Gas chromatographic method for the determination of hexaconazole residues in black tea

Karthika et a/. /J Zhejiang Univ Sci B 2008 9(2): 160-164Joumal of zhejiang UnSCIENCE BISSN 1673-1581(Print): ISSN 1862-1783(Onlinewww.zju.edu.cn/jzuswww.spningerlink.comGas chromatographic method for the determination ofhexaconazole residues in black teaChinnachamy KARTHIKA, Paul James SACHIN(United Planter s Association of South india (UPASI) Tea Research Foundation, Tea Research InstituteNirar Dam Branch Post Ofice(BPO), Valparai 642127, Coimbatore District, Tamil Nadu, India)E-mail:kartheecm(arediffmail.comReceived Nov. 24, 2007; revision accepted Dec 24, 2007; published online Jan. 16, 2008Abstract: A highly reliable, quantitative and sensitive analytical method for determining the residues of the fungicide, hex-aconazole in black tea is described. The proposed method is based on liquid-liquid extraction followed by gas chromatographicletermination, using nitrogen phosphorus detector(GC-NPD) for the identification and quantitation of hexaconazole. the mostappropriate solvent mixture for extracting hexaconazole residues from black tea was n-hexane: acetone at 1: l(v/v). The extractwas cleaned up by adsorption column chromatography using activated florisil. Performance of the method was assessed byaluating quality parameters such as recovery value, repeatability, reproducibility, linearity and limits of detection and quanti-tation. When the method was assessed for repeatability, the percentage of recovery ranged between 86% and 96% while therelative standard deviation was between 0. 30% and 2.35%. In studies on reproducibility the recovery ranged from 81%to 85%andrelative standard deviation from 1.68% to 5. 13%, implying that the method was reliable. A field trial was conducted to verify theapplication of this method with real samples. Results prove that the validated method was suitable for extracting hexaconazoleresiduesKey words: Hexaconazole, Residues, Black tea, Florisil, Gas chromatography-nitrogen phous detector(GP-NPD)doi:10.1631 jus. B0720262Document code: ACLC number:NTRODUCTIONanalyzed by gas-liquid chromatography with nitrogen(1H-1, 2, 4-triazole-1-yl-hexane-2-ol] is a systemic matography-tandem mass spectrometry for confir-fungicide which is being used widely in India for mation and quantitation(Bernal et al., 1997; Otero etcontrolling the blister blight (Exobasidium vexans) al. 2003: Schermerhorn and Golden. 2005: Troskendisease of tea( Premkumar and Baby, 2005)et al., 2005). Data are available on the hexaconazoleSeveral multi-residue methods are available for residues, but there is only limited information on thehe determination of residues of different triazoles in validation of the analytical method for its residues invarious food products, such as processed fruits, black tea(Kumar et al, 2004; Manikandan et alvegetables,grapes,must, wine and strawberries 2006). The objective of the present study is to give a(Garland et al., 1999; Sannino, 2004; Zambonin et al., detailed report on the simple and sensitive gas chro-2002)involving intensive sample preparation such as matography-nitrogen phosphorus detector(GC-NPD)solid-phase extraction which is time consuming andlabor intensive. The residues of these fungicides are中国煤化 Titation of hexaconaCNMHGating the validatioand reproducibilityProject supported by the National Tea Research Foundation, Kolkata, Quality parameters such as precision, linearity anddetection limits were also evaluatedKarthika et al. /J Zhejiang Univ Sci B 2008 9(2): 160-164MATERIALS AND METHODSleaves and a bud )were harvested from each plot andbrought to the laboratory on the same day(3 h afterChemicals, solvents and reagentsspraying) and on Days 3, 5, 7, 10 and 14 after im-Analytical standard of hexaconazole (purity posing treatments. The harvested shoots were sepa99.5%)was obtained from Dr. Ehrenstorfer (Augs- rately manufactured as described belowburg, Germany) and the formulated product, ContafThe leaves were processed in the InstitutesSEC(hexaconazole 5EC)from Rallis India Ltd., India. miniature CTC( Crush, Tear and Curl) tea manufacDichloromethane, n-hexane, acetone and ethyl acetate turing unit as per standard practice(Ramasamy, 1996)required for liquid and gas chromatography were The shoots were spread out in a withering trough andfrom Merck(Darmstadt, Germany), while other re- allowed to wither for 16-18 h. Withered leaves wereagents such as sodium chloride and anhydrous so- then passed six times through a rotorvane for crushingdium sulfate were from SD Fine Chemicals(India). and mixing leaves with the exuded juice which wasAlumina(acidic, basic and neutral), florisil (60-100 then passed through a CTC machine. The cut"dhoolmesh)and silica gel used as sorbents for purification was then allowed to undergo fermentation for onethrough column chromatography were from M/s. hour by spreading it in a tray and maintaining a relaSigma-Aldrich, USA.tive humidity of 90%-95%. The fermented"dhoolwas dried in a mini-fluid bed drier to attain a finStock standard solutionsmoisture content of 2%-3%. the drier mouth blackA stock standard solution of hexaconazole (1000 tea samples were used for analysismg/L) was prepared in acetone by weighing 0.025 gof the analyte into a 25 ml volumetric flask. Various Extraction and purificationconcentrations of hexaconazole at 0. 05.0.1.0.2. 0.4Ten grams of control black tea sample was0.8 and 1.0 mg/L were prepared by serial dilutions weighed in a 250 ml conical flask and 100 ml offrom the stock solution for linearity study and to n-hexane: acetone (1: l, v/v) was added, and thenprepare calibration curve. The standard and various tightly sealed. The mixture was shaken on a me-dilutions were stored in the dark at 4Cchanical shaker for 2 h. Supernatant was filteredthrough Whatman No. I filter paper and the contentsChromatographic columnsof the flask were washed twice with 25 ml of the exGlass columns(60 cm lengthxl I cm i d ) with tracting mixture. The filtrate and the washings wereteflon stopcocks were packed from the bottom with a pooled and transferred to a 500 ml separatory funnelglass wool plug, and 10 g of various activated ad- One hundred ml of saturated sodium chloride wassorbent materials were packed between two layers of added to the funnel and was vigorously shaken. Theanhydrous sodium sulfatelower aqueous layer was discarded and the upper or-ganic layer was once again extracted with 50 ml ofField trial for black tea preparationsaturated sodium chloride. Upper hexane layer wasA field trial was carried out during the wet sea- allowed to pass through a layer of anhydrous sodiumson at United Planter's Association of South India s sulfate to remove moisture into a 500 ml round bottomexperimental farm at Valparai, Coimbatore District flask. The extracted residue was then concentrated to(1150 m above mean sea level), India, during the year dryness using a rotary vacuum evaporator at 60C2006. There were 3 treatments each replicated in 6 The residue obtained was dissolved in 10 ml hexaneplots(for harvest on 6 different days), each plot withrification of the extract was performed by100 bushes. The plots were separated by guard rows. column chromatography using preactivated adsorbentHexaconazole formulation( Contaf 5EC)was sprayed florisil. Before use, the columns were conditionedat 200 ml/ha(the recommended dosage)and 400 with 50-mlane withont allowing them to drmIha(double the recommended dose)through a hand out. T中国煤化工10 hoperated knapsack sprayer with the recommended tractCNMHGo ethyl acetate inspray volume of 175 L water/ha. Untreated block was dichloromethane Eluate was evaporated to dryness atkept as control. about 2 kg of the green shoots(three 60C on the rotary evaporator. The residue was fi-162Karthika et al. /J Zhejiang Univ Sci B 2008 9(2): 160-164nally redissolved in 5 ml of acetone and filteredSolvents such as hexane. acetone. acetone-through a 22 um nylon filter, prior to chroma- dichloromethane and acetone-hexane are known to betographic analysisused for extracting several commonly used fungicidesHowever, for hexaconazole there was a need toInstrumentation and operatinchange the solvent by taking into consideration itsGas chromatographic analysis was carried out on solubility in solvents such as ethyl acetate and dia Hewlett Packard GC(5890 series Il) and HP (3396 chloromethane the amount of solvent used and, if aseries III) integrator equipped with DB-5 column mixture was chosen, the ratio between the compo-(phenyl methyl siloxane wide bore capillary column; nents needed to be fixed. Based on the solubility of30 m lengthx053 mm i.d. x0. 25 Hm film thickness), hexaconazole, the extraction efficiency of solventscoupled with NPD. The GC was operated at the oven such as dichloromethane and acetone was studiedtemperature 225C, injector temperature 220C, The efficiency of two individual solvents in extractdetector temperature 250C and carrier flow(nitro- ing hexaconazole from a black tea matrix was lessgen)5 ml/minsignificant and the recovery was very low. Therefore,mixtures of organic solvents such as ace-tone: dichloromethane at 1: 1 and 3: 1 (v/v) andRESULTS AND DISCUSSIONmethanol: water at 2: 1 (v/v)were evaluated. The re-covery percentage was 36%, 48% and 54%, respecGas chromatography-nitrogen phosphorus detec- tively, when the above combinations were used. But ator(GP-NPD) performancecombination of 200 ml of acetone and n-hexane(1: IThe hexaconazole residue to be analyzed by v/v)was found to be appropriate for extraction ofGC-NPD was dissolved in 10 ml of acetone and the hexaconazole from the tea matrixinjection volume was 0.5 ul. The peak resolution andappearance were poor in such cases. Therefore, the Extract purification performancefinal dilution volume was fixed at 5 ml and the injecOrganic solvent extracts from black tea containtion volume raised to 1 ulmany interfering compounds from the matrix. Toremove matrix interferences, purification efficiencySom bla.u pole from the untreated control plot by alumina(acidic, basic and neutral) and silica wasple extraction performanceof alumina, silica and florisil was tested. Purificatiowas spiked with hexaconazole at 0.5, 0.1, 1.0, 2.0 and not efficient for hexaconazole due to low retention4.0 mg/kg. The contents were allowed to equilibrate The eluate obtained with such adsorbing materialsfor about 15 min so that the fungicide spread uni- was less transparent and matrix interferences wereformly within the matrix. Subsequently, the spiked also noted during gas chromatographic analysisblack tea sample was extracted with 150 ml of However, activated florisil was found to be effectiven-hexane: acetone(L: 1, v/v) by shaking it for 2 h on a in purifying the sample since the eluate was moremechanical shaker. The contents were filtered transparent. The chromatograms had less peak interthrough Whatman No 1 filter paper and transferred to ferences from the matrixa 500 ml separating funnel. One hundred ml of satu-rated sodium chloride was added to the filtrate, shaken Sample clean up performancevigorously and allowed to settle. The aqueous phaseAfter choosing a suitable adsorbing material, thewas removed and the organic phase was again parti- next step was to select a suitable eluant or a mixture oftioned with 50 ml of saturated sodium chloride. After solvents which could successfully elute the targetpartitioning, the hexane layer was passed through an fungicide from florisil. Since the solubility of hex-anhydrous sodium sulfate layer and collected in a 500 aconml round bottomed flask. The extract was evaporated metto dryness on a rotary vacuum evaporator at 60C, and birH中国煤化 Ts such as dichlorond n-hexane. com.CNMHGn-hexane: dichloro-the residue was dissolved in 10 ml hexane. Each methane (4: 6, v/v), 5% ethyl acetate in hexane(150sample was replicated three timesml), 5% diethyl ether in hexane (150 ml),ace-Karthika ef al. /J Zhejiang Univ Sci B 2008 9(2): 160-164tone: dichloromethane(1: 3, v/v)and 5% ethyl acetate fied up to a level of 0.06 mg/kg, which proved that thein dichloromethane(200 ml) were tested. Among newly developed method was suitable for quantifyingthese. the last combination was found to be the most the hexaconazole residues even when present in traceefficient since the recovery percentage was >80%for amounts in the black tea matrix. Dissipation of hexall the fortified samplesaconazole at 200 and 400 ml/ha followed first orderkinetics. The correlation coefficient values and re-Method performancegression equations are presented in Table 4. TheEvaluation of quality parameters such as the re- chromatograms are presented in Fig. 1covery percentage, repeatability, reproducibilitynearity and limits of detection and quantitation Table 1 Repeatability for the fungicide hexaconazole in(Zanella et al., 2000)is essential to assess the method black tea spiked at five levelsperformance. Calibration curve for all chroma-Found(mg/kg) Recovery RSD,tographic analysis was prepared by plotting detector concentration(mg/kg)( mean+SD)(%)(%)response () and analyte concentration(r)expressed0.504678±000993.55191y the equation: y=c+mx. The regressed linear equa0.9357±0.01793.571tion obtained by the selected chromatographic condi-2.019198±0.00695990.29tions for hexaconazole was3.8387±0.01295980.30Number of replicates at each level (n 6(three extractions with4513.8+3979.lx(=0.99two injections each); All made under the same conditions on thesame day: RSDr: Relative standard deviation for repeatabilityThis equation was obtained by injecting a seriesof hexaconazole dilutions of 0.051.0 mg/L prepared Table 2 Reproducibility for the fungicide hexaconazoleas described in the section of stock standard solutions in black tea spiked at five levelsmentioned above. The correlation coefficient valueHexaconazole Found(mg/kg) Recovery RSDRconcentration(mg/kg)(mean+SD)(%)(%indicated perfect linearityThe limit of quantitation(LoQ)of hexaconazole04177±0.01483.533.28was established by fortifying control black tea samples0.8588±002685883.06to the lowest level of the analyte from which the1.7281±0.0518641296residues could be extracted with acceptable precision3.4159±0.05785401.68and accuracy under the established experimental con-Number of replicates at each level (n)6(three extractions withditions. The instrumental limit of detection of hexwo injections each): Each extraction and injection series wasaccomplished on 3 consecutive days; RSDR: Relative standardaconazole was 0.05 mg/kg and the amount of hex- deviation for reproducibilityaconazole that can be extracted with acceptable prcision from the black tea matrix is 0. 1 mg/kg(loQ)Table 3 Residues of hexaconazole in black tea at dif-ferent harvest intervalsThe repeatability of the developed analyticalResidues of hexaconazole(mg/kg)method was determined by adding hexaconazole in Day200 ml/ha400 mahafive different concentrations to control black tea. The02.19repeatability of spiked hexaconazole in black tea atBDL1.37the levels of 0.1, 0.5, 1.0, 2.0 and 4.0 mg/kg are5summarized in Table I0.27The reproducibility of hexaconazole was deter-BDL0.060.19mined by analyzing the fortified control black tea 14 BDLsamples with hexaconazole over three consecutive BDL: Below detectable limit(<0.05 mg/kg)days and by two different analysts as presented inTable 4 Kinetics of dissipation of hexaconazole in teaTable 2suh中国煤化工 CorrelationcoefficientVerification of methodCNMHG0.982Black tea samples prepared were subjected to400logy=0.3690.1150.977residual analysis for hexaconazole. Results(Table 3)Log y=a+b, where y is the residue level in x10, r is the harvestindicate that hexaconazole residues could be identiinterval in days, and a and b are regression constantsKarthika et al. /J Zhejiang Univ Sci B 2008 9(2): 160-164ReferencesBernal. J. L. del Nozal M.J. Jimenez, J.J. Rivera. M.J. 1997Matrix effects in the determination of acaricides andfungicides in must by gas chromatography with electroncapture and nitrogen phosphorus detection. J. Chroma-togr.A,78(1-2ll-l17.doi10.1016/s0021-9673(97)Garland, S M., Menary, R C, Davies, N.W., 1999. Dissipationof propiconazole and tebuconazole in peppermint crops( Mentha piperita(Labiatae) and their residues in dis-tilled oils. J. Agric. Food Chem., 47(1): 294-298. [doi10.1021小1980120eKumar, V, Ravindranath, S D, Shanker, A, 2004. Fate of人人hexaconazole residues in tea and its behavior duringhemical Health and Safery, 11(1):21-25.doi:10.1016chs200309018]Manikandan. K.N. Karthika. C. Muraleedharan. N. Seenivasan, S, Selvasundaram, R, 2006. Studies on the residues of copper and hexaconazole during their combinedapplication and their subsequent transfer into tea infusionJ. Plant Crop, 34(3): 405-409Otero, R. R, Grande, B. C, Gandara, J.S., 2003. Multiresiduemethod for fourteen fungicides in white grapes by liquid-liquid and solid-phase extraction followed by liquidromatography-diode array detection. J Chromatogr992(1-2):121-131.[doi:10.1016s0021-96730300317-0Premkumar, R, Baby, U L, 2005. Blister blight control-AFig 1 GC-NPD chromatogram of hexaconazole resi-review of current recommendations. Planter's Chronicledues in black tea samples101(5):26-34represents hexaconazole peak. (a)Standard hexaconazole; Ramasamy, V, 1996. CTC Tea Manufacture. The United(b)Control black tea;(c) Fortified black tea; (d) Black teaPlanters Association of Southern India, Coonoor, p Ion Day 1;(e) Black tea on Day 5Sannino, A, 2004. Evaluation of a method based on liquidchromatography/electrospray tandem mass spectrometryfor analyzing eight triazolic and pyrimidine fungicides inCONCLUSIONextracts of processed fruits and vegetables, J. AOAC int87(4)991-996A simple gC-NPd method for the determinationchermerhom, P G, Golden, P. E, 2005. Determination of 22of the triazole fungicide hexaconazole was developedtriazole compounds including parent fungicides and me-tabolites in apples, peaches, flour and water by liquidand applied for the analysis of its residues in black teachromatography/tandem mass spectrometry. J. AOAC Inisamples. The method is simple and highly reliable,88(5):1491-1502.and is exclusively applied for the determination of Trosken, E.R., Bittner, N, Volkel, W, 2005. Quantitation of 13hexaconazole residues in black tea. The test resultsazole fungicides in wine samples by liquid chromatog-for calibration, linearity, repeatability and reproduciaphy-tandem mass spectrometry. J. Chromatogr: Ability show that this was a rapid and efficient method1083(1-2):113-119.[doi:10.1016 chroma200506.020]the quantification of hexaconazole in black teaZambonin, G, Cilenti, A, Palmisano, F, 2002. Solid-phasemicro extraction and gas chromatography-mass spetrometry for the rapid screening of triazole residueswine and straw berries. Chromatogr. A, 967(2): 255-260ACKNOWLEDGEMENTdo:10.1016/50021-9673(0200780Zanella, R, Prime, E. G, Goncalves, FF, Martins, A FThanks are due to Dr. n. Muraleedharan, Di-中国煤化工 gh performandetermination of clo-rector. United Planter's Association of South IndiaCNMH Gr J Chromatogr.ATea Research Foundation for2.[do:10.1016/s0021-967300)009122]manuscript and offering valuable suggestions