1、BRITISH STANDARDBS EN 15054:2006Non fatty foods Determination of chlormequat and mepiquat LC-MS method The European Standard EN 15054:2006 has the status of a British StandardICS 67.060; 67.080.01g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g
2、38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN 15054:2006This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 June 2006 BSI 2006ISBN 0 580 48617 6National forewordThis British Standard is t
3、he official English language version of EN 15054:2006.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 secret
4、ary.Cross-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
5、 or of British 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 te
6、xt; present 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, a
7、n inside front cover, the EN title page, pages 2 to 18, an inside back cover 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 150
8、54May 2006ICS 67.060; 67.080.01English VersionNon fatty foods - Determination of chlormequat and mepiquat -LC-MS methodAliments non gras - Dtermination du chlormquat et dumpiquat - Mthode CL-SMFettarme Lebensmittel - Bestimmung von Chlormequat undMepiquat - LC-MS-VerfahrenThis European Standard was
9、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 EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationa
10、lstandards 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 other language made by translationunder the responsibility of a CEN member into its own language and notified
11、 to the Central Secretariat 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, N
12、orway, Poland, Portugal, 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
13、and by any means reservedworldwide 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 Procedure .5 6 Evaluation of results .6 7 Confirmatory tests.7 8 Precision.7 9 Test report 8 Annex A (informative) Exa
14、mples 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 Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Sta
15、ndard 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 15055 “Non fatty foods - Det
16、ermination of chlormequat and mepiquat - LC-MS/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,
17、France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EN 15054:2006 (E) 4 1 Scope This draft European Standard specifies a method using high p
18、erformance liquid chromatography/mass spectrometry (LC-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 collabo
19、ratively 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 centrifuged. An aliquot portion of the supernatant is filtered. An aliquot portion of the filtrate is analyzed by hig
20、h 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 otherwise specified, use reagents of recognized analytical grade. Take every precaution to avoid possible contaminati
21、on 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 safety problems associated with its use. It is the responsibility of the user of this standard to establish appro
22、priate 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 acetic acid, mass fraction w at least 96 g/100 g 3.5 Acetonitrile, HPLC quality 3.6 Methanol, HPLC quality 3.7 Water,
23、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 Mepiquat chloride stock solution, (C7H16NCl) = 4,37 g/ml in methanol This stock solution contains 3,33 g/ml mepiquat cation
24、. 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 be sho
25、wn 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 16,67 g/ml d4-chlormequat cation. For the availability of the standard, contact your national standardisation institute.
26、 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 standard, contact your national standardisation institute. 3.12 Calibration solutions Prepare six calibration solution
27、s (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 of mepiquat chloride stock solution (3.9). To all flasks add 60 l of the internal standard solution 1 (3.10) and 60 l
28、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 calibration solutions contain 100 ng/ml of d4-chlormequat cation and 100 ng/ml of d3-mepiquat cation. Solutions no. 1 to no. 6
29、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 cation
30、 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 of at
31、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 system, equipped with electrospray interface 5 Procedure 5.1 Preparation of the samples Where possible, carry out th
32、e 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 the laboratory sample to which the maximum residue level applies. No further plant-parts may be removed. A record of
33、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 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
34、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 are in larger unit
35、s, 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 stones should be remov
36、ed. 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). If samples have to be stored for more than 3 days, they shall be deep-frozen at 20 C.
37、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 out test portions of masses of 20 g to an accuracy of 1 %. 5.2 Extraction Transfer a representative test portion of mA=
38、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 (3.10) and 360 l of internal standard solution 2 (3.11). Add so much water, that a total volume (added and natural) of
39、 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 extract (taking into account the natural water content of the sample) is 60 ml. Centrifuge an aliquot portion at approxim
40、ately 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 (3.3) onto the bottom of the
41、 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-MS system. The selected ions shall be appropriate for the sensitive and selective determination of chlormequat, mepiqua
42、t 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 interfering pe
43、aks 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 out spiked recovery tests at levels appropriate to the maximum residue level. The chromatogram of the reagent blank shou
44、ld 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 obtained from the sample test solution with those obtained from the calibration solutions. Positive findings are conf
45、irmed 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 ratios from the standards. If the peak ratio of a residue EN 15054:2006 (E) 7 peak differs more than 30 % from the ex
46、pected 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 based on the response ratio of chlormequat/d4-chlormequat and mepiquat/d3-mepiquat, respectively. As units on the x-axis
47、 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 should be obtained with the calibration solution no. 4 (corresponding to a mass fraction of 0,3 mg/kg for the 20 g test por
48、tion). 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, it may be necessary to construct more than one calibration graph from the results of calibration measurements. Measure
49、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 milligrams per kilogram of sample, using equation (1): AIstdARmbaAAwg20)/(= (1) where: AAis the peak response of chlormequat cation
