StressDegradationStudiesonDutasteride

andDevelopmentofaStability-Indicating

HPLCAssayMethodforBulkDrug

andPharmaceuticalDosageForm

D.V.SubbaRao1,2,&,P.Radhakrishnanand1,2

1ReferenceStandardLaboratory,UnitedStatesPharmacopeia-IndiaPrivateLimited,ICICIKnowledgePark,Turkapally,Shameerpet,Hyderabad500078,India;E-Mail:d_venkatasubbarao@yahoo.co.in2DepartmentofChemistry,JawaharlalNehruTechnologicalUniversity,Kukatpally,Hyderabad500072,India

Received:2October2007/Revised:25December2007/Accepted:30January2008Onlinepublication:17April2008

Abstract

Asimplestability-indicatingLCmethodhasbeendevelopedforthequantitativedetermina-

tionofdutasterideinbulkdrugsamplesandinpharmaceuticaldosageformsinthepresence

ofdegradationproducts.Theretentiontimeofdutasterideisabout7min.Thedrugwas

subjectedtostressconditionsofhydrolysis,oxidation,photolysisandthermaldegradation.

Degradationwasfoundtooccurunderhydrolysisandtoalesserextentunderoxidation

conditionsbutthecompoundwasstabletophotolyticandthermalstress.Theassayofstress

sampleswascalculatedagainstareferencestandardandthemassbalancewasfoundclose

to99.3%.Thedevelopedmethodwasvalidatedwithrespecttolinearity,accuracy,precision

andruggedness.

Keywords

ColumnliquidchromatographyStability-indicatingassayStressstudiesDutasteride

Introduction

Dutasteride,(5a,17b)-N-(2,5-bis(trifluo-

romethyl)phenyl)-3oxo-4-azaandrost-l-ene-

17-carboxamide(Fig.1),isapotentand

specificdual5alpha-reductaseinhibitor

forthetreatmentofbenignprostatic

hyperplasia(BPH)andlowerurinary

tractsymptoms(LUTS)[1,2].ItwasapprovedinOctober2002byUSFDA

andhasbeenapprovedinseveralcoun-

tries[3,4].

Dutasterideinhibitstheconversionof

testosteroneto5a-dihydrotestosterone

(DHT)[2].DHTistheandrogenpri-

marilyresponsiblefortheinitialdevel-

opmentandsubsequentenlargementof

theprostategland.DHTisconvertedfromtestosteronebytheenzyme

5a-reductase,whichexistsastwo

isoforms,Type1andType2.Type1

5a-reductaseisfoundprimarilyinthe

skinandliver,buthasalsobeenfound

inprostatictissueinBPH.Type2

5a-reductaseisfoundintheprostate[2].

Rapidliquidchromatography–

tandemmassspectrometryassayof

dutasterideinhumanplasma[5]and

massspectralfragmentationreactionsof

atherapeutic4-azasteroidandrelated

compounds[6]havebeenreported.

Thecurrentdrugstabilitytestguide-

lineQ1A(R2)issuedbytheInterna-

tionalConferenceonHarmonization

(ICH)[7]suggeststhatstressstudies

shouldbecarriedoutonadrugto

establishitsinherentstability,leadingto

identificationofdegradationproducts

andhencesupportingthesuitabilityof

proposedanalyticalprocedures.Italso

requiresthatanalyticaltestprocedures

shouldbestability-indicatingandthat

theyshouldbefullyvalidated.

Accordingly,theaimofthepresent

studywastoestablishthestabilityof

dutasteridethroughstressstudiesunder

avarietyofICH-recommendedtest

conditions[7–9]andtodevelopasta-

bility-indicatingassaymethod[10–12].

Sofar,toourknowledgenostability-

indicatingLCassaymethodfor

dutasteridehasbeendeveloped.The

aimofthepresentworkwastodevelopa

stability-indicatingLCmethodfor

dutasterideinbulkdrugandpharma-

ceuticaldosageformsandcapable

ofseparatingvariousdegradationprod-

ucts.

Experimental

Chemicals

Samplesandstandardweresuppliedby

GansenLaboratoriesLimited;Mumbai,

India;commerciallyavailable0.5mg

dutasteridesoftgelatincapsules(Dutas)

werepurchased.LCgradeacetonitrile,

analyticalreagentgradesodiumdihy-

drogenphosphatemonohydrateand

phosphoricacidwerepurchasedfrom

Merck,Darmstadt,Germany;high

puritywaterwaspreparedwithaMilli-

poreMilli-Qplussystem.

Equipment

AWaters2695binarypump,autosam-

pleranda2996photodiodearraydetec-

torwereused.Theoutputsignalwas

monitoredandprocessedusingEmpower

softwareonaPentiumcomputer(Digital

EquipmentCo.),waterbathsequipped

withMVcontrollers(Julabo,Seelabach,

Germany)wereusedforhydrolysis

studies.Stabilitystudieswerecarriedout

inahumiditychamber(ThermoLab,

India).Thermalstabilitystudieswere

performedinadryairoven(MACK

Pharmatech,Hyderabad,India).Intermediateprecisionofthemethod

wasevaluatedbyusingadifferentsystem

(Agilent1100series,AgilentTechnolo-

gies,Waldbronn,Germany)withadiode

arraydetector(DAD).Theoutputsignal

wasmonitoredandprocessedusing

Chemstationsoftware(Agilent)ona

Pentiumcomputer.

ChromatographicConditions

A3lm,100·4.6mmPhenomenex

LunaC-18,columnwasusedforsepara-

tions.Themobilephasecontaineda

mixtureofbufferandacetonitrileinthe

ratioof40:60(v/v).Thebufferconsistedof

10mMsodiumdihydrogenphosphate

monohydrate,pHadjustedto3.0using

dilutedphosphoricacid(1mLphospho-

ricacidin10mLwater).Theflowrateof

themobilephasewas1.0mLminÀ1.The

columntemperaturewasmaintainedat

27°Candtheeluentwasmonitoredata

wavelengthof210nm.Theinjection

volumewas10lL.

PreparationofStockSolutions

Stocksolutionsofdutasteridestandard

andsamples(1.0mgmLÀ1)werepre-

paredbydissolvingappropriateamounts

inacetonitrile.Workingsolutionsof

0.1mgmLÀ1werepreparedfromthe

stocksolutionforassaydetermination.

PreparationofSample

Solution

Twentysoftgelatincapsuleswere

weighedandthecontentsweregroundin

acleandrymortar.Thenequivalentto

10mgofdrugwastransferredtoa

100mLvolumetricflask,80mLoface-

tonitrileaddedandtheflaskplacedona

rotatoryshakerfor10mintodisperse

thematerialcompletely,sonicatedfor

10min.Thecontentswerethendiluted

to100mL(1,000lgmLÀ1).Theresult-

ingsolutionwascentrifugedat

3,000rpmfor5min.Aftercentrifuging,thesupernatantsolutionwasfiltered

usinga0.45lmnylon66-membranefil-

ter.Thissolutionwasusedforanalysis.

StressStudies/Specificity

Stresstestingofthedrugsubstance

canhelptoidentifythelikelydegra-

dationproducts,whichcaninturn

helpestablishthedegradationpath-

waysandtheintrinsicstabilityofthe

molecule.Specificityistheabilityofthe

methodtomeasuretheanalyte

responseinthepresenceofitspotential

impurities[10].

Allstressdecompositionstudies

wereperformedataninitialdrugcon-

centrationof1mgmLÀ1(1,000lg

mLÀ1).Acidhydrolysiswasperformed

in1NHClat27°Cfor48handin1N

HClat80°Cfor3h.Thestudyin

basicsolutionwascarriedoutin1N

NaOHat27°Cfor48handin1N

NaOHat80°Cfor3h.Forstudyin

neutralsolution,thedrugdissolvedin

waterandacetonitrile(6:4,v/v)was

heatedat80°Cfor3h.Oxidation

studieswerecarriedoutatroomtem-

peraturein6%hydrogenperoxidefor

48handin6%hydrogenperoxideat

80°Cfor2h.Photodegradationstud-

ieswerecarriedoutaccordingto

Option2ofQ1BinICHguidelines[9].

Sampleswereexposedtolightforan

overallilluminationof1.2millionlux

hoursandanintegratednearultravio-

letenergyof200Whm2.Thedrug

powderwasexposedtodryheatat

60°Cfor10days.Sampleswerewith-

drawnatappropriatetimesandsub-

jectedtoLCanalysisaftersuitable

dilution(0.1mgmLÀ1).

MethodValidation

Precision

Theprecisionofthemethodwas

evaluatedbycarryingoutsixinde-

pendentassaysofasampleofdutaste-

ride(100lgmLÀ1)againsta

reference