Quantification of the antimalarial drug pyronaridine in whole blood using LC–MS/MS — increased sensitivity resulting from reduced non specific binding

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Original citation:

Blessborn, Daniel, Kaewkhao, Karnrawee, Song, Lijiang, White, Nicholas J., Day, Nicholas P. J.

and Tarning, Joel. (2017) Quantification of the antimalarial drug pyronaridine in whole blood

using LC–MS/MS — increased sensitivity resulting from reduced non-specific binding.

Journal of Pharmaceutical and Biomedical Analysis, 146. pp. 214-219.

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Journal

of

Pharmaceutical

and

Biomedical

Analysis

jou rn a l h om ep a g e :w w w . e l s e v i e r . c o m / l oc a t e / j p b a

Quantification

of

the

antimalarial

drug

pyronaridine

in

whole

blood

using

LC–MS/MS

—

Increased

sensitivity

resulting

from

reduced

non-specific

binding

Daniel

Blessborn

_,

_Karnrawee

_Kaewkhao

_,

_Lijiang

_Song

_,

_Nicholas

_J.

_White

_,

Nicholas

P.J.

Day

_,

_Joel

_Tarning

a_{Mahidol-OxfordTropicalMedicineResearchUnit,FacultyofTropicalMedicine,MahidolUniversity,Bangkok,10400,Thailand} b_{CentreforTropicalMedicineandGlobalHealth,NuffieldDepartmentofClinicalMedicine,UniversityofOxford,Oxford,UK}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15June2017

Receivedinrevisedform7August2017 Accepted17August2017

Availableonline26August2017

Keywords:

Pyronaridine Pyramax Malaria Wholeblood Bioanalysis

a

b

s

t

r

a

c

t

Malariaisoneofthemostimportantparasiticdiseasesofman.Thedevelopmentofdrugresistancein malariaparasitesisaninevitableconsequenceoftheirwidespreadandoftenunregulateduse.Thereis anurgentneedfornewandeffectivedrugs.Pyronaridineisaknownantimalarialdrugthathasreceived renewedinterestasapartnerdruginartemisinin-basedcombinationtherapy.Tostudyits pharmacoki-neticproperties,particularlyinfieldsettings,itisnecessarytodevelopandvalidatearobust,highly sensitiveandaccuratebioanalyticalmethodfordrugmeasurementsinbiologicalsamples.Wehave developedasensitivequantificationmethodthatcoversawiderangeofclinicallyrelevant concentra-tions(1.5ng/mLto882ng/mL)usingarelativelylowvolumesampleof100␮Lofwholeblood.Totalrun timeis5minandprecisioniswithin±15%atallconcentrationlevels.Pyronaridinewasextractedona weakcationexchangesolid-phasecolumn(SPE)andseparatedonaHALORPamidefused-corecolumn usingagradientmobilephaseofacetonitrile–ammoniumformateandacetonitrile-methanol.Detection wasperformedusingelectrosprayionizationandtandemmassspectrometry(positiveionmodewith selectedreactionmonitoring).Thedevelopedmethodissuitableforimplementationinhigh-throughput routinedruganalysis,andwasusedtoquantifypyronaridineaccuratelyforupto42daysafterasingle oraldoseinadrug-druginteractionstudyinhealthyvolunteers.

©2017TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).

1. Introduction

Malariaisoneofthemostimportantinfectiousdiseasesinthe world,resultinginan estimated214millioncasesand 438,000 deathsin2015[1].Childrenundertheageoffivebearthemain burdenofmalaria-attributeddeaths.Artemisinin-based combina-tiontherapy(ACT)istheWorldHealthOrganizationrecommended first-line treatment against P. falciparum malaria [2]. The fast-actingartemisinincomponentkillsthemajorityofparasitesduring the first days of treatment. As, this class of drug has a short biologicalhalf-life[3,4],itisnecessarytocombineitwitha long-actingpartnerdrugtoeliminateresidualparasitesandensurecure.

∗_{Correspondingauthorat:Mahidol-OxfordTropicalMedicineResearchUnit,}

Fac-ultyofTropicalMedicine,MahidolUniversity,Bangkok,10400,Thailand.

E-mailaddress:[email protected](D.Blessborn).

1 _{Currentaddress:DepartmentofChemistry,UniversityofWarwick,Coventry,}

UK.

Pyronaridine is a synthetic antimalarial drug,developed in the 1970s,whichhasbeenusedasa mono-therapyin China.It has alongbiologicalhalf-life(13–17days)andisthereforesuitableto

useinACTs[5–7].Thefixed-dosecombinationofartesunateand

pyronaridineisanewlydevelopedanddeployedACT,whichhas shownexcellent efficacyagainstbothP.falciparum andP.vivax

malaria[8].

Sensitiveand accuratebioanalytical methods are crucial for high-qualitypharmacokineticstudies.Therearea few methods publishedforthequantificationofpyronaridineinplasma[9–12], andmostoftheserequireasamplevolumeof200–250␮Lofplasma and present a quantitation limit of 10ng/mL or higher [11,12]. Hodeletal.reportedaLC–MS/MSmethodabletoquantify pyronar-idineat1ng/mL[10],buttheruntime wasreportedtobeover 20minmakingit unsuitablefor highthroughputroutine analy-sisofclinicalstudies.Aninvivostudyinrabbitsshowedahigh blood-to-plasmadistributionratio(4.9-17.8)forpyronaridine[13], suggesting that whole blood rather than plasma would bethe preferredsample matrix fordrugmeasurements [6,14]. Several

http://dx.doi.org/10.1016/j.jpba.2017.08.023

Fig.1. Chemicalstructureofpyronaridinetetraphosphate.

quantificationmethodshavebeenreportedforwholeblood,but witharelativelypoorsensitivity(i.e.20–30ng/mL)[13,15,16].A recentLC–MS/MSmethod[17]usedasamplevolumeof300␮L, resultingin alower limitof quantification(LLOQ) of 5.7ng/mL. However,liquid-liquidextractionwas employedwhich is labo-riousanddifficulttoautomate.Anothercrucialdrawbackinthe previouslyreportedLC–MS/MSmethodsisthatnoneofthemused stableisotope-labeledinternalstandards,andcouldthereforebe compromisedbymatrixeffects.

Theaimofthisworkwastodevelopasensitiveandaccurate quantificationmethodofpyronaridineinlowvolumewholeblood samplesthatissuitableforimplementationintohigh-throughput routinedrugquantificationinclinicaltrials.

2. Materialsandmethods

2.1. Materials

Pyronaridine tetraphosphate (Fig. 1) and its stable isotope labeled internal standard, pyronaridine-13_C

2D4 (SIL-PYN), was

providedbytheWorldwideAntimalarialResistanceNetwork Ref-erence program [18]. LC–MS grade of water, acetonitrile and methanol were obtainedfrom JT Baker (Phillipsburg, USA). All other chemicals and reagents were of analytical grade. Formic acid(98%),ammoniumformateandammoniumcarbonatewere from Sigma–Aldrich (St. Louis, USA). Acetic acid was obtained fromMerck(Darmstadt,Germany).Blankbloodinfluoride-oxalate, EDTA,Na-heparin,Li-heparin,andfluoride-heparinwascollected fromhealthyvolunteersattheFacultyofTropicalMedicine, Mahi-dolUniversity,Thailand.

2.2. Samplepreparation

2.2.1. Preparationofstandardsandworkingsolution

Stocksolutions(1mg/mL)of pyronaridineand SIL-PYNwere prepared in methanol-formicacid 1%(50:50v/v) and stored in methanol-washed cryovials at−80◦C. Working solutionswere preparedinhumanplasma-water(50:50v/v)andusedforspiking ofwholeblood.Allsolutionswereallowedtoequilibratetoroom temperaturebeforeuse.

2.2.2. Preparationofcalibratorsandqualitycontrolsamples

Freshfluoride-oxalatewholebloodwasusedtoprepare cal-ibration standards at a concentration range of 1.47–882ng/mL andLLOQat1.47ng/mL,upperlimitofquantification(ULOQ)at 882ng/mL and over-curve samples(OC)at 1807ng/mL.Quality

control(QC)samplesat4.67,57.2and452ng/mLwereprepared fromasecondstocksolution.Thefinalvolumeofworkingsolution inwholebloodwaslessthan5%inallsamples.

2.2.3. Extractionprocedure

SamplepreparationandSPEwasperformedonaFreedomEvo 200platform(TECAN,Mannedorf,Switzerland).Pipettetips, 96-wellplatesandsealmatswereallmethanol-washedbeforeuse. Wholebloodsamples(100␮L)weremanuallyaliquotedtothe 96-wellplate.Then,byliquidhandler,100␮Lplasma-water50:50v/v wasaddedinthefirstwell(i.e.doubleblank)and100␮Lof inter-nalstandard(30ng/mL)inplasma-water50:50v/vwasaddedto allotherwells.Fourhundredmicrolitersofammonium carbon-ate(20mM,pH8.8)wasaddedtoallwellsandthencoveredwith a sealmat.The96-wellplatewasmixedfor2minat 1000rpm and centrifugedfor 2minat 1100×g.Then500␮L of the cen-trifuged samplewas transferredto a 96 wellplate solid phase CBA extraction plate (Biotage, Uppsala, Sweden) and extracted accordingtothefollowingprocedure;(1)conditioningoftheSPE cartridgewith1mLmethanolfollowedby1mLammonium car-bonate (20mM,pH8.8),(2)loadingofthewholebloodsample (500␮L),(3)washingwith1mLammoniumcarbonate(20mM,pH 8.8)followed by1.5mLmethanol-ammoniumcarbonate20mM (80:20v/v)andfinally1mLmethanol-water (50:50v/v),(4) elu-tionwith0.9mLmethanol-formicacid(98:2v/v).Theeluateswere evaporated under a gentle streamof nitrogen at 70◦C (Caliper TurboVap® 96)untildrynessandreconstitutedin400_␮Lmobile phaseA(seebelowsection2.3.1).Theextractedandreconstituted samplesweremixedfor2minat1000rpm,centrifugedfor2min at1100×gandplacedintheautosamplertoequilibrateforatleast 30minbeforeanalysis.

2.3. Instrumentationandchromatographicconditions

2.3.1. Chromatography

for-dientswitch)and total run-timeof 5min.Thesampleinjection volumewas2␮L.

2.3.2. Massspectrometry

An API 5000 triple quadrupole mass spectrometer (Applied Biosystems/MDSSciex) withTurboVionizationsourceinterface operatinginthepositiveionmodewasusedfortheMS/MSanalysis. Ionsprayvoltagewassetto5500V,withadryingtemperatureat 650◦C.Thecurtaingaswas30psiandthenebulizer(GS1)and aux-iliary(GS2)gases45and55psi,respectively.Quantificationwas performedusingselectedreactionmonitoringforthetransitions m/z518.10–447.15and524.25–453.15(collisionenergyof23V) forpyronaridineandSIL-PYN,respectively.

2.4. Validation

TheassaywasvalidatedaccordingtotheUS Foodand Drug Administration(FDA)guidelinesonbioanalyticalmethod valida-tion[19].Accuracyandprecisionofthemethodwasdetermined byanalysisof5replicatesetsof5concentrations(1.47,4.67,57.2, 452and882ng/mL)analyzed in 4separateruns.The abilityto dilute over-curve sampleswas investigated at 1807ng/mL and 1:4dilutionwithblankblood.Overallaccuracywascalculatedas themeanoftherelativeaccuracies(i.e.relative errorcompared tothenominalconcentration)ateach QClevel.Precisionofthe method(within-run,between-runandtotal-variability)was cal-culatedusinganalysisofvariance(ANOVA)andexpressedasthe relativestandarddeviation(%RSD).

2.4.1. Linearity,selectivityandrecovery

Linearitywasassessedusingthecalibrationstandardsfromthe fourseparateruns.Themostsuitableregressionmodelwaschosen basedontheaccuracyoftheback-calculatedconcentrationsofthe calibrationcurvesandQCsamples[20].

Selectivity was evaluated by analysis of six blank samples from six different blood donors, and using different antico-agulants (fluoride-oxalate, EDTA, Na-heparin, Li-heparin and fluoride-heparin). Potentiallyinterfering antimalarial drugs (i.e. chloroquine, amodiaquine, artesunate, dihydroartemisinin, pri-maquine and piperaquine) were evaluated using post-column infusionandalsoaconventionalLCrun.

RecoverywasdeterminedbycomparingthepeakareaofQC samples(×5ateachlevel)withthatofextractedblankblood sam-plespost-spikedtocontainthesamenominalconcentrationasthe QCsample,simulatinga100%extractionrecovery.

2.4.2. Matrixeffectandcarry-over

Post-columninfusionwasperformedwithinjectionofblank extractedbloodsamplesfromsixdifferentdonors.Bloodfroma singledonorwasalsocollectedwithdifferentanticoagulantsto evaluatepotentialmatrixeffectsfromthedifferentanticoagulants. Tocalculatethematrixeffect,theMSresponsefromextractedblank samples(post-spiked)wasdividedwiththeMSresponseofthe analyteinneatsolution,atthesamenominalconcentration.A cal-culatedmatrixeffectbelow0.85orabove1.15wouldimplythata matrixeffectwaspresent.Carry-overeffectswerealsoinvestigated byinjecting3blanksamples(mobilephase)directlyafterfive injec-tionsofextractedsamplesattheULOQ.Ifthesignalwasmorethan 20%ofthatofLLOQitwouldbeclassifiedascarry-overeffects.

2.4.3. Stability

Stabilityofpyronaridineinwholebloodwasinvestigated dur-ing3 freeze/thaw cycleswhere sampleswere frozenat −80◦C

ityat−80◦Cfor1yearforbothspikedsamplesandstocksolutions wereevaluated. Stability wasalsoinvestigated duringdifferent stepsintheextractionprocesse.g.bufferpre-treatedblood(before SPEextraction)4hat22◦C,extractedsamples(inelutionsolution) at4◦Cfor24h,asevaporatedsamplesstoredat4◦Cforupto72h aswellasstabilityinLCautosamplerat4◦Cforupto72h.Aheat deactivatingtestwasevaluatedat60◦Cfor1htoinactivateany HIVvirusesinthebloodsamples[21–23].

2.5. Clinicalpharmacokineticstudywithincurredsample reanalysis

Thisassaywasusedtoquantifypyronaridineinwholeblood samplesaspartofanopen-labelcross-overstudy(NCT01552330) toevaluatethepotentialpharmacokineticdrug-druginteractions betweenpyronaridine-artesunateandprimaquinein 17healthy Thaivolunteers[5].Thedrugwasgivenasa singleoral doseof artesunate-pyronaridine(Pyramax®,ShinPoongPharmaceuticals, Republicof Korea),withandwithoutprimaquine(Thai Govern-mentPharmaceuticalOrganization[ThaiGPO],Bangkok,Thailand). Bloodsampleswerecollectedatdifferenttimeintervalsuntilday 42.Atotalof680sampleswereanalyzed.Atotalof73sampleswere selectedforincurredsamplereanalysis.

3. Resultsanddiscussion

Therangeofthecalibrationcurve1.47–882ng/mLwasbased onpublishedclinicalpharmacokineticpatientdata[14,17]withan extendedrangetocoverbetween-patientdifferences.Inthe drug-druginteractionstudyinhealthyvolunteers[5],fluoride-oxalate wasusedasananticoagulantforalldrugsinthestudyandthis anticoagulantwasthereforeselectedforthevalidationof pyronar-idine.

Pyronaridineadsorbsreadilytodifferentsurfacematerials, par-ticularlytoglassbutalsotosomeextenttoplasticsurfaces.This isamajorproblemwhen dilutingstocksolutionswithwaterat lowconcentrations,affectingworkingsolutionsandinternal stan-dardswhichcouldleadtoseriouslybiasedresultsifnottakeninto consideration[24–26].Theadsorptionissuewaseliminatedinthe developedmethodbyusingdilutedplasmaforbothworking solu-tionsandinternalstandarddilutions.

3.1. Samplepreparation

Itisimportantthatthespikedsamples,whichareusedto quan-tify theunknownclinicalsamples,mimic thestudysamplesas closely as possible. A shift in pH or too much organicsolvent may precipitate some of the blood components. Plasma-water (50:50v/v)wasusedtopreparetheworkingsolutionsfromastock solutioncontainingmethanol-formicacid1%.Theamountofstock solutionaddedwaskeptlow(<5%)toavoidanyprecipitationof plasmainthedilutionstep.Thegeneratedplasma-waterworking solutionwouldthenhaveminimaleffectonthespikedwholeblood. Forsampleextraction,proteinprecipitationisquickandeasy but it also releases many other components from the blood sample,potentially contaminating thedetectorand causingthe LC–MS/MS system to suffer from matrix effects. Liquid-liquid extractionhasbeenusedinmanypreviousmethodsfor

pyronari-dine[9,11,13,16,17]butitcanbecomplicatedandlaboriouswhen

Table1

Accuracyandprecisionwhenquantifyingpyronaridineinwholeblood.

Sample Concentration(ng/mL) Within-runprecision(%) Between–runprecision(%) Total-runprecision(%) Accuracy(%)

LLOQ 1.47 9.83 14.8 10.9 1.45

QC1 4.67 7.23 8.42 7.44 −1.87

QC2 57.2 4.56 9.27 5.57 −0.38

QC3 452 3.93 2.56 3.74 3.36

ULOQ 882 3.26 4.38 3.46 −3.27

OC(1:4) 1807 3.64 4.53 3.80 −0.89

Resultsarepresentedfromtheanalysisof5replicates/dayover4daysateachconcentrationlevel,usingspikedfluoride-oxalatewholebloodsamples.Within-,between-and total-runprecisionwereobtainedfromanANOVA.Accuracywascalculatedasthemeanoftherelativeaccuracies(i.e.relativeerrorcomparedtothenominalconcentration) ateachQClevel.Precisionandaccuracymustbewithin±15%forallcontrollevelsexceptatLLOQwhichshouldbewithin±20%.

highthroughputanalysiswhenoperatedinthe96wellplate

for-mat.TheSPEwashingconsistedofthreesteps;(1)bufferonlyto

washoutremainingbloodcomponentsbefore(2)ahighmethanol

washstepandfinally(3)water-methanoltowashoutremaining

bufferbeforeaddingtheelutionsolvent.Thisgeneratedaveryclean

extractsuitableforLC–MS/MSanalysis,thuspreventingdowntime

oftheinstrument.

3.2. Instrumentationandchromatographicconditions

ItwaschallengingtofindasuitableLC-columnforpyronaridine

thatproducedasymmetricalpeakshape.Inthepublished

litera-ture,mostLC-UVmethodsusedion-pairingreagentstoimprove

thepeakshape,butthisisoftennotcompatiblewithMS-detection

[27].MostC18andCNcolumnsthatwerescreenedshowedvarious degreesofpeaktailing,butagoodpeakshapewasobtainedfrom theamidefused-coreHALOcolumnwithoutusinganyadditives. The MS was operated in the positive ion mode, which gener-atedseveralabundantfragmentionsofpyronaridine(518.1m/z); 447.2m/z,376.2,378.2and341.2m/z(listedindecreasingorderof abundance).

3.3. Validation

Theassaywasvalidated accordingtotheUSFDA guidelines on bioanalytical method validation. Accuracy and precision of themethodwasdeterminedbyANOVAandthemethodshowed satisfactoryperformance(RSDbelow15%atallconcentration lev-els)whenquantifyingpyronaridine-spikedfluoride-oxalatewhole bloodsamples(Table1).

3.3.1. Linearity,selectivityandrecovery

Different calibration models were evaluated. The simplest modelthatdescribedthecalibrationcurveadequatelyandreturned highaccuracy forback-calculatedQCsampleswasordinary lin-earregressionwith1/xweighting.Thedevelopedmethodshowed goodselectivitywithnointerferingpeakswhenanalyzingblank blood from six different donors and also when using different anticoagulants(fluoride-oxalate,EDTA,Na-heparin,Li-heparinand fluoride-heparin).Furthermore,injectionofhighconcentrationsof commonlyusedantimalarialdrugs(i.e.chloroquine,amodiaquine, artesunate, dihydroartemisinin, primaquine and piperaquine) did not interfere with thequantification of pyronaridine (data notshown).Recoveries(extractedsample/post-extractionspiked sample)wereintherangeof65–75%forpyronaridinein fluoride-oxalatewholeblood,similartothatpreviouslyreported[13,15–17].

3.3.2. Matrixeffectandcarry-over

Matrixeffectinvestigationwithpost-columninfusiondidnot showanyincreaseordecreaseof thepyronaridinesignalwhen injectingextractedblankbloodfrom6differentdonorsorwhen using different anticoagulants. Matrix effects were also

inves-tigated by extracting blank blood that was post-spiked and comparingtheresponsewithspikedneatsolution.Thecalculations showedapost-spiked/spikedneatsolutionratiocloseto1forall samples,provingnoionsuppressionorenhancementfromanyof the6differentblooddonorsorfromthedifferentanticoagulants.

Carry-overwasnotedafter150–250samples(Fig.2)andcould not beeliminatedwitha more potentneedlewash solutionor longerwashtime.Theinjector usedaflow throughdesignthat shouldtheoreticallyeliminatepotentialcarry-overeffects. How-ever,thecarry-overoriginatedfromcontaminationoftheinjection needletip,andtosolvethisproblem,acustomcleaningprogram wasimplementedbetweensampleinjections.Theinjectionneedle aspiratedanddispensedawashingsolution4timesfromeachof the4wash-vialsbeforeaspiratingthesample,followedbystandard needlewashinaflushportfor15swithacetonitrile-water-acetic acid(70:29:1v/v/v)andinjectionofthesampleontothecolumn. The4wash-vialscontainedthefollowingsolutions;Vial1: acetoni-trile:methanol25:75;Vial2:acetonitrile-formicacid(2%inwater) 50:50v/v; Vial3:wateronly; Vial 4:mobilephase A.This cus-tomwashsuccessfullyremovedthecarry-overproblemsbutadded almost1mintotheruntimeofeachsample.

3.3.3. Stability

Freeze/thawstabilityoverthreecyclesshowedgoodstabilityfor spikedQCsamplesinfluoride-oxalateandEDTA.Fluoride-heparin barelypassedthestabilitycriteria,whileNa-heparinandLi-heparin resultedina10–15%lossineachfreeze-thawcycleresultingin atotallossof40–50%afterthreefreeze-thawcycles.Therewere novisualdifferences(precipitationorclogging)betweencyclesor samplesthatwouldexplainsuchasignificantdrop.Thus,duetothe poorfreeze-thawperformance,Na-heparinandLi-heparinshould notbeusedasanticoagulantsformeasurementofpyronaridinein bloodsamples.

Short termstability for upto 48hin fluoride-oxalate blood at22◦Candat4◦Cshowedgoodstabilitywithoutany degrada-tionoftheanalyte.Long-termstabilityat−80◦Cinthedifferent anticoagulantsshowedgoodstabilityfor upto6months.At12 monthstheQC3levelwasstillwithinthe15%limit,butalmost allmeasurementsattheQC1levelwerejustoutsidethe15%limit

(Table2).However,after50monthsfluoride-oxalatesamplesare

stillwithintheacceptablerangeindicatingsomeofthedropat12 monthscouldhavebeeninfluencedbyvariationinthe prepara-tionofthefreshstandards.Unfortunatelynootheranticoagulant sampleswereleftforanalysisatthattime.

Fig.2. Overlayofblankhumanfluoride-oxalatebloodandpyronaridine(1.47ng/mL)withinternalstandard(SIL-PYN;30ng/mL)showingearlysignsofcarry-overinthe blanksampleafterabout80sampleinjections.

Table2

Long-termstabilityofpyronaridineat−80◦C.

Fluorideoxalate EDTA Li-heparin Na-heparin Fluoride-heparin

QC1 QC3 QC1 QC3 QC1 QC3 QC1 QC3 QC1 QC3

Day0 100 100 100 100 100 100 100 100 100 100

2months 106 101 107 103 100 103 99 104 101 105

6months 89 95 96 100 98 99 89 99 92 93

12months 83 97 89 97 84 87 80 86 81 92

50months 89 90 N/A N/A N/A N/A N/A N/A N/A N/A

Thecalibrationcurvewaspreparedinfluoride-oxalate.Allotheranticoagulantcontrolsamplesweremeasuredusingthiscalibrationcurve,thereforeallvalueshasbeen normalizedvsday0QCconcentrationmeasurementsforeachanticoagulantandarepresentedaspercentages.ConcentrationlevelofQC1is4.67ng/mLandQC3is 452ng/mL.N/A,Notavailable.

alltypesofvirusesbutitwilldeactivateHIVvirusesandhopefully

reducemostothervirusesmakingbioanalysissaferforlaboratory

workers[21–23].BothpyronaridineandSIL-PYNstocksolutions

showedgoodstabilitywhenstoredat−80◦Cforupto12months.

3.4. Clinicalapplicabilityofthedevelopedmethod

Thedeveloped andvalidated methodwasusedtoanalyze a pharmacokineticstudyin17healthyThaivolunteers[5].Frequent blood samples were collected for up to 42days after a single oraldosetoevaluatethepotentialdrug-druginteractionbetween artesunate-pyronaridineandprimaquine.Thedevelopedmethod wasimplemented ina high-throughputsetting,capableof ana-lyzingupto300samplesperday.Themethodshowedexcellent clinicalapplicability,coveredthetherapeutic rangeof observed concentrations,andwasabletoquantifyclinicalsamplesaccurately withhighsensitivity.Totalprecision(%CV)ofqualitycontrol sam-plesduringanalysiswere3.53%,4.16%and4.20%forQC1,QC2and QC3.Allday42samples(n=31)wereabovetheLLOQconcentration level.Aconcentration-timeprofileforasinglehealthyvolunteer isshowninFig.3.Amaximumconcentrationof464ng/mLwas reachedafter1handthedrugwaseliminatedfromthebodywith aneliminationhalf-life of17days,resultingin a totalexposure (AUC)of11,100(hr×ng/mL)inwholeblood.Similar pharmacoki-neticprofilescouldbeseenforallvolunteers.

Fig.3.Observedconcentration–timeprofileofpyronaridineafterasingleoraldose of3Pyramax®

tablets(180mgpyronaridinetetra-phosphateand60mgartesunate pertablet)wasgiventoahealthyadultThaivolunteer.

4. Conclusions

The analytical method for pyronaridine in fluoride-oxalate wholeblood passedtheFDA validationcriteriasuccessfully and showedexcellentaccuracyand precision.Thenewmethodhad muchshorterruntime(5min)comparedtopreviouslypublished methods and used only 100␮L of whole blood. It provided a lower quantification limit of 1.5ng/mL. However, there were a fewfindingsthatneedtobehighlighted.Toavoidadsorptionof pyronaridine orinternal standard tosurfaces,allworking solu-tions used for dilution needed to contain a small volume of dilutedplasma.Fluoride-oxalateandEDTAshowedgood freeze-thawstabilitybutLi-heparin andNa-heparinshouldbeavoided duetothedecreasedrecoverywithsubsequentfreeze-thawcycles. Carry-overissuesintheHPLC-autosamplerwereovercomewith thedevelopedcustom cleaningprogram thatwasimplemented betweensampleinjections.

Wholebloodsamplesfromahealthyvolunteerstudyshowed thatthecalibrationcurvecoveredthewholeconcentrationrange from the study subjects and that the method could quantify pyronaridineforupto42daysafterasingleoraldose.Thesmaller samplevolumeof100_␮Lcomparedtopreviouslypublished meth-odswillbeasignificantadvantageinfuturefieldpharmacokinetic studies,particularlyinthestudyofyoungchildren.

Acknowledgement

ThisworkwassupportedbytheWellcomeTrustofGreatBritain [GrantCode:091901/Z/10/Z].

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