CleaningupyoursampleprepprocessesJuliaC.Drees,Ph.D.,DABCCScientificDirector—ChemistryKaiserPermanenteRegionalLaboratoriesBerkeleyandRic
Extractionmethods’relativesimplicity/ro bustnessSPE PPT LLESimpler MoreRobustBestWorstAdaptedfromJudyStone,Ph.D.
Regardlessofextractionmode,dothis: Addinternalstandard(IS)asFIRSTstepinextraction Evaluateforionsuppression: Qualitative(post‐colu
Quantitativecharacterizationofyourextraction:recovery,efficiency,matrixeff ects SampleA:Neatsample(exhibits100%recovery,nomatrixeff
Automation Onlineextraction Automatedliquid handlers Casehistoriesinautomation
OnlineSPEskipsevaporationandreconstitutionstepsSymbiosisproductliterature,Spark‐Holland
Onlineex traction‐ mechanicsSchebb,Anal.Methods 2011;3:4206‐portswitchingvalve
Considerationsforfeasibilityofonlineextraction Evaluatecycletimesofextraction&LCrunwithcare:Isthroughput adequate? Aretwoextr
ConsiderationsforOnlineExtraction‐ AnalyticalIssues # ofsolvents,solventtypesforwash®enerationofSPEcolumn(solventselectvalve
AutomatedLiquidHandling
AdaptedfromJudyStone,Ph.D.
FinancialdisclosuresNothingtodisclose
AutomatedLiquidHandling1,2,4,8,&/or96pipettingchannelsTubesand96wellplatesCandotubetoplate(startingwithpatientsampletube)
ConsiderationsforbestvaluefromALH Disposabletipsvs.washable(consumablecostvs.carryoverrisk,speedofeach?) #ofpipettechannels(>
ConsiderationsforAutomatedLiquidHandling– AnalyticalIssues Minimumandmaximumpipettable volumes(andwithwhatprecision)? Workingwithdiff
Auto mationchoices OnlineextractionVendor“complete”solutionsThermo‐Scientific/CohesiveTechnologiesTLXTurbo‐FlowonlineSPEextractionSpark‐H
OnlineExtractionvs.AutomatedLiquidHandling(ALH)Parameter Online ALHTech handsontime/sample Less MoreLCsophisticationrequired More LessFlexib
AutomatedLiquidHandling–CaseHistoriesSampleprepfor25‐OHVitaminDanalysisbyLC‐MSMSThankstomycolleagueswhohelpedtroubleshoottheseca
1. Transfer 50 µL IS from reservoir to plate (8 tips, pick up & dispense 12 times)IS reservoir2. Transfer serum from tubes to plate (8x12 tips) &a
3. Move plate to 2ndALH (96 channel head) 4. Transfer serum + IS to PPT/phospholipid removal plate5. Transfer precipitating reagent to extraction plat
Case1–TheTouch‐OffTaskPipetserumandinternalstandard(IS)toa96‐wellplateDesiredintra‐assayprecision(withinplate)=<5%CV
DevelopmentHistoryOriginalProtocol:50µLISdispensedintoplate50µLserumadded,mixwithtipsIntra‐assayprecision4‐6%RevisedProt
LearningObjectivesAfterthispresentationyouwillbeableto: Liststrengthsandweaknessesofcommonsamplepreparationtechniquesusedforclinic
Problem:%CV↑to6‐9%whenISvolume↓from50µLto25µLRe‐measurebottomofplate dimensionswcalipers&adjustheightofdispense–notfi
SolutionDispenseserum1stDispenseIS2nd‐ touchofftoserum(liquid)insteadofquestionabletouchoffto(dry)plateIntra‐assay%CVdecreasedt
2. Transfer 50 25 µL IS from reservoir to plate (8 tips, pick up & dispense 12 times) (8x12 tips & mix)IS reservoir1. Transfer serum from tube
Case2– M issing filtratesTaskPrecipitateserum+ISinahybrid(protein&phospholipidremoval)filtrationplate–transferfiltratetocolle
ProblemFirstfewplateslookgoodThenafewwellsineveryplate–nofiltratetransferredtocollectionplateAllotherwe llsinthoseplateshave
InvestigationChangeofplatelot?– notfixedBreakthroughoffiltrationmembraneorquickflowthroughinsurroundingwellscausinginadequatevacu
Eureka!InitialstudiesusedfrozenaliquotsofpooledserumHadrecentlyswitchedtofreshpatientsamplesVendorvalidatedwithfrozenratserumHyp
Solution:crash,wait,then“sip”&transferCrashinplain,conicalbottom,1mL,plateINSTEADofcrashingINSIDEhybridfiltrationplateWait4
3. Move plate to 2ndALH (96 channel head) 5. Wait 4 min for ppt to settle. Transfer supernatant to extraction plate4. Transfer precipitating reagent t
Lessonslearned Givethelowtechsolutionatry Extractionautomationisaboutsurfacechemistry:CharacteristicsofdifferentplasticsSurfacet
CommonSamplePrepOptionsDilutionProteinCrashorUltrafiltration(PPT)Liquid‐Liquidextraction(LLE)SolidPhaseextraction(SPE)–canbeoffline
Case3–staticclingoftipstopipettehead(tipsnotshucked)
SolutionPurc hasetipswithoutplasticfilmwrappingofindividualtipboxe sOnlyre‐usereagenttipsafewtimes(staticbuildsupwithre‐use)Sta
InconclusionAtte ntiontotrivialdetail&persistence wasthekeytosuccessAutomationmadepossibleextractionof2,500samples/daybytwot
Thanksforyourattention
DilutionUsuallyforurinedrugconfirmationsTypically10‐20xdilutionAddafiltrationstepforadded robustness
Proteinprecipitation(PPT)Avoidaddinginternalstandard(IS)toprecipitationsolventIdeallyaratioof3:1precipitant:sampleOptionstoinclude
Liquid‐LiquidExtraction(LLE)BestforneutralanalytesIdeally10:1solvent:sampleMixtureofpolarandnonpolarsolventsMorepolar→morejunkPha
SolidPhaseExtraction(SPE)MostchancesforusingscientificapproachforoptimizationIonexchangeSPEismostselective(chargedanalytes)Washag
ComparingextractionmodesRankordered 1=best,4=worst.*=IonexchangeSPEmodeetc.Differentiator SPE PPT LLE OnlineCharged Anal
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