1、 DEUTSCHE NORM August 2006DIN EN 15054 ICS 67.050 Non fatty foods Determination of chlormequat and mepiquat LC-MS method English version of DIN EN 15054:2006-08 Fettarme Lebensmittel Bestimmung von Chlormequat und Mepiquat LC-MS-Verfahren Englische Fassung DIN EN 15054:2006-08 Document comprises 20
2、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 12 www.din.de www.beuth.de !,pLD“01.07 977413
3、3DIN EN 15054:2006-08 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 Lebensmittel und land
4、-wirtschaftliche 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 396/2005. Accor
5、dingly, it is appropriate 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 follows: ISO 5725-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO 5725-2 Annex A of this s
6、tandard includes unit “psi” which is not permitted in German standards. Users of this standard should note that the Gesetz ber Einheiten im Messwesen (German Law on units in metrology) prohibits the use of unit “psi” for official and commercial purposes in Germany. National Annex NA (informative) Bi
7、bliography 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 repeatability and reproducibil
8、ity of a standard measurement method EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15054 May 2006 ICS 67.060; 67.080.01 English Version Non fatty foods - Determination of chlormequat and mepiquat - LC-MS method Aliments non gras - Dtermination du chlormquat et du mpiquat - Mthode CL-SM Fettar
9、me Lebensmittel - Bestimmung von Chlormequat und Mepiquat - LC-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 Standard the status of a national
10、 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, French, German). A version in any ot
11、her 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 Republic, Denmark, Estonia, Finland,
12、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 NORMALISATION EUROPISCHES KOMITE
13、E 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 15054:2006: EEN 15054:2006 (E) 2 Contents Page Foreword3 1 Scope 4 2 Principle4 3 Reagents.4 4 Apparatus .5 5
14、Procedure .5 6 Evaluation of results .6 7 Confirmatory tests.7 8 Precision.7 9 Test report 8 Annex A (informative) Examples of experimental conditions 9 Annex B (informative) Precision data. 16 Bibliography. 18 EN 15054:2006 (E) 3 Foreword This document (EN 15054:2006) has been prepared by Technical
15、 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 November 2006, and conflicting national standar
16、ds shall be withdrawn at the latest by November 2006. As an alternative, there is also EN 15055 “Non fatty foods - Determination of chlormequat and mepiquat - LC-MS/MS method“ available. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countrie
17、s 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, Romania, Slovakia, Slovenia, Spain, Sweden, Sw
18、itzerland and United Kingdom. EN 15054:2006 (E) 4 1 Scope This draft European Standard specifies a method using high performance liquid chromatography/mass spectrometry (LC-MS) for the determination of the growth regulators chlormequat and mepiquat in non fatty foods as chlormequat and mepiquat cati
19、on, respectively. The method is applicable to all kinds of fruits, vegetables and cereal products. It has been collaboratively studied on mushrooms, pears, wheat flour and fruit puree, see 1. 2 Principle The sample is mixed with deuterated internal standards, water and methanol and the homogenate is
20、 centrifuged. An aliquot portion of the supernatant is filtered. An aliquot portion of the filtrate is analyzed by high performance liquid chromatography (HPLC) with mass spectrometric detection after positive electrospray ionisation, see also 2 to 7. 3 Reagents 3.1 General and safety aspects Unless
21、 otherwise specified, use reagents of recognized analytical grade. Take every precaution to avoid possible contamination of water, solvents, inorganic salts, etc. WARNING The use of this standard may involve hazardous materials, operations and equipment. This standard does not purport to address all
22、 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. 3.2 Ammonium acetate 3.3 Filter aid, for example Celite5451)3.4 Glacial
23、acetic acid, mass fraction w at least 96 g/100 g 3.5 Acetonitrile, HPLC quality 3.6 Methanol, HPLC quality 3.7 Water, suitable for HPLC 3.8 Chlormequat chloride stock solution, mass concentration (C5H13NCl2) = 4,30 g/ml in methanol This stock solution contains 3,33 g/ml chlormequat cation. 3.9 Mepiq
24、uat chloride stock solution, (C7H16NCl) = 4,37 g/ml in methanol This stock solution contains 3,33 g/ml mepiquat cation. 1) Celite 545 is a product supplied by Celite Corp. (World Minerals Inc., Santa Barbara, CA, USA). This information is given for the convenience of users of this European Standard
25、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 15054:2006 (E) 5 3.10 Internal standard solution 1 (d4-Chlormequat chloride), (C5H9D4NCl2) = 21,33 g/ml in methanol This stock solution contains 16
26、,67 g/ml d4-chlormequat cation. For the availability of the standard, contact your national standardisation institute. 3.11 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
27、standard, contact your national standardisation institute. 3.12 Calibration solutions Prepare six calibration solutions (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 (3.8) and
28、of mepiquat chloride stock solution (3.9). To all flasks add 60 l of the internal standard solution 1 (3.10) and 60 l of the internal standard solution 2 (3.11). 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 calibrati
29、on solutions contain 100 ng/ml of d4-chlormequat cation and 100 ng/ml of d3-mepiquat cation. Solutions no. 1 to 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, t
30、his 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 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 prod
31、ucing a centrifugal acceleration of at least 3 000 g at the bases of the centrifuge tubes 4.3 Syringe, capacity 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-M
32、S system, equipped with electrospray interface 5 Procedure 5.1 Preparation of the samples Where possible, carry out the analysis of samples immediately on their arrival in the laboratory. Do not analyse a laboratory sample which is wholly or extensively spoiled. For analysis take only the portion of
33、 the laboratory sample to which the maximum residue level applies. No further plant-parts may be removed. A record of the plant-parts which have been removed shall be kept. The sample thus prepared is the test sample. EN 15054:2006 (E) 6 If the sample cannot be analysed immediately, store it at 0 C
34、to 5 C for no longer than 3 days before analysis. The reduction of the test sample shall be carried out 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
35、), the test sample shall be thoroughly mixed before weighing out the test portion. When the samples are 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 th
36、ose parts which would interfere with the analytical procedure. In the case of stone fruits, the stones 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 orig
37、inal test sample (with stones). If samples have to be stored for more than 3 days, they shall 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. Chop the test sample and weigh ou
38、t test portions of masses of 20 g to an accuracy of 1 %. 5.2 Extraction Transfer a representative test portion of mA= 20 g into the blender cup (4.1). For dry sample materials like cereal products, weigh a homogenised test portion of 10 g (mA) into the cup. Add 360 l of internal standard solution 1
39、(3.10) and 360 l of internal standard solution 2 (3.11). Add so much water, that a total volume (added and natural) of 20 ml water is obtained. In the case of dry sample materials wait 10 min after the addition of water. Add 40 ml of methanol (3.6) and blend for 2 min. The total volume of liquid ext
40、ract (taking into account the natural water content of the sample) is 60 ml. Centrifuge an aliquot portion at approximately 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
41、 the filter is likely to be blocked by suspended matter, place a 1 cm layer of filter aid (3.3) onto the bottom of the 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 (3.12) into the LC-M
42、S system. The selected ions shall be appropriate for the sensitive and selective determination of chlormequat, mepiquat and the internal standards (see Annex A). Make sure that the liquid chromatographic conditions (column length, stationary phase type, injection volume, column temperature, electros
43、pray interface parameters, etc.) are such that the separation of chlormequat and mepiquat from possible interfering peaks originating from the samples is as complete as possible. Typical LC/MS conditions are given in Annex A. 5.4 Test for interference and recovery Prepare reagent blanks and carry ou
44、t spiked recovery tests at levels appropriate to the maximum residue level. The chromatogram of the reagent blank should not show any significant peak at the retention time of chlormequat or mepiquat. 6 Evaluation of results To identify residues of chlormequat or mepiquat compare the retention times
45、 obtained from the sample test solution with those obtained from the calibration solutions. Positive findings are confirmed by comparing the peak intensity ratios of the first and second compound specific ions (e. g. ions m/z 122 and m/z 124 for chlormequat, see A.8) with the expected peak intensity
46、 ratios from the standards. If the peak ratio of a residue EN 15054:2006 (E) 7 peak differs more than 30 % from the expected response ratio, additional measures are necessary, e.g. the use of other LC column. Use the calibration solutions to check linearity and to determine the calibration graphs ba
47、sed on the response ratio of chlormequat/d4-chlormequat and mepiquat/d3-mepiquat, respectively. As units on the x-axis use the corresponding mass fraction of chlormequat cation or mepiquat cation respectively in 20 g of the test portion (see 3.12). NOTE 1 Typically, a response ratio of about 1 shoul
48、d be obtained with the calibration solution no. 4 (corresponding to a mass fraction of 0,3 mg/kg for the 20 g test portion). The resulting slope of the calibration graphs should be approximately 3,3 kg/mg. NOTE 2 Since the calibration range shall be appropriate to the residue concentrations found, i
49、t may be necessary to construct more than one calibration graph from the results of 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 corresponding internal standard (d4-chlormequat or d3-mepiquat, respectively). Based on the resulting response ratios and the two calibration graphs calculate the mass fraction wRof chlormequat cation and mepiquat cation, in millig
