BS EN 12393-2:2013 BSI Standards Publication 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 BS EN 12393-2:2013 BRITISH STANDARD National foreword This British Standard is the UK implementation of EN 12393-2:2013 It supersedes BS EN 12393-2:2008 which is withdrawn The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods A list of organizations represented on this committee can be obtained on request to its secretary This publication does not purport to include all the necessary provisions of a contract Users are responsible for its correct application © The British Standards Institution 2013 Published by BSI Standards Limited 2013 ISBN 978 580 77444 ICS 67.080.01 Compliance with a British Standard cannot confer immunity from legal obligations This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 November 2013 Amendments issued since publication Date Text affected BS EN 12393-2:2013 EN 12393-2 EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM November 2013 ICS 67.050 Supersedes EN 12393-2:2008 English 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 CLSM/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: E BS EN 12393-2:2013 EN 12393-2:2013 (E) Contents Page Foreword Introduction Scope Normative references Principles General: Summary of procedures Method M: Extraction with acetone and liquid-liquid partition with dichloromethane/light petroleum, if necessary clean-up on Florisil® Method N: Extraction with acetone, liquid-liquid partition with dichloromethane or cyclohexane/ethyl acetate, clean-up with gel permeation and silica gel chromatography 10 Method P: Extraction with ethyl acetate, and if necessary, clean-up by gel permeation chromatography 35 Annex A (informative) Average water content of crops and foods 41 Bibliography 42 BS EN 12393-2:2013 EN 12393-2:2013 (E) Foreword This document (EN 12393-2:2013) 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 2014, and conflicting national standards shall be withdrawn at the latest by May 2014 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 12393-2:2008 In comparison with EN 12393-2:2008, the following significant technical changes have been made: a) implementation of liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS) for the quantification and/or confirmation of pesticide residues; b) incorporation of information on GC-MS/MS; c) deletion of method L as no longer in use; d) editorial updating of the document according to references, etc; e) enlargement of scope of method N concerning number of pesticides and validation data EN 12393, Foods of plant origin – Multiresidue methods for the determination of pesticide residues by GC or LC-MS/MS is divided into three parts: Part "General considerations" provides general considerations with regard to reagents, apparatus, gas chromatography, etc., applying to each of the analytical selected methods; Part "Methods for extraction and clean-up" presents methods M, N and P for the extraction and cleanup using techniques such as liquid-liquid partition, adsorption column chromatography or gel permeation column chromatography, etc.; Part "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, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom BS EN 12393-2:2013 EN 12393-2:2013 (E) 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 BS EN 12393-2:2013 EN 12393-2:2013 (E) 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 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 chromatography [6] with ethyl acetate, and if necessary, clean-up by gel permeation 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 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 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 BS EN 12393-2:2013 EN 12393-2:2013 (E) General: Summary of procedures 4.1 Extraction The extraction procedures are summarized in Table Table — Extraction procedures Methods Mass of samples (MS) Volume of solvent (VS) Ratio (MS / VS) g ml g/ml 100 Acetone: 200 ½ Acetone: 200 ½ Ethyl acetate: 20 ½ M a 4.2 N 100 P 10 a Only relevant if the water content of the matrix is greater than 70 % Clean-up 4.2.1 Liquid-liquid partition The liquid-liquid partition procedures are summarized in Table Table — Liquid-liquid partition Method a Aliquot portion of extract Volume of added water Volume of solvent Ratio (AE) ml (VW) ml (VS) ml AE / VW M 80 200 -a N 200 xa 100 -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-X3 2) 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 BS EN 12393-2:2013 EN 12393-2:2013 (E) Method M: Extraction with acetone and liquid-liquid partition with dichloromethane/light petroleum, if necessary clean-up on Florisil® 5.1 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 Either mixtures of diethyl ether and light petroleum (method M1) or mixtures of dichlormethane, light petroleum and acetonitrile (method M2) are used for the elution of analytes from Florisil® The eluates are concentrated for examination by GC 5.2 5.2.1 Reagents General All reagents shall be suitable for the analysis of pesticide residues and in accordance with EN 12393-1:2013, Clause 5.2.2 Acetone 5.2.3 Light petroleum, boiling range 40 °C to 60 °C 5.2.4 Sodium chloride Heat at 500 °C for at least 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 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 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 % (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) BS EN 12393-2:2013 EN 12393-2:2013 (E) 5.3 Apparatus 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 Danish 3) 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 5.4.2 The general water content of some crops and foods is given in Table A.1 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 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 l separating funnel with 100 ml of dichloromethane and 100 ml of light petroleum (5.2.3) Shake for and leave to separate layers Transfer the lower aqueous layer to a second l separating funnel Dry upper organic layer from the first separatory funnel by passing through cm of sodium sulfate (5.2.7) supported on washed glass wool in 10 cm funnel collecting in a round-bottomed flask Add 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 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 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 ml Add 100 ml of light petroleum and again concentrate to e.g ml and again until all dichloromethane disappears Add 20 ml of acetone and reconcentrate to 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 ml of acetone (instead of ml) and diluted to a volume of 10 ml with light petroleum 5.4.3 5.4.3.1 Clean-up 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 cm to 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 BS EN 12393-2:2013 EN 12393-2:2013 (E) Table (continued) + Diethofencarb + + + + Difenoconazole + + + + Diflubenzuron x Diflufenican + + + + x x x x Dimethoat x Dimetilan x x x Dimoxystrobin x x x x + + + + Disulfoton x Ditalimfos x Diuron x β-endosulfan + + Endosulfan sulfate + + Endrin x x x x x x x x Ethion + Ethofumesate + x x x + + + Famoxadone x x x x Fenamidone + + + + Fenarimol + Fenazaquin x + Fenhexamid + x + + + x + + + + + Fenpiclonil + + + + x x x x + Fenpropimorph x Fenvalerat x Fluazifop-P-butyl x Flucythrinat x 30 x x Fenoxycarb Fludioxonil x + Ethiofencarbsulfone Fenpropathrin x + α-endosulfan Fenitrothion x x Dimethachlor Diphenylamin raisins + yellow peas nuts, avocado cereals citrus fruits cucumber + tomato + spinach + savoy cabbage + potato carrot Dieldrin + x + + x + BS EN 12393-2:2013 EN 12393-2:2013 (E) Table (continued) + + + Fluquinconazole + + + + + + + Flurtamone + Flusilazol x Flutriafol + Fluvalinat + + x HaloxyfopEthoxyethyl x Haloxyfop-methyl x + + + + α-HCH + ß-HCH x + + + + + + Heptachlor Heptachlor epoxide + + Furathiocarb Lindane (γ-HCH) x x Folpet HCB + x + + Hexaconazol x Hexaflumuron x x Hexythiazox x Imazalil x x Imidacloprid x x x Indoxacarb + + + + Iprovalicarb + + + + Isoproturon x x x Iprodione x + Isoxaflutole x x x Jodfenphos x Kresoxim-methyl + Linuron x Malathion + Mecarbam + Mefenpyr-diethyl raisins + yellow peas nuts, avocado cereals citrus fruits cucumber tomato spinach savoy cabbage potato carrot Fluoroglycofen-ethyl x + + x x + x x + + + + + Mepanipyrim x x x x Mepronil x x x x x 31 BS EN 12393-2:2013 EN 12393-2:2013 (E) Table (continued) + + + Methabenzthiazuron x Methacrifos x Methamidophos x x Methidathion x x Methiocarb x x Methomyl x x Methoxychlor x Metolachlor x x x Metolcarb x x x Metrafenon x x Metribuzin x x Mevinphos x x Monocrotophos x x Napropamide + x + x x + x x x x x x x x x + x x + x Nicosulfuron x Nuarimol + Oxadixyl + x + x + + + x + Parathion-methyl x x PCB 28 x x PCB 138 x x Penconazol x x Pendimathalin x Permethrin x + x x + x Picoxystrobin + Pirimicarb x + + x + Phosphamidon 32 x + Phenmedipham Pirimiphos-methyl x x Oxydemeton-methyl Phosalone + x Oxamyl Parathion raisins Metconazole x yellow peas x x nuts, avocado Metazachlor cereals x citrus fruits cucumber tomato spinach savoy cabbage potato carrot Metalaxyl + + + + x x x + BS EN 12393-2:2013 EN 12393-2:2013 (E) Table (continued) + Profenofos x x Promecarb x x Propiconazol x x Propoxur x x x x Propyzamid x x x x Proquinazid x x x x + + + x + Prosulfocarb + Prosulfuron x Pyridaben + Pyrifenox + x x + + + + + + Pyrimethanil x x x Pyriproxyfen + + + Quinalphos x Quinmerac x Quinoxyfen x x + x x x x x + + Quizalofop-ethyl x Silthiofam + Simazin x + + + x Spinosyn A (Spinosad) x Spirodiclofen x x x x Spiroxamine + + + + Sulfotep x Tebuconazol x Tebufenpyrad + Tebutam x x x x x x + + + x x Teflubenzuron x Terbacil x x Terbuthylazin x x Tetradifon x x Pyrazophos Quintozene raisins x Procymidone Propham yellow peas nuts, avocado cereals citrus fruits cucumber tomato spinach savoy cabbage potato carrot Prochloraz + 33 BS EN 12393-2:2013 EN 12393-2:2013 (E) Table (continued) raisins yellow peas nuts, avocado cereals citrus fruits cucumber tomato spinach savoy cabbage potato carrot Thiabendazol x Thiacloprid x x x x Thiamethoxam x x x x Thifensulfuronmethyl x x Thiodicarb x Thiofanox-sulfone x x Thiofanox-sulfoxide x x Triadimefon x x Triadimenol x x Triallat x Tolclofos-methyl + Triasulfuron x Triazophos x Trichloronat x Trifloxystrobin + Triflumizol x Trifluralin x Triflusulfuron-methyl x x x + + + x x x x Triforin x Triticonazole Vinclozolin x + + + + + + + + + + x + Zoxamide + Key + = data obtained from proficiency tests; x = data obtained from inter-laboratory method validation studies Validation data from the study which was the basis for [7]; individual recovery data listed in [7] 6.9 Applicability The pesticides that can be analysed by this method are listed in Table Crops and foods on which the method was tested are listed in Table 34 BS EN 12393-2:2013 EN 12393-2:2013 (E) Table — Crops and foods tested with Method N Apples Coffee, raw Nuts Spices Avocados Cucumbers Onions Spinach Bananas Curly kale Peaches Strawberries Beans Currants, red Peanuts Sugar beets Beer Grapes Pears Sweet peppers Carrots Head cabbage Peas Tea and tea-like products Cauliflower Hops Pineapples Tobacco Cereals Kohlrabi Plums Tomatoes Cherries Lettuce Potatoes Wine Citrus fruit Melons Raisins Cacao products Must Savoy cabbage Method P: Extraction with ethyl acetate, and if necessary, clean-up by gel permeation chromatography 7.1 Principle A portion of the chopped and homogenized sample is extracted with ethyl acetate together with sodium hydrogen carbonate and sodium sulfate in a sample tube After shaking and ultrasonication, the sample tube is centrifuged An aliquot portion of the crude extract can be injected directly without clean-up into a gas chromatograph or liquid chromatograph, or, if necessary, purified by gel permeation chromatography on Bio-Beads®S-X3 using a mixture of cyclohexane and ethyl acetate as eluent The eluate is concentrated for examination by GC or LC 7.2 7.2.1 Reagents General All reagents shall be suitable for the analysis of pesticide residues and in accordance with EN 12393-1 7.2.2 Ethyl acetate 7.2.3 Sodium sulfate Heat at 500 °C for at least h, allow cool, and store in a stoppered bottle 7.2.4 Cyclohexane 7.2.5 GPC eluting mixture: cyclohexane/ethyl acetate 1+1 (V/V) 7.2.6 Bio-Beadsđ S-X3, 38 àm to 75 àm (200 mesh to 400 mesh) 7.2.7 Sodium hydrogen carbonate 7.3 Apparatus Usual laboratory equipment in accordance with EN 12393-1 and in particular, the following: 35 BS EN 12393-2:2013 EN 12393-2:2013 (E) 7.3.1 Homogenizer 7.3.2 Solvent evaporator, with water bath 7.3.3 Instrument for GPC, e.g GPC Autoprep® 1001 or 1002, equipped with chromatographic column, 25 mm internal diameter, 50 cm length, and ml sample loops (VR1); column packing 50 g of Bio-Beads® S-X3 resin, pre-swelled overnight in the GPC eluting mixture, approximately 32 cm column height, packed as described in 7.4.3.1 7.3.4 Centrifuge tubes, e.g of polypropylene with screw caps 7.3.5 Centrifuge 7.3.6 Syringes, e.g 10 ml disposable syringes, and Syringe filters, PTFE 0,20 µm pore size 7.4 Procedure 7.4.1 Preparation of the sample Chop the sample and homogenate (7.3.1) carefully 7.4.2 Extraction Weigh 10 g (m) of the prepared sample into a centrifuge tube (7.3.4), add g of sodium hydrogen carbonate (7.2.7), a suitable amount of at least 10 g of sodium sulfate (7.2.3) in order to absorb the sample water (see Table A.1 for water content) and 20 ml (VEx) of ethyl acetate Shake vigorously for and ultrasonicate for If the sample’s degree of comminution is insufficient or the residues not readily extract from the matrix, the extraction time may be prolonged (e.g to 20 using a mechanical shaker) or assisted by a high-speed disperser (e.g Ultra-Turrax®) Centrifuge (7.3.5) for at 000 g Isolate and filtrate the crude extract through a 0.20 µm PTFE filter (7.3.6) In some cases when interferences occur, typically for high fat commodities such as avocado, clean-up by GPC is recommended 7.4.3 7.4.3.1 Clean-up by gel permeation chromatography (if required) Packing gel permeation column Allow the Bio-Beads® (7.2.6) (approximately 50 g) to swell overnight in the GPC eluting mixture (7.2.5) 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 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) 7.4.3.2 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, and on appropriate crude extracts To so, load the sample loop with a mixture of standard solutions or crude extracts, elute as described in 7.4.3.3 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 columns after they have been in use for a lengthy period 36 BS EN 12393-2:2013 EN 12393-2:2013 (E) 7.4.3.3 Clean-up of crude extracts To 5,0 ml (VR1) of ethyl acetate extract (7.4.2) add 5,0 ml (VR2) of cyclohexane Shake for approximately 20 s, filter through a fast flow rate filter paper and inject 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 Usually the setting is as follows: dump switch to 16 to discard 80 ml; collect switch to 22 to collect 110 ml; wash switch to for column rinse with 10 ml Concentrate the collected volume to approximately ml on a solvent evaporator (rotate slowly, immerse flask only slightly), pipette into a ground-stoppered graduated test tube, rinse the evaporator flask with ethyl acetate and dilute with ethyl acetate to a volume of 2,5 ml (Vend) This addition of ethyl acetate shall not be omitted on any account in order to ensure complete dissolution of the residue If analytes are analysed which elute early in GPC, the use of a crude extract or a wider GPC collect fraction is necessary 7.5 Gas chromatography Use a suitable gas chromatographic system as described in EN 12393-3 and inject an aliquot of the extract from 7.4.2 or 7.4.3.3 7.6 Liquid chromatography Use a suitable liquid chromatographic system as described in EN 12393-3 and inject an appropriate aliquot of e.g µl or less of the extract from 7.4.2 or 7.4.3.3 7.7 Calculation of results The residue R, expressed in milligrams per kilogram, of an identified analyte is calculated from Formula (4): R = FA × WSt × VEX × f FSt × Vi × m (4) in which either f4 is (if 7.4.3 is not required) or f4 is calculated by Formula (5) in the case of clean-up of crude extracts by GPC: f4= (VR1 + VR ) × Vend VR1 × VR (5) where FA is the peak area obtained from Vi; WSt is the amount of compound injected with standard solution, in nanograms; VEx is the volume, in millilitres, of ethyl acetate added in extraction step 7.4.2; 37 BS EN 12393-2:2013 EN 12393-2:2013 (E) FSt is the peak area obtained from WSt; Vi is the portion of volume injected into the gas chromatograph or the liquid chromatograph, respectively, in microlitres; m is the mass of the test portion, in grams; VR1 is the portion of volume VEx used in 7.4.3.3, in millilitres; VR2 is the volume of cyclohexane added to VR1 in 7.4.3.3, in millilitres; Vend is the final volume obtained in 7.4.3.3 after re-dissolvation, in millilitres; VR3 is the portion of volume VR1 + VR2 injected for gel permeation chromatography (volume of sample loop), in millilitres 7.8 Proficiency testing data All couples of the following matrices and pesticides were proven by proficiency tests without clean-up: Matrices: Leek, mandarin Pesticides: See Table Table — Pesticides proven by proficiency tests without clean-up Aldicarb Ethion Methidathion Pyriproxyfen Azinphos-methyl Fenpropathrin Methomyl Spinosyn A Carbendazim Imazalil Orthophenylphenol Spinosyn D Chlorpyrifos Imidacloprid Oxamyl Thiabendazole Deltamethrin Indoxacarb Pendimethalin Thiacloprid Diazinon Kresoxim-methyl Phosalone Tolylfluanid Dimethoate Malathion Prochloraz Triflumuron EPN Methamidophos Prothiofos 7.9 Applicability Crops and foods on which the method was tested are listed in Table Table — Crops and foods tested with Method P Apples Carrots Head cabbage Oranges Strawberries Avocados Cucumbers Leeks Peaches Tomatoes Bananas Grapes Lettuce The pesticides and metabolites listed in Table can be analysed with GC- or LC-MS/MS by this method 38 BS EN 12393-2:2013 EN 12393-2:2013 (E) Table — Pesticides and metabolites that can be analysed with GC- or LC-MS/MS by Method P Abamectin Acephate Acetamiprid Acibenzolar-S-methyl Aclonifen* Acrinathrin ** Aldicarb Aldicarb-sulfone Aldicarb-sulfoxide Aminocarb Aspon Atrazine Atrazine-desethyl Atrazine-desisopropyl Azinphos-ethyl Azinphos-methyl Azoxystrobin Benalaxyl Bendiocarb Benfuracarb Bentazone Bifenthrin Binapacryl Biphenyl Bitertanol Boscalid Bromophos Bromophos-ethyl Bromopropylate Bromuconazole Bupirimate Buprofezin Butocarboxim Butocarboxim-sulfoxide Butoxycarboxim Butralin Cadusafos Carbaryl Carbendazim Carbofuran Carbofuran-3OH Carbophenothion Carbosulfan Carfentrazone-ethyl Chinomethionat Chlordane, cisChlordane, transChlordimeform Chlorfenapyr Chlorfenson Chlorfenvinphos Chlorfluazuron Chlormephos Chlorobenzilate Chlorobromuron Chloropropylate Chlorothalonil Chlorpropham Chlorpyrifos Chlorpyrifos-methyl Chlorpyrifos-O-Analogue Chlorthal-dimethyl Chlozolinate Clofentezine Clomazone Clothianidin Coumaphos Cyanazin Cyanofenfos Cyanofos Cyazofamid Cyfluthrin Cyfluthrin, betaCypermethrin Cyproconazole Cyprodinil Danifos DEET Deltamethrin Demeton Demeton-S-methyl Demeton-S-methyl-sulfone Demeton-S-methyl-sulfoxide Desmethyl pirimicarb Desmetryn Dialifos Diazinon Dichlobenil Dichlofluanid Dichloroaniline, 3,5Dichlorvos Dicloran Dicrotophos Dieldrin Diethofencarb Difenoconazole Diflubenzuron Dimethoate Dimethomorph Dinobuton Dinocap Dinoseb Dinoterb Dioxathion Diphenamid Diphenylamine Disulfoton Disulfoton-Sulfone Disulfoton-sulfoxide DMSA DMST DNOC Endosulfan, alphaEndosulfan, betaEndosulfansulfate Endrin EPN Epoxiconazole Esfenvalerate Ethiofencarb Ethiofencarb-sulfone Ethiofencarb-sulfoxide Ethion Ethofumesate Ethoprophos Etofenprox Etrimfos Famoxadone Fenamiphos Fenamiphos-Sulfone Fenamiphos-Sulfoxide Fenarimol Fenazaquin Fenbuconazole Fenchlorphos Fenhexamid Fenitrothion Fenoxycarb Fenpiclonil Fenpropathrin Fenpropimorph Fenpyroximate Fenson Fensulfothion Fensulfothion-oxon Fensulfothion-oxon-sulfone Fensulfothion-sulfone Fenthion Fenthion-oxon Fenthion-oxon-sulfone Fenthion-oxon-sulfoxide Fenthion-sulfone Fenthion-sulfoxide Fenvalerate Fipronil Fluacrypyrim Fluazifop-P-butyl Fluazinam Flucythrinate Fludioxinil Fludioxonil Flufenoxuron Flumetralin Fluquinconazole Flusilazole Flutriafol Fonofos Formothion Fosthiazate Furalaxyl Furathiocarb Haloxyfop Haloxyfop-Ethoxyethylester Haloxyfop-Methyl 39 BS EN 12393-2:2013 EN 12393-2:2013 (E) Table (continued) HCH, alphaHCH, betaHCH, deltaHCH, gammaHeptachlor Heptachlor epoxide Heptenophos Hexachlorobenzene Hexaconazole Hexazinone Hexythiazox Imazalil Imidacloprid Indoxacarb Iprodione Iprovalicarb Isofenphos Isofenphos-methyl Isoprocarb Isopropalin Isoproturon Isoxaben Jodfenphos Kresoxim-methyl Lambda-Cyhalothrin** Leptophos Linuron Lufenuron Malaoxon Malathion Mecarbam Mepanipyrim Mephosfolan Metaflumizone Metalaxyl Metconazole Methabenzthiazuron Methamidophos Methiocarb Methiocarb-sulfone Methiocarb-sulfoxide Methomyl Methoxychlor Methoxyfenozide Metidathion Metribuzin * Mevinphos Monocrotophos Myclobutanil Napropamide Nitrofen Omethoate Orthophenylphenol Oxadixyl Oxamyl Oxamyl-Oxime Paclobutrazol Paraoxon Paraoxon-Methyl Parathion Parathion-methyl Penconazole Pencycuron Pendimethalin Pentachloroaniline Pentachloroanisole Pentachlorobenzene Permethrin Phenmedipham Phenothrin Phenthoate Phorate Phorate-O-Analogue Phorate-Sulfone Phorate-Sulfoxide Phosalone Phosmet Phosmet oxon Phosphamidon Phoxim Piperonyl Butoxide Pirimicarb Pirimiphos-Ethyl Pirimiphos-methyl Prochloraz Procymidone Profenofos Promecarb Propamocarb Propaquizafop Propargite Propetamphos Propham Propiconazole Propoxur Propyzamide Prosulfocarb Prothioconazole-desthio Prothiofos Pymetrozine Pyraclofos Pyraclostrobin Pyrazophos Pyridaben Pyridaphenthion Pyrifenox Pyrimethanil Pyriproxyfen Quinalphos Quinoxyfen Quintozene Quizalofop Simazine Spinosyn A Spinosyn D Spiroxamine Sulfentrazone Sulfotep tau-Fluvalinate TCNB, 2,3,4,5Tebuconazole Tebufenozide Tebufenpyrad Tecnazene Teflubenzuron Tefluthrin TEPP Tepraloxydim Terbufos Terbufos Sulfone Terbufos Sulfoxide Terbufos-O-sulfone Terbufos-oxon Terbufos-oxon-sulphoxid Terbuthylazine Terbutryn Tetrachloranilin, 2,3,5,6Tetrachlorvinphos Tetraconazole Tetradifon Tetrasul Thiabendazole Thiacloprid Thiametoxam Thiodicarb Thiometon Thiometon-sulfone Thiometon-sulfoxide Thionazin Thiophanate-methyl Tolclofos-methyl Tolylfluanid Triadimefon Triadimenol Triamiphos Triazamate Triazofos Trichlorfon Trichloronat Trichlorophenol, 2,4,6Trifloxystrobin Triflumizole Triflumuron Trimethacarb, 2,3,5Triticonazole Vamidothion Vamidothion-sulfoxide Vinclozolin Zoxamide Substances shown in italics have been evaluated using GC and all other using LC ** It has to be considered that this substance elutes very early in GPC (near to the dump volume) and thus losses of recovery can be observed 40 BS EN 12393-2:2013 EN 12393-2:2013 (E) Annex A (informative) Average water content of crops and foods Table A.1 — Average water content of crops and foods Crops and foods Average water content (in g/100 g) Cocoa powder Cereals, spices (herbs), coffee (raw), tea, tea-like products 10 Bananas, horse radish 75 Peas, currants(black), potatoes, parsley, grapes 80 Pineapples, apples, pears, currants(red), cherries, oranges, plums, chives 85 Cauliflower, beans (green), broccoli, strawberries, grapefruit, curly kale, kohlrabi, melons, carrots, must, pepper, peaches, mushrooms, red beet, red cabbage, spinach, head cabbage, lemons, sugar beet (edible root), onions 90 Witloof chicory, endives, cucumbers, radish, rhubarb, lettuce, celeriac, asparagus, tomatoes 95 41 BS EN 12393-2:2013 EN 12393-2:2013 (E) Bibliography [1] Luke, Milton A.; Froberg, Jerry E.; Masumoto, Herbert T., Extraction and clean-up of organochlorine, organophosphate, organonitrogen and hydrocarbon pesticides in produce for determination by gasliquid chromatography J Assoc Off Anal Chem 58, 1020 - 1026, 1975 [2] Luke, Milton A.; Froberg, Jerry E.; Doose, Gregory M.; Masumoto, Herbert T Improved multiresidue gas chromatographic determination of organophosphorus, organonitrogen and organohalogen pesticides in produce, using flame photometric and electrolytic conductivity detectors J Assoc Off Anal Chem 64, 1187 - 1195 (1981) [3] Pesticide Analytical Manual -Vol.I, Multiresidue methods, Section 302, 3rd Edition, 1994 [4] Specht, W.: Organochlorine, organophosphorus, nitrogen-containing and other pesticides In DFG, Manual of Pesticide Residue Analysis, VCH Weinheim, Method S 19, in Vol.1 (1987), pp 383, and Vol.2 (1992), pp 317 [5] Specht W., Pelz, S., Gilsbach, W.: Gas-chromatographic determination of pesticide residues after clean-up by gel-permeation chromatography and mini-silica gel-column chromatography Replacement of dichloromethane by ethyl acetate/cyclohexane in liquid-liquid partition and simplified conditions for extraction and liquid-liquid partition, Fresenius J Anal Chem 353, 183-190 (1995) [6] Analytical Methods for Residues of Pesticides in Foodstuffs, Sixth edition, The Hague (1996) [7] Methode L 00.00-34 Untersuchung von Lebensmitteln - Modulare Multimethode zur Bestimmung von Pflanzenschutzmitteln in Lebensmitteln, Erweiterte Neufassung der DFG-Methode S 19, Amtliche Sammlung von Untersuchungsverfahren nach § 64 Lebensmittel-, Bedarfsgegenstände- und Futtermittelgesetzbuch LFGB (Method L 00.00-34 Food analysis - Modular Multiple Analytical Method for the Determination of Pesticide Residues in Foodstuffs, Extended and Revised Version of the DFG Method S 19, Collection of Official Methods under Article 64 of the German Federal Food and Feed Code LFGB) 42 This page deliberately left blank NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW British Standards Institution (BSI) BSI is the national body responsible for preparing British Standards and other standards-related publications, information and services BSI is incorporated by Royal Charter British Standards and other standardization products are published by BSI Standards Limited About us Revisions We bring together business, industry, government, consumers, innovators and others to shape their combined experience and expertise into standards -based solutions Our British Standards and other publications are updated by amendment or revision The knowledge embodied in our standards has been carefully assembled in a dependable format and refined through our open consultation process Organizations of all sizes and across all sectors choose standards to help them achieve their goals Information on standards We can provide you with the knowledge that your organization needs to succeed Find out more about British Standards by visiting our website at bsigroup.com/standards or contacting our Customer Services team or Knowledge Centre Buying standards You can buy and download PDF versions of BSI publications, including British and adopted European and international standards, through our website at bsigroup.com/shop, where hard copies can also be purchased If you need international and foreign standards from other Standards Development Organizations, hard copies can be ordered from our Customer Services team Subscriptions Our range of subscription services are designed to make using standards easier for you For further information on our subscription products go to bsigroup.com/subscriptions With British Standards Online (BSOL) you’ll have instant access to over 55,000 British and adopted European and international standards from your desktop It’s available 24/7 and is refreshed daily so you’ll always be up to date You can keep in touch with standards developments and receive substantial discounts on the purchase price of standards, both in single copy and subscription format, by becoming a BSI Subscribing Member PLUS is an updating service exclusive to BSI Subscribing Members You will automatically receive the latest hard copy of your standards when they’re revised or replaced To find out more about becoming a BSI Subscribing Member and the benefits of membership, please visit bsigroup.com/shop With a Multi-User Network Licence (MUNL) you are able to host standards publications on your intranet Licences can cover as few or as many users as you wish With updates supplied as soon as they’re available, you can be sure your documentation is current For further information, email bsmusales@bsigroup.com BSI Group Headquarters 389 Chiswick High Road London W4 4AL UK We continually improve the quality of our products and services to benefit your business If you find an inaccuracy or ambiguity within a British Standard or other BSI publication please inform the Knowledge Centre Copyright All the data, software and documentation set out in all British Standards and other BSI publications are the property of and copyrighted by BSI, or some person or entity that owns copyright in the information used (such as the international standardization bodies) and has formally licensed such information to BSI for commercial publication and use Except as permitted under the Copyright, Designs and Patents Act 1988 no extract may be reproduced, stored in a retrieval system or transmitted in any form or by any means – electronic, photocopying, recording or otherwise – without prior written permission from BSI Details and advice can be obtained from the Copyright & Licensing Department Useful Contacts: Customer Services Tel: +44 845 086 9001 Email (orders): orders@bsigroup.com Email (enquiries): cservices@bsigroup.com Subscriptions Tel: +44 845 086 9001 Email: subscriptions@bsigroup.com Knowledge Centre Tel: +44 20 8996 7004 Email: knowledgecentre@bsigroup.com Copyright & Licensing Tel: +44 20 8996 7070 Email: copyright@bsigroup.com