1、BSI Standards PublicationBS EN 12393-2:2013Foods of plant origin Multiresidue methods for thedetermination of pesticideresidues by GC or LC-MS/MSPart 2: Methods for extraction and clean-upBS EN 12393-2:2013 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 12393-2
2、:2013.It supersedes BS EN 12393-2:2008 which is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not
3、purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2013. Published by BSI StandardsLimited 2013ISBN 978 0 580 77444 7ICS 67.080.01Compliance with a British Standard cannot confer immunity fromlegal obligat
4、ions.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 30 November 2013.Amendments issued since publicationDate Text affectedBS EN 12393-2:2013EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 12393-2 November 2013 ICS 67.050 Supersedes EN 12
5、393-2:2008English 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 dorigine vgtale - Mthodes multirsidus de dtermination de rsidus de pesticides par CPG ou CL-SM/SM - Partie 2: Mt
6、hodes dextraction et de purificationPflanzliche Lebensmittel - Multiverfahren zur Bestimmung von Pestizidrckstnden 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/CENEL
7、EC 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
8、 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 offi
9、cial 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,
10、 Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2013 CEN All rights of
11、exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12393-2:2013: EBS EN 12393-2:2013EN 12393-2:2013 (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 M: Extracti
12、on with acetone and liquid-liquid partition with dichloromethane/light petroleum, if necessary clean-up on Florisil 7 6 Method N: Extraction with acetone, liquid-liquid partition with dichloromethane or cyclohexane/ethyl acetate, clean-up with gel permeation and silica gel chromatography 10 7 Method
13、 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 12393-2:2013EN 12393-2:2013 (E) 3 Foreword This document (EN 12393-2:2013) has been prepared by Technical Comm
14、ittee 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 b
15、e 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 co
16、mparison 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;
17、 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
18、by GC or LC-MS/MS is divided into 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 M, N and P f
19、or 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 re
20、sidues 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
21、 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 12393-2:2013EN 12393-2:2013 (E) 4 Int
22、roduction 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 wide
23、ly 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
24、improve method performance, see Clause 3. BS EN 12393-2:2013EN 12393-2:2013 (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. Th
25、ese 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
26、 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 cycloh
27、exane/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 f
28、ood 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 be
29、en 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
30、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 P
31、art 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 t
32、he 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 obt
33、ain 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
34、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
35、 product. BS EN 12393-2:2013EN 12393-2:2013 (E) 6 4 General: Summary of procedures 4.1 Extraction The extraction procedures are summarized in Table 1. Table 1 Extraction procedures Methods Mass of samples (MS) Volume of solvent (VS) Ratio (MS/ VS) g ml g/ml M 100 Acetone: 200 N 100 aAcetone: 200 P 1
36、0 Ethyl acetate: 20 aOnly 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 s
37、olvent Ratio (AE) ml (VW) ml (VS) ml AE/ VWM 80 0 200 - aN 200 x a100 - aaDepends 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 ad
38、sorbents: 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 stan
39、dard and does not constitute an endorsement by CEN of this product. BS EN 12393-2:2013EN 12393-2:2013 (E) 7 5 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
40、 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
41、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 Reagents 5.2.1 General All re
42、agents shall be suitable for the analysis of pesticide residues and in accordance with EN 12393-1:2013, Clause 4. 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 4 h, allow to cool, and store in a stoppered bottle. 5.2.5 Dichloromethan
43、e 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 m to 250 m (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 airt
44、ight 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, p
45、eroxide-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 petrol
46、eum 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 12393-2:2013EN 12393-2:2013 (E) 8 5.3 Apparatus Usual laboratory equipment in accordance with
47、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 s
48、ample 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 1
49、00 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 Bchner funnel containing a filter paper moistened with acetone, filter under suction into the Bchner 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 s
