Bioanalysis of Deltamethrin from GC

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1 Bioanalysis of Deltamethrin from GC/MS on Enhanced ChemStation John Tang, Dr. Michael Bartlett, Dr. Darren Gullick 07/24/2015

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  1. 1. 1 Bioanalysis of Deltamethrin from GC/MS on Enhanced ChemStation John Tang, Dr. Michael Bartlett, Dr. Darren Gullick 07/24/2015
  2. 2. 2 Table of Contents: Procedure 1: CollectingDatafromGC/MS.3 Steps1: LoadingMethod File.....3 Step2: VerifyMethod...3 Procedure 2: Interpretingdata..6 Step1: LoadingGraph...6 Step2: Setup ParametersforIntegration .6 Step3: IntegratingaPeak..8 Step4: ObtainResultsof Integration .10 Step5: OrganizingResultsof Integration..11 Step6: CreatingCalibrationCurve....11 Step7: FindingPercentError.16 Procedure 3: Validation16 References.19
  3. 3. 3 The programs usedinthisprocedure are MSD ChemStationD.0100 Build75 by AgilentTechnologies, GraphPadPrismVersion3.03, and MicrosoftExcel 2007. The purpose of the procedure istomeasure Deltamethrinaspartof a pyrethroidsprojectsponsoredby CAPHRA,Council forthe Advancementof PyrethroidHumanRiskAssessment.The projectistotest presence of pyrethroidsandtheirneurotoxicityindifferentrattissue withdifference circumstances. Deltamethrin appearswith twopeakswhenreadthroughthe GC/MS.Because of this, the automatic quantitationfunctioninChemStationcannotbe usedandthusmanual quantitationmethodisrequired. Thisreportseeksto listoutthe stepstakento integrate the twopeaksand ultimately determinepercent error throughMicrosoftExcel. Procedure 1: CollectingData from GC/MS Step1: LoadingMethodFile FindMethodindrop downmenus.Itis the firstoptiononthe top menu. Method> Load The methodshowninthisreportis DLM CP NOV 2014.M. Step2: VerifyMethod Method> EditEntire Method Hit OK forthe firsttwowindowsthatcome up.The nextwindow (Figure 1) thatappearsisInletand InjectionParameters.Make sure the Sample InletisGC,InjectionSourceisExternal Device, InjectionLocationisFront,andthe Use MS optionischecked. HitOK. Thiswill bringupa windowwithInstrument| Edit| Inlets:shown onthe followingpage.There are fouraspectsto thiswindowthatare important:Inlet,Columns,Oven,andthe OvenTempgraph. Picturesof eachare shownand shouldmatch Figures2,3 and 4. The numbershave beenoptimizedto maximize the GCperformance. Figure 1: InletandInjectionParametersWindow
  4. 4. 4 Figure 2: Inletssubwindowinthe EditInstrumentWindow Figure 3: Columnssub windowinthe EditInstruments Window Figure 4: Ovenand OvenTempgraphin EditInstrumentWindow
  5. 5. 5 ThenhitOK for the nextwindow.The nextwindowislabeledMSTune File.In thisexperiment,the file ncich4.uwasused.ClickOKfor the nexttwowindows.Forthe thirdmenu,the programistrying to finda printerto print the information.Becausedeltamethrinresultsneedtobe manuallyquantitated, any optionisfine. ClickSelect.Thenthe programgivesthe window labeledSave MethodAs.If thisis justa verificationof method,nothingneedstobe changedandit isrecommendedtosimplycancel out of the windowanddontsave.If the methodfile name isnt changed,the changesdone tothe methods will overwritethe previousmethod.If the methodfilename ischanged,thenitisokayto justsave but knowa newfile withwhateverchangesmade willbe creates. In the lab,a PAL auto samplersystemwasusedwhichworksexternallyandindependentlyfromthe GC. The purpose of the PALsystemisnecessarytorun multiple sampleswithouthavingtomanuallyinject samplesintothe inlet.The methodof the PALautosamplerusedinthe procedure waslabeled under MethodNameas DG Dir Inj Pyrethroids(Figure 5) andmustbe consistentwiththe sequence used for the EnhancedChemStation.The systeminjects1.5 L of the sample torun inthe GC. The GC isnow readyto use. Figure 5: PAL MethodFile Settings
  6. 6. 6 Procedure 2: Interpretingdata Step1: LoadingGraph Load up EnhancedData Analysis (Figure 6).Thiswill bringupadifferentwindow thanthe previous,but isstill EnhancedChemStation. File > Load data file Thiswill bringupthe windowinFigure 7.Path showsfile director. The box below Path showsdifferent file namessavedinside thatfolder.Clickinganoptionhasa preview of the graphas well assome simple information. Generally,aLOQ or LLOQ shouldbe chosenfirstbecause those peakscouldbe more difficulttosee withinthe backgroundnoise. Step2: Setup ParametersforIntegration FindChromatograminthe dropdownmenus Chromatogram> SelectIntegrator>RTE Integrator Chromatogram> MS Signal IntegrationParameters Thiswill bringupa windowlabeledRTEIntegratorParameters showninFigure 8.Most of thiswindow can stay default.The aspectsthatare generallychanged inordertocreate a more accurate integration Figure 6: Icon forEnhancedData Analysis Figure 7: Data File Window
  7. 7. 7 are Start threshold,Maximumnumberof peaks,andBaseline Preference.Note thatchangesin the parametersmustremainconsistentthroughoutadata setexceptforMaximumnumberof peaks. The followingisafewnotesof whateach optionsfunction is: Start thresholdcanbe loweredtomake the peakstobe identifiedbythe program.The thresholdis mostimportantwhenthe programis unable tointegrate the smallerpeaksfromthe backgroundnoise because of howclose the baseline istothe peak.Thisnumberisa 0.200 default,butcanbe changed from0.200 to 0.001. Baseline Preference (Figure 9) isrelevantdependingonthe slopesof the graphespeciallyif thereare twoor more peaks.The baseline isthe generallineof where the backgroundnoisefluctuatesaround. WhenusingBaseline dropelse Tangent,the integrate functionwill draw aline fromthe baseline where the peakstartsexactly horizontallytowardsthe nextpeakorthe baseline.Whenencountering anotherpeak,the otherpeakwill be consideredseparate whenintegratingsothe areaof integration will endwithastraightline upwardstowhere the peaksmeetandseparate the areaswiththe basisthat the area of frombelowthe peakall the wayto the baseline.The TangentelseBaselinedrop option functionsdifferently.Insteadof assumingwhere the baseline iscreatingintegrationoff of the peak above the baseline,the programsimplydrawsabestfitline fromwhere the peakisstartingtowhere it stops.Thismeansthat these integration numbersare lessthanthe Baselinedropelse Tangent. Figure 8: RTE IntegratorParameters
  8. 8. 8 Maximumnumberof peaksisresourceful whenthe backgroundnoise isinterferingwiththe signalof the peaksdesired.Thisfunctiondoesnotchange anythingintermsof the functionof integration,but insteadshowsmore peaks.Thisiswhythisaspectcan be changedbetweengraphsof the same data set. Step3: IntegratingaPeak A fewnotesonhowto maneuveraroundthe graph. A left-click-hold-and-dragwill zoominonthe box created. A double leftclickwill zoomoutthe graphto the previouszoomview used. Rightclickcan be chosento be eitherManual Integration orMS graph view (Figure 9).Thisisdone by findingthe Toolsdropdownmenu.Tools> Optionswill bringupaSelectDA Optionsand here Manual Integrationcanbe checkedto change the rightclick,or unselectedtoshow aMS graph of whereverselectedonthe graph. Whenusingthe Manual integration,a right-click-hold-and-drag betweenlineswill findthe areaof the curve above towardsthe peakwhere the line thatwascreatedis. Whennot usingManual integration,avertical line will presidewhere the mousecursoris.A double- right-clickbringsupa graphunderneaththe initial graphtoshow the Mass Spectroscopypoints (Figure 10). Figure 9: Tangentelse BaselinedropVersusBaselinedropelse Tangent Tangentelse Baselinedrop Baseline dropelse Tangent
  9. 9. 9 Figure 10: Right-ClickOptionsFunctions Mass SpectroscopyandGC Graph Example of Manual integration (Line wasdrawnmanually)
  10. 10. 10 To integrate apeak,simplyuse the integrate button.Thisbuttonisshown inFigure 11highlightedin red. Note that thiscan alsobe done throughthe Chromatogramdropdownmenu. Chromatogram> Integrate It is possible thatinthe firstintegrationof lowerconcentrationgraphsmaynotresultinidentificationof the peaksdesired.If thisoccurs,referbackto Step2 to go back intothe parametersof integrationto change the Start ThresholdorMaximumNumberof Peaks.Make sure that if Start Thresholdis changed.Do notchange againwhile usingthe same setof tests. Forexample,the settingsusedforthe CalibrationandQCshouldalsobe usedfor the validationof samples. Step4: ObtainResultsof Integration If integrationof the peakissuccessful,the areaof specificpeaksisneededforfuture calculations.Thisis done throughthe Chromatogramdrop downmenu. Anexample of the formattingforthis information isshownin Table 1. Chromatogram> Integrate Results Figure 11: Integrate Button Table 1: Example of PeakIntegrationResults
  11. 11. 11 Thisinformationisto be copiedintoMicrosoftExcel foreach concentrationpoint. Step5: OrganizingResultsof Integration Switchback andforth fromEnhanced ChemStationandMicrosoftExcel totransferoverthe integration resultslike shownabove.Onlythe peaksdesiredshouldbe kept. Thesepeaksare the Cis-permethrin, and the twodeltamethrinpeaks. Delete the linesof peakswhichwere pickedupbythe programthat are notthe desiredpeaks. Forexample,if the maximumnumberof peaksissetto4, the program will find4 peaksno matterwhat.There are onlythree desiredpeaksthough,butthe otherpeakwill be automaticallyselectedandneedstobe deleted. Anexampleof how adata set lookslike isshown in Table 1. Step6: CreatingCalibrationCurve To create a calibrationcurve,achart of data needstobe made.Anexample of thischartis shown in Table 3 withthe calibrationgraph.Thischart contains concentrationlevel,response ratio(RR),the cis- permethrinpeakarea,deltamethrin1and 2 peakareas,the calculatedng/mL,andthe calculated percenterror. The response ratio,ng/mL,andpercenterrorare all calculatedthroughexcel. The response ratioiscalculatedwiththe equation: (+2) . Thatis cis-permethrinpeakareadivided by the sumof the deltamethrinpeakareas. Thisequationcanbe formattedintoExcel forconvenience. Aftercalculatingthe RR,the calibrationcurve isneededtoobtainthe y-interceptandslope tocalculate ng/mLand percenterror.To calculate a weighted curve,GraphpadPrismwasusedinorder give the curve a weightof 1 2 . Weighted Calibration Curvein Graphpad Prism AfteropeningupPrism (Figure 12),starta new project. File > NewProject Figure 12: PrismIcon
  12. 12. 12 The settingscan be leftasdefault.Thiswill bringupablankdata table. Take the firsttwo columnsof the excel chart,whichshouldbe the concentrationspointsandthe RR,andcopy themontothe data table on the firsttwocolumns. Make sure to omitthe zeroconcentrationasthiswill interfere withthe weightingfeature of thisprogram. Table 2 showshow the data shouldbe formatted. NextfindAnalyzeina small box above the mainscreenandclickon it (highlightedinredinFigure 13). Thiswill bringupa windowlabeledAnalyzeData. In the Type:box,selectCurves&RegressionandselectNonlinearregression(curve fit)inthe box immediatelytothe right.All otheroptionscanstaydefault.AfterclickingOkay,anew screenlabeled Parameters:Nonlinearregressionwill appearlike inFigure 14.Inthe Choose anequationbox find the Polynomial:FirstOrder(straightline)option.NextinOptionsunderneaththe previousbox,find Table 2: PrismData Table Figure 13: PrismAnalyze Button
  13. 13. 13 the Method button.Whenclickingonthisbutton,the programwill give options foraWeighting methodalsoshowninFigure 13. SelectWeightby1/2, thenclickOK. Thiswill bringyouto yourresultspage showninFigure 15. Copyall of the valuesfromthisresultspage intothe Excel page. The graph can also beenseen inthe side toolbarbyclickingonthe Graphsfolder and selectingthe file withthe graph (Figure 16).Inthe results,the valuesdesiredare the bestfitvalues. A representsthe y-interceptandB representsthe slope. Figure 14: WindowforNonlinearregression (left)andMethodswindow toweighdata (right).
  14. 14. 14 Figure 15: PrismResultsPage Figure 16: PrismGraph Page
  15. 15. 15 Unweighted Calibration Curvein MicrosoftExcel An unweightcalibrationcurve (Table3) can be createdin Microsoft Excel easilytogive ageneral ideaof the plotof where pointslie.The unweightedcurve inExcel ismore influencedbyhigherXvaluesmaking it lessaccurate. To create thiscurve,select the firsttwocolumnslike beforeexceptthe zerovalue can be selected.Gotothe Inserttab of the menuandselectScatterinthe groupof Charts.When clickingScattera drop downof a fewscatterplotcharts will appear.Selectthe firstone graph.This will create ascatteredplotof the differentdatapointsselected.Highlightone of these datapointsand thenright-clickthe pointinordertobringup a drop downmenu.SelectAddTrendlinetobringup the Format Trendlinewindow.Select Linearandlooktowardsthe bottomat the final three options. Checkthe final twooptionslabeledDisplayEquationonchart andDisplayR-squaredvalue onchart. ThenhitClose.Thiswill complete the unweightedcalibrationcurve forMicrosoftExcel.Forthis experiment,these valuesare notused. The unweightedcurve made inExcel issimplyto reference to see where the pointslie onthe graph. HighlightDataPoints> Insert> Scatter > FirstGraph > Right-ClickaData Point> AddTrendline >Linear> DisplayEquationonchart and DisplayR-squaredvalueonchart Table 3: Excel Cal and QC Graph andChart
  16. 16. 16 Step7: FindingPercentError The ng/mL iscalculatedthrough usingthe response ratio,y-intercept,andslope (Table4).The equation is = () . In Excel,these calculationscanbe done easilybysettingupanequation.Inorder to make Excel pull fromthe same cell whenworkingwithmore thanone setof points,use the $ sign inbetweenthe rowandcolumnvalue. To calculate percenterror,the ng/mLvalue isneeded.The equationis = / / / 100% The / value inthe equationrepresentsthe concentrationvalue inthe columnbefore RR. Thisconcentrationvalue isthe expectedvalue tobe measuredfromthe sample.Thisequation comparesthe percentdifference betweenthe ng/mLmeasuredandthe ng/mLthatis expected.Referto Table 3 fora viewof a completedCal andQCtable. Perthe Universityof GeorgiaPhysical Pharmacy requirements,the percenterrorhastobe lessthan 25% for the resultstobe consideredviable.If anon-range definingpointhasa percenterrorover25%, the pointcan be takenoutand the overall percenterrorcan be recalculatedwithoutthe point. Procedure 3: Validation Afterrunninga Cal and QC,validationof specificconcentrationsforsamples isnecessary. Validation informationisaverysimilartorunningCal and QC exceptthere are more factorsto calculate thanjust percenterrorand ng/mL.A validationrunwill generallyinclude fourgroupsof concentrationpoints: LOQ, LQC, MQC, and HQC. Each of these groupshas six samples,fourof whichhave topass.Validation requiresdifferentfactorstopass.It adds an average ng/mL,a standarddeviation,accuracy,anda precisionfactor.Accuracyand precision bothhave tobe lessthan25% to pass the criteriaasacceptable. Calculating avg ng Take the average of the entire ng/mLcolumn.Thiscan be done easilyinExcel withthe AVERAGE functionandhighlightingthe portionof the column. Table 4: Findingng/mLfromRR,y-interceptandslope
  17. 17. 17 Calculating stdev Take the standarddeviationof the entire ng/mLcolumn.Thiscanbe done easilyinExcel withthe STDEV function. Calculating accuracy Take the average of the %error column.Again,thiscanbe done withthe AVERAGEfunctionand highlightingthe portionof the column. Calculating precision Precisioniscalculatedbyusingthe stdevandavgng. The formula: = 100%
  18. 18. 18 Table 5: Example of aBlankValidationTable
  19. 19. 19 References: All informationwastaught byDr. DarrenGullick,anddictatedandreported byJohnTang. Resources providedbyDr.Michael Bartlettin the Universityof GeorgiaCollege of Pharmacy. All figures,tables,and pictureswere takenonthe GC1 computerinthe Physical Pharmacylabat the Universityof Georgia, College of Pharmacy.