aliquots

JournalofChromatographyB,868(2008)70–76

ContentslistsavailableatScienceDirect

JournalofChromatographyB

journalhomepage:/locate/chromb

DevelopmentandvalidationofahighlynsitiveandrobustLC–MS/MS

withelectrosprayionizationmethodforsimultaneousquantitationof

itraconazoleandhydroxyitraconazoleinhumanplasma:

Applicationtoabioequivalencestudy

Bharathia,b,KishoreKumarHothab,agarb,SanagapatiSirishKumarb,

PanduRangaReddyb,,RameshMullangic,∗

aDepartmentofChemistry,JawaharlalNehruTechnologicalUniversityKukatpally,Hyderabad500072,India

bBioanalyticalDepartment,IntegratedProductDevelopment,’sLaboratoriesLtd.,Bachupalli,Hyderabad500072,India

cBiopharmaceuticsDepartment,IntegratedProductDevelopment,’sLaboratoriesLtd.,Bachupalli,Hyderabad500072,India

articleinfo

Articlehistory:

Received23January2008

Accepted17April2008

Availableonline25April2008

Keywords:

Itraconazole

Hydroxyitraconazole

Humanplasma

Methodvalidation

LC–MS/MS

Pharmacokinetics

Humans

abstract

AhighlynsitiveandspecificLC–MS/MSmethodhasbeendevelopedforsimultaneoustimationof

itraconazole(ITZ)andhydroxyitraconazole(OH-ITZ)with500␮Lofhumanplasmausingfluconazoleas

aninternalstandard(IS).TheAPI-4000LC–MS/MSwasoperatedunderthemultiplereaction-monitoring

mode(MRM)haextractionprocesswasudto

extractITZ,alruntimewas3.0minandtheelutionofITZ,

OH-ITZandISoccurredat2.08min,1.85minand1.29min,respectively;thiswasachievedwithamobile

phaconsistingof0.2%(v/v)ammoniasolution:acetonitrile(20:80,v/v)ataflowrateof0.50mL/minon

aHyPurityC

18

(50mm×4.6mm,5␮m)elopedmethodwasvalidatedinhumanplasma

withalowerlimitofquantitationof0.50ng/rresponfunctionwas

establishedfortherangeofconcentrations0.5–263ng/mL(r>0.998)ra-

andinter-daypOH-ITZ

werestableinthebatteryofstabilitystudies,viz.,bench-top,auto-sampler,dryextractandfreeze/thaw

elopedassaymethodwasappliedtoanoralbioequivalencestudyinhumans.

©htsrerved.

uction

Itraconazole(ITZ,CASno.84625-61-6,Sporanox)isanorally

activetriazoleantifungaldrugwithabroadspectrumactivity

hanismofactionis

inhibiting14-alpha-demethyla,acytochromeP

450

enzymethat

catalysthesynthesisofergosterol,amajorcomponentofcell

membraneofyeastandfungalcells[1,2].Followingoralabsorp-

tion,itixtensivelymetabolizedbysidechainhydroxylation(by

CYP3A4)toformhydroxyitraconazole(OH-ITZ).OH-ITZ,whichis

themajormetabolite,isbiologicallyactiveanditsplasmaconcen-

trationistwofoldhigherthanparentatsteadystate[3].

FewmethodshavereportedforthequantitationofITZ[4]or

ITZandOH-ITZ[5]usingLC–thereareonlyfewbioan-

alyticalmethodsusingLC–MS/MSreportedforthesimultaneous

estimationofITZandOH-ITZ[6,7].Vogeretal.[6]havereported

∗

.:+9;fax:+9.

E-mailaddress:mullangiramesh@(gi).

anautomatedSPEmethodforsamplesprocessingwithalower

limitofquantification(LLOQ)of10ng/mLforbothITZandOH-ITZ.

Kousoulotal.[7]havereportedaLLOQof2ng/mLand4ng/mL

forITZandOH-ITZ,respectivelyinhumanplasmabyusingauto-

wearereporting

ahighlynsitiveandruggedLC–MS/MSmethod,whichhasover-

comethedrawbacksofthepreviouslyreportedmethods,viz.,usage

ofautomatedmethodsandenabledustogetareproducibleLLOQof

0.5ng/mL(fourfoldandeightfoldlessthanpreviouslyreportedlow-

estLLOQforITZandOH-ITZ,respectivelybyKousoulotal.[7])for

bothITZandOH-ITZandapplicationofthismethodtoabioequiv-

alencestudyinhealthyvolunteersfollowingoraladministrationof

100mgITZcapsule.

mental

alsandreagents

ITZwasobtainedfromNeulandLaboratories,Hyderabad,

-ITZwasobtainedfromSyncom,ATGroningen,The

1570-0232/$–efrontmatter©htsrerved.

doi:10.1016/b.2008.04.029

hietal./togr.B868(2008)70–7671

azole(IS)wasobtainedfromUnit-II,Dr.

Reddy’sLaboratoriesLtd.,compoundswere

foundtobe>98.5%puritydeterminedbychromatographic(HPLC,

LC–MS/MS)adeofacetonitrile;analyticalgrade

30%(v/v)ammoniaandhydrochloricacidwerepurchadfrom

.,Mumbai,eoussolutions

includingthebufferforthemobilephawerepreparedwithMilli

Q(Millipore,Milford,MA,USA)trolK2EDTA

humanplasmawaspurchadfromCauveryDiagnosticsandBlood

Bank,Secunderabad,India.

mentationandchromatographicconditions

AnAgilent(AgilentTechnologies,Waldbronn,Germany)1100

riesLCsystemequippedwithdegasr(G1322A),isopump

(G1310A)alongwithauto-sampler(G13167B)wasudtoinject

20-␮LaliquotsoftheprocesdsamplesonaHyPurityC

18

column(4.6mm×50mm,5␮m,ThermoElectronCorporation,

Cheshire,UK),whichwaskeptatroomtemperature(24±2

◦

C).

Theisocraticmobilepha,amixtureof0.2%(v/v)ammoniaand

acetonitrilemixture(20:80,v/v)wasdeliveredat0.50mL/min

intothemassspectrometer’lectrosprayionizationcham-

ber.

QuantitationwasachievedbyMS/MSdetectioninpositiveion

modeforbothITZandOH-ITZandIS,usingaMDSSciex(Foster

City,CA,USA)API-4000massspectrometer,equippedwitha

TurboionsprayTMinterfaceat400

◦

sprayvoltagewast

monparameters,viz.,nebulizergas,curtaingas,

auxillarygasandcollisiongasweretat30psi,10psi,40psiand

5psi,poundparameters,viz.,declustering

potential(DP),collisionenergy(CE),entrancepotential(EP)and

collisionexitpotential(CXP)were124V,48V,10V,14VforITZ;

120V,49V,10V,12VforOH-ITZand65V,25V,10V,15VforIS.

Detectionoftheionswasperformedinthemultiplereaction-

monitoring(MRM)mode,monitoringthetransitionofthem/z

705.3precursoriontothem/z392.3forITZ,m/z721.4precursor

iontothem/z408.3forOH-ITZandm/z307.0precursorionto

them/polesQ1andQ3weret

lyticaldatawereprocesdbyAnalyst

software(Version1.4.2).

rdsolutions

PrimarystocksolutionsofITZandOH-ITZforpreparationof

standardandqualitycontrol(QC)sampleswerepreparedbyweigh-

marystocksolution(1.00mg/mL)ofITZand

OH-ITZandISwerepreparedinmethanolandstoredat−20

◦

C,

whichwerefoundtobestablefor1month(datanotshown).

AppropriatedilutionsweremadeinmethanolforITZandOH-

ITZtoproduceworkingstocksolutionsof26.4␮g/mL,23.2␮g/mL,

19.7␮g/mL,12.8␮g/mL,3.84␮g/mL,1.34␮g/mL,0.25␮g/mL,

0.10␮g/mLand0.05␮g/mLonthedayofanalysisandthestocks

wereudtopreparecalibrationcurve(CC).Anothertofworking

stocksolutionsofbothITZandOH-ITZweremadeinamix-

tureof0.1NHCl:methanol(30:70,v/v)(fromprimarystock)at

23.3␮g/mL,12.8␮g/mL,1.41␮g/mLand0.05␮g/mLforprepa-

gstocksolutionswere

storedatapproximately5

◦

Cforaweek(datanotshown).Indi-

viduallystockQCandCCtwo-in-oneworkingsolutionsofITZand

OH-ITZweremadebeforespikingintoQCandCCsamplesaccord-

ngISsolution(300ng/mL)wasalsopreparedin

ationsampleswerepreparedbyspiking490␮L

ofcontrolhumanplasmawiththeappropriateamountofana-

lytes(10␮L)andIS(50␮L)sforthe

determinationofrecovery,precisionandaccuracywereprepared

byspikingcontrolhumanplasmainbulkatappropriateconcen-

trationsand500␮Lvolumeswerealiquotedintodifferenttubes

anddependingonthenatureofexperimentsampleswerestored

at−80±10

◦

Cuntilanalysis.

preparation

Toanaliquotof500␮Lhumanplasmasample,ISsolution

(50␮L)wasadded;dilutedwith500␮LofMilliQwaterandvor-

texmixedfor30sonacyclomixer(RemiInstruments,Mumbai,

India).Thissamplemixturewasloadedonpre-conditioned(1mL

acetonitrilefollowedby1mLwater)OasisHLBcartridges(1cm3,

30mg)andwashedwith0.5mLwaterfollowedby1mL10%ace-

tonitrileinwaterandfinallyelutedwith1mLofmobilepha.

Fromtheeluate20␮LwasdirectlyinjectedontoLC–MS/MSsys-

tem.

validation

Themethodwasvalidatedtomeettheacceptancecriteria

ofindustrialguidanceforthebioanalyticalmethodvalidation

[8].

Thespecificityofthemethodwasdeterminedbyanalyzingsix

differentbatchesofhumanplasmaasis,todemonstratethelack

ofchromatographicinterferencefromendogenousplasmacom-

spikedstandardsandQCsamples(n=6ateach

concentration)werepreparedandanalyzedonfourdifferentocca-

sionstoevaluatelinearity,ionand

accuracywasalsoassdatthelowestconcentrationofthestan-

dards(0.5ng/mL),reprentingtheLLOQfortheassay.

TherecoveryofITZ,OH-ITZandISwasdeterminedbycom-

paringtheresponsoftheanalytextractedfromreplicate

QCsamples(n=6)withtheresponofanalytesfrompost-

extractedplasmastandardsampleatequivalentconcentrations

[9].Recoverywasdeterminedatlow,midandhighqualitycon-

trolconcentrations,whereastherecoveryoftheISwasdetermined

atasingleconcentrationof300ng/ectofplasma

constituentsovertheionizationofanalytesandISwasdeter-

minedbycomparingtheresponsofthepost-extractedplasma

standardQCsamples(n=6)withtheresponofanalytesfrom

neatsamplesatequivalentconcentrations[9,10].Matrixeffect

wasdeterminedatLLOQforITZandOH-ITZandforISat

300ng/mL.

ThestabilityofanalytesandISintheinjectionsolventwasdeter-

minedperiodicallybyinjectingreplicatepreparationsofprocesd

samplesupto18h(inauto-sampler)

peakareasoftheanalytesandISobtainedatinitialcyclewereud

asthereferencetodeterminetherelativestabilityoftheanalytes

ityofanalytesinthebiomatrixafter

10hexposureinanicebath(bench-top)wasdeterminedattwo

rstabilityoftheanalytes

inbiomatrixwasassdbyanalyzingtheQCsamplesstoredat

−80±10◦

bilityofanalytesinbiomatrix

followingrepeatedthreefreeze/thawcycles(storedat−80±10

◦

C

betweencycles)wasassdusingQCsamplesspikedwithana-

swereprocesdasdescribedunderSection2.4.

Sampleswereconsideredtobestableifassayvalueswerewithin

theacceptablelimitsofaccuracy(i.e.,±15%S.D.)andprecision(i.e.,

15%R.S.D.)[8].

cokineticstudy

Abioequivalencestudywasperformedinhealthymalesubjects.

Theethicscommitteeapprovedtheprotocolandthevolunteers

ampleswere

hietal./togr.B868(2008)70–76

lMRMchromatogramsofITZ(leftpanel)andIS(rightpanel)in(a)humanblankplasma(b)humanplasmaspikedwithITZatLLOQ(0.50ng/mL)andIS(c)a

3.0-hplasmasampleshowingITZpeak(46.9ng/mL)obtainedfollowingoraldoofITZcapsuletohealthyvolunteeralongwithIS.

obtainedfollowingoraladministrationof100mgofITZcapsule

intopolypropylenetubescontainingK2EDTAsolutionasananti-

coagulantatpre-do,1h,2h,3h,4h,5h,6h,7h,8h,9h,10h,12h,

24h,36h,washarvestedbycentrifugingthe

bloodusingBiofuge(Hereaus,Germany)at1760×gfor5minand

storedfrozenat−80±10

◦

Cuntilanalysis.

Analiquotof500␮Lofthawedplasmasampleswerespiked

ith

hietal./togr.B868(2008)70–7673

lMRMchromatogramsofOH-ITZ(leftpanel)andIS(rightpanel)in(a)humanblankplasma(b)humanplasmaspikedwithOH-ITZatLLOQ(0.50ng/mL)and

IS(c)a3.0-hplasmasampleshowingOH-ITZpeak(62.8ng/mL)obtainedfollowingoraldoofITZcapsuletohealthyvolunteeralongwithIS.

studysamples,QCsamplesatlow,mediumandhighconcentration

wereassayedinduplicateandweredistributedamongunknown

teriaforacceptanceoftheana-

lyticalrunncompasdthefollowing:(i)notmorethan33%of

theQCsamplesweregreaterthan±15%ofthenominalconcentra-

tionand(ii)notlessthan50%ateachQCconcentrationlevelmust

concentration–timedataof

ITZandOH-ITZwasanalyzedbynon-compartmentalmethodusing

WinNonlinVersion5.1(PharsightCorporation,MountainView,CA,

USA).

hietal./togr.B868(2008)70–76

Table1

Intra-andinter-dayprecisiondeterminationofITZandOH-ITZqualitycontrolsinhumanplasma

Theoreticalconcentration(ng/mL)RunMeasuredconcentration(ng/mL)(ITZ)Measuredconcentration(ng/mL)(OH-ITZ)

cy(%)cy(%)

Intra-dayvariation(sixreplicatesateachconcentration)

ITZ:0.51,OH-ITZ:0.49

10.530.024.601040.490.0715.099.4

20.500.023.8698.40.480.059.3798.4

30.540.047.441050.440.036.9790.5

40.520.024.561020.480.047.5297.7

Average0.520.035.121020.470.059.7296.5

ITZ:1.41,OH-ITZ:1.37

11.310.064.2992.81.450.042.90106

21.320.042.6693.81.350.043.0198.4

31.250.043.5788.91.280.107.8493.4

41.360.043.0296.21.390.085.77101

Average1.310.053.3992.91.370.074.8899.7

ITZ:128,OH-ITZ:125

11173.843.2891.21134.183.7190.4

21243.843.0997.01155.805.0692.1

31206.555.4493.91144.804.2191.4

41231.741.4195.91182.782.3694.6

Average1213.993.3194.51154.393.8492.1

ITZ:233,OH-ITZ:226

12198.673.9594.01944.642.3986.5

223511.04.671012015.872.9288.7

32337.563.241002115.052.3993.4

424716.66.7310623911.04.77101.9

Average23411.04.651002116.643.1292.6

Inter-dayvariation(18replicatesateachconcentration)

ITZ:0.51,OH-ITZ:0.490.520.046.801030.480.0611.598.7

ITZ:1.41,OH-ITZ:1.371.270.075.1990.21.380.1410.3101

ITZ:128,OH-ITZ:1251185.364.5292.31144.443.9091.5

ITZ:233,OH-ITZ:2262248.653.8696.22134.552.1494.0

R.S.D.:relativestandarddeviation(S.D.×100/mean).

s

development

pre-treatment

Differentmethodsofsamplepre-treatmentwereinvestigated.

Proteinprecipitationandliquid–liquidextractionwithvarious

organicsolventsandtheirmixturesresultedinnon-reproducible

recoveriesandinterferencesfromthesamplematrixwiththechro-

matographyoftheanalytes(datanotshown).Subquently,SPE

wasinvestigatedassamplespre-treatmenttechnique,whichhas

alsobeenudbyotherinvestigators[6,7].Followingoptimization

ofvarioustypesofSPEsandveraldilution,conditioning,washing

andelutionreagents,wehavefinallylectedOasisHLBcartridges

(1cm3,30mg)spre-conditioned(1mLace-

tonitrilefollowedby1mLwater)werewashedwith0.5mLwater

followedby1mL10%acetonitrileinwaterandfinallyelutedwith

iluatewasdirectlyinjectedinto

theLC–MS/MSsystem.

chromatography

Inpursuitofsymmetricpeakshapeandretentiontimeof

∼3.0min,feasibilityofvariousmixture(s)ofsolventssuchasace-

tonitrileandmethanolusingdifferentbufferssuchasammonium

acetate,ammoniumformateandformicacidwithvariablepH

rangeof4.0–7.0,alongwithalteredflowrates(intherangeof

0.3–1.0mL/min)weretestedforcompletechromatographicreso-

lutionofITZ,OH-ITZandIS(datanotshown).Theresolutionof

peakswasachievedwith0.2%(v/v)ammoniaandacetonitrilemix-

ture(20:80,v/v)withaflowrateof0.5mL/min,onaHyPurity

C

18

columnandwasfoundtobesuitableforthedeterminationof

electrosprayresponforITZ,OH-ITZandIS.

ectrometry

InordertooptimizeESIconditionsforITZ,OH-ITZandIS,

quadrupolefullscanswerecarriedoutinpositiveiondetection

adirectinfusionexperiment,themassspectraforITZ,

OH-ITZandISrevealedpeaksatm/z705.3,721.4and307.0,respec-

tivelyasprotonatedmolecularions[M+H]+.Followingdetailed

optimizationofmassspectrometryconditions(providedinSection

2.2)m/z705.3precursoriontothem/z392.3andm/z721.4precur-

soriontothem/z408.3wereudforquantificationforITZand

OH-ITZ,rly,forISm/z307.0precursoriontothe

m/z220.1wasudforquantificationpurpo.

ficityandlectivity

Atypicalchromatogramforthecontrolhumanplasma(freeof

analyteandIS)andhumanplasmaspikedwithITZandOH-ITZat

LLOQalongwithISareshowninFigs.1and2,

interferingpeaksfromendogenouscompoundsareobrvedatthe

entiontimeofITZ,OH-ITZ

andISwere2.08min,1.85minand1.29min,al

chromatographicruntimewas3.0min.

ry

Recoverywasfoundtobe84.37±2.81,79.53±5.07and

75.54±2.92atLQC,MQCandHQC,respectivelyforITZ;

93.06±2.10,88.08±6.93and85.56±2.73atLQC,MQCandHQC,

hietal./togr.B868(2008)70–7675

Table2

StabilitydataofITZandOH-ITZqualitycontrolsinhumanplasma

Nominal

concentration

(ng/mL)

StabilityITZOH-ITZ

Mean±=6(ng/mL)Accuracy(%)bPrecision

(%CV)

Mean±=6(ng/mL)Accuracy(%)bPrecision

(%CV)

ITZ:

1.41,

OH-

ITZ:

1.37

0h(forall)1.36±0.0496.23.031.39±0.081015.78

3rdfreeze/thaw1.35±0.0595.43.721.38±0.111004.77

10h(bench-top)1.27±0.0789.95.681.48±0.091085.91

18h(in-injector)1.47±0.061044.351.45±0.081065.63

15daysat−80

◦

C1.27±0.0990.57.271.37±0.1710012.1

ITZ:

233.22,

OH-

ITZ:

26.36

0h(forall)246±16.61056.73230±10.91014.77

3rdfreeze/thaw231±13.399.35.78220±10.497.44.72

10h(bench-top)221±10.495.04.73226±4.8796.92.22

18h(in-injector)249±22.51069.03210±12.393.05.86

15daysat−80

◦

C227±1.4897.40.65210±1.9993.10.94

aBack-calculatedplasmaconcentrations.

b(Meanassayedconcentration/meanassayedconcentrationat0h)×100.

nrecoveryforITZandOH-ITZwas

foundtobe79.81±4.42and87.23±6.29,ov-

eryofISwas86.70±2.95%.

effect

Inthisstudy,thematrixeffectwavaluatedbyanalyzingLLOQ

ematrixfactorvalues(matrixfactor=respon

ofpost-spikedconcentrations/responofneatconcentrations)

obtainedforITZandOH-ITZwere+1.13(CV6.72%,n=6)and+1.07

(CV3.20%,n=6),respectivelyatLLOQlevel,whereasonISitwas

foundtobe+0.99(CV0.78%,n=6)%attestedconcentrationof

300ng/effectwasnotobrvedatanalytesandISreten-

tiontimes.

ationcurve

Theplasmacalibrationcurvewasconstructedusingcalibration

standardsof0.5–263.63ng/mLand0.49–255.88ng/mLforITZand

OH-ITZ,ationcurvewaspreparedbydetermin-

asmaconcentration–timeprofileofITZandOH-ITZinhumanplasma

followingoraldosingof100mgITZcapsuleto24subjects.

ingthebestfitofpeakarearatios(peakareaanalyte/peakarea

IS)versusconcentration,andfittedtothey=mx+cusingweighing

factor(1/X2).Theaverageregression(n=4)forITZandOH-ITZwas

foundtobe≥0.998and0.997,estconcentra-

tionwiththeR.S.D.<20%wastakenasLLOQ[8]andwasfoundto

be0.50ng/%accuracyobrved

forthemeanofback-calculatedconcentrationforfourlinearities

waswithin94.17–105.02%,and93.94–103.21%forITZandOH-ITZ,

cision(%CV)valuesrangedfrom0.98%to4.68%

and1.06%to8.81%forITZandOH-ITZ,respectively.

ionandaccuracy

Theaccuracy,intra-andinter-assayprecisionwhichwas

determinedbyanalyzingsixreplicatesofQCsamplesatfourcon-

centrationsonfourdifferentdaysareshowninTable1.

ity

ThepredictedconcentrationsforeachanalyteatLQCandHQC

samplesdeviatedwithin±15%ofthenominalconcentrationsin

abatterofstabilitytests,viz.,in-injector(18h),bench-top(10h),

repeatedthreefreeze/thawcyclesandat−80±10

◦

Cforatleastfor

15days(Table2).Theresultswerefoundtobewithintheassay

variabilitylimitsduringtheentireprocess.

cokineticstudy

Theprentmethodwasappliedtotheanalysisofplasmasam-

plesobtainedfrom24healthyhumanvolunteersfollowingoral

administrationof100mgofITZcapsuleasapartofbioequiva-

sitivityandspecificityoftheassaywerefound

tobesufficientforaccuratelycharacterizingtheplasmapharma-

.3depictsthemean

plasmaconcentrationversustimeprofileofITZandOH-ITZin

ingtheoraladministrationof100mgof

ITZcapsule(testandreferenceformulations)tovolunteers,the

meanmaximumplasmaconcentrations(C

max

)forITZ(45.75ng/mL

and49.94ng/mLfortestandreferenceformulations,respectively)

wereattainedat∼4h(T

max

)forbothtestandreferenceformu-

stheC

max

valuesforOH-ITZwere105ng/mLand

117ng/mLfortestandreferenceformulations,respectivelyandthe

T

max

wasfoundtobe∼5hand∼6hfortestandreferencefor-

mulation,f-life(t

1/2

)ofITZandOH-ITZwas

foundtobe∼29hand14h,respectivelyinbothreferenceandtest

hietal./togr.B868(2008)70–76

theAUC

(0–∞)

valuesfortestandreferencefor-

mulationswerefoundtobe699ngh/mLand798ngh/mL,whereas

forOH-ITZthevalueswere2347ngh/mLand2801ngh/mLfortest

andreferenceformulations,respectively.

sion

SofartherewereonlytwoLC–MS/MSmethods[6,7]published

forthesimultaneousdeterminationofITZandOH-ITZandthe

reportedlowestLLOQforITZandOH-ITZwas2ng/mLand4ng/mL,

portedmethodshaveutilizedeitherfullyor

mi-automatedsamplepreparationandhandlingprocessusing

homologderivativeofITZasanIS,whichisnotcommerciallyavail-

estofourknowledge,wehavedeveloped

anLC–MS/MSmethodforthedeterminationofITZandOH-ITZ

simultaneously,whichoffersthehighestnsitivity(0.5ng/mL)

comparedtoothermethodsdescribedintheliteratureusingasim-

LLOQreportedbyusisfourfoldandeightfoldlowerthantheearlier

reportedLLOQforITZandOH-ITZ,respectively.

sions

Insummary,wehavedevelopedandvalidatedahighlynsitive,

specificandreproducibleLC–MS/MSassaytoquantifyITZandOH-

eresultsofallthe

validationparameters,wecanconcludethattheprentmethod

canbeufulforbioequivalencestudieswithdesiredprecisionand

accuracy.

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