1、February 2009DEUTSCHE NORM English price group 22No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67.050!$V(K“1510540ww
2、w.din.deDDIN EN 15637Foods of plant origin Determination of pesticide residues using LC-MS/MS followingmethanol extraction and clean-up using diatomaceous earthEnglish version of DIN EN 15637:2009-02Pflanzliche Lebensmittel LC-MS/MS-Verfahren zur Bestimmung von Pestizidrckstnden mit Methanolextrakti
3、onund Reinigung an DiatomeenerdeEnglische Fassung DIN EN 15637:2009-02www.beuth.deDocument comprises 65 pagesDIN EN 15637:2009-02 2 National foreword This standard has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). Preliminary work was
4、 done by Working Group WG 4 “Pesticides in foods of plant origin”. The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-01-08 AA Pestizide. Maximu
5、m residue levels of pesticides in or on food and feed of plant and animal origin are specified in EC Regulation no. 396/2005. The determination of these residues is therefore to be harmonized within the Community. This is the purpose of the present standard. In this standard, the unit “psi” is used.
6、 It should, however, be noted that the Gesetz ber Einheiten im purposes in Germany. Conversion: 1 psi = 6 894,757 Pa The DIN Standards corresponding to the International Standards referred to in the EN are as follows: ISO 5725 (all parts) DIN ISO 5725 (all parts) National Annex NA (informative) Bibl
7、iography DIN ISO 5725 (all parts), Accuracy (trueness and precision) of measurement methods and results (all parts) Messwesen (German Law on units in metrology) prohibits the use of the unit “psi” for official and commercial EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15637November 2008ICS 67.
8、050English VersionFoods of plant origin - Determination of pesticide residues usingLC-MS/MS following methanol extraction and clean-up usingdiatomaceous earthAliments dorigine vgtale - Dtermination des rsidus despesticides par LC-MS/MS aprs extraction mthanolique etpurification sur terre de diatomes
9、Pflanzliche Lebensmittel - LC-MS/MS-Verfahren zurBestimmung von Pestizidrckstnden mitMethanolextraktion und Reinigung an DiatomeenerdeThis 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 conditio
10、ns for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three offi
11、cial versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, B
12、elgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR
13、 STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2008 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15637:2008: EEN 15637:2008 (E) 2 Contents Page Fo
14、reword3 1 Scope 4 2 Principle4 3 Reagents.4 4 Apparatus .6 5 Procedure .7 6 Evaluation of results .12 7 Confirmatory tests.16 8 Precision.16 9 Test report 16 Annex A (informative) Example for appropriate experimental conditions17 Annex B (informative) Precision data21 Bibliography 63 DIN EN 15637:20
15、09-02 EN 15637:2008 (E) 3 Foreword This document (EN 15637:2008) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an ide
16、ntical text or by endorsement, at the latest by May 2009, and conflicting national standards shall be withdrawn at the latest by May 2009. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held respon
17、sible for identifying any or all such patent rights. WARNING The application of this standard may involve hazardous 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 safet
18、y and health practices and determining the applicability of regulatory limitations prior to use. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech
19、 Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15637:2009-02 EN 15637:2008 (E) 4 1 Sco
20、pe This European Standard describes a method for the analysis of pesticide residues in foods of plant origin, such as 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 combinati
21、ons. 2 Principle The sample is extracted with methanol after addition of some water. After partition into dichloromethane the organic phase is evaporated and the residue is reconstituted with methanol. Quantification of pesticide residues is performed by liquid chromatography with tandem mass spectr
22、ometric detection, using electrospray ionisation. To achieve the required selectivity the mass spectrometer is operated 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
23、 precaution to avoid possible contamination of water, solvents, inorganic salts, etc. 3.2 Ammonium formate 3.3 Sodium 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
24、1 shows a list of potential internal standards that may be used in this method. The concentrations listed refer to the 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 Nam
25、e of the compound Log P (octanol-water partion coefficient) Chlorine atoms Concentration C ISTDg/ml Triphenyl phosphate 4,59 - 20 Tris-(1,3-dichlorisopropyl)-phosphate 3,65 6 50 Bis-nitrophenyl urea (nicarbazin) 3,76 - 10 1) = mass concentration DIN EN 15637:2009-02 EN 15637:2008 (E) 5 3.8 Pesticide
26、 stock solutions Prepare individual stock solutions of analytical standards at concentrations that are sufficiently high to allow the preparation of complex pesticide mixtures. The solvent used should not negatively influence the stability of the pesticides employed. NOTE Usually, store stock soluti
27、ons at -18 C. Check the stability of stock solutions during storage regularly. In some cases the addition of acids or bases 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 div
28、ergent pH-stability of pesticides, analyte mixtures of different composition can be needed. These are prepared by mixing together defined volumes of the required analyte stock solutions (3.8) and appropriately diluting them with methanol. The analyte concentrations in this mixture should be sufficie
29、nt to allow the preparation of the required matrix matched standards (see 3.10.3) with moderate dilution of the blank sample 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
30、 has to be checked regularly. In some cases the addition of acids or bases can be helpful to enhance stability and extend acceptable 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 ce
31、rtain volume of methanol with known amounts of pesticide mixtures (3.9). The preparation of multiple standards of different 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
32、 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 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
33、mixtures (3.9) and a fixed volume of internal standard solution (3.7). The volume used shall result in that concentration of 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)
34、can be calculated using Equation (1). The preparation of multiple standards of different pesticide concentration but with constant ISTD concentration is useful to cover a broad concentration range. endexISTDISTDsampleISTDVVVVVVCVC=2312)(1) where: V ISTDis the volume of internal standard solution (3.
35、7) added to the test portion; C ISTDis the concentration of internal standard solution (3.7); V1is the volume of NaCl solution (2,5 ml); V2is the volume of measuring flask used in 5.2 (10 ml); DIN EN 15637:2009-02 EN 15637:2008 (E) 6 V3is the volume used for solid supported liquid/liquid extraction
36、(5 ml); Vexis the total volume of extraction solvents and natural water (30 ml); Vendis the final volume of extract obtained after 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 meth
37、anol during preparation of the final extract and fluctuations of instrument sensitivity during a batch of measurements. However, validation results in Annex B were obtained without internal standards. 3.10.3 Standard solutions prepared in blank matrix extracts (matrix-matched standards) Prepare matr
38、ix-matched standards in the same way as the solvent-based standards, however, instead of pure methanol use extracts of blank samples (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 comm
39、odities (e.g. apple for apple samples, carrots for carrot samples, etc.). The stability of pesticide in matrix-matched standards 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 samp
40、le volume, diatomaceous earth, for example ChemElut CE 10052)3.12 20 ml cartridge for solid supported liquid/liquid extraction, 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,
41、 for chopping up food samples for analysis 4.2 Homogenizer or high speed blender, fitted with jar 4.3 Laboratory balance 4.4 Measuring 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 (a
42、t 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 product 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 b
43、y CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. DIN EN 15637:2009-02 EN 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 s
44、ize, 4 mm diameter, polytetrafluoroethylene (PTFE) membrane 4.13 Glass vials and caps, 1,8 ml volume, suitable for an autosampler 4.14 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 processi
45、ng and storage procedures should be demonstrated to have no significant effect on the residues present in the test sample (sometimes 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
46、analytical portion is unlikely to be representative of the test sample, larger or replicate portions shall be analysed, to provide a 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
47、extensively spoiled or degraded should not be analysed. When possible, prepare laboratory samples immediately after arrival and in any 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 appro
48、priate conditions to keep it fresh and to avoid deterioration. Generally, laboratory samples should not be stored longer than 3 days 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 pa
49、rtly-prepared test sample take only the portion of the laboratory sample to which the maximum residue level applies. No further plant-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 sample shall be thoroughly mixed before weighing out the partly-prepared test sample. When the samples are made up of
