1、BRITISH STANDARDBS EN14185-2:2006Non fatty foods Determination of N-methylcarbamate residues Part 2: HPLC method with clean-up on a diatomaceous earth columnThe European Standard EN 14185-2:2006 has the status of a British StandardICS 65.060g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g5
2、4g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN 14185-2:2006This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 June 2006 BSI 2006ISBN 0 580 486
3、19 2National forewordThis British Standard is the official English language version of EN 14185-2:2006.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 c
4、ommittee can be obtained on request to its secretary.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
5、“Search” facility of the BSI Electronic Catalogue 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 lega
6、l obligations. aid enquirers to understand the text; 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
7、 of pagesThis document comprises a front cover, an inside front cover, the EN title page, pages 2 to 17 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 EUR
8、OPENNEEUROPISCHE NORMEN 14185-2May 2006ICS 65.060English VersionNon fatty foods - Determination of N-methylcarbamate residues -Part 2: HPLC method with clean-up on a diatomaceous earthcolumnAliments non gras - Dosage des N-mthylcarbamates -Partie 2 : Mthode CLHP avec purification sur colonne deterre
9、 de diatomesFettarme Lebensmittel - Bestimmung von N-Methylcarbamatrckstnden - Teil2: HPLC-Verfahren mitReinigung auf einer Kieselgur-SuleThis 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 conditio
10、ns 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 Central Secretariat or to any CEN member.This European Standard exists in three offici
11、al 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 Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgi
12、um, 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, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATI
13、ONCOMIT 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 reservedworldwide for CEN national Members.Ref. No. EN 14185-2:2006: EEN 14185-2:2006 (E) 2 Contents Page Foreword3 1
14、Scope 4 2 Principle4 3 Reagents.4 4 Apparatus .6 5 Sampling.7 6 Preparation of the samples.7 7 Procedure .8 8 Calculation of the results9 9 Confirmatory tests 10 10 Precision, Repeatability and Reproducibility 10 11 Test report . 12 Annex A (informative) Precision Data 13 Bibliography. 17 EN 14185-2
15、:2006 (E) 3 Foreword This document (EN 14185-2:2006) 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
16、or by endorsement, at the latest by November 2006, and conflicting national standards shall be withdrawn at the latest by November 2006. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: A
17、ustria, 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, Switzerland and United Kingdom. EN 14185-2:2006 (E)
18、4 1 Scope This draft European Standard specifies a high performance liquid chromatographic (HPLC) method for the determination of residues of N-methylcarbamate pesticides in fruits and vegetables and is based on the method of Krause 1. The method has been validated by collaborative study for aldicar
19、b, carbofuran, furathiocarb, methomyl, oxamyl, propoxur and thiodicarb parent compounds and for the metabolites aldicarb sulfoxide, aldicarb sulfone (aldoxycarb) and 3-hydroxy-carbofuran in tomatoes and oranges at levels between 0,04 mg/kg and 0,25 mg/kg. No collaborative data are available for the
20、performance of the method in the determination of other significant compounds although it is known that the method is unsatisfactory for benfuracarb. 2 Principle The sample is homogenized with dichloromethane, the homogenate is filtered and the filtrate evaporated. For citrus fruits only, the sample
21、 is homogenized with acetonitrile, the filtrate is washed with n-hexane and evaporated. In both cases, the residue is dissolved in an aqueous sodium chloride solution. The solution is transferred to a diatomaceous earth column and the column is eluted with dichloromethane. The eluates are evaporated
22、 and the residue is dissolved in methanol. In this solution, the N-methylcarbamates are determined by reversed-phase high-performance liquid chromatography (HPLC) with post-column hydrolysis. The methylamine formed is allowed to react with o-phthaldialdehyde and 2-mercaptoethanol and the derivatives
23、 are detected with a fluorescence detector 2. 3 Reagents 3.1 General Unless otherwise specified, use reagents of recognized analytical grade, preferably for HPLC and pesticide residue analysis, and distilled water or water of equivalent purity. Label all standard containers with name and purity of t
24、he pesticides. For the full chemical names and structures, seeISO 1750. 3.2 Safety aspects 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 o
25、f the user of this standard to establish appropriate safety and health practices and to determine the applicability of regulatory limitations prior to use. 3.3 Acetonitrile 3.4 n-Hexane 3.5 Acetonitrile, saturated with n-hexane In a separating funnel, shake 400 ml of acetonitrile (3.3) with 40 ml of
26、 n-hexane (3.4) for 3 min and use the lower layer. EN 14185-2:2006 (E) 5 3.6 Dichloromethane1)3.7 Methanol 3.8 Sodium chloride solution, saturated 3.9 Sodium hydroxide solution, c = 0,05 mol/l NOTE Sodium hydroxide was found to be a possible source of contamination. Suitable products are e.g. Hydrol
27、ysis Reagent for Carbamate Pesticide Analysis CB 1302)or sodium hydroxyde solution (1 mol/l) for HPCE, Merck no. 1022513). 3.10 Filter aid, e.g. Celite 5454)3.11 Solid support material, Extrelut NT2) refill packs 3.12 o-Phthaldialdehyde 3.13 Tetraborate buffer solution, 38,1 g/l sodium tetraborate d
28、ecahydrate in water 3.14 2-Mercaptoethanol 3.15 Mobile phase A Water, purified by a LC-grade water purification system. Prior to use, filter it with gentle suction through a membrane filter (4.13). 3.16 Mobile phase B Acetonitrile (3.3). Prior to use, filter it with gentle suction through a membrane
29、 filter (4.13). 1)Due to environmental reasons, also ethyl acetate can be used, however, only the use of dichloromethane has been tested in the inter-laboratory study, and therefore, the applicability of ethyl acetate has to be proven in an appropriate manner. 2)Hydrolysis Reagent for Carbamate Pest
30、icide Analysis CB 130 and Derivatization Reagent 2 are products supplied by Pickering Laboratories, Mountain View, California, 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
31、 may be used if they can be shown to lead to the same results.3)Sodium hydroxide solution for HPCE, Extrelut NT and RP Select B are products supplied by E. Merck, Darmstadt, Germany. This information is given for the convenience of users of this European Standard and does not constitute an endorseme
32、nt by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results.4) 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 doe
33、s 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 14185-2:2006 (E) 6 3.17 OPA reagent Dissolve 1 g of o-phthaldialdehyde (3.12) in 10 ml of methanol (3.7) and add 0,4 ml of 2-mercaptoethanol (3.14). In a
34、1 000 ml volumetric flask, dilute the mixture and make up to the mark with tetraborate buffer solution (3.13). Adjust the pH value of the solution to pH 10. Store under a nitrogen atmosphere. NOTE Alternatively the “Derivatization Reagent 2“1)may be used. 3.18 Standard materials N-methylcarbamate pe
35、sticides such as aldicarb, bendiocarb, bufencarb, butocarboxim, carbaryl, carbofuran, dioxacarb, ethiofencarb, furathiocarb, methiocarb, methomyl, oxamyl, promecarb, propoxur, thiodicarb, thiofanox, trimethacarb, the metabolites 3-hydroxy-carbofuran and 3-keto-carbofuran and the sulfoxide and sulfon
36、e metabolites of aldicarb, butocarboxim and thiofanox. 3.19 Pesticide stock solutions, mass concentration = 2,0 g/ml. In a 25 ml volumetric flask, dissolve 50 mg of a standard material (3.18) in acetonitrile (3.3) and make up to the mark with acetonitrile. 3.20 Pesticide standard solutions Prepare a
37、ppropriate standard solutions by diluting suitable amounts of pesticide stock solutions (3.19) with methanol, e.g. to 1 g/ml or to 0,1 g/ml, if necessary. 4 Apparatus 4.1 General Glassware shall be thoroughly cleaned. Hot detergent solution may be used for cleaning, but afterwards the glassware shal
38、l be well rinsed with distilled water and acetone before drying. Usual laboratory apparatus is used and, in particular, the following. 4.2 Food chopper 4.3 Homogenizer or high speed blender 4.4 Glass bottle, 500 ml 4.5 Rotary evaporator with a water bath, capable of being maintained at 35 C 4.6 Buch
39、ner porcelain funnel, 9,5 cm diameter 4.7 Filtration flask, 1 l 4.8 Glass fibre filter, 9 cm diameter 4.9 Separating funnel, 1 l EN 14185-2:2006 (E) 7 4.10 Column filled with diatomaceous earth, applicable for a volume of 20 ml solution, e. g. pre-packed polypropylene columns Extrelut NT 20 2)(see p
40、age 5), Chem Elut 20 ml5)or Chromabond XTR (70 ml) 6). Used Extrelut NT 20 columns may be refilled with Extrelut NT refill packs (3.11). 4.11 High performance liquid chromatograph, equipped with 4.11.1 Binary pumping system with six-port injection valve with a 20 l sample loop, a fluorescence detect
41、or and a quantification unit with an integrating system. 4.11.2 HPLC analytical column, stainless steel cartridge, 250 mm long, 4,0 mm inner diameter (e.g. packed with RP Select B 2)(see page 5) (particle size 5 m). 4.11.3 Post-column system, consisting of two reaction coils in different thermostats
42、, two low-dead volume T-pieces, and two pulse-free reagent pumps. NOTE A complete post-column LC-system for the analysis of N-methylcarbamates is commercially available. 4.12 Ultrasonic bath 4.13 Membrane filter, pore size 0,45 m 5 Sampling Prepare the laboratory sample according to a generally reco
43、mmended method of sampling to achieve a representative part of the product to be analyzed. 6 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 ana
44、lysis 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 sample cannot be analysed immediately, store
45、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. small fruits, legumes,
46、 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. 5)Chem Elut 20 ml is a pro
47、duct supplied by Varian Inc., Palo Alto, 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 shown to lead to the same results.6)Chromabond XTR is
48、a product supplied by Macherey-Nagel, Dren, Germany. 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 shown to lead to the same results.EN 14185-2:2006 (
49、E) 8 From 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 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 18 C or lower. To ensure that even after thawing representative samples can be taken, prepare portions of
