1、 DEUTSCHE NORM August 2006DIN EN 15055 ICS 67.060; 67.080.01 Non fatty foods Determination of chlormequat and mepiquat LC-MS/MS method English version of DIN EN 15055:2006-08 Fettarme Lebensmittel Bestimmung von Chlormequat und Mepiquat LC-MS/MS-Verfahren Englische Fassung DIN EN 15055:2006-08 Docum
2、ent comprises 21 pages No part of this standard may be reproduced without prior permission of DIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany, has the exclusive right of sale for German Standards (DIN-Normen). English price group 13 www.din.de www.beuth.de !
3、,pLF“01.07 9774135DIN EN 15055:2006-08 2 National foreword This standard has been prepared by CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: Germany), Working Group WG 4 “Pesticides in non fatty foods”. The responsible German body involved in its preparation was the Normenausschuss Lebe
4、nsmittel und landwirtschaftliche Produkte (Foodstuffs and Agricultural Products Standards Committee), Technical Committee Pestizide. The maximum residue levels of pesticides (including chlormequat and mepiquat) in non fatty foods to be observed throughout the Community are laid down in EC Regulation
5、 396/2005. Accordingly, it is appropri-ate that harmonised methods of determining chlormequat and mepiquat be specified at Community level. The DIN Standards corresponding to the International Standards referred to in this document are as fol-lows: ISO 5725-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO 5725-2
6、 Annex A of this standard includes unit “psi” which is not permitted in German standards. Users of this stan-dard should note that the Gesetz ber Einheiten im Messwesen (German Law on units in metrology) pro-hibits the use of unit “psi” for official and commercial purposes in Germany. National Annex
7、 NA (informative) Bibliography DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions DIN ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatabil
8、ity and reproducibility of a standard measurement method EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15055 May 2006 ICS 65.060 English Version Non fatty foods - Determination of chlormequat and mepiquat - LC-MS/MS method Aliments non gras - Dtermination de la teneur en chlormequate et mepiq
9、uate - Mthode LC-MS/MS Fettarme Lebensmittel - Bestimmung von Chlormequat und Mepiquat - LC-MS/MS-Verfahren This European Standard was approved by CEN on 20 April 2006. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European S
10、tandard 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 Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, Fren
11、ch, 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 Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Repub
12、lic, 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 STANDARDIZATION COMIT EUROPEN DE
13、NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2006 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15055:2006: EEN 15055:2006 (E) 2 Contents Page Foreword3 1 Scope 4 2 Principle
14、4 3 Reagents.4 4 Apparatus .5 5 Procedure .5 6 Evaluation of results .7 7 Confirmatory tests.7 8 Precision.8 9 Test report 8 Annex A (informative) Examples for appropriate experimental conditions . 10 Annex B (informative) Precision data. 18 Bibliography. 19 EN 15055:2006 (E) 3 Foreword This documen
15、t (EN 15055:2006) 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
16、 November 2006, and conflicting national standards shall be withdrawn at the latest by November 2006. As an alternative, there is also EN 15054 “Non fatty foods - Determination of chlormequat and mepiquat - LC-MS method“ available. According to the CEN/CENELEC Internal Regulations, the national stan
17、dards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, R
18、omania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EN 15055:2006 (E) 4 1 Scope This draft European Standard specifies a method using high performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the determination of the growth regulators chlormequat and mepiq
19、uat in non-fatty foods as chlormequat and mepiquat cation, respectively. The method is applicable to all kinds of fruits, vegetables and cereal products. It has been collaboratively studied on mushrooms, pears, wheat flour, fruit puree and, additionally, on infant formula, see 1. 2 Principle The sam
20、ple is mixed with deuterated internal standards, water and methanol and the homogenate is centrifuged. An aliquot portion of the supernatant is filtered. An aliquot portion of the filtrate is analysed by liquid chromatography with tandem mass spectrometric detection after positive electrospray ionis
21、ation. To achieve the required selectivity the mass spectrometer is operated in the multi reaction mode, see 2 to 7. 3 Reagents 3.1 General and safety aspects Unless otherwise specified, use reagents of recognised analytical grade. Take every precaution to avoid possible contamination of water, solv
22、ents, inorganic salts, etc. WARNING The use of this standard may involve hazardous materials, operations and equipment. This standard does not purport 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
23、 health practices and to determine the applicability of regulatory limitations prior to use. 3.2 Ammonium acetate 3.3 Ammonium formate 3.4 Filter aid, for example Celite5451)3.5 Glacial acetic acid, mass fraction w at least 96 g/100 g 3.6 Acetonitrile, HPLC quality 3.7 Methanol, HPLC quality 3.8 Wat
24、er, suitable for HPLC 3.9 Chlormequat chloride stock solution,mass concentration (C5H13NCl2) = 4,30 g/ml in methanol This stock solution contains 3,33 g/ml chlormequat cation. 1) Celite 545 is a product supplied by Celite Corp. (World Minerals Inc., Santa Barbara, CA, USA). This information is given
25、 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. EN 15055:2006 (E) 5 3.10 Mepiquat chloride stock solution, (C7H16NCl) = 4,37 g/ml in methanol
26、This stock solution contains 3,33 g/ml mepiquat cation. 3.11 Internal standard solution 1 (d4-Chlormequat chloride), (C5H9D4NCl2) = 21,33 g/ml in methanol This stock solution contains 16,67 g/ml d4-chlormequat cation. For the availability of the standard, contact your national standardisation instit
27、ute. 3.12 Internal standard solution 2 (d3-Mepiquat iodide), (C7H13D3NI) = 34,70 g/ml in methanol This stock solution contains 16,67 g/ml d3-mepiquat cation. For the availability of the standard, contact your national standardisation institute. 3.13 Calibration solutions Prepare six calibration solu
28、tions (no. 1 to no. 6) in 10 ml volumetric flasks as follows. Into the flasks, pipette 10 l, 50 l, 100 l, 300 l, 1 000 l or 3 000 l each of chlormequat chloride stock solution (3.9) and of mepiquat chloride stock solution (3.10). To all flasks add 10 l of the internal standard solution 1 (3.11) and
29、10 l of the internal standard solution 2 (3.12). Dilute the solutions no. 1 to no. 5 to the mark with a methanol/water mixture 1 + 1 (V+V) and solution no. 6 with water. All six calibration solutions contain 16,7 ng/ml of d4-chlormequat cation and 16,7 ng/ml of d3-mepiquat cation. Solutions no. 1 to
30、 no. 6 contain 3,3 ng/ml, 16,7 ng/ml, 33,3 ng/ml, 100 ng/ml, 333 ng/ml and 1 000 ng/ml each of chlormequat cation and of mepiquat cation respectively. On the basis of a 20 g test portion, this corresponds to 0,01 mg/kg, 0,05 mg/kg, 0,10 mg/kg, 0,30 mg/kg, 1,00 mg/kg or 3,00 mg/kg each of chlormequat
31、 cation and mepiquat cation. 4 Apparatus Usual laboratory apparatus and, in particular, the following: 4.1 Homogenizer or high speed blender, fitted with jar 4.2 Centrifuge, capable of producing a centrifugal acceleration of at least 3 000 g at the bases of the centrifuge tubes 4.3 Syringe, capacity
32、 of at least 2 ml 4.4 Syringe filter, pore size 0,45 m (polyamide or polytetrafluoroethylene) 4.5 Glass vials, 1,8 ml volume; applicable for automatic injection into an autosampler 4.6 LC-MS/MS system, equipped with electrospray interface 5 Procedure 5.1 Preparation of the samples Where possible, ca
33、rry out the analysis of samples immediately upon their arrival in the laboratory. Do not analyse a laboratory sample which is wholly or extensively spoiled. EN 15055:2006 (E) 6 For analysis take only the portion of the laboratory sample to which the maximum residue level applies. No further plant-pa
34、rts may be removed. A record of the plant parts which have been removed shall be kept. The sample thus prepared is the test sample. If the test sample cannot be analysed immediately, store it at 0 C to 5 C for no longer than 3 days before analysis. The reduction of the test sample shall be carried o
35、ut in such a way that representative portions are obtained (e. g. by division into four and selection of opposite quadrants). When the samples are in small units (e. g. small fruits, legumes, cereals), the test sample shall be thoroughly mixed before weighing out the test portion. When the samples a
36、re in larger units, take wedge-shaped sections (e. g. large fruits and vegetables) or cross sections (e. g. cucumbers) which include the outer surface from each unit. From each test sample, remove those parts which would interfere with the analytical procedure. In the case of stone fruits, the stone
37、s should be removed. Care shall be taken that as little as possible of the remainder such as juice or flesh is lost. The basis for the calculation of the residue mass fraction is the mass of the original test sample (with stones). Chop the test sample and weigh out test portions of masses of 20 g to
38、 an accuracy of 1 %. If samples have to be stored for more than 3 days, they should be deep-frozen at 20 C. To ensure that, even after thawing, representative samples can be taken, prepare portions of the product which are each sufficient for one analysis. 5.2 Extraction Transfer a representative te
39、st portion of mA= 20 g into the blender cup (4.1). For dry sample materials like cereal products, weigh a homogenised portion of 10 g (mA) into the cup. Add 60 l of internal standard solution 1 (3.11) and 60 l of internal standard solution 2 (3.12). Add sufficient water, that a total volume (added a
40、nd natural) of 20 ml water is obtained. In the case of dry sample materials wait 10 min after addition of water. Add 40 ml of methanol (3.7) and blend for 2 min. The total volume of liquid extract (taking into account the natural water content of the sample) is 60 ml. Centrifuge an aliquot at approx
41、imately 3 000 g (4.2), transfer approximately 2 ml of the supernatant into the syringe (4.3) and filter the solution through the syringe filter (4.4) into a 1,8 ml glass vial (4.5). If the filter is likely to be blocked by suspended matter, place a 1 cm layer of filter aid (3.4) onto the bottom of t
42、he syringe before filling it with the extract. 5.3 Determination Inject equal volumes of the sample test solution derived from 5.2 and of the calibration solutions (3.13) into the LC-MS/MS system. The LC-MS/MS system shall be operated in the multi reaction mode with transitions selective for chlorme
43、quat, mepiquat and the internal standards (see Annex A). Make sure that the liquid chromatographic conditions (column length, stationary phase type, injection volume, column temperature, electrospray interface parameters, etc.) are such that the separation of chlormequat and mepiquat from possible i
44、nterfering peaks originating from the samples is as complete as possible. Typical LC-MS/MS conditions are given in Annex A. 5.4 Test for interference and recovery Prepare reagent blanks and carry out spiked recovery tests at levels appropriate to the maximum residue level. The chromatogram of the re
45、agent blank should not show any significant peak at the retention time of chlormequat or mepiquat. EN 15055:2006 (E) 7 6 Evaluation of results To identify residues of chlormequat or mepiquat compare the retention times obtained from the sample test solution with those obtained from the calibration s
46、olutions. Positive findings are confirmed by comparing the peak intensity ratios of the first and second compound specific transition (e. g. transition 122 58 and 124 58 for chlormequat) with the expected peak intensity ratios from the standards. If the peak ratio of a residue peak differs more than
47、 30 % from the expected response ratio, additional measures are necessary, e.g. the use of other LC column, another eluent or an additional transition. Use the calibration solutions to check linearity and to determine the calibration graphs based on the response ratio of chlormequat/d4-chlormequat a
48、nd mepiquat/d3-mepiquat. As units on the x-axis, use the corresponding mass fraction of chlormequat cation or mepiquat cation respectively in the test portion (see 3.13). NOTE 1 Typically, a response ratio of about 1 should be obtained with the calibration solution no. 2 (corresponding to a mass fra
49、ction of 0,05 mg/kg for the 20 g test portion). The resulting slope of the calibration graphs should be approximately 20 kg/mg. NOTE 2 Since the calibration range shall be appropriate to the residue concentrations found, it may be necessary to construct more than one calibration graph from the results of the calibration measurements. Measure the peak height (or peak area) obtained for chlormequat and mepiquat cation in the sample test solution and divide it by the peak height (or peak area) obtained for the peak of the c
