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

This draft European Standard specifies a high performance liquid chromatographic (HPLC) 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 not determined with the method.
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.

Fettarme Lebensmittel - Bestimmung der Benzimidazol-Fungizide Carbendazim, Thiabendazol und Benomyl (als Carbendazim) - Teil 3: HPLC-Verfahren mit Reinigung durch Flüssig/Flüssig-Verteilung

Diese Europäische Norm legt ein hochleistungs-flüssigchromatographisches Verfahren zur Bestimmung der Benzimidazol-Fungizide Carbendazim und Thiabendazol in Obst, Gemüse und deren Verarbeitungsprodukten fest.
Wenn Benomyl anwesend ist, wird es vollständig zu Carbendazim abgebaut und ebenfalls als Carbendazim bestimmt. Thiophanat-methyl wird mit dem Verfahren nicht bestimmt.
Das Verfahren ist für Carbendazim und Thiabendazol durch einen Ringversuch mit Homogenaten von Äpfeln, grünen Bohnen, Pilzen, Zitronen und Kindernahrung aus Fruchtzutaten validiert worden.

Aliments non gras - Détermination des benzimidazoles antifongiques : le carbendazime, le thiabendazole et le bénomyl en tant que carbendazime - Partie 3: Méthode CLHP avec purification par séparation liquide/liquide

Le présent projet de Norme européenne décrit une méthode de chromatographie en phase liquide à haute performance (CLHP) pour la détermination du carbendazime et du thiabendazole, benzimidazoles antifongiques, dans les fruits, les légumes et les produits transformés.
Si du bénomyl est présent, celui-ci est complètement dégradé en carbendazime et dosé en tant que du carbendazime. Le thiophanate-méthyle n'est pas dosé par cette méthode.
La présente méthode a été validée pour le carbendazime et le thiabendazole lors d'un essai interlaboratoires réalisé à l'aide de broyats de pommes, de haricots verts, de champignons et d'aliments pour bébé à base de citrons et de fruits.

Nemastna živila - Določevanje benzimidazolnih fungicidov: karbendazim, tiabendazol in benomil (kot karbendazim) - 3. del: Metoda HPLC s čiščenjem s porazdelitvijo tekoče-tekoče

General Information

• Status: Withdrawn
• Publication Date: 05-Oct-2004
• Withdrawal Date: 31-Dec-2014
• ICS: 67.080.01 - Fruits, vegetables and derived products in general
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 4 - Pesticides in foods of plant origin
• Current Stage: 9960 - Withdrawal effective - Withdrawal
• Start Date: 01-Jan-2015
• Completion Date: 01-Jan-2015
• Ref Project: SIST EN 14333-3:2005 - Non fatty foods - Determination of benzimidazole fungicides carbendazim, thiabendazole and benomyl (as carbendazim) - Part 3: HPLC method with liquid/liquid-partition clean up

Standards Content (Sample)

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 3: HPLC-Verfahren mit Reinigung durch Flüssig/Flüssig-VerteilungAliments non gras - Détermination des benzimidazoles antifongiques : le carbendazime, le thiabendazole et le bénomyl en tant que carbendazime - Partie 3: Méthode CLHP avec purification par séparation liquide/liquideNon fatty foods - Determination of benzimidazole fungicides carbendazim, thiabendazole and benomyl (as carbendazim) - Part 3: HPLC method with liquid/liquid-partition clean up67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 14333-3:2004SIST EN 14333-3:2005en01-januar-2005SIST EN 14333-3:2005SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14333-3October 2004ICS 67.080.01 English versionNon fatty foods - Determination of benzimidazole fungicidescarbendazim, thiabendazole and benomyl (as carbendazim) -Part 3: HPLC method with liquid/liquid-partition clean upAliments non gras - Détermination des benzimidazolesantifongiques : le carbendazime, le thiabendazole et lebénomyl en tant que carbendazime - Partie 3: MéthodeCLHP avec purification par séparation liquide/liquideFettarme Lebensmittel - Bestimmung der Benzimidazol-Fungizide Carbendazim, Thiabendazol und Benomyl (alsCarbendazim) - Teil 3: HPLC-Verfahren mit Reinigungdurch Flüssig/Flüssig-VerteilungThis 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 14333-3:2004: ESIST EN 14333-3:2005

Precision data.9 Annex B (informative)
Alternative HPLC operating conditions.12 Bibliography.13
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 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 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 determined 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 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 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 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. SIST EN 14333-3:2005

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 ml 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,
(thiabendazole) = 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 chopper 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 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, e.g. 250 mm long, 4,6 mm inner diameter, packed with ODS-120T®, TSK-GEL® 1), particle size 5 µm. 4.7 Membrane filter, pore size 0,45 µm, suitable for water and methanolic solutions 4.8 Glass fibre filter, 90 mm diameter 4.9 Syringe filter, pore size 0,45 µm, 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 CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 14333-3:2005

5.2.2 Lemons, limes 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 (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 (3.5) 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 (V2) of the solution 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 hydrochloric 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 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 using a gentle stream of nitrogen. To the residue, add 5 ml ± 0,2 ml (V3) 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 µl of this sample test solution into the HPLC system (4.6), applying a flow 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 excitation 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. NOTE 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. SIST EN 14333-3:2005

Measure the peak heights or peak areas obtained for carbendazim and thiabendazole 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
AV
V
C A
w2St 31St×××××= (1) where A is the peak height or peak area obtained from the sample test solution; ASt is the peak height or peak area obtained from the standard solution;
CSt is the mass concentration of carbendazim or thiabendazole in the standard solution, in micrograms per millilitre; V1 is the volume of ethyl acetate used for extracti
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