capillary electrophoresis and by stability-indicating HPLCmethods Determination and of tetrahydrozoline antazoline in ophthalmicsolutions Journal of Pharmaceutical and Biomedical Analysis

ContentslistsavailableatScienceDirect

Journal

of

Pharmaceutical

and

Biomedical

Analysis

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

Determination

of

antazoline

and

tetrahydrozoline

in

ophthalmic

solutions

by

capillary

electrophoresis

and

stability-indicating

HPLC

methods

夽

Mehmet

Gumustas

_,

_Usama

_Alshana

_,

_Nusret

_Ertas

_,

_Nilgun

_Gunden

_Goger

_,

Sibel

A.

Ozkan

_,

_Bengi

_Uslu

a_{DepartmentofAnalyticalChemistry,FacultyofPharmacy,AnkaraUniversity,06100Ankara,Turkey} b_{DepartmentofAnalyticalChemistry,FacultyofPharmacy,GaziUniversity,06330Ankara,Turkey} c_{DepartmentofAnalyticalChemistry,FacultyofPharmacy,NearEastUniversity,99138Mersin10,Turkey}

a

r

t

i

c

l

e

i

n

f

o

Received16October2015

Receivedinrevisedform19February2016 Accepted23February2016

Availableonline27February2016 Keywords: Antazoline Capillaryelectrophoresis Coreshell HPLC Ophthalmicsolutions Tetrahydrozoline

a

b

s

t

r

a

c

t

Capillaryelectrophoretic(CE)andhighperformanceliquidchromatographic(HPLC)methodswere

devel-opedandoptimizedforthedeterminationofantazoline(ANT)andtetrahydrozoline(TET)inophthalmic

formulations.Optimumelectrophoreticconditionswereachievedusingabackgroundelectrolyteof

20mMphosphatebufferatpH 7.0,acapillarytemperatureof 25◦_{C,aseparation voltageof22kV}

andapressureinjectionofthesampleat50mbarfor17s.HPLCanalysiswasperformedwith

Kine-tex(150×4.6mmID×5␮m)(Phenomenex,USA)analyticalcolumnwith1mLmin−1flowrateofmobile

phasewhichconsistedof0.05%TFAinbidistilledwater(pHadjustedto3.0with5MNaOH)and

acetoni-trile/bufferintheratioof63:37(v/v)atroomtemperature.Injectionvolumeofthesampleswas10␮L

andthewavelengthofthedetectorwassetat215nmformonitoringbothanalytes.

Calibrationgraphsshowedagoodlinearitywithacoefficientofdetermination(R2_{)ofatleast0.998}

forbothmethods.Intradayandinterdayprecision(expressedasRSD%)werelowerthan2.8%forCEand

0.92%forHPLC.Thedevelopedmethodsweredemonstratedtobesimpleandrapidforthedetermination

ofANTandTETinophthalmicsolutionsprovidingrecoveriesintherangebetween97.9and102.70%for

CEandHPLC.

©2016ElsevierB.V.Allrightsreserved.

1. Introduction

Antazoline(ANT)(Scheme1a)isahistamineH1receptor antag-onist.Itcanpreventhistaminefromactingontargetcellsthrough areversiblecompetitioneffectonhistaminereceptorsitesofthose cells[1–4].Itisclassifiedasafirstgenerationantihistaminehaving anticholinergicpropertiesusedtoreducenasalcongestionandin eyedrops,usuallyincombinationwithnaphazolineand/or tetrahy-drozoline(TET)(Scheme1b)toreducethesymptomsofallergic conjunctivitis.TETisaderivativeofimidazolinethatisfoundin over-the-counter eye drops and nasal sprays. It is a sympath-omimeticagentwith␣-adrenergicactivityanditsmainmechanism ofactionistheconstrictionofconjunctionalbloodvesselsanditacts asalocalvasoconstrictor.Thisservestoreducetherednessofthe

夽 Selectedpaperfrom26thInternationalSymposiumonPharmaceuticaland BiomedicalAnalysis(PBA2015),July5thto8th,2015,Tbilisi,Georgia.

∗ Correspondingauthorat:AnkaraUniversity,FacultyofPharmacy,Department ofAnalyticalChemistry,06100,Tandogan/Ankara,Turkey.

E-mailaddress:buslu@pharmacy.ankara.edu.tr(B.Uslu).

eyecausedbyminorocularirritants.Solutionsandsuspensionsof TETareusedasaconjunctivaldecongestant[1–5].Acombination ofthesetwodrugsisnowavailableinthepharmaceuticalmarket forophthalmicuseintheworldsuchasGermany,India,Nigeria, Malaysia,etc.

High-performanceliquidchromatography(HPLC)iscommonly reported for thedetermination of ANT and TET [6,7]. Capillary electrophoresis(CE)isacomplementarytechniquetoHPLCwhich offers several unique characteristics that make it particularly attractive,whichincludeitshighresolvingpower,minimalreagent consumption,rapidnessandlowanalysescost.

Tothebestofourknowledge,neitherCEnorstability-indicating HPLCmethodcanbefoundinliteratureforthesimultaneous deter-minationofANTandTETinpharmaceuticaldosageforms.Afew methodsusingHPLC[6,8]andonlyonehigh-performancethinlayer chromatographymethod[9]havebeenreportedforthe simultane-ousdeterminationofthesedrugs.Theproposedmethoddescribes theoptimizationofthetwomethodsanditpresentstheeffectof core-shellparticlesonthechromatographicseparations.

http://dx.doi.org/10.1016/j.jpba.2016.02.032

M.Gumustasetal./JournalofPharmaceuticalandBiomedicalAnalysis124(2016)390–398 391

Core–shellparticlesarethebest-suitedmaterialsforrapid anal-ysesbecausetheiroptimumlinearvelocityissignificantlyhigher thanthatoffullyporousparticles.Ontheotherhand,these particle-packedcolumnsshowapressure dropthatcanbehalforeven lessthanthatoffullyporouspackedcolumns.Thistypeof mate-rialprovideshigherplatenumber (efficiency)than fullyporous stationaryphasesunderthesameconditionsandwiththesame instrumentation.Asaresultofthehigherefficiency,better resolu-tion,narrowerandmoresymmetricalsharppeakscanbeobserved. Analyzingcomplexmatricesandimpurity-profilingstudiesrequire thatthemethodbesensitive[10,11].Inaddition,consumptionof organicsolventsshouldbeminimizedforthemethodtobe inex-pensiveandtofitintogreenchemistryregulations.Accordingly, core-shellmaterialscanbeidealforqualitycontrolandresearch anddevelopmentlaboratories,wherecostandtimeareofessential consideration,sincetheydecreasetheruntimesperanalysis.

Thegoalofthisstudyistopresentthedevelopmentand vali-dationofrapidandeasyCEandstability-indicatingHPLCmethods forthesimultaneousdeterminationofANTandTETinraw materi-alsandpharmaceuticalpreparations.Theproposedmethodsoffer automaticsampleprocessing,aswellasimprovedselectivityand efficiencythatmaybeadequateforsensitivepharmaceutical stud-ieswithsmallsamplesizeinadditiontominimumconsumption oforganicsolvents, symmetricalpeaksand highresolution val-ues.BothmethodswerevalidatedaccordingtotheUnitedStatesof Pharmacopeia(USP32)andguidelinesoftheInternational Confer-enceonHarmonization(ICH)[12–15].Forceddegradationstudies arealsopresentedtoshowthestability-indicatingcapacityofthe developedHPLCmethod.

2. Materialsandmethods

2.1. Chemicalsandreagents

ANT phosphate,TET Hydrochlorideand methanolwere pur-chasedfromSigma-Aldrich(St.Louis,MO,USA).Quetiapinethat wasusedasaninternalstandard(IS)forHPLCstudywaskindly suppliedfromMustafaNevzatIlacSan.Tic.A.S.(Istanbul,Turkey). Potassiumdihydrogenphosphate(KH2PO4), dipotassium

hydro-genphosphate(K2HPO4),sodiumhydroxide(NaOH),hydrochloric

acid (HCl), trifluoro acetic acid (TFA) and hydrogen peroxide (H2O2) werepurchased from Merck (Darmstadt, Germany). All

reagentswereatleastofananalyticalgrade.Deionized(DI)water (18.2M.cm)whichwastreatedwithMillipore(Simplicity,185 waterpurificationsystem)wasusedunlessotherwisestated.

2.2. Equipmentandconditions

HP3D_{CE(AgilentTechnologies,Waldbronn,Germany)equipped}

withanonlinediode-arrayUVdetector(DAD)wasoperatedata wavelengthof192nmformonitoringtheanalytes,anoptimum wavelengthasdeterminedusing‘Isoabsorbance’and‘3D’plotsin theinstrument’s‘Data Analysis’ software (AgilentTechnologies, Waldbronn,Germany),wasused.Separationswereachievedusing anuncoatedfused-silicacapillary(AgilentTechnologies,USA)of

Scheme1.ChemicalstructuresofANT(a)andTET(b).

75␮mi.d.and 64.5cm totallengthwitheffectivelengthtothe detectorof56cm.

AgilentTechnologies1100HPLCsystem(Wilmington,USA)was usedformethoddevelopment,forceddegradationandmethod val-idationstudies.This systemequippedwitha G1379Adegasser, G1311Aquaternarypump,G1313autoinjectorandG1315BDAD. The chromatogramswererecorded andthepeakswere quanti-fiedandintegratedusingChemstation®_{softwareandpHoftheall}

buffersolutionswasmeasuredwithaThermoScientificBenchtop pHmeter(Orion3StarTM_{Plus,USA)usingacombinedelectrode}

withanaccuracyof±0.05pH.

HPLC analyses were performed with Kinetex (150×4.6mm ID_×5_␮m)(Phenomenex,USA)analyticalcolumnthathave core-shellparticles.Aflowrateof1.0mLmin−1wasappliedforpumping of themobilephase which consistedof0.05%TFA inbidistilled water(pHadjustedto3.0with5MNaOH)andacetonitrileinthe ratioof63:37(v/v)atroomtemperature.Sampleswereinjected withthevolumeof10␮Landthewavelengthofthedetectorwas setupat215nm.

2.3. Samplepreparation

2.3.1. Standardandworkingsolutions

Individual stocksolutions of ANTand TET werepreparedin methanolataconcentrationof2000␮gmL−1andstoredat−15◦_C.

Mixed standardsolutions werefreshlyprepared fromthestock solutionsbyproperdilutionswithDIwaterforCEexperiments. ForHPLCstudy,stocksolutionsofANTandTETwerepreparedas (1000␮gmL−1)andISwaspreparedinmethanolat100␮gmL−1in ultrasonicbathfor10minandwasthenkeptinthedarknessat4◦C. Workingsolutionswerepreparedbydilutionofthestocksolutions inthemobilephase.

2.3.2. Preparationofforceddegradationsolutions

Thestability-indicatingwereperformedunderalkaline,acidic, oxidative,photolyticandthermalstressconditionsatafinal con-centrationof50␮gmL−1.

Forceddegradationwasachievedbytreatingthestocksolution with0.1Mand1MHClforacidichydrolysis,0.1Mand1MNaOHfor alkalinehydrolysis,and3%and30%H2O2foroxidativedegradation

forbothanalytes.ThesolidformoftheanalyteswasexposedtoUV lightat254nmandroomtemperature,andthermaldegradation wasrealizedinanoventhatwasadjusted100◦Cfor6and24h. Purityresultsofeachpeakwerethenscreenedonthesoftware. 2.3.3. Analysisofpharmaceuticaldosageformsandrecovery assay

For the analysis of pharmaceutical preparations with HPLC, Allergoconjunct® _{solutionwhichcontainedANTat0.15mgmL}−1

andTETat0.50mgmL−1wasdilutedwithmethanoltothemarkina 25-mLvolumetricflask.Thecontentsoftheflaskweresonicatedfor 10mintoachievecompletedissolution.Appropriatesolutionswere preparedbytakingsuitablealiquotsanddilutingwiththemobile phase.ConcentrationoftheISwaskeptconstantinalldilutionsand thechromatographicprocedurewasapplied.ForCEexperiments, dilutedsolutionsoftheabove-mentionedformulationwerefreshly preparedbyproperdilutionswithDIwater.

2.4. Validationofthemethods

HPLCandCEmethodswerevalidatedtoquantifyANTandTET in thecommercialformulationaccordingtoparameterssuchas specificity,linearity,lineardynamicrange(LDR),precision, accu-racy, limit ofdetection(LOD) and limitof quantification (LOQ). Priortothevalidationstudies,theseparationconditionswere care-fullyoptimized.Todoso,systemsuitabilitytestswereperformed

Fig.1.Optimizationofcapillaryelectrophoresisparameters.Effectofbuffertype(a),Effectofbufferconcentration(b),Effectoforganicmodifier(c),Effectofseparation voltage(d),Effectofseparationtemperature(e)andEffectofinjectiontime(f).

Table1

Parametersofsystemsuitabilitytests.

Technique HPLC CE

Compounds TET IS ANT TET ANT

Retention/MigrationTime 1.785 2.819 3.165 3.80 4.05

SelectivityFactor(␣) – 1.580 1.120 – 1.07

ResolutionFactor(Rs) – 7.330 2.190 – 6.370

TheoreticalPlateNumbers(Plate/column) 3369 5053 6525 114770 231831

RSD%ofRetentionTimea _{0.047 0.135 0.149 0.850 0.798}

a_{Eachvalueisthemeanoffiveexperiments.}

inordertoensurethatthemethodcangenerateresultsof accept-ableaccuracyandprecision.Testparametersincludedtheoretical platenumber,resolutionfactorandselectivity.

2.4.1. Specificity

Specificityofthemethodcanbedescribedbasedontheability of the methodto verify the analyte in the presence of excipi-ents, matrix andimpurities [15–17]. Accordingtothe obtained chromatograms,thepharmaceuticalpreparationanditsplacebo (mixtureoftheactivepharmaceuticalingredient(API)and excipi-ents)showedalmostnointerferingpeakswithintheretentiontime ranges.Inaddition,forceddegradationstudieswereperformedto provethespecificityofthechromatographicmethod.

2.4.2. Linearityandlineardynamicrange

InCEstudies,concentrationsoftheanalyteswerevariedwithin therangeof5.3–100_␮gmL−1 and2.3–125_␮gmL−1 forANTand TET,respectively.InHPLC,widerLDRswereachievedwithinthe concentrationrange of0.5–200␮gmL−1 forboth analytes. Cali-brationgraphswereconstructedbyplottingtheratioofthepeak areaofthedrugtothatoftheISagainsttheactivepharmaceutical ingredient(API)concentrationforHPLCandplottingconcentration versuspeakareaforCEwithrealizingrepeatedanalysis(Itwas checkedoverthesameconcentrationrangeonthreeconsecutive days).Thelinearregressionwasdemonstratedandtabulatedbythe necessaryparameters.

M.Gumustasetal./JournalofPharmaceuticalandBiomedicalAnalysis124(2016)390–398 393

Fig.2.OptimizationofHPLCmethod.(a)Optimizationofmobilephase,column:KinetexC18(150×4.6mm;5␮m),pHofthemobilephaseadjustedto3.0,25◦Ctemperature withtheflowrateof1mLmin−1_{,(b)Choiceofcolumn,pHofthemobilephaseadjustedto5.30,withaflowrateof1mLmin}−1_{,compositionofmobilephase60:40(v/v)}

ACN:Buffer(0.05%TFA),andtemperature25◦_C.

2.4.3. Limitsofdetectionandquantification

LODandLOQwerecalculatedfromtheequationsinwhichthe standard deviationof response andtheslope of thecalibration curvewereused[18].

2.4.4. Precisionandaccuracy

Theintermediateprecisionwasassessedbycarryingout the analysisofatleastthreeworkingsolutionsinthreeconsecutive daysforinter-dayrepeatabilityanditwasevaluatedbyassayingat leastfiveinjectionswithusingoneconcentrationfromtheworking rangewithinthesamedayforintra-dayunderthesame experimen-talconditions.

Accuracy is one of the main requirements of any chro-matographicorcapillaryelectrophoreticmethodespeciallywhen workingon pharmaceuticals which is defined astheproximity oftheexperimentalvaluetothetruevaluefortherealsamples

[17,18].Recoverywascarriedoutbyspikingthealreadyanalyzed samplesofdosageformwiththeknownamountsofstandard solu-tionsofpharmaceuticalactivecompoundsindosageforms.

3. Resultsanddiscussion

3.1. OptimizationofCEconditions 3.1.1. Selectionofwavelength

OptimizationofCEconditionsaswellastheselectionof opti-mumdetectionwavelengthwasbasedoncorrectedpeakarea(CPA) whichofferedtheadvantageofhavingahigherreproducibilitythan wouldbeobtainedusingpeakareaorpeakheight.Absorptionof ANTandTETwasscannedintherangeof192–600nmanda3D absorptionspectrum(absorption-migrationtime-wavelength)was recorded.Itwasfoundthatbothanalytesabsorbedintherangeof 192–200nmwithabsorptionmaximabeingat192nm.

3.1.2. Typeofbufferanditsconcentration

Acidic,neutralandbasicbuffers(i.e.,acetatepH4.6,phosphate pH7.0andboratebufferatpH9.2)wereevaluatedtoachieve opti-mumefficiency.AlthoughpHofthebackgroundelectrolyte(BGE) had nonoticeableeffectonresolution(datanotshown), migra-tiontimeaswellaspeakareasdecreasedwithincreasingthepH whichwasthoughttobeduetothelowermagnitudeofthe elec-troosmoticflow(EOF)insidethecapillaryunderacidicconditions.

Fig.3.TypicalHPLCchromatogramsofpurebulksampleof50␮gmL−1TET(a)andmildstressedconditionsofsamples:in0.1MHClfor60min(b);in0.1MNaOHfor60min (c);in3%H2O2for60min(d);underUVlightat254nmfor6h(e);andat100◦Cfor6h(f).

Thus,thehighestCPAwasachievedusingacetatebufferatpH4.6 (Fig.1a).Despitethis,phosphatebufferatpH7.0waspreferredas acompromisebetweenpeakareaandmigrationtime.Theeffectof bufferconcentrationonCPAwasstudiedintherangeof5–50mM. Itwasobservedthatthehigheststabilitywasobtainedwithinthe rangeof10–25mMabovewhichcausedahighcurrentandwasthus avoided.Therewasnoconsiderableeffectofbufferconcentration onmigrationtimewithinthewholerange(i.e.,5–50mM).Above 20mMpeakareastartedtodecreasewhicheventuallydecreased CPA(Fig.1b).Thus,20mMphosphatebufferatpH7.0wasused throughouttheexperiments.

3.1.3. Organicmodifier

Addingasmallamountofanorganicsolvent(modifier)suchas methanol,ethanoloracetonitriletotheBGEiscommoninCEto improveresolution.Inthisstudy,theireffectwasstudiedwithno modifieraddedtotheBGEaswellasintherangeof5–15%(v/v). Itwasnoticedthatwithallofthesesolvents,theresolutionand thepeakareaswerealmostthesame.Thus,CPAdecreasedwith

increasingtheorganicpercentage(Fig.1c).Hence,nomodifierwas usedinthisstudy.

3.1.4. Separationvoltageandtemperature

Separationvoltagewasinvestigatedintherangeof18–27kV. Increasingtheseparationvoltagewithinthisrangedecreasedboth the migration time and peak areas. Calculating CPA, however, revealed that it increasedgradually up to22kV after which it startedtodecrease(Fig.1d).Thus,22kVwasappliedthereafter.

Capillarytemperaturewastestedovertherangeof12–30◦C. CPAincreasedgraduallyupto25◦Candthenstartedtodecrease. Increasingtheseparationtemperaturedecreasedboththe migra-tiontimeandpeakareas.ItwasseenthatCPAincreasedrapidlyup to25◦Candthenstartedtodecreaseafterwards(Fig.1e).Hence, thisvaluewasconsideredoptimumforfurtherexperiments.

3.1.5. Injectiontime

Theeffectofinjectiontimewasinvestigatedbyapplyinga pres-sureof50mbarfor5–17s.Although,increasingtheinjectiontime

M.Gumustasetal./JournalofPharmaceuticalandBiomedicalAnalysis124(2016)390–398 395

Fig.4.TypicalHPLCchromatogramsofpurebulksampleof50␮gmL−1ANT(a)andmildstressedconditionsofsamples:in0.1MHClfor60min(b);in0.1MNaOHfor60min (c);in3%H2O2for60min(d);(e)underUVlightat254nmfor6h;andat100◦Cfor6h(f).

withinthisrange hadnonoticeableeffectonmigration time,it eventuallyincreasedpeakareasandhenceCPA(Fig.1f).Despitethe factthatevenata17-sinjectiontimeagoodresolutionbetweenthe peakswasstillobserved,higherinjectiontimeswerenotapplied duetostartofdeteriorationofpeaksymmetry.Hence,aninjection timeof17swasconsideredoptimum.

Undertheelectrophoreticconditionswerealloptimized,the twoanalyteswereseparatedandsimultaneouslydeterminedin thecommercialpharmaceuticalpreparations.

3.2. OptimizationofHPLCconditions

ToobtainthebestHPLCconditions,stepbystepoptimization wasadopted. The point of anHPLC methoddevelopment is to achievesufficientresolutionofthetargetanalytesfromallother excipients,interferencesandmatrixeffectwithinashortanalysis timeandtoobtainsuitablepeaksymmetrywithacceptable effi-ciency[15,19].Differentnewgenerationstationaryphaseswhich werepackedas fullyporousand core-shellmaterialswere pri-marily tested. Using the same isocratic conditions, an efficient separationwasobtainedusingKinetexC18(150mm×4.6mmi.d., 5␮m)astheanalyticalcolumn.Fig.2ademonstratesthatwhen

core-shellparticleswereused,sharperpeakswereobtainedwhen comparingwithothercolumnsthathavetraditionalfullyporous particleswhichprovidedmuchmoreefficientanalysisinaddition of reduced analysistime.Poor separation wasobservedwithX Selectcolumnwhichnotonlyincreasedtheanalysistimebutalso producedbackpressurethatwasalmostthreetimesthatofthe other.XBridgecanalsobechosenforthesimultaneous determina-tionofANTandTETbutafteroptimizingallconditions,theelution timeswiththiscolumnwerelongerthanthoseobtainedwiththe core-shellpackedKinetexcolumn.

Theeffectofdifferentorganicsolventswithvariedcompositions inthemobilephasewasevaluatedwithdifferentbuffers.Forthis reason,acetonitrileandmethanolwereinvestigatedastheorganic solvents.Becauseofthenarrowandsharppeakshapesobtained withacetonitrile,thissolventwasselected.Theeffectof acetoni-trilecompositioninthemobilephasewastestedintherangeof 35–55%and theresultsare shownin Fig.2a. pHof themobile phaseisanotherremarkablepointforthemethoddevelopment. Becauseofthesharperandefficientsignals,TFA(0.05%)wasadded tothebufferandthepHwasadjustedto3.0with5MNaOH. Tem-peratureofthecolumnovenwasadjustedandstudiedwithinthe rangeof25–40◦C.Noremarkableeffectofcolumntemperaturewas

Fig.5. Representativeelectropherogram(a)andchromatogram(b)ofunspikedophthalmicsolutionsample.100␮gmL−1TETand30␮gmL−1ANTrespectively.2␮gmL−1 ISwasusedforHPLCanalysis.

observed.Therefore,25◦Cwaschosenastheoptimum tempera-ture.Thesystemsuitabilitytestresults,evaluatedaccordingtothe ICH,showedthatthedevelopedmethodwassuitableforthe simul-taneousdeterminationandquantificationofANTandTET.Under optimizedconditions,retentiontimesobtainedwere1.785,2.819 and3.165minforTET,ISandANT,respectively(Table1).

3.3. Analyticalperformanceandvalidation

Forpresentingthestabilityindicatingcapabilityofthe devel-opedHPLCmethod,forceddegradationstudieswereperformed. ThechromatogramsareshowninFigs.3and4anddegradation per-centagesweretabulatedaftereachtreatmentasshowninTable2. ThestocksolutionsofthecompoundsweredilutedwithHCl, NaOHandH2O2to50␮gmL−1andwaitedfor1h.Asamild

con-ditiontherewasveryfewamountofTETdegraded(2.57%)(Fig.3)

with0.1MHClandnodegradationobservedonANT(Fig.4).When applyingdrasticconditions(e.g.,1MHCl),3.37%ofTETand1.21% ANTdegraded.Incontrast,basichydrolysiswasmoreeffectivethan theacidicone.Asaresultofmildalkalinehydrolysis,8.43%ANT and5.38%TETdegradedafter1hoftreatment.Ontheotherhand, 1MofNaOHdegradedTETcompletelyand80%ofANT.The addi-tionof3%H2O2didnotshowanyeffectontheanalytesbutpeak

broadeningofTETwasobserved.Aftertreatmentwith30%H2O2,

theresultswerefoundas13.42%and5.24forTETandANT, respec-tively.SolidformsofbothcompoundswerekeptunderUVlightat 254nmforphotolyticandat100◦Cinanovenforthermal degrada-tion.Nodegradationwasobservedontheshortterm(6h)butafter 24h,theresultswithphotolyticdegradationwerefoundas27.76% and10.39%andtheresultofthermaldegradationwerefoundas 17.27%and35.50%forTETandANT,respectively.Asaresultofthese

M.Gumustasetal./JournalofPharmaceuticalandBiomedicalAnalysis124(2016)390–398 397

Table2

Resultsofhydrolytic,oxidizing,thermalandphotolyticstressconditions.

Stressconditions %DegradationofTET %DegradationofANT

Mildconditions HCl(0.1M) 2.57 ND NaOH(0.1M) 8.43 5.38 H2O2(3%) NDa 0.49 6hUV ND ND 6h100◦_{C ND ND} Drasticconditions HCl(1M) 3.37 1.21 NaOH(1M) 100 80.63 H2O2(30%) 13.42 5.24 24hUV 27.76 10.39 24h100◦_{C 17.27 35.50}

Eachvalueisthemeanoffiveexperiments.ND:Nodegradation.

a_{Peakshapegettingbroader.}

Table3

RegressiondataofthecalibrationgraphsforquantitativedeterminationofTETand ANTbyHPLCandCE.

Compounds HPLC CE

TET ANT TET ANT

Linearityrange(␮gmL−1) 0.5−200 0.5−200 2.3–100 5.3–100 Slope 0.155 0.171 6.060 4.830 Intercept 0.023 0.038 −1.130 −7.440 Correlationcoefficient 0.999 0.999 0.999 0.998 SEofslope 1.16×10−3 _6.63×10−4 _{0.050 0.080} SEofintercept 1.05×10−1 _6.01×10−2 _{1.130 2.420} Limitofdetection(␮gmL−1) 0.068 0.078 0.70 1.60 Limitofquantification(␮gmL−1) 0.206 0.238 2.30 5.30 Within-dayprecisiona_{(RSD%) 0.426 0.388 2.10 1.80} Between-dayprecisiona_{(RSD%) 0.918 0.517 2.80 1.90} a_{Eachvalueisthemeanoffiveexperiments.}

experiments,thedevelopedHPLCmethodwasfoundasspecificfor theanalyzedAPIs.

Standard calibrationgraphs were constructed as mentioned above.Underoptimizedconditions,LODswerefoundas1.6and 0.7␮gmL−1 byCEand0.068and0.078␮gmL−1forTETandANT byHPLC,respectively(Table3).LOQswerecalculatedas5.3and 2.3␮gmL−1withCEand0.206and0.238␮gmL−1forANTandTET, respectively.Theresponsewaslinearovertheconcentrationranges of2.3–100,5.3–100withCEand0.5–200␮gmL−1withHPLCfor ANTandTET,respectively,withR2_{notlowerthan0.998withboth}

methods.Reproducibilitywasevaluatedintermsofintradayand interdayprecision,byinjectingthestandardsatfiveconcentration levelsinthesamedayandinthreeconsecutivedays,respectively. AnacceptableprecisionwasobtainedinbothcasesRSD%values below2.8%forinterdayand2.1%forintradayexperimentswithCE and0.426%forinterdayand0.918%forintradayassayswithHPLC.

3.4. Analysisofpharmaceuticaldosageforms

In order to evaluatetheapplicability, recovery and possible matrix effectof theproposedCEmethod,thecommercial oph-thalmicsolution(Allergoconjunct®_{)withreportedconcentration}

of0.15mgmL−1ANTand0.50mgmL−1TETwasexamined.Itwas ensuredthattheremovaloftheexcipientswithanextractionstep beforeanalysiswasunnecessary.AsindicatedbyTable4,the recov-eryresultswerefoundintherangebetween97.90–102.87%for bothmethods.Itwasconcludedthattheproposedmethodswere sufficiently accurateand precisefor thepharmaceutical dosage formofTETandANT.Theproposedmethodswerecompared sta-tistically.Bothmethods showedsimilaraccuracyand precision. A statistical comparison wasperformed ondataobtained from bothtechniques.Student’st-andFtestrevealednostatistically significant difference between methods with regard to accu-racyandprecision(Table4)[16–19].Typicalchromatogramand

Table4

AssayresultsandmeanrecoverystudiesofTETandANTinpharmaceuticaldosage forms.

Compounds HPLC CE

TET ANT TET ANT

Labeledclaim(mgmL−1) 0.500 0.150 0.500 0.150 Amountfound(mgmL−1₎a _{0.484 0.152 0.489 0.147} RSD(%)a _{1.029 0.390 97.80 98.00} Bias(%) 3.200 −1.333 2.20 2.00 Added(mgmL−1_{) 0.250 0.075 0.250 0.075} Found(mgmL−1₎a _{0.254 0.077 0.246 0.073} Recovery(%) 101.565 102.870 98.20 97.90 %RSDofrecoverya _{0.246 0.282 1.90 2.15} Bias(%) −1.565 −2.870 1.80 2.10

a_{Eachvalueisthemeanoffiveexperiments.}

electropherogram of ophthalmic solution sample are shown in

Fig.5aandb.Theanalyteswerebaselineseparatedinless than 4.5minwithbothtechniques.

4. Conclusion

Neither CE norstability-indicating HPLC methods are found intheliteratureforthesimultaneousdeterminationofANTand TETfrompharmaceutical dosageforms. For thisreason, CEand stability-indicatingHPLCmethodswerefullyvalidatedaccording totheICHguidelinesandwerepresentedforthedeterminationof ANTandTETinophthalmicformulationswhichoffersnumerous advantages,suchasrapidity,useofminimumamountsoforganic solvents, simplicity,low cost,easeofoperation,and high selec-tivity.Goodrecoveries,highreproducibilityandinterference-free electropherogramsandchromatogramswerealsoachieved.Even CEisstudiedasacomparisonmethod,itshowedbetterpeak capac-ityandefficiency.Furthermore,theconditioningtimeofCEshowed anadvantageoverHPLC.

The proposed methods present a step by step optimization procedure.Inthisstudy,newgenerationstationaryphaseswere comparedforthefirsttimewiththenewcolumnpacking mate-rialsespeciallythecore-shellparticles.Asa resultofthisstudy, theproposedmethodsare suitableforquality control laborato-ries,whereeconomy andtimeareessential.Highpercentageof recoveryresultsshowedthattheproposedmethodswerefreefrom interferences ofcommonlyusedexcipientsandadditives inthe formulations.

Acknowledgment

The authorsarethankful toGaziUniversity for thefinancial supportofthiswork“ProjectNo:BAP-02/2010-02’.

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