Determination of Herbicide Residuesin Garlic by GC–MSZilei Chen1,2,Hong Zhang2,Bing Liu1,Guosheng Yang1,Hassan Y.Aboul-Enein3,&,Wenbo Wang2, Ruiyan Ding2,Hongxia Du2,Huidong Li21School of Chemistry and Chemical Engineering,Shandong University,Jinan250100,China2Central Laboratory of Shandong Academy of Agricultural Science,Jinan250100,China3Pharmaceutical and Medicinal Chemistry Department,Pharmaceutical and Drug Industries Research Division,National Research Centre,Dokki,Cairo12311,Egypt;E-Mail:enein@Received:31May2007/Revised:27August2007/Accepted:3September2007Online publication:19October2007AbstractA rapid multi-residue method has been developed for the determination of16herbicides ingarlic.The analytical procedure is based on prior microwave heating prevention of the gen-eration of sulfur-containing compounds.The garlic samples which had been pretreated wereextracted with acetonitrile and cleaned up by solid-phase extraction.The herbicide residueswere detected by gas chromatography–mass spectrometry with selected ion monitoring.Therecoveries of16herbicides ranged from69.0to105.4%with the relative standard deviations(RSD)below10.1%.The limit of quantification(LOQ)ranged from0.003to0.015mg kgÀ1.KeywordsGas chromatography–mass spectrometryMicrowave sulfur removalGarlicHerbicide residuesIntroductionChina produces more than6million tons of garlic per year.Herbicides,such as Chlorotoluron and Acetochlor,find widespread use during the planting of the garlic crop.Although herbicides have low immediate toxicity,they are easy to accumulate in the body,they may be carcinogenic and have a high endocrine disrupting potential[1–5].Hence,many countries have established maximum recommended limits(MRL)for herbicideresidues in foodstuffs[6].Currently,the methods used for thedetermination of herbicide residues invegetables are mainly based on analysisby GC or HPLC[7–9].However,thereare many sulfur compounds in garlic,which mask the detection of herbicidesresidues[10,11].Supercriticalfluidextraction(SFE)can remove sulfurcompounds prior to analysis[12]andWang et al.detected four organochlorinepesticide residues in garlic with SFE andGC-ECD[13],but the assay is lengthyand time consuming.Free sulfur compounds are presentonly when the garlic bulbs are crushed orcut[14].The vacuolar enzyme,allinase,transform alliin into the very unstablethiosulphinate allicin which is rapidlydegraded to form secondary productsconsisting of various sulfides,sulfonicesters,and sulfonic ethers which contrib-ute to the characteristicflavor and odorof garlic[15].Unfortunately,the presenceof these sulfur compounds affects thedetermination of herbicide residues.Onlywhen the allinase activity is destroyedbefore the garlic cloves are crushed andhomogenized,can the interference causedby the sulfur compounds be successfullyeliminated.Allinase activity changeswith the temperature,pH and the pres-ence of metal ions[16,17,18].This paper describes various methodsto eliminate the action of allinase andthus effectively minimize the interferencescaused by sulfur compounds on theanalysis of various herbicides residues byGC–MS.ExperimentalMaterialsHerbicide standards at1,000l g mLÀ1,were obtained from the EnvironmentInspect Department of the China Agri-cultural Ministry.A mixed standard stock solution(10g mLÀ1)was obtained by diluting the standard solutions in actone,and storing atÀ20°C.All organic solvents and chemical re-agents were analytical grade(acetonitrile, acetone,n-hexane,sodium chloride),and purchased either from Concord Tech-nology Co.or Tianjin Chemical Reagent Company.The solid phase extraction (SPE)columns(Florisil,6mL,1g)were purchased from Supelco(USA).Uncontaminated garlic samples pro-duced in our own garden were used as mercial samples were ob-tained from a garlic plantation in Shan-dong province of China.The samples were stored at4°C in the dark place until analysis.InstrumentationA12-port vacuum manifold Visiprep (Supelco,Madrid,Spain)was employed for SPE.The microwave oven was a WG800TL23-2W(Galanz Co.,China). The GC–MS system was a Hewlett-Packard6890GC equipped with a5973 mass selective detector and a7683auto-sampler.The capillary column was 30m·0.25mm I.D·0.25l m DB-5MS (Agilent Technology,USA)The water bath was HH-W420(Jingbo Instrument Co.,China).The oven temperature program was as follows:initial temperature isothermal at50°C for1min,then to200°C at 15°C minÀ1,with a hold of5min at 200°C,then from200to280°C at10°C minÀ1,with afinal hold for5minat250°C.Injector temperature was280°C.Carrier gas was helium at aflowrate of1.0mL minÀ1.Injection volumewas 1.0l L with split less mode.Tem-perature of transfer line was260°C.Ionsource was EI(70eV,230°C).Temper-ature of quadrupole was1,500°C andelectron multiplier voltage was1,800V.Scan mode was full scan(35–500m/z)and selected ion monitoring(SIM)mode.Retention times and selected ion of the16herbicides are listed in Table1and thechromatogram of the standard solutionof the herbicides is shown in Fig.1.Sample Preparationand ExtractionThirty grams of intact garlic cloves wereweighed into a jar and heated in amicrowave oven for30s,then rapidlycooled in ice-water andfinally homoge-nized at18,000rpm for1min in ahomogenizer.The resulting extract wasfiltered into a100mL graduatedflaskcontaining15g NaCl,shaked vigorouslyfor1min,then allowed to stand for30min to separate.The50mL upperlayer was evaporated in a rotary evapo-rator to near dryness for clean up.Clean-UpThe Florisil cartridge for SPE wereloaded with the extract after beingactivated with10mL offa mixture ofacetone/n-hexane(1:9v/v)The cartridgewas then eluted with10mL of acetone/n-hexane(1:9v/v).Finally,the eluate wasevaporated to less than5mL with agentle nitrogen stream and was made upto5mL with n-hexane for analysis.Result and DiscussionThe Conditions for InactivatingAllinaseThe interference of sulfur-containingcompounds in garlic would disappear ifallinase was inhibited.When the pHwas less than2or more than10,theallinase may lose its activities.Never-theless,the herbicides were not stableunder the acid/basis conditions.So thechange of pH was not successful duringthe assay herbicides in garlic.The metalions such as Cu had a strong inhibitoryeffect on allinase.On the other hand,allinase shows the highest activity at30°C,and this slowly disappear with adecrease of temperature to0°C.Whenthe temperature is greater than60°C,the allinase activity decreases sharply.We describe below three possiblemethods to deactivating the allinaseenzyme,namely through freezing,heat-ing and the use of metallic inhibitors.The untreated garlic was used as ablank during the experiments(Fig.2).Allinase activity was assayed by deter-mination of3-vinyl-1,2-dithiacyclohex-5-ene(Fig.3).Table1.Retention time and selected ions of 16herbicides using GC–MSCompound t R(min)Monitor ions(m/z)a Chlorotoluron8.25132,167,104 Molinate11.06126,187,55 Trifluralin11.87306,264,335 Simazine13.02201,186,173 Atrazine13.09200,215,173 Acetochlor14.94146,223,162 2,4-D-Butylate15.11276,175,220 Alachlor15.27160,188,237 Prometryn15.72241,184,226 Metolachlor16.77162,238,211 Pendimethalin18.07252,281,220 Butachlor19.24176,160,188 Pretilachlor19.85238,162,176 Oxadiazon20.06175,258,344 Oxyfluorfen20.26252,300,361 Nitrofen20.92283,202,253 a Thefirst ion is the quantitativeionDeactivation of FreezingThe garlic cloves were kept atÀ20°C overnight,and then were crushed in a mortar for extraction with acetonitrile. Assayed3-vinyl-1,2-dithiacyclohex-5-ene showed that the content of this com-pound was reduced by about25–50%. However this reduction proved insuffi-cient for the determination of herbicide residues in garlic and hence the method was not suitable.Deactivation with a Metallic InhibitorAbout100g garlic cloves were steeped in 100mL CuSO4solution(0.1mol LÀ1) for1h,1,3,5,7,and14days,then cra-shed in a high speed tissue homogenizer then analyzed for3-vinyl-1,2-dithiacy-clohex-5-ene.The results of this assay of this compound,shown in Fig4indicate that the content gradually decreased with time.The results indicated that the sulfur compounds produced had an appreciable effect on the assay of the herbicides for at least7days but decreased sufficiently after14days.The process required a long time because the Cu2+ion slowly pene-trates the cell tissues to inhibit the enzyme and this method is too time consuming for routine deactivation. Deactivation by HeatingTwo simple,heating methods were applied:heating in a water-bath and microwave heating.Water-BathAbout100g garlic cloves were thor-oughly mixed with120mL distilled water in glass beakers and immersed in a water-bath at80and100°C respectively.When the sample was incubated for beyond 30min in80°C or for more than3min at100°C,the allinase lost its activity (Table2).Heating by MicrowaveAbout20,50,and100g intact garlic cloves were heated in microwave oven(800W) for different times(Table3).The allinase inactivation depends on the amount ofsample and the time of microwave heating.When the ratio of microwaved heatingtime and sample amount was higher than1g sÀ1at(800W),the enzyme lost itsactivity completely.Since heating was effective in destroy-ing the allinase activity,the optimumconditions for the two heating methodswere investigated.For heating in a waterbath,100g of intact garlic cloves spikedwith working standard solutions ofher-Original Chromatographia2007,66,December(No.11/12)889bicides at a level of5l g mLÀ1were placed inflasks and120mL redistilled water was added into eachflask in a water bath.The heating times were30min at 80°C and3min at100°C.Table4shows the recoveries of the16herbicides.Table4data indicates that the meth-od was suitable for triazine herbicides such as simazine,atrazine,prometryn,and amide herbicides such as acetochlor,alachlor,metolachlor but could not beapplied to other kinds of herbicides thatwere easy to hydrolyze under such con-ditions.For microwave heating,25and50gof intact garlic cloves were spiked withthe mixed standard solution of herbicidesat concentrations of0.1mg kgÀ1each.After the solvent was removed by naturalevaporation,the samples were heatedusing a microwave oven(800W)for dif-ferent times and the recoveries deter-mined.The data are presented inTables5and6.The results indicate thatthe recoveries are dependent on thesample quantity and the microwaveheating time.The recoveries all decreasedwith the increase of heating time,espe-cially for the compounds molinate,tri-fluralin,2,4-D-butylate,pendimethalin,oxyfluorfen and nitrofen.All the recov-eries were higher than75%when theratio was1.0s gÀ1and all the recoveriesexcept2,4-D-butylate were more than70%when the ratio was1.2s gÀ1.Whenratio was higher than 1.2s gÀ1,mostanalytical recoveries were lower than70%,because some herbicides hydro-lyzed rapidly as the microwave heatingtime increased.Moreover,the sampleswere chilled to room temperature quicklyby the ice-water bath after heated bymicrowave,otherwise,the recoveries werelow.From the data obtained for the allin-ase deactivation and herbicides stability,one can conclude that both methods ofheating can deactivate allinase efficiently.However,the water-bath heating was notchosen because of the hydrolysis of someherbicides.Microwave heating providessatisfactory recoveries,if the factorsinfluencing the enzyme inactivation wereoptimized as shown in Fig.5.So theoptimum microwaving time of1–1.2sgÀ1of sample was sufficient to eliminatethe interferences and yielded goodrecoveries.Recovery,LOD and LOQ,LinearityBlank samples(30g)were spiked with16herbicides at concentrations of0.02,0.1,and0.5mg kgÀ1.The samples wereheated in a microwave oven for30s,thenextracted with acetonitrile and cleaned upwith a Florisil SPE cartridge andfinallyanalyzed by GC–MS.The recovery ofeach herbicide was replicatedfive times.The data are presented in Table7whichshows that the recoveries were in therange69.0–105.4%,the relative standarddeviation(RSD)varied from 1.0to10.4%.The method is applicable for theanalysis of16herbicides in garlic used inthis study.The limit of detection(LOD)of theproposed method was determined byconsidering a value three times thebackground noise obtained for blanksamples,whereas the limits of quantifi-cation(LOQ)were determined consider-ing a value10times the backgroundnoise.Table7summarizes the detectionand quantification limits obtained foreach herbicide.Table2.Activity of allinase in garlic after pretreatment by heating in a water bathTime of heating (min)Temperature of water bath80°C100°C1–25%2–1%3–0.1% 1065%–2015%–300.1%–Table3.Activity of allinase in various weights of garlic after microwave oven heatingHeating time(s)Weight of garlic25g50g100g1520%––201%––250.1%35%–300.1%20%–40–2%–50–0.1%–60–0.1%33%80––5% 100–– 2.5% 120––0.1% Table4.Recoveries(%)of herbicides for samples heated in a water bath at80°C (30min)and100°C(3min)Compound Temperature ofwater bath80°C100°C Chlorotoluron72.470.7 Molinate23.835.8Trifluralin22.515.8 Simazine80.977.5 Atrazine85.480.6 Acetochlor88.988.82,4-D-Butylate55.833.3 Alachlor86.086.6 Prometryn84.388.9 Metolachlor88.291.8 Pendimethalin63.462.3 Butachlor78.378.0 Pretilachlor85.582.8 Oxadiazon86.475.2 Oxyfluorfen64.366.5 Nitrofen53.771.6Table5.Recovery(%)of16herbicides in 25g garlic samples pretreated by microwave ovenCompound Heating time25s30s40s50s Chlorotoluron94.585.575.376.4 Molinate83.899.463.555.8 Trifluralin84.377.665.635.6 Simazine88.390.680.576.1 Atrazine103.896.495.692.6 Acetochlor95.886.188.990.8 2,4-D-Butylate78.672.553.233.3 Alachlor98.688.086.080.5 Prometryn95.190.584.384.6 Metolachlor98.895.388.290.5 Pendimethalin87.675.361.962.3 Butachlor93.085.378.370.0 Pretilachlor95.795.381.080.6 Oxadiazon91.288.378.885.2 Oxyfluorfen93.285.368.165.0 Nitrofen94.493.273.569.5 Table6.Recovery(%)of16herbicides in 50g garlic samples pretreated by microwave ovenCompound Heating time50s60s80s100s Chlorotoluron90.680.671.666.5 Molinate80.775.867.563.4 Trifluralin82.074.165.040.8 Simazine93.583.678.476.0 Atrazine93.586.488.482.6 Acetochlor92.282.381.584.8 2,4-D-Butylate76.667.163.243.4 Alachlor95.592.687.784.6 Prometryn92.096.383.582.3 Metolachlor95.292.390.591.2 Pendimethalin88.877.868.746.7 Butachlor94.787.575.474.4 Pretilachlor92.492.382.075.7 Oxadiazon85.380.570.475.3 Oxyfluorfen75.472.363.155.0 Nitrofen93.790.477.567.2890Chromatographia2007,66,December(No.11/12)OriginalThe linearity of all the herbicides was in the range of0.01–5.0l g mLÀ1with r2>0.995.Correlation coefficients of each herbicide are presented in Table7.Application to Real Samples Six samples from garlic plantation in Shandong province were analyzed by the method described above.Four herbicides have been detected.The herbicide con-centrations were0.020mg kgÀ1for acetochlor(sample5),0.017mg kgÀ1for prometryn(sample6),0.068(sample5) and0.010mg kgÀ1(sample6)for pentadimethalin and0.014mg kgÀ1for nitrofen(sample2).ConclusionA rapid multi-residue method has been developed for the determination of16 herbicides in garlic.The proposed method is based on deactivation of allinase enzyme in garlic cloves using microwave heating, thus effectively eliminating interference from sulfur compounds in the analysis of herbicides residues.Strict microwave processing time keeps the herbicides stable during the heating.Treated garlic was ex-tracted with acetonitrile and clean-up by SPE,GC–MS/SIM was used to determine the pesticide residues.All herbicides recoveries ranged from69.0to105.4%and RSD were in the range 1.0–10.4%for spiked samples.LOQ below0.02mg kgÀ1 were achieved with this procedure.This validated analytical method is fast andconvenient for the determination of her-bicide residues in garlic. AcknowledgmentsThis work was supported by the Young Scientist Research Foundation of Shan-dong Academy of Agricultural Science. Bing Liu is currently working in Chem-istry Department of Xinxiang Teachers’College,Henan Province,People’s Republic of China.References1.Hurley PM,Hill RN(1998)EnvironHealth Persp106:437–4452.Ashby J,Tinwell H,Lefevre PA,WilliamsJ,Kier L,Adler ID,Clapp MJ(1997)Mutat Res-Gen Tox En393:263–2813.Lee WJ,Hoppin JA,Blair A,Lubin JH,Dosemeci M,Sandler DP,Alavanja MC(2004)Am J Epidemiol159:373–3804.Health Effects Division,Office of PesticidePrograms,U.S.Environmental ProtectionAgency(2004).List of Chemicals Evalu-ated for Carcinogenic Envi-ronmental Protection Agency,Washington,DC5.Ma J,Wang S,Wang P,Ma L,Chen X,XuR(2006)Ecotox Environ Safe63:456–4626.California Department of Food and Agri-culture.International Maximum ResidueLimits Database[DB/OL](2007).http:///mrl_databases.asp7.Tadeo JL,Sa nchez-Brunete C,Pe rez RA,Ferna ndez MD(2000)J Chromatogr A882:175–1918.Albero B,Sa nchez-Brunete C,Donoso A,Tadeo JL(2004)J Chromatogr A1043:127–1339.Aramendıa MA,Borau V,Lafont F,Marinas A,Marinas JM,Moreno JM,Urbano FJ(2007).Food Chem105:855–86110.Ahmed FE(2001)Trends Anal Chem20:649–66111.Tekel J,Hatrık S(1996)J Chromatogr A75:397–41012.Tilio R,Kapila S,Nam KS,Bossi R,Facchetti S(1994)J Chromatogr A662:191–19713.Wang JH,Xu Q,Jiao K(1998)J Chro-matogr A818:138–14314.Stoll A,Seeback E(1949)Helv Chim Acta32:197–20515.Yan X,Wang Z,Barlow P(1992)FoodChem45:135–139ncaster JE,Collin HA(1981)Plant SciLett22:169–176wson LD,Hughes BG(1992)PlantaMed58:345–35018.Sunyoo K,Pike LM(2001)Sci Hort89:249–256Table7.Recovery(%)±RSD(%),LOD,LOQ and correlation coefficients(r2)of the herbicides studiedCompound Recovery(%)±RSD(%)(n=5)LOD(mg kgÀ1)LOQ(mg kgÀ1)r20.02mg kgÀ10.1mg kgÀ10.5mg kgÀ1Chlorotoluron82.1±7.285.4±5.294.5±3.30.0030.0100.9991 Molinate76.1±10.480.2±4.688.3±5.20.0020.0060.9983 Trifluralin80.5±8.579.2±7.388.0±6.10.0030.0100.9958 Simazine89.3±6.5102.2±6.689.9±2.00.0020.0060.9972 Atrazine100.5±7.792.9±2.595.6±4.30.0020.0060.9989 Acetochlor92.3±5.596.7±6.194.4±2.80.0020.0060.9993 2,4-D-Butylate69.0±8.779.2±4.678.3±4.00.0050.0150.9976 Alachlor87.5±3.392.0±3.798.6±3.00.0020.0060.9988 Prometryn94.3±4.6104.9±4.595.0±1.30.00150.0050.9995 Metolachlor91.4±7.393.9±4.097.5±1.00.0010.0030.9985 Pendimethalin72.6±9.383.0±7.281.4±3.50.0030.0100.9978 Butachlor93.3±5.798.8±1.1105.4±3.40.00150.0050.9990 Pretilachlor90.0±2.595.5±2.2103.2±2.70.0010.0040.9966 Oxadiazon81.3±8.083.7±3.491.5±2.40.0020.0060.9968 Oxyfluorfen90.8±2.386.9±4.693.3±3.90.0030.0100.9992 Nitrofen78.4±6.983.6±5.590.1±4.10.0030.0100.9979 parison of chromatograms:chromatogram a(upper)0.02mg kgÀ1spiked blank sample(pretreated by microwave oven);chromatogram b(lower)0.02mg kgÀ1spiked blank sample(untreated)Original Chromatographia2007,66,December(No.11/12)891。
