Development of an UPLC-MS/MS method for the determination of antibiotic ertapenem on dried blood spots

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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

Development

of

an

UPLC–MS/MS

method

for

the

determination

of

antibiotic

ertapenem

on

dried

blood

spots

Giancarlo

la

Marca

a,b,∗,1

_,

_Elisa

_Giocaliere

a,1

_,

_Fabio

_Villanelli

a,1

_,

_Sabrina

_Malvagia

a

_,

_Silvia

_Funghini

a

_,

Daniela

Ombrone

a

_,

_Luca

_Filippi

c

_,

_Marina

_De

_Gaudio

d

_,

_Maurizio

_De

_Martino

d

_,

_Luisa

_Galli

d

a_Meyer_Children’s_Hospital,_Mass_Spectrometry_Lab,_Florence,_Italy b_Department_of_{Pharmacology,}_University_of_Florence,_Florence,_Italy c_Meyer_Children’s_Hospital,_Neonatal_Intensive_Care_Unit,_Florence,_Italy

d_Department_of_Sciences_for_Woman_and_Child’s_Health,_University_of_Florence,_Florence,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received7October2011 Receivedinrevisedform 14December2011 Accepted14December2011 Available online 23 December 2011

Keywords: Ertapenem Carbapenem DBS UPLC–MS/MS

a

b

s

t

r

a

c

t

Ertapenem(Invanz®₎_is_a_newly_developed_carbapenem_␤-lactam_{antimicrobial}_agent._The_drug_usage inpediatricageneedsanaccuratedrugmonitoringforeffectivepatientmanagement.Theaimofthis studywastoevaluatetheuseofdriedbloodspot(DBS)specimenstomeasureertapenemconcentration duringtreatment.TheanalysiswasperformedbyUPLC–MS/MSoperatinginmultiplereactionmonitoring (MRM)mode.Thecalibrationcurveinmatrixwaslinearintheconcentrationrangeof0.5–100mg/Lwith correlationcoefﬁcientvaluehigherthan0.997.Performanceparametersofthismethodlikelowerlimitof detection(LLOD,0.2mg/L),lowerlimitofquantiﬁcation(LLOQ,0.5mg/L),matrixeffect(20%),intra-and inter-dayimprecision(CVwithinthan15%)andaccuracy(between94and155%)ofdrugconcentrations havebeenevaluated.Thedrugstabilityatdifferenttemperatureswastestedforonemonth,toevaluate therisksofsampledeliveryatdifferentclimaticconditions.

Thereportedmethodallowsnowertapenemanalysisandoffersmanyadvantagesforpatientsincluding thepossibilityofcollectingsamplesathome.

Thisnewassayisbothpreciseandaccurateandisespeciallysuitablefortherapeuticdrugmonitoring andpharmacokineticstudiesinneonatesinwhomobtaininglargerbloodsamplesisnotconvenientor possible.

1. Introduction

Ertapenemisacarbapenemantibioticwithabroadspectrum

activityandstabilityagainstawiderangeof␤-lactamases.Ithas

limitedactivityagainstPseudomonasaeruginosa,enterococciand

methicillin-resistantStaphylococcus aureusand therefore itsuse

issuggestedinmoderate-severecommunity-acquiredinfections,

ratherthaninnosocomialinfections.Sinceertapenemhasagood

penetrationintointra-abdominalorgansandinterstitialﬂuid,ithas

beensuggestedasatherapeuticoption inpatientswith

compli-catedbacterialinfections[1].Recently,internationallyguidelines

recommendedertapenem(asothercarbapenems)asanalternative

optionforthetreatmentofcomplicatedintra-abdominalinfections

(cIAIs)inadultsandchildrenaged≥3months[2].

∗ Correspondingauthorat:MeyerChildren’sHospital,MassSpectrometryLab, Florence,Italy.Tel.:+390555662988;fax:+390555662489.

E-mailaddress:g.lamarca@meyer.it(G.laMarca).

1 _These_authors_equally_contributed_to_this_work.

Theroleofertapeneminthetreatmentofcomplicated

bacte-rialinfectionsinchildrenis,uptonow,verylimited.Sinceithas

invitroactivityagainstthemajorityoftheextended-spectrum

beta-lactamase(ESBL)-producingEscherichiacoliandKlebsiellaspecies,

itsuseincomplicatedurinarytractinfections(cUTIs),otherthan

in cIAIs, is under investigation [3].At the present, some

infec-tiousdiseasespecialistssuggestthatertapenemcouldbeagood

therapeuticoptionincomplicatedcommunity-acquiredbacterial

infections, reserving the use of antipseudomonal carbapenems

forinfectionswherePseudomonasaeruginosaisfrequently

impli-cated, thus reducing the selective pressure on theselection of

carbapenem-resistantPseudomonasspp.

Ertapenemhasafavorable pharmacokinetic(PK)proﬁlethat

allowsonce-daily(OD)administrationinadultsafterintravenous

andintramuscularadministration.DataonPKproﬁleinchildren

youngerthan12yearsshowedthatclearanceofertapenemisabout

2-foldhigherascomparedtoadults[4].Consequently,the

recom-mendedintravenousdosageis1gODinpatientsaged≥13years

and15mg/kgtwice-daily(toamaximumof1g/day)inchildren

aged3monthsto12years[4].

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Tothebestofourknowledge,nopediatricdataonertapenem

PKproﬁleinnewbornsandchildrenyoungerthan3months,nor

PKdataafterintramuscularadministrationareavailable.

SeveralLC-UVmethodshavebeenreportedinliteratureforthe

determinationofcarbapenemantibioticsbothinbiological

sam-plessuchasplasma,serum,urine,bronchialsecretionsandintissue

samples.Ontheotherhand,onlyfewLC–MSmethodshavebeen

publishedfortheidentiﬁcationandquantizationofcarbapenem

compoundsortheirdegradationproducts[5–8].Asiswellknown,

PKstudiesaredifﬁculttoperforminyoungerchildren,sincethey

requirecollectionofmanybloodsamplesinthesamedaywitha

considerablediscomfortforthepatients.Thus,anattractingand

potentialmethodtoeasilyobtainsamplesforPKstudiesin

chil-dreniscollectionofwholebloodsamplesontoﬁlterpaper(dried

bloodspotorDBS).DBSspecimensarecommonlyusedforneonatal

screeningofmetabolicdiseases,cysticﬁbrosisandhypothyroidism

inmicro-bloodsamples.Somestudiesrecentlyevaluatedtheuse

ofDBSfortherapeuticdrugmonitoring(TDM)andtoxicologyin

adults[9,10].Somestudies,alsofromourgroup,appliedtheDBS

techniquetoevaluatedrugconcentrationsinnewborns,infantsand

children[11–15].ThesereportsshowedthattheaccuracyofTDM

studiesusingDBSiscomparabletothatoftraditionalTDMstudies

onplasmawhichrequirelargerbloodvolumes.TheUPLC–MS/MS

method, here described, represents a robust, highly speciﬁc

and sensitive approach to quantify ertapenem on dried blood

spots. Sensitivity enhancement, required in quantitative

analy-sis,andalsoselectivityareobtainedacquiringmultipletransition

pairs.

2. Materialsandmethods

2.1. Standards

ErtapenemwasprovidedbyMerckSharpandDohme(Rome,

Italy).Astocksolutionof1g/Lwaspreparedinwaterandstored

at−80◦_C_in_different_aliquots_for_no_longer_than_six_months.

Suc-cessivedilutionsweremadeusingHPLCgradewater.Allchemicals

andsolventswereofthehighestpurityavailablefromcommercial

sourcesandusedwithoutanyfurtherpuriﬁcation.Analyticalgrade

methanol,acetonitrileand water werepurchased fromPanreac

(Barcelona,Spain).

2.2. Samplepreparation

Spikingstudieswereconductedusingapoolofhumanblood

fromhealthydonors;we evaluatedlinearity byanalyzing

forti-ﬁed3.2mm dried blood spots preparedat 0.5, 1.5, 10, 50 and

100mg/Lonﬁlterpaper(903®_,_Whatman_GmbH,_Dassel_Germany).

Theamountofbloodusedtopreparespotswas20␮Linorderto

producehomogeneousspots[16].

DBS samples were stored at −20◦_C _in _a _sealed _plastic _bag

containingdesiccantuntilanalysis.One3.2mmdiameterdisk

(con-tainingabout3.3–3.4␮Lofblood) waspunched fromeachDBS

sampleandextractedwith200_␮Lofa30/70ofwater/methanol

(v/v)solution.Sampleswereputinanorbitalshakerandkeptat

37◦Cfor25min.Theextractsweretransferredintoanew96-well

plateandanalyzedimmediately.

2.3. Validationprocedures

Themethodwasvalidatedintermsoflinearity,precision,

accu-racy,extractionrecovery,matrixeffectand stability.Calibration

curvewaspreparedbyspottingonﬁlterpaperspikedhuman

con-trolbloodtoobtainconcentrationsof0,0.5,1.5,5,10,20,50and

100mg/L.Intra-dayprecisiondatawereevaluatedbytenreplicate

analysisofﬁvedifferentertapenemconcentrationsonthesame

day.Inter-dayprecisiondataweredeterminedbytheanalysisof

ﬁvedifferentertapenemconcentrationson10differentdays.

Tocalculatethelinearregression, thepeak areawasplotted

againstthedrugconcentrationinmilligramsperliter.

ThestabilitystudyonDBSsampleswasevaluateduptoone

monthafterstorageat−20◦_C,₊₄◦_C,_room_temperature_and₊₃₇◦_C.

2.4. Massspectrometry

Thesamplesandthecalibrationcurvesampleswereanalyzed

onaAgilent(Waldbronn,Germany)6430bench-topTriple-Quad

MassSpectrometerequippedwithanElectrospray source(ESI);

theESIsourceoperatedinpositiveionmode,usingthefollowing

setting:capillaryvoltage5000V,fragmentorvoltage80Vforeach

transition,dryinggasﬂow9L/minofnitrogen,temperature325◦C,

nebulizergas35psi.Thefollowingtransitionsweremonitoredin

MRMmode:m/z476.2>432.2(quantiﬁer),m/z476.2>390.1

(qual-iﬁer)andm/z476.2>233.1(qualiﬁer).Optimalcollisionenergies

werefoundat20,18,and18V,andtheresultingcellacceleration

voltagewas+7Vforalltransitions.

The quantitation experiments were undertaken by using a

Series1290InﬁnityLCSystem(AgilentTechnologies,Waldbronn,

Germany)UHPLCCapillaryPumpcoupledtoanAgilent6430,both

beingfullycontrolledfromtheMassHunterdatasystem.

TheanalyticalcolumnwasanAgilentZorbaxEclipsePlusC18,

RapidResolution1.8␮m,2.1mm×50mm(AgilentTechnologies,

Waldbronn,Germany)operatingat0.4mL/minﬂowrateandusing

water(A)andacetonitrile(B)bothcontaining0.1%formicacidas

mobilephase.Thecolumnwasmaintainedat60◦Cduringtherun.

Thechromatographicseparationwasobtainedusingafast

gradi-entstartingfroma 95%ofsolutionA and5%ofsolutionB. The

95%oforganicsolventwasreachedin1minandmaintainedfor

15s;initialconditionswererestoredin5sandthecolumn

equi-libratedfor1min.Thetotalrunningtimewas2.2minlongand

theertapenemretentiontime wasﬁxedto1.04min.Theeluent

fromthecolumnwasdirectedintotheElectrospraysource

with-outsplit.Threemicrolitersoftheextractedsamplewereinjected

fortheUPLC–MS/MSexperiments.

Systemcontrol,dataacquisitionandinterpretationweremade

with the Agilent Mass Huntersoftware (Version B.04.00)

soft-wareincludingtheQualitativepackage(forchromatographicand

spectralinterpretation)andtheQuantitativeSoftware(for

quanti-tativeinformationgeneration).Calibrationcurvesweresetupwith

theMassHunterQuantitativeprogramusingalinearleast-square

regressionnon-weighted.

3. Resultsanddiscussion

Dried blood spot sampling is increasingly becoming a good

alternativetotraditionalplasmasamplesforbiochemicalanalysis.

Theemergingtechnologyoffersgreatopportunitiesforimproved

patientcareespeciallyinthepediatricpopulationwhereis

gen-erallydifﬁcultandunethicaltoobtainsufﬁcientnumberofblood

samples.

Inthiswork,wehavedevelopedamethodfortheanalysisof

ertapenemconcentrationsinUPLC–MS/MSperformedwithonlya

fewdropsofbloodandthereforeveryappealingforits

applica-tioninpharmacokineticstudiesinvolvinginfantsandveryyoung

children.Fig.1showstheMS/MSspectrumobtainedby

fragment-ingtheprecursor ion(476.2 Th)ofertapenem undertheabove

describedconditions.Fromtheseexperiments,theresultingmost

selectiveion-pairtransitionforthequantitativeexperiment(MRM)

was476.2>432.2while476.2>390.1and476.2>233.1wereused

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Fig.1.Productionscanandmolecularstructureofertapenem.

Fig.2.Comparisonamongthechromatogramsfromablanksolventsamplespikedwithertapenem(5mg/L)(C)versusaertapenemstandardsolution(5mg/L)70/30of methanol/wateraddedtoanextractedDBSblanksample(B)andanextractedDBSfortiﬁedwith5mg/Lofertapenem(A).

Oncethetransitionswerechosen,samplepreparationwas

opti-mizedadjustingmanyvariablesintheextractionprocesssuchas

extractionsolutioncomposition,volume,timeandtemperature.

Different extractionsolutionswere examinedand compared

withtheobjective ofchoosing thebest extractionmixture.The

organicsolventsusedweremethanoloracetonitrilewithdifferent

percentagesofwater(from50to10%).Themoreefﬁcientextraction

solution,withthehighestextractionyield,wasfoundtobe30/70of

water/methanol(v/v).Thevolumeofextractionsolution(200␮L)

waschoseninordertocombinehighextractioncapacitywitha

minimalsampledilution.Asuccessiveextractionusingthesame

conditionsontheexhaustedspotproducedlessthan10%compared

totheﬁrstone.Westudiedtheextractiontimebetween10and

60minbutnosubsequentincreaseinthepeakareawasreached

after25min(datanotshown).Additionally,extraction

tempera-turewastestat25◦C, 37◦Cand 60◦C.Thebestextractionyield

occurredat37◦C.

EvaluationofdrugextractionprocedurefromDBS,expressedas

theratiooftheareasofthedrugfortiﬁedDBSextracttothatof

blankDBSspikedafterextractionwasdeterminedtobe15%(Fig.2,

tracesAandB).

Table1

Intra-andinter-dayertapenemreproducibilityonDBSassessedby%CVcalculations.

Intraday Expectedconcentration (mg/L) Mean(n=10) SD %CV Accuracy% 0.5 0.78 0.04 5.12 155 1.5 1.70 0.12 7.05 113 10 10.47 0.68 6.49 105 50 47.44 2.04 4.30 95 100 102.56 7.53 7.34 103 Interday Expectedconcentration (mg/L) Mean(n=10×3) SD %CV Accuracy% 0.5 0.53 0.05 9.43 105 1.5 1.49 0.22 14.77 99 10 10.70 1.59 14.86 107 50 46.90 1.60 3.41 94 100 99.44 3.31 3.32 99

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Fig.3.Stabilityofertapeneminvestigatedat4differenttemperatures:−20◦_C,₊₄◦_C,_room_temperature_and₊₃₇◦_C_for_a_month_by_repeat_injection_of₃_DBS_spiked_samples (5,20,and50mg/L).

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Thedifferencebetween(B)and(A)couldbeduetoa

signiﬁ-cantportionofdrugboundedtothebloodproteins,asreported

from Musson et al. [17]. This suggested us that the

extrac-tion solution is able to extract the total free ertapenem from

DBS.

Wetestedalsodifferenttimestoequilibratethesampleafterthe

ertapenemstandardspiking(datanotshown)toverifythe

inﬂu-enceoftimeintheproteinbindingonthefreedrugportion.The

equilibrationtimerangeinvestigatedwasfrom20to120minat

roomtemperaturebutanychangesintheextractedconcentration

wereobserved.Thereforeafter20minofequilibration,eachspiked

bloodsamplewasspottedonﬁlterpaper.

Matrixeffect isshown bytheratioof extractedDBSsample

spikedpost-extractionandspikedsolventsample(Fig.2,tracesB

andC);thisvaluerepresentsalossof20%oftheanalytesignal(ion

suppression)duetoalterationsinionizationefﬁciency.

Oneof themostimportantfeatures forscreening methodis

a fast runtime analysis. It allows processing many samples in

ashort periodoftime. Moreovertheresolution isanother

fun-damental parameter and the column we used was a 1.8␮m

stationary phasecolumn, coupledto a superior speciﬁcity of a

triplequadrupolesystemoperatinginMS/MSmode.Theinjection

volumewasmaintainedlowtoavoid thesystemcontamination

fromthematrix interferences and columnover loading.In this

newmethodtheoriginalsampleisapproximately60timesdiluted

andthesampleamountinjectedis0.172␮L.Intheseconditions

thesensitivityachievedisenoughtoaffordtheanalyzedsamples,

obtainingrobustperformances,withthelowestsample

prepara-tionpossible.Ertapenemwasretainedbythecolumnenoughtobe

separatedfromthesalts,whichrepresentsthemainion

suppres-sionsourceforbiologicalsamplesanalysis.Inter-assayvariability

ofcalibrationdataobtainedoverconcentrationsof0.5–100mg/L

wasmonitoredon4consecutiveweeks.Theaverageslope,

inter-cept,andcoefﬁcientoflinearregression(r2₎_were_964,₋₄₂₇_and

0.9987(SD±0.001,range0.9970–0.9999).Acorrelationcoefﬁcient

>0.9950isgenerallyconsideredastheevidenceofanacceptableﬁt

ofthedatatotheregressionline.

Inordertoevaluatepunchinglocationimpactandthe

homo-geneityofdrugdistribution,experimentsonseveralspotsfromthe

samedropofbloodhavebeenperformed;nosigniﬁcantdifferences

weredetected.Thisfactisduetothelowspottedvolumeonpaper

(20␮L)aselsewherereported[17].

Inordertoassesstherobustnessofthemethod,ﬁvespikedlevels

wereused(0.5,1.5,10,50and100mg/L)andeachwasprocessed

tentimesinoneday,resultinginanintra-dayrepeatabilitybelow

7%forallvalues(Table1).Theinter-dayrepeatabilityobtainedin

fourseparateassaysforfourweekswasbetterthan15%(Table1).

Thelimitofdetection(LOD)andlowerlimitofquantiﬁcation(LLOQ)

ofthismethodweredeterminedfromthecoefﬁcientofvariation

ofaknownconcentrationofreferencestandards.TheLODforthis

assay,calculatedfrom5timesthenoiseleveloftheresponse,in

DBSwas0.2mg/L.TheLLOQforthisassaycalculatedfrom15times

thenoiseleveloftheresponse,is0.5mg/L.

Weinvestigatedtheeffectofstoragetimeandstorage

tempera-tureatfourdifferentconcentrationlevels(0.5,5,20and50mg/L)on

spikedDBSsamples.Ertapenemseemstobenotstableunderthe

testedtemperatures(+4◦C, roomtemperatureand+37◦C),with

theexceptionof−20◦_C ₍_Fig.₃_)._In_literature_data_related_to_the

ertapenemstabilityreportedarapiddegradationinsolutionandfor

long-termstoragefreezeat₋₂₀◦Cisrequired[7,16].Datarelated

totheLLOQ(0.5mg/L)stabilityarenotshowninFig.3,because

of thedrug concentration after24hdecreased underthe limit

ofquantiﬁcation.UsuallytheDBSsamplingprocedurehelpsthe

moleculestabilization,allowingcollection,shippinganddeliveryat

roomtemperature[16,18].InthiscasetheDBSsamplingadvantage

remains,buttheshipmentmustbeperformedat−20◦_C.

Fig.4.Relationshipbetweenertapenemlevelsinplasmaandcorrespondingdried bloodspots.Valuesrepresentthemeanofduplicateexperiments.

Toverifythecorrelationbetweendataobtainedondriedblood

spot and plasma, since no patients receiving ertapenem were

available,threedifferentbloodsamplesatdistincthematocrit

lev-els(low,mediumandhigh)fromhealthydonorswereselected.

Foreachbloodsamplethreealiquotswerepreparedandspiked

at5,10and20mg/Lofertapenem.Sampleswereequilibratedfor

20min,then20␮Lofbloodwerespottedonpaperandthe

remain-ingbloodwascentrifugedtoobtainplasma.TheDBSsampleswere

extractedasreportedinSection2;200␮Lofextractionmixture

wereaddedto2␮Lofplasma,vortexedandcentrifugedto

pre-cipitateproteins.Threemicrolitersofsupernatantwereinjected.

Agoodcorrelation(R2₌_0.9850)_between_ertapenem_plasma_and

ertapenem levelsmeasuredinthecorrespondingDBSafterreal

haematocritcorrectionwasobserved (Fig.4).Thisconﬁrmsthe

validityofDBSanalysisasanalternativetoplasmadrug.

Themethodwillbehelpfulinprocessinglargenumberof

sam-plesforpatient’streatmentmonitoringatfollow-up.UseofDBSfor

samplingrequireslittlevolumeofbloodspottedanddriedoncard,

withgreatadvantagesoverconventionalplasmasamplingbeing

lessinvasive,particularlyinpediatricpopulation.

4. Conclusions

TheuseofDBSoffersaneasy wayofcollecting,storing, and

shippingsamplesandisanon-invasivemethodbecausebloodcan

beobtainedfromﬁngerprickssampling.

In order to keep stability for long term storage, ertapenem

shouldbestoredatlowtemperature(−20◦_C).

Agoodcorrelationbetweendruglevelsmeasuredinplasmaand

inthecorrespondingDBSaftercorrectionforrealhematocritwas

observed.

Inthis work,wedevelopedamethodforthe“onDBSassay”

ofertapenembyusingUPLC–MS/MS.Itcouldbeconsideredvery

appealingforitsapplicationforTDMorinpharmacokineticstudies

involvinginfantsandveryyoungchildren.

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