MeasuringRateandEquilibriumConstantsofβ‐Cyclodextrin‐SmallMoleculeDrug

Non‐CovalentInteractionswithCapillaryElectrophoresis

By

JenniferLogie

HonorsthesissubmittedtotheFacultyofScienceDepartmentofChemistryInpartialfulfillmentoftherequirementsoftheB.Sc.Degree

Supervisor:MaximBerezovski

DepartmentofChemistry

UniversityofOttawa

Ottawa,Canada

©April2011,JenniferLogie

ii

Abstract

Cyclodextrinisacircularoligosaccharidemadeof6‐8glucosemonomers,givingtheideal

conformationtoforminclusioncomplexeswithmanydifferentsmallmolecules.Inthe

pharmaceuticalindustry,itiscommonlyusedasanexcipienttoincreasethesolubilityofsmall

moleculedrugs.Thepurposeofthisresearchwastomeasureequilibriumandrateconstants

betweensmallmoleculesandcyclodextrinsusingtheEquilibriumCapillaryElectrophoresisof

EquilibriumMixtures(ECEEM)method.InECEEM,anequilibriummixtureofsmallmolecule,

cyclodextrinandcomplexisinjectedintoacapillaryandsubjectedtoelectrophoreticseparation.

Therunningbuffercontainsthesameconcentrationofcyclodextrinasintheequilibriummixture.

Bindingparametersofcomplexescanbefoundbyusingthetimepropagationpatternandshapes

ofthepeaksobtainedwhentheconcentrationofcyclodextrinisgraduallyincreased.Inthisstudy,

eightsmallmoleculedrugs:ibuprofen,s‐flurbiprofen,4,4’‐(propane‐1,3‐diyl)dibenzoicacid,

resveratrol,naproxen,diclofenac,folicacidandphenylbutazonewerestudiedfortheir

complexationwithβ‐cyclodextrin.TheECEEMmethodprovedtobeavaluabletechniquefor

determiningthepracticalityofcyclodextrinasadrugdeliverysystemforspecificdrugs.

iii

Acknowledgements

WithoutthehelpandsupportoftheBerezovskilab,thisworkcouldnothavebeencompleted.

Particularlythesupervision,guidanceandinsightofDr.MaximBerezovski;themathskillsofDr.

VictorOkhonin;andfinallytheinstructionandhelpofGlebMironov.

iv

TableofContents

ListofFigures......................................................................................................................................v

StatementofContribution................................................................................................................vi

1.Introduction....................................................................................................................................11.1Rationale.................................................................................................................................................11.2CyclodextrinasaDrugDeliveryAgent....................................................................................................21.3BindingConstantsofCyclodextrin‐SmallMoleculeComplexes..............................................................51.4LiteratureReviewofKDDeterminationMethods...................................................................................61.5PrinciplesofCapillaryElectrophoresis....................................................................................................9

2.ResultsandDiscussion..................................................................................................................132.14,4’(Propane‐1,3Diyl)DibenzoicAcidModel......................................................................................132.2MultiplexApplication............................................................................................................................23

3.References....................................................................................................................................31

4.Experimental..................................................................................................................................334.1ChemicalsandMaterials.......................................................................................................................334.2ExperimentalConditions.......................................................................................................................34

v

ListofFigures

1.StructuresofSmallMoleculeDrugs................................................................................................3

2.β‐cyclodextrinStructureandInclusionComplexation....................................................................4

3.SchematicofECEEMSet‐up..........................................................................................................11

4.LimitofDetectionofPDDAElectropherogram.............................................................................15

5.DegradationofPDDAElectropherogram......................................................................................17

6.PDDAwithvaryingβ‐cyclodextrinconcentrationsElectropherogram.........................................18

7.VelocityofEquilibriumMixtureasaFunctionofβ‐cyclodextrinConcentration..........................20

8.ECEEM‐MSSchematic...................................................................................................................22

9.3‐DAbsorptionPlotsofSmallMolecules......................................................................................24

10.2‐Dand3‐DAbsorptionPlotsofMultiplex.................................................................................25

11.Multiplexwithvaryingβ‐cyclodextrinconcentrationsElectropherogram.................................27

12.MultiplexKDdeterminationGraphs...........................................................................................29

vi

StatementofContribution

Dr.SergeGorelskydidtheDFTcalculationstodeterminethemodeloftheinclusioncomplex

betweenβ‐cyclodextrinandibuprofen.VictorOkhonincreatedthemathematicalmodelusedto

calculatebindingparametersforthePDDA‐β‐cyclodextrininclusioncomplex.GlebMironovdidall

CE‐MSworkthatisreferencedinSection2.

1

1.Introduction

1.1Rationale

Pharmaceuticalcompaniesspendbillionsofdollarseachyearresearchingnewandexcitingdrug

candidates.Gettingthesedrugsintothebodyisanongoingbattleduetotheoften‐lowsolubility

ofthesemolecules.Oftendrugsareformulatedwithexcipients,whichusevariousmethodsto

increasethebioavailabilityofthedrugs(1).Inthisstudy,severalcommondrugsonthemarketwill

beusedtocreateamodelfortheevaluationofdrugdeliverysystems.

Someoftheoldestandmostcommonlyuseddrugsavailablearethenon‐steroidalanti‐

inflammatoryclass(NSAIDS).Althoughthestructuresofthesedrugsvary,theyallserveas

analgesicsandantipyretics,andathighdosesmayhaveanti‐inflammatoryeffects.Theyworkby

non‐specificallyinhibitingtheenzymecyclooxygenase,whichpreventstheproductionof

prostaglandinsandthromboxanesfromarachidonicacid(2).Naproxen,Ibuprofen,andS‐

FlurbiprofenallbelongtotheclassofNSAIDsderivedfrompropionicacid.Diclofenacisanacetic

acidderivative.PhenylbutazoneisanNSAIDderivedfromenolicacidthatisnotcommonlyusedin

humans,howeveritisveryprevalentinveterinarymedicine(2).

TheotherdrugsthatwerestudieddonotbelongtotheNSAIDclass,butwerechosentoshowthe

potentialdiversityofthemethod.FolicAcidisanessentialvitaminfortetrahydrofolateproduction

inthebody,anddietsupplementsareusuallyrecommendedaspartofapre‐natalvitamin

regimen(3).Resveratrolisacommonantioxidantfoundinmanyfoodproducts,andiscurrently

beingtestedforitsvaryinghealthbenefits(4).Finally,4,4’‐(Propane‐1,3‐Diyl)DibenzoicAcid

(PDDA),asmallmoleculewhichdoesnotcurrentlyhaveaclinicaluse,wasselectedduetoits

2

structuralpropertieswhichwillbedescribedindetailinSection2.1.Alldrugstructuresarefound

inFigure1.

1.2CyclodextrinasaDrugDeliveryAgent

Theexcipientsusedtoincreasethesolubilityofdrugsvarywidely,inthisstudy,onesuch

excipient,cyclodextrin,willbediscussed(5).Cyclodextrinsarecyclicoligosaccharidesmadeof6‐8

glucosemonomers,withmolecularweightsrangingbetween1000and2000g/mol.Theyforma

toroid‐truncatedcone‐likestructureduetothechairformationoftheglucopyranoseunits(6).

Thehydroxylgroupsareattheexteriorofeitherendofthecone,makingtheexteriorofthe

moleculeveryhydrophilic.Theinteriorofthecyclodextrinisslightlyhydrophobic,makingitan

excellentcavityforhydrophobicsmallmoleculestoenterandformacomplex(6).Theformation

ofthisinclusioncomplexsignificantlyincreasesthewatersolubilityofthedrug,andallowsittobe

transportedthroughthebodytothelipidcellmembraneofthedrugstarget(6).Thestructureof

β‐cyclodextrinandaninclusioncomplexwithibuprofenareshowninFigure2.Thelarge

hydrophiliccyclodextrinmoleculeisnotabletopenetratethroughthehydrophobiccell

membrane,soitisessentialthattheformedinclusioncomplexesareinarapidequilibriumwith

thefreedrug.Thisenablesthefreedrugtogothroughthemembraneandreachitstarget(6).

Differentcyclodextrins(α,βorγ)areuseddependingonthesizeofthesmallmolecule,aseach

cyclodextrinhasadifferentsizedinternalcavity.

Currentlycyclodextrinsareonthemarketforawiderangeofapplications.Thisincludes

householdproducts(suchasFebreeze),cosmetics,andfoods‐wheretheyarenotablyableto

encapsulateflavormolecules.Themostprevalentpharmaceuticalapplicationsareinnasalsprays

andeyedrops,andthereareover30pharmaceuticalproductsonthemarketwhichuse

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cyclodextrinasanexcipient(6).Inthecaseofsmallmoleculedrugswithinthemolecularweight

rangeof100‐400g/mol,beta‐cyclodextrin,whichhassevenglucopyranoseunits,isthebestdrug

deliverymolecule.Thisisduetoaninternalcavitydiameterof5.3Åatthebottomand7.0Åatthe

top(7).Thiscavitydiametermeansthereisgoodmolecularsizeandshapecomplementationto

formastablecomplex(7).ADFTcalculatedmodeldonewithibuprofenshowedthatthe

cyclodextrinholdsthedruginsidethecavitybytwohydrogenbondsbetweenaterminalcarboxyl

groupofibuprofenandtwohydroxylgroupsonthebottomofthecyclicoligosaccharide(7).

1.3BindingConstantsofCyclodextrin‐SmallMoleculeComplexes

Inordertoevaluatethepracticalityofcyclodextrinasadrugdeliverysystemforaparticulardrug,

informationaboutthenatureofthecomplexandthebindingconstantsisnecessary.

Determinationofthedissociationconstant(KD)ofcyclodextrin‐smallmoleculeinclusion

complexesallowsforbettercharacterizationofthecomplex.TheKDisanequilibriumconstant

whichmeasuresthepropensityofacomplextofallapartintosmallercomponents.Forthe

generalreaction

AB A+B

Thedissociationconstantisrepresentedbytheequation:

𝐾! =𝐴 𝐵𝐴𝐵

Itcanalsobeexpressedastheratiooftheon‐rateandtheoff‐rate,whosevaluesgiveevenmore

informationaboutthenatureofthecomplex.

𝐾! =!!""!!"

6

DeterminingtheKDofthenon‐covalentcomplexesbetweencyclodextrinsandsmallmoleculeshas

beenwellstudied,andgenerallyanymethodthatallowstheobservationofchangesin

physiochemicalpropertiesthathappenwithcomplexationcanbeusedtoquantifythedissociation

constant.Althoughthemethodsshouldgiveconsistentresults,oftenthereisalotofdiscrepancy.

Methodsthathavebeenprevalentintheliteraturearesummarizedinthefollowingpages.

1.4LiteratureReviewofKDDeterminationMethods

NuclearMagneticResonance(NMR)hasbeenusedforthecharacterizationofprotein‐ligandnon‐

covalentcomplexestocalculateKD(8),howevernotmuchworkhasbeendonecalculatingthe

bindingconstantsofcyclodextrin‐smallmoleculeinteractions.Theworkthathasbeendone

generallylooksat1Hchemicalshiftchangesofthebeta‐cyclodextrinandcalculatestheKDbased

ontheHildebrand‐Benesimodel(9).Thismodelworksonthebasisthatoneofthereactantsis

presentinexcessamountsoftheother,andyoucanonlytakethatoneintoaccount.Italsoworks

solelyforcomplexeswitha1:1stereochemistry(9).OneotherstudyusingNMRcalculatedtheKD

onthebasisofliganddissociationkinetics.Inthiscase,itmadetheassumptionthattherateof

association,kon,isdiffusionlimitedandequalto1x109M‐1sec‐1.Itthenmonitoredthereaction

byquenchingthereactionatvarioustimepointsandtakinganNMRspectra.Bydoingalineshape

analysisofindividualNMRpeaks,theycalculatedtherateofdissociation,koff,anddeterminedaKD

basedonthisvalue(8).UsingNMRtofindthesebindingparametersmakesalotofassumptions.

Firstly,itisnotalwaysthecasethattheβ‐cyclodextrinisinexcessofthesmallmolecule.Secondly,

inthecaseofcalculatingtheKDonthebasisofliganddissociationkinetics,itislargelyincorrectto

assumetherateofassociationisdiffusionlimited.Finally,youcannotassumethatformed

7

complexeswillalwayshave1:1stoichiometry,especiallyathighconcentrations,whenaggregation

ismorelikelytooccur(8).

OneofthemostpopularmethodsfordeterminationoftheKDisequilibriumdialysis(11).Like

NMR,thishasbeenwidelystudiedforprotein‐ligandinteractionsandexamplesarelimitedforits

useincyclodextrin‐smallmoleculeinteractions(10).Thismethodreliesontheuseofamembrane

thattheligandcanpassthroughbutthereceptorcannot.Ononesideofthemembranethe

receptorisplaced,whileontheotherthereisaknownconcentrationoftheligand.Thesystemis

thenallowedtoreachequilibrium.Thehighertheaffinitytheligandhasforthereceptor,the

highertheconcentrationofligandwillbebound.Atequilibrium,theconcentrationofthefree

ligandwillbethesameonbothsidesofthemembrane.Knowingtheoriginalconcentrationused,

enoughinformationcanbeobtainedinordertocalculatetheKD.Althoughthismodelisvery

practicalbecauseofitssimplicity,likeNMRitcanbehinderedbytheassumptionsitmakes.The

modelassumesthatallreceptorsareequallyaccessibletoligands,thatreceptorsareeitherfreeor

boundtoaligand,thatbindingdoesnotaltertheligandorreceptor,andthatthebindingis

reversible(11).Despitetheseassumptions,equilibriumdialysisremainsoneofthemostaccurate

methodsforKDdetermination(10).

Phase‐solubilitydiagramshavebeenextensivelyusedforstudyingthebindingwithcyclodextrins

(12).ThismethodwasdevelopedbyHiguchiandConnorsandisbasedonhowcomplexation

affectstheaqueoussolubilityofdrugs(13).Plottingthedrugsolubilityagainsttheconcentration

ofcyclodextrincreatesaphasesolubilitydiagram.Thenoveltyofthismethodcomparedtothose

previouslymentionedisthatitgivesinformationregardingthestoichiometryofthecomplex

throughthelinearityofthecurve(14).Ifitisverylinear,itsuggeststhatthecomplexisfirstorder

8

withrespecttoboththedrugandcomplexingagent.Ifthestoichiometryisgreaterthan1:1,it

becomesmoredifficulttocalculateanaccurateKD,butimpliesahigherorderofcomplexation(14).

Themaindownsideofthephase‐solubilitydiagramapproachisthelargequantitiesofcyclodextrin

anddrugrequiredinordertomeasurethesolubility.

Amoreefficientmethodtoevaluatetheinteractionisasurfaceplasmonresonanceassay(15).

Thisassayworksbyimmobilizingligands,inthiscaseβ‐cyclodextrin,onagoldsurfaceand

monitoringthechangesinrefractiveindex.Themagnitudeofthechangeisdirectlyproportional

tothemasschangeatthesurface(15).Monitoringthesignalproducedinrealtimeallowsforthe

calculationofassociationanddissociationrates(usedtocalculatedthedissociationconstant)and

canalsoshowthestoichiometry(16).Thismethodisstillbeingoptimizedandimprovedforuse

withcyclodextrins,particularlyobtainingbettercouplingefficiencyofthecyclodextrintothe

immobilizedsurface,andincreasingthesensitivityoftheassay,asaddingasmallmolecular

weightdrugtoalargemolecularweightcyclodextringivesonlyasmallchangeinsignal(16).

Stopped‐flowisafrequentlyemployedtechniquetostudythekineticsofareaction,particularly

whenlookingatenzymes.Fewstudieshavebeendoneusingstoppedflowtolookatthebinding

ofcyclodextrins,butthiscouldbeduetothenatureoftheequilibrium(17).Cyclodextrin‐small

moleculecomplexesareinarapidequilibrium,andtheratesareveryfast.Themixingdead‐time

andre‐dissociationofthereagentsmeansthattheserapidratesarenotmeasured(7).Thevalues

obtaineddonotrepresentthesystematequilibrium(7).

Electrosprayionizationmassspectrometry(ESI‐MS)isausefultoolforworkingwith

macromoleculesandnon‐covalentcomplexes,asitionizesthemolecules/complexeswithout

fragmentingthem(18).ThegreatestadvantageofESI‐MSisthatitmakesitpossiblytodirectly

9

obtainstoichiometricinformation.Byevaluatingthepresenceofcomplex,freedrug,andfree

cyclodextrinusingtheintensitiesoftheirrespectivem/zpeaks,andcalculatingaresponsefactor

tocorrespondtheintensitiestotheconcentrations,theKDcanbecalculated(19,20).Ithaslargely

beenthroughMSthattheaggregationofcyclodextrininsolutionhasbeenverified(18).Although

ESI‐MSelegantlycalculatestheKDbasedontheintensities,thestabilityoftheionizationisahuge

problem,andleadstoalotoferrorintheresult(19).Thebehaviorofthenon‐covalentcomplexes

inthegasphasemaynotrepresentwhatishappeninginsolution(18).Althoughitsusefulnessfor

identificationofcomplexesisnotable,adirectcorrespondencebetweenintensityand

concentrationleavesmuchtobedesired.

AllofthesemethodstodeterminetheKDhavespecificadvantagesanddisadvantages,however

theproblemwithmostofthem,theexceptionbeingMS,isthattheycanonlyquantitatively

evaluatethecomplexationbetweencyclodextrinandasinglesmallmolecule.Thisisaproblemin

thepharmaceuticalindustry,wheremanydrugformulationshavemultipleactiveingredients,and

manydrugsaretakenincombinationdoseswithotherdrugs(21).Aseparationtechniqueis

crucialtothecharacterizationofcomplexesformingbetweencyclodextrinsanddifferentsmall

moleculessimultaneously.

1.5PrinciplesofCapillaryElectrophoresis

Capillaryelectrophoresis(CE)isaseparationtechniquethatseparatesionsbasedontheircharge

andfrictionalforcesaswellastheirhydrodynamicradius.Whenusinganumberofdrugs,their

uniqueabsorptionspectraandelutiontimescanbeusedtoidentifythemindividually.This

separationisdoneunderaninducedelectricfield,whichcausesanelectroosmoticflowofthe

liquidmovingfromthepositiveelectrodetothenegativeelectrode(21).Thisbulkflowofliquid

10

causesallspecies(positive,neutralornegative)tomoveinonedirectionduetothesurfacecharge

onsilanolgroupsoftheinteriorcapillarywall.Theelectroosmoticflowisaffectedbyseveral

factorsincludingthepHandtheionicstrengthofthesolution(21).Inthiswork,Equilibrium

CapillaryElectrophoresisofEquilibriumMixtures(ECEEM),atypeofKineticCapillary

Electrophoresis(KCE),willbeappliedinordertostudytheequilibriumbetweenthecyclodextrin

andthechosensmallmoleculedrugs.KCEisageneralconceptusedtodescribeCEseparationof

speciesthatinteractduringelectrophoresis(22).Bychangingtheconditions,specificmethods

undertheumbrellaofKCEhavebeendevelopedtomeasureaffinities(includingbinding

parameters)aswellaspurifyaninteractingmixture(22).

InECEEM,aquasi‐equilibriumsystemofthesmallmolecule,cyclodextrin,andcomplexisinjected

intothecapillaryandsubjectedtoelectrophoreticseparation.AschematicofECEEMisshownin

Figure3.Theconcentrationofcyclodextrinismaintainedintherunningbuffer,soitiseffectively

thesamethroughoutthewholesystem(22).UniquetoECEEMarethreemaincharacteristicsfor

rapidequilibriummixtureslikethatofsmallmoleculesandcyclodextrins.(i)Changingthe

concentrationoftheβ‐cyclodextrinwillchangethemigrationtimeoftheequilibriummixture.(ii)

Thefreedrugandthecomplexwillmigratetogetherbecauseoftherapidequilibriumbetween

them.(iii)Finally,thewidthofthepeakfortheequilibriummixtureisdependentonthe

concentrationofβ‐cyclodextrin,therelaxationtime,andtheseparationtime(7).Thisis

fundamentaltostudyingaveryfastinteraction,asitwillnotseparatethecyclodextrinfromthe

drug,butratherseparatedrugswithdifferentKDsfromeachother.Inpreviousworkdonebythe

Berezovskilab,ECEEMwasprovenforthedeterminationofkineticparametersoffastnon‐

covalentinteractions,andaparameterbasedmethodforthequantificationofrateconstants

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ofcomplexformationanddissociationwasdeveloped(7).Inthismethod,thebindingconstantsof

theinteractionsbetweenβ‐cyclodextrinandfoursmallmoleculedrugs(ibuprofen,s‐flurbiprofen,

salicylicacidandphenylbutazone)weredetermined.

Furthermore,bycouplingECEEMwithESI‐MS,thebenefitsofboththeseparationtechnique

(ECEEM)andthecomplexidentification(MS)canbecapitalizeduponforabetterunderstandingof

thenon‐covalentbinding.

ThegoalofthisworkwastousethepreviouslyestablishedtechniqueofECEEMtofurtheranalyze

theinteractionsbetweenβ‐cyclodextrinsandsmallmoleculesofgreatercomplexity,aswellas

developamethodfortheanalysisofamultiplexofeightdrugs.Withthisstudy,itisthehopethat

capillaryelectrophoresismaybecomeanimportanttoolfortheevaluationofβ‐cyclodextrinasa

drugdeliveryagentfordrugs.

13

2.ResultsandDiscussion

2.1ProofofPrinciple4,4’‐(Propane‐1,3‐Diyl)DibenzoicAcidModel

PreviousworkdonebytheBerezovskigroup(7),showedthedeterminationofbindingconstants

betweenβ‐cyclodextrinandfoursmallmoleculedrugs.Eachofthesesmallmoleculedrugs

structurallyhadthepropensitytobindina1:1stoichiometricratiowithβ‐cyclodextrin,andthey

eitherhadasinglephenylringortwophenylringsincloseproximity.

Inthisresearch,amodelfordeterminingtheKDsofgreaterstoichiometrywasdesired,soasmall

moleculethathadtheabilitytoforma2:1stoichiometricsandwichcomplexwiththeβ‐

cyclodextrinwasselected.Carboxylicacidgroupswerealsopreferred,meaningthedrugwould

haveanegativechargeinsolution.ThisisidealfortheCEmethodalreadyused,asthenegative

drugswillmoveslowerthantheneutralcyclodextrin,andthustheformationofthecomplexwill

causeafastervelocityofthedrugandashortermobilitytime.4,4’‐(Propane‐1,3‐Diyl)Dibenzoic

Acid(PDDA)wasselectedasithadbothoftheseproperties.Itsstructurehadtwophenylrings

withcarboxylicacidgroupsattachedbyathree‐carbonlinker.Becausethissmallmoleculeis

symmetrical,itwillbeeasiertodevelopamodelformultipleKDcalculationsthanitwouldbefora

morecomplicatedstructure.

ThechangeinthemobilitybetweenthefullycomplexedPDDAandthefreeformalsoservesto

maketheanalysiseasy.Thischangeinmobility,ofapproximately3minfora90cmcapillaryin10

mMAmmoniumAcetatebufferseparatedwith26kVfor12min,wasmuchgreaterthanallother

smallmoleculesevaluatedinthisresearch.ThiscanbeattributedtothefactthatPDDAhastwo

negativecharges,soitwillbeslowermovinginthecapillarywheninitsfreeform.ThePDDAis

fullysaturatedatβ‐cyclodextrinconcentrationsofaround2500µM.Inordertousecapillary

14

electrophoresistocalculatetheKD,itisessentialthatthedrugsbesaturatedwithinthesoluble

concentrationofβ‐cyclodextrin(upto17,000µM).

Methoddevelopmentofthecapillaryelectrophoresistookalargeamountoftimeinthisresearch.

Initially,thelimitofdetectionofthePDDAneededtobedetermined.Thiswasdoneusinga30cm

capillary,andthemethodpreviouslydevelopedbytheBerezovskilab(7).Thelimitofdetection

wasdeterminedtobe5µMina25mMTris‐Acetatebuffer,asshownintheelectropherogramin

Figure4.Movingforward,aconcentrationofapproximatelysixtimesthelimitofdetection,30µM

ofPDDAwasused,asthisprovidedagoodsignalthatwaseasytoanalyze.Usingthephotodiode

array(PDA)detector,severalwavelengthswerecheckedtofindthewavelengthatwhichthe

maximumintensityofsignalwasseen.Sinceithastwophenylrings,twomainareasofabsorbance

onthespectrawereseen:around200nmand250nm.Themostintensesignalwasseenaround

200nm,soinallfutureseparations,oneofthechannelswassetat200nmforevaluation.

Toensurethecurrentremainedstablethroughouttheseparation,severalparameterswere

changed.Initially,ahighconcentrationofβ‐cyclodextrinof5000µMwaspre‐injectedintothe

capillary,followedbytherunbufferandthenthesampleinjection.Thismethodtookalotoftime,

andwouldhaveaddedalotofextratimewhendoingmultiplerunsforbindingparameter

determination.Instead,rinsingthecapillaryforalongerlengthoftime(approximately3min)with

therunbuffergaveastablecurrentwithouthavingtodoapre‐injection.

OneofthemainproblemswithPDDAwasthataftersampleshadbeenleftforseveralhoursthe

migrationtimechangeddrastically.Althoughthisproblemcouldbesolvedbyusingonlyfresh

samples,thecostofthissmallmoleculewouldhavemadethisimpractical.Tosolvethisproblem,

15

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16

afreshsamplewasrunandthencomparedtosampleskeptovernightatroomtemperature,+4°C

and‐20°C.AsshowninFigure5,Samplesat‐20°Cgaveresultsconsistentwithfreshsamples,soall

PDDAsampleswerefrozenat‐20°Candthenthawedbeforeuse.

WiththeoptimizedmethoddescribedinSection4.2,thePDDAwassubjectedtoelectrophoretic

separationwithincreasingconcentrationsofβ‐cyclodextrinintheequilibriummixture.The

electropherogramshowingtheresultsoftheseexperimentsisshowninFigure6.Themobility

shiftofthepeakforthePDDAinrapidequilibriumwithβ‐cyclodextringraduallydecreasesas

higherconcentrationsareused.Thiscanbeexplainedbythefactthatathigherconcentrations

moreofthePDDAisinthecomplex,whichhasaneutralchargeandwillthusmovefasteralong

thecapillarytowardthenegativeelectrodethanthefree,negativelychargedformofthedrug.

IftheproposedsandwichcomplexbetweenPDDAandβ‐cyclodextrinwasformed,itwouldbe

anticipatedthattherewouldbetwoareasofalargemobilityshiftontheelectropherogram,and

anareainthemiddlewherethepeak’smobilitydoesnotchangeinwhichthedrughadbeen

saturatedasa1:1complex,butnotenoughβ‐cyclodextrinwasaddedtoforma2:1complex.This

wasnotseen,sotocalculatethebindingparametersofthiscomplex,themathematicalmodelfor

1:1stoichiometrydevelopedpreviously(7)wasapplied.

UsingtheshiftinmobilityofthePDDAallowsforthecalculationoftheKD,howevermore

informationaboutthenatureofacomplexrequiresthedeterminationofthekonandkoff.This

mathematicalmodelcalculatesthebindingparametersbasednotjustontheshiftinmobility(and

thusthevelocityoftheequilibriummixture)withthechangeinconcentration,butalsowiththe

shapeofthepeak.Whenthedrugisonlyinoneform(eitherthefreeformorthecomplexed

17

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18

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Figure6:RepresentativeECEEMelectropherogramsofPDDA(30μM)forvaryingconcentrationsofβ‐cyclodextrin.

19

form),seenatverylowandveryhighconcentrationsofβ‐cyclodextrin,verysymmetricalpeaksare

seen.Howeverwhentheβ‐cyclodextrinconcentrationisaroundthatoftheKD(sothedrugcanbe

foundinbothformssimultaneously),thepeakisasymmetricandwider.Aschematicofthisis

showninFigure3b.Thisreflectsthefactthatwithintheequilibriumcomplexthereisagradientof

thedrug,andthiscausesnonlinearcomplexformation.Essentially,becausethecomplexeddrug

movesfaster,thisgradientofdrugcausesonepartofthepeaktomovefasterthantheotherpart.

Usingthismodel,theKDfortheβ‐cyclodextrin‐PDDAcomplexwascalculatedtobe56.6µMwitha

koffof14.7sec‐1andakonof2.6x105M‐1sec‐1.Theseconstantswerecalculatedbasedondata

collectedfrom20experimentsrepeatedthreetimeseach.Theconcentrationsofβ‐cyclodextrin

usedtocalculatetheKDwerechosenbecausetheywereapproximately0.5‐5timestheKD,

providingthemostpreciseresults.Thevelocityvsβ‐cyclodextrinconcentrationgraphisshownin

figure7.Thedatafitcloselywiththistheoreticalcurve.Unfortunately,thedatacollectedwasnot

consistentwiththetheoreticalpatternforthechangesintheasymmetryandthewidthofthe

peak.Thissuggestedthattherewereperhapscomplexesofgreaterstoichiometricratiosforming,

butthattheycouldnotbedetectedwithcapillaryelectrophoresis,whichgivesnostructural

informationaboutthecomplexesformed.

InformationcalculatedusingtheCEwascomparedtoinformationfoundbyotherlabmembers

workingonthesamecomplexesusingCE‐MStogainabetterinsight.UsingMSasadetectorgives

muchmoreinformationaboutwhatisintheequilibriummixtureasitshowstherespectiveions

fordifferentcomplexesinsolution.Ifthereare2:1complexesforming,itisthoughtthatthey

20

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21

wouldbemoreprevalentathigherconcentrationsofβ‐cyclodextrin.TheideaofanECEEM‐MS

separationofacyclodextrin‐druginteractionwith2:1stoichiometryissummarizedinFigure8.In

fact,usingCE‐MS,wewereabletodeterminethat2:1complexesdoform,butnottheexpected

sandwichcomplex,rathercomplexesbetweenthedrugandadimerofcyclodextrin(Gleb

Mironov,unpublisheddata).Athigherconcentrations,thereismoreβ‐cyclodextrininthe

dimerizedform.KDvaluesforthetwodifferentcomplexeswerecalculatedtobe83.65µMforthe

monomericcomplexand39.08µMforthedimericcomplex(GlebMironov,unpublisheddata).

Interestingly,PDDAhasahigheraffinityforthedimerofβ‐cyclodextrin,whichmaybeattributable

tomorehydrogenbondingwithintheinclusioncavityduetomorealcoholgroupswhentwo

cyclodextrinscombine.ThesevaluesaveragetoaroundtheapparentKD(56.55µM)calculated

usingthecapillaryelectrophoresisdata,verifyingbothmethodsaccuracy.

Insummary,capillaryelectrophoresisworksverywellforcalculatingtheKDandbindingconstants

forcomplexesof1:1stoichiometry.Forcomplexeswithhigherstoichiometry,itwillgiveaKDthat

isrepresentativeoftheweightedaverageforallcomplexesformed,informationthatcanstillbe

usedfordrugdeliverydesigninwhichlowconcentrationsofβ‐cyclodextrinareused(andthus1:1

complexeswillbedominant).Alloftheothercommonlyusedmethods(exceptforMS)for

cyclodextrincomplexbindingcalculationsalsocalculateaweightedaverageKDassuming1:1

stoichiometry,highlightingthatcomplexstoichiometryisabigproblemincyclodextrininclusion

complexcharacterization.CE‐MScanbeusedtocharacterizethebindingofthecomplexingreater

stoichiometricdetail,howevercurrentlynomathematicalmodelhasbeendevelopedtocalculate

otherbindingparametersofthesecomplexessuchaskonandkoff.

22

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.%

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0&$$%,&12%

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Figure8:(A)SchematicrepresentationofECEEM‐MSseparationwithstoichiometryofthecomplexwithincreasedβ‐cyclodextrinconcentrations.(B)ComplexformationwithPDDAaccordingtoMSresults.

23

2.2MultiplexApplication

AfteroptimizingtheCEmethodwithPDDAandshowingitwasaviablemethodofaweighted

averageKDdetermination,themethodwasappliedtoamultiplexofeightdrugs:ibuprofen,(s)‐

flurbiprofen,PDDA,naproxen,folicacid,diclofenac,phenylbutazoneandresveratrol.Thisexploits

thepropertyoftheECEEMmethodtoseparatedrugswithdifferentKDs,andprovidesamore

accuratemodelofamoderndrugformulation(21).

WithaPDAdetector,individualdrugscanbeidentifiedbasedontheiruniqueabsorptionspectra

andshiftinmobility.The3‐Dabsorptionspectrasofeachoftheeightdrugsaresummarizedin

Figure9.Theabsorptionspectrathatwouldbeseenwhenalldrugsarecombinedintoone

formulationisshowninFigure10.Itshouldbenotedthatforabettervisualseparationandless

overlapalongercapillary(90cm)andincreasedseparationtimewasusedforthemultiplex

experiments.Allofthedrugscanbevisualizedat200nm,sothischannelwaschosenforpeak

analysisandcalculations.ThemainproblemwithusingaPDAdetectorasopposedtoaMS

detectoristhatthedrugmustabsorbinauniquerange.Conveniently,themajorityof

pharmaceuticals,suchastheonesusedinthiswork,havearomaticringsinthemthatwillabsorb

athigherwavelengths.Absorbancesunder200nmcangenerallynotbeidentifiedasthe

backgroundfromhydrocarbonsthatmaybecomponentsofthebuffer,includingthecyclodextrin,

istoohigh.

Peakmobilitydatawascollectedfrom17experimentsrepeated4timeseachtoensureaccuracy.

Theelectropherogramswereconsistentwhenalignedusingphenylbutazone,apreviously

determinedinternalstandardfortheseexperiments(7).Phenylbutazonehasanegativecharge

whichisdelocalizedoverfiveatoms,andisverystericallyhindered,meaningthatitcannotforma

24

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26

complexwithcyclodextrin.Ifphenylbutazonewasnotused,itispossibletoalignallrunsusingthe

dipinsignalbytheelectroosmoticflow,howeverifthereisanychangeinthebufferthiscanlead

toinaccurateresults.Overthecourseofthefourrepeats,theammoniumacetatebufferchanged

whichcausedalargechangeinthemobilityshiftforalldrugs.Theuseofaninternalstandard

ensuresthatexperimentsareveryreproducible.Anelectropherogramofoneofthetrialsisshown

inFigure11.Nouniquepeakwasseenforresveratrol.Thisisbecauseresveratrolisaneutral

drug,anddoesnothavethecarboxylategroupsthatgivethenegativechargeoftheotherdrugs.

Becauseofitsneutralcharge,acomplexwithresveratrolwillnotmovefasterwhencomplexed

withcyclodextrin,ascyclodextrinisalsoaneutralmolecule.Resveratrolcomesoutwiththepeak

associatedwiththeelectroosmoticflow,verifiedbyitsabsorptionspectra.

Duetothelargenumberofdrugsused,asimplermethodforKDdeterminationwasappliedthat

reliedonlyontheshiftinmobilityoftheindividualequilibriummixturesandnotthepeak’swidth

andasymmetry.Thiswasdonebecausethereisalotofoverlapinpeaksatdifferent

concentrations,assomecomplexesmovefasterthanothers,anditisimpossibletomeasurethe

peakwidthsaccuratelywhenthisisthecase.AcalculationmethoddevelopedbyKargeretal.(23)

formeasuringthebindingconstantsofpeptideswasappliedtothemultiplexsystem.

𝐾! = 𝛽𝐶𝐷𝑡! − 𝑡!𝑡! − 𝑡!

wheretisthemigrationtimeandthesubscript0isthefreedrug,tisequilibriummixture,andcis

thedrugcomplex.t0wastakenasthemobilitywhentheconcentrationofβ‐cyclodextrinwaszero,

whiletcwastakenasthemobilitywhenthesystemwassaturated.Thiscouldcauseproblemswith

drugsthataredidnotsaturatewithinasolublerangeofcyclodextrin.Thisformulawas

27

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Figure11:RepresentativeECEEMelectropherogramsofmultiplex(15μMofeachdrug)forvaryingconcentrationsofβ‐cyclodextrin.Drugsinthemultiplexare:Phenylbutazone(1),Diclofenac(2),Ibuprofen(3),s‐Flurbiprofen(4),Naproxen(5),FolicAcid(6),PDDA(7)andResveratrol(8).Resveratrolemittedwiththeelectroosmoticflow.

28

appliedtoallmobilityshifts,andtheaverageofthosewithintherangeoftheKD(determinedby

themovementofthepeakontheelectropherogram)wastakentogiveavalueoftheKD.The

calculatedKDsandtherepresentativegraphsofthesixdrugsforKDdeterminationaresummarized

inFigure12.

Phenylbutazoneisknowntonotcomplexwithβ‐cyclodextrin(7),andwasusedastheinternal

standard,sonoKDwascalculated.Resveratrolisaneutraldrug,sousingthismethoditis

impossibletodetermineitscomplexationwithβ‐cyclodextrin,ascomplexationwouldnotincrease

itsvelocity.ResveratrolhighlightsthemainproblemwiththeECEEMmethodforKD

determination.Thistechniqueworksverywellfornegativelychargeddrugs,howeveritdoesnot

workwithneutraldrugs,ascomplexationhasnoaffectonthesedrugsmobility.Forpositively

chargeddrugs,thisparticularmethodofECEEMwouldnotworkaspositivelychargeddrugswould

moveveryquicklytowardsthenegativeelectrode,andtheareaofthemobilityshiftwouldbe

smallanddifficulttoanalyze.Presumably,byreversingtheconditions,anECEEMmethodcouldbe

utilizedforKDdeterminationofpositivelychargeddrug‐cyclodextrininteractions.

TheKDvaluescalculatedforthedrugsDiclofenacandFolicAcidareveryhigh,andhavelarge

errorsassociatedwiththem.Thishighlightstwolimitationsofthismethod.Firstly,veryhigh

concentrationsofβ‐cyclodextrinbegintoinfluencetheelectroosmoticflowbyinterferingwiththe

buffer,whichinturnaffectsthemobilityshiftsoftheequilibriummixturesofdrugsaswellasthe

shapesofthepeaks.Thepeakstendtogetverybroadandshortathighconcentrations,andthus

shouldnotbeusedforKDcalculations.Thesecondlimitationisthesaturationoftheindividual

systems.Evenatveryhighconcentrationsofβ‐cyclodextrin,thepeaksofdiclofenacandfolicacid

continuedmovinganddidnotsaturate.Withoutsaturation(meaningallofthedrugcomplexed

29

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30

withβ‐cyclodextrin),themathematicalequationcannotbeused,asnotcexists.Inthesecases,the

KDforthisinteractionisverylarge,andtheβ‐cyclodextrinisusuallynotarelevantexcipientto

makethedrugmoresoluble.Onthisbasis,thislimitationisnotabigprobleminpracticalsettings‐

inwhichtheuseofcyclodextrinasanexcipientisbeingevaluated.

ItshouldbenotedthattheKDsforPDDA,Ibuprofen,ands‐Flurbiprofenaresmallerthanthose

previouslyreportedinSection2.1(PDDA)andpreviousworkbytheBerezovskigroup(7).Thisis

attributabletothechangeofbuffer.Ammoniumacetatewasusedforthemultiplexexperiments,

sothepHislowerthanwhenTrisAcetatebufferisused,asinSection2.1andpreviouswork.

LowerpHconditionshavebeenshowntoincreasetheaffinityofcyclodextrin‐smallmolecule

interactions(6).

Themainbenefitofthismodelisasapreliminarystudyforwhetherornotcyclodextrinwouldbe

agooddrugdeliveryagentforaspecificdrug.Although,asmentionedinsection2.1,capillary

electrophoresiscannotgiveinformationaboutstoichiometrygreaterthan1:1,itcangiveavalue

fortheapparentKDbetweenasmallmoleculeandcyclodextrin.Fordrugsthatdonotsaturate,or

shiftonlyatveryhighconcentrationsofβ‐cyclodextrin,cyclodextrinislikelyabaddrugdelivery

agent.SmallerKDvaluessuggestthepotentialofthisdrugdeliverysystem,andfurtheranalysis

usingCE‐MScanbeappliedtogiveevenmoreinformationaboutthecomplexation.

31

3.References

1.Heimbach,T.,Fleisher,D.andKaddoumi,A.(2007).Overcomingpooraqueoussolubilityofdrugsfororaldelivery.Biotechnology:PharmaceuticalAspects.5:p.157‐215.

2.Smith,W.,DeWitt,D.,andGaravito,R.(2000).Cyclooxygenases:Structural,cellularandmolecularbiology.AnnualReviewofBiochemistry.69:p.145‐182.

3.Wald,N.(1991).Preventionofneuraltubedefects.Lancet.338:p.121‐137.

4.Baur,J.andSinclair,D.(2006).Therapeuticpotentialofresveratrol:theinvivoevidence.NatureReviewsDrugDiscovery.5:p.493‐506.

5.Kata,M.andSelmeczi,B.(1987).Increasingthesolubilityofdrugsthroughcyclodextrincomplexation.JournalofInclusionPhenomenaandMacrocyclicChemistry.5:p.39‐43

6.Loftsson,T.andBrewster,M.(1996).Pharmaceuticalapplicationsofcyclodextrins.JournalofPharmaceuticalSciences.85:p.1017‐1025.

7.Mironov,G.,Okhonin,V.,Gorelsky,S.,andBerezovski,M.(2011)Revealingequilibriumandrateconstantsofweakandfastnoncovalentinteractions.AnalyticalChem.83:p.2364‐2370.

8.Fielding,L.(2007)NMRmethodsforthedeterminationofprotein‐liganddissociationconstants.ProgressinNuclearMagneticResonanceSpectroscopy.51:p.219‐242

9.Sompornpisut,P.,Deechalao,N.,andVongsvivut,J.(2002)Aninclusioncomplexofb‐cyclodextrin‐L‐phenylalanine:1HNMRandmoleculardockingstudies.ScienceAsia.28:p.263‐270.

10.Ogwu,S.,Alcala,M.,Bhardwaj,R.andBlanchard,J.(1996)Theapplicationofequilibriumdialysistothedeterminationofdrug‐cyclodextrinstabilityconstants.JournalofInclusionPhenomenaandMolecularRecognitioninChemistry.25:p.173‐176

11.HarvardApparatus.(2002).GuidetoEquilibriumDialysis.HarvardBioscience,Inc.p.2‐9.

12.Loftsson,T.,Masson,M.andBrewster,M.(2003)Self‐associationofcyclodextrinsandcyclodextrincomplexes.JournalofPharmaceuticalSciences.93:p.1091‐1097.

13.Marques,H.,Hadgraft,J.andKellaway,I.(1990)StudiesofcyclodextrininclusioncomplexesbyphasesolubilityandDSC.InternationalJournalofPharmaceutics.63:p.259‐266.

14.Kurkov,S.,Ukhatskaya,E.andLoftsson,T.(2011)Drug/cyclodextrin:beyondinclusioncomplexation.JournalofInclusionPhenomenaandMacrocyclicChemistry.69:p.297‐301.

15.Brown,S.,Easton,C.andKelly,J.(2003)Surfaceplasmonresonancetodetermineapparentstabilityconstantsforthebindingofcyclodextrinstosmallimmobilizedguests.JournalofInclusionPhenomenaandMacrocyclicChemistry.46:p.167‐173.

32

16.Kobayashi,H.,Endo,T.,Ogawa,N.,Nagase,H.,Iwata,M.andUeda,H.(2011)Evaluationoftheinteractionbetweenb‐cyclodextrinandpsychotropicdrugsbysurfaceplasmonresonanceassaywithaBiacoresystem.JournalofPharmaceuticalandBiomedicalAnalysis.54:p.258‐263.

17.Zheng,P.,Li,Z.,Tong,L.,andLu,R.(2002)StudyofinclusioncomplexesofcyclodextrinswithOrangeII.JournalofInclusionPhenomenaandMacrocyclicChemistry.43:p.183‐186.

18.Zhang,H.,Zhang,H.,Jin,W.andDing,L.(2006)Determinationofdissociationconstantsofcyclodextrin‐ligandinclusioncomplexesbyelectrosprayionizationmassspectrometry.EuropeanJournalofMassSpectrometry.12:p.291‐299.

19.Gabelica,V.,Galic,N.,Rosu,F.,Houssier,C.andDePauw,E.(2003)Influenceofresponsefactorsondeterminingequilibriumassociationconstantsofnon‐covalentcomplexesbyelectrosprayionizationmassspectrometry.JournalofMassSpectrometry.38:p.491‐501.

20.Mohamed,M.,Wilson,L.,Headley,J.,andPeru,K.(2009)Electrosprayionizationmassspectrometrystudiesofcyclodextrin‐carboxylateioninclusioncomplexes.RapidCommunicationsinMassSpectrometry.23:p.3703‐3712.

21.Neubert,R.andRuttinger,H.(2003)Affinitycapillaryelectrophoresisinpharmaceuticsandbiopharmaceutics.NewYork:MarcelDekker.

22.Krylov,S.(2007)Review:KineticCE:Foundationforhomogeneouskineticaffinitymethods.Electrophoresis.28:p.69‐88.

23.Dunayevskiy,Y.,Lyubarskaya,Y.,Chu,Y.,Vouros,P.andKarger,B.(1998)Simultaneousmeasurementofnineteenbindingconstantsofpeptidestovancomycinusingaffinitycapillaryelectrophoresis‐massspectrometry.JournalofMedicinalChemistry.41:p.1201‐1204.

33

4.Experimental

4.1ChemicalsandMaterials

Chemicalswerepurchasedfromthefollowingcompanies:β‐cyclodextrin(Sigma‐Aldrich,Canada,

EC231‐493‐2),Naproxen(Sigma‐Aldrich,Canada,N8280),4,4’‐Propane‐1,3‐DiylDibenzoicAcid

(SigmaAldrich,Canada,SS499455),Ibuprofen(SigmaAldrich,Canada,I4883),Diclofenac(Sigma

Aldrich,Canada,D6899),Phenylbutazone(SantaCruzBiotechnology,USA,SC‐204843),FolicAcid

(SigmaAldrich,Canada,F7876),(S)‐Flurbiprofen(CaymanChemical,USA,10004207),and

Resveratrol(OntarioHealthResearchInstitute).ForthePDDAmodelexperiments,a25mMTris‐

Acetatebuffer,pH7.80wasusedastheincubationandrunbuffer.Thiswaspreparedbydilutinga

stockbuffer,whichwaspreparedbydissolving12.11gofTris‐base(BioBasicInc,Canada,77‐86‐1)

and2.86mLofaceticacid(BioBasicInc.,Canada,1000)in500mLofddH2O.Multiplex

experimentsweredonein10mMAmmoniumAcetate,pH6.6.Thiswaspreparedfroma100mM

stocksolutionmadebydissolving3.85gofAmmoniumacetate(FisherScientific,Canada,639‐500)

in500mLddH2O.

ForthePDDAmodelingexperiments,theequilibriummixtureofPDDAandcyclodextrinwas

preparedintheincubationbufferwith30uMPDDAand10uMto2500uMofβ‐cyclodextrin.A1

mMstocksolutionofPDDAwasprepareddirectlybydissolving0.0028gofthedrugin10mLof

theincubationbuffer.Allsolutionswerefilteredthrough0.22‐umporesizemembranefilters

(Millipore,Nepean,ON,Canada).ThebaresilicacapillarywaspurchasedfromPolymicro(Pheonix,

AZ,USA)

Forthemultiplexseparationexperiments,theequilibriummixturewaspreparedwith15uMof

eachoftheeightdrugs:PDDA,Ibuprofen,Diclofenac,Naproxen,s‐Flurbiprofen,Phenylbutazone,

34

FolicAcid,andResveratroland10uMto15,000uMβ‐cyclodextrin.Stocksolutionsofalldrugs

(exceptphenylbutazone)werepreparedbydissolvingaweighedamountofthedrugsin10mLof

theincubationbuffer.Inthecaseofphenylbutazone,a10mMstocksolutionwaspreparedby

dissolvingtheweighedamountin95%ethanolandthendilutingintheincubationbuffer.Allof

thesesolutionswerealsofilteredthroughthe0.22umporesizemembranefilters.Thesamebare

silicacapillarywasused.

4.2ExperimentalConditions

ECEEMexperimentswerecarriedoutusingaPA800MDQPharmaceuticalAnalysisCEsystem

(BeckmanCoulter,USA)equippedwithaPDAdetector.

ForthePDDAmodelingexperiments,thefollowingconditionsweremaintained.Thesample

storageandcapillarytemperatureweremaintainedat25°C±0.5°C,anelectricfieldof26kVwith

apositiveelectrodeattheinjectionend,therunbufferwithcyclodextrinintheinletreservoir,and

theincubationbufferintheoutletreservoir.Theelectricfieldappliedcausestheelectroosmotic

flow,abulkflowoftheliquidduetothecationsformingadiffuselayerbeingattractedtowardthe

cathode.Theconcentrationofthecyclodextrinintheequilibriummixtureandtherunbufferwas

thesameforindividualECEEMexperiments.Thecapillarywas29cmlong(20cmtothedetection

window)withaninnerdiameterof50umandanouterdiameterof360um.A5.03mmlongplug

oftheequilibriummixturewasinjectedintothecapillaryfromtheinletendbyapressurepulsefor

20sat1psi.Beforeeachexperimentthecapillarywasrinsedby20psipressurewith0.1MHClfor

3min,0.1MNaOHfor3min,ddH2Ofor3min,25mMTris‐Acetatebufferfor3min,andthe

incubation/runbufferwithcyclodextrinfor1min.Theoutputdatawasabsorbanceintensityinthe

detectionpointasafunctionoftimepassedsincetheapplicationoftheelectricfield.

35

Multiplexexperimentsweredoneunderthesameconditionsexceptwitha89cmlong(80cmto

thedetectionwindow)capillary.A3.35mmplugoftheequilibriummixturewasinjectedintothe

capillaryfromtheinletendbyapressurepulsefor5secat1psi.Beforeeachexperimentthe

capillarywasrinsedby50psipressurewith0.1MHClfor5min,0.1MNaOHfor5min,ddH2Ofor

5min,10mMAmmoniumAcetatebufferfor5minandtheincubationbufferwithcyclodextrinfor

1min.

CE‐MSdatawasobtainedusingaSynapt‐G2‐HD‐MS(Waters,USA)coupledwithaPA800+

PharmaceuticalAnalysisCEsystem(BeckmanCoulter,USA).