1、BS EN 15637:2008ICS 67.050NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDFoods of plant origin Determination ofpesticide residuesusing LC-MS/MSfollowing methanolextraction and clean-up using diatomaceousearthThis British Standardwas published underthe authority
2、 of theStandards Policy andStrategy Committee on 30November 2008 BSI 2008ISBN 978 0 580 58242 4Amendments/corrigenda issued since publicationDate CommentsBS EN 15637:2008National forewordThis British Standard is the UK implementation of EN 15637:2008.The UK participation in its preparation was entru
3、sted to TechnicalCommittee AW/-/3, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct applica
4、tion.Compliance with a British Standard cannot confer immunityfrom legal obligations.BS EN 15637:2008EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15637November 2008ICS 67.050English VersionFoods of plant origin - Determination of pesticide residues usingLC-MS/MS following methanol extraction an
5、d clean-up usingdiatomaceous earthAliments dorigine vgtale - Dtermination des rsidus despesticides par LC-MS/MS aprs extraction mthanolique etpurification sur terre de diatomesPflanzliche Lebensmittel - LC-MS/MS-Verfahren zurBestimmung von Pestizidrckstnden mitMethanolextraktion und Reinigung an Dia
6、tomeenerdeThis European Standard was approved by CEN on 13 September 2008.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliogra
7、phical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CE
8、N member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Ital
9、y, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Br
10、ussels 2008 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15637:2008: EBS EN 15637:2008EN 15637:2008 (E) 2 Contents Page Foreword3 1 Scope 4 2 Principle4 3 Reagents.4 4 Apparatus .6 5 Procedure .7 6 Evaluation of results .12 7 Conf
11、irmatory tests.16 8 Precision.16 9 Test report 16 Annex A (informative) Example for appropriate experimental conditions17 Annex B (informative) Precision data21 Bibliography 63 BS EN 15637:2008EN 15637:2008 (E) 3 Foreword This document (EN 15637:2008) has been prepared by Technical Committee CEN/TC
12、275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by May 2009, and conflicting national standards shall be withdrawn a
13、t the latest by May 2009. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. WARNING The application of this standard may involve hazardo
14、us materials, operations and equipment. It does not claim to address all safety issues associated with its use. The user of this standard is responsible for establishing appropriate safety and health practices and determining the applicability of regulatory limitations prior to use. According to the
15、 CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuani
16、a, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 15637:2008EN 15637:2008 (E) 4 1 Scope This European Standard describes a method for the analysis of pesticide residues in foods of plant origin, such as
17、fruits, vegetables, cereals, nuts as well as processed products including dried fruits. The method has been collaboratively studied on a large number of commodity/pesticide combinations. 2 Principle The sample is extracted with methanol after addition of some water. After partition into dichlorometh
18、ane the organic phase is evaporated and the residue is reconstituted with methanol. Quantification of pesticide residues is performed by liquid chromatography with tandem mass spectrometric detection, using electrospray ionisation. To achieve the required selectivity the mass spectrometer is operate
19、d in the selected reaction monitoring mode (SRM). 3 Reagents 3.1 General and safety considerations Unless otherwise specified, use reagents of recognised analytical grade. Take every precaution to avoid possible contamination of water, solvents, inorganic salts, etc. 3.2 Ammonium formate 3.3 Sodium
20、chloride 3.4 Water, HPLC quality 3.5 Dichloromethane, for residue analysis 3.6 Methanol, HPLC quality 3.7 Internal Standard (ISTD) solutions in methanol, = 10 g/ml to 50 g/ml1)Table 1 shows a list of potential internal standards that may be used in this method. The concentrations listed refer to the
21、 ISTD solutions that should be added at the first extraction step (5.2) and to standard solutions. Table 1: Potential internal standards (ISTDs) or quality control (QC) standards Name of the compound Log P (octanol-water partion coefficient) Chlorine atoms Concentration C ISTDg/ml Triphenyl phosphat
22、e 4,59 - 20 Tris-(1,3-dichlorisopropyl)-phosphate 3,65 6 50 Bis-nitrophenyl urea (nicarbazin) 3,76 - 10 1) = mass concentration BS EN 15637:2008EN 15637:2008 (E) 5 3.8 Pesticide stock solutions Prepare individual stock solutions of analytical standards at concentrations that are sufficiently high to
23、 allow the preparation of complex pesticide mixtures. The solvent used should not negatively influence the stability of the pesticides employed. NOTE Usually, store stock solutions at -18 C. Check the stability of stock solutions during storage regularly. In some cases the addition of acids or bases
24、 can be helpful to enhance stability and extend the acceptable storage period. 3.9 Pesticide mixtures Because of the broad applicability of this method and due to the partly divergent pH-stability of pesticides, analyte mixtures of different composition can be needed. These are prepared by mixing to
25、gether defined volumes of the required analyte stock solutions (3.8) and appropriately diluting them with methanol. The analyte concentrations in this mixture should be sufficient to allow the preparation of the required matrix matched standards (see 3.10.3) with moderate dilution of the blank sampl
26、e extract (e.g. less than 20 %). Usually, store pesticide mixtures at -18 C. Since the stability of the pesticides in the mixture may be lower than in stock solutions, stability has to be checked regularly. In some cases the addition of acids or bases can be helpful to enhance stability and extend a
27、cceptable storage times. 3.10 Standard solutions 3.10.1 Standard solutions prepared in pure solvent (solvent-based standards) Solvent-based standards are prepared by mixing a certain volume of methanol with known amounts of pesticide mixtures (3.9). The preparation of multiple standards of different
28、 pesticide concentration is useful to cover a broad concentration range. NOTE An analyte concentration of 1 g/ml correlates to a residue level of 0,4 mg/kg when a 10 g sample is employed (e.g. samples with water content 30 %) or 0,8 mg/kg when a 5 g sample is employed (e.g. cereals). 3.10.2 Standard
29、 solutions with internal standard prepared in pure solvent Solvent-based standards with ISTD are prepared by mixing a certain volume of methanol with known amounts of pesticide mixtures (3.9) and a fixed volume of internal standard solution (3.7). The volume used shall result in that concentration o
30、f ISTD which is obtained in the final extracts after sample extraction and clean-up (see 5.2 and 5.3). The concentration of internal standard in the final extract (CsampleISTD) can be calculated using Equation (1). The preparation of multiple standards of different pesticide concentration but with c
31、onstant ISTD concentration is useful to cover a broad concentration range. endexISTDISTDsampleISTDVVVVVVCVC=2312)(1) where: V ISTDis the volume of internal standard solution (3.7) added to the test portion; C ISTDis the concentration of internal standard solution (3.7); V1is the volume of NaCl solut
32、ion (2,5 ml); V2is the volume of measuring flask used in 5.2 (10 ml); BS EN 15637:2008EN 15637:2008 (E) 6 V3is the volume used for solid supported liquid/liquid extraction (5 ml); Vexis the total volume of extraction solvents and natural water (30 ml); Vendis the final volume of extract obtained aft
33、er clean-up (0,5 ml). NOTE The internal standard may correct for deviations from the correct extraction volume, a wrong estimate of water content of samples, losses of methanol during preparation of the final extract and fluctuations of instrument sensitivity during a batch of measurements. However,
34、 validation results in Annex B were obtained without internal standards. 3.10.3 Standard solutions prepared in blank matrix extracts (matrix-matched standards) Prepare matrix-matched standards in the same way as the solvent-based standards, however, instead of pure methanol use extracts of blank sam
35、ples (prepared as described in 5.2, but without ISTD addition). To minimize errors caused by matrix induced effects during chromatography, it is best to choose similar commodities (e.g. apple for apple samples, carrots for carrot samples, etc.). The stability of pesticide in matrix-matched standards
36、 may be lower than that of standards in pure acetonitrile and has to be checked more thoroughly. 3.11 5 ml cartridge for solid supported liquid/liquid extraction, 5 ml sample volume, diatomaceous earth, for example ChemElut CE 10052)3.12 20 ml cartridge for solid supported liquid/liquid extraction,
37、20 ml sample volume, diatomaceous earth, for example ChemElutCE 10202)4 Apparatus Usual laboratory apparatus and, in particular, the following: 4.1 Carving board and knife, for chopping up food samples for analysis 4.2 Homogenizer or high speed blender, fitted with jar 4.3 Laboratory balance 4.4 Mea
38、suring flasks, 10 ml and 20 ml 4.5 Ultrasonic bath 4.6 Centrifuge tubes, 80 ml 4.7 Centrifuge, capable of producing a relative centrifugal force (RCF) of at least 3000 g (at the bottom of the tube) 4.8 Round bottom flasks, 50 ml and 250 ml 4.9 Glass syringe, minimum volume 2 ml 2) ChemElut is a prod
39、uct supplied by Varian, Inc. (Palo Alto, CA, USA). This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. BS EN 15637:2008EN
40、 15637:2008 (E) 7 4.10 Microliter syringes, for sample fortification 4.11 Rotary evaporator, with temperature-controlled water bath 4.12 Syringe filters, 0,45 m pore size, 4 mm diameter, polytetrafluoroethylene (PTFE) membrane 4.13 Glass vials and caps, 1,8 ml volume, suitable for an autosampler 4.1
41、4 LC-MS/MS system, triple quadrupole mass spectrometer with electrospray interface 5 Procedure 5.1 Preparation and storage of the samples 5.1.1 General Sample processing and storage procedures should be demonstrated to have no significant effect on the residues present in the test sample (sometimes
42、also called “analytical sample”). Processing should also ensure that the test sample is homogeneous enough so that sub-sampling variability is acceptable. If a single analytical portion is unlikely to be representative of the test sample, larger or replicate portions shall be analysed, to provide a
43、better estimate of the true value. The degree of comminution supports a quantitative residue extraction. 5.1.2 Laboratory sample A laboratory sample that is wholly or extensively spoiled or degraded should not be analysed. When possible, prepare laboratory samples immediately after arrival and in an
44、y event, before any significant physical or chemical changes have taken place. If a laboratory sample cannot be prepared without delay, it should be stored under appropriate conditions to keep it fresh and to avoid deterioration. Generally, laboratory samples should not be stored longer than 3 days
45、before preparation. Dried or similarly processed samples should be analysed within their stated shelf life. 5.1.3 Partly-prepared test sample For preparation of the partly-prepared test sample take only the portion of the laboratory sample to which the maximum residue level applies. No further plant
46、-parts may be removed. The reduction of the laboratory sample shall be carried out in such a way that representative portions are obtained (e. g. by sub-division into four and selection of opposite quarters). For samples of small units (e. g. small fruits such as berries, legumes, cereals), the samp
47、le shall be thoroughly mixed before weighing out the partly-prepared test sample. When the samples are made up of larger units, take wedge-shaped sections (e. g. melons) or cross sections (e. g. cucumbers) that include the skin (outer surface) from each unit 1. 5.1.4 Test sample From each partly-pre
48、pared test sample, any parts that would cause difficulties with the homogenisation process should be removed. In the case of stone fruits, the stones shall be removed. A record of the plant-parts that have been removed shall be kept. Precautions should be taken to avoid any losses of juice or flesh.
49、 This is the test sample. Calculation of the residue shall be based on the mass of the original test sample (including the stones). Where the homogeneity of the test sample is not sufficient or the extraction of residues may be significantly compromised due to large particle sizes, intensive comminution should be performed using appropriate means. This is possible at ambient temperature, if separation of flesh and juice or degradation of target BS EN 15637:2008EN 15637:2008 (E) 8 pesticides does not occur to a significant extent. Comminution of sa
