1、BRITISH STANDARD BS EN 14333-2:2004 Non fatty foods Determination of benzimidazole fungicides carbendazim, thiabendazole and benomyl (as carbendazim) Part 2: HPLC method with gel permeation chromatography clean up The European Standard EN 14333-2:2004 has the status of a British Standard ICS 67.080.
2、01 BS EN 14333-2:2004 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 44606 9 National foreword This British Standard is the official English language version of EN 14333-2:2004. The UK participa
3、tion in 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 Europe
4、an 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 British Standards Online. This publication does not purport to include all
5、 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; present to the responsible international/European committee any enquiries on
6、 the interpretation, 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 15 and a back cover. T
7、he BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM EN143332 October2004 ICS67.080.01 Englishversion NonfattyfoodsDeterminationofbenzimidazolefungicide
8、s carbendazim,thiabendazoleandbenomyl(ascarbendazim) Part2:HPLCmethodwithgelpermeationchromatographyclean up AlimentsnongrasDterminationdesbenzimidazoles antifongiques,lecarbendazime,lethiabendazoleetle bnomylentantquecarbendazimePartie2:Mthode CLHPavecpurificationparchromatographiepar permationdege
9、l FettarmeLebensmittelBestimmungderBenzimidazol FungizideCarbendazim,ThiabendazolundBenomyl(als Carbendazim)Teil2:HPLCVerfahrenmitReinigung durchGelpermeationschromatographie ThisEuropeanStandardwasapprovedbyCENon29July2004. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipula
10、tetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheCentralSecretariatortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French
11、,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheCentralSecretariathasthesamestatusast heofficial versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,Cyprus,CzechRepublic,Denmark,Estonia,Finland,France, Germany,G
12、reece,Hungary,Iceland,Ireland,Italy,Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,Poland,Portugal, Slovakia, Slovenia,Spain,Sweden,SwitzerlandandUnitedKingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Bru
13、ssels 2004CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.EN143332:2004:EEN 14333-2:2004 (E) 2 Contents Page Foreword3 1 Scope 4 2 Principle4 3 Reagents.4 4 Apparatus .5 5 Procedure .6 6 Calculation7 7 Confirmatory tests.8 8 Precision.8 9 Test report
14、9 Annex A (informative) Precision data10 Annex B (informative) Alternative GPC column .13 Annex C (informative) HPLC with reversed-phase column.14 Bibliography 15 EN 14333-2:2004 (E) 3 Foreword This document (EN 14333-2:2004) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horiz
15、ontal 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 or by endorsement, at the latest by April 2005, and conflicting national standards shall be withdrawn at the latest by April 20
16、05. EN 14333 consists of the following parts, under the general title Non 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 cl
17、ean up; Part 3: HPLC method with liquid/liquid-partition clean up. 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 stand
18、ard to establish appropriate safety and health practices and determine 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,
19、Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EN 14333-2:2004 (E) 4 1 Scope This do
20、cument specifies a high performance liquid chromatographic method for the 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.
21、 Thiophanate-methyl is partly decomposed and therefore not quantitatively determined. The method has been 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 homogeni
22、zed with ethyl acetate, sodium hydroxide solution and anhydrous sodium sulfate and the homogenate is filtered. An aliquot portion of the ethyl acetate extract is cleaned up by gel permeation chromatography (GPC) on a polystyrene gel using a cyclohexane/ethyl acetate mixture for elution. In the GPC e
23、luate, carbendazim and thiabendazole are determined by high performance liquid chromatography (HPLC) on a normal phase column and 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 pesticid
24、e residue analysis, and only distilled or demineralized water. 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.
25、Carbendazim and thiabendazole are toxic; avoid contact with skin and eyes. 3.3 Ethyl acetate 3.4 Cyclohexane 3.5 Sodium sulfate, anhydrous 3.6 Sodium hydroxide solution, mass concentration (NaOH) = 26 g/100 ml 3.7 Diluted sodium hydroxide solution, (NaOH) = 2,6 g/100 ml 3.8 GPC eluting mixture: Cycl
26、ohexane (3.4) / ethyl acetate (3.3) 1 + 1 (V/V) 3.9 Solvent mixture: dilute 5 ml ethyl acetate (3.3) with cyclohexane (3.4) to 100 ml 3.10 Mobile phase A for HPLC: Cyclohexane (3.4) 3.11 Mobile phase B for HPLC: Cyclohexane (3.4) / isopropanol / methanol 85 + 15 + 5 (V/V/V) To 250 ml of this mixture
27、, add one drop of ammonia solution (25 g/100 g). Prior to use, filter the mixture through a membrane filter (4.7). EN 14333-2:2004 (E) 5 3.12 Mobile phase C for HPLC: Isopropanol 3.13 Carbendazim stock solution, (carbendazim) = 25 mg/100 ml in methanol 3.14 Thiabendazole stock solution, (thiabendazo
28、le) = 65 mg/100 ml in acetone 3.15 Standard solutions Dilute the carbendazim stock solution (3.13) or the thiabendazole stock solution (3.14) with ethyl acetate (3.3) and cyclohexane (3.4) to obtain appropriate dilutions in cyclohexane / ethyl acetate of about 80 + 20 (V/V) to 75 + 25 (V/V). When in
29、jected into the HPLC system (4.6), the dilutions should contain less than 10 ml/100 ml of methanol and less than 2 ml/100 ml of acetone. 4 Apparatus 4.1 General Usual laboratory equipment and in particular the following : 4.2 Food chopper 4.3 High speed blender or homogenizer 4.4 Rotary evaporator w
30、ith a water bath 4.5 Instrument for GPC equipped with glass column with two adjustable end pieces, 500 mm long, 10 mm inner diameter, and with at least one sample loop (1 ml), column packing BioBeads 1)S-X3 resin, pre- swelled overnight in the GPC eluting mixture (3.8) and packed as described in 5.3
31、.1. NOTE Apparatus for automated GPC is commercially available. 4.6 High performance liquid chromatograph equipped with 4.6.1 Pumping system, with three solvent reservoirs, an injection valve for 15 l, a UV detector and a fluorescence detector connected in series and a quantification unit with an in
32、tegrating system; 4.6.2 HPLC analytical column, stainless steel cartridge, 150 mm long, 4,6 mm inner diameter, packed with a suitable diol-bonded silica for normal phase chromatography, particle size 3 m. 4.7 Membrane filter, pore size 0,45 m, suitable for organic solutions 4.8 Glass fibre filter, 9
33、0 mm diameter 4.9 Syringe filter, pore size 0,45 m, suitable for organic solutions 1) BioBeads is a trade name of a product supplied by Bio-Rad Laboratories, Richmond, CA, USA. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by
34、CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. EN 14333-2:2004 (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 portion is tak
35、en. 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 200 ml (V 1 ) of ethyl acetate (3.3) and 3,0 ml sodium hydroxide solution (3.6) and homogenize the mixtur
36、e for 30 s. Add 40 g of sodium sulfate (3.5) 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. From the ethy
37、l acetate solution, take an aliquot portion of 100 ml (V 2 ) and concentrate it to approximately 1 ml in a rotary evaporator (4.4) with the water bath temperature set at 37 C. Transfer the concentrate to a 5 ml calibrated test tube, rinsing the flask with ethyl acetate, and dilute the solution to 2,
38、5 ml with ethyl acetate. Rinse the flask again with cyclohexane (3.4), add the rinsing to the test tube and adjust with cyclohexane to a volume of 5 ml (V 3 ). 5.2.2 Lemons, limes, plums Proceed as described in 5.2.1, but add 6,0 ml sodium hydroxide solution (3.6) instead of 3,0 ml. 5.2.3 Juices Che
39、ck which volume (x ml) of diluted sodium hydroxide solution (3.7) is required to adjust a portion of 7,5 g of the juice to approximately pH 10. Weigh a test portion of 75 g (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
40、 solution (3.6) and homogenize the mixture for 30 s. Proceed as described in 5.2.1. 5.3 Gel permeation chromatography 5.3.1 Packing gel permeation column Allow approximately 9 g of BioBeads S-X3 resin (4.5) to swell overnight in the GPC eluting mixture (3.8). Then pour the suspension all at once int
41、o the column. As soon as the gel bed has settled (free from air bubbles) to a level of approximately 40 cm, insert the plunger, lower it down to the bed level and screw it into place. Start the flow of GPC eluting mixture at a low rate and gradually increase it to 1 ml/min. If the gel bed sinks to a
42、 still lower level, adjust the plunger accordingly (observe manufacturers instructions). NOTE For alternative GPC column, see Annex B. 5.3.2 Checking elution volumes Load the sample loop with 1 ml of appropriately diluted standard solutions in cyclohexane (3.4) / ethyl acetate (3.3) 1 + 1 (V/V), elu
43、te as described in 5.3.3 and determine, whether carbendazim and thiabendazole are completely recovered. Collect fractions of 0,5 ml around the expected starting and end points of the elution range. NOTE In general, the elution range is 20 ml to 27 ml for carbendazim and 24 ml to 30 ml for thiabendaz
44、ole. EN 14333-2:2004 (E) 7 5.3.3 Clean-up Filter the solution derived from 5.2 through a syringe filter (4.9) and inject 1 ml (V 4 ) of the filtrate into the sample loop of the gel permeation chromatograph (4.5). Elute with the GPC eluting mixture (3.8) at a flow rate of 1 ml/min and collect the fra
45、ction during the elution ranges for carbendazim and thiabendazole, which were determined in 5.3.2. Concentrate this fraction to approximately 1 ml in a rotary evaporator (4.4) with the water bath temperature set at 37 C. Transfer the concentrate to a 3 ml (V 5 ) volumetric flask, rinsing the evapora
46、tion flask with solvent mixture (3.9), and dilute the solution to the mark with solvent mixture. 5.4 HPLC measurement Filter the solution derived from 5.3.3 through a syringe filter (4.9) and inject 15 l of this sample test solution into the HPLC system (4.6). For quantitation, inject also the same
47、volume of appropriately diluted standard solutions (3.15). Apply the following ternary gradient programme, at a flow rate of 0,65 ml/min : 88 % mobile phase A (3.10), 10 % mobile phase B (3.11) and 2 % mobile phase C (3.12) for 30 s, then linearly to 78 % mobile phase A, 20 % mobile phase B and 2 %
48、mobile phase C from 30 s to 3 min and linearly to 54 % mobile phase A, 20 % mobile phase B and 26 % mobile phase C from 3 min to 35 min. The gradient programme may be shortened if no late eluting peaks are expected. Pass the HPLC column eluate first through a UV detector set at 285 nm and, if both detectors are used, then through the fluorescence detector set at excitation and emission wavelengths of 285 nm and 315 nm, respectively. NOTE 1 For UV detection, other suitable wavelengths are 240 nm for carbenda
