Rapid and sensitive LC-MS/MS method for the analysis of antibiotic linezolid on dried blood spot.

ContentslistsavailableatSciVerseScienceDirect

Journal

of

Pharmaceutical

and

Biomedical

Analysis

jo u r n al h om epag 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

Rapid

and

sensitive

LC–MS/MS

method

for

the

analysis

of

antibiotic

linezolid

on

dried

blood

spot

Giancarlo

la

Marca

_,

_Fabio

_Villanelli

_,

_Sabrina

_Malvagia

_,

_Daniela

_Ombrone

_,

_Silvia

_Funghini

_,

Marina

De

Gaudio

_,

_Stefania

_Fallani

_,

_Maria

_Iris

_Cassetta

_,

_Andrea

_Novelli

_,

_Elena

_Chiappini

_,

Maurizio

de

Martino

_,

_Luisa

_Galli

a_{MassSpectrometryLaboratory,ClinicofPediatricNeurology,A.MeyerChildren’sHospital,Florence,Italy} b_{DepartmentofPharmacology,UniversityofFlorence,Italy}

c_{DepartmentofSciencesforWomanandChild’sHealth,UniversityofFlorence,Italy}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11January2012

Receivedinrevisedform4April2012 Accepted7April2012

Available online 16 April 2012 Keywords:

Linezolid Antibiotics

Liquidchromatography–massspectrometry Driedbloodspot

a

b

s

t

r

a

c

t

Linezolidisanewdrugfromtheoxazolidinoneclassofantibioticsusedagainstmycobacteriaand

multi-drugresistant(MDR)Gram-positivebacterialinfections,whichmayarealsoglycopeptide-resistant.The

drugusageinpediatricageneedsanaccuratedrugmonitoringforeffectivepatientmanagement.The

aimofthisstudywastoevaluatetheuseofdriedbloodspot(DBS)specimenstodeterminatelinezolid

levelsduringtreatment.AdvantagesofDBSincludeshortcollectiontime,lowinvasiveness,easeandlow

costofsamplecollection,transportandstorage.TheanalysiswasperformedinLC–MS/MSoperatingin

positiveionmodeandmultiplereactionmonitoring(MRM)mode.Thecalibrationcurveinmatrixwas

linearintheconcentrationrangeof1–100mg/Lwithcorrelationcoefficientvalueof0.9987.Intradayand

interdaycoefficientsofvariationwerewithin3.6%and13.0%,respectively.Wealsotestedthethermaland

temporaldrugstabilityindriedbloodspotsatfourdifferenttemperaturestoevaluatetherisksofsample

deliveryindifferentconditions.Theshorttermstabilitystudiesshowedthatlinezolidconcentration

remainedstableforatleastonemonthunderalltheconditionstested.

Thisnewassayhasfavorablecharacteristicsbeinghighlypreciseandaccurateandallowsafastlinezolid

analysiswithatotalruntime22minlong,ingradientanalysis.ConcentrationdataforplasmaandDBS

samplesfrompatientsaftertreatmentwerecomparedshowingagoodcorrelation.

CorrelationbetweenDBSdataandserumsamplesmeasuredbyHPLC–UVwassatisfactory.

Thebenefitforpatientsistheabilitytomonitorthetreatmentwithasimpleandconvenientsample

collectionathome.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Linezolid is the first member of a new class of antibiotics, the oxazolidinones [1]. Linezolid exhibits a broad spectrum of activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS), glycopeptide-resistant enterococci and penicillin-resistantStreptococcuspneumoniae[2,3].Itisalsoactive againstmycobacterialspecies,includingMycobacterium tuberculo-sisandNocardiaspp[2].Anadvantageoflinezolidonglycopeptides intheclinicalpracticeisitsavailabilityasintravenous(iv)andoral

∗ Correspondingauthorat:DepartmentofPharmacology,UniversityofFlorence, VialePieraccini,24,50134Florence,Italy.Tel.:+390555662988;

fax:+390555662489.

E-mailaddresses:g.lamarca@meyer.it,giancarlo.lamarca@unifi.it(G.laMarca). 1 _{Theseauthorscontributedequallytothiswork.}

formulationswhich allowtheswitchtoanoral treatmentafter inductionivtreatment.

IntheUnitedStates,linezolidwaslicensedbytheFDAinadults and childrenin 2002.On thecontrary,in mostEuropean coun-trieslinezoliduseinthepediatricsettingremainsoff-labelandthe clinicalexperiencewiththisantibioticisstilllimited[2].Recent data suggestthat linezolidis a safeand effectiveagent for the treatmentofseriousGram-positivebacterialinfectionsinneonates andchildren,however,atpresent,linezolidisreservedforthose neonatesandchildrenwhoareintoleranttoorfailconventional agents [2].Linezolid resistance even though is rare, withrates lowerthan0.1%,itisalready describedinthepediatric popula-tion,sothis treatmentshouldbechosenforselectedconditions [2].

Alinezolid-containingregimencanbealsoa valuableoption for treating MDRand extensively drug-resistanttuberculosisin childrenaswellasdisseminatednon-tuberculousmycobacterial infections[2,4].

0731-7085/$–seefrontmatter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.04.007

Mostinformationregardingthepharmacokinetic(PK)profile, efficacy,andtolerabilityoflinezolidisderivedfromadultstudies [3].

RecentresultsininfantsandchildrenindicatethatPKof line-zolidisstronglyage-relatedsincechildren<12yearshaveafaster clearanceandshortereliminationhalf-lifethanchildren>12years andadults.Moreover,therearedataonPKininfants<1yearofage andinpreterminfantsbutresultshavealimitedsignificancedueto thelownumberofsubjectsevaluated.Asaresult,thereisan agree-mentthatlinezolidshouldbeadministeredatadosageof10mg/kg every8hinchildren≥1week-11yearsofageandatadosageof 10mg/kgevery12hinpreterminfants<1weekofage[3].Since linezolidisknowntobe100%availablefollowingoral administra-tion,thedosagedoesnotneedtobechangedwhenswitchingivto oraladministration[5].However,linezolidPKstudiesinchildren showedaninter-individualvariabilityinchildren,particularlyafter oraladministration[6,7].

Therefore,atherapeuticaldrugmonitoring(TDM)maybevery usefulinchildrenreceivingoraladministrationofthisdrug.

ThepublishedHPLC–UVandLC–MS/MSmethodsprovedtobe preciseandaccurate,butrequiredlargevolumeofbiologicalfluids andarethereforeunsuitableforsamplecollectioninneonatesin whomobtainingbloodsamplesisnotconvenientorpossible.

Anattractingandpotentialmethodtoeasilyobtainsamplesfor PKstudiesandTDMinchildreniscollectionofwholeblood sam-plesontofilterpaper(driedbloodspotorDBS)[7–12].DBSare commonlyusedforneonatalscreeningofmetabolicdiseases,cystic fibrosisandhypothyroidisminmicro-bloodsamples.Somestudies recentlyevaluatedtheuseofDBSforTDMandtoxicologyinadults [7–12].Someauthors,alsofromourgroup,appliedtheDBS tech-niquetoevaluatedrugconcentrations innewborns,infantsand children[7–12].ThesereportsshowedthattheaccuracyofTDM studiesusingDBSiscomparabletothatoftraditionalTDMstudies onplasmawhichrequirelargerbloodvolumes.

InthepresentstudywedevelopedandvalidatedaLC–MS/MS basedmethodonDBSforthequantificationoflinezolidinchildren’s bloodsamples,anditwascomparedwithapreviousvalidatedHPLC methodusedinareferenceclinical–toxicologicallaboratory. 2. Experimental

2.1. Patientsandsamplescollection

Patientswereenrolledinthisprospective,open-labelstudy fol-lowed fromthe PediatricInfectious Diseases Unit,AnnaMeyer Children’sUniversityHospital,Florence,Italy.Onlychildrenwith infectionsunresponsivetotreatmentandwhoneedtobetreated withlinezolid for clinical reasons were enrolled.Paired serum andDBSsamplesbeforeand/orafteradministrationoflinezolid (10mg/kgevery8hadministeredintravenouslyororally). Sam-pleswerecollectedafter3daysfromstartinglinezolidhematocrit wasobtainedfromeachpatientsimultaneouslytoserum/DBS col-lection.Inallindividualslinezolidwasusedadd-onmedication. ThestudyprotocolwasapprovedbythelocalEthicalCommittee andwritteninformedconsentwasobtainedfromchildren’s par-ents/guardians.

2.2. Standards

ChemicalstandardoflinezolidwaskindlysuppliedbyPfizer (Groton,CT,USA).Stocksolutionof2g/Lwasmadeinwaterand storedindifferentaliquotsat−20◦_{C.Dilutionsweremadeusing} HPLCgradewater.Allchemicalsandsolventswereofthehighest purityavailablefromcommercialsourcesandusedwithoutany furtherpurification.

2.3. Samplepreparation

Bloodspotsampleswerestoredat4◦Cinasealedplasticbag containingdesiccantuntilanalysis.One32mmdiameterdisk (con-tainingabout3.3–3.4␮Lof blood)waspunchedfromeachDBS sampleandextractedwith300␮Lofamixture30:70:0.05(v/v/v) ofwater,acetonitrileandformicacid,respectively.Sampleswere extractedinanorbitalshakerandkeptat37◦Cfor25min.

Forthesetting-upofthisstudy,apooledmixtureofblood sam-pleswasspikedwithlinezolidand 20␮Lwerespottedonfilter paper(Whatman903,GmbH,Dassel,Germany).

2.4. Validationprocedures

Inordertoachieveaccuratequantizationofanalytesin com-plexmatrices astableisotope-labeledinternalstandard maybe moreappropriated,butunfortunatelylabeledlinezolidisnot com-merciallyavailable.Inadditiontheuseofstructuralanalogousas internal standard couldresultsin a differentdetector response becauseofionsuppressionfrommatrixcomponentsorlow extrac-tionrecoveries.

Therefore weused a calibrationcurvepreparedin duplicate byspottingonfilterpaperspikedhumancontrolbloodtoobtain concentrationsof0,15,5,10,20,50and100mg/L.

Asreportedinthebioanalyticalvalidationprocedure[13],three replicates of three different concentrations of linezolid (1, 50, 100mg/L),wereanalyzedintendifferentrunsfordeterminingthe accuracyandprecision.Theseconcentrationlevelsrepresentthe entirecalibrationcurverangeinparticular:threetimestheLLOQ (lowQC),nearthecenterofthestandardcurve(middleQC)closeto thehighestconcentrationlevel(highQC).Averagerecoveryof line-zolidfromDBSsampleswasdeterminedbycomparingresponses withthoseobtainedbyinjectionofthesameamountofdrug,added beforeoraftertheextractionattwo differentconcentrations(5 and50mg/L).Tocalculatethelinearregression,thepeaksizewas plottedagainstthedrugconcentrationinmg/L.

Theshort-termstabilitystudyonDBSsampleswasevaluated uptoonemonthafterstorageat−20◦_C,+4◦_{C,roomtemperature} and+37◦C.

2.5. Massspectrometry

The samples were measured using an Agilent (Waldbronn, Germany) 6430 bench-top Triple-Quad Mass Spectrometer equippedwiththeeletrospraysourceoperatedinMRMinpositive ionmode.Thecapillaryvoltageofthemassspectrometerwassetto 4000VandtheFragmentorvoltagewassetto110V,foreach tran-sition,thedryinggasflowwas9L/minofnitrogenheatedat325◦C. Thefollowingtransitionsweremonitored:m/z338.3>235.2 (quan-tifier),m/z338.3>296.2(qualifier)andm/z338.3>195.1(qualifier). Optimalcollisionenergieswerefoundat20,18,18V,andthe result-ingcellaccelerationvoltagewas+7Vforalltransitions.

The quantitation experiments were undertaken by using a Series1290InfinityLCSystem(AgilentTechnologies,Waldbronn, Germany)UHPLCcapillarypumpcoupledtoanAgilentMicroALS autosampler,bothbeingfullycontrolledfromtheMassHunterdata system.LiquidchromatographywasperformedusinganAgilent ZorbaxEclipsePlusC18,rapidresolution18␮m,21mm×50mm HPLCcolumn (AgilentTechnologies,Waldbronn,Germany).The columnwasmaintainedat60◦Cduringtherunandthecolumn flowwassetat0.4mL/min.Thechromatographicseparationwere obtainedusingafastgradientstartingfroma90%aqueous solu-tioncontaining0.1%formicacidand10%acetonitrilecontaining 0.1%formicacid.The90%oforganicsolventwasreachedin15min andthesystemreconditionedtothestartingconditionin0.7min. Thetotalruntimewas22minlongandthelinezolidretentiontime

wasfixedto115minTheeluentfromthecolumnwasdirectedto theelectrospraysourcewithoutsplitratio.One␮Loftheextracted samplewasinjectedfortheLC–MS/MSexperiments.Allsamples werekeptstoredat+4◦Cintheautosamplertraytoavoid degra-dation.

SystemcontrolanddataacquisitionwereperformedwithMass Hunter(VersionB.0400)softwareincludingtheQualitative pack-age (for chromatographic and spectral interpretation) and the Quantitative Software(forquantitativeinformation generation). CalibrationcurvesweresetupwiththeMassHunterQuantitative programusingalinearleast-squareregressionnon-weighted.

2.6. HPLC–UV

Linezolidserumconcentrationsweredeterminedbyavalidated HPLC–UV assay [14]. Briefly samples wereprepared by mixing aliquots(50:50)ofthespecimenwithacetonitrileand centrifug-ingat5000×gfor5min.Theeluentwasevaporatedtodryness, theresiduewasreconstitutedinmobilephaseand100␮Lwere injectedintotheHPLC.

The stationary phase was a Pinnacle 2 C18 5ODS, 100mm×46mm (Restek Corporation, Bellefonte, PA, USA). Themobilephasewas1%ortho-phosphoricacid,30%methanol, 2g/L heptanesulphonic acid, adjusted topH5 by theaddition of 10M sodiumhydroxide, witha flow rate of 10mL/min and UV absorbance detection at 254nm. The validated lower limit of quantificationwas0.06mg/L.A Series200 auto-sampler,UV detectorandpumpwereused(PerkinElmerInstruments,Shelton, CT,USA).Assayreproducibilitywas:intra-day<6%;inter-day<10%. Thecorrelationbetweendrugconcentrationand areavaluewas goodforbothaqueousandserumsamplesacrosstheconcentration range(r=0.995forboth).

3. Resultsanddiscussion 3.1. Patients

FifteenpairedserumandDBSsampleswereobtainedfrom9 patients,6(67%)males,medianage(7years).Onechildpresented MRSAorbitalcellulitis,onewastreatedforMRSAmastoiditis,two for MRSApneumonia, and one had a CoNS superinfection dur-ingseverechickenpoxLinezolidwasalsousedinthree patients withMDRtuberculosisandinanotheroneforthetreatmentofan intracranialabscessnotrespondingtoconventionaltherapy.

3.2. OptimizedextractionfromDBS

TheDBSuseisbecomingavalidalternativecomparedtoa tradi-tionalbloodsample,inparticularforpediatricapplication,where isusuallystressfulandunethicaltoobtainasufficientnumberof bloodsamples.Inthisscenariotheanalysisbasedonfewdropsof bloodcanbeappealingalsoforpharmacokineticstudiesinvolving children.

Theextractionmixtures wereselected aftertest ofdifferent solventcompositionsandthe70–30acetonitrile/waterproportion yieldthebestextractionrecovery.EachDBSwastwiceextracted sequentiallyandthesecondextractionrepresentslessthan5%of thefirstone.Wetestedalsodifferentextractiontimes(25,45and 60min)andnovariationswererecorded.Additionally,extraction temperaturewastestat25◦C,37◦Cand60◦C.Thebestextraction yieldoccurredat37◦C.

Tobeabletoevaluatepunchinglocationimpactandthe homo-geneityofdrugdistribution,experimentsonseveralspotsfromthe samedropofbloodhavebeenperformed;nosignificantdifferences

weredetected.Thisfactisduetothelowspottedbloodvolumeon paper(20␮L)generatinganhomogenousspot[15,16].

3.3. Chromatographicconditions

Thechromatographicconditionswereselectedthrougha num-berofpreliminarystudiesofoptimizationofparametersinorder tospeed-uptherunningtime(22min)ingradientmode.To main-taintheneededresolutiona18␮mstationaryphasecolumnwas used,andthecolumnovenwassetto60◦C.Sampledilutionrate(90 times)andinjectionvolume(1␮L)werechosentopreventcolumn overload.Thesampleinjectionvolumewasenoughtoachievethe neededsensitivity,reducingthematrixinjectionintothesystem.

Under the conditions of our assay, the dead-volume of the entiresystem(includingthetubing)waslessthan130_␮L,andthe retentiontimeofthesolventfront(unretainedpeak)was0.3min. ConsideringtheLinezolidretentiontime(115min),itwasretained longenoughfromthecolumntoavoidthesaltsuppression. More-overthecolumnshowedrobustperformancesregardlessofthesalt oranyotherinterferingcomponentandnodeteriorationincolumn efficiencywasobservedaftertheanalysisof500DBSsamples.

3.4. Methodvalidation

ThespecificityisprovidedbytheMS/MSmeasurement com-binedtothehighretentiontimestability.Fig.1showstheMS/MS spectrumobtainedbyfragmentingtheprecursor ion(338.3Th) of linezolid under the above described conditions. From these experiments,theresultingmostselectiveion-pairtransitionfor thequantitativeexperiment(SRM)is338.3>235.1.Wehave cho-sen two additional transitions as qualifiers: 338.3>296.1 and 338.3>194.9.

AssuggestedfromLiandTse[16]weevaluatedtherecoveryas showninFig.2.Wecomparedtwoconcentrations(5and50mg/L) inthreereplicates,obtaining15%recoveryforbothlevels.

The non-weighted regression equation for our LC–MS/MS methodwasy=258x+184;R2_{=0.9980.Themeancorrelation} coef-ficient for regression lines,generated on 10 differentdays was R2_{=0.9987(SD±0.0009,range0.9973–0.9996).Acorrelation} coef-ficient of >0.995 is generally considered as theevidence of an acceptablefitofthedatatotheregressionline.

Inordertoassessthemethodsuitabilityonawiderangeof con-centrations,tenreplicatesofthreedifferentcalibratorswereused (1,50and100mg/L)resultinganintra-dayrepeatabilitybelow11% forallvalues (Table1).Theinter-day repeatability,obtainedby processingthreereplicatesofeachconcentrationlevel(1,50and 100mg/L)intenseparateassaysfortwoweeks,wasbetterthan 13%(Table1).

Using theproposedmethodtheestimatedlimit ofdetection (signaltonoiseratio>5)inDBSwas0.2mg/L,thelimitof quan-titation(signaltonoiseratio>15)was0.4mg/L.

3.5. Shorttermstabilitystudies

Stabilitystudiesshowednosignificantdifferencesbetweenthe roomandrefrigeratedtemperatures,highlightingthatmoleculeis stabilizedonpaper.Also,linezolidconcentrationsondriedblood spotwerenotsignificantlydifferentunderconditionsofstorage at37◦Cfor amonth (Table2).Thismeansthat,fortherapeutic linezolidmonitoring,theDBS couldbesent bymail even from tropical countries where ambient temperature is elevated. The overallresultsindeterminingstabilityatdifferenttemperatures arereportedinFig.3.

Fig.1. Productionscanandmolecularstructureoflinezolid.

Fig.2. ComparisonbetweenthechromatogramfromextractedDBSfortifiedwith5mg/Loflinezolid(a)versusalinezolidstandardsolution(5mg/L)inacetonitrile:water (70:30,v/v)addedtoanextractedDBSblanksample(b),consideringthesamematrixeffectsonbothsamples.

3.6. Evaluationofhematocritimpactinlinezolidvalueobtained onDBS

Inthisstudy,15DBSandmatchedplasmasamples,collected from 9 patients, were compared. For each samples hemat-ocrit (HCT) was measured and hematocrit-corrected DBS was

calculated according to the equation Cplasma=CDBS× (100−HCT)/100. This equation is used as a conversion factor toexpresslevels determinedonDBS in theequivalentplasma. In addition, DBS values were corrected using the theoretical hematocrit(meanperage)inordertodeterminateifthemethod could beused when real HCTis not available. We observed a Table1

Inter-dayandintra-dayimprecisionoflinezolidmeasurementsonDBS.Inter-dayandintra-dayimprecisionofthedriedbloodspotassaysweredeterminedasdescribedin Section2.

Expectedconcentrationng/␮L Mean Standarddeviation CV% %Accuracy

Inter-day(n=10) 1 1.2 0.2 13.0 119.0 50 49.7 0.8 1.5 99.4 100 100.7 0.9 0.9 100.7 Intra-day(n=10) 1 0.9 0.1 5.1 94.0 50 44.1 2.3 5.2 88.3 100 96.0 4.7 4.9 96.0

Fig.3.Stabilityoflinezolidinvestigatedat4differenttemperatures:−20◦_C,+4◦_{C,roomtemperatureand+37}◦_{Cforamonthbyrepeatinjectionof3DBSspikedsamples(5,}

20,and50mg/L).

good correlation between linezolid and the levels measured

in the corresponding DBS both if the real HCT (R2_=0.9268)

(Fig. 4a) or the theoretical one have been used (R2_=0.9849) (Fig. 4b).Fig. 5 shows a Bland–Altman plotof thepercent dif-ference in linezolid concentration (DBS vs plasma) between the two matrices versus the mean linezolid concentration. All the differences in concentration fell within ±196 standard deviations.

Table2

Stabilityoflinezolidindriedbloodspotatroomtemperature,+4◦_C,+37◦_Cand −20◦_{C.Valuesarethemeanoftriplicatemeasurements.}

Expected concentration (mg/L) Storage temperature Average,analyses intriplicatefor 30days(mg/L) DS CV% Accuracy 5 −20◦_{C 4.6 0.2 5.1 92.2} 20 −20◦_{C 21.9 0.7 3.2 109.6} 50 −20◦_{C 49.2 2.6 5.3 98.4} 5 +4◦_{C 5.1 0.4 8.3 100.9} 20 +4◦_{C 21.7 1.3 5.9 108.4} 50 +4◦C 51.4 3.4 6.5 102.8 5 Room 4.8 0.5 10.4 95.0 20 Room 21.9 2.1 9.7 109.5 50 Room 49.0 2.5 5.1 97.9 5 +37◦_{C 4.9 0.7 15.2 97.2} 20 +37◦_{C 21.7 2.9 13.4 108.5} 50 +37◦_{C 48.0 4.7 9.7 96.0}

Fig.4.Comparisonbetweenthevaluesconcentrationobtainedfromtheplasma andcalculatedafterDBSanalysis,correctedusingthehematocritratio.Intheupper partthemeasuredhematocritwasusedandinthelowerpartthecalculationwere performedusingtheoreticalhematocrit(meanperage).

Fig.5. Percentdifferenceinlinezolidconcentrationmeasuredinplasmaandfrom DBSversusmeanconcentration.Themeandifferenceis−695%.Alldifferencesare containedwithin196standarddeviations(S.D.).

Fig.6.Bland–AltmanplotbetweenHPLCandLC–MS/MSvaluesobtainedinplasma andDBScorrectedbyhematocritvalue.Themeandifferenceis+764%.Alldifferences arecontainedwithin196standarddeviations(S.D.).

3.7. ComparisonbetweenLC–MS/MSandHPLC–UV

measurements

Tocomparethetwomethodswe usedaBland–Altmanplot,

wherethepercentdifferencesinlinezolidconcentrationfellwithin

196standard deviations(Fig.6).On thebasisofourstudydata

theDBStechniquewaswellcorrelatedwithastandardanalytical methodsuchasHPLC.

4. Conclusions

DBSsamplingoffersmanyadvantagesforcollection,handling, shipmentandstorageofspecimens.Bloodsamplesareeasily recov-ered,involvingminimalsamplemanipulationandmakingiteasy theiruseinnumerousclinicalapplicationsespeciallyfor therapeu-ticdruginpediatricpatients.

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