Simplicity 1690843 Manuel d'utilisateur

CleaningupyoursampleprepprocessesJuliaC.Drees,Ph.D.,DABCCScientificDirector—ChemistryKaiserPermanenteRegionalLaboratoriesBerkeleyandRic

Extractionmethods’relativesimplicity/ro bustnessSPE PPT LLESimpler MoreRobustBestWorstAdaptedfromJudyStone,Ph.D.

Regardlessofextractionmode,dothis: Addinternalstandard(IS)asFIRSTstepinextraction Evaluateforionsuppression: Qualitative(post‐colu

Quantitativecharacterizationofyourextraction:recovery,efficiency,matrixeff ects SampleA:Neatsample(exhibits100%recovery,nomatrixeff

Automation Onlineextraction Automatedliquid handlers Casehistoriesinautomation

OnlineSPEskipsevaporationandreconstitutionstepsSymbiosisproductliterature,Spark‐Holland

Onlineex traction‐ mechanicsSchebb,Anal.Methods 2011;3:4206‐portswitchingvalve

Considerationsforfeasibilityofonlineextraction Evaluatecycletimesofextraction&LCrunwithcare:Isthroughput adequate? Aretwoextr

ConsiderationsforOnlineExtraction‐ AnalyticalIssues # ofsolvents,solventtypesforwash&regenerationofSPEcolumn(solventselectvalve

AutomatedLiquidHandling

AdaptedfromJudyStone,Ph.D.

FinancialdisclosuresNothingtodisclose

AutomatedLiquidHandling1,2,4,8,&/or96pipettingchannelsTubesand96wellplatesCandotubetoplate(startingwithpatientsampletube)

ConsiderationsforbestvaluefromALH Disposabletipsvs.washable(consumablecostvs.carryoverrisk,speedofeach?) #ofpipettechannels(>

ConsiderationsforAutomatedLiquidHandling– AnalyticalIssues Minimumandmaximumpipettable volumes(andwithwhatprecision)? Workingwithdiff

Auto mationchoices OnlineextractionVendor“complete”solutionsThermo‐Scientific/CohesiveTechnologiesTLXTurbo‐FlowonlineSPEextractionSpark‐H

OnlineExtractionvs.AutomatedLiquidHandling(ALH)Parameter Online ALHTech handsontime/sample Less MoreLCsophisticationrequired More LessFlexib

AutomatedLiquidHandling–CaseHistoriesSampleprepfor25‐OHVitaminDanalysisbyLC‐MSMSThankstomycolleagueswhohelpedtroubleshoottheseca

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

Case1–TheTouch‐OffTaskPipetserumandinternalstandard(IS)toa96‐wellplateDesiredintra‐assayprecision(withinplate)=<5%CV

DevelopmentHistoryOriginalProtocol:50µLISdispensedintoplate50µLserumadded,mixwithtipsIntra‐assayprecision4‐6%RevisedProt

LearningObjectivesAfterthispresentationyouwillbeableto: Liststrengthsandweaknessesofcommonsamplepreparationtechniquesusedforclinic

Problem:%CV↑to6‐9%whenISvolume↓from50µLto25µLRe‐measurebottomofplate dimensionswcalipers&adjustheightofdispense–notfi

SolutionDispenseserum1stDispenseIS2nd‐ touchofftoserum(liquid)insteadofquestionabletouchoffto(dry)plateIntra‐assay%CVdecreasedt

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

Case2– M issing filtratesTaskPrecipitateserum+ISinahybrid(protein&phospholipidremoval)filtrationplate–transferfiltratetocolle

ProblemFirstfewplateslookgoodThenafewwellsineveryplate–nofiltratetransferredtocollectionplateAllotherwe llsinthoseplateshave

InvestigationChangeofplatelot?– notfixedBreakthroughoffiltrationmembraneorquickflowthroughinsurroundingwellscausinginadequatevacu

Eureka!InitialstudiesusedfrozenaliquotsofpooledserumHadrecentlyswitchedtofreshpatientsamplesVendorvalidatedwithfrozenratserumHyp

Solution:crash,wait,then“sip”&transferCrashinplain,conicalbottom,1mL,plateINSTEADofcrashingINSIDEhybridfiltrationplateWait4

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

Lessonslearned Givethelowtechsolutionatry Extractionautomationisaboutsurfacechemistry:CharacteristicsofdifferentplasticsSurfacet

CommonSamplePrepOptionsDilutionProteinCrashorUltrafiltration(PPT)Liquid‐Liquidextraction(LLE)SolidPhaseextraction(SPE)–canbeoffline

Case3–staticclingoftipstopipettehead(tipsnotshucked)

SolutionPurc hasetipswithoutplasticfilmwrappingofindividualtipboxe sOnlyre‐usereagenttipsafewtimes(staticbuildsupwithre‐use)Sta

InconclusionAtte ntiontotrivialdetail&persistence wasthekeytosuccessAutomationmadepossibleextractionof2,500samples/daybytwot

Thanksforyourattention

DilutionUsuallyforurinedrugconfirmationsTypically10‐20xdilutionAddafiltrationstepforadded robustness

Proteinprecipitation(PPT)Avoidaddinginternalstandard(IS)toprecipitationsolventIdeallyaratioof3:1precipitant:sampleOptionstoinclude

Liquid‐LiquidExtraction(LLE)BestforneutralanalytesIdeally10:1solvent:sampleMixtureofpolarandnonpolarsolventsMorepolar→morejunkPha

SolidPhaseExtraction(SPE)MostchancesforusingscientificapproachforoptimizationIonexchangeSPEismostselective(chargedanalytes)Washag

ComparingextractionmodesRankordered 1=best,4=worst.*=IonexchangeSPEmodeetc.Differentiator SPE PPT LLE OnlineCharged Anal