1. PainManagement&
DrugsofAbuse
MeasuredinUrine
and/orOralFluid
Measuring all the drugs at
all the relevant concentrations in a
single unified method / workflow using
an ITSP solution for on-line
SPE-LC/MS/MS
Developed in collaboration with
Assurance Scientific Laboratories
Instrument Top Sample Prep
Aconsumablesolutionforautomationand
thebestmeasurementperformance!
71+Drugs, 4.5 MinutesInject-to-Inject,
Two 96-position traysperinstrumentovernight

2. Themeasurementofdrugsofabuseinurineand/ororalfluid
iscommonforpre-employmentscreeningandDOT/federally
mandatedtesting.However,themostrapidexpansionoftest-
inghasbeeninthefieldsoflawenforcementandcompliance
monitoring/diagnosticevaluationbyphysicians.Althoughmany
methodsareavailableforthesemeasurements,thefastestgrow-
ingandpreferredapproachisliquidchromatographytandemmass
spectrometry(LC/MS/MS)becauseofthehighdegreeofcertainty
itaffordsdeterminingconcentrationandidentificationofcom-
pounds. WhilethecontinuedgrowthoftheuseofLC/MS/MSfor
themeasurementofdrugsofabuseinurineandoralfluidseems
certain,therearestillseveraltechnicalchallengesthatneedtobe
met. Theseneedsincludebeingabletoeasilymeasurelow-dose
drugsatornear1ng/gconcentration(formedicalpurposes,Pesce,
et.al.2012AACCconferenceandzerotolerancetesting),simplicity
forperformingmeasurementsbylabtechnicianswithrelatively
littletraining,andtheabilitytoachievehighthroughputforallwork
whileminimizingthelaborandnumberofworkflowsrequired.
Inanefforttomeettheseneeds,InstrumentTopSamplePrep
Solutions,Inc.(ITSP)alongwithAssuranceScientificLaboratories
hasdevelopedtheautomatedon-lineITSP-LC/MS/MSmethod
describedhere. ThisITSPsolutionuseson-lineSolidPhase
Extraction(SPE)tocleanandpre-concentrateurineororalfluid
(usingtheQuantisalOFsamplecollectiondevice)samplesso
thatlow-dosedrugsatornear1ng/gconcentrationareeasily
measuredatS/N≥20. Atthesametime,themethod’sdesignis
balancedtoidentifyandmeasureallofthedrugs(acidicandbasic
drugsaswellaspolarandnon-polardrugsinurineororalfluid)in
oneITSPSPEmethod,allinoneunifiedLC/MS/MSworkflow. Itis
simple,robust,andcanbeperformedbylabtechnicianswithMS
familiarity.Itiscompletelyautomatedfromsampleplatesorvials
toresults(usingthenativeMSsoftware)andcanprocesstwo
96-wellplatesofsamplesovernightperLC/MS/MS. Theresults
willbewaitingforyouinthemorning.
TotalautomationisachievedusingthePALSystemLCsample
handler(themostcommonlyusedautosamplerforGC/MS/MS
andLC/MS/MSworldwide). Sinceonemustinvestinan
autosamplerasaprerequisitetoLC/MS/MS,werecommend
choosingonethatcanpreparethesampleaswellasinjectit.
Thecycletimeachievedforon-lineITSP-LC/MS/MSis4.5minutes
for71drugsinurine(opiates,metabolites,illicits,opioids,barbs,
benzos,andTHCA)andarepresentative4.1minutechromato-
gramisshownbelow.
Introduction
Figure 1: Total automation of the ITSP Solid Phase Extraction then analysis by LC/MS/MS (ITSP-LC/MS/MS) is routinely performed in the clinical
production environment for 71 drugs (basic and acidic) in one injection in 4.5 minutes, injection to injection. Additional compounds can be added or
removed based on the individual needs of the lab.

3. SamplePreparation-ITSPReverse Phase SPE Conditions
Sample preparation
Oralfluidsamplesrequireeithercentrifugationorfiltering
(Quantisalfilter)andonoccasion,urinesamplesmayrequirethis
too. Allstandardsandtheirisotopicallylabeledinternalstandards
maybeobtainedfromCerilliant. Standardsofthedrugsshould
bepreparedinurine(ororalfluid)atappropriateconcentrations
acrosstheanalyticalrange. Isotopicallylabeledinternalstandards
alsoshouldbeaddedtothesample.ß-Glucuronidase(5000
FishmanUnits)in1MpH4.5buffershouldbeaddedtoeachurine
sample(notoralfluid)andheatedat60°Cfor3hoursusingthese
steps:
Step-by-Step Process
1)Toeachwellorvial(exceptforthosecontainingdouble
blanks)inthe96-wellplate(choosecarefullytooptimize
heating,volume,andadsorptionproperties),add25μl
(per200μlsample)internalstandardworkingsolution
(Concentration:0.1-1ng/μl)
2)Toeachwell/vial,addanaliquot(200-1200μL)oftheappli-
cablesample(i.e.,patientspecimen,standard,QCorblank)
3)Toeachwell/vial,add25μl(per200μlsample)
ß-Glucuronidasein0.1MpH4.5NH4OAcbuffer
4)Sealplates/vials(heat-sealedfoilpreferredonplates)
5)Mixgentlyandheatat60˚Cforapproximately3hours
6)Alternatively,consideruaingprifiedrecombinentß-Gluc-
uronidaseat55˚Cfor30minutes.
7)Centrifugeforapproximately5minutesatapproximately
2000g
ITSP – SPE Solvent Preparation
Wash Solvent: MeOH(Honeywellpartno.:230-4(VWR))with
0.2%NH4OAc.
Conditioning Buffer: traceanalyticalgradewaterfreshfrom
aMilliporeIntegralWaterPurificationSystemw/BioPak(or
equivalent)bufferedwith20%solidNH4OAc(atomicanalysis
grade,Aldrichpartno.:372331-100G).Donotusebufferfrom
ß-Glucuronidasestep3above.
Buffered Elution Solvent: MeOHwitheither2%HOAc
(atomicanalysisgrade,Flukano.:07692)whenusingabiphenyl
LCcolumnor0.2%NH4OAc(atomicanalysisgrade,Aldrichpart
no:372331-100G)whenusingaC18 LCcolumn(SeetheLC
injectionnotesintheLCmethodbelow).
The ITSP Solution for RP-SPE Sample Preparation
EachsampleispreparedusingSPEindividuallyforLC/MS/MS
analysis. Thisprocessisperformedforeachwellorvial(steps
7-15continuedinthenextcolumn)beforemovingtothenextwell
orvial. Themethodoperatesconcurrentlywithallsuccessive
LC/MS/MSanalyseswithinalist,inparallel,asshowninFigure2.
Cleansamplesareelutedinto96-wellplatesor2mlvialsandthen
injectedbythePALSystemintotheLC/MS/MS.
Reverse Phase ITSP SPE Cartridges
ITSPProductNumber:10S-UC18EC-T-UnitedChemical
Technologies(UCT),end-cappedC18,10mg,96/Tray
Continued Step-by-Step Process
7) BuffertheurineororalfluidusingtheConditioningBuffer
byaddingavolumeequalto10%oftheurinesamplevolume
andmix(typically25µLaddedto250µLurineor100µladded
to1000µloralfluidfromtheQuantisalsamplingdevice,2%
finalconcentration).SufficientNH4OAcconcentrationis
requiredtoachievehighrecoveriesforopiates.Thiscanbe
performedbythePALSystemormanuallyaddedtoplate
justpriortoSPEcleanup(resealplateifdonemanually).
8) WashITSPcartridgewith100μlWashSolvent
9) ConditionITSPcartridgewith100μlConditioningBuffer
[steps8-9canbecombinedusingtheDLWoption]
10) Load200μLofurineat5μl/sontoITSPcartridge(match
volumetosensitivityneeds:250μlperfectformostTriple
Quadrupoles(QQQs), 100μlcanbeusedwithtopend
QQQs,upto400μlcanbeusedwitholderQQQs,seeFigure
3onfollowingpage)Alternatively,load1000μLoforalfluidat
5μl/sontoITSPcartridge
11) Washthecartridgewith100μlConditioningBuffer
12) Eluteat5μl/swith75μlBufferedElutionSolventinto
well/vial
13) Mixelutionthoroughlywithsyringe(5xat50%volume
[minimum]at20μl/s)
14) InjectontoLC/MS/MSsystemforanalysis(>3xoverfill
recommendedforprecision;seeLCmethodnotesbelow)
15) RinsesyringeandLCinjectionvalvewithACN/formicacid
(99/1)andwater/formic(99/1),cleaningshouldconsistof
atleast3fullsyringevolumerinsesand>1mleachpushed
throughtheLCinjectionvalve,thenprepare(SPE)next
sampleinparalleltotheongoingLC/MS/MSanalysis.
ITSPSPE1
ITSPSPE2
ITSPSPE3
LC/MS/MS1
LC/MS/MS2
4.5minutes 4.5minutes 4.5minutes
Figure 2: An ITSP solution uses the analytcal instrument’s look-ahead
feature embedded into the LC/MS/MS operating software to prepare
samples while the instrument is analyzing the prior sample. Notice how
ITSP elutes the sample just-in-time for injection.
ITSP-LC/MS/MSRuntime

4. 0
0.5
1
0 200 400 600 800 1000
MDMA
Fentanyl
Amphetamine
Diazepam
Oxymorphone
Pentobarbital
THCA
Recommended Work Flow:
Presumelowconcentrations. Fortheinitialsampleexamination,
loadalargeenoughsamplevolumetoeasilyhitallstatedcut-offs
foralldrugs. Reviewdata. Inthefewcaseswherehighdose
drugsaredetected(exceedingtheupperend[+20%]ofthevalid
LC/MS/MSmethodrange),thesamesamplescanbererunusing
smallvolumeloading(25μldilutedto250μlwithConditioningBuf-
ferand100μlloadedoncartridgeusingthePALSystem)tobring
theresponseintothevalidmeasurementrange(whereneeded).
Thisapproachisdesirableintheclinicallabtoavoidtheintroduc-
tionoferrorthroughmanualdilution. Also,where
desirable,thePALSystemcanbeusedforaddinginternal
standardtotheselower-volumesamples.
PALSystemConfiguration:DLW2Optionwith500µlFEP
sampleloopand22gneedleforurineonlyor1000µlFEPsample
loopand22gneedlefororalfluidandurinesamples.
ThefollowingfigureshowstheLC/MS/MSdrugresponseasa
functionofurinesamplevolume. Thelinearresponserangefor
71painmanagementdrugs(drugsandISsspiked500ng/g)is
100to500μldeliveredwitha500μlsyringe.
Notethatcartridgecapacityhasnotbeencompletely
depletedat500μlofurine(benzos,opioids,andTHCAcontinue
tobeadsorbedbeyond500μl).This,inpart,canbemitigatedby
usinglowerconcentrationinternalstandardsforbenzos/opioids
and/orincreasingtheamount(concentration)ofNH2
,OACadded
tosample.Thisalsomaybeanimportantconsiderationifitis
desirabletoincreasethenumberofdrugsto≥100. Ifoneaims
tomeasure≥100drugs,thenuseofthe30mgITSPcartridge
(Productnumber:30S-UC18EC-T)with100μlelutionBuffered
ElutionSolventisrecommended. Thissameapproach(30mg
ITSPcartridge)alsocanbeusedfortracedruganalysis(<1ng/g)
whenloading400-1200μlurine.
SampleloadinginITSPSPE:
Theidealwaytoadjustassay sensitivityvia sample amount
Linear Range
Current
LC/MS/MS
Systems
Older
LC/MS/MS
Systems
Opioidsandbenzosare
mostoptimizedbydesign
(recoveries>90%)
NormalizedLC/MS/MSresponse
Volume(µl)ofurineloadedonITSPSPEcartridge
Figure3:Thisillustrationshowshowthesameanalyticalmethodcanbeusedarossavarietyofinstrumentsensitivityranges.ByadjustingthesampleloadontoITSP
SPEcartridges,lesssensitiveinstrumentscanachieveexcellentanalysisofeventhemostdifficultsamples.
MDMA
Fentanyl
Amphetamine
Diazepam
Oxymorphone
Pentobarbital
THCA
10008006004002000
0
0.5
1

5. Mobile Phases:
A–TraceanalyticalgradewaterfreshfromaMilliporeIntegral
WaterPurificationSystemw/BioPak(orequivalent)buffered
with1%HOAc(atomicanalysisgrade,Flukaproduct
no.:07692)
B–ACN(LC/MSgrade,Honeywellproductno.:015-4)
Column:
GLSciencesInertsilC18 ODS3,3µmparticles,2.1x50mmorsame
sizebiphenylcolumn(RestekRaptor)heldat50°Cusingaheat
exchangerincolumnoventopreheatmobilephase.
Gradient (1 ml/min):
Time= 0.00: 97%A,3%B(start)
Time= 0.05: 97%A,3%B(hold)
Time= 0.60: 80%A,20%B(lineargradient)
Time= 3.00: 40%A,60%B(lineargradient)
Time= 3.20: 5%A,95%B(lineargradient)
Time= 3.35: 5%A,95%B(hold)
Time= 3.36: 100%B(columncleanup)
Time= 3.50: 100%B(columncleanup)
Time= 3.51: 97%A,3%B(columnconditioning)
Time= 4.00: 97%A,3%B(columnconditioning)
Dependingonthedrugsmeasured,gradienttransitionpointsand/
orrampratesmayhavetobeadjustedtoseparateisobaricdrugs
and/ortoachieveatleast16datapointsacrosseachLCpeak.
These(andallLC/MS/MSparameters)shouldbeoptimized,fully
functional,andmaderoutinebasedonsolvent/standards-only
solutionspriortoproceedingwithon-lineSPEwithITSP.
LC/MS/MSconditions tocoverawide range ofdrugsafterITSP RP/SPE
Sample Injection by PAL System Relative to
Mass Spec Sensititvity
1)Fillvalve/loopwithtraceanalyticalgradewaterprior
toinjection.
2)Inallcases,apre-cutandpolishedSSloopisused.
3)UsingordinaryLC/MS/MSs: 5μlloopusing>3xoverfill.
Ifpeakshapeand/orretentionareinsufficientforearly
elutingpeaks,checkbufferinginSPEeluent(seeFigure4
below).
4)Usingtop-endLC/MS/MSs: 1-2μlloopusing>3xoverfill
(usenarrowerLCpeakstoimprovespeedand/or
separation).
5)Injectionvolumeshouldbeheldtoamaximumof5μl
whenusinga2.1mmdiameterLCcolumn. Largervolumes
increasepeakareaprimarilyinwidth,notheight,andthus
deterioratetheLCseparationwithlittle,ifany,gainin
sensitivity. AlwaysoperateLC/MS/MSatoptimalconditions.
6) Chemical presentation of the sample from ITSP SPE to
the LC is important. In LC analysis, control of the pH
(ionization state) controls peak shape (see Figure 4).
Elution in 80% ACN limits LC injection volume to ~2µL
(2.1 x 50mm column). Elution in 100% MeOH (buffered)
allows 5µL LC injection. Viscosity has an equally impor-
tant role in LC injection along with pH.
MS/MS Conditions:
UsealltheusualMRMsforalldrugs. BarbituratesandTHCAuse
(–)ionMRMs(Figure5),allothers(+)ion.
Figure 4: This chromatogram illustrates two ITSP SPE elutions, one using
a buffer and the second without.
Pentobarbital
Secobarbital
THCA
Figure 5: This chromatogram illustrates that YES, acidic drugs can be
measured by LC conditions used for basic drugs.

6. ITSPusesPALSystemshardware,themostcommonlyused
autosamplerforGC/MS/MSandLC/MS/MSworldwide,andisfully
abletoseamlesslyintegrateintoallLC/MS/MSand
GC/MS/MSsoftwaresystems.
WhereassomeITSPtemplates(providedatnocostwiththe
PAL-xthardwarekit)arestandardscriptssuppliedwithnewPAL
RTCSystems,ahighdegreeofcustomizationispossible,includ-
ingbarcodereading(chainofcustody),allsampleformats
(96&384wellplates/vials/12x75mmtubes),filtration,and
multidimensionalSPE.
The PAL RTC
TheCTCAnalyticsPALRTC(Figure7)canchangebetweenupto
sixdifferentsyringeoptions(1µlto10ml)automatically.The
DilutorToolspeedsuptheconditioning/elutionsteps.
ThisRTCisshownwithasinglelocationfor96ITSPSPE
samples.ThereisamplespaceontherailtoaddadditionalITSP
stationstoexpandthenumberofsamplespersamplerun.
ITSPSolutions,Inc.SPEsamplepreparationwasusedto
introducethisplatformatASMSin2013.AllITSPobjectsand
templatemethodsareembeddedand/orprovidedbyCTC
AnalyticsValue-AddedResellers(VARs)andbymanymajor
analyticalinstrumentmanufacturers(OEMs)globally.
The PAL-xt with DLW2 Option
TheCTCAnalyticsPAL-xtSystems(Figure8)arethemost-used
autosamplersinthemarket.Theflexibility,precisionanddepend-
abilityoftheinstrumentareunmatchedandarethereforesoldby
allOEMsandCTC’sglobalnetworkofVARs.
WhencoupledwithanITSPsolutionlaboratoriescanincrease
theefficiencyofthelabbyautomatinglabor-intensivesample
preparationprocessesontoaninstrumentthatmustalreadybe
purchasedforautomationofanLCorGCanalyticalinstrument.
ITSPrecommendsadding(ifitisn’talreadyequippedwith
one)theDynamicLoadandWash(DLW)option.Thisallowsfor
moreefficientsolventdispensing,andmostimportantly,cleaning
ofthesyringeandvalveinletstoalmostcompletelyeliminate
carryover.ITSPoperatesduringtheLC/MS/MSanalysisofa
sampletoextractcompoundsofinterestfromthenextsample.
ThereisnocarryoverassociatedwithITSPsolutionsforsample
prepandnolossofthechainofcustody.ThePALSysteminjects
theeluateontotheinstrumentandcleansthesyringeandthe
valveinletbeforepreparingthenextsample.Chainofcustodyis
enhancedduetotheremovalofmanualinteraction.
ITSPSolutions’patentedcartridgeforautomatedSPEuses
precisecontrolofflowratesandvolumesforsolventsand
samplesthroughthesorbentbythePALSystemwhilethe
LC/MS/MSorGC/MS/MSisanalyzingasampleinparallel.
ThePALSystemusesordinaryautosamplertrayholders,
trays,syringesandsolventreservoirstowhichweadaptoursingle
positionsolutiontoautomateSPE,thegold-standardforsample
preparationforLiquidandGasChromatography.
Byaspiratingasolventorsample,thenpenetratingthe
septumontheITSPcartidge,thesyringeplungercanpassboth
throughthesorbentbedathigherprecisionthanachievableby
ordinarymanualandautomatedvacuum-basedandpneumatic
systems(noneofwhichhaveflowcontrol).Figure6detailsthe
constructionoftheITSPcartridge.
Themechanics ofITSP’s patented
cartridgesforsolidphaseextraction
Theplungerofthesyringe drives
thesolventsandsamplethrough
theupperchamberofthe
cartridgeandthroughthesorbent
atpreciseflowratesthatcannot
beachievedbymostmanualand
automatedSPEhardware.
ITSPHardwareKitincludesalower
needleguidethatcentersthecartridge
beforepiercingtakesplacetoallowthe
sameholeintheseptumtobeaccessed
multipletimes.
TheITSPcartridgeincorporatesan8mm
crimpsealtoallowtheneedletopierce
theseptumthensealaroundtheneedle.
Whentheplungerdispensessolvents
andsample,hydraulicpressureinside
thecartridgedrivesthesesolutions
throughthemedia.
Theupperneedleguideremovesthevolume
abovethemediacuptoincreaseefficiency
andincreasehydraulicpressure.
Theneedleremainsfullyinsertedin
thecartridgetokeepapreciseverticalposi-
tion requiredfortransportingthecartridge
toanylocationonthePALSystemplatform.
Sorbentsandwichedbetweenfritsorfilter
membrane(s).StandardSPEcolumncon-
struction,standardSPEconventions.
TheSPEcolumnasyouwouldnormallyknow
it,justmicro-volumescale.
Figure6
www.PALSystem.com

7. Figure7:ThePALRTCpicturedhereisoutfittedwithproductnumber:HW3-KITwhichincludesasingle96-positionITSPSolidPhaseExtractionworkstation
(ITSPlowerneedleguideforasingleRTCSyringetoolofyourchoice,a3-positiontrayholderandawastereceptaclewithhosebib).Unlikeotherdedicatedon-line
SPEsystems,ITSPusesstandardCTCtraysandoccupiesonlyonepositiononthedeck,allowingforotherapplicationstorunusingthePALRTCaswell.
Figure8:ThePAL-xtwithDLW2optionpicturedhereisoutfittedwithHW-KIT-APMwhichincludestwo96-positionITSPSolidPhaseExtractionworkstations(ITSP
lowerneedleguide,three2-positiontrayholders,two3-positionsolventreservoirsandtwowastereceptacleswithhosebibs).AswiththeRTCaboveand unlike
otherdedicatedon-lineSPEsystems,ITSPusesstandardCTCtraysandoccupiesonlytwopositionsonthedeck,allowingforother applicationstorunusingthePAL
Systemaswell.

8. 212NorthlakeDrive,HartwellGA30643•10SouthCarolinaStreet,Hartwell,GA30643
855-395-8300Toll-Free•706-395-8300International•706-395-4946FAX
www.ITSPsolutions.com
US Patents: 6,859,615 & 7,001,774 • European Patents: EP 1 174 701 • Canadian Patent: 2,316,648
727MemorialDrive,Suite103,Bessemer,Alabama35022•855-319-4459
www.AssuranceScientific.com
SmartSPE
80
85
90
95
100
0 1 2 3 4 5 6 7 8
SCX %-Recovery in 20% Water
SCX %-Recovery in 80% Water
Reverse Phase %-Recovery
Flow (l/s) [5 l/s = 1.5 mm/s]
%Recovery
SPEisChromatography!
Optimizedoutcomesrequireaccurateflow
Oxycodone
Van Deemter curves
for SPE by ITSP
SCX optimum velocity = 0.37 mm/s (1.2 µl/s by ITSP) with little room of error!
Reverse Phase optimum velocity = 1.5 mm/s (5 µl/s by ITSP) 4x higher than SCX!
SPE flow driven pneumatically or by vacuum cannot achieve and maintain optimum flow!
™