Method Validation for Qualitative and Quantitative Analysis of Pesticide Residues in Tomato
et al .ȱ 2011)ȱ mayȱ poseȱ healthȱ hazardsȱ toȱ consumersȱ withȱ 1%ȱ phenyl-methylȱ polysiloxane)ȱ maintainedȱ
(Elliionȱ et al .ȱ2000;ȱMukherjeeȱandȱGopal,ȱ2003).ȱTheȱ theȱ columnȱ temperaturesȱ startingȱ fromȱ 50°Cȱ toȱ
determinationȱofȱpesticideȱresiduesȱinȱvegetablesȱandȱ 290°Cȱwithȱfourȱrampsȱwithȱaȱtotalȱprogramȱofȱ50ȱ
fruitsȱisȱofȱgreatȱconcernȱforȱallȱcountriesȱtoȱstudyȱtheȱ minutes.ȱMassȱspectraȱdetectorȱ(TripleȱQuadrupole)ȱ
riskȱanalysisȱandȱtakeȱupȱfoodȱsafetyȱmeasuresȱforȱ withȱmassȱrangeȱ50ȱtoȱ400ȱwasȱoperatedȱatȱ250°Cȱofȱ
bothȱexportȱandȱdomesticȱtradeȱpurposes.ȱTheȱaimȱ transferȱline,ȱ220°Cȱofȱsource,ȱandȱ40°Cȱofȱmanifoldȱ
ofȱthisȱworkȱisȱtoȱdevelopȱproceduresȱforȱtheȱanalysisȱ temperatures.ȱLimitȱofȱdetectionȱ(LOD),ȱLOQȱ(Limitȱ
ofȱmultiȱclassȱpesticidesȱandȱitsȱmetabolitesȱthroughȱ ofȱ Quantification)ȱ and%RSDȱ (Relativeȱ standardȱ
bestȱextractionȱmethods,ȱandȱbyȱgasȱchromatographyȱ deviation)ȱvaluesȱwereȱcalculatedȱforȱeachȱpesticideȱ
massȱ spectroscopy.ȱ Theȱ studiesȱ focusȱ onȱ sampleȱ underȱtheȱstandardȱconditions.
preparation,ȱsampleȱextractionȱfollowingȱQuEChERSȱ
methodȱ andȱ instrumentalȱ analysisȱ usingȱ MRMȱ Field samples and extraction methods
methodsȱinȱGC-MS/MS.
Tomatoesȱ (untreated)ȱ wereȱ collectedȱ fromȱ theȱ
supervisedȱ fieldsȱ ofȱ Studentȱ farm,ȱ Collegeȱ ofȱ
Materials and Methods
Agriculture.ȱQuEChERSȱ(QuickȱEasyȱCheapȱEffectiveȱ
Chemicals and Reagents
Ruggedȱ Safe)ȱ methodȱ forȱ extractionȱ andȱ cleanȱ upȱ
wasȱvalidatedȱasȱperȱSANCO/12571/2013ȱguidelines.ȱ
Solventsȱ likeȱ n- hexane,ȱ acetone,ȱ tolueneȱ andȱ Tomatoȱ fruitsȱ (5ȱ kg)ȱ collectedȱ fromȱ controlȱ plotsȱ
acetonitrileȱ(HPLCȱgrade)ȱwereȱpurchasedȱfromȱM/Sȱ wereȱhomogenizedȱwithȱRobotȱCoupeȱBlixer,ȱfromȱ
Merckȱ India.ȱ Anhydrousȱ sodiumȱ sulfateȱ (Na 2 SO 4 )ȱ whichȱ15ȱgȱwasȱtakenȱinȱtoȱ50ȱmLȱcentrifugeȱtubes.ȱ
andȱ anhydrousȱ magnesiumȱ sulfateȱ (MgSO 4 )ȱ wereȱ Theȱrequiredȱquantitiesȱofȱ64ȱpesticidesȱintermediaryȱ
purifiedȱ withȱ acetoneȱ andȱ bakedȱ forȱ 4ȱ hȱ atȱ 600°Cȱ standardsȱ areȱ addedȱ toȱ eachȱ 15ȱ gȱ sampleȱ toȱ getȱ
inȱ muffleȱ Furnaceȱ toȱ removeȱ possibleȱ phthalateȱ fortificationȱlevelsȱofȱ0.05ȱmgȱkg -1 ,ȱ0.25ȱmgȱkg -1 ,ȱandȱ
impurities.ȱPrimaryȱsecondaryȱamineȱ(PSA)ȱbondasilȱ 0.5ȱmgȱkg -1 ,ȱinȱthreeȱreplicationsȱeach.ȱAcetonitrileȱ
40ȱ µmȱ wasȱ purchasedȱ fromȱ M/SȱAgilent.ȱ Certifiedȱ (30ȱ±ȱ0.1ȱmL)ȱwasȱaddedȱtoȱtube,ȱhomogenizedȱforȱ
Referenceȱ Materialsȱ (CRMs)ȱ ofȱ highȱ purityȱ (≥98%)ȱ 1-3ȱminȱusingȱHeidolphȱsilentȱcrusherȱ(lowȱvolumeȱ
wereȱprocuredȱfromȱM/SȱSigmaȱAldrich,ȱUSA.
homogenizer).ȱ Thenȱ 3±0.1ȱ gȱ sodiumȱ chlorideȱ wasȱ
addedȱ toȱ tubeȱ andȱ mixedȱ byȱ shakingȱ gently,ȱ andȱ
Calibration standards and Linearity studies
centrifugedȱforȱ3ȱminȱatȱ2500-3000ȱxgȱwithȱRemiȱR-238ȱ
Primaryȱ(500-1000ȱppm)ȱandȱintermediaryȱstandardsȱ toȱseparateȱtheȱorganicȱlayer.ȱTheȱtopȱorganicȱlayerȱ
(50ȱ ppm)ȱ ofȱ pesticidesȱ (Tableȱ 1)ȱ wereȱ preparedȱ inȱ ofȱaboutȱ16ȱmLȱwasȱtakenȱintoȱtheȱ50ȱmLȱcentrifugeȱ
calibratedȱ volumetricȱ flasksȱ fromȱ theȱ CRMsȱ usingȱ tubeȱtoȱwhichȱ9ȱ±ȱ0.1ȱgȱanhydrousȱsodiumȱsulphateȱ
GCȱ PRȱ gradeȱ acetoneȱ andȱ hexaneȱ solvents.ȱ Sixȱ wasȱaddedȱtoȱremoveȱtheȱmoistureȱcontent.ȱExtractȱ
calibrationȱ pesticideȱ solutionsȱ (workingȱ standards)ȱ (9ȱmL)ȱwasȱtakenȱinȱtoȱ15ȱmLȱtubeȱcontainingȱ0.4ȱ±ȱ
wereȱpreparedȱinȱtheȱrangeȱofȱ0.01ȱppmȱtoȱ0.5ȱppmȱinȱ 0.1ȱgȱPSAȱsorbentȱ(forȱdispersiveȱsolidȱphaseȱd-SPEȱ
calibratedȱgraduatedȱvolumetricȱflasksȱusingȱdistilledȱ cleanup)ȱ andȱ 1.2ȱ ±ȱ 0.01ȱ gȱ anhydrousȱ magnesiumȱ
n-hexaneȱ asȱ solvent.ȱ Eachȱ concentrationȱ levelȱ wasȱ sulphate,ȱandȱtheȱsampleȱtubeȱwasȱvortexedȱforȱ30ȱ
injectedȱ(1ȱµL)ȱsixȱtimesȱinȱBrukerȱScionȱ436ȱGC-MS/
secȱfollowedȱbyȱcentrifugationȱforȱ5ȱminȱatȱ2500-3000ȱ
MSȱTripleȱQuadrupoleȱDetectorȱ(EI)ȱusingȱMultipleȱ xg.ȱ Theȱ extractȱ ofȱ (2ȱ mL)ȱ wasȱ transferredȱ intoȱ testȱ
Reactionȱ Monitoringȱ (MRM)ȱ methodȱ (Tableȱ 1).ȱ tubesȱandȱevaporatedȱtoȱdrynessȱusingȱconcentrationȱ
Standardsȱwereȱinjectedȱinȱsplitȱmodeȱ(1:ȱ10)ȱatȱ260°Cȱ workȱstationȱ(TurbovapȱLVȱofȱCapilerȱlifeȱsciences)ȱ
injectorȱtempȱatȱcolumnȱflowȱofȱ1ȱml/minȱ(Heliumȱ withȱ nitrogenȱ gasȱ andȱ reconstitutedȱ withȱ 1mLȱ
99.99%ȱpurity)ȱusingȱZebronȱMRȱ2ȱcapillaryȱcolumnȱ n-Hexane:Acetoneȱ (9:1)ȱ forȱ analysis.ȱ Theȱ meanȱ
(30ȱ mmȱ length,ȱ 0.25ȱ mmȱ ID,ȱ 0.20ȱ µmȱ filmȱ coatedȱ recoveryȱofȱtheȱresiduesȱwasȱcalculatedȱtoȱjudgeȱtheȱ
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