1、BRITISH STANDARDBS EN 15055:2006Non fatty foods Determination of chlormequat and mepiquat LC-MS/MS methodThe European Standard EN 15055:2006 has the status of a British StandardICS 65.060g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g
2、55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN 15055:2006This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 June 2006 BSI 2006ISBN 0 580 48618 4National forewordThis British Standard is the offici
3、al English language version of EN 15055:2006.The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Food analysis Horizontal methods, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary.Cross
4、-referencesThe British Standards which implement international or European publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of Br
5、itish Standards Online.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; prese
6、nt to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep UK interests informed; monitor related international and European developments and promulgate them in the UK.Summary of pagesThis document comprises a front cover, an inside
7、front cover, the EN title page, pages 2 to 19 and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments issued since publicationAmd. No. Date CommentsEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15055May 2006ICS 65.060English Ver
8、sionNon fatty foods - Determination of chlormequat and mepiquat -LC-MS/MS methodAliments non gras - Dtermination de la teneur enchlormequate et mepiquate - Mthode LC-MS/MSFettarme Lebensmittel - Bestimmung von Chlormequat undMepiquat - LC-MS/MS-VerfahrenThis European Standard was approved by CEN on
9、20 April 2006.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 bibliographical references concerning such nationalstandards may be o
10、btained 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 other language made by translationunder the responsibility of a CEN member into its own language and notified to the Central Sec
11、retariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Port
12、ugal, 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 Brussels 2006 CEN All rights of exploitation in any form and by any means re
13、servedworldwide for CEN national Members.Ref. No. EN 15055:2006: EEN 15055:2006 (E) 2 Contents Page Foreword3 1 Scope 4 2 Principle4 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 appropria
14、te experimental conditions . 10 Annex B (informative) Precision data. 18 Bibliography. 19 EN 15055:2006 (E) 3 Foreword This document (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 Standa
15、rd 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 standards shall be withdrawn at the latest by November 2006. As an alternative, there is also EN 15054 “Non fatty foods - Determ
16、ination of chlormequat and mepiquat - LC-MS method“ available. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France
17、, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EN 15055:2006 (E) 4 1 Scope This draft European Standard specifies a method using high perform
18、ance liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the determination of the growth regulators chlormequat and mepiquat 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 col
19、laboratively studied on mushrooms, pears, wheat flour, fruit puree and, additionally, on infant formula, see 1. 2 Principle The sample is mixed with deuterated internal standards, water and methanol and the homogenate is centrifuged. An aliquot portion of the supernatant is filtered. An aliquot port
20、ion of the filtrate is analysed by liquid chromatography with tandem mass spectrometric detection after positive electrospray ionisation. 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 oth
21、erwise specified, use reagents of recognised 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 the
22、 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 Ammonium formate 3.4 Filter aid, for example Celite
23、5451)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 Water, 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 c
24、ation. 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 and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can b
25、e 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 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 sol
26、ution contains 16,67 g/ml d4-chlormequat cation. For the availability of the standard, contact your national standardisation institute. 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 ava
27、ilability of the standard, contact your national standardisation institute. 3.13 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 so
28、lution (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 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.
29、 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 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
30、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 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 Centrif
31、uge, capable of producing 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
32、autosampler 4.6 LC-MS/MS system, equipped with electrospray interface 5 Procedure 5.1 Preparation of the samples Where possible, carry 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 15055:200
33、6 (E) 6 For analysis take only the portion of 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. If the test sample cannot be analysed
34、 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 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.
35、small fruits, legumes, cereals), 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. Fr
36、om each test sample, remove those 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 fr
37、action 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 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 ca
38、n be taken, prepare portions of the product which are each sufficient for one analysis. 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 portion of 10 g (mA) into the cup. Add 60 l of inte
39、rnal standard solution 1 (3.11) and 60 l of internal standard solution 2 (3.12). Add sufficient 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 addition of water. Add 40 ml of methanol (3.7) and blend for 2 min. The tot
40、al volume of liquid extract (taking into account the natural water content of the sample) is 60 ml. Centrifuge an aliquot 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
41、 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 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 (3.13
42、) into the LC-MS/MS system. The LC-MS/MS system shall be operated in the multi reaction mode with transitions selective for chlormequat, mepiquat and the internal standards (see Annex A). Make sure that the liquid chromatographic conditions (column length, stationary phase type, injection volume, co
43、lumn temperature, electrospray 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/MS conditions are given in Annex A. 5.4 Test for interference and recovery Prepar
44、e reagent blanks and carry out 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. EN 15055:2006 (E) 7 6 Evaluation of results To identify residues of ch
45、lormequat or mepiquat compare the retention times 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 transition (e. g. transition 122 58 and 124 58
46、 for chlormequat) with the expected peak intensity ratios from the standards. If the peak ratio of a residue peak differs more than 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 calibrati
47、on solutions to check linearity and to determine the calibration graphs based on the response ratio of chlormequat/d4-chlormequat and 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)
48、. NOTE 1 Typically, a response ratio of about 1 should be obtained with the calibration solution no. 2 (corresponding to a mass fraction 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
49、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 corresponding internal standard (d4-chlormequat or d3-mepiquat, respectively). Based on the resulting response ratios and the two calibration graphs calculate the mass fraction wR
