Fast and reliable quantification of busulfan in blood plasma using two-channel liquid chromatography tandem mass spectrometry: Validation of assay performance in the presence of drug formulation excipients

ContentslistsavailableatScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

jo u r n al ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p b a

Fast and reliable quantification of busulfan in blood plasma using two-channel liquid chromatography tandem mass spectrometry:

Validation of assay performance in the presence of drug formulation excipients

Anders Mikal Andersen

, Stein Bergan

, Tobias Gedde-Dahl

, Jochen Buechner

, Nils Tore Vethe

aDepartmentofPharmacology,OsloUniversityHospital,Oslo,Norway

bDepartmentofPharmacy,UniversityofOslo,Oslo,Norway

cDepartmentofHematology,SectionforStemCellTransplantation,OsloUniversityHospital,Oslo,Norway

dInstituteforClinicalMedicine,UniversityofOslo,Oslo,Norway

eDepartmentofPediatricHematologyandOncology,OsloUniversityHospital,Oslo,Norway

a rt i c l e i nf o

Articlehistory:

Received31March2021

Receivedinrevisedform11June2021 Accepted15June2021

Availableonline17June2021

Keywords:

Liquidchromatography Massspectrometry Busulfan

Therapeuticdrugmonitoring Matrixeffects

Polyethyleneglycol

a b s t ra c t

Afastandreliablemethodbasedontwo-channel liquidchromatographycoupled totandemmass spectrometrywasdevelopedandsuccessfullyvalidatedforquantificationofbusulfan.Thedrugvehi- clepolyethyleneglycol400wasquantifiedsimultaneouslyinpatientsamples.Thesamplepreparation consistedofsimpleproteinprecipitationusingamixtureofmethanolandzincsulphatecontaining busulfan-d8asinternalstandard.Chromatographicseparationwasperformedonashortbiphenylcol- umn(30mm×3.0mm,5␮mparticles)usingastepgradientfrom30%to85%methanol,ensuring co-elutionoftheanalyteandinternalstandard.Quantificationwasperformedusingthemasstransition of264.1>151.1forbusulfanand272.1>159.1fortheinternalstandard.Usingonly20␮Lofplasma sample,thelowerlimitofquantificationwas25ng/mL.Signaltonoiseratioatthelowerlimitofquan- tificationexceeded300.Theassayperformancewasnotadverselyaffectedbymatrixeffectsoriginating fromdrugformulationexcipientsorothersamplecomponents.Thecoefficientofvariationwas≤4% andthemeanaccuracy101–108%acrossthecalibrationrange25–5000ng/mL.Chromatographicrun timewas2minand8s,allowinganeffectiverun-timeof1minand10swhenusingtwoalternating LC-channels.TheassayhasbeenimplementedinroutinepracticewithaccreditationaccordingtotheISO 15189standard,andperformswellinexternalqualitycontrolassessments.Wepresentforthefirsttime thatshortlyafteranIVinfusionofbusulfan,theplasmalevelsofpolyethyleneglycol400maybeinthe rangeof400−800mg/L.Thepresenceoftheselevelsofdetergentinpatientsamplesmayhavedetri- mentaleffectsonassayperformanceinLC–MS/MS,notlimitedtobusulfanassays.Thismaybeaconcern foranyLC–MS/MSanalysisperformedonsamplescollectedwithinthefirst24hafteranIVinfusionof busulfan.

©2021TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).

1. Introduction

Followingdecadesofuseinchemotherapy,thealkylatingagent busulfanisnowadaysmainlyusedfor myeloablationinprecon- ditioningbeforehematopoieticstemcelltransplantation(HSCT).

Forseveralyears,onlyformulationsfororaladministrationwere available.However,duringthelasttwodecades,busulfanforintra-

∗Correspondingauthorat:DepartmentofPharmacology,OsloUniversityHospi- tal,Rikshospitalet,P.O.Box4950Nydalen,0424Oslo,Norway.

E-mailaddress:[email protected](N.T.Vethe).

venoususehasgraduallybeenintroduced[1].Withoraluse,the erraticbioavailabilityofbusulfanwasaproblemwhichwasrein- forcedbytheemeticeffectofthedrug.Hepaticfirstpassandhigh plasmaconcentrationsfollowingoraldosingprobablycontributed to severe adverse effects like sinusoidal obstruction syndrome (SOS)andmucositis.Inordertoreducetreatmentrelatedmortal- ity,inourcenterasinmanyothers,therapeuticdrugmonitoring (TDM)ofbusulfanwasregularlyperformed,ideallyfromthefirst dayinorder topreventcontinuedexposuretohighconcentra- tions[2].Withtheintroductionofintravenous(IV)busulfanone mightquestionwhetherTDMwouldshowadvantageoverdosing adjustedonlytobodyweightorsurfacearea.Alargeretrospective https://doi.org/10.1016/j.jpba.2021.114216

0731-7085/©2021TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).

multicenterstudyshowedthattheprognosis,bothintermsofout- comeandtoxicity,isindeedrelatedtoanidentifiedbusulfantarget exposureincontextofIVdrugadministration[3],andasimilarcon- clusionwaspresentedinarecentreview[4].Suchstudieshavealso highlightedtheimportanceofadequatemethodsforthebioanal- ysisofbusulfanandfortheappropriatecalculationofindividual pharmacokineticcharacteristicsasprerequisitesforthesuccessful personalizationofbusulfandosing.

Dimethylacetamide(DMA)andpolyethyleneglycol400(PEG 400)arefrequentlyusedexcipientsinIVbusulfanformulations.

Each vialof 60 mg busulfancontains 3.3 mL DMAand 6.7 mL PEG 400 [5]. The toxicological aspects of IV DMA in busulfan patientshaveattractedsomeattention[6],whereasthishasnot beenaddressedforPEG400.Anotheraspectrelatestothepoten- tialuntowardeffectstheseexcipientsmayhaveontheanalytical performance when busulfan is quantifiedin blood plasma. The European Medicines Agency (EMA) guideline on bioanalytical methodvalidationstatesthatpotentialmatrixeffects causedby drugformulationexcipientsshouldbeinvestigatedasapartofthe methodvalidation[7].Anumberofanalyticalmethodsforbusul- faninbloodplasmahavebeenpublishedoverthelasttenyears [8–16],butthepotentialinfluenceofDMAandPEG400ontheana- lyticalperformancehastothebestofourknowledgeneitherbeen examinednordiscussed.

Of22analyticalmethodsinuseatTDMcentersforthedeter- minationofbusulfan(reviewedin [17]),fivewerebasedongas chromatography,fourwerebasedonHPLCwithUVdetection,and 12onLC–MS/MS.Allofthe16liquidchromatographymethodsused alkanetypereversedphasecolumns(C-8toC-18)forseparation.

Samplevolumesrangedfrom50to700␮L,onemethoduseda5␮L driedplasmaspot.Chromatographicruntimerangedfrom2to20 min.Thetimerequiredforsamplepreparationortotalturnaround timeforacompletesetofsampleswasnotlisted.Twomethods reportedusingsimpleproteinprecipitationforsamplepreparation followedbyachromatographicruntimeof2min,acombination thathasapotentialforshortturnaroundtime.

Theaimofthisstudywastodevelopaliquidchromatography tandemmassspectrometry(LC–MS/MS)methodforthereliable quantificationofbusulfaninpatientplasma,providingsufficient performance even in the low concentration range of pre-dose samplesduringonce-dailyIVdosingregimens.Aprioritywasto establishanassaysuitableforeveryday routineuse,withafast turnaroundtimeandusingasmallsamplevolume.

Wedescribehereafastandsensitivemethodforthedetermi- nationofbusulfan,usinganovelseparationmechanismandusing only20␮Lofsample.Themethodalsoallowsforthequantification ofPEG400inpatientsamplesinthesamerun.Thepresentmethod wasvalidated inaccordance withtheEMAandFDA guidelines, andithasbeenimplementedinroutinepracticewithaccreditation accordingtotheISO15189standard.

2. Materialsandmethods 2.1. Chemicalsandreagents

Busulfan(purity99.7%)forthepreparationofcalibratorsand qualitycontrols(QC)waspurchasedasacertifiedsolutionof1.000

± 0.005 mg/mL from Sigma-Aldrich (Oslo, Norway). Busulfan- d8 (purity 99 %) from C/D/N Isotopes Inc. (Quebec, Canada) was usedasinternal standard (IS). HPLC-gradeacetonitrileand methanolwereobtainedfromRathburnChemicals(Walkerburn, Scotland). Ethylenediaminetetraacetic acid (EDTA) solution 500 mmol/L,LC–MSgradeammoniumacetate,HPLC-gradewater,zinc sulphate0.10mol/L,DMAandPEG400solutionwerepurchased fromSigma-Aldrich.MobilephaseswerepreparedwithUHPLC-MS gradewaterandmethanolwith0.10%formicacidfromHoneywell

(MorrisPlains,NJ).Autonormdrug-freeanimalserumwasaprod- uctfromSeroAS(Billingstad,Norway).Autonormwasdissolvedas describedbythesupplier.

AstocksolutionofdeuteratedIS(0.05mg/mL)waspreparedby dissolving2.5mgofthesubstancein50mLofacetonitrile.Thesolu- tionwasdividedinto2mLaliquotsandstoredat−70^◦C.Onetube atthetimewasstoredat−20^◦Cforuptooneyear,andusedforthe preparationoftheprecipitationreagent.Theprecipitationreagent wasmadeupbyblending240mLmethanoland120mLzincsul- phatesolutionina500mLglassbottle.Thesolutionwascooledto +4^◦Cbeforetheadditionof200␮LofISsolution.Thisprecipitation reagentwasstoredat +4^◦Cfor upto 8weeks. Theconcentra- tionofbusulfan-d8inthesolutionwas28ng/mL,corresponding toapproximately700ng/mLofISperplasmasample.

2.2. Preparationofcalibratorsandqualitycontrolsamples

Asthestabilityofbusulfaninplasmaorserumisdependent ontemperature,thepreparationandaliquotingofcalibratorsand controlswasperformedasacontinuousprocess,i.e.thetimeat roomtemperaturebeforealiquotingandfreezingat−70^◦Cwas keptataminimum.

Thethreehighcalibrators(5000,2000and1000ng/mL)were madeupbypipettingthecertifiedbusulfansolutionintothreesep- aratevolumetricflasks(20mL,50mLand50mL,respectively)using acalibrated100␮LHamiltonsyringe.Theflaskswerefilledtovol- umewithAutonorm.Calibratorsat667,222,74.1,and24.7ng/mL werepreparedbyserialthreefolddilutionsofthe2000ng/mLcali- bratorinAutonorm.Thus,atotalofsevennon-zerocalibratorlevels andonecalibratorblank(neatAutonorm)wereincluded.QCswere madeupthesamewayfromthesamevialofcertifiedbusulfan solution.ThehighandmediumQCs(4000and1000ng/mL)were preparedbypipettingthecertifiedbusulfansolutionintotwosepa- ratevolumetricflasks(25mLand50mLrespectively),andfillingthe flaskstovolumewithAutonorm.ThelowlevelQC(50ng/mL)was obtainedbydilutingthemediumcontroltwentyfoldinAutonorm.

NeatAutonormwasusedasQCblank.AseparateQCatthelower limitofquantification(LLOQ,25ng/mL)foruseduringthevali- dationprocess,exclusively,waspreparedbydilutingthelowQC twofoldinAutonorm.Allcalibratorsandcontrolswerealiquoted andstoredinanultrafreezer(≤−70^◦C)untilthedayofuse.Cali- bratorsin300␮LaliquotsandQCsin500␮Laliquots,bothin0.5 mLpolypropylenetubeswithO-ringsealedscrewcaps(Sarstedt# 72.730.406).

ExternalQC:Ourlaboratoryparticipatedinabusulfancrossver- ificationexerciseorganizedby theCansearchlaboratory(DrM.

Ansari,Geneva,Switzerland)[17],andalsointheexternalquality assessmentschemeorganizedbytheDutchFoundationforQuality AssessmentinMedicalLaboratories(SKML,https://www.skml.nl).

CalibratorsandQCforPEG400weremadebyserialdilutionof stocksolutionsof10g/LofPEG400dissolvedinAutonorm.The stocksolutionswerepreparedbypipetting100mgofPEGsolution intotwoseparate10mLvolumetricflasks(oneforcalibrators,and oneforQCs),andfillingtheflaskstovolumewithAutonorm.Cal- ibratorlevelswere50,30,10,7.5,5.0,3.0and1.0mg/L.QClevels were50,25and2.5mg/L.Thesolutionswerealiquotedandstored at−70^◦Cuntilthedayofuse.

2.3. Samples;handlingandpreparation

Patientsamplesusedfordevelopment,validationandquality assessmentofthepresentanalyticalassaywereeitherprocessed accordingtocurrentstandardpracticeandprotocolsforbusulfan treatmentatourinstitution,oranonymizedsurplusofsuchsam- ples.Noextrasamplewasdrawnforthepurposeofthisproject.

Venousblood frompatientsweresampledin EDTAvacutainers

Table1

Conditionsforchromatography.

Starttime(seconds) Duration(seconds) Flowrate(mL/min) MobilephaseB(%) Stepname Stepvol(␮L)

0 25 0.750 30 Preelutewash 320

25 8 0.750 85 Preelutewash 100

33 50 0.400 85 Elutinganalytes 330

83 20 1.200 99 Postelutewash 410

103 10 1.000 30 Re-equilibration1 160

113 10 0.850 30 Re-equilibration2 150

123 5 0.750 30 Re-equilibration3 60

128 – 0.750 30 Runend –

Themobilephasewasdirectedtothemassspectrometerbetween42and78s.

Table2

Selectivereactionmonitoring.

Analytesandinternalstandard Precursor(m/z) Product(m/z) Collisionenergy(V) Dwelltime(ms)

Busulfan 264.1 151.1 13 100

Busulfan-d8 272.1 159.1 13 100

PEG400 344.3 89.2 20 5

Argoncollisiongas1.5mTorr.Resolution0.7Da(fullwidthathalfmaximum).RFLens30V.

withoutgel,andcentrifugedat4^◦Cfor10minat2000g.Theresult- ingplasmawasstoredinarefrigeratoruntilanalysiswithin24h.

CalibratorsandQCswerethawedatroomtemperaturefor15 minandthereafterkeptat4^◦Cforupto48h.Onesetofcalibrators andQCswasusedforuptotwoconsecutivedaysofanalysis,but fornolongerthan48hafterthawing.Timeatroomtemperature waskeptataminimum,andlimitedtothetimeofpreparationof samples.Oneachanalysisday,onefreshtubeofin-houselongterm QC(patientpool)wasthawedfrom−70^◦C.

Samplesfrompatientsreceivingbusulfanorallywereprepared undiluted.Samplesfrompatientsreceivingbusulfanintravenously werepre-preparationdilutedinAutonormcontaining5mmol/L EDTA(beforeuse,50␮Lofa500mmol/LEDTA-solutionwasadded toeach 5mLvialof Autonorm).EDTAwasusedtopreventthe formationofclotswhendilutingplasmasamples.Thedilutionsfol- lowedapredefinedprotocol.Thedilutionschemewascustomized tofitsamplesfrompatientsadministeredbusulfanintravenously asa3hinfusionaimingforaconcentrationofdrugatsteadystate (Css)of750–900ng/mL.Atotalof8–9sampleswerecollectedat each doseinterval:Pre-dose,andthen 5min,30 min,1h, 2,3, 5,7,and10hafterendofinfusion.Sample1(pre-dose),8and9 wasdilutedtwofold(200␮L+200␮L)withAutonormcontain- ingEDTA,theremainingsamplesweredilutedeleven-fold(20␮L +200␮L).Correspondingdilutionfactorswereembeddedinthe batchtemplateintheanalysissoftware.

Samplepreparation wasperformedin2mLdeepwellplates withroundwells(ThermoScientific,catno278752).Twentymicro- liters of calibrator, QC orpatient samplewas pipetted intothe wells.Then,500␮LcoldprecipitationsolutionwithISwasadded toeachwell,andtheplatewasheat-sealedwithaluminumfoil (Porvair#229572)usingaPorvairMinisealIIheatsealer(Porvair Sciences,King’sLynn,UK).Theplateswereshakenforsixminutes (1400rpm,3mmorbit;High-SpeedMultiPlateShaker,BioSan), andcentrifugedfor10min(2000g,4^◦C;Rotanta,Hettich).Subse- quently,theplatesweretransferredtotheLC–MS/MSautosampler thermostatedat10^◦C.

3. LC–MS/MSinstrumentationandanalyticalmethods

Massspectrometricconditionsfortheanalytes(busulfanand PEG 400) were optimized manually using syringe pump side- infusionofanalytesolutionsintoamobilephasestreamofrelevant compositionandflowrate.Sampleanalysiswasperformedona TranscendIILX-2UHPLC-systemincludingtwo-channelLCwith separate injectionports and exact lengthtubing (Viper Finger-

tightFittings,Thermo).Theinjectionswerealternatelydirectedto themassspectrometer,therebyallowingoverlappingchromato- graphicrunsandreducedbatchanalysistime.Massspectrometric detection wasperformed ona TSQ Quantiva triple quadrupole massspectrometer(ThermoFisherScientific).Theinletsystemwas controlledbyAriaversion2.1,andthemassspectrometerwascon- trolledusingTuneApplicationversion1.1.Ourlaboratoryhastwo identicalsystemsofthisLC–MS-MSinstrumentation.Thebusul- fanassaywasvalidatedonbothsystems,andrunsseamlesslyon thetwosystemsintheroutine.Chromatographicseparationwas performedatroomtemperature(23−25^◦C)onRaptorBiphenyl columns(30xID3.0mm,particlesize5␮m;Restek).Thecolumns were protected by a 0.5 ␮min-line filter placed between the injectorsandthecolumns.Noguardcolumnswereused.Mobile phaseswere(A) water and (B)methanol,both with0.10 %v/v formicacidand2.0mmol/Lammoniumacetate.Theanalyteswere stackedonthecolumnat30%mobilephaseB,and elutedbya stepgradientto85%mobilephaseB.Chromatographydetailsare specifiedinTable 1.Evaporation andionizationwasperformed usingelectrosprayinpositivemodewithasprayvoltageof3400 V.Nitrogengasflow:Sheathgas60AU,auxiliary gas8AU and sweepgas4 AU. Vaporizerand ion transfer tubetemperatures were470^◦Cand270^◦C,respectively.Settingsforselectivereac- tionmonitoringarelistedinTable2.Thedatawereacquiredand processedusingTracefinderSoftwareversion3.2.Thepeakarea ratiobetweenanalyteandISwasusedassignalresponse.Alin- earcalibrationmodelwithorigoexcludedand1/x²weightingwas applied.Twomicroliterssupernatantfromtheupperpartofthe wellwasinjected.AstheLC–MS/MSsystems weresetupwith 25 ␮L sample loopsfor theanalysis ofimmunosuppressants, a front volume of 20 ␮L wasused toavoid carry over. Between injections,theneedleandinjectionportwerewashedwithacetoni- trile:methanol:isopropanol50:25:25andwater:acetonitrile95:5.

Adetaileddescriptionoftheinjectionprogrammaybefoundin supplementaryfile-1.

PEG400andbusulfanmaybequantifiedsimultaneouslyinthe samerun. The massspectrometric conditions for PEG400was basedontheworkofVijayaBhaskaretal[18],withsomemodi- fications:Quantificationwasperformedusingtheprecursormass 344.3(ammonium adductoftheoligomermass326) producing thefragment89.2,andusingbusulfan-d8asIS.Qualitatively,the wholePEG400envelopewasdepictedusingprecursorionscansto thefragment89.2.

Phospholipidswerequalitativelyassessedbothbymasstransi- tions,specificorgeneral(184.1>184.1),andbyprecursorionscans

tothefragment184.1,methodologicallybasedontheworkbyXia andJemal[19].

Asa non-accreditedbackup-assay, thebusulfanmethodwas also cross-validated on two identical Xevo TQ-S micro IVD LC–MS/MSsystemsfromWaters.TheinletsconsistedofAcquity UPLCI-classsystems(binaryhighpressuremixing).Theconditions ofchromatographyontheWaterssystemswereadirectcopyofthe conditionsontheThermosystemswithtwoexceptions:1)Compo- sitionofthestrong-washsolution,and2):Asthesesystemswere lesstoleranttoinjectingsamplescontaininghighlevels(67%)of methanol,thesampleswerepostpreparationdiluted,andthesam- ples wereinjected asapartialloop injectionof5␮L.Postprep dilution wasperformedbypipetting 20␮Lofeach wellfroma platepreparedasdescribedaboveintowellscontaining500␮L of coldmobilephaseA ona new96deepwellplate.Theplate washeatsealedandshakenasdescribedabove,andplacedinthe autosamplerwithoutpriorcentrifugation.

DetailsofanalyticalconditionsontheWaterssystemsaregiven insupplementaryfile-1.AstheseWaterssystemsdonothavethe two-channelchromatography option,theeffectiverun-timeper batchwastwiceaslong.

4. Methodvalidation

Measurements of PEG400in plasma wereperformedsemi- quantitativelyafterasimplifiedone-dayvalidationprocedure.

The busulfan assay was validated with respect to limits of quantification,selectivity,carry-over,presenceofghostpeaks,cal- ibration curve,accuracyand precision,dilutionintegrity,matrix effectsandstabilityinaccordancewiththeguidelinesonbioan- alytical methodvalidation providedbytheEuropeanMedicines AgencyandtheUSFoodandDrugAdministration[7,20].Allval- idationexperiments,exceptforexperimentsrelatedtostability, wereperformedonbothoftheThermoLC–MS/MSsystems.

Analytical selectivity was assessed using ten anonymized samples from patients conditioned for HSCT without busul- fan. Selectivity was also investigated by analysis of a com- mercial QC containing 22 therapeutic drugs (amikacin, caf- feine,carbamazepine,chloramphenicol,clonazepam,cyclosporine, desipramine,diazepam,digoxin,disopyramide,ethosuximid,fle- cainide, gentamicin, haloperidol,imipramine,lidocaine, lithium, methotrexate,nortriptyline,paracetamol,phenobarbitone,pheny- toin,primidone,procainamide,quinidine,salicylate,theophylline, tobramycin,valproicacid,vancomycin;SeronormPharmacaL-2, Sero).TheISselectivitywasadditionallyinvestigatedbyanalyzing 8samples(collectedduringacompletedoseinterval)fromapatient receivingbusulfanorallyusingaprecipitationreagentwithoutIS added.

Weassessedcarry-overbetweensubsequentinjectionsbyanal- ysis of blanks after QCs spiked with analyte concentrations at the upperlimit ofquantification (ULOQ)and IS at regularcon- centration.PotentialanalyteandISresponsesintheblankswere calculatedrelativetotheresponsesinQCsattheLLOQaddedreg- ularISconcentration.

Thepresenceofpotentiallate-elutingpeaksaftertheanalysisof patientsampleswasexaminedbysequentialanalysisof47wells containingmobilephase(30%B)followingtheinjectionofabusul- fansamplefromamalepatient.Thesamesetupwasalsoperformed witha samplefromafemalepatient.Thepresence ofpotential late-elutingpeaksaftertheanalysisofcalibratorsandcontrolswas examinedbythesequentialanalysisof36wellsofmobilephase (30%B)followingtheanalysisofafullsetofcalibratorsandquality controls.

Threeseparatelypreparedcalibratorcurvesfromthesamelot of calibratorswereusedfor selectionof calibrationmodel.Cal-

ibration curveswere examinedwithrespecttotheaccuracy of back-calculatedconcentrationsvs.nominalconcentrationsateach level.Calibratorcurvesfromtwoadditionallotsofcalibratorswere usedforverificationofthecalibrationmodel.

Within-andbetween-seriesaccuracyandprecisionweredeter- minedwithspikedQCsatfourlevels.Between-series(apparent) accuracyandprecisionwerealsodeterminedwithrepeatedanaly- sisofourin-houselongtermQC(patientpool)inseriesperformed duringandafterthetimeperiodofvalidation.Signaltonoisewas calculatedattheLLOQ.Thecoefficientofvariation(CV)wasused asmeasureofprecision.

Dilutionintegritywasassessedattwodilutionlevels:by2-fold and11-folddilutionofthehighQC(4000ng/mL)inAutonormcon- tainingEDTA. Asrecommended inthe EMAguidelinesin cases whereexcipientsarelikely toberesponsibleformatrix effects, dilutionintegrityofpatientsampleswasfurtherassessedbyserial dilutionintriplicateofa patientsamplecollectedattheend of infusion,containing4052ng/mLofbusulfanand659mg/LofPEG 400.

Matrix effects were assessed using six anonymized patient samplesfromtransplanted patientsnot receivingbusulfan. The post-precipitationaddition method wasused; withanalytes at threefoldLLOQandatULOQ,andISatregularconcentrations.The matrixfactor(MF)wascalculatedversusnon-matrix(mobilephase, 30 %B), and theIS-normalized MF wascalculated as theratio betweentheanalyteMFandtheISMF.Thecompletematrixeffect setupwasrepeatedwithpatientsamplesspikedwithPEG400to alevelof9.1mg/Land45.5mg/L(equivalenttoaPEG400level of100mg/Land500mg/Lrespectively,inpatientsamplesdiluted 11-foldbeforepreparation).

Stabilityoftheprecipitationreagentstoredinarefrigerator(5–8

◦C)wasinvestigated at24 h, at4 and7 days,and at 3,4,7, 8 and12weeks.Acceptancecriterionwasabusulfan-d8-response (chromatographicpeak area)nolowerthanmedian70 %ofthe busulfan-d8-responsefroma freshlyprepared bottle.Thechro- matogramswerealsoexaminedforanyrisingsignaloftheanalyte (busulfan-d0)asafunctionofstoragetime.

Stabilityofbusulfaninthehigh,mediumandlowQCstoredin arefrigeratorwasinvestigatedfor24h,and2,3,4,5and6days.

Stabilityofbusulfaninpatientsamplesstoredinarefrigeratorwas investigatedafter2.5h,24hand3daysusingsamplescollected duringtwooraldoseintervals,atotalof15patientsamples.Sta- bilityofbusulfaninpatientsamplesatroomtemperature(23–24

◦C)wasinvestigatedfor2.5hand24husingsamplesfromtwooral doseintervals,atotalof15patientsamples.

Long-termstabilityofbusulfancalibrators,QCsandpatientpool (thein-houselongtermQC)storedat≤−70^◦Cwasinvestigatedfor upto27months.Thestabilityofbusulfanpatientsamplesstoredat

≤−70^◦Cwasinvestigatedforupto12monthsusingsamplesfrom threecompleteIVdoseintervals,atotalof22patientsamples.

Post-preparativestabilityofbusulfanpatientsamplesinarefrig- eratorforupto24hwasinvestigated.Patientsamplesfromtwo completeoraldoseintervalswereincluded,atotalof15patient samples.Stabilitywasinvestigatedbothusinginternalcalibration (quantificationperformedagainstcalibratorsonthesameplate) andbyexternalcalibration(quantificationperformedagainstcali- brationperformedattimezero).

Forcomparisonwithourpreviousin-houseHPLC-UV-method (basedon[21]),atotalof200patientsamplesfrom27patientseries and84parallelsofthein-houselongtermqualitycontrol(Lot#9, target776ng/mL)wereanalyzed.Thecomparisonwasperformed withinthequantificationlimitsfortheHPLC-UVmethod,andusing leftoverpatientsamplesstoredat-70^◦Cuntilthedayofanalysis byLC–MS/MS.

Cross-validationbetweentheThermo-instrumentsandourtwo Waterssystemswasperformedbyanalyzingcalibrators,controls

andatotalof9patientseries(67patientsamples)onthreesepa- ratedays.TheplateswerefirstanalyzedontheThermo-systems, thereaftersplittotwoseparateplates,andanalyzedonbothWaters systemsatthesameday.Instrument-relateddifferencesinboth singlesampleresultsandCss-valueswascalculated,andalsocarry- over,accuracyandprecisionontheWatersinstruments.

5. Resultsanddiscussion

Unlessstatedotherwise,theresultsarepresentedasmean± 1SD.

5.1. Chromatographicandmassspectrometricconditions

ThelowlogPvalueofbusulfan(approximately-0.5)indicates thatchromatographicretention ofthiscompoundontraditional reversedphasecolumnsischallenging.Busulfantendstoelutewith alowretentionfactor(k-value)andwithlowamountsoforganic modifierinthemobilephase.Aloworganiccontentinthemobile phasehasanegativeimpactbothoncompoundstackingonthecol- umnandontheeffectivenessoftheelectrosprayionization.Elution oftheanalyteatlowk-valuesmayalsocontributetocontamination oftheionsourcefrommatrixcomponents.Therefore,anumber of differentstationaryphasesweretested fortheirretention of busulfan.Resultsfromthetestingofchromatographycolumnsare presentedinTable3.Allofthetestedalkanetypecolumnsproduced limitedretentionofbusulfan,whereasthephenylorbiphenyltypes showed superiorretentionproperties, facilitating theelutionof busulfanathighmethanolconcentrations.Attemptsatdevelop- ingmethodsbasedonisocraticorshallowgradientelutionwere abandonedduetochromatographicseparationbetweenbusulfan andthedeuteratedIS,resultinginpooraccuracyandprecisionfor patientsamplescontaininghighlevelsofPEG400.Thisproblematic deuteriumisotopeeffectisaknownissue,andhaspreviouslybeen describedbyothers[22].Theanalytevs.ISseparationproblemwas minimizedbyusingthecombinationofashortcolumnpackedwith largeparticles(the3.0mmIDx30mmRaptorbiphenylcolumn fromRestek,with5␮mparticles)andahighstep-gradientforelu- tionofanalytes.Ashortcolumnwithlargeparticleshadadditional benefitsforthisapplicationasitreducedtherun-timebyallow- inghighflowduringloading,washingandre-equilibrationwithout producingexcessiveback-pressure(Fig.1a).Lowtemperature(no columnheating)allowedforhighermethanolcontentinthemobile phaseduringstackingofthecompoundsonthecolumn.Elutionof thecompoundsofinterestwasperformedatlowflow,facilitating aneffectiveevaporationandionizationintheionsource.Eluting bya stepgradientfrom30 to85%methanolresultedinanear completeco-elutionoftheanalyteanddeuteratedIS(Fig.1c)with ak-valueof5(calculatedbasedonvolume,nottime).Thisprin- cipleforloading,washingandre-equilibrationhasprovenrobust, andhasalsobeenadaptedtotheanalysisofatorvastatinusingthe samepairofcolumns[23].

Thechromatographicrun-timeperinjectionwas2minand8s, allowinganeffectiverun-timeof1minand10spersampleina batchwhenusingtwoalternatingLC-channels.Thestepheightof thegradientwaslimitedto85%methanoltopreventphospholipids andotherlipophiliccompoundsenteringthemassspectrometer.

Allmajorphospholipidselutedbetween1.4and1.7min,i.e.eluted towasteinthewashoutbetweeninjections(datanotshown).As thePEG400envelopecoversawiderangeofpolarities,itproved difficulttocompletelyseparatebusulfanfromPEG400.Busulfan eluted inthesame timeframeasthelower massesofPEG400, partlyco-elutingwiththeoligomerproducingaprecursoratmass 344(ammoniumadductoftheoligomermass326).Aprecursorion scantofragment89.2depictedtheelutionprofileofthewholePEG

Fig.1.Chromatography.

a.Chromatographicconditionsduringsampleanalysis.

b.Unsmoothedchromatogramsofblankplasma,LLOQ[25ng/mL],andapatient samplecontaining49ng/mLofBusulfan.

c.Chromatogramsofabusulfan(BUS)calibrator[667ng/mL]andtheISBUS-d8, showinganegligiblechromatographicseparationofthetwopeaks.

400envelope,andalsothecharacteristic44amuspacingbetween thePEGoligomers(Fig.2).Toreducetheionsuppressionandthe potentialionsourcecontaminationfromPEG400,thesamplesfrom patientsreceivingIVbusulfanwerepre-preparationdilutedwith Autonorm.

Conditionsforthemasstransitions arepresentedinTable2.

Fragmentationoftheprecursoratm/z264.1producedamajorfrag- mentatm/z151.1(272.1>159.1forbusulfan-d8).Noqualifierions wereusedinthismethod,thedwelltimeforbusulfanandtheISwas settoobtain20–25datapointsacrossthechromatographicpeaks.

PEG400(344.3>89.2),producingmorethan2E5countspersecond (CPS)atLLOQ,wasgranted5msofdwelltime.Thecombinationof

Table3

Chromatographycolumnstestedfortheretentionofbusulfan.

Supplier Column Particles MobilephaseB(%)

1)ThermoFS AccucoreC8 2.6␮msolidcore 10

2)“ AccucorePolarPremium “ 10

3)“ AccucoreAQ “ 10

4)“ AccucorePhenyl-X “ 10

5)“ AccucorePFP “ 12

6)ACELTD ACE3Phenyl 3.0␮mfullyporous 25

7)Restek RaptorBiphenyl 2.7␮msolidcore 30

8)Restek RaptorBiphenyl 5.0␮msolidcore 42

Chromatographicconditionsused:Methanolcontent(%mobilephaseB)adjustedtoachievearetentiontimeofbusulfanof1.0+/−0.1min(retentionfactorof4)under isocraticconditions.

Columns1to7:2.1mmIDx50mm.Flow0.5mL/minat60^◦C.

Column8:3.0mmIDx30mm.Flow0.5mL/minatroomtemperature(23−25^◦C).

Fig.2.PEG-400inpatientsamples.

a.Chromatogramsfromapatientsampletaken5minafterendofinfusion.Showing thebusulfanIS,andthewholePEG-400envelope,depictedbyaprecursorionscan frommass130tomass680measuringthefragment89.2.ThePEG-400signalwas attenuatedx100tofitonscale.

b.Themassesproducedfrom0.9to1.3minbytheprecursorionscaninFig.2a, showingthecharacteristic44amuspacingbetweenthePEGoligomers.

thesemassspectrometricconditionsandthehighmethanolcon- tentinthemobilephaseallowedfortheinjectionofalowsample volume(equivalentto77nLofplasma)whilestillproducingasig- naltonoiseforbusulfan(ascalculatedbytheTracefindersoftware) exceeding300attheLLOQ(Fig.1b).

5.2. Samplepreparation

AsthebusulfanassayweretorunonLC–MS/MSsystemsrou- tinely analyzing 200samplesperdayfor immunosuppressants, thesample preparationwasdesigned tofitthesame formatas ourimmunosuppressants-assay[24].Theprecipitationreagentwas alsothesameinthetwoassays,exceptfordifferentinternalstan- dards.SamplesfrompatientsreceivingIVbusulfanweredilutedto decreasethecontentofPEG400(severalhundredmg/L,Fig.3)in

thefirstsamplesaftertheendofinfusion.Pre-preparationdilution waspreferredoverpost-preparationdilutionastheformerpro- ducedmoreconsistentISresponsesinthepre-validationtests(data notshown).Inourexperience,theISresponseisavaluablediag- nosticparameterinaroutineassay.Dilutionandpipettingontothe 96wellplatewasperformedfollowingapredefined,colour-coded platemap(Showninsupplementaryfile-2).Thedilutionscheme wascustomizedtofitsamplesfrompatientsadministeredbusulfan intravenouslyasa3hinfusionaimingforaCssof750–900ng/mL.

Foranassayrequiringafastturnaroundtime,samplepreparation basedonasinglestepproteinprecipitationon96wellplates,and adirectinjectionofthesupernatantwastheobviousmethodof choice,avoidingtime-consumingsolventevaporationandrecon- stitution.Duringthefirstyearofroutineuse(59analysisdays),the medianturnaroundtimefromthestartofsamplepreparationuntil theanalyticalresultsweresignedforandreleasedfromtheinstru- mentwas1h56minforanalysiscontainingonepatientseries, and2h17minforanalysiscontainingtwopatientseries(9sam- plesperpatient).Dansoetalpresentedanassaywithextremely fastchromatographicruntime,andatotalturnaroundtimeofless thanonehourforacompletesetofsamples[9].Thissetuphowever requiresequipmentnotcommonlyfoundinroutinelabs,andalso theevaporationof3mLofn-butylchloridepersamplefollowedby reconstitutionbeforeinjection.

5.3. QuantificationofPEG400inpatientsamples

Severalstudiesonthepotentialionsuppressioneffectsfrom drugformulationexcipientshavebeenpublished[25,26],butto ourknowledgethisissuehasnotbeendiscussedinrelationtoTDM ofIV-busulfan.Asatoolinthedevelopmentofthebusulfanassay, wevalidatedthequantificationofPEG400inthepatientsreceiving IV-busulfan.QuantificationofPEG400andbusulfanwasdoneinthe samerun,usingbusulfan-d8asinternalstandardforbothanalytes.

ThequantificationofPEG400inplasmawasvalidatedforthe LLOQ, carry-over, within day accuracy and precision. The vali- dationwasperformedonbothThermoLC–MS/MSsystems and the results were considered satisfactory for performing semi- quantitativemeasurementsofPEG400inpatientsamples(Data showninsupplementaryfile-3).

Shortly after end of infusion, the PEG-400 concentration in plasmawas400−800mg/L(Fig.3).Theeliminationof PEG400 paralleledthatofbusulfanwithahalf-lifeof1.6–3.0h,inlinewith thefindingsbyShaffer[27].Thisparalleleliminationcouldentaila specialriskastheionsuppressioneffectofPEG400islikelytobe concentration-dependentanditcanthusexertadecreasinginflu- enceonthesignalresponsealongthetimecurve.Ifnotadequately correctedby theIS, sucha systematically varyingmatrix effect wouldleadtoincorrectcalculationsofpharmacokineticparame- ters[25,26].Larger[25]createdadecisionaltreethatmaybea

Fig.3.EliminationofbusulfanandPEG-400inpatients.

Patient1,2,3,4and6wereadministeredbusulfanintravenouslyasa3hinfusionaimingforaCssof900ng/mL.Patient5hadatargetCssof750ng/mL.Busulfanand PEG-400werequantifiedsimultaneouslyinthesamerun.

usefultoolinrevealingsignalsuppressionissuescausedbydrug formulationexcipientsinLC–MS/MSanalyticalmethods.

PEG 400 is considered to be relatively non-toxic, but PEG- containing busulfan-formulationsfor IV useare amongthefew drugsthatmayproducecumulativePEGexposuresthatapprox- imates those thatare associatedwithtoxicity (mainlyrenal)in humans[28].

5.4. Selectivity

NoanalyteorISinterferenceswereobservedinbusulfan-free samplesfromtransplantpatients(n=10)andneitherinthecom-

mercialQCwiththerapeuticdrugs.Nointerferencefrombusulfan ormetaboliteswasobservedwithrespecttotheISsignalinthe8 patientsamplespreparedandanalyzedwithoutIS.

5.5. Carry-over

Thecarry-overwasunacceptablyhighduringtheearlystages ofmethodsdevelopment,probablyasaresultofthecombination ofawide-ranging calibrationcurveandtheinjectionof asmall samplevolumeintolargesampleloops.Thecarry-overwasmini- mizedbyusingafrontvolumeof20␮Ltowashthesamplepath beforeloading2␮Lofsampleintotheloop.Adetaileddescription

Table4

Calibrationcurvesandlinearity.

Nominal[ng/mL] Run1 Run2 Run3 Accuracy(n=3) Run1 Run1

Callot1 Callot1 Callot1 Callot1 Callot2 Callot3

24.7 25.1 25.1 25.1 101.6 25.1 24.7

74.1 71.7 71.0 71.8 96.5 71.2 74.1

222 211 213 212 95.5 218 220

667 665 678 662 100.2 666 671

1000 1065 1006 1025 103.2 1012 1000

2000 1999 2070 2075 102.4 2011 2004

5000 5039 5030 5028 100.6 5131 5007

Slope 0.00152 0.00150 0.00151 0.00145 0.00152

Intercept −0.00375 −0.00319 −0.00251 −0.00025 −0.00317

RSQ 0.9999 1.000 1.000 1.000 0.9999

of theinjectionprogrammaybefoundinsupplementaryfile-1.

Usingthesesettings,thecarry-overbetweensubsequentinjections wasequaltozero(undetectable)forbusulfanandbusulfan-d8(n= 6)

5.6. Presenceofghostpeaks

Nolate-elutingpeaksweredetectedfollowingtheinjectionof patientsamplesnorafterthecalibratorsandqualitycontrols.

5.7. Calibrationcurvesandlinearity

Usingsevennon-zerocalibratorsincludingLLOQandULOQ,the simplestcalibrationmodelthatadequatelydescribedtherelation- ship betweentheinstrument response (peakarearatioBUS/IS) and the busulfanconcentrationwas a linearcalibration model, weighted1/x².Origowasnotincludedinthemodel.Thecalibration modelprovedrobustoverseverallotsofcalibrators(Table4).The accuracies ofallindividualback-calculatedconcentrations were within94.8%–107%.

5.8. Accuracyandprecision

ThemeanaccuracyofQCsamplesrangedfrom101to108%, andCVswere≤3.4%,verifiedontwoLC–MSsystems(detailsin Tables 5aand5b).During theperiodofvalidation ofthebusul- fanassay,atotalof84determinationsover24analysisdaysofthe in-houselongtermqualitycontrol(Lot#9,target776ng/mL)pro- ducedameanapparentaccuracyof98.7%andaCVof3.5%.Duringa totalof64analysisdaysoveraperiodof18monthsofroutineuse, andwithsevendifferentoperators,thein-houselongtermqual- itycontrol(Lot#10,target955ng/mL)producedameanapparent accuracyof101%andaCVof3.7%(n=64).Analysisoftheexter- nalqualitycontrolseriesfromtheCansearchlaboratoryproduced ablankresultforthenullsample,andamedianaccuracyof95.3% (range92.9%–99.6%)forthespikedsamples,withdeviationsevenly distributedovertheconcentrationrange125ng/mLto4000ng/mL.

The2019yearreportfromSKMLshowedatruenessof+8.1%and aprecisionof1.4%.

Externalqualitycontrolschemesareimportanttoolsindocu- mentingandmaintainingassayquality.Wefoundnoquantifiable amountsofPEG400inanyofthesamplesfromtheexternalquality control series from the Cansearch laboratory[17] or the sam- plesreceivedfromSKMLin2019.Theseobservationssuggestthat noneoftheexternalcontrolsoriginatedfrompatientsreceiving IV-busulfan.Theclinicalrepresentativenessoftheexternalcontrol materialwouldbeimprovedifitalsoincludedpatientsamplescol- lectedduringthefirsthoursafteranintravenousbusulfaninfusion.

Potentialmatrixeffectchallengesthatwillnotbedetectedbycom- parisonofspikedsampleresultscouldthenhavebeenidentifiedby participationintheproficiencytestingprogram.

5.9. Limitsofquantification

Theupperlimitofquantificationwassetat5000ng/mL,and LLOQwassetat25ng/mLasthisrangewasconsideredadequate byusersofourbusulfananalyticalservice.Theresultsforaccu- racy,precision,selectivityandcarry-overallallowedforanLLOQ at25ng/mL.Signal-to-noiseratiosatLLOQwere≥300(n=12in twoseries),suggestingthatanLLOQlowerthanthismaybepos- sible(Fig.1b).Thisoptionwasnotpursuedfurtherinthismethod validation.

5.10. Dilutionintegrity

ThehighlevelQCanalyzedundilutedwasmean4049ng/mL (n=8,CV2.9%).Thesamecontroldilutedtwofoldbeforeanalysis wasmean4017ng/mL(n=8,CV2.1%)anddilutedeleven-foldthe measuredconcentrationwasmean3836ng/mL(n=8,CV1.9%).

Ameanrecoveryof95.9%forthesampledilutedeleven-foldwas wellwithintherecoverylimit85–115%asrecommendedbyEMA.

Matrixeffectsfromdrugformulationexcipientsmaypotentially compromisethedilutionintegrityofpatientsamples.Aspointed outbyLargeretal.[25]andintheEMAguidelines[7],spikingexper- imentsmaynotfullyrevealthepotentialforionsuppressioncaused byformulationagentsduetometabolismorinvivodegradationof theformulationexcipients.Asabout25%ofanintravenousdose ofPEG400ismetabolized[27],dilutionexperimentswithactual patientsamplescollectedshortlyafterendofinfusionshouldbe performedtorevealdilutionorsuppressionissueswithbusulfan patientssamples.Fig.4showsstrongionsuppression,calculated asrelativeISarea(ISareaofsamplex100/meanISareaofcalibra- tors).IntheundilutedsampletherelativeISresponsewasmean34

±6%,risingwithincreasingdilutionfactorto93±4%atadilution factor11.Thestrongsuppressioneffectdidnot,however,haveany negativeimpactonthequantificationofbusulfan,indicatingthat theionsuppressiononthebusulfansignalisfullycorrectedbythe co-elutingIS.

5.11. Matrixeffects

Significant ion enhancement (matrix factor 1.2 ± 0.2) was demonstratedforbusulfanathighanalyteconcentration.Thiswas howevercorrectedbytheIS.TheIS-normalizedmatrixfactorwas 1.00±0.04atlowanalyteconcentrationand0.98±0.06athigh analyteconcentration.TheadditionofPEG400toalevelof9.1mg/L and45.5mg/Lproducednomatrixeffects,butattenuatedtheion enhancementathighanalyteconcentrationtoamatrixfactorof 1.00±0.02.TheIS-normalizedmatrixfactorinthesamplesspiked with45.5mg/LofPEG400(equivalenttoaPEG400levelof500mg/L inapatientsamplediluted11-foldbeforepreparation)was0.96± 0.07(CV7.5%)atlowanalyteconcentrationand0.94±0.08(CV 8.9%)athighanalyteconcentration.Theseresultsindicatethatthe

Table5a

AccuracyandprecisiononLC–MS/MSSystem1.

Withinseries Betweenseries

Nominalconc.(ng/mL) Meanaccuracy(%) CV(%) n Meanaccuracy(%) CV(%) n[numberofseries]

25 104 1.9 6 106 1.5 9[3]

50 108 3.5 6 105 2.9 9[3]

1000 105 3.2 6 103 1.1 9[3]

4000 102 1.3 6 102 2.0 9[3]

Table5b

AccuracyandprecisiononLC–MS/MSSystem2.

Withinseries Betweenseries

Nominalconc.(ng/mL) Meanaccuracy(%) CV(%) n Meanaccuracy(%) CV(%) n[numberofseries]

25 107 2.0 6 106 2.3 9[3]

50 108 3.4 6 106 2.9 9[3]

1000 101 1.6 6 101 1.5 9[3]

4000 101 3.6 6 101 2.1 9[3]

Fig.4.Dilutionintegrity.

Apatientsamplecollectedattheendofinfusion,containing4052ng/mLofbusulfan and659mg/LofPEG400,wasseriallyserialdilutedinautonormbeforesample preparation.TheexperimentwasperformedintriplicateontwodifferentLC–MS/MS instruments.Dataarepresentedasmean±1SD,n=6.

eleven-foldprepreparationdilutionofpatientsamplesissufficient toavoidsignificantionsuppressionbyPEG400.

DMAelutesat0.4mininthisassay,i.e.towastebeforetheMS isswitchedin-line.Spikingexperimentsforstudyingsuppression effectsfromDMAwasthereforenotincludedinthisstudy.

While formulationsof busulfanforIVadministrationcontain detergentsgivingrisetomatrixeffects,acommerciallyavailable 13C-based IS would probably facilitate robustquantification of busulfan by providing a betterchromatographic overlap under more flexiblegradientsthanwhat ispossiblewithd8-busulfan, whichcurrentlyistheonlycommerciallyavailablebusulfanisotope IS.

5.12. Stability

Thestabilityofbusulfaninplasmaistemperaturedependent [29].Our labanalyzes40–60busulfanpatientseriesperyearas a servicefortheclinicalroutine.Tofacilitateroutineuseofthis assay,weprefertopreparebatchesofready-to-useseriesofcali- bratorsandcontrolsinsufficientquantitiesforatleastoneyear.To maximizelongtermstability,thesebatchesarestoredat≤−70^◦C, hencewedidnotvalidatestabilityinordinaryfreezers(−18to−20

◦C).Storingorhandlingbusulfancalibratorsandcontrolsatroom temperatureshouldbeminimized,thisstoragetemperaturewas thereforevalidatedonlytoalimitedextent.

BusulfanQCsstoredinarefrigeratorforupto6daysshowed alossof2.1±1.8%at48h,4.3±1.9%at3daysand9.5±2.8% at6days(144h)(n=3ateachconcentrationlevel).Althoughthe stabilitywasdocumentedtobe6daysinarefrigeratoraccording totheformalacceptancelimit(lossoflessthan15%),intheroutine laboratorytherecommendedshelflifeinarefrigeratorwassetto 48h,or2consecutiveanalysisdays.

Busulfan patient samples in plasma stored in a refrigerator showedalossof1.3±5.4%at2.5h,1.9±5.1%at24hand8.3±4.5

%at3days(72h)(n=15ateachtimepoint).Intheroutineanaly- sissetting,therecommendedshelflifeforpatientplasmasamples storedinarefrigeratorwassetto24h.Storageforupto72hwas allowed,thenconsideringanexpected8%lossofbusulfan.

Busulfanpatientsamplesinplasmastoredatroomtemperature (23–24^◦C)showedalossof2.5±5.0%at2.5hand23.4±3.5%at24 h(n=15ateachtimepoint).Intheroutineanalysis,themaximum shelflifeforpatientplasmasamplesstoredatroomtemperature wassetto2.5h,witharecommendationtominimizeanystorage ofbusulfanpatientsamplesinplasmaatroomtemperature.

BusulfanQCsatthreelevelsstoredinanultrafreezershoweda lossof1.5±2.1%after12months,1.4±3.4%after15months, 0.1±4.9%after20monthsand0.2±6.7%after27months(n= 18ateachtimepoint).Theformalmaximumshelflifeforquality controlsstoredinanultrafreezerwassettotwoyears(24months).

Busulfancalibratorsstoredinanultrafreezershowedalossof 3.7±2.5%after12months,1.6±3.8%after15months,3.1±3.9

%after20monthsand5.5±4.9%after27months(n=21ateach timepoint).Therecommendedshelflifeforcalibratorsstoredinan ultrafreezerwassettooneyear(12months).Theformalmaximum shelflifeforcalibratorsstoredinanultrafreezerwassettotwoyears (24months).

The QCs were more stable during long term storage in an ultrafreezerthanthecalibrators,eventhoughtheywereprepared simultaneouslyandinthesamematrix.Theonlydifferencebeing thefillgradeofthetubes,60%forthecalibrators(300␮Laliquots in0.5mLtubes)vs100%fortheQCs(500␮Laliquotsin0.5mL tubes).

Thein-houselongtermQC(patientpool,Lot#9,target776) storedat≤−70^◦Cshowedalossof2.1±1.4%(n=12)at26months.

Lot#10,target955ng/mLshowedalossof0.7±1.6%at1month, 0.0±0.7%at10monthsandanincreaseof1.0±2.1%at17months (n=6ateachtimepoint).Theformalmaximumshelflifeforthein-

houselongtermQCstoredinanultrafreezerwassettotwoyears (24months).

Busulfanpatientsamplesstoredat≤−70^◦Cshowedalossof1.8

±4.5%after12months(range−11.2to+5.0%,n=22).Theshelf lifeforpatientsamplesstoredinanultrafreezerwassettooneyear (12months).

Inthepresentstudy,patientsamples(singlesamplesorpools) weremorestableduringlongtermstorageinultrafreezersthan whathavebeenreportedinotherstudies.Balasubramanian[30]

foundalossof7%afteroneyearand8%aftertwoyears.Choong [17]foundalossof14%aftertwoyearsand15%afterfouryears.The reasonsforthesediscrepanciesremainunknown,butreferringto ourfindingsregardingthestability-differencesbetweencalibrators andcontrols,itistemptingtospeculatethatfillgradeinthetubes usedforstoragemaybeafactor.

Preparedpatientsamplesofbusulfanwerestableinarefrig- erator for up to 24 h. Reanalysis using internal calibration (quantification performed against reinjected calibrators on the sameplate)producedanaccuracyof98.3±4.9%(n=15).Reanal- ysisusingexternalcalibration(quantificationperformedagainst calibrationperformedattimezero)producedanaccuracyof97.5± 4.7%(n=15).Giventhattheplatewithpreparedbusulfanpatient samplesisstoredcold,samplesontheplatemaybereinjectedand quantifiedforupto24hwithoutrunninganewcalibration.Also theplatemay,ifnecessary,bestoredundercoolconditionsforup to24hawaitinganalysis.

Repeatedfreezingandthawingofpatientsampleswasnotval- idatedbyus.Nadella[15]foundbusulfantobestableforatleast 5freeze/thawcycles,Danso[9]foundbusulfantobestableforat least6freeze/thawcycles,andFrench[11]foundbusulfantobe stableforatleast8freeze/thawcycles.

5.13. MethodscomparisonUHPL-MSMSvsHPLC-UV

Themeandifferencebetweentheanalyticalmethods(UHPLC- MSMSvsHPLC-UV)for200patientsampleswas+0.5%witha95

%confidenceintervalfrom−2.5%to+3.4%.TheHPLC-UVin-house longtermQCanalyzedbyLC-MSMSproducedaninaccuracyof−1.3

%witha95%confidenceintervalfrom−2.1%to−0.6%(n=84).

5.14. Cross-validationonwatersacquityUPLCI-class/XevoTQ-S systems

Busulfanlevelsinthepatientsamplesweremean4.5%higher (range-12.8%to+15.1%,n=67)whenthesamesampleswereana- lyzedontheWaterssystems.TheCss-valuescalculatedbasedon theresultsfromtheWatersinstrumentswerealsoslightlyhigher, mean difference+3.9 %(range+2.5 %to+5.6 %,n=9 foreach instrument).Thesedifferenceswerehoweverconsideredtobeof minorimportanceinabusulfanTDMsetting.Carry-overwasunde- tectable,accuracyrangedfrom101%to105%fortheQCsandallCVs were≤5.0%.Basedontheseresults,busulfananalysisonthetwo Waterssystemswasapprovedasanon-accreditedbackup-assay.

5.15. Clinicalapplication

Thepresentassayhasbeenappliedinclinicalroutinefortheper- sonalizationofbusulfandosinginatotalof113pediatricandadult patientswhowereadministeredIVbusulfanforpre-conditioning priortoHSCT.Accordingtocurrentprotocols,busulfanisgivenfor fourdaysasa2–3hinfusion.Individualpharmacokineticparame- tersarecalculatedbasedontheanalysisofninesamplescollected during the first dose interval. The reliability and speed of the presentassayallowedresultsincludingdoseadjustmentrecom- mendationstobereportedwellinadvanceofthenextdose.When largedoseadjustmentsweremade(>20%),attainmentoftarget

concentrationscouldbeconfirmedfollowingsubsequentsampling andanalysis.Therobustnessofthemethodallowedavailabilityof suchservicesevendaysperweek.

6. Conclusions

Wehavedevelopedafastandsimplemethodforquantifying busulfanacrossawideconcentrationrangerequiringonly20␮Lof bloodplasma.Themethodhasbeenvalidatedinaccordancewith theEMAandFDAguidelines,toourknowledgethefirstbusulfan assayvalidationthattakesintoaccountthepotentialionsuppres- sioneffectsfromdrugformulationexcipients.Theassayperforms well across instrument platforms, it has been implemented in routinepracticewithaccreditationaccordingtotheISO15189stan- dard,andperformswellinexternalqualitycontrolassessments.

Authorstatement

Anders MAndersen: Conceptualization,Investigation,Writ- ing-originaldraft,Methodology.SteinBergan:Conceptualization, Investigation,Writing - original draft, Methodology, Resources, Supervision. Tobias Gedde-Dahl: Conceptualization, Writing - review&editing,Resources.JochenBuechner:Conceptualization, Writing-review&editing,Resources.NilsToreVethe:Concep- tualization,Investigation,Writing - originaldraft,Methodology, Supervision.

Previouspresentationofmanuscript None.

Funding None.

DeclarationofCompetingInterest

Theauthorsreportnodeclarationsofinterest.

Acknowledgments

TheauthorsarethankfultoThaiTran,AnneRandiGryvilland therestoftheskillfulstaffattheanalyticalpharmacologyunitat Rikshospitalet,OsloUniversityHospitalfortheirimportantcontri- butionstolaboratoryorganizationandoperations.

AppendixA. Supplementarydata

Supplementarymaterialrelated tothis article canbe found, in theonline version, at doi:https://doi.org/10.1016/j.jpba.2021.

114216.

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