Capillary gas chromatographic determination of dimethachlon residues in fresh tobacco leaves and cut

272Liu et al. / J Zhejiang Univ Sci B 2007 8(4):272-276Journal of Zhejang University SCIENCE BISSN 1673-1581 (Print); ISSN 1862-1783 (Online)www.zju.edu.cn/jzus; www.springerlink.comE-mail: jzus@zju.edu.cnJzusCapillary gas chromatographic determination of dimethachlonresidues in fresh tobacco leaves and cut-tobacco^LIU Hong-cheng', LI Qi-wan't, TANG Li-bin2(' Supervision and Testing Center for Farm Product Quality, Ministry of Agriculture (Kkunming),Yunnan Academy of Agriculture Science, Kunming 650223, China)(Kunming Institute of Physics, Kunming 650223, China)E-mail: cmliu. _0@sina.comReceived June 14, 206; revision accepted Oct. 17, 2006Abstract: Simple procedures for extraction and chromatographic determination of dimethachlon residues in fresh tobacco leavesand cut-tobacco are described. The determination was carried out by capillary gas chromatography (GC) with electron capturedetection (ECD) and confirmed by GC-MS. The mean recoveries and relative standard deviation (RSD) were 93.2%~112.9% and3.5%~ 6.7%, respectively at levels ranging from 0.01 to 0.1 mgkg. The limit of determination was 0.001 mg/kg. Tobacco samplesin routine check were successfully analyzed using the proposed method.Key words: Dimethachlon residues, Tobacco, Gas chromatography (GC)doi: 10.163 1/jzus.2007.B0272Document code: ACLC number: 0657.7" 1INTRODUCTIONin a pesticide manual (Wang and Zhang, 1994). Ef-fective analytical methods for determineation 0Myzus persica (Sulzer) is a serious diseasemany pesticides are more developed (Janska et al,caused by Alternaria alternata (Fries) Keissler af- 2006; Huang et al, 2006; Lehotay et al, 2005;fecting the Ieaves of tbacco, resuting in important Mastovska and Lehotay, 2004; Mastovska et al,economic lss No efective treatment against 2004), Zhang et al.(2006) determines 405 pesticidesMyzus persica has so far been devised, although theresidues. But no method has been published for de-fungicide dimethachlon [N-(3,5-dichlorophenyl)-termining dimethachlon residues in tobacco or othersuccinimide] (Fig.1) seemed the most suitable plants. Shi (1996) reported the analysis procedure forchemical for contolling the disease (Zhang et al, dimetachlon by GC-lame ionization detection (FID),2003; Li et al, 2003; Ma et al, 2002; Chen et al, which is used to evaluate the purity of commerial2001; Shi et al, 2000), in China. Dimethachlon ispesticide containing dimethachlon (95%).mainly used in Y unnan, a Chinese province, which isthe largest producer of tobacco in China. This has ledCto the problem of declining tobacco quality due to thesoils' dimethachlon residues. So it is imperative tosearch for a simple method to determine the dimeth-cachlon residues in tobacco. Dimethachlon's physical,chemical and toxicological properties are summarizedFig.1 Structure of dimethachlon$ Corresponding authorIn this work, simple analysis procedures have* Project (No. 2006NG01) supported by the Agriculture Breakthrough been develope中国煤化工hachlon iProgram of Yunnan Province, Chinatobacco sample:d includesYHCNMHGLiu et al. / J Zhejang Univ Sci B 2007 8();:272-276273solvent extraction, solid phase extraction (SPE) Extractionclean-up and determination by GC with electronTwenty-five grams samples were homogenizedcapture detection (ECD). Our work aimed at (1) de- by high-speed refiner with 50 ml acetonitrile for 3veloping a simpler, faster and sensitive methodology min; the mixture was filtered through a 8.9 cm highfor determining dimethachlon residues in tobacco by speed filter paper into a 100 ml graduated flask (withGC-ECD, considering aspects such as quantification, tap) into which had been placed 10 g sodium chloride.recoveries and identification problems and alsoThe graduated flask was shaken vigorously for 5 mincharacterization by GC-MS; (2) studying the appli- and was left to stand for 30 min until the filtrate wascability of the proposed method in routine analysis of transparent. Ten milliliters organic phase was accu-tobacco samples containing dimethachlon residues.rately transferred to a 250 ml breaker. The acetonitrilephase was evaporated nearly to dry by blowing ni-trogen on an 80 °C water-bath. The residues wereEXPERIMENTAL DETAILSdissolved in 1 ml of n-hexane.ReagentsClean-upDimethachlon certified standards were providedThe residues were then transferred to the LC-by Chemservice (West Chester, PA, USA). Standard Florisil (1 g/6 ml), which was conditioned with 3 mlstock solutions in acetone (1 mg/ml) were prepared acetone:n-hexane (1:10, V:V) and 3 ml n-hexane. Theand stored at -18 °C for three months. Various stan- pesticides were eluted with 10 ml of acetone:dards for standard curved (0, 0.005, 0.01, 0.05, 0.1 n-hexane (1:10, v:v), and collected ina 10 ml gradu-and 0.5 μg/ml) were prepared from the standard stock ated test tube with a tap. The elution was concentratedsolution by serial dilutions. All the solvents used were to 5 ml by nitrogen evaporator (N-EVAP IHI, Or-of analytic-grade and redistilled.ganomation Associates Inc., USA) on a 40 °C wa-Cartridges repacked with SPE-Florisil (SU- ter-bath, and its volume was accurately made up to 5PELCO, USA) 1.0 g/6 ml.ml with n-hexane. A 1 μl portion was analysed by GC.InstrumentationGC determination: ECDAnalysis was carried out using an Agilent 6890NThe operating conditions were as follows: cap-GC system consisting of a 7683 automatic liquid illary column DB-1 (0.25 um filmx0.25 mm i.d.x30samples tray 100, a micro electron capture detector m length, J&W Scientific, USA). The detector and(m-ECD). Data acquisition and processing were per- injection temperatures were 320。C and 200。C, re-formed with an HP computer using Agilent Tech- spectively. The injection volume was 1 ul.nologies Chemstation Software (A.09.03， WindowsThe oven temperature was maintained at 150 °C2000).for 2 min after injection, then programmed at 6Mass spectrometric analysis was carried out °C/min to 270 °C, which was held for 8 min, and afterusing a Shimadzu GC/MS-QP2010 system, with 8 °C/min to 280 °C, which was held for 5 min. TheDI-2010 automatic liquid samples. Data acquisition makeup gas was nitrogen (99 999%) with flow rate ofand processing were performed with a computer us- 1 ml/min, and splitless.ing GC-MS solution (Ver. 2, Windows 2000).Confirmation by GC-MSPreparation of samplesMass spectrometry analysis was used to verifyFresh tobacco leaves chopped in a high speed the dimethachlon in the extract. Mass spectra wereblending jar were homogenized for 2 min and stored obtained on a GC/MS-QP2010, a capillary columnat -18。C. To analyze the cut-tobacco samples, a 100 DB-1 (0.25 μm flmx0.25 mm i.d.x30 m length, J&Wg sample was first cut into pieces in a high speed Scientific, USA). The oven temperature was main-blending jar and homogenized for 5 min, then the tained at 50 °C for 1 min after injection, then pro-sample dropped into 200 ml water for 10 min before grammed at 20 °C/min to 150 °C, which was held forextraction.2 min, and after中国煤化工h was heldfYHCNMHG274Liu et al. / J Zhejiang Univ Sci B 2007 8(4):272-276for 3 min. The MS operation conditions were 70-eV isotope ions m/z 243:245:247=9:6:1, included twoelectron ionization (EI, 200 °C) at scan mode, scan- chlorines. A fragmentation pattern at m/z 215 corre-ning between m/z 45 to 475. Helium at flow rate of 1 sponding to [M-CO]， m/z 187 corresponding tom/min was used. The injection temperatures was 250 [M-COC2H4]", the split ions m/z 152 were the ions。C, and injection volume was 1 μl and splitless.m/z 187 leaking out one chlorine (CI), the split ion m/z124 was then leaking out carbon oxide (CO). ThroughRecovery studiesthe split ions peaks, we confirmed that the molecularThe proposed samples preparation method was particle was the dimethachlon.validated by carrying out a recovery study on freshtobacco leaves and cut-tobacco samples, which were Linearity, reproducibility and recoverynot treated with the pesticide studied, and fortifiedThe linear dynamic range was checked for to-following the procedure described above with 1 ml of bacco extracts fortified with dimethachlon by inject-the working standard mixture solution. These values ing an absolute amount of 0.005~0.5 ng and the cali-represent 0.01~0.1 mgkg of dimethachlon in the bration graph showed good linearity with asamples. The recovery assays were replicated fivetimes.550500]50月RESULTS AND DISCUSSION400300A different chromatographic profile was ob-50|tained for fresh tobacco leaves and cut-tobacco, re-=Msulting in the most complex chromatogram. Fig.2111213141516171819shows typical chromatograms of standard dimeth-Time (min)achlan, fresh tobacco leaves and cut-tobacco samples(afortified with dimethachlon at 0.1 mgkg and thespectral matches were obtained at 18.474 min, theretention time of dimethachlon. The background level002for cut-tobacco was more complicated than that at00)fresh tobacco leaves, but it did not disturb the deter-mination of dimethachlon, the other peak was 18.598昌300)min; two peaks could be completely separated. It wasimportant to drop cut-tobacco in water for 10 minbefore it was extracted by acetonitrile, because thetissue of cut-tobacco is soluble in water and the con-(btent extracted by acetonitrile is lttle. It was also im-portant that the graduated flask was shaken vigor-ously for 5 min and left in place >30 min until thefiltrate was transparence. This can ensure that theratio of extraction was more than 95% and that the400acetonitrile completely evaporated.Mass spectrometric studyThe presence of dimethachlon in the extracts ofsamples was confirmed by GC-MS in scan mode as(cshown in Fig.3. We could not find the dimethachlonin the standard datum (NIST datum). The molecularFig.2 Chromatogram of standard (a), tobacco leavesion peak was m/z 243, corresponding to [M-H]", the(b) and cut-tob中国煤化Iichlon1HCNMHGLiu et al. / J Zhejang Univ Sci B 2007 8();:272-276275100邕80手60司)斗46218Time (min)RT: 13.52 min80 -60+40- |员20-|是月司量6(12140160180202224260 280 300/zFig.3 Mass spectrum of dimethachlon. (a) Scan mode; (b) Bar chromatogramcorrelation coefficient of 0.9997; y=2475.5035xTable 1 Recovery and RSD obtained on fresh tobaccoAmt+ 194.25589. Non fortified samples were alsoleaves and cut-tobacco sample after applying the meth-analyzed in order to ensure that no peak was obtainedods (n=5)FortificationDimethachlonat the retention time of the analyte. A concentration ofSample(mgkg)Recovery (%) RSD (%)0.001 mg/kg can be considered as the detection limitTobacco leaves0.0193.25.2of the method as lower concentrations did not always0.0596.73.5allow a proper definition of the spectrum. This level0.1098.54.6was low enough for monitoring dimethachlon in to-bacco.Cut-tobacco112.9105.63.8The retention time was 18.474 min and the re-producibility of the retention time was tested by102.35.4analyzing five fortified tobacco samples.The procedure was validated by carrying out a the average recovery and RSD were replicated fiverecovery study on tobacco samples, fortified at three times respectively at levels ranging from 0.01 to 0.1levels, 0.01, 0.05 and 0.1 mg/kg. Table 1 shows the mgkg.average recovery and relative standard deviation(RSD) obtained in the application of the proposed Application of the method to real samplesprocedures on the fresh tobacco leaves andRoutine analysis of 50 tobacco leaves and 50cut-tobacco sample spiked with dimethachlon. The cut-tobacco samples (from three areas in Y unnanrecovery of dimethachlon was complete and inde- Province, China) were selected to evaluate the effec-pendent of the commodity and fortification level, and tiveness of the中国煤化工ues of di-YHCNMHG276Liu et al. / J Zhejiang Univ Sci B 2007 8(4):272-276methachlon were found in 5 tobacco leaves in thebuffering and other means to improve results of prob-range of 0.01~0.08 mgkg. Fig.4 shows the chroma-lematic pesticides in a fast and easy method for residueanalysis of fruits and vegetables. J. AOAC Int, 88(2):tograms of sample with 0.06 mg/kg dimethachlon615-629.residue.Li, YJ, Chen, H.M, Zhou, L, 2003. Experiment on mixedpesticides for controlling tobacco brown spot and MyzusPersicae (Sulzer). Chin. J. Tobacco Sci Technol./Dis.700Pest Control, 10(2):45-47 (in Chinese)Ma, J., Zheng, R., Xu, L, Wang, S., 2002. Diffrential sensi-600tivity of two green algae, Scenedesmus obliqnus andChlorella pyrenoidosa, to 12 pesticides. Ecotoxicol. En-00 |viron. Saf, 52(1):57-61. [doi: 10.106/eesa.2002.2146]Mastovska, K, Lehotay, SJ, 2004. Evaluation of commonorganic solvents for gas chromatographic analysis andstability of multiclass pesticide residues. J Chromatogr.A, 1040(2):259-272. [doi:10.1016/j.chroma 2004.04.017]00Mastovska, K., Hajslova, J. Lehotay, SJ, 2004. Ruggedness10111213141516171819and other performance characteristics of low-pressure gaschromatography-mass spectrometry for the fast analysisof multiple pesticide residues in food crops. J. Chroma-Fig.4 Chromatograms of sample with dimethachlontogr. A, 1054( 1-2):335-349. [doi:10.1016/j.chroma .2004.residue08.061]Shi, J.H., 1996. 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BYERSISSN 1673-1581 (Print); ISSN 1862-1783 (Online), m onthlyJournal of Zhejiang UniversitySCIENCE Bwww.zju.edu.cn/jzus; www.springerlink.comjzus@ zju.edu.cnJZUS-B focuses on“Biomedicine, Biochemistry & Biotechnology"JZUS-B online in PMC: htp://ww.pubedcentral.ih.gov/tocrenderfqi/iowrna1=37 18ontin=archive中国煤化工fYHCNMHG