1、BS EN 15662:2008ICS 67.050NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDFoods of plant origin Determination ofpesticide residuesusing GC-MS and/orLC-MS/MS followingacetonitrile extraction/partitioning and clean-up by dispersive SPE QuEChERS-methodThis British
2、Standardwas published underthe authority of theStandards Policy andStrategy Committee on 32008 BSI 2008ISBN 978 0 580 58441 1Amendments/corrigenda issued since publicationDate CommentsBS EN 15662:2008National forewordThis British Standard is the UK implementation of EN 15662:2008.The UK participatio
3、n in its preparation was entrusted 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 respo
4、nsible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.1DecemberBS EN 15662:2008EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15662November 2008ICS 67.050English VersionFoods of plant origin - Determination of pesticide residues usingGC
5、-MS and/or LC-MS/MS following acetonitrileextraction/partitioning and clean-up by dispersive SPE -QuEChERS-methodAliments dorigine vgtale - Mthode polyvalente dedtermination des rsidus des pesticides par CG-SM etSL/SM(SM) avec extraction/partition avec de lactonitrile etnettoyage par SPE disperss -
6、Mthode QuEchERSPflanzliche Lebensmittel - Bestimmung vonPestizidrckstnden mit GC-MS und/oder LC-MS/MS nachAcetonitril-Extraktion/Verteilung und Reinigung mitdispersiver SPE - QuEChERS-VerfahrenThis European Standard was approved by CEN on 13 September 2008.CEN members are bound to comply with the CE
7、N/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 bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to
8、 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 CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.
9、CEN members are the national standards bodies of Austria, Belgium, 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, Sw
10、eden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR 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.Re
11、f. No. EN 15662:2008: EBS EN 15662:2008EN 15662:2008 (E) 2 Contents Page Foreword3 1 Scope 4 2 Principle4 3 Reagents.4 4 Apparatus .8 5 Procedure .10 6 Evaluation of results .16 7 Confirmatory tests.21 8 Precision.21 9 Test report 21 Annex A (informative) Examples of experimental conditions 22 Annex
12、 B (informative) Precision data25 Annex C (informative) Procedure schematically (for 10 g sample)75 Annex D (informative) Complementary information 76 Bibliography 81 BS EN 15662:2008EN 15662:2008 (E) 3 Foreword This document (EN 15662:2008) has been prepared by Technical Committee CEN/TC 275 “Food
13、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 at the late
14、st 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 hazardous materia
15、ls, operations and equipment. This standard does not claim to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and to determine the applicability of regulatory limitations prior to use.
16、 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 Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy,
17、 Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 15662:2008EN 15662:2008 (E) 4 1 Scope This European Standard describes a method for the analysis of pesticide residues in foods of plant
18、 origin, such as fruits (including dried fruits), vegetables, cereals and processed products thereof. The method has been collaboratively studied on a large number of commodity/pesticide combinations. 2 Principle The homogeneous sample is extracted with the help of acetonitrile. Samples with low wat
19、er content ( 95 %) to 10 ml with acetonitrile (3.3). 3.14 Primary secondary amin sorbent For example, Bondesil-PSA40 m Varian No. 122130231). Other amino sorbents may be used, but investigations may be necessary to prove equivalency especially regarding analyte losses and pH value of the end extract
20、s. 3.15 Graphitised Carbon Black sorbent (GCB), e.g. Supelco Supelclean Envi-Carb1)SPE Bulk Packing, No. 57210U Other graphitised carbon sorbents may be used, but investigations will be necessary to prove equivalency especially regarding analyte losses. 3.16 Sorption mixture 1: GCB (3.15)/ magnesium
21、 sulfate anhydrous fine powder (3.7)-mixture, 1 + 59 mass portions Mix the two components intensively to form a visually homogeneous mixture. 3.17 Sorption mixture 2: GCB (3.15)/ magnesium sulfate anhydrous fine powder (3.7)-mixture, 1 + 19 mass portions Mix the two components intensively to form a
22、visually homogeneous mixture. NOTE It is highly advisable to prepare the sorption mixtures 1 (3.16) and 2 (3.17) in advance and store them in sealable vessels. For the extract clean-up according to 5.4.3 the pre-mixed sorption mixtures 1 or 2 are weighed into the centrifuge tubes (4.4). 1) Bondesil-
23、PSAis a product supplied by Varian, Inc. (Palo Alto, CA, USA). Envi-Carb is a product supplied by Supelco. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the products named. Equivalent products may be used if they can
24、 be shown to lead to the same results. BS EN 15662:2008EN 15662:2008 (E) 6 3.18 C-18-sorbent (Octadecyl-silyl-modified silica gel), Bulk material 50 m 3.19 Internal standard and quality control standard solutions in acetonitrile, = 10 g/ml to 50 g/ml Table 1 shows a list of potential internal standa
25、rds (ISTDs) and quality control (QC) standards that may be used in this method. The suggested concentration values (CISTD) listed refers to the ISTD solutions that should be added at the first extraction step (5.2). An appropriate dilution of this solution (mixcalISTDC ) should be prepared to be use
26、d for the preparation of the standard solutions. For more details see 3.22. Table 1 Potential internal standards (ISTDs) or quality control (QC) standards GC LCName of the compound Log P (octanol-water partition coefficient) Chlorine atoms Sugges-ted concen-trationCISTD g/mlaECD NPD MSD EI (+) MSD C
27、I (-) MS/MS ESI (+) MS/MS ESI (-) Potential Internal Standards PCB 18 5,55 3 50 + - + + - - PCB 28 5,62 3 50 + - + + - - PCB 52 6,09 4 50 + - + + - - Triphenyl phosphate 4,59 - 20 - + + - + - Tris-(1,3-dichlorisopropyl)-phosphate 3,65 6 50 + + + + + + Triphenylmethane 5,37 - 10 - - + - - - Bis-nitro
28、phenyl urea (nicarbazin) 3,76 - 10 - - - - - + Potential Quality Control Standards (may be contained in the same mixture as the other ISTDs used or added at a different stage of analysis to detect and localize sources of error) PCB 138b6,83 6 50 + - + + - - PCB 153b7,75 6 50 + - + + - - Anthracene (
29、or its d10 analogue)c4,45 - 100 - - + - - - a Exemplary concentrations of the ISTD solutions to be added to the test samples in 5.2, use acetonitrile as solvent. b Recoveries of PCB 138 and 153 drop as lipid amount in the sample increases, recoveries of those two compounds exceeding 70 % indicate th
30、at no unacceptable partitioning losses have occurred even for the most lipophilic pesticides. c Recoveries of anthracene exceeding 70 % will indicate that no unacceptable losses of pesticides with high carbon affinity have occurred during dispersive SPE with GCB. 3.20 Pesticide stock solutions Prepa
31、re individual stock solutions of analytical standards at concentrations that are sufficient to allow the preparation of complex pesticide working solutions (3.21) that are used for the preparation of standard solutions. Usually, store stock solutions at -18 C. Check the stability of stock solutions
32、during storage regularly 2. In some cases the addition of acids or bases can be helpful to enhance stability and extend the acceptable BS EN 15662:2008EN 15662:2008 (E) 7 storage period. Before withdrawing any aliquot from this solution redissolve any precipitation that may have occurred. 3.21 Pesti
33、cide working solutions Because of the broad applicability of this method and due to the partly divergent pH-stability of pesticides, more than one working solution each containing one or more pesticides can be needed to cover the entire pesticide spectrum of interest. These are prepared by mixing to
34、gether defined volumes of the required pesticide stock solutions (3.20) and appropriately diluting them with acetonitrile. The pesticide concentrations in these mixtures should be sufficient to allow the preparation of the required matrix matched standards (see 3.22.2) with moderate dilution of the
35、blank sample extract (e.g. less than 20 %). Usually, store pesticide working solutions at -18 C. Check the stability of pesticides contained in these mixtures during storage regularly 2. In some cases the addition of acids or bases can be helpful to enhance stability and extend acceptable storage ti
36、mes. 3.22 Standard solutions (calibration mixtures) 3.22.1 Solvent-based standards Solvent-based standards are prepared by mixing known volumes of the pesticide working solutions (mixcalpestV see 3.21) and the ISTD solution (mixcalISTDV see 3.19) and filling up to volume with acetonitrile. The volum
37、e of the ISTD solution to be employed (mixcalISTDV ) will depend on the volume of the standard solution to be prepared (mixcalV ) and should be such to ensure an ISTD concentration similar to that in the sample test solutions (5.3, 5.4). EXAMPLE If 1 ml solvent-based standard is prepared the volume
38、of ISTD solution to be added should contain a mass of ISTD (mixcalISTDmixcalISTDmixcalISTDVxCm = ) which is 10-fold smaller than the mass of ISTD added to the test portions in 5.2.3, where 10 ml of acetonitrile are used for extraction. It is thus indicated to appropriately dilute the concentration o
39、f internal standard solution (in this case mixcalISTDC = 0,1 C ISTD). Then the same pipette volume can be used to add ISTDs to spike test samples and for the preparation of standard solutions. Table 2 shows exemplarily the ratio of the ISTD mass that should be added to the test portions (5.2.3) and
40、the standard solutions (3.22). The preparation of multiple standard solutions covering a broad concentration range will allow the construction of a calibration curve (see 6.2). NOTE A pesticide concentration of 1 g/ml correlates to a residue level of 1 mg/kg when a 10 g sample is employed (e.g. samp
41、les with water content 30 %) or 2 mg/kg when 5 g sample is employed (e.g. cereals). 3.22.2 Matrix-matched standards Prepare matrix-matched standards in the same way as solvent-based standards, however, instead of pure acetonitrile use extracts of blank samples (prepared as described in 5.1 to 5.4, b
42、ut 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, carrot for carrot samples, etc.). Should the dilution of the blank sample extract upon addition of the pesticide working solut
43、ions exceed 20 %, a volume adjustment may be necessary to avoid errors caused by differences in the matrix-induced enhancement effect between sample extract and matrix-matched standard. The stability of pesticides in matrix-matched standards can be lower than that of standards in pure acetonitrile a
44、nd has to be checked more thoroughly. BS EN 15662:2008EN 15662:2008 (E) 8 Table 2 Ratios of the masses of ISTD added to the test-portion and to the standard solutions (calibration mixtures) Volume of standard solution V cal mix ml mixcalISTDsampleISTDmm= calmixISTDmix calISTDsampleISTDISTDx V Cx V C
45、1 10 2 5 5 2 10 1 NOTE The values given in this table refer to sample extract volumes of ca. 10 ml (i.e. following addition of 10 ml acetonitrile in 5.2.3). The blank sample employed to prepare the matrix-matched standard should be extracted in the same way as the sample. 3.23 Cold water ( 3 000 g.
46、NOTE When dealing with highly acidic commodities, such as lemons, limes, currants and raspberries, use buffer-salt mixtures to which NaOH was added as stated in 3.12. In the presence of water, magnesium sulfate tends to form lumps, which can harden rapidly. This can be avoided, if immediately after
47、the addition of the salt mixture the centrifuge tube is shaken vigorously for few seconds. The 1 min extraction of the entire batch may be performed in parallel after the salts have been added to all the samples. Pesticides with acidic groups (e.g. phenoxyalcanoic acids) interact with amino-sorbents
48、 such as PSA. Thus, if such pesticides are within the scope of analysis, their determinative analysis (preferably via LC-MS/MS ESI BS EN 15662:2008EN 15662:2008 (E) 15 neg.) should be performed directly from the raw extract after centrifugation but prior to clean-up (5.4). For this, fill an aliquot
49、of the raw extract into a vial and employ for LC-MS/MS analysis. Typical LC-MS/MS conditions for acidic pesticides are given in Annex A. 5.4 Clean-up 5.4.1 Removal of co-extracted fat, wax, sugars (e.g. for cereals, citrus fruits) Transfer an aliquot of 8 ml of the acetonitrile phase (5.3) into a centrifuge tube (4.5) and store overnight in a freezer (for flour, 2 h are sufficient), wherewith the major part of fat and waxes solidify and precipitate. Following a short centrifugation (where ne
