Table Of ContentDEVELOPMENTOFLIQUIDCHROMATOGRAPHYTANDEMMASS
SPECTROMETRYINSTRUMENTATIONANDMETHODOLOGYFORTHE
INVESTIGATIONOFBIOLOGICALLYACTIVECOMPOUNDS
By
KEVINJOSEPHMCHALE
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOLOFTHE
UNIVERSITYOFFLORIDAINPARTIALFULFILLMENTOFTHE
REQUIREMENTSFORTHEDEGREEOFDOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
2002
.
Thisdissertationisdedicatedtothememoriesofthemen,womenandchildrenwhowere
lostintheGreatAmericanSiegeofSeptember11,2001
“IfIhaveseenfurther,itisbystandingontheshouldersofgiants.”
-SirIsaacNewton
ACKNOWLEDGMENTS
Foremost,Iwishtothankmyparents,JosephandKayMcHale,forallthe
support,encouragementandlovetheyhavebestowedonmeovertheyears. Theywere
theoneswhoinstilledinmefromaveryyoungagethemeritsofhardworkand
perseverance. Foralltheirsupport,Ishallbeeternallygrateful.
IextendmymostsincerethankstomygoodfriendAndyOttensforbeingtheone
reliableconstantthroughtheyearshereinGainesville. Withouthisexpertisein
electronicsandcomputersIwouldprobablystillbetryingtomakethenewinstrument
operational. Iwillrememberthemanytimeswetriedtomakesenseofitalloverapint
ofKillians. Forhiswordsofreasonandencouragement,Iaminhisdebt.
ManythanksalsoaregiventomyfriendJoeMulholland. Beingfromsimilar
backgroundsandhavingsimilarwaysofthinking,JoeinstantlyrelatedtowhoIwasand
hewasabletobringoutthebestinme. Joe’scounselonmattersofprofessional,
personalorscientificimportance,althoughsometimesbluntinnature,wasalwaystaken
toheart. Additionally,hewasoftenquickwithawhimsicalremark,bringingasmileto
myfacenomatterwhatmoodIwasinatthetime.
IwishtoexpressmygratitudetoJoshCoonforalwaysbeingwillingtoextenda
helpinghandinmytimeofneed. FromthetimeJoshextendedaninvitationfor
Thanksgivingdinnerin1999tothetimehewatchedovermethedayIhadsurgery,Josh
hasstoodbymysideinthesamewayabrotherwould. EventhoughIwassometimes
harshinmycriticismofJosh’sefforts,itwasonlydonetomakehimintoabetter
IV
scientist. IhopehewillalwaysrememberthatIwillalwaysbeathisdisposalshouldhe
requiremyassistance.
Forallhishelpfuldiscussionsandforthewordsofencouragement,Iespecially
wanttothankWillHaskins. JustwhenIthoughtmyresearchwouldneveryieldany
successfulresults,Willwasalwayswillingandabletooffermethekeyinformation
neededtosolvetheproblemathand. IcanonlyhopethatIprovidedafractionofthe
helpthatWillgavetome.
AspecialacknowledgementisextendedtoJoeShalosky,MikeHerlevich,
RaymondFrier,andToddProxofthemachineshopandtoSteveMilesandLarryHartley
oftheelectronicsshop. Theirwillingnesstoprovideassistanceandtoendurewithmy
inexperienceinmechanicalandelectricalengineeringisimmenselyappreciated.
IoffermyappreciationtoArtEdison,mycollaboratorattheUniversityofFlorida
BrainInstitute,fortheopportunitytoworkwithhimandhisresearchgroup. Dr.Edison
wasalwayswillingtodiscussmyresearcheffortswhenIshowedupunexpectedlyathis
door. Dr.Edisonwasalsoforthcomingwithacomplimentonmyresearchprogressand
encouragedmetolookatthepositiveaspectsofmyresults. MyonlyregretisthatIwas
unabletoyieldconclusiveanswersconcerninghisresearchproject.
IextendmymostgratefulappreciationtoDr.SteveRobertsandJohnMunsonof
theUniversityofFloridaCenterforEnvironmentalandHumanToxicology. Their
enthusiasminworkingwithmeonthenorcocainenitroxideprojecttrulyinspiredmeto
“gotheextramile”attheendofmydissertationresearch. Ihopemyeffortsin
developingtheLC/MS/MSmethodwillbefruitfultotheirfutureresearchendeavorsin
thisarea.
v
IwishtoacknowledgethemanyYostgroupmembers,bothpastandpresent,who
providedinspirationandassistanceinmytenureattheUniversityofFlorida. Whiletoo
numeroustoadequatelyrecognizehere,Idowanttobrieflythankthosewhomadethe
greatestimpactonmydoctoralexperience. Foremost,mysincerethanksgotoRick
Troendlewhowasalwayswillingtodiscussanyscientifictopicinamostinsightful
manner. Healwayskeptmehonestinsuchmattersandheforcedmetopushthelimitsof
myknowledge. Forhiseagernesstoassistmewithinstrumentalproblems,Iexpressmy
gratitudetoBrodyGuckenberger. Evenafterhegraduatedandwenttoworkat
ThermoFinnigan,Brodyalwaysansweredmyquestionsconcerningthenewinstrumentin
atimelymanner. Also,IextendmanythankstoScottQuarmbyforprovidingkey
informationontheLCQ/GCQsystems,whichmadeitpossibletobringthenew
instrumenttooperationalstatus.
Lastly,IwanttothankmyadvisorRickYostforgivingmetheopportunityto
obtainmydoctorateunderhisdirection. Heaffordedmethefreedomtochooseany
researchprojectandgavemetheflexibilitytocarryoutthatproject. Rickinstilledinme
asenseofself-confidencethatIcanachievewhateverIsetmymindto. Withthat,Iwill
nowtrytoinstillthatattributeinothers.
vi
TABLEOFCONTENTS
page
ACKNOWLEDGMENTS iv
ABSTRACT ix
CHAPTER
INTRODUCTION
1 1
QuadrupoleIonTrapMassSpectrometer 2
Background 2
EquationsofIonMotion 7
BasicOperatingPrinciplesoftheIonTrap 14
ElectrosprayIonization 20
History 20
TheoryandFundamentalsofESI-MS 25
LowFlowESI-MS 35
DissertationOverview 38
2 DEVELOPMENTOFTHEQITMSWITHMICROELECTROSPRAY
IONIZATIONINTERFACE 40
PreliminaryMicroelectrosprayIonizationExperiments 40
ShortcomingsoftheITMS 42
ConstructionoftheNewQITMS 48
TheMainRFCoil 50
ElectronicsIssues 55
OptimizationoftheQITMS 60
MassCalibration 60
MassRangeofPHOENIX 64
IonTransmissionandIonInjection 65
GatingIonstotheQIT 70
OptimizationinMS/MSMode 75
SensitivityoftheNewQITMS 78
vii
7
3
INCORPORATIONOFCAPILLARYLIQUIDCHROMATOGRAPHY 114
IntroductiontocLC/MS 114
ConstructionofCapillaryColumns 116
High-PressurePackingChamber 116
InitialCapillaryLCColumns 11
PreparationofPorousFrits 119
cLC/MSApparatus 123
cLC/MSofTrypticDigestandOtherPeptideStandards 128
InitialcLC/MSExperiments 128
RevisedInjectionApparatus 132
AssessmentoftheGradientElutionProblem 134
AnalysisofTrypticDigest 135
SensitivityofcLC/MS 137
DualMS/MSMonitoring 138
4 INVESTIGATIONOFNEUROPEPTIDESINCAENORHABDITISELEGANS.162
cLC/MSofC.elegansExtracts 163
cLC/MS/MSofC.elegansExtracts 165
5 CHARACTERIZATIONOFTHETOXICCOCAINEMETABOLITE
NORCOCAINENITROXIDEBYLC/MS/MS 175
MassSpectrometryofNitroxides 177
StabilityofNorcocaineNitroxideinAqueousSolutions 179
LC/MSandLC/MS/MSofNorcocaineNitroxide 181
EffectofMobilePhaseCompositiononSeparationEfficiency 187
EvaluationofaPolymer-BasedColumn 189
ExtractionofN-OxidativeMetabolitesofCocaine 191
InVitroIncubationsofNorcocaineandN-hydroxynorcocaine 196
6 CONCLUSIONSANDFUTUREWORK 242
REFERENCES 251
BIOGRAPHICALSKETCH 266
viii
AbstractofDissertationPresentedtotheGraduateSchooloftheUniversityofFloridain
PartialFulfillmentoftheRequirementsfortheDegreeofDoctorofPhilosophy
DEVELOPMENTOFLIQUIDCHROMATOGRAPHYTANDEMMASS
SPECTROMETRYINSTRUMENTATIONANDMETHODOLOGYFORTHE
INVESTIGATIONOFBIOLOGICALLYACTIVECOMPOUNDS
By
KevinJosephMcHale
August2002
Chair: RichardA.Yost
MajorDepartment: Chemistry
Anewversatilequadrupoleiontrapmassspectrometer(QITMS)wasdeveloped
withtheabilitytoyieldreliablecapillaryliquidchromatographytandemmass
spectrometry(cLC/MS/MS)data. ThiswasrealizedbyadaptingaThermoFinniganGCQ
seriesQITMStoahome-builtvacuumchamber. Accesstotheinstrument’ssourcecode
allowedtheupgradedQITMStobecustomizedtomeettheneedsofanyresearchproject.
SomeofthekeychangesmadetotheQITMSincludeincreasingthenominalmassrange
from1000to1800atomicmassunits,alteringtheinstrument’soctopolecircuitrytobe
underdirectcontroloftheCiCQsystemanddevisingasubroutinefordualMS/MS
monitoring.
Integrationofalow-flowmicroelectrosprayionization(pESI)sourceforthe
QITMSyieldedamassdetectionlimitof12attomolesbyinfusionofthepeptide
angiotensinI. Additionally,thepESIsourceprovidedadirectcouplingoftheQITMS
withcLC.
IX
Theintrinsicpropertiesofliquidchromatographyforprovidingsample
concentrationandanalyteseparationmaketheincorporationofcLCwithMSavaluable
meansoffurtherimprovingthesensitivityandselectivityofanalyses. Thiswas
demonstratedbyexaminingastandardpeptidemixture,whereangiotensinIwas
observedataconcentrationof2.5nMbycLC/MSandat10pMwhencLC/MS/MSwas
used.
TobetterunderstandthefunctionofFMRFamide-likepeptides(FLPs),extracts
fromthenematodeCaenorhabditiseleganswereexaminedviacLC/MS/MStoidentify
whichoftheputativepeptideswereexpressed. Unfortunately,preliminaryeffortswere
notsuccessfulinconfirmingthepresenceofanyFLPsintheC.elegansextracts.
However,theinternalstandardangiotensinIwasobservedwithinthesecomplexsamples
atthelevelof335attomoles,validatingthesensitivityoftheQITMSandthe
cLC/MS/MSmethodologyforidentifyinganalytesattracelevelsinabiologicalmatrix.
ToassaythehepatotoxicmetabolitenorcocainenitroxideandotherN-oxidative
metabolitesofcocaine,anewLC/MS/MSmethodwasestablished. Norcocaine
nitroxide,astableradical,wasdiscoveredtobereducedinthepresenceofacid,thereby
requiringthemobilephasetobebufferedtonearpH7usingammoniumacetate. The
productionofnorcocainenitroxideinvitrowasconfirmedbyLC/MS/MSafter
incubationof500pMN-hydroxynorcocaineinratlivermicrosomes.
x
