Analytical sample preparation strategies for the determination of

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JournalofChromatographyB

journalhomepage:www.elsevier.com/locate/chromb

Review

Analyticalsamplepreparationstrategiesforthedeterminationofantimalarialdrugsinhumanwholeblood,plasmaandurine

MonicaEscolàCasasa,MartinHansenb,c,KristineA.Kroghd,BjarneStyrishaved,ErlandBjörklunde,∗

DepartmentofEnvironmentalScience,FacultyofScienceandTechnology,AarhusUniversity,Frederiksborgvej399,4000Roskilde,Denmark

DepartmentofCivil&EnvironmentalEngineering,Yang&YamazakiEnvironment&EnergyBldg.,StanfordUniversity,473ViaOrtega,Room259,Stanford,CA94305,UnitedStatesc

DepartmentofGrowthandReproduction,CopenhagenUniversityHospital,Blegdamsvej9,DK-2100,Denmarkd

ToxicologyLaboratory,AnalyticalBiosciences,DepartmentofPharmacy,FacultyofHealthandMedicalSciences,UniversityofCopenhagen,Universitetsparken2,DK-2100Copenhagen,Denmarke

SchoolofEducationandEnvironment,DivisionofNaturalSciences,KristianstadUniversity,SE-29188Kristianstad,Sweden

a

b

article

info

abstract

Articlehistory:

Received7May2013

Receivedinrevisedform25February2014Accepted28February2014

Availableonline10March2014

Keywords:

SamplepreparationAntimalarialsBloodPlasmaUrine

Antimalarialdrugscommonlyreferredtoasantimalarials,includeavarietyofcompoundswithdifferentphysicochemicalproperties.Thereisalackofinformationonantimalarialdistributioninthebodyovertimeafteradministration,e.g.thedrugconcentrationsinwholeblood,plasma,andurine,whichmustbeimprovedinordertoadvancecuringtheparasiticdiseasemalaria.Akeyproblemalsoliesinthatphar-macokineticstudiesnotalwaysareperformedinpatientgroupsthatmaybenefitmostofthetreatmentsuchaschildren,pregnancyandlower-weightethnicpopulations.Herewereviewtheavailablesamplepreparationstrategiescombinedwithliquidchromatographic(LC)analysistodetermineantimalarialsinwholeblood,plasmaandurinepublishedoverthelastdecade.Samplepreparationcanbedonebyproteinprecipitation,solid-phaseextraction,liquid–liquidextractionordilution.AfterLCseparation,thepreferreddetectiontoolistandemmassspectrometry(MS/MS)butotherdetectionmethodshavebeenusede.g.UV,fluorescenceandelectrochemicaldetection.Majortrendsforsamplepreparationofthedifferentgroupsofantimalarialsforeachmatrixanditsdetectionhavebeensummarized.Finally,themainproblemsthattheresearchershavedealtwitharehighlighted.Thisinformationwillaidanalyticalchemistsinthedevelopmentofnovelmethodsfordeterminingexistingantimalarialsandupcomingnewdrugs.

©2014ElsevierB.V.Allrightsreserved.

Contents1.2.3.4.

Introduction.........................................................................................................................................Physicochemicalpropertiesofantimalarials.......................................................................................................Overviewofantimalarialmethodsandmetabolitesdeterminedindifferentmatrices...........................................................Samplepreparationstrategies......................................................................................................................4.1.Wholebloodsamplepreparation...........................................................................................................

4.1.1.RegularbloodsamplingandSPEorLLE...........................................................................................4.1.2.DBSandLLEorSPE.................................................................................................................

4.2.Plasmasamplepreparation..................................................................................................................

4.2.1.Proteinprecipitation...............................................................................................................4.2.2.SPE.................................................................................................................................4.2.3.Liquid–liquidextraction...........................................................................................................

110

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∗Correspondingauthor.Tel.:+46707319244.

E-mailaddress:erland.bjorklund@hkr.se(E.Björklund).

http://dx.doi.org/10.1016/j.jchromb.2014.02.0481570-0232/©2014ElsevierB.V.Allrightsreserved.

110

M.E.Casasetal./J.Chromatogr.B962(2014)109–131

5.

6.

Urinesamplepreparation...................................................................................................................4.3.1.SPE.................................................................................................................................4.3.2.Liquid–liquidextraction...........................................................................................................4.3.3.Dilution............................................................................................................................

Applicationofinternalstandards,liquidchromatographyandfinaldetection....................................................................5.1.1.Internalstandards(IS).....................................................................................................................5.1.2.Separationbyliquidchromatography.....................................................................................................5.1.3.Finaldetection.............................................................................................................................Concludingremarks.................................................................................................................................References...........................................................................................................................................

4.3.

129129129129129129129130130130

1.Introduction

Malariaisahealthproblemofmajorconcernworldwide.In2009therewereanestimated247millioncasesofmalariaandnearlyonemilliondeaths[1,2].ThepathogenicagentofmalariaisfourspeciesofPlasmodiumparasites[3,4].Sexualreproductionoccursinmosquitoes,actingasparasitevectors,whileasexualreproductionoccursinhumansactingashostsfortheparasite[5].Malariacanbecategorizedasuncom-plicatedorsevere.Theformerisdefinedasasymptomaticinfectionwithoutsignsofseverityorevidenceoforgandys-function[2],whilethelatterislife-threateningandresultsfromorgandysfunction.Oncehumansareinfectedandthesymptomsofmalariaobserved,antimalarialpharmaceuticals(antimalarials)areprescribedfortreatment[4,6].Currentlyusedantimalarialscanbeclassifiedintosevengroups[4](Table1);sesquiter-peneendoperoxides(artemisininbaseddrugs),4-aminoquinolines,arylaminoalcohols,8-aminoquinolines,antifolates,hydroxynaph-toquinones,andtetracyclines.Tetracyclines,however,havebeenexcludedfromthispapersincethesehavebeencoveredelse-where[7,8].Todaymostmalariatreatmentshavechangedfromthelesseffectivechloroquine/sulfadoxine–pyrimethaminemixtotheartemisinin-basedcombinationtherapies(ACT’s)recommendedbytheWorldHealthOrganization(WHO)[9].Theseconsistofartemisinin-basedcompoundscombinedwithavarietyofantimalarialsthathavelostefficacyasmonother-apeutics[10].AvailableACT’sare;artemether/lumefantrine,artesunate/amodiaquine,artesunate/mefloquine,andartesunate/sulfadoxine–pyrimethamine[11].Othercommontreatmentsarecombinationsofsulfadoxine–pyrimethaminewithchloroquine,amodiaquineorquinine[11].Quininemonotherapy(appliedfor300years)isstillinusefortreatmentofcertainresistantpara-sites,butshowstrongsideeffects[2,10].Additionally,quininecanbecombinedwithclindamycin,doxycyclinortetracyclineasrecommendedbytheWHO[10,11].Themixturechlor-proguanil/dapsone,wasprovenefficient,butwithdrawnin2008duetoitshaematologicalside-effects[12].Insteadthecombi-nationofproguanil/atovaquonehaslatelyprovedtobeefficientbuthighcostslimitsitsaccessibility,beingmostlyusedfortreat-mentbyWesterntravellersandmilitarypersonnel[10,11].Despitethelargenumberofantimalarialsprescribedworldwideformanyyears,thereisstilltodayoftenalackofknowledgeonantimalarialconcentrationsreachedinthebodyovertimeaftertheadminis-tration,whichisessentialtounderstandpharmacokinetics[13].Thereisalsodatadeficiencyontherelationshipbetweenbloodconcentrationsandthetherapeuticresponseinpatientswhichiscrucialforevaluatingthecauseoftreatmentfailuresuchaspar-asiteresistanceortoinsufficientdruglevelsintheblood[13].Additionally,themajorityoftheantimalarialshavenotbeendevel-opedusingstringentpharmacokinetic–pharmacodynamicstudies,asthismostoftenisnoteasilyperformedinfieldtrials.Con-sequently,somepatientsaremostlikelynotadequatelydosed.Thismayleadtounder-dosingpromotingparasiteresistanceor

itmayleadtoover-dosingtriggeringintoxication[14].Oneobsta-cleindeterminingpatientantimalariallevelsduringtreatmenthasbeenthelackofsensitive,reliableandrobustanalyticalmethods[14].However,theimprovementofliquidchromatography–massspectrometry(LC–MS)systemshasaidedingeneratingresultsforpharmacokineticstudiesthataremorereliablethanprevi-oussimplerchemicalorchromatographicassaysandbioassays[13].Withinthelastdecadeseveralanalyticalmethodshavebeenpublishedforthedeterminationofantimalarialsinhumanbodyfluids.Theaimwiththisreviewistoidentifyandassessrecentscientificarticlesapplyingliquidchromatographic(LC)meth-odsforthedeterminationofantimalarialdrugsinwholeblood,plasmaandurine.Majorfocusisgiventosamplepreparationstrategies,whichoftenisoverseendespitethatthisoftenisthepartoftheanalyticalchainmostpronetoerrorssuchaspoorrecoveriesduetolossesduringsampling,storage,extractionandevaporation.

2.Physicochemicalpropertiesofantimalarials

Thechemicalstructureandsizeoftheantimalarialsdiffertoalargeextentcausingagreatvariationinphysicochemicalparameters(Table1).Twokeyparametersarewatersolubility(Sw)andoctanol–waterpartitioningcoefficient(Kow)showingthesubstancesabilitytobesolubilizedinhydrophilicandlipophiliccompartments,respectively.ThelargedifferencesbetweenthelogKowandwatersolubilityforantimalarialsareillustratedinFig.1.ForamajorityoftheantimalarialsinTable1theidentifiedphysi-cochemicaldataarerelativelyconsistent,howeverafewofthemshowrelativelylargespansofSwandKow,forthesamecom-pound,dependingonsource.Thereasonforsuchdeviationsisnotalwaysobviousbutmightbearesultofthenatureofthemolecules,

10Sesquiterpeneendoperoxidesa)4-Ami noquinolines8Arylaminoalcohols8-Ami noquinolinesb)AntifolatesowK6Hydroxynaphtoquing oneol42r2 = . 720-2024log Swater (mg/L)Fig.1.RelationbetweenwatersolubilityandlogKowforvariousgroupsofanti-malarials,showingthelargevariationinphysicochemicalpropertiesforthis

heterogeneousgroupofpharmaceuticals.DatafromTable1.(a)Swwasfixedto0.01mg/Lforlumefantrine.(b)Swwasfixedto0.50mg/Lforpiperaquine.

Table1

Compoundname,CASnumber,structure,molecularweight,pKa,watersolubilityandlogKowofantimalarialpharmaceuticals.

Compound[CAS]

Structure

Molecularweight(g/mol)

pKa

Watersolubilitymg/L

LogKow

Sesquiterpeneendoperoxides/artemisininbaseddrugsArtemisinin[63968-64-9]

Dihydroartemisinin[71939-50-9]

Artesunate[88495-63-0]

Artemether[71963-77-4]

282.2284.4384.4298.4Noionizablegroup[74]

12.6[78]

4.6[74]

4.35[15]

Noionizablegroup

52[17]

62[75]50[76]8.4[77]168[79]

565[77]

56[15]

12[17]

117[77]457[16]

2.20[74]

2.90[17]2.90b

2.35[77]

2.73[77]2.19[80]2.77[77]

1.59[77]2.61[15,77]3.53[17]

2.05[80]3.53[15]3.02[16]3.48[16]

M.E.Casasetal./J.Chromatogr.B962(2014)109–131111Table1(Continued)

Compound[CAS]

4-Aminoquinolines

Amodiaquine[86-42-0]

Chloroquine[54-05-7]

Piperaquine[4085-31-8]

Structure

Molecularweight(g/mol)

355.9319.9535.5

pKa

7.1and8.1[81]

7.1and8.1[82]

10.1[15–17]9.5[84]

8.4[85]10.2[85]

5.39,5.72,6.24,6.88[86]

Watersolubilitymg/L

2.8[17]

2.8[15]8.8[16]

10.6[17]

0.14[15]17.5[16]

Poorlysoluble[87]

LogKow

3.99[17]

3.99[15]3.76[16]4.83[16]3.01[83]4.63[15,17]

3.93[16]5.28[16]4.72[85]

6.11[74,75,80,86]

112M.E.Casasetal./J.Chromatogr.B962(2014)109–131Pyronaridine[74847-35-1]

Arylaminoalcohols

Mefloquine[53230-10-7]

Quinine[130-95-0]

518.1

7.08,7.39,9.88,10.25[88]

378.38.7[15]

8.6[89](pKa2)

324.45.1,9.7[15]

210[88]

0.26[88]

22[15]

3.85[15]

38[16]

3.1[89]4.1[89]4.49a

500[17]

3.44[17]

M.E.Casasetal./J.Chromatogr.B962(2014)109–131113Table1(Continued)

Compound[CAS]

Halofantrine[69756-53-2]

Lumefantrine[82186-77-4]

8-AminoquinolinesPrimaquine[90-34-6]

Structure

Molecularweight(g/mol)

500.4

582.9

259.4pKa8.18[18](40%MeOHinwater)

–

10.39[90]

Watersolubilitymg/L

334[16]

0.11[16]

Insoluble[16]

1290[17]

114LogKow

2.51[16]2.82[16]7.34[16]

M.E.Casasetal./J.8.06[16]

Chromatogr.B962(2014)9.19[16]

109–1313.15[17]

Tafenoquinea[106635-80-7]Antifolates

Sulfadoxine[2447-57-6]

Dapsone[80-08-0]

Pyrimethamine[58-14-0]

Proguanil[500-92-5]

Chlorproguanil[537-21-3]

463.50.86,3.49,10.20[58]

310.36.01[64]

248.302.41[16,17]

248.77.34[16,17]

253.710.6,12.6[40]

288.2

–

56[16]

1.64[16]2.76[16]

3.00[76,82,91]34.18b

5.81[58]

6.34b

2700[17]

0.70[17]

296[16]

0.58[16]0.72[16]380[17]

0.97[17]

284[16]

1.19[16]1.27[16]121[17]

2.69[17]

179[16]

2.62[16]2.75[16]156[17]

2.53[17]

286[16]

1.89[16]1.9[16]

28[17]

3.17[17]

M.E.Casasetal./J.Chromatogr.B962(2014)109–131115116

M.E.Casasetal./J.Chromatogr.B962(2014)109–131

]w7o1K[g6o9.L0L/gmytilibul]o7s1r[et0a5W81]04[6.21,a6K.0p1)lom/g(thgiewraluce7l.o1M52erutcurtS]2-12)]-dS6eA1uC5n[[iltdinnnouaCou(g1polemclboyaCCT]61[]4671[.45]51[elbulosniyll]a6c1it[ca0r8.P0AN8.663.esabatadetiuSipEmorflatne.m)0ir1.e4p]4xv(-e,ee89tin10uo-n30Si2Iu3tPq2sEio5lt9ryh[abpetanSdenoetyutaxqyobmiraotdvisyotBEHAabdifferencesbetweenmethodologiesorvariationsinexperimentalconditionssuchastemperatureandpH,oracombinationsofsev-eralofthementionedfactors.

Sesquiterpeneendoperoxideshavewatersolubilityfromaround50mg/Luptoseveralhundredmg/Landamoderatelipophilicity(logKowintherange2–3.5).AtphysiologicalpHthemajorityofthemareonneutralformduetohighpKavaluesorlackofionizablegroup.ArtesunateisanexceptionwithapKavalueof4.6(carboxylicacid),makingartesunaterelativelywatersolu-ble(500mg/ml).Inaddition,artesunateisanunstabledrugthatrapidlywilltransformintodihydroartemisinin[10].Itshouldbenotedthatdihydroartemisinininitselfalsoisaprescribedanti-malarial.

4-Aminoquinolinesingeneralvarygreatlyinwatersolubil-ityfrompoorlysoluble(piperaquine)tofairlysoluble(200mg/L,pyronaridine).LikewisethelogKowspanmanyordersofmagnitudefromaround0.3upto6.

Arylaminoalcoholsalsohavelargedifferencesinwatersolubil-ityfromtheinsolublelumefantrinetothefairlysolublequinine(>300mg/L).AsaconsequencethelogKowvaluesspanmanyordersofmagnitudefromaround3(quinine)upto9(lumefantrine).ItshouldbenotedthatthehighvalueforlumefantrineisanestimatedlogKowwhileanexperimentalvaluemightbelower.ForexamplehalofantrinehasareportedexperimentallogKowvalueof3.5[18]whiletheestimatedvaluesare>7.However,itwasnotpossibletoassessthestudyin-depth[18],thereforetheexperimentalvaluewasomittedfromFig.1.

8-Aminoquinolinesconsistsofprimaquineandtafenoquine,andarebothtosomeextentsolubleinwater(>30mg/L),thoughpri-maquineseemstohaveasomewhathigherabilitytodissolveinaqueousphases.However,itshouldbenotedthatthereisasubstan-tialdiscrepancyinreportedwatersolubilitydataforprimaquinedependingonsource,varyingbyafactor20(Table1).Addition-ally,thereportedlogKowforprimaquinealsodiffersbynearlyafactorof30,probablyreflectingthedifficultiesoftenencounteredwhendeterminingphysicochemicalpropertiesofcompounds.YetitseemsreasonabletobelievethatprimaquineissomewhatmorewatersolublethantafenoquinewhencomparinglogKowvaluesforthetwocompounds,TheformerhavelogKowvaluesatleast2ordersofmagnitudelowerthanthelatterdemonstratingamuchlowerlipophiliccharacter.

Antifolatesareallingeneralhighlysolublewithseveralhundredmg/Luptotheg/Llevel.Consequently,thisisalsothegroupwiththelowestlipohilicity(logKow)rangingfrom0.5to3.

Thehydroxynapthoquinone,atovaquonehasfairlylowwatersolubilityandanestimatedlogKowofalmost6.Atphysiologi-calpH-levels,thismoleculewillmainlybefoundonitsneutralform.Thefirstobservationtobemadeisthatthesesquiterpeneendoperoxidesandtosomeextenttheantifolatesareveryhomoge-nousgroupswithalargeabilitytodissolveinwater,exceeding10mg/L(lowerrightofFig.1).Furthermore,thegroupshaverel-ativelylowlipophilicityshowinglogKowvaluesof3orbelow.InsharpcontrasttothesetwogroupsarethearylaminoalcoholswithgreatvariationinbothKowandwatersolubility.Likewisethe4-aminoquinolonesshowlargedifferencesasdothetwo8-aminoquinolones.Acleartrendintermsofwatersolubilityforthesegroupscanthereforenotbegiven.IntermsofhydrophobicityontheotherhandthereisatendencytowardshigherlogKowvaluesforthesethreegroupscomparedtosesquiterpenesandantifo-lates.ThemajorityoftheaminoquinoloneshavealogKowabove3demonstratingarelativelyhighlipophilicity,withtheexcep-tionofpyronaridine(logKow0.26).AfinalobservationtobemadefromFig.1isthewell-knowncorrelation(r2>0.70)betweenlow-eredwatersolubilitybeingaccompaniedbyincreasedlipophilicity(logKow).

M.E.Casasetal./J.Chromatogr.B962(2014)109–131

117

Fig.2.OverviewofLC–MSmethodsforthedeterminationofvariousantimalarialsseparatedasblood,plasmaandurineapplications.

3.Overviewofantimalarialmethodsandmetabolitesdeterminedindifferentmatrices

Anoverviewofantimalarialmethodspublishedforeachanti-malarialisshowninFig.2.Plasmaisbyfarthemostcommonmatrixinvestigatedwithatotalof34methods[14,19–51]followedbywholeblood[48,49,52–59]andurine[22,36,44,48,50,60–63]with11and9methods,respectively.Themajorityofthemethodsaredevelopedforfourcompoundsorless.Mostofthesemeth-odsfocusontheanalysisof2–4compoundsandatleastoneofitsmetabolites[14,19,22,24,30–35,37,39–48,50,52–56,59,63],oth-ersanalyzedrugspertainingtothesamegroup[52,56]oranalyzeanexistingmarketdrugcombination[21,24,26,34,35,51,64].Onlythreemulti-methodshavebeenpublished,analysingmorethanfourantimalarialsinoneinjection[14,22,59].

ThelargestnumberofHPLCmethodsexistsforthesesquiter-peneendoperoxides.TheWHO,whichadvocatestheusageofACTs[11],in2006requestedpharmaceuticalcompaniestoendmarketingandsaleofsingleartemisinin-basedantimalarials[65]andmanycountriesadoptedACTsasamaintherapy.Inlinewiththis,Souppartandco-workerspublishedin2002thefirstLC–MSvalidatedmethodforartemetheranddihydroartemisinininplasma[43].Between2005and2011,another12methods[19,30–33,41,42,44–47]werepublishedfortheanalysisofonlyartemetherorartesunateandtheircommonmetabolitedihy-droartemisinin.Surprisingly,merelytwomethodsexisttoanalyzeartemisinin,whichisnotbeingappliedasanACT[29,52].

The4-aminoquinolineshasseveralmethodsavailableandforallmatrices.Amodiaquineadministratedalonehasahighnumberoftreatmentfailuresandmaycausehepatotoxicity[3].However,itisstillefficientagainstchloroquineresistantstrainsandcheap[10].Consequentlyitisusedincombinationwithartesunateorsulfadoxine–pyrimethamine.Sixmethodspublishedbetween2002and2007includetheanalysisofamodiaquine[14,48,53,54,56].Chloroquinewaseffectiveandwidelyusedinthepast,howevernowadaysPlasmodiumfalciparumisresistanttothisdruginallendemicareasexceptCentralAmerica[11].Between2002and2010,sevenmethodsthatincludetheanal-ysisofchloroquinehavebeenpublished[14,24,48,56,59–61].Piperaquineisusedincombinationwithdihydroartemisininandthiscombinationprovidescost-effectiveandwell-toleratedtreatment[3].Fivemethodsincludedtheanalysisofthisdrug[14,20,36,57,66].Pyrimethamine,incombinationwithartesunate,isbeingusedforthetreatmentofacuteuncomplicatedmalaria[3]andfourmethodsincludedthisdrug[14,38,49,67].

Arylaminoalcoholsincludesmefloquine,quinine,halofantrineandlumefantrine.Mefloquineisaneffectivedrugbutitisfre-quentlybadtolerated.Ithasfourmethodspublished[14,34,59,60]during2005and2010.Quinineisstillimportantforthetreatment

ofuncomplicatedmalaria,andoftentheonlyoptionforthetreat-mentofseveremalaria[11].Sincesomestrainsofmalariaparasitestodayareresistanttothenewerantimalarials,theimportanceofquinineisincreasing[10,60]andsixmethods[14,22,24,55,59,60]werepublishedbetween2001and2010.Nomethodshavebeenidentifiedthelastdecadeforhalofantrine,whilefourmethodsexistforlumefantrine[14,23,37,59]publishedbetween2005and2010.Halofantrine,asimilardrugtolumefantrinebutwithhigherantimalarialactivity,hasbeenwithdrawnfromthemarketinsev-eralcountriesduetocardiotoxicsideeffects[10].Fataleffectsofthisdrugoccurredonpatientswithpredisposingfactorforcardiotoxicityorpatientswhowereadministratedhigherthanrec-ommendeddosesortookanotherconcomitantdrug.Thereforefollowingmanufacturerrecommendationscoulddecreaseadverseeffects.Halofantrinemayhowever,againbeneededifnewanti-malarialarenotsuccessful.

Thegroupof8-aminoquinolinescoversprimaquineandtafeno-quine.Primaquine,istheonlycompoundcurrentlyinuse,andislicensedforradicalcureofPlasmodiumvivaxinfectionsortoavoidrelapse[2].However,itispotentiallytriggeringhaemolyticanaemiainhumanswithG6PDdeficiency[2].Tafenoquineisinthelaststageofevaluationwiththesamerisksasprimaquinebutithasashortertreatmentcourse[58].Wehaveonlyidentifiedasinglemethodfrom2011[58]fortheanalysisoftafenoquine,whichisadruginthefinaltestingphaseoftheclinicaltrial.

Theantifolatesgroupconsistofsulfadoxine,pyrimethamine,dapsone,proguanilandchlorproguanil.Sulfadoxineismainlyusedcombinedwithpyrimethamine[11],andthreemethodsthatana-lyzesulfadoxineandpyrimethaminewiththesamemethodhavebeenpublished[14,26,59].Inthelate1990s,acombinationofdap-soneandchlorproguanil(namedLapdap)wasintroduced[68].Bothlow-costdrugswereexpectedtopreventresistancetogether.How-ever,haemolyticanaemiaoccurredasasideeffect,moresevereinpeoplewhohadgeneticdeficiencyofglucose6-phosphatedehydrogenase(G6PD),whichiscommoninareaswithendemicmalaria.Nevertheless,LapdapremainedonthemarketinmanyAfricancountriesuntil2008[12].Nomethodshavebeenfoundforthestudiedperiodfordapsoneandchlorproguanil.Proguanil,likechlorproguanil,hasstructuralsimilaritiestopyrimethamineanditismostlyusedcombinedwithatovaquone[11].Fourmethodshavebeendevelopedfortheanalysisofproguanil[39,40,50]between2005and2009.

Hydroxynaphtoquinonesisrepresentedbyatovaquone.Thisdrugisadministratedincombinationwithproguanilfortheprophylaxisandtherapyofmalaria[11].Threemethods[27,28,40]werepublishedbetween2005and2010fortheanalysisofatovaquone.

Severalmethodsincludethedeterminationofantimalarialpharmaceuticalstogetherwithoneormoremetabolites.Table2

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M.E.Casasetal./J.Chromatogr.B962(2014)109–131

Table2

Analyticalstudieswithsimultaneousdeterminationofantimalarialsandmajormetabolitesthereofinwholeblood,plasmaandurine.

Antimalarialdrugs

Metabolites

References[52][52]

[48,53,54,56][48,56,59][55]

[14,19,30–32,34,35,41,42][14,33,43–47][14,35,48][22][22][22][22][37]

[39,40,50][40,50][44][48][22,63][22][22][22][50][50]

WholebloodArtemisininArtesunateAmodiaquineChloroquineQuinine

DihydroartemisininDihydroartemisininDesethylamodiaquineDesethylchloroquine3-hydroxyquinine

PlasmaArtesunateArtemetherAmodiaquineQuinineQuinineQuinineQuinine

LumefantrineProguanilProguanil

DihydroartemisininDihydroartemisininDesethylamodiaquine(3S)-3-Hydroxyquinine2󰀇-Quininone

(10R)10,11-dihydroxydihydroquinine(10S)10,11-dihydroxydihydroquinineDesbuthylumefantrineCycloguanill

4-Chlorophenylbiguanid

Urine

ArtemetherAmodiaquineQuinineQuinineQuinineQuinineProguanilProguanil

DihydroartemisininDesethylamodiaquine3s-3-Hydroxyquinine2󰀇-Quininone

(10R)10,11-Hydroxydihydroquinine(10S)10,11-HydroxydihydroquinineCycloguanill

4-Chlorophenylbiguanid

givesanoverviewofanalyticalstudiesweresuchmetaboliteshavebeenstudiedtogetherwiththemothercompoundinwholeblood,plasmaorurine.Well-knownmetabolitessuchasdesethylamodi-aquine,desethylchloroquine,dihydroartemisininandcycloguanilarealsoactiveantimalarialcompounds[10,39,48,69].Determi-nationofthesemetabolitesinbiologicalmatricescanbeusefultodevelopbettertherapeuticdosesoftheparentcompound.Desbutyl-lumefantrineisapotentialactivemetaboliteoflume-fantrine,butitsformationhastoourknowledgenotbeenreportedinvivo.Presently,thereisasinglemethoddevelopedforthedeter-minationofthesetwocompoundsinplasma[37].Asinglemethodisavailabletoelucidatetheeliminationpathwaysofquinineandfourofitsmetabolitesinplasmaandurine[22].Itislikelythatthenumberofstudiesinvolvingmetaboliteswillincreasethecomingyearsasmoredetailedresearchstudiesareconductedtoinvesti-gatetheproblemsrelatedtoantimalarialsresistance.TechnologyimprovementsthelastdecadeespeciallyrelatedtocheaperandmoreavailableLC–MS/MSequipmentworld-widewillaidinthisprocess.

ormaybetheonlystepperformedpriortoinjection[22,63].UrinecanalsobedirectlyextractedbySPE[36,60]orLLE[44,50,61,62].Ingeneral,urineisusedinpharmacokineticstudiestoquantifytheexcretionlevelsofantimalarial,includingmetabolites.Urineassaysarenon-invasiveandallowslargesamplevolumes,whichmayleadtoimprovedsensitivityandlongerquantificationovertime.Itshouldbeemphasizedthatnearlyallmethodspublishedforthethreematricesmentionedincludeeitherliquid–liquidextrac-tion(LLE)orsolid-phaseextraction(SPE)aspartofthesamplepreparationstrategy(Fig.3).Thevariousmethodsarepresentedforwholeblood,plasmaandurineinTables3–5,andreviewedinthefollowingsections.

4.1.Wholebloodsamplepreparation

4.Samplepreparationstrategies

Differentsamplepre-treatmentsandextractiontechniqueshavebeenappliedforthedeterminationofantimalarialsasvisual-izedforwholeblood,plasmaandurineinFig.3.Forwholeblood,extractioncanbedoneeitherdirectlyonblood[48,49,52]orbythedriedbloodspot(DBS)technique[48,53–59].Plasmacanbedirectlyanalyzedafterproteinprecipitation[14,19–24,26–28]orfurtherextractedafteraproteinprecipitation[37,40].PlasmacanalsobedirectlyextractedbySPE[29–39]orLLE[41–51].

Ithasbeenarguedthatantimalarialplasmaconcentrationsnotalwaysrevealtheactualconcentrationinwholeblood,whichmaybehigherinbloodcellsthaninplasma[49,70].Thus,forpharma-cokineticstudiesitmightbepreferabletoanalyzewholeblood.However,asAshleyetal.experiencedforpiperaquine,thelimitofquantificationmaybehigherinwholebloodthaninplasmabecauseofsmallersamplevolumesandlowerrecoveries[70].Forurine,asimpledilutionissometimesappliedasapre-extractionstep[48]

4.1.1.RegularbloodsamplingandSPEorLLE

Therearethreemethodsdevelopedusingordinarybloodsamplingcoveringpyronaridine[49],amodiaquineanditsmetabo-litedesethylamodiaquine[48],andtheartemisinin-baseddrugsartemisininandartesunateandthemetabolitedihydroartemisinin[52](Table3).

Chenetal.[49]applied200␮lbloodsamplesdilutedinphos-phatebufferandextractedpyronaridinewithdiethylether.Spikedbloodshowedabsoluterecoveriesbetween73and101%forana-lytesand85%forIS.Minzietal.[48]collectedbetween100and1000␮lblood(frozenuntilanalysis)dilutedwithwaterandextractedamodiaquineanditsmetabolitedesethylamodiaquineintwostepswithdi-isopropyletherfollowedbyphosphatebuffer.Absoluterecoverieswere82–103%foramodiaquine,68–78%fordesethylamodiaquine,and73–92%forIS.Animportantobservationwasthelowstabilityofanalytesinwholebloodsamples.Maxi-mumstoragetimerecommendedwas7daysat4◦C.In2011thefirstanalyticalmethodforthemeasurementofartesunate,dihy-droartemisininandartemisinininwholebloodwaspresented[52].Iron-mediateddegradationoccurswhenhaemoglobinisprecipi-tatedbyorganicsolvents,releasingFe2+thatdegradesthedrugs.Thiswaspreventedbyarelativelycomplicatedtreatmentpro-cedureapplyingpotassiumdichromateforoxidationofFe2+to

M.E.Casasetal./J.Chromatogr.B962(2014)109–131

119

Table3

Analyticalmethodsforthedeterminationofantimalarialsinwholeblood.

Analyte[Ref.]

Extraction

Regularbloodsamplingandliquid–liquidextraction

Pyronaridine[49]

Sample:200␮lblood+400ngIS+500␮l500mMPBpH=10.3Extraction:3mldiethylether.Centrifugation,transferorganicphase.

100␮lEtOHaddedtoorganicphase.Driedandreconstitutedwith100␮lACN:PB(pH=2)

AmodiaquineSample:100␮l

Desethylamodiaquineblood+900␮l+water+50␮l[48]1.6␮MIS+2000␮lCBpH=9.5.

Extraction1:7mldi-isopropylether.

Extraction2:150␮lPBpH=4Sample:1000␮lblood+1000␮lwater+50␮l1.6␮MIS+2000␮lCBpH=9.5.Proceedasabove

Regularbloodsamplingandsolid-phaseextraction

Artemisinin(ARN)

Sample:2mlblood+potassiumDihydroartemisinindicromate.Storageat-80◦C.

(DHA)

HLBSPE96-wellplate:70␮lArtesunate(ARS)

blood+50␮lpotassium[52]dichromate0.008M+50␮l

deferoxamine20mg/ml+250␮lice-coldIS(SILDHAandARS).Offline-SPEwithhalfofthetotalsamplevolume.

Elution:100␮lEtOH:ACN(90:10,v/v)+50␮lwater

Driedbloodspotandliquid–liquidextractionAmodiaquineDBS:200␮lbloodonglassDesethylamodiaquinemicrofibrefilterstrips(GF/C[53]Whatman).

Extraction:DBS+50␮lMeOHcontaining500ngIS+2.5mldistilledwater.Additionof5mldimethylether.Centrifuged.Upperorganicphasetransferred,

evaporatedandreconstitutedwith100␮lMP

Amodiaquine

DBS:100␮lblood+100␮lPA.DesethylamodiaquineSpottedonfilterpaper.[54]Extraction:DBS+CB(2ml,2M,pH

9.7)+KOH(120␮l,1M)+8mldi-isopropylether.

Organicphasebackextractedwith150␮lPB(0.1M,pH=4).Centrifugation.Waterphaseinjected

Chloroquine

DBS:100␮lofbloodspottedonDesethylchloroquinefilterpaper.Mixedwith500␮l[48]0.5%DEAinwater+50␮lor75␮l

ofIS+2000␮lKOH1M.Extraction1:7mldi-isopropylether.

Extraction2:150␮lPBpH=2.5

QuinineDBS:100␮lcapillaryor200␮l3-venousbloodspottedoncellulosehydroxyquininefilterpaper(Whatman3MMChr).[55]

Extraction:1ml0.1M

NaOH+100␮lIS.Vortexed.

Additionof2mltoluene/butanol(75:25,v/v)mixedbyrotation.Centrifugation.Upperorganic

phasetransferred,evaporatedandreconstitutedwith100␮lof10%ACNinAB(pH=4).

Separation

ZorbaxBonusRP;

250mm×4.6mm,5␮m

MP:IE,ACN:0.08MPB(13:87,v/v)adjustedtopH=2.8

FR:1.0ml/min;RT:aprox.20min;IV:50␮l

IS:Amodiaquine

ZorbaxSBC18;75mm×4.6mm,3.5␮m

MP:IE,MeOH:0.1MPB:PCA(250:747.5:2.5,v/v)

FR:1.5ml/min;RT:aprox12.min,IV:100–120␮l

IS:4-(4-dimethylamino-1-methylbutylamino)-7-chloroquinoline

HypersilGoldC18;

150mm×2.1mm,3␮m

MP:IE,ACN:AmAc10mMpH3.5(40/60,v/v)

FR:0.5ml/min,RT:N/A,IV:5␮lIS:SILARN,DHAandARS

Synergi4␮M

Polar-RP;150mm×4.6mm

MP:25mMKH2PO4:MeOH80:20(v/v)with1%(v/v)triethylamineadjustedtopH=2.8ortophosphoricacid.

FR:1.2ml/min,RT:aprox.20min,IV:50␮l

IS:Quinidinebase

ZorbaxSBC1875mm×4.6mm;3.5␮m

MP:IE,MeOH:PB(0.1M,

pH=2.7):PCA(250:747.5:2.5,v/v/v)

FR:1.5ml/min,RT:N/A;IV:130␮lIS:4-(4-dimethylamino-1-methylbutylamino)-7-chloroquinoline

ZorbaxSBC18;75mm×4.6mm,3.5␮m

MP:IE;MeOH:PB(0.1M,

pH=3):PCA(250:747.5:2.5,v/v/v)FR:1.5ml/min,RT:aprox.12min,IV:100–120␮l

IS:4–4dimethylamino-1-methylbutylamino)-7-chloroquinoline

ZorbaxEclipseXDBphenyl;150mm×4.6mm,5␮m

MP:A:8%ACNin0.1MABpH=3.9,B:24%ACNin0.1MABpH=4.3FR:1.0ml/min,RT:aprox.15min,IV:5–70␮lIS:Quinidine

Detection

UV(275nm)

UV(333nm)

ESI(+)LC–MS/MS,MRM

m/z:ARN:300>209

DHA:302>163SIL-DHA:307>166ARS:402>267SIL-ARS:406>163

UV(340nm)

UV(333nm)

UV(333nm)

FL(EX/EM)(350/450nm)

RecoveryRange(%)(ng/ml)73–101a

29–1140

82–103–

68–78a

>90b

1.4–8502.0–25001.4–850

8430–250074c30–2500

4917.8–71248d16.4–656

72–920.79–1279101–105e0.73–1167.3

79–1033.2–32496–109a3.4–3404

120

M.E.Casasetal./J.Chromatogr.B962(2014)109–131

Table3(Continued)

Analyte[Ref.]

Extraction

Separation

Detection

Recovery(%)44–5672–8241–5775–78f

Range(ng/ml)36–106732–6033–98329–876

Driedbloodspot+solid-phaseextractionAmodiaquineDBS:100␮lofbloodspottedonChloroquinefilterpaper(31ETCHRWhatman)DesethylamodiaquineExtraction:2ml0.3MPCA.

Vortexed.1000␮lACNDesethylchloroquine

[56]added+5000␮lISinPBpH=2.

LiquidphaseplacedonSPE

cartridgesPRS(cationexchange).Elution:4×450␮l

MeOH:Triethylamine(98:2,v/v)DBS:100␮lofbloodspottedonPiperaquine

[57]filterpaper(31ETCHRWhatman)

Extraction:2ml0.3M

PCA+1000␮lACNand5000␮lPBpH=2withIS.LiquidphaseplacedonSPEcartridgesMPC/C8(cation/RP).

Elution:4×450␮l

MeOH:triethylamine(98:2,v/v).DBS:100␮lofbloodspottedonTafenoquine[58]

filterpaper(31ETCHRWhatman).Samplespre-treatedwith0.6Mtartaricacid.

Extraction:2000␮lACN(0.01MIS)+2000␮lAA.LiquidphaseplacedonSPEcartridges(RP/cation/anion).Elution:2×1000␮l

MeOH:Triethylamine(80:20,v/v).Eluatesevaporatedand

reconstitutedin50␮lACN:FA(0.1M)(50:50,v/v)

Screeningmulti-methodQuinine

DBS:100␮lbloodspottedonfilterMefloquine

paper(31ETCHRWhatman).Sulfadoxine

Extraction1:1.5mlMeOH:AAPyrimethamine

(20:80,v/v).LiquiddecantedandChloroquine

1mlAA(0.5M)added.PlacedonDesethylchloroquine

SPEcartridgesmixed-mode.Lumefantrine

[59]Extraction2:1.5mlACN:AA

(50:50,v/v).Liquiddecantedand0.7mlAA(0.5M)added.PlacedonSPEdisksC8.

Eluatesfrombothextractionsdried.

1stextractioneluates+100mlMeOH:HCl(0.01M)10:90,v/v2ndextractioneluates+100mlMeOH:HCl(0.01M)60:40,v/v

ZorbaxSB-CN;250mm×4.6mm;5␮m

ACN:PB(0.1M,pH=2):Sodiumperchlorate(1.0M)(13:86:1,v/v)FR:1ml/min,RT:aprox.40min,IV:100␮l

IS:Chloroquinemethylatedinthequinolinegroup

UV(342nm)

ChromolitPerformance;200mm×4.6mm

ACN:PB(0.1M,pH=2.5)(8:92,v/v)FR:3.5ml/min,RT:aprox.2min;IV:100␮l

IS:3-methyl-4-(3-hydroxy-4-dimethylaminopropyl)-7-chloroquine

UV(345nm)

48–65i27–1204

ZorbaxSB-CN;150mm×3.5mm;3.5␮m

MP:ACN:FA(0.1M)(63:37,v/v)FR:0.45ml/min;RT:aprox.5min;IV:20␮l

IS:(N-(2.6-dimethoxy-4-methyl-5-(3-rifluoromethyl-phenoxy)-quinolin-8-yl)acetamid

FL(EX/EM)(262/470nm)

36–49b23–695

PhenomenexGeminiC18150mm×2mm;5␮m

MP:GE,A:ACN:Ammonium

formiate(20mMwith1%vol.FA)(80:20)(v/v),B:ACN:Ammoniumformiate(10mMwith1%vol.FA)(80:20)(v/v)

FR:0.3ml/min,RT:15min,IV:10␮lIS:NoIS

IontrapMS(+)mode

75–8540–5025–3560–7560–7560–75Ca.20g

250–2000250–2000500–50000250–800250–800250–800250–2000

AA:aceticacid,A:mobilephaseA,AB:acetatebuffer,ACN:acetonitrile,AmAc:ammoniumacetate,B:mobilephaseB,Cond:conditioning,EM:emission,ESI:electrosprayionization,EtOH:ethanol,EX:excitation,FA:formicacid,FL:fluorescence,FR:flowrate,GE:gradientelution,IE:isocraticelution,IS:internalstandard,IV:injectionvolume,MeOH:methanol,MP:mobilephase,MRM:multi-reactionmonitoring,MTBE:methyltert-butylether,PB:phosphatebuffer,PCA:perchloricacid,RT:runtime,SIM:singleionmonitoring.a

Quantifiedcomparingspikedwholebloodsamplespeakheightsorareaswiththoseofdirectlyinjectedstandards.b

QuantifiedcomparingqualitycontrolsamplestodirectlyinjectedsolutionscontainingthesamenominalconcentrationoftheanalyteafterSPE.c

Quantifiedcomparingspikedwholebloodspottedonfilterpapertoequivalentamountsofanalytesspikeddirectlyintheorganicphase.d

Quantifiedcomparingspikedwholebloodspottedonfilterpapercomparedtodirectlyinjectedreferencesolutionspreparedin0.01Mhydrochloricacid.e

Quantifiedcomparingspikedwholebloodspottedonfilterpapertodirectlyinjectedstandardsolutionsinwatercontaining0.5%DEA.f

Absoluterecoveryofspikedwholebloodspottedonfilterpapercomparedtoblanksampleeluatespost-spikedatthesamenominalconcentration.g

Quantifiedcomparingspikedwholebloodspottedonfilterpapertodirectlyinjectedstandardpreparedinreconstitutionsolventatthesamenominalconcentration.

Fe3+andadditionofdeferoxaminetochelateFe3+[52].Tomini-mizecontactwithorganicsolventsandtoensuredrugssolubilitytheaddedISsolutionswerepreparedinplasma/water(50:50).Elutionwasmadewithmethanol/acetonitrilemixtureandwater,andabsoluterecoveriesweredemonstratedlargerthan90%forallcompounds,includingtheinternalstandard.Otherstrategieshavealsobeentestedtostabilizeartemetheranddihydroartemisinininhemolyzedsamples,includingoxidationofFe2+toFe3+,using3Msodiumnitritecontaining1%aceticacid[92].Averyrecentapproachutilizedhydrogenperoxidetoprotectthetwodrugsfromdegradationinhumanplasma[93].

4.1.2.DBSandLLEorSPE

ThecombinationDBS/LLEhasbeendevelopedfortheanalysisofamodiaquineandthemetabolitedesethylamodiaquine[53,54],chloroquineandthemetabolitedesethylchloroquine[48]andqui-nineandthemetabolite3-hydroxyquinine[55].Thesemoleculeshavestructuralsimilaritiesandthereforethesethreemethodshavefeaturesincommon(Table3).Thebloodsamplesizesallrangedfrom100to200␮l.Oncedried,bloodspotsweremixedwithwaterandextractedbyshakingwith5–8mldi-isopropyletherordimethylether.Sincequinineand3-hydroxyquininedifferinpolarity,theywereextractedwithamixtureoftoluene/butanol.

M.E.Casasetal./J.Chromatogr.B962(2014)109–131

121

Fig.3.Samplepreparationstrategiesforthedeterminationofantimalarialsinblood,plasmaandurine.

Bloodspottingandextractionfromglassmicrofibrefiltersgaveabsoluterecoveriesof84%and74%foramodiaquineanddesethy-lamodiaquine,respectively,whilecellulose-basedfilterpapers,showedverypoorrecoveries,below20%[53].Asimilarstudyonamodiaquineanddesethylamodiaquinerevealedlowabso-luterecoveriesforthesametwocompounds,lessthan50%,butthefilterpapertypewasnotstated[54].PoorrecoverieswereexplainedasacompromiseofoptimalpHforextractingamodi-aquineanddesethylamodiaquineinordertoobtainsimultaneousandcomparableextractionefficienciesofbothcompounds.Abso-luterecoveriesofapproximately80%and100%wereobtainedforchloroquineanddesethylchloroquine,respectivelyonanunspeci-fiedfilterpaper[55].Incontrasttothelowrecoveriespresentedaboveforamodiaquineanddesethylamodiaquineoncellulosebasedfilterpapers,quinineand3-hydroxyquininecouldbequali-tativelyrecoveredfromsuchfilterpapers[22,55].

FourmethodscombineDBSwithSPE[56–59](Table3).Threeofthesehavebeenvalidatedforamodiaquine,desethylamodi-aquine,chloroquineanddesethylchloroquine[56],piperaquine[57]andtafenoquine[58].Theseantimalarialsareallamino-quinolinesandshowstructuralsimilaritiesandconsequentlythethreemethodsfollowalmostthesameprotocolapplyingsimilarsolvents.Bloodsample(100␮l)werespottedonfilterpaperanddried.Amodiaquine,chloroquineandpiperaquineweremixedwithperchloricacid,acetonitrileandphosphatebuffer(pH=2)[56,57],orinthecaseoftafenoquine[58]withace-tonitrileandaceticacid.Centrifugationfollowedandtheliquidphasewasrunthroughacation-exchange-SPEorcombinedwithanionexchangerand/orreversedphasegroups.Overallrecov-eriesforamodiaquine,chloroquine,desethylamodiaquine,anddesethylchloroquinerangedfrom48to77%,whenusingcellulosebasedfilterpapers[56].Thisisinlinewithpreviousdiscussionthatsuchfilterpapersmaycauselosses[53,54],thoughitshouldbenotedthattheextractionsolventappliedherewasacetoni-trileandnotethers.Lossesduringfiltrationwereinvestigatedinastudyonpiperaquinesincethecellulosebasedpaperresultedin

recoveriesbetween56and88%[57].NolosseswerefoundinSPE,evaporationorreconstitution.Piperaquinebindingtoplasmapro-teinswasinvestigatedthroughbindingtoredbloodcells,howeverthesedifficultexperimentsdidnotshowconcludingresults.Twoothertypesofcellulose-basedmaterialswereinvestigatedwith-outimprovedrecoveries.Theseauthorsconcludedthatbindingtoeitherfilterpaperorbloodcellscouldcausetheselosses,butthecausativeagentforlowrecoveriescouldnotbefullyidentified.Lowrecoverieswerealsoobservedfortafenoquinespikedonuntreatedcellulosebasedfilterpaperwithrecoveriesaslowas10–15%[58]whilepapertreatedwithtartaricacidincreasedrecovertoca.40%.

Ascreeningmethodforsevenantimalarials:pyrimethamine,sulfadoxine,lumefantrine,quinine,mefloquine,chloroquineanddesethylchloroquine,butunfortunatelywithoutIS,waspublishedin2010[59].Blood(100␮l)wasspottedoncellulose-basedfilterpaper.Recoverieswereanalytedependedandmefloquine,sul-fadoxineandlumefantrinehadrecoveriesbelow50%,onceagaindemonstratingpotentiallossestosuchfilterpapers[53,56].Overallitisclearthatthechoiceoffilterpapermaterialmayhavesignifi-canteffectsonrecoveriesincaseswhereproteinbindingorSPEmaybetheactualcauseforthelowrecoveries.Scientistsshouldeval-uatetheDBSmethodologyonacase-by-casebasis,butingeneralcellulosebasedmaterialsseemstosufferpoorextractability.

4.2.Plasmasamplepreparation

4.2.1.Proteinprecipitation

Tenmethodsuseproteinprecipitationasasinglesampleprepa-rationstepprecipitation[14,19–24,26–28]whiletwomethodscombineproteinprecipitationwithSPE[37,40](Table4).Whenproteinprecipitationwasappliedasasinglestep,organicsol-vents,mainlyacetonitrileormethanol,wereaddedto25–500␮lofplasma(dilutionfactor2–10).TheIS’swereeitheraddedtosamplepriortoprecipitationortotheorganicsolventbeforebeingaddedtotheplasma.Samplesweremixed,centrifugedandmostoftenthe

122Table4

Analyticalmethodsforthedeterminationofantimalarialsinplasma.

Analyte[Ref]

ProteinprecipitationArtesunate

Dihydroartemisinin[19]

Piperaquine(PQ)[20]

Artemether

Lumefantrine(LMF)[21]

Quinine

3s-3-Hydroxyquinine2󰀇-quininone(10R)-10,11-dihydroxydihydroquinine(10S)-10,11-dihydroxydihydroquinine[22]

Lumefantrine[23]

ChloroquineQuinine[24]

DihydroartemisinArtesunateArtemetherLumefantrine

Desbutyl-lumefantrineMefloquineSulfadoxinePyrimethaminePiperaquinePyronaridine

Desethyl-amodiaquineChloroquineAmodiaquineQuinine[14]

Extraction

100␮lplasma+4␮lIS15␮M

Precipitation:200␮lACN(ice-cold)

50␮lplasma

Precipitation:300␮lISsolutioninMeOHSupernatanttransferredtoinjection.

250␮lofplasma+50␮lISinMeOH

Precipitation:450␮lofglacialAAinMeOHSupernatanttransferredtoinjection.

100␮lhumanplasma

Precipitation:200␮lcoldMeOH

100␮lplasma

Precipitation:1.0mlIS(0.5%,v/vFAinMeOH)Supernatanttransferredtoinjection.

HPLC)Sample:100␮lplasma+20␮lISsolutionPrecipitation:300␮lACN.DilutionwithMPuntil0.5ml.Supernatanttransferredtoinjection

␮HPLC)Sample:40␮lplasma+10␮lISsolutionPrecipitation:120␮lACN.DilutionwithMPuntil0.2ml.Supernatanttransferredtoinjection.

Sample:200␮lplasma+100␮lISPrecipitation:700␮lACN

Supernatantevaporatedtodrynessandreconstitutedin150␮lMeOH:Ammoniumformate20mM(1:1,v/v)adjustedtopH4withFA.Supernatanttransferredtoinjection.

Separation

XterraRP18;50mm×2.1mm,3.5␮m

MP:GE,A:ACN:6.25mMAmAcbufferpH=4.5:water(40:8:52,v/v/v),B:ACN:0.5mMAmAcbufferpH=4.5:water(60:8:32,v/v/v)

FR:0,4ml/min;RT:12min;IV:5␮lIS:Artemisinin

CromolithSpeedRODRP-18e;50mm×4.6mmMP:IE,10mMAmAcbuffer:MeOH:FA:ammoniasolution(25:75:0.2:0.15,v/v)

FR:0.8ml/min;RT:2.5min;IV:10␮lIS:Piperazinebischloroquinoline

ZorbaxSB-Cianocolumn;150mm×4.6mm,5␮mMP:GE,A:MeOH,B:10mMAmAc0.2%(v/v)AA,0.1%(v/v)FA.

FR:1ml/min;RT:9min;IV:50␮lIS:Artesunate

ZorbaxEclipseXDBphenyl;50mm×4.6mm,5␮mMP:GE,A:8%ACNin0.1MABpH3.9,B:24%ACNin0.1MABpH4.2

FR:1ml/min;RT:22min,IV:10–30␮lIS:Nointernalstandard

HypurityAdvanceC8;50mm×4.6mm,5␮mMP:IE,10mMAmAcbuffer/ACN/0.05%AA(10:85:5,v/v/v)

FR:0.6ml/min;RT:2.9min;IV:5␮lIS:Piperazine-bis-chloroquinoline

HPLC)SynergiMax-RP80A,

˚C12;150mm×4.6mm,4␮m

MP:IE,MeOH:CAN:0.5AmAc(50:10:40,v/v/v)pH=7.4FR:0.7ml/min;RT:aprox10min;IV:10␮l

␮HPLC)ZorbaxSBC18;150mm×0.3mm,3.5␮mMP:MeOH/0.2MAmAc(60:40,v/v),pH=7.4FR:7␮l/min;RT:aprox.5min;IV:200nlIS:VitaminB6

AtlantisdC18␮m;50mm×2.1mm,3␮m

MP:GE,A:20mMammoniumformateinultrapurewaterwith0.5%FA,B:ACNwith0.5%FAFR:0.3ml/min;RT:21min;IV:10␮l

IS:artemisinin(ART)andtrimipramine-D3

Detection

ESI(+),LC–MS,SIM,m/z:ARS:402,DHA:302,ARN:300.

ESI(+),LC–MS/MS,MRMm/z:PQ:535.3>288.2IS:409.1>205.2

ESI(+),LC–MS/MS,MRMm/z:AM:316>267,ARS:402>267,LMF:530>348.

FL:EX/EM(350/450nm)

ESI(+),LC–MS/MS,MRM,m/z:LMF:528.2>510.3

FL(325nm)He-Cd

Laser-inducedfluorescenceLengthcapillarydetectioncell=20cm

ESI(+),LC–MS/MS,MRM,(2segments)

RecoveryRange(%)(ng/ml)≥95%b

1.2–11501.5–1420

93±10.a

1–250

>8310–1000>81a10–18000

>104356.4–3564>90139.5–1395>920.043–1.2␮M>850.034–2.1␮M>85a0.024–1.5␮M

>51.45a210–25050

>10095–1600nM>94a65–1030nM>981.9fmolto>94

1360nM1.3fmolto640nM

>961–2000>1022–2000>855–2000>784–4000>1104–4000>1102.5–5000>1040.5–1000>1060.5–1000>952–4000>971–1000>1020.3–600>1032.5–5000>1080.3–600>105d2.5–5000

M.E.Casasetal./J.Chromatogr.B962(2014)109–131PyrimetamineSulfadiazine

N-acetyl-sulfadiazine[25]

Pyrimethamine(PM)Sulfadoxine(SD)[26]

Atovaquone(ATQ)[27]

Atovaquone[28]

SPE

Artemisinin(ARN)[29]

Artesunate(ARS)

Dihydroartemisinin(DHA)[30]

Artesunate

Dihydroartemisinin[31]

Artesunate

Dihydroartemisinin[32]

Artemether

Dihydroartemisinin[33]

Sample:25␮lplasma+25␮lISinMeOHPrecipitation:20␮l5%PCA

Supernatanttransferredtoinjection.

Sample:250␮lplasma+100␮lISsolutionPrecipitation:1650␮lofACN.

Supernatantevaporatedandreconstitutedin500␮lof0.1%(v/v)FAina15:85(v/v)ACN:water.Supernatanttransferredtoinjection

Sample:100␮lplasma

Precipitation:1000␮lofISsolution(0.2%FAinACN)

Sample:500␮lplasma+20␮lISsolution+100␮lAmAc(5mM).

Precipitation:ethylacetate(4ml)

Supernatantevaporatedtodrynessandreconstitutedin1mlMP.

HLBSPE96-wellplate

Sample:50␮lofplasma+150␮lISsolutioninplasma/water.

Cond:750␮lACN+750␮lMeOH+200␮lwaterWash:300␮lwater

Elution:100␮lMeOH:ACN(90:10,v/v)+100␮lwaterOASISHLB1cc

Sample:0.5mlplasma+12␮lISsolutionCond:1mlMeOH+1mlof1MAA

Wash:2mlof1MAA+1ml20%MeOHin1MAAElution:2ml40%ethylacetateinbutylchloride.

Eluentevaporatedandreconstitutedwith200␮lofMPHLBSPE96-wellplate

Sample:50␮lplasma+150␮licecoldISsolutionCond:750␮lACN+750␮lMeOH+200␮lwaterWash:300␮lwater

Elution:300␮lMeOH:ACN(90:10,v/v)+100␮lwater.Eluatesmixed,centrifuged,storedat4◦Cfor15hStrata-X33␮mPolymericRP

Sample:980␮lblankplasma+20␮lstandard+20␮lISCond:1mlMeOHand1mlAA1M

Elution:3ml40%ethylacetateinbuthylchlorideEluentevaporated,reconstitutedwith100␮lACNOasisHLB

Sample:0.5mlplasma+50␮lISCond:1mlMeOH+1mlwater

Wash:1mlwater×3and0.5mlACN10%Elution:ACN-Methylacetate(9:1,150␮l)Supernatanttransferredtoinjection

PhenomenexC18,AQUA,100mm×4.6mm,3␮mMP:GE,A:MeOH+water+conc.FA(50:950:1,v/v/v).B:MeOH+water+conc.FA(500:500:1,v/v/v).FR:0.5ml/min;RT:12min;IV:2␮lIS:Sulfamethoxazole

HypersilBDSphenyl;100mm×2.1mm,3␮m

MP:GE,A:0.1%(v/v)FAinwater.B:0.1%(v/v)FAina80/20(v/v)ACN/watermixture.

FR:0.3ml/min;RT:10min;IV:10␮lIS:Sulfamerazine

SynergiPolar-RP80A(C18);150mm×2.0mm,4␮mMP:IE,0.1%FA(v/v:ACN(20:80,v/v)

FR:0.5ml/min;RT:aprox.2min);IV:10␮lIS:Laphacol

HyPURITYC18;50.9mm×4.6mm,5␮mMP:IE,2mMAmAc:ACN(20:80,v/v)FR:0.8ml/min;RT:2.5min;IV:10␮lIS:Chlorothalidone

HypersilGoldC18;100mm×2.1mm,5␮mMP:GE,A:ACN:AmAc10mM(50:50,v/v),B:MeOH:ACN(75:25,v/v)

FR:0.5ml/min;RT:5.1min;IV:5␮lIS:Artesunate(ARS)

SynergiMax-RP80A(C18);75mm×4.6mm,4␮mMP:IE,AA(0.1%):MeOH:ACN(38:46.5:15.5,v/v/v)FR:0.5ml/min;RT:21min;IV:25–5␮l(lower-highercalibrationlevelsrespectively)IS:Artemisinin(ARN)

HypersilGoldC18;100mm×2.1mm,5␮mMP:GE,A:ACN:AmAc10mM(50:50,v/v),B:MeOH:ACN(75:25,v/v)

FR:0.5ml/min;RT:5.5min;IV:5␮l

IS:SILArtesunateandSILDihydroartemisinin

SynergiMax-RP80a;75mm×4.6mm,4␮m

MP:IE,AmAc(adjustedtopH=4withAA)andACN(53:47,v/v)

FR:0.4ml/min;RT:N/A;IV:20␮lIS:Artemisinin

SymmetryC18;150mm×4.6mm,5␮m

MP:IE,A:aqueous10mMNH4FA:ACNwith0.1%FA(20:80,v/v)

FR:1ml/min;RT:6.5min(fromchromatogram);IV:50␮l

IS:Artemisinin

UV(269nm)

ESI(+),LC–MS/MS,MRM,m/z:PM:249.10>233.10,198.19,177.30

SD:311.10>254.15,156.0,108.0

IS:265>190.05,172.00,110.0APCI,LC–MS/MS,CEM(-),MRMm/z:ATQ:365.2>337.1IS:240.9>185.7

ESI(-),LC–MS/MS,MRM,m/z:ATQ:365.0>171.1,IS:337.2>189.7

ESI(+),LC–MS/MS,MRM,300>209forARN,402>267forARS

APCI,LC–MS,SIM(+),221forARSandDHA,283forARN

ESI(+),LC–MS/MS,MRM,402>267and406>163forARSandSIL-ARS,302>163and307>166forDHAandSIL-DHA.

ESI(+),LC–MS

ESI(+),LC–MS/MS,MRMm/z:AM:316>267,DHA:302>267,IS:300>209

>9020–5000>90200–200000>90a200–50000

9710–1000

94a

2700–270000

AtLOQ

>84a50–23900

85±6a

50–2000

110–125a1.0–762

>801–3000>91a1–3000

>921.2–728>100a2.0–2500

>94±3.95–1000>78±5.0c

5–1000

>732–200>90a2–200

M.E.Casasetal./J.Chromatogr.B962(2014)109–131123Table4(Continued)

Analyte[Ref]

Artesunate(ARS)

Dihydroartemisinin(DHA)

Mefloquine(MQ)[34]

Artesunate(ARS)

DihydroartemisininAmodiaquine(AQ)Desethylamodiaquine(DeAQ)[35]

Piperaquine[36]

Lumefantrine

Desbutyl-lumefantrine[37]

Pyronaridine(PND)[38]

Proguanil(PRO)

Cycloguanil(CYC)[39]

Atovaquone(ATQ)Proguanil(PRO)Cycloguanil(CYC)4-Chlorophyenylbiguanid(4-CPB)[40]

Extraction

SPE:SupelcleanLC-18

Sample:0.5mlplasma+IS+ionizedwaterupto1mlCond1:1mlACN+1mlMeOH+1mldeionizedwaterWash:1ml0.05MAB(pH5.2)+1mlACN10%indeionizedwater.

ElutionARS/DHA:2×0.5mlACN:MeOH(90:10,v/v)Cond.2:1ml0.05PB(7.4)+1mlof10%ACNElution(MQ):2×0.5ml1%NH3inMeOH.

SupelcleanLC-18

Sample:0.5mlplasma+0.5mlPB(pH=4;0.05M)Cond:1mlACN+1mlMeOH+1ml1MPB(pH=4;0.05M)

Wash:1ml1MPB(pH4;0.05M)+1ml10%ACNindeionisedwater

ElutionAS/DHA:2×0.5mlACN:MeOH(95:5,v/v)ElutionAQ/DeAQ:3×0.5ml5%NH3inMeOHMPC-SDSPE96-wellplate

Sample:50␮lplasma+50␮lPB(pH2,0.05M)+IS+700␮lPB(pH2,0.05M)Cond:950␮lMeOH

Wash:950␮lMeOH:PB(pH2,0.05M)(80:20,v/v)Elution:MeOH-triethylamine(98:2,v/v).

ProteinPrecipitation:0.250mlplasma+0.5mlACN:AAglacial(99:1,v/v).Supernatantdecanted,960␮lHPLCwateradded.

SPE:octylsylica3MEmpore

Cond:0.5mlMeOH+0.5mlACN:water:AA(30:69.5:0.5)Wash:0.5mlACN:water:AA

Elution:MeOH:trifluoroaceticacid(99.9:0.1)Eluatesevaporatedandreconstitutedin100␮lMeOH:sodiumPB(pH2.0;0.05M)(70:30,v/v)SupelcleanLC-18

Sample:0.25mlplasma+IS10␮l+distilledwaterupto1ml.

Cond:1mlMeOH+1mlwater

Wash:1mlMeOH:water(50:50,v/v)

Elution:3×0.5mlMeOH:0.1MHCl(99:1,v/v)Eluentdriedandreconstitutedin100␮lMeOHOasisHLB30mg/1cc

Sample:0.5mlplasma+20␮l+0.2mlNaOH0.1NCond:1mlMeOH+1ml0.1NhydrochloricacidWash:1ml0.1NhydrochloricacidElution:3mloftheMP

Proteinprecipitation:500␮lplasma+1000␮lATQ-ISinACNicecold.Aqueousphasetransferredand1000␮lCYC-ISinPB(pH6.8,0.01M)added.SPE:HCX-Q

Activation:2mlMeOH

Cond:ACN:PB(pH6.8,0.01M)(40:60,v/v)Wash:MeOH-PB(pH6.8,0.005M)(30:70,v/v)Elution:MeOH:AA,glacial(98:2,v/v)

Eluatesevaporatedandreconstitutedin40␮lMeOH:water(90:10,v/v)

Separation

ARS/DHA:HypersilC4;250mm×4.6mm,5␮mMP:IE,ACNand0.05MAAadjustedpH=5.2with0.1MNaOH(42:58,v/v)

FR:1.5ml/min;RT:14min(fromgraph);IV:20␮lIS:Artemisinin(ART)

MQ:InertsilC8-3;150mm×4.6mm,5␮m

MP:IE,MeOH:ACN:0.05MPBadjustedtopH=3withortophosphoricacid(50:8:42,v/v/v)

FR:1ml/min;RT:aprox.14min;IV:50␮lIS:Chlorpromazinehydrocholoride(CPM)

ARS/DHA:HypersilC4;250mm×4.6mm,5␮mMP:ACNand0.05MAA(42:58,v/v)

FR:1.5ml/min;RT:14.5min;IV:50␮lmanual(loop)IS:Artemisinin

AQ/DeAQ:InertsilC4;4.6mm×100mm,5mmMP:ACN:PB(pH=4.0;0.05M)(11:89,v/v)

FR:1ml/min;RT:17min;IV:20␮lmanual(loop)IS:isobutylanalogueofDeAQ(IB-DeAQ)GeminiC18;50mm×2.0mm

MP:IE,ACN-ammoniumbicarbonate2.5mM(85:15,v/v)

FR:0.5ml/min;RT:aprox.2.5min;IV:5␮lIS:LabelledPiperaquineD6(PQD6)

SB-CN;250mm×4.6mm

MP:ACN:PB(pH=2.0,0.1M)(55:45,v/v)andsodiumperchlorate0.05.M

FR:1.2ml/min;RT:25min(fromgraph);IV:50␮lIS:IS1andIS2(showsstructure,nameNA)

C18Partisil10ODS;250mm×4.6mm

MP:IE,MeOH:0.05MAmAcbufferadjustedtopH=4.0withglacialAA(50:50,v/v)

FR:1.2ml/min;RT:aprox25min(fromgraph);IV:50␮l

IS:Quinidine(QN)

HyPURITYAdvanceC18;50mm×4.0mm,5␮mMP:ACNand5mMAmAc(90:10,v/v)FR:0.6ml/min;RT:2.5min;IV:10␮lIS:Riluzole(RIL)

ZorbaxSB-CN;250mm×4.6mm,5␮mMP:GE,A:ACN,B:PB(pH=2.6,0.03M)FR:1ml/min;RT:35min;IV:20␮lIS:AtovaquoneISandCycloguanilIS

Detection

ARS/DHA:ECD:reductive(Ag/AgCl).

MQ:DualwavelengthAbsorbance(284nm)

ECD(Ag/AgCl)

AS/DHA:reductiveAQ/DeAQ:oxidative

ESI(+),LC–MS/MS,MRM,m/z:PQ:535>288,D6PQ:541>284for

UV(350nm)

FL:EX/EM(267/443nm)

ESI(+),LC–MS/MS,MRM,m/z:PRO:254.10>170.10,CYC:252.10>195.00,RIL:235>166

UV(245nm)

RecoveryRange(%)

(ng/ml)86±5.520–160089±8.920–160079±3.1a

50–3200

>7920–1600>8920–1600>8520–1600>80a20–1600

>63±1.5a

1.5–500

>6324–20000>61a21–1010

90–99a40–600

1031.5–150.0107a0.5–50.0

∼9050–14830nM∼6025–2000nM∼6025–2000nM∼60a25–2000nM

124M.E.Casasetal./J.Chromatogr.B962(2014)109–131Liquid–liquidextractionArtesunate

Dihydroartemisinin[41]

Artesunate

Dihydroartemisinin[42]

Artemether(AM)

Dihydroartemisinin[43]

Artemether

Dihydroartemisinin[44]

Artemether

Dihydroartemisinin[45]

Artemether

Dihydroartemisinin[46]

Artemether

Dihydroartemisinin[47]

Amodiaquine

Desethylamodiaquine[48]

Pyronaridine(PND)[49]

Sample:50–100␮lplasmafortifiedwithARSandDHA.Extraction:ethylacetate

Extracts,reconstitutedwithACN:waterwithIS(50:50,v/v).Supernatantcombinedwith50␮lofwater.Sample:1mlplasma

Extraction:3.5mlDCM:MTBE(8:2,v/v).Yellow

supernatantdiscarded.Remainingfluidtransferred,evaporatedandreconstitutedwith60␮lMeOHSample:0.5mlplasma+0.125mlNaCl

Extraction:2.5mlof1-chlorobutane-isooctane(55:45,v/v).Organicphasetransferred,evaporatedand

reconstitutedwith100␮lEtOH:0.1%glacialAA(50:50,v/v)

Sample:1mlplasma+50␮lIS+0.5mlsaturatedNaCl.Extraction:5ml2,2,4-trimethylpentane:ethylacetate(7:3,v/v).4.5mloforganiclayertransferred,

evaporatedandreconstitutedin0.5mlofMeOH:AmAc10mM(1:1,v/v)

Sample:0.1mlplasma+20␮lIS

Extraction:1mlMTBE.Organicphasetransferred,evaporatedandreconstitutedin100␮lMP

LPME

Sample:1mlplasma+deionizedwaterupto4ml.Acceptorsolution:toluene:n-octanol(1:1,v/v).Evaporationofacceptorsolutionundervacuum.Residuereconstitutedwith100␮lorganicphaseSample:100␮lplasma

Extraction:2mlEthylacetate.

1.6mlorganicphasetransferred,evaporatedandreconstitutedin100␮lofMP

Sample:100␮lplasma+900␮lwater+50␮lIS+2000␮lCBpH9.5.

Extraction:7mlofdiisopropylether.

Organicphasetransferredandextractedwith150␮lofPB.

Sample:1000␮lplasma+1000␮lwater+50␮lIS+2000␮lCBpH9.5.Proceededasabove

Sample:200␮lplasma+400ngIS+500␮lPB(pH=10.3)

Extraction:3mldiethylether.

100␮lMeOHaddedtoorganiclayer.MixturedriedandreconstitutedwithACN:0.02MKH2PO4(27:73,v/v)adjustedtopH2withortophosphoricacid.

VarianPursuitC18,150mm×2mm,5␮mMP:GE,A:ACN,B:10mMAmAcinwater.FR:0.2ml/min;RT:10min;IV:N/AIS:Indomethacin

ElipseXDB-C18;150mm×4.6mm,5␮m

MP:IE,ACNand0.003MglacialAA(62:83,v/v)FR:0.5ml/min;RT:12min;IV:N/AIS:Artemisinin

AltimaC18;150mm×4.6mm,5␮mMP:IE,ACN-glacialAA0.1%(66:34)FR:1ml/min;RT:14min;IV:N/AIS:Artemisinin

UltrasphereC18,150mm×4.6mm;5␮m

MP:GE,A:AmAc10mMin0.1%(v/v)glacialAA.B:ACNFR:1–1.5ml/min;RT:20min;IV:50␮lIS:Artemisinin

GeminiC18;150mm×4.6mm,5␮mMP:IE,ACN:FA0.1%(80:20,v/v)FR:1ml/min;RT:N/A;IV:20␮lIS:Artemisinin

Laboratorycolumn:poly(methyltetradecylsiloxane)ontozirconized-silicasupport;150mm×3.9mm,5␮mMP:IE,MeOH:AmAc(10mmol/L,pH=5.0,80:20,v/v)FR:0.1ml/min;RT:N/A;IV:50␮lIS:Artemisinin

PhenomenexLuna,PFP(2)100A50mm×2.0mm,5␮m

MP:GE,A:MeOH:10mMAmAc0.1%AA(65:35,v/v),B:MeOH:10mMAmAc0.1%AA(90:10,v/v).FR:0.5ml/min;RT:8min;IV:10␮lIS:SILArtemetherandSILArtemisininZorbaxSBC18,75mm×4.6mm,3.5␮m,

MP:IE,MeOH:PB(0.1M,pH3):PCA(250:747.5:2.5,v/v)

FR:1.5ml/min;RT:12min;IV:100–120␮l

IS:4–4dimethylamino-1-methylbutylamino)-7-chloroquinoline

ZorbaxBonus-RP(14alkylchain);250mm×4.6mm,5␮m

MP:IE,ACN-0.08MpotassiumdihydrogenPB(13:87,v/v),pH=2.8adjustedwithortophosphoricacid.FR:1ml/min;RT:aprox.15IS:Amodiaquine

ESI(+),LC–MS/MS,MRM(notspecifiedm/ztransitions)

ESI(+),LC–MS,Iontrap,m/z:ARS:407,DHA:307and261for,ARN:305

APCI,LC–MS,SIM,m/z:AM:267and22,DHA:267and221

ESI(+)

LC–Q-TOF,scanmode100–700m/z

APCI,LC–MS/MS,MRM,m/z:AM/DHA:221>163,IS:283>219

ESI(+),LC–MS/MS,MRM,m/z:AM/DHA:263>163,IS:283>209

ESI(+),LC–MS/MS,MRM,m/z:AM:316.2>163.1DHA:302.2>163.0,SIL-AM:320>163.1,SILDHA:307.2>168.2

UV–visabsorbance(333nm)

UV(275nm)

>574–400>76a2–200

>89±1320–3200>80±3.3a

20–3200LOQ:10forboth765–20084a

5–200

10410–100098a10–1000

>915–400>75a5–400

335–100026a5–1000

>772–500>76a2–500

>87±2100␮l>75±6asample:From100-100␮l1000nMsamples

100-1000nM1000␮lsample:10-1000nM10-1000nM>81

a

29–1140

M.E.Casasetal./J.Chromatogr.B962(2014)109–131125126

M.E.Casasetal./J.Chromatogr.B962(2014)109–131

c:0i0mBP70r,0or)06fel081:e0hgm–21Atn/1––Felag010,Rn.7(112nytouitba-ttircextelyy:eeere395Xht...v677cEeo,lmc±±±155o:.e)6920R%1nE(767>>ahBTteM:,HgOnitrEo,t,i,M,nMPISSIonoiRrtMororamzffofin,10noiS.0.M88.ot980i0cy/aS111aeM,,rr)30,m0..p-i–76.6stlnnC755oo4L111rutit5,)>>cme:c>e2+193l((...teMeVI901:RDUS480E223ISME,,ensoaishrsPigentameliiwebn:oia/tMmnmNE:Co␮A,Pgmc)3)nMi,l␮5,vnom/vo.55m(linS:0taI,i␮m5f:120dhtom04.:nell(202oa6␮×8:Vcmn.cA0::mnIg4i;dHim2s×:mAnnOAVioeemI0FmC0)Mh%tm;5A,1v5B,/.eh.t00/);ev2.i5l)(v1smn2:2Nae/%:uowN.2:yiv2Tni(.Rlhltd,8CTe=po%0;avce1ARnneiazC;eri:H2)nxlntlSHpnmhpi.iBio0.boxanDO(i)rmni/otemOeAmahS/tDoAetluimctrMClePtmmBeuoll,ajiions,:aE2iEmqanrrsBih,reIM.:taGw3fl.inp:1yrPe,p:n0uieVeP:StIwoPm:piytHM5a,rcS7R:FSyIHMAR:FFSItderoarcat.adh)txmnviea/utvwre,Sisnl0Itfnioa5ea:fo,Admnr0htdlFmeme5fet(okianrr%i:.eaps:t1r2eee.cn+f0sASgInarsiim,nnnlrwaell:epnaoa␮kirAootNhiioltt0,tCtmu0iatleeunAoalolotsurs5ilesahretmsfodicmShtisnIpwnioeottnrSluoaofysla␮cI(Hidtriaelece.snnr␮rO0neeaetcol5guasgaoiaa0hiet+rt:nircoht1tMelaOitN:Esaawi+CI:fmn.sgrttwao␮0mA,l0soMnonMnxeii.ImatCc),kehnotis1l5roiSidtpueet,a3prnflDr1elfusnilol:laoc.fap×ue␮smdemd5l28binrvttm0oastaxr.eHn4a(ienu:)t5O:dtmeen1ns2anetencscaiuaronuNlaertdraotC:i:eNotiaearp:ttkonocrTxceioittiAlctsl:apaipMtcroragmrdnrom1arpeRemafieet::-opbar,ttnctBEdtxaxoesrbfediEa0ta.xSES0vaEewAGilcRco,nheka,Aeapotictcdifptosleaicasrrnassnaoalroerhwhetilepoxtpetdeflcrrpearmnie:ieggannliipgabRnnnsnF)z:iiiauorrf,gMarmeAraaot)iPycCapppssdb(pP:nlAe,mmmanieaeuyeylcroorg,eocccaan]dinnxiosfnefifieddgdtenRahmhtcrudeenele]o[iupaaobeufififiirfiiiingohtm1cCeitttatiflt(etaorla5[tnnpn4yuco]elfl)lgye:anaaacLhauumueah0uIaFpuloCC5mi[rSS(,sQQQoCQbnry:daAPPAiobcdeTAchaapsupernatantwascollectedanddirectlyinjectedor,alternatively,evaporatedandreconstituted[28].

Foranalysisoffullblood(Section4.2),samplingandmalariainfectiongeneratedhaemolyticproductsperse,degradingartemisinincompoundswhenincontactwithorganicsolvents.Thus,theapplicationofproteinprecipitationtoartemisininbaseddrugshasbeenproblematic[19,71].Fortheanalysisofartesunateanddihydroartemisinin,Teja-Isavadharmetal.[19]proposedpro-teinprecipitationwithacetonitrilegivingrecoveriesoftheanalytesfromplasmaabove95%.However,plasmawithmorethan0.09g/Lofhaemoglobincompromisedthequantification.Toovercomethis,theuseofstableisotopiclabelledISwassuggested,howevertheseareexpensiveandnotreadilyavailable.Furthermore,partialsepa-rationbetweenthelabelledISandtheanalytemayoccurandtheionsuppression/enhancementmaythennotbefullycompensatedbytheIS[71].Finally,amulti-methodincludingartemisininderiva-tivedrugsusingproteinprecipitationwasdevelopedbyHodeletal.[14].Here,signalintensitywasdecreasedbyca.20%forartemisinininthepresenceof0.2%ofhaemolysisbutsignalintensitywasnotconsiderablydecreasedforitsderivatives.

Proteinprecipitationhasbeenusedsuccessfullytoanalyzecom-poundspertainingtoallgroups(Table4)withrecoveriesmostoftenexceeding80%exceptforasinglestudy[23]weretherecoveryoflumefantrinewasapproximately50%.Itshouldbenotedthatinsomemethods,especiallyforantifolates,thepHmustbeloweredpriortoproteinprecipitationtorecoveranalytesquantitatively.Fortheanalysisofatovaquone,tworecentmethodsapplyingpro-teinprecipitationhasbeenpublished[27,28]Acetonitrileorethylacetatewasaddedtotheplasma(dilutionfactor8–10)withrecov-eriesofapproximately80%forbothmethods.

4.2.2.SPE

Proteinprecipitationisusedinacoupleofmethodasapre-vioussteptoSPE[37,40].However,elevenmethodsapplythesamplesdirectlyontothesorbent[29–39](Table4).SPEisfre-quentlyusedforextractingartemisininanditsderivates,reducingcontactwithorganicsolvents.Acommonsolid-phasematerialforthissubgroupofantimalariasisHLB[29–31,33],whichpro-videsgoodextractionefficienciesincombinationwithanumberofelutionsolventssuchasethylacetate/buthylchloride[30,32],methanol/acetonitrile[29,31]andmethylacetate/acetonitrile[33].Recoveriesrangedfrom75to125%intheabove-mentionedstudies.HLBcartridgeswerealsosuccessfullyappliedtoextractthemoder-atelylipophilicmoleculesproguanilandcycloguanil(Table1)[39].Inasinglecase,StrataXwithethylacetate/buthylchlorideassol-vent[32]wasappliedforartesunateanddihydroartemisiningivingquantitativerecoveries>78%.OccasionallyextractionofthesetwocompoundshasbeenperformedonC18cartridges,butthenalwaysincombinationwithotherantimalarialgroups,mefloquine[34]oramodiaquine/desethylamodiaquine[35].Insuchcases,atwo-stepelutionprocedurewasadoptedfirstelutingartesunateanddihy-droartemisininwithamixtureofacetonitrile/methanol,followedby1–5%ammoniainmethanolforextractingtheremaininganti-malarials.Thisassuredquantitativerecoveriesofallcompounds(>80%).

C18materialwasappliedtoextractpyronaridine[38],whichisslightlylipophilic(Table1),obtaininghighextractionrecover-ies(90–99%).InordertoavoidsecondaryinteractionsofthesilanolgroupsintheSPEmaterialandpyronaridine,elutionwasperformedwith1%hydrochloricacidinmethanol.InonecasetheC8cartridge[37]wasutilizedtoextracttheverylipophiliccompoundlume-fantrine(Table1)anditsmetabolitedesbutyl-lumefantrine.Sincelipophilicdrugstendtobindtoplasmaproteinsandareveryinsol-ubleinwater,therecoveryofthesecompoundswasimprovedwithproteinprecipitationpriortoSPE,andelutionwasperformedwithmethanoladjustedtopH2.TheacidicpHincreasedlumefantrine

Table5

Analyticalmethodsforthedeterminationofantimalarialsinurine.

Analyte[Ref]

SolidphaseextractionQuinine

Chloroquine[60]

Piperaquine[36]

Liquid–liquidextractionAmodiaquine

Desethylamodiaquine[48]

␤-Artemether

Dihydroartemisinin[44]

Chloroquine[61]

ProguanilCycloguanil

4-Chlorophenylbiguanide[50]

Pyronaridine(PND)[62]

Dilution

Quinine(QN)

(3S)-3-hydroxyquinine(3HQN)[63]

Extraction

NexusAbsolut

Sample:100␮lurine+100␮laqueousISCond:2mlMeOHand2mlAmAc0.1MWash:NA

Elution:1.3mlMPand0.7mlAA1%

ASPECXLSPErobotwithMPCcartridges

Sample:1mlurine+1mlofPBcontainingIS.Centrifugation.Supernatantsdilutedwith2mlofPB.Cond:MeOHandPB(pH=2.15;0.05M)Wash:Water+PB:MeOH(20:80,v/v)

Elution:MeOH:triethylamine(98:2,v/v)(0.7ml).

Eluatesdriedandreconstitutedin100␮lPB:ACN(95:5,v/v)

Sample:100␮lurine(diluted10–100times)+900␮lwater+50␮l1.6␮MI.S+2000␮lCBpH9.5.

Extraction:7mldi-isopropylether.Transferorganicphase.Backextraction:150␮lPBpH=4

Sample:2mlurine+50␮lISand0.5mlsaturatedNaCl.

Extraction:5mlof2,2,4-trimethylpentane:ethylacetate(7:3,v/v).Centrifugation.Organictransferred,evaporatedtodrynessandreconstitutedin0.5mlofMeOH:AmAc10mM(1:1,v/v)

Liquid–Liquid–LiquidMicroExtraction

Sample:2mlurineadjustedtopH=12withNaOH0.5MandspikedwithChloroquineandIS

Extraction:250␮lcyclohexane:2-ethyl-1-hexanol(1:1,v/v).Microdropacceptorphase:7␮lofaqueoussolution0.02MPA(pH=2)

Sample:1mlofurine+20␮lIS+0.5mlNaOH2M.Extraction:2×3mlofether

Organicphasetransferred,evaporatedandreconstitutedin100␮lMeOH

Sample:200␮lofurine+10␮lIS+500␮lSodiumphosphatetribasicdodeca-hydratebuffer(pH=10.3with85%orthophosphoricacid).

Extraction:3mlofether.Organicphasetransferred,

evaporatedandreconstitutedwith200␮lACNand0.02MPB(27:73,v/v)

Sample:50␮lofurine+50␮lISand+900␮lMeOH:water(50:50,v/v)

Vortexedandcentrifuged.

50␮lsupernatantdilutedwith950␮lMeOH:water(50:50,v/v)

Separation

Kromasil,C18,250mm×4mm,5␮m

MP:IE,MeOH:ACN:0.1MAmAc(45:15:40,v/v)FR:1ml/min,RT:3.5min,IV:50␮lIS:Salicylicacid

ChromolithPerformanceRP-18e,100mm×4.6mmMP:IE,PB(pH2.5;0.1M):ACN(92:8,v/v)FR:3ml/min,RT2.5min,IV:15or50␮l

IS:3-methyl-4-(3-hydroxy-4-diethylaminopropyl)-7-chloroquinoline

ZorbaxSBC18,75mm×4.6mm,3.5␮m

MP:MeOH:PB(0.1M,pH3):PCA(250:747.5:2.5,v/v)FR:1.5ml/min,RT:12min,IV:100–120␮lIS:4–4

dimethylamino-1-methylbutylamino)-7-chloroquinolineUltrasphereC18,150mm×4.6mm;5␮m

MP:GE,A:AmAc10mMinwatercontaining0.1%(v/v)glacialAA.B:ACN

FR:1–1.5ml/min,RT:20min;IV:50␮lIS:Artemisinin

CLC-ODS-C18,250mm×6mm;5␮m

MP:ACN/EtOH/0.05MPA(13:2:85,v/v/v)adjustedtopH=2.1with0.05MNaOH

FR:1ml/min,RT:NAIV:NA

IS:Hydroxychloroquinesulfatesalt

HypersilODSC18,250mm×4.6mm,5␮m

MP:IE,MeOH:ACN:0.5%:AmAc(40:5:55)with75mMPCA(pH=2.2)

FR:1.2ml/min,RT:NA,IV:20␮l,IS:PyrimethamineGeminiC18,150mm×3.0mm,5␮m

MP:GE,A:2mMperflurooctanoicacid.B:ACN

FR:0.5ml/min,RT:14min;IV:30␮l,IS:Amodiaquine

Acquitybridged-ethylenhybridC18,50mm×2.1mm,1.7␮m.MP:GE,A:10mMammoniumbicarbonate(pH=10)withammoniumhydroxidesolution.B:MeOH.FR:0.6ml/min,RT:2.5min,IV:20␮lIS:quinine-D3

Detection

FL:EX/EM(325/375nm)

UV(347nm)

UV(333nm)

ESI(+)

LC–Q-TOF,scanmode100–700m/z

UV(254nm)

UV(254nm)

APCI(+)

LC–MS,SIM,m/z:PND=518.2

ESI(+),

UPLC–MS/MS,MRM,m/z:

QN=325>160;3HQN=341>160

RecoveryRange(ng/mL)

(%)10210–70091b

10–500

86b9–10000

>910.5–400nM

>73f

1035–500102a5–500

>95d1–200

>9010–3000

>60>88e>86e

14.3–1425

>1011–20

>97e

M.E.Casasetal./J.Chromatogr.B962(2014)109–131127128

M.E.Casasetal./J.Chromatogr.B962(2014)109–131

t.ng)eniiLmMMMdr45ao/␮␮rtig69␮gnn53215...:o(31121Ee–––––Gmg45344..,nn69432eoa53000tiaR...31000relwgnoifls::yRMrce7FIS,v1o1e,cece)-nmR%9eit(8csneuror:uTflR:,:hLd)tFicmd,inaocinitroMnwiE0daolt/5nticX4ahcceE/0bmtxr:5eeLMneD8:pF3(E1X:EAC,nPo,irteafzfiunboietyamah␮rpps5soo,mrhtpc:melB6eP,.:4Ig×9lSn.Eirm3␮,=0notmHo3i–in0p20so5.si1BA41mm,en=:lyMHVIo:inp,e1.nMtch0BiEapnAme,iBgr-DNM22nitilnXC1:.TuoeA0Ris%n,tmi:p8indiinl:NnMocmoRiEAC/,lctMax:EA,ramdenabG%4s:1pr–:oaeoP2ZM:BR:ChSFSIP,Beleisbaohpm:.edPleitbMco,lejnmon:iadBhtn,ee.atSsamIt:edemcHeaOtictmee0j0uMn,i1ine.–yomel0t1muvrclmoued.rscieavantdu:neocorihlitAitat.idmcsegearweAjnvbslpnirikule,ilmeica:plpcisnl.arVImsti,noaei.aendotenvrsianortrvruditacbretredaiucetnUcnrlcnincoaata:aitxcndoiter:tclpNseontial-dtarxarmCateeAnsabrohcrnidtxEa,SptboliAetaitalicndalaeninmcddasa:a-xlaandeehSIeip,rrndreekit,enniinparrteanpseonnnligtiiiiitnuubunmxdiaenanganiiolruqqmeatnttrqoorrcpsassa)dydd:iexAtmniahgnpitnaogiiamuoyyrehh,rcawsugoacn]ddn1i-id1dcifioddddddteyo1y1ynRcseeeeeeo[Hn,x,xi0aI:fifififififi,-o0oiiiiiie3n1cttttttCi-r1riE(etn-u)d-)dtInnnnnni)yS]e,5yaaaaaalnSqR-0h0y2cnuuuuuueai3󰀇iaolu(21(d1hi2(d[iQQQQQQbnQ:taAAuabcdefTAlesolubilityinthemethanolandeliminatedsecondaryinteractionswiththesolidphase.DuetodifferenceinlipophilicitybetweentheanalyzedcompoundsandIS,thedevelopmentofasuitableSPEmethodwascomplicated.ThereforetheauthorsusedanotherISwithhigherlipophilicity,similartotheanalyzedcompounds.Finallyobtainedextractionrecoverieswereabout60%.

Twomethodsareusingmixed-modecartridges.MPC-SDisaC8cartridgewithstrongcationexchangegroupsusefulfortheextractionofpiperaquine[36]whileHCX-QisaC8cartridgewithweakcationexchangegroupssuitablefortheextractionofato-vaquone,proguanilanditsmetabolites[40].Themaindifferencebetweenthesetwomethodsistheinitialproteinprecipitationinthelattermethod,whichisusefulforavoidingthebindingofato-vaquonetoplasmaproteins.Extractionrecoveriesofproguanilanditsmetaboliteswerearound60%andforatovaquoneca.90%.Lowrecoveriesofproguanilandmetabolitesmightbeduetolossesofthesecompoundsduringtheloadingandwashsteps[40].Thesameexplanationcanbegivenforpiperaquine,whichatpH2isveryhydrophilicgivingarecoveryof60%.

4.2.3.Liquid–liquidextraction

LLEhasbeenemployedasextractionprocedureinelevenmethods[41–51].Sevenmethodsdetermineartemisininderiva-tives[41–47]withsolventssuchasethers,ethylacetate,dichloromethaneandmixturesofthesewithotherorganicsol-vents.

Fortheextractionofartemisinin,artesunate,artemetheranddihydroartemisinin,tert-methyl-buthylether(MTBE)combinedwithdichloromethane[45]andpureMTBE[42]wereappliedtoplasma(100␮lto1ml)withrecoveriesofca.80%.Twometh-odsusedethylacetateforthesamecompoundsin50–100␮lofplasmawithrecoveriesof56–76%[41]and75%[47].Peysetal.[44]developedamethodusingtrimethylpentane/ethylacetatebasedonanothermethodbySouppartetal.whoutilizedchlorobu-tane/isooctane[43].Bothmethodsusedsaltationof1mland0.5mlplasma,respectively,priortoextractiondecreasingthesolubilityoftheartemisininderivativesandfacilitatingthemigrationtotheorganicsolvents.Thelattermethodobtainedrecoverieshigherthan75%,whilePeysandco-workersachievednearly100%recovery.FinallyfortheabovecompoundsMagalhaesetal.[46]proposedaLiquidPhaseMicroExtraction(LPME),whichisaminiaturizedapproachsimilartoconventionalliquid–liquidextraction.Thismethodwasdevelopedtominimizetheconsumptionofsolvent.Applyingtoluene/n-octanol(1:1,v/v)theyobtainedrecoveriesaround30%.

Twomethodshavebeenpublishedfortheextractionof4-aminoquinolines,[48,49]amodiaquineanddesethylamodiaquineandpyronaridine.Bothmethodsextract100–200␮lofplasmaapplyingdi-isopropyletherordiethylether,respectively.Both4-aminoquinolinesareweakbases(Table1)sorisingthepHoftheplasmabyadding2mlofcarbonatebuffer(pH=9.5)priortoextractionensuredthedeprotonatedformofthesecompoundsandtherebyextractiontotheorganicphase.Minzietal.[48]furtherappliedabackextractionwithphosphatebuffer(pH4).Theextrac-tionrecoveriesofthesemethodswereingeneralabove80%.Ethersarealsousefulfortheextractionofproguanil,cycloguanilandchlorphenylbiguanideasseeninthemethodproposedbyEbeshietal.[50]withrecoveriesofabout70%.Thereisasinglemethodpublishedfortheextractionofacombinationofpyrimethamineandsulfamethoxypyrazine[51]usingdichloromethane(250␮lplasma).Likeforthe4-aminoquinolines,abasicpHfacili-tatesthemigrationofanalytestotheorganicphase.Overallrecoveriesofpyrimethamineandsulfamethoxypyrazinewererel-ativelylow,25and0.5%respectively,makingthismethodlessuseful.

M.E.Casasetal./J.Chromatogr.B962(2014)109–131

129

4.3.Urinesamplepreparation

4.3.1.SPE

TwoSPEmethodshavebeendevelopedforthedeterminationofquinineandchloroquine[60]aswellaspiperaquineinurine[36],thelatterinvolvinganautomatedprocedure(Table5).Qui-nineandchloroquineweredeterminedin100␮lsampleputonpolymericcartridgesandelutedwithmethanol/acetonitrile/0.1Mammoniumformiate,followedby1%aceticacid,givingrelativerecoveriesof102%and91%,respectively.Piperaquinewasdeter-minedinurineusingamixedphasematerialofcation-exchangeandoctylsilica.UrinesamplesweredilutedwithphosphatebufferatpH2.15beforeloadingontheSPE.AtthispH,thebasicmoleculepiperaquineisprotonated(Table1)andmaybindtoeitherthecation-exchangesitesorthelipophilicbindingsitesofthemixedphase.Amixtureofmethanol/triethylaminewasusedforelution,witharecoveryof85%.

4.3.2.Liquid–liquidextraction

FourmethodsapplyLLEtourine[44,50,61,62](Table5)withsamplesizesfrom100␮lto2ml.Inthreemethods,sampleswerealkalinizedbyaddingbufferstoextractamodiaquine[48]andpyronaridine[62]orNaOHtoextractproguanilandmetabolites[50].Asaprimaryorsingleextractionthepreferredsolventsareetherordi-isopropylether.Samplesmaythenbebackextractedwithphosphatebuffer[48]oralternativelyasecondextractionwithethermaybeperformed[50].Asstatedabove(Section4.2)raisingsamplepHupto9.5willdeprotonateamodiaquine[48]andfacil-itateitsmigrationintotheorganicphase,whilebackextractiontotheaqueousphaseisachievedwithphosphatebufferatapHof4.Averagerecoveriesusingtheseapproachesweredeterminedtobeabove70%.Pyronaridinehasbothacidicandbasefunctionalgroups,andcannotbeobtaineduncharged(Table1).

DaneshfarandcolleaguesusedLiquid–Liquid–LiquidMicroExtraction(LLLME)todeterminechloroquine[61]andadjustedpHtoalkalinepriortoextractionintotheorganicphase.Atthesametimetheorganicphasewasextractedbyanacidicaqueousacceptorphasepriortoinjection,givinganoverallrecoveryof96%.

4.3.3.Dilution

Twomethodsapplieddilutionanddirectanalysisoftheurinesamples.Mirghanietal.proposedasimplesamplepreparationpro-cedureforanalyzingquinineandfourofitsmetabolites[22].Urinesampleswerediluted10–100timesbeforeinjection.Obtainedrecoverieswerebetween89and117%(Table5).Analysingquinineand(3S)-3-hydroxyquinineinurine,amoreadvanced2-stepdilu-tionprocedurewasdevelopedbyHeatonetal.[63].First,sampleswerediluted20timesinamixtureofmethanol/waterfollowedbycentrifugation.Then,asubsampleofthesupernatantwasdiluted1:20withthesamemixtureandtheobtainedsamplewasinjected.Recoverieswerecloseto100%forbothcompounds.Ingeneralthese“dilute-and-shot”approachesaresimpleandapparentlyrobustbuttodateveryfewstudiesusingthisapproachhavebeenperformedonantimalarials,andmightbeworthamorethoroughevaluation.

5.Applicationofinternalstandards,liquidchromatographyandfinaldetection

Oncesuitablesamplepreparationandpre-concentrationhasbeenperformedthesamplesshouldbeinjectedontothechromato-graphicsystemandthereafterbedetectedforaqualitativeandquantitativedeterminationoftheantimalarials.Akeyparameterintheanalyticalchainisthechoiceofproperinternalstandardsasdiscussedbelow.Additionallyanumberofstationaryphasesaretodayavailabletoassurethattheanalyteofinterestissepa-ratedfromotheranalytesaswellasco-elutingmatrixcomponents

thatmighthaveslippedthroughthesamplepreparationprocedure.Majortrendsinchosenstationaryphasesforthedifferentanti-malarialsareoutlinedbelow.ThefinaldetectionsystemsavailablerangefromsimplerandlessexpensiveUV-detectorstoexpen-siveMS-systems,wherethelatterhavebeenusedinmoststudiespublishedwithinthelastdecade.Themostcommonlyapplieddetectorsforthevariousdrugsareoutlinedbelow.Foramorecompletedescriptionofchromatographicseparationanddetec-tionsystemsthereadershouldconsultarecentcomprehensivepaperonbioanalysisofantimalarialsusingliquidchromatography[94].ThereviewsummarizesHPLCandLC–MSmethodspub-lishedsince1988forthebioanalysisof15antimalarialcompounds(chloroquine,atovaquone,quinine,sulfadoxine,pyrimethamine,artemisinin,artemether,dihydroartemisinin,amodiaquine,arte-sunate,mefloquine,piperaquine,proguanil,pyronaridine,andlumefantrine)usedasfirst-linedrugsinmalariatherapy,butonlyaddressessamplepreparationstrategiestoaminorextent.

5.1.1.Internalstandards(IS)

ArtemisininhasbeenusedasISinseveralassaysofartemisininderivatesanalysis[14,19,30,32,33,42–47].Althoughartemisininisstructurallyrelatedtoitsderivatives,itdoesbehavelikethemtowardshaemoglobin[14].Apparently,plasmasamplesfromsomemalariapatientsarehaemolytic,andusingartemisininasISmaythereforeaffectanalyticalresults.Inaddition,previouspatientself-treatmentwithArtemisiaannuaherbalpreparationsthatcontainartemisinincouldbiastheanalyticalresultswhenusingartemisininasIS[14]

Biascausedbyself-treatmentcouldalsooccurinothermethodsthatmakeuseofnon-labelledornon-modifiedantimalarialdrugsasanIS[21,29,30,38,49,50,55].Forthisreason,samplesfromself-treatedpatientsshouldbecompletelydiscardedwhenapplyingmethodsusingnon-labelledornon-modifiedantimalarialdrugsasISforpharmacokineticstudies.Avoidingsamplesfromself-treatedpatientsmaybequitedifficultconsideringthegreatamountofcounterfeitsmedicineincirculationincountrieswithmostmalariaincidence[72].

Widelyusednon-steroidalanti-inflammatorydrugssuchasindomethacine[41]orsalicylicacid(mainmetaboliteofacetylsali-cylicacid)[60]arenotsuitableasISduetoextensiveself-treatment.Drugsthatarelessusedsuchasriluzol[39],orchlorthalidone[28]maybebetterchoices.Obviously,thebestchoiceforISwouldbelabelleddrugswithpropertiessimilartotheanalytebutsuchchem-icalsareratherexpensiveandthetechniquesabletomeasuresuchISareexpensive.Goodalternativescouldbemodifiedmoleculesthatresemblethestructureoftheanalytebutarenotcommercial-izedasdrugs.

5.1.2.Separationbyliquidchromatography

Fortheseparationofartemisinincompounds,biguanideantifo-latesandatovaquone,C18hasbeenusedascolumnmaterial.Bothgradientelutionandisocraticelutionhasbeenappliedfortheanalysisofartemether/dihydroartesmisininandarte-sunate/dihydroartemisinin[14,19,29–33,41–45,52].MostofthemethodsareusingmixturesofacetonitrileandaqueousphaseadjustedtolowpHwithaceticorformicacid.Whenanalyzingproguanilanditstwometabolitesthemobilephasecontainedper-chloricacidascounterioninordertoseparatetheanalytesonaC18column[50].Alsoforthe4-aminoquinolinesC18isacom-moncolumntype[14,38,48,53,54,59,62].However,cyano-columnsmayalsobeused[56].Likewisethe8-aminoquinoline,tafenoquine,wasseparatedinacyano-column[58].ForpyronaridineaC14-columnhasbeenapplied[49],whileforartemisininderivatesand4-aminoquinolinestwodifferentC4columnswithmobilephases

130

M.E.Casasetal./J.Chromatogr.B962(2014)109–131

containingacetonitrileandaqueousacidicphasehavebeenused[35].

Arylaminoalcoholscanbeseparatedwithdifferentcolumntypes.Quinineandseveralofitsrelatedmetabolitespossesstwobasicfunctionalgroups(Table1)thatcaninteractwithacidicsilanolgroupsfromthecolumnandproducepeaktailing.Usinganacidicmobilephasethatsuppressessilanolionizationcanmin-imizethisphenomenon.Forthisreason,theseparationofquinineanditsmetabolitesmaybeperformedusingphenyl-bonded-phasecolumnsatlowpHvalues[22],[55].Anotherwaytoavoidsilanolinteractionistodeprotonatethebasicgroupbyperformingchro-matographyinhighlybasicmedia.Thiscanbeachievedusingethylene-bridged-hybridcolumns,whicharestableathighpHvalues[63].HoweverwithincreasingpH,quininebecomesmorehydrophobicleadingtoincreasedretentiononreversed-phasecol-umn.MefloquinewhichhasarelativelyhighlogKowvalue∼4(Table1),afactoroftengreaterthanquinine,hasbeenanalyzedwithaC8columnandamobilephasecontainingmethanol,acetoni-trileandammoniumformiatesolution[34].InthiscaseitmaybespeculatedthatC8waschosentogiveshorterretentiontimescom-paredtoregularC18materials.Finally,severalcompoundshavebeenseparatedusinganumberofstationaryphaseswithoutagen-eraltrendamongmethods.ForexampleLumefantrinehasbeenanalyzedondifferentkindsofcolumnssuchasC18[14,59],C8[23]andcyano-columns[21,37].WhilesulfadrugshavebeenanalyzedonbothC18columns[51]andonphenyl-columns[26,51].

5.1.3.Finaldetection

Generally,thedetectionofantimalarialsinthematricesreviewedhereisperformedusingtandemquadrupolemassspectrometry(MS/MS).Inthiscasetheionizationisconductedpreferablywithelectro-sprayionization(ESI)inpositivemode(Tables3–5),exceptforatovaquonethatusesESIinnegativemode[28].Itshouldbenotedthoughthatafewmethodsuseatmosphericpressurechemicalionization(APCI)[27,30,43,45,67].

Ingeneral,artemisininderivatesdonotabsorbUVlightandthereforetheanalysisofthesecompoundshastobecarriedoutwithotherdetectorspreferablywithMS/MS.

Fortheanalysisofnon-artemisininderivates,someofthemeth-odsuseUVorfluorescencedetection.ThesedetectorsarelessexpensivethanMS,butthelimitofdetectionishigherthanforMS.Finally,twomethodsbyLaietal.[34,35]analyzedartesunate,dihydroartemisinin,mefloquine,amodiaquineanddesethylamodi-aquinewithelectro-chemicaldetection(ECD).ECDmayallowfortheconductingofaffordablepharmacokineticstudieswithoutcompromisingthesensitivityandtherebydetectionlimits.

Methodsonwholebloodandurinemainlyfocusingontheanalysisof4-aminoquinolinesandquinineprimarilyuseUV-VISabsorbancedetectionorFluorescenceexcitation-emission(Tables3and5).UVandFluorescencedetectorsarecheaperthanMSand,sincewholeblood(especiallyDBS)andurineanalysisaresuitablematricesforpharmacokineticstudiesinausterecondi-tions,thesedetectionsmethodsareappropriatedforthat.

6.Concludingremarks

Toimprovethetreatmentandcureofmalariasounddescrip-tionsofadsorption,distribution,metabolismandeliminationofantimalarialdrugsinthehumanbodyareneeded.Aprerequisiteforthisarehighqualityanalyticalmethodologiesfortheanalysisofantimalarialsinsamplesfromhumans.Inbloodanalysis,DBSsam-plinghassomeadvantagesoverregularbloodsamplingduetoeasystorageandtransport.Samplesarealsooftenmorestableoncesam-pledasdriedbloodspotswithlessneedtobefrozen[56],however

stabilitywilldependonthetypeofantimalariasampled.Further-more,incontrasttoregularbloodsamples,DBSarenotinfectious,avoidingtransmittingHIVandotherpathogens,andcanbeshippedatroomtemperatureasnon-infectiousmaterial[73].Therefore,DBS’aremoresuitableforfieldstudies,whichareusuallycon-ductedwithfewresourcesandwithfewsafetyprecautions.How-ever,theDBSmethodalsohassomeweaknesses;Samplingmustbecarriedoutavoidingcontaminationofthefilterpaper[73].Also,drugextractionfromthepapermatrixmaysometimesresultinlowrecoveries[53,56,59].Thebindingofantimalarialsonfilterpapersseemstodependontheparticulardrugandtypeoffilterpaperused.Thereforeitshouldbeevaluatedonacase-by-casebasis.

Forplasmasamplesproteinprecipitationhavesomeadvan-tages.Itisfastandtheprocessisusuallysimplesincethesupernatantcanoftenbeinjecteddirectly.Proteinprecipita-tionwithorganicsolventisalsoagoodchoicewhenextractingverylipophiliccompoundssuchaslumefantrineandatovaquoneDisadvantagesmayincludesampledilution.Theapplicationofproteinprecipitationtoartemisinin-baseddrugsmaybeproblem-aticbecausebloodhaemolyticproductsdegradethecompoundswhenbeingincontactwithorganicsolvents.SPEisfrequentlyusedfortheextractionofartemisininanditsderivatesbecauseitminimizescontactwithorganicsolvents.Polymericreversedphasesorbentsaremostcommonlyusedfortheextractionofartemisininderivates.Whenapplyingthistechniquetolipophilicdrugs,itisimportanttotakeintoaccountthatthesedrugsbindtoplasmaproteinsandneedorganicsolventtostayinsolution.Therefore,aproteinprecipitationstepwithorganicsolventpriortoSPEmayimprovetherecoveryofanalytessuchaslumefantrine,halofantrineandatovaquone.Finally,whenusingmixed-modecar-tridges,hydrophiliccompoundsmaygivelowrecoveriesduetolossesduringtheloadingandwashsteps.WhenusingLLE,thedegradationofartemisininderivatesincontactwithorganicsol-ventapparentlyisaproblem.Inordertofacilitatethemigrationoftheweakbasestothedesiredsolvent,increasingthepHinthesolventpriortoLLE,andloweringthepHincaseofbackextractionisagoodprocedure.

Forurine,therecoveriesobtainedforthepublishedmethodsarequitehighanditseemsthatthedifferencesontheextrac-tionmethoddonothaveamajorinfluencetherecovery.LLEisthemostappliedmethodanditisutilizedtoextractratherdif-ferentantimalarials.Liquid–Liquid–LiquidMicroExtractionhastheadvantagethatextractionandconcentrationofthecompoundana-lyzediscarriedoutatthesametime,whileSPEhavetheadvantageoftheavailabilityoflargeurinesamplesthatcanbeconcentratedandleadtoimprovedassaysensitivity.

Overthelastdecadethenumberofmethodsdealingwithanti-malarialsanalysisinvariousbodyfluidshasbeenincreasingataslowpace.Malariaisstillamajorproblemandcostefficientproceduresmustbeintroducedcontinuously.Thismayincludeinformation,mosquitonets,butalsoprovidingsafeantimalari-alsaswellasavoidcounterfight.Asrichcountrieswemustalsoaidintheprocessofdevelopingbetterantimalarials,whichfromapharmaceuticalperspectivemeansunderstandingpharmacoki-netics/dynamics.Thisrequiresvalidatedmethodsandespeciallyrobust,accurateandprecisesamplepreparationstrategiessup-portingthegrowingnumberofLC–MS/MSmethodsslowlybeingdevelopedaroundtheworld.AmajorstepforwardinthisrespectistheExternalQualityControlProgramforantimalarialdrugsthathasbeensetupsince2010bytheWorldWideAntimalarialResistanceProgram(WWARN)[95].

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