SIST EN 14333-2:2005

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.D]LPFettarme Lebensmittel - Bestimmung der Benzimidazol-Fungizide Carbendazim, Thiabendazol und Benomyl (als Carbendazim) - Teil 2: HPLC-Verfahren mit Reinigung durch GelpermeationschromatographieAliments non gras - Détermination des benzimidazoles antifongiques, le carbendazime, le thiabendazole et le bénomyl en tant que carbendazime - Partie 2: Méthode CLHP avec purification par chromatographie par perméation de gelNon fatty foods - Determination of benzimidazole fungicides carbendazim, thiabendazole and benomyl (as carbendazim) - Part 2: HPLC method with gel permeation chromatography clean up67.080.01Sadje, zelenjava in njuni proizvodi na splošnoFruits, vegetables and derived products in general67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 14333-2:2004SIST EN 14333-2:2005en01-januar-2005SIST EN 14333-2:2005SLOVENSKI
STANDARD



SIST EN 14333-2:2005



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14333-2October 2004ICS 67.080.01 English versionNon fatty foods - Determination of benzimidazole fungicidescarbendazim, thiabendazole and benomyl (as carbendazim) -Part 2: HPLC method with gel permeation chromatography cleanupAliments non gras - Détermination des benzimidazolesantifongiques, le carbendazime, le thiabendazole et lebénomyl en tant que carbendazime - Partie 2: MéthodeCLHP avec purification par chromatographie parperméation de gelFettarme Lebensmittel - Bestimmung der Benzimidazol-Fungizide Carbendazim, Thiabendazol und Benomyl (alsCarbendazim) - Teil 2: HPLC-Verfahren mit Reinigungdurch GelpermeationschromatographieThis European Standard was approved by CEN on 29 July 2004.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 nationalstandards 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 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, Norway, Poland, Portugal, Slovakia,Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2004 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14333-2:2004: ESIST EN 14333-2:2005



EN 14333-2:2004 (E) 2 Contents Page Foreword.3 1 Scope.4 2 Principle.4 3 Reagents.4 4 Apparatus.5 5 Procedure.6 6 Calculation.7 7 Confirmatory tests.8 8 Precision.8 9 Test report.9 Annex A (informative)
Precision data.10 Annex B (informative)
Alternative GPC column.13 Annex C (informative)
HPLC with reversed-phase column.14 Bibliography.15
SIST EN 14333-2:2005



EN 14333-2:2004 (E) 3 Foreword This document (EN 14333-2:2004) 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 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 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. 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 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, 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. SIST EN 14333-2:2005



EN 14333-2:2004 (E) 4 1 Scope This document 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. 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 homogenized 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 eluate, 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 pesticide 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. 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: Cyclohexane (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, add one drop of ammonia solution (25 g/100 g). Prior to use, filter the mixture through a membrane filter (4.7). SIST EN 14333-2:2005



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,
(thiabendazole) = 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 injected 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 with 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
5.3.1. 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 µl, a UV detector and a fluorescence detector connected in series and a quantification unit with an integrating 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 µm. 4.7 Membrane filter, pore size 0,45 µm, suitable for organic solutions 4.8 Glass fibre filter, 90 mm diameter 4.9 Syringe filter, pore size 0,45 µm, 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 CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 14333-2:2005



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 taken. 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 (V1) of ethyl acetate (3.3) and 3,0 ml sodium hydroxide solution (3.6) and homogenize the mixture 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 ethyl acetate solution, take an aliquot portion of 100 ml (V2) 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 2,5 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 (V3).
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 Check 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 (V1) of ethyl acetate (3.3) and x ml of sodium hydroxide 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 into 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 still lower level, adjust the plunger accordingly (observe manufacturer’s 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
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