EN 14333-1-2004 en Non fatty foods - Determination of benzimidazole fungicides carbendazim thiabendazole and benomyl (as carbendazim) - Part 1 HPLC method with solid phase extracti.pdf

1、BRITISH STANDARD BS EN 14333-1:2004 Non fatty foods Determination of benzimidazole fungicides carbendazim, thiabendazole and benomyl (as carbendazim) Part 1: HPLC method with solid phase extraction clean up The European Standard EN 14333-1:2004 has the status of a British Standard ICS 67.080.01 BS E

2、N 14333-1:2004 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 15 October 2004 BSI 15 October 2004 ISBN 0 580 44607 7 National foreword This British Standard is the official English language version of EN 14333-1:2004. The UK participation in

3、 its preparation was entrusted to Technical Committee AW/-/3, Horizontal analysis, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publ

4、ications 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 British Standards Online. This publication does not purport to include all the ne

5、cessary 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; present to the responsible international/European committee any enquiries on the in

6、terpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 12, an inside back cover and

7、a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM EN143331 October2004 ICS67.080.01 Englishversion NonfattyfoodsDeterminationofbenzimi

8、dazolefungicides carbendazim,thiabendazoleandbenomyl(ascarbendazim) Part1:HPLCmethodwithsolidphaseextractioncleanup AlimentsnongrasDterminationdesbenzimidazoles antifongiques:lecarbendazime,lethiabendazoleetle bnomylentantquecarbendazimePartie1:Mthode CLHPavecpurificationparextractionenphasesolide F

9、ettarmeLebensmittelBestimmungderBenzimidazol FungizideCarbendazim,ThiabendazolundBenomyl(als Carbendazim)Teil1:HPLCVefahrenmitReinigungdurch Festphasenextraktion ThisEuropeanStandardwasapprovedbyCENon29July2004. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditio

10、nsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheCentralSecretariatortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aver

11、sioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheCentralSecretariathasthesamestatusast heofficial versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,Cyprus,CzechRepublic,Denmark,Estonia,Finland,France, Germany,Greece,Hungary

12、,Iceland,Ireland,Italy,Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,Poland,Portugal, Slovakia, Slovenia,Spain,Sweden,SwitzerlandandUnitedKingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2004CEN

13、 Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.EN143331:2004:EEN 14333-1:2004 (E) 2 Contents Page Foreword3 1 Scope 4 2 Principle4 3 Reagents.4 4 Apparatus .5 5 Procedure .6 6 Calculation7 7 Confirmatory tests.7 8 Precision.8 9 Test report 9 Annex A (in

14、formative) Precision data10 Bibliography 12 EN 14333-1:2004 (E) 3 Foreword This document (EN 14333-1:2004) 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

15、 standard, either by publication of an identical text or by endorsement, at the latest by April 2005, and conflicting national standards shall be withdrawn at the latest by April 2005. EN 14333 consists of the following parts, under the general title Non fatty foods Determination of benzimidazole fu

16、ngicides carbendazim, thiabendazole and benomyl (as carbendazim): Part 1: HPLC method with solid phase extraction clean up; Part 2: HPLC method with gel permeation chromatography clean up; Part 3: HPLC method with liquid/liquid-partition clean up. WARNING The use of this standard may involve hazardo

17、us 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 health practices and determine the applicability of regulatory limitations prio

18、r to use. 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, Germany, Greece, Hungary, Iceland, Ireland, Italy,

19、Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EN 14333-1:2004 (E) 4 1 Scope This document specifies a high performance liquid chromatographic method for the determination of the benzimidazole fungicides

20、 carbendazim and thiabendazole in fruits and vegetables. When benomyl is present, it is completely degraded to carbendazim and is also determined as carbendazim. Thiophanate-methyl is not determined with the method. The method has been validated for carbendazim and thiabendazole in an interlaborator

21、y test with homogenates of apples and oranges. 2 Principle The sample is homogenized with acetone, dichloromethane and light petroleum and the homogenate is centrifuged to yield two layers of the supernatant. An aliquot portion of the upper layer is evaporated to dryness and the residue is dissolved

22、 in methanol containing benzimidazole as the internal standard. The solution is cleaned up by solid phase extraction (SPE) using a cartridge packed with diol-bonded silica. In the cartridge eluate, carbendazim and thiabendazole are determined by reversed-phase high performance liquid chromatography

23、(HPLC) with UV or UV and fluorescence detectors. 3 Reagents 3.1 General Unless otherwise specified, use reagents of recognized analytical grade, preferably for HPLC and pesticide residue analysis, and only distilled or demineralized water. 3.2 Safety aspects associated with reagents Vapours from som

24、e volatile solvents are toxic. Several of these solvents are readily absorbed through skin. Use an effective fume hood to remove vapours of these solvents as they are set free. Carbendazim and thiabendazole are toxic; avoid contact with skin and eyes. 3.3 Acetone 3.4 Dichloromethane 3.5 Light petrol

25、eum, boiling range 40 C to 60 C 3.6 Methanol 3.7 Sodium hydroxide solution, mass concentration (NaOH) = 4,0 g/100 ml 3.8 Ortho-Phosphoric acid, at least (H 3 PO 4 ) = 85 g/100 g 3.9 Phosphoric acid solution: To a 100 ml volumetric flask, transfer 0,6 ml ortho-phosphoric acid (3.8) and dilute to the

26、mark with water. EN 14333-1:2004 (E) 5 3.10 SPE eluting mixture: Phosphoric acid solution (3.9) / methanol (3.6) 1 + 1 (V/V) 3.11 Phosphate buffer solution (pH 7): In a 500 ml volumetric flask, dissolve 1,76 g of potassium dihydrogen phosphate and 3,63 g of disodium hydrogen phosphate in water and d

27、ilute to the mark with water. 3.12 Mobile phase for HPLC: Methanol (3.6) / phosphate buffer solution (3.11) 7 + 3 (V/V). Prior to use, filter the mixture with suction through a membrane filter (4.9). 3.13 Carbendazim stock solution, (carbendazim) = 10 mg/100 ml in methanol (3.6) 3.14 Thiabendazole s

28、tock solution, (thiabendazole) = 10 mg/100 ml in methanol (3.6) 3.15 Benzimidazole stock solution, (benzimidazole) = 10 mg/100 ml in methanol (3.6) 3.16 Internal standard solution, (benzimidazole) = 0,50 g/ml In a 100 ml volumetric flask, dilute 500 l of the benzimidazole stock solution (3.15) to th

29、e mark with methanol (3.6). 3.17 Standard solution, (carbendazim) = 0,25 g/ml, (thiabendazole) = 0,25 g/ml, (benzimidazole) = 0,50 g/ml Transfer 250 l of carbendazim stock solution (3.13), 250 l of thiabendazole stock solution (3.14) and 500 l of benzimidazole stock solution (3.15) to a 100 ml volum

30、etric flask and dilute to the mark with methanol (3.6) / phosphate buffer solution (3.11) 1 + 1 (V/V). 4 Apparatus 4.1 General Usual laboratory equipment and in particular the following: 4.2 Food chopper 4.3 Homogenizer or high speed blender 4.4 Centrifuge, provided with polytetrafluoroethylene tube

31、s of capacity 250 ml, and capable of producing a rotational speed of at least 4000 min -14.5 Water bath, capable of being maintained at 60 C 4.6 SPE cartridges, packed with 500 mg of the diol-bonded silica Bond Elut 2OH 1) , 40 m irregular particles NOTE Several other brands have given unacceptable

32、low recoveries, which is why the brand in this application is critical. 4.7 Device for eluting SPE cartridges (4.6), with suction NOTE Apparatus for automated SPE elution is commercially available. 4.8 High performance liquid chromatograph, equipped with EN 14333-1:2004 (E) 6 4.8.1 Pumping system, a

33、n injection valve for 100 l, a UV detector and a fluoresence detector connected in series and a quantification unit with an integrating system. 4.8.2 HPLC analytical column, stainless steel cartridge, 150 mm long, 6,0 mm inner diameter, packed with Shodex DE-613 1) (poly alkyl methacrylate), particl

34、e size 6 m. 4.9 Membrane filter, pore size 0,45 m, suitable for water and methanolic solutions 4.10 Syringe filter, pore size 0,45 m, for water and methanolic solutions 5 Procedure 5.1 Preparation of test sample Prepare a homogenate from the laboratory sample, for example by chopping (4.2), from whi

35、ch a representative test portion is taken. 5.2 Extraction From the test sample (5.1), weigh a test portion of 15 g (m) to the nearest 0,1 g into a centrifuge tube (4.4), add 30 ml of acetone (3.3) and homogenize the mixture for 30 s using the homogenizer (4.3). Add 30 ml of dichloromethane (3.4) and

36、 30 ml of light petroleum (3.5) and continue to homogenize the mixture for 30 s. Centrifuge the tube for 5 min at a rotational speed of 4000 min -1 . Decant the upper organic layer into a conical flask and measure its volume. From this layer (V 1 ), transfer an aliquot portion of 3 ml (V 2 ) to a te

37、st tube. Place the test tube in a water bath set at 60 C (4.5) and gently evaporate the solution to near dryness. Allow the remaining solvent to evaporate in air, and dissolve the residue in 2 ml 0,1 ml (V 3 ) of internal standard solution (3.16). 5.3 Solid phase extraction Attach a SPE cartridge (4

38、.6) to a suitable eluting device (4.7). Aspirate the liquids through the cartridge giving a dropwise flow or a flow of at most 5 ml/min. Pass 2 ml of SPE eluting mixture (3.10) followed by 2 ml of methanol (3.6), and discard the eluates. Transfer an aliquot portion of 1 ml 0,05 ml of the solution de

39、rived from 5.2 to the SPE cartridge, pass 1 ml of methanol (3.6) through the cartridge and discard the eluate. Dry the cartridge with a stream of air. Next, pass 2,0 ml of SPE eluting mixture (3.10) through the cartridge and immediately start to collect the eluate in a test tube. To the eluate, add

40、100 l of sodium hydroxide solution (3.7) and mix. 5.4 HPLC measurement Filter the solution derived from 5.3 through a syringe filter (4.10) and inject 100 l of this sample test solution into the HPLC system (4.8). For quantitation, inject also the same volume of the standard solution (3.17). Apply a

41、 column oven temperature of 40 C and a flow rate of 0,75 ml/min of the mobile phase (3.12). Pass the HPLC column eluate first through a UV detector set at 285 nm and then through a fluorescence detector set at excitation and emission wavelengths of 285 nm and 315 nm, respectively. NOTE 1 For UV dete

42、ction, other suitable wavelengths are 240 nm for carbendazim and 300 nm for thiabendazole. For the fluorescence detection of thiabendazole, the optimum wavelengths are 295 nm for excitation and 350 nm for emission. NOTE 2 The retention times obtained under these conditions are approximately 8,5 min

43、for the internal standard benzimidazole, 11,5 min for carbendazim and 14 min for thiabendazole. 1) Bond Elut 2OH is a trade name of a product supplied by Varian Sample Preparation Products, Harbor City, CA, USA. Shodex DE-613 is a trade name of a product supplied by Showa Denko, Japan. These informa

44、tions are given for the convenience of users of this European Standard and do not constitute an endorsement by CEN of the products named. Equivalent products may be used if they can be shown to lead to the same results. EN 14333-1:2004 (E) 7 6 Calculation For quantitation, use, if possible, several

45、wavelengths for the UV absorption and the fluorescence measurement. Measure the peak heights or peak areas for carbendazim, thiabendazole and the internal standard benzimidazole obtained from the standard solution (3.17). Calculate the response factor f, using equation (1): A C A Cf St St = (1) wher

46、e C is the mass concentration of carbendazim or thiabendazole in the standard solution, in micrograms per millilitre; C St is the mass concentration of benzimidazole in the standard solution, in micrograms per millilitre; A is the peak height or peak area obtained for carbendazim or thiabendazole; A

47、 St is the peak height or peak area obtained for benzimidazole. Measure the peak heights or peak areas for carbendazim, thiabendazole and the internal standard benzimidazole obtained from the sample test solution. Calculate the mass fraction w of carbendazim or thiabendazole, in milligrams per kilog

48、ram of sample, using equation (2): m V A f V V C A w 2 St3 1 St = (2) where A is the peak height or peak area obtained for carbendazim or thiabendazole; A St is the peak height or peak area obtained for benzimidazole; C St is the mass concentration of benzimidazole in the internal standard solution, in micrograms per millilitre; V 1 is the volume of the organic layer obtained from extraction, in millilitres; NOTE Under the conditions described in 5.2, V 1 has be