 0 Fenamiphos 105 to 140 0 0 0 4 2 Fenarimol 125 to 150 0 0 0 4 0 Fenazaquin 105 to 145 0 0 3 2 0 Fenchlorphos 120 to 150 4 2 0 0 0 Fenhexamid 100 to 140 0 0 1 4 1 Fenitrothion 120 to 150 0 4 0 0 0 Fenoxycarb 120 to 145 0 0 4 0 0 Fenpiclonil 100 to 130 0 0 3 3 0 Fenpropathrin 100 to 120 0 5 1 0 0 Fenpropimorph 90 to 120 0 0 4 2 0 Fenson 130 to 160 0 5 0 0 0 Fensulfothion 120 to 150 0 0 0 3 3 Fenthion 130 to 160 0 3 0 0 0 Fenvalerate 105 to 135 0 4 1 0 0 Fluazifop-P-butyl 105 to 130 0 0 5 0 0 Flubenzimine j a 085 to 120 0 5 0 0 0 Fluchloralin 100 to 120 5 1 0 0 0 Flucythrinat a 090 to 120 0 5 0 0 0 Fludioxonil a 090 to 120 0 0 5 0 0 Fluoroglycofen-ethyl a 090 to 115 0 0 5 0 0 Fluotrimazole 100 to 140 0 0 4 2 0 Fluquinconazol 095 to 125 0 0 3 3 0 Flurtamone a 085 to 105 0 0 0 5 0 Flusilazol 105 to 135 0 0 0 4 2 Flutriafol 115 to 135 0 0 0 3 3 Fluvalinate 095 to 120 0 5 0 0 0 Folpet 140 to 180 0 3 4 0 0 Fonofos 120 to 150 0 4 1 0 0 Formothion 120 to 150 0 0 4 1 0 Fuberidazole m 120 to 160 0 0 0 5 1 SIST EN 12393-2:2014
ml 1 t 2 u 3 v 4 w 5 x Furathiocarb 105 to 135 0 0 5 1 0 Genite 135 to 165 0 5 0 0 0 Haloxyfop-ethoxyethyl 70 to 110
Haloxyfop-methyl 100 to 160
α-HCH 120 to 150 5 0 0 0 0 ß-HCH 100 to 130 5 0 0 0 0 δ-HCH 100 to 130 5 0 0 0 0 ε-HCH 105 to 135 5 0 0 0 0 Heptachlor 110 to 140 5 0 0 0 0 cis-Heptachlor epoxide 125 to 155 3 3 0 0 0 trans-Heptachlor epoxide 125 to 155 3 3 0 0 0 Heptenophos 120 to 150 0 0 1 4 0 Hexachlorobenzene 140 to 165 5 0 0 0 0 Hexaconazol 105 to 135 0 0 0 4 2 Hexaflumuron a 70 to 110 0 0 5 1 0 Hexythiazox 120 to 140 0 0 4 0 0 Imazalil i m 120 to 150 0 0 0 0 5 Imidacloprid 120 to 160 0 0 0 1 1 Indoxacarb 95 to 120 0 0 5 0 0 Iodofenphos 120 to 150 4 2 0 0 0 Ioxynil
125 to 155 0 5 1 0 0 Iprodione 115 to 145 0 0 5 1 0 Iprovalicarb a 085 to 110 0 0 0 5 0 Isobenzan 105 to 140 5 0 0 0 0 Isocarbamid 130 to 165 0 0 0 1 5 Isodrin 120 to 150 5 0 0 0 0 Isopropalin 110 to 135 5 0 0 0 0 Isoproturon 115 to 135
Isoxaflutole 100 to 140 0 0 5 0 0 Iodfenphos 120 to 150 4 2 0 0 0 δ-Keto-endrin 135 to 165 3 4 0 0 0 Kresoxim-methyl 120 to 155 0 0 5 0 0 Lenacil 130 to 160 0 0 0 5 0 Leptophos 120 to 150 5 1 0 0 0 Lindane (γ-HCH) 110 to 140 5 0 0 0 0 SIST EN 12393-2:2014
ml 1 t 2 u 3 v 4 w 5 x Linuron 120 to 140 0 0 4 1 0 Malaoxon 110 to 140 0 0 0 4 0 Malathion 110 to 140 0 0 4 0 0 MCPA-(2-butoxyethyl) ester f 115 to 145 0 0 5 0 0 Mecarbam 105 to 145 0 0 4 0 0 Mefenpyr-diethyl 100 to 130 0 0 5 0 0 Mepanipyrim 110 to 140 0 0 5 0 0 Mepronil 110 to 140 0 0 5 0 0 Mephosfolan 140 to 170 0 0 0 2 4 Merphos o
125 to 145 5 0 0 0 0 Metalaxyl 115 to 150 0 0 0 5 1 Metazachlor 125 to 150 0 0 4 2 0 Metconazol 85 to 115 0 0 0 4 1 Methabenzthiazuron 150 to 180 0 0 0 5 0 Methacrifos 125 to 165 0 0 4 0 0 Methamidophos 120 to 150 0 0 0 0 4 Methidathion 130 to 165 0 0 4 0 0 Methiocarb 120 to 150 0 0 4 0 0 Methomyl 95 to 175 0 0 0 5 0 Methoprotryne 115 to 140 0 0 0 4 1 Methoxychlor 125 to 155 0 5 0 0 0 Metolachlor 130 to 160 0 0 5 1 0 Metolcarb
Metrafenon a 90 to 140 0 0 5 0 0 Metribuzin 125 to 150 0 0 3 1 0 Mevinphos 120 to 150 0 0 0 5 0 Mirex 130 to 160 5 0 0 0 0 Monocrotophos 115 to 140 0 0 0 0 5 Monolinuron 125 to 150 0 0 4 2 0 Morphothion 130 to 170 0 0 0 5 0 Naled p
115 to 155 0 0 4 1 0 Napropamid 135 to 165 0 0 2 4
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