EN 14333-3-2004 en Non fatty foods - Determination of benzimidazole fungicides carbendazim thiabendazole and benomyl (as carbendazim) - Part 3 HPLC method with liquid liquid-partit.pdf

1、BRITISH STANDARD BS EN 14333-3:2004 Non fatty foods Determination of benzimidazole fungicides carbendazim, thiabendazole and benomyl (as carbendazim) Part 3: HPLC method with liquid/liquid-partition clean up The European Standard EN 14333-3:2004 has the status of a British Standard ICS 67.080.01 BS

2、EN 14333-3:2004 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 44605 0 National foreword This British Standard is the official English language version of EN 14333-3:2004. The UK participation i

3、n 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 pub

4、lications 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 n

5、ecessary 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 i

6、nterpretation, 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 13 and a back cover. The BSI

7、 copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM EN143333 October2004 ICS67.080.01 Englishversion NonfattyfoodsDeterminationofbenzimidazolefungicides carb

8、endazim,thiabendazoleandbenomyl(ascarbendazim) Part3:HPLCmethodwithliquid/liquidpartitioncleanup AlimentsnongrasDterminationdesbenzimidazoles antifongiques:lecarbendazime,lethiabendazoleetle bnomylentantquecarbendazimePartie3:Mthode CLHPavecpurificationparsparationliquide/liquide FettarmeLebensmitte

9、lBestimmungderBenzimidazol FungizideCarbendazim,ThiabendazolundBenomyl(als Carbendazim)Teil3:HPLCVerfahrenmitReinigung durchFlssig/FlssigVerteilung ThisEuropeanStandardwasapprovedbyCENon29July2004. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthi

10、sEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheCentralSecretariatortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyother

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

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

13、ploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.EN143333:2004:EEN 14333-3:2004 (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.7 9 Test report 8 Annex A (informative) Pre

14、cision data9 Annex B (informative) Alternative HPLC operating conditions 12 Bibliography 13 EN 14333-3:2004 (E) 3 Foreword This document (EN 14333-3:2004) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European

15、Standard shall be given the status of a national 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 N

16、on fatty foods Determination of benzimidazole fungicides 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. WARN

17、ING 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 appropriate safety and health practices and determine

18、the applicability of regulatory limitations prior 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, Ger

19、many, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EN 14333-3:2004 (E) 4 1 Scope This document specifies a high performance liquid chromatographic method for t

20、he determination of the benzimidazole fungicides carbendazim and thiabendazole in fruits, vegetables and processed products. 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 b

21、een validated for carbendazim and thiabendazole in an interlaboratory test with homogenates of apples, French beans, mushrooms, lemons and fruit based infant food. 2 Principle The sample is homogenized with ethyl acetate, sodium hydroxide solution and anhydrous sodium sulfate and the homogenate is f

22、iltered. An aliquot portion of the ethyl acetate extract is partitioned with hydrochloric acid solution; the aqueous phase is made alkaline and partitioned with ethyl acetate. The organic layer is evaporated and the residue is dissolved in the HPLC mobile phase. Carbendazim and thiabendazole are det

23、ermined by reversed-phase high performance liquid chromatography (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 wa

24、ter. 3.2 Safety aspects associated with reagents Vapours from some 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 wit

25、h skin and eyes. 3.3 Ethyl acetate 3.4 Methanol 3.5 Sodium hydroxide solution, mass concentration (NaOH) = 26 g/100 ml 3.6 Diluted sodium hydroxide solution, (NaOH) = 2,6 g/100 ml 3.7 Sodium sulfate, anhydrous 3.8 Hydrochloric acid solution, (HCl) = 0,1 mol/l 3.9 Alkaline solution, pH 13,4 Dissolve

26、33 g of anhydrous sodium acetate, 200 g of sodium chloride and 40 g of sodium hydroxide in 1 l of water. 3.10 Phosphate buffer solution, pH 7,2 to 7,5 Dissolve 2 g of dipotassium hydrogen phosphate trihydrate and 0,5 g of potassium dihydrogen phosphate in 1 l of water. EN 14333-3:2004 (E) 5 3.11 Mob

27、ile phase for HPLC: Methanol (3.4) / phosphate buffer solution (3.10) 55 + 45 (V/V). Prior to use, filter the mixture through a membrane filter (4.7). 3.12 Carbendazim stock solution, (carbendazim) = 10 mg/100 ml In a 50 ml volumetric flask, weigh 5 mg, to the nearest 0,1 mg, of carbendazim. Add 5 m

28、l of the hydrochloric acid solution (3.8) and allow the flask to stand in an ultrasonic bath for 5 min to 10 min. Dilute the solution with 40 ml of water, allow the flask to stand again in the ultrasonic bath for 10 min and dilute to the mark with water. 3.13 Thiabendazole stock solution, (thiabenda

29、zole) = 50 mg/100 ml in methanol (3.4) 3.14 Standard solutions Dilute the carbendazim stock solution (3.12) or the thiabendazole stock solution (3.13) as appropriate with the mobile phase for HPLC (3.11). 4 Apparatus 4.1 General Usual laboratory equipment and in particular the following: 4.2 Food ch

30、opper 4.3 High speed blender or homogenizer 4.4 Separatory funnels, capacity 100 ml 4.5 Rotary evaporator, with a water bath 4.6 High performance liquid chromatograph, equipped with 4.6.1 Pumping system, an injection valve for 50 l, a UV detector and a fluorescence detector connected in series and a

31、 quantification unit with an integrating system. 4.6.2 HPLC analytical column, stainless steel cartridge, e.g. 250 mm long, 4,6 mm inner diameter, packed with ODS-120T , TSK-GEL 1) , particle size 5 m. 4.7 Membrane filter, pore size 0,45 m, suitable for water and methanolic solutions 4.8 Glass fibre

32、 filter, 90 mm diameter 4.9 Syringe filter, pore size 0,45 m, suitable for water and methanolic solutions 1) TSK-GEL is a trade name of a product supplied by Tosoh Biosep, USA. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by

33、CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. EN 14333-3:2004 (E) 6 5 Procedure 5.1 Preparation of test sample Prepare a homogenate from the laboratory sample, for example by chopping (4.2), from which a representative test sample is take

34、n. 5.2 Extraction 5.2.1 Commodities except lemons, limes, plums and juices From the test sample (5.1), weigh a test portion of 75 g (m) to the nearest 0,5 g into the jar of a blender (4.3). Add 150 ml (V 1 ) of ethyl acetate (3.3) and 3 ml sodium hydroxide solution (3.5) and homogenize the mixture f

35、or 30 s. Add 30 g of sodium sulfate (3.7) and continue to homogenize the mixture for 2,5 min. Filter the homogenate with gentle suction through a glass fibre filter (4.8) topped with 20 g of sodium sulfate. To the filtrate, add 10 g of sodium sulfate, and allow it to stand for 3 min. 5.2.2 Lemons, l

36、imes and plums Proceed as described in 5.2.1, but add 6,0 ml sodium hydroxide solution (3.5) instead of 3,0 ml. 5.2.3 Juices Check which volume (x ml) of diluted sodium hydroxide solution (3.6) is required to adjust a portion of 7,5 g of the juice to approximately pH 10. Weigh a test portion of 75 g

37、 (m) to the nearest 0,5 g into the jar of a blender (4.3). Add 200 ml (V 1 ) of ethyl acetate (3.3) and x ml of sodium hydroxide solution (3.5) and homogenize the mixture for 30 s. Proceed as described in 5.2.1. 5.3 Liquid-liquid partitioning Transfer an aliquot portion of 50 ml (V 2 ) of the soluti

38、on derived from 5.2 to a separatory funnel (4.4). Add 10 ml of hydrochloric solution (3.8), shake the funnel for 2 min and allow the layers to separate. Drain the lower aqueous layer to a second separatory funnel and repeat the extraction of the upper organic layer twice, using 10 ml and 5 ml of hyd

39、rochloric acid solution, respectively. Collect all aqueous layers in the second separatory funnel and discard the organic layer. To the combined aqueous layers, add 5 ml of the alkaline solution (3.9) and 15 ml of ethyl acetate and shake the funnel for 2 min. Allow sufficient time for the layers to

40、separate and discard the lower aqueous layer. Shake the upper organic layer with 10 ml of water and discard the aqueous layer. Concentrate the upper organic layer to approximately 2 ml in a rotary evaporator (4.5) with the water bath temperature set at 35 C and evaporate the remaining ethyl acetate

41、using a gentle stream of nitrogen. To the residue, add 5 ml 0,2 ml (V 3 ) of the mobile phase for HPLC (3.11) and mix well. 5.4 HPLC measurement Filter the solution derived from 5.3 through a syringe filter (4.9) and inject 50 l of this sample test solution into the HPLC system (4.6), applying a flo

42、w rate of 1,0 ml/min of the mobile phase (3.11). For quantitation, inject also the same volume of appropriately diluted standard solutions (3.14). Pass the HPLC column eluate first through a UV detector set at 285 nm and, if both detectors are used, then through a fluorescence detector set at excita

43、tion and emission wavelengths of 285 nm and 315 nm, respectively. NOTE 1 For UV detection, 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. N

44、OTE 2 The retention times obtained under these conditions are approximately 6 min for carbendazim and 8,5 min for thiabendazole. NOTE 3 For alternative HPLC operating conditions, see Annex B. EN 14333-3:2004 (E) 7 6 Calculation Measure the peak heights or peak areas obtained for carbendazim and thia

45、bendazole in the sample test solution and the standard solution. Use, if possible, several wavelengths for the UV absorption and the fluorescence measurement. Calculate the mass fraction w of carbendazim or thiabendazole, in milligrams per kilogram of sample, using equation (1): m V A V V C A w 2 St

46、3 1 St = (1) where A is the peak height or peak area obtained from the sample test solution; A Stis the peak height or peak area obtained from the standard solution; C Stis the mass concentration of carbendazim or thiabendazole in the standard solution, in micrograms per millilitre; V 1 is the volum

47、e of ethyl acetate used for extraction (5.2), in millilitres; V 2 is the aliquot portion of V 1taken for partitioning (5.3), in millilitres; V 3 is the volume of the final sample test solution obtained from step 5.3, in millilitres; m is the mass of the test portion, in grams. 7 Confirmatory tests A

48、nalyses for confirming the identity and quantity of the carbendazim and thiabendazole should be performed, particularly in those cases in which it appears that a maximum residue limit (MRL) has been exceeded. The identity of carbendazim and thiabendazole can be confirmed by comparing the absorption spectra of sample test solution and standard solution by using a diode array detector. The results can also be confirmed by using part 1 or part 2 of this European Standard. For thiabendazole, gas c