A novel stability-indicating UPLC method development and validation for the determination of seven impurities in various diclofenac pharmaceutical dosage forms | [Développement et validation d'une nouvelle méthode indicatrice de stabilité par UPLC pour la

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AnnalesPharmaceutiquesFrançaises(2016)74,358—369

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ORIGINAL ARTICLE

A novel stability-indicating UPLC method development and validation for the

determination of seven impurities in

various diclofenac pharmaceutical dosage forms

Développement et validation d’une nouvelle méthode indicatrice de stabilité par UPLC pour la détermination de sept impuretés de diclofénac dans

plusieurs formes pharmaceutiques

M. Azougagh

^a^,∗

, M. Elkarbane

^a

, K. Bakhous

^b

,

S. Issmaili

^b

, A. Skalli

^c

, S. Iben Moussad

^d

, B. Benaji

^c

aInstitutsupérieurdesprofessionsinﬁrmièresettechniquesdesanté,Rabat,Morocco

bPhysico-chemicalservice,drugsqualitycontrollaboratory,divisionofdrugsandpharmacy, MinistryofHealth,Rabat,Morocco

cÉcolenormalesupérieuredel’enseignementtechniquedeRabat,Rabat,Morocco

dFacultédemédecine,universitéHassanII,Casablanca,Morocco

Received22May2016;accepted1stJuly2016 Availableonline27July2016

KEYWORDS

Methoddevelopment;

Validation;

Indicatingstability;

UPLC;

Diclofenac;

Impurities

Summary An innovative simple,fast, precise and accurateultra-high performance liquid chromatography(UPLC)methodwasdevelopedforthedeterminationofdiclofenac(Dic)along withitsimpuritiesincludingthenewdimerimpurityinvariouspharmaceuticaldosageforms.

AnAcquityHSST3(C18,100×2.1mm,1.8␮m)columningradientmodewasusedwithmobile phasecomprisingofphosphoricacid,whichhasapHvalueof2.3andmethanol.Theﬂowrate andtheinjectionvolumeweresetat0.35ml·min⁻¹and1␮l,respectively,andtheUVdetec- tionwascarriedoutat254nmbyusingphotodiodearraydetector.Dicwassubjectedtostress conditionsfromacid,base,hydrolytic,thermal,oxidativeandphotolyticdegradation.Thenew developedmethodwassuccessfullyvalidatedinaccordancetotheInternationalConferenceon

∗Correspondingauthor.

E-mailaddress:azougaghma@yahoo.fr(M.Azougagh).

http://dx.doi.org/10.1016/j.pharma.2016.07.001

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Harmonization(ICH)guidelineswith respecttospeciﬁcity,limitofdetection,limitofquan- titation, precision, linearity,accuracy androbustness.The degradation products were well resolvedfrommainpeakanditssevenimpurities,provingthespeciﬁcitypowerofthemethod.

ThemethodshowedgoodlinearitywithconsistentrecoveriesforDiccontentanditsimpurities.

Therelativepercentageofstandarddeviationobtainedfortherepeatabilityandintermediate precisionexperimentswaslessthan3%andLOQwaslessthan0.5␮g·ml⁻¹forallcompounds.

Thenewproposedmethodwasfoundtobeaccurate,precise,speciﬁc,linearandrobust.Inaddi- tion,themethodwassuccessfullyappliedfortheassaydeterminationofDicanditsimpurities intheseveralpharmaceuticaldosageforms.

MOTSCLÉS Développementde méthode;

Validation;

Méthodeindicatrice destabilité; UPLC; Diclofénac; Impuretés

Résumé Unenouvelleméthodesimple,rapide,préciseetexacteparchromatographieliquide ultrahauteperformance(UPLC)aétédéveloppéeetvalidéepourladéterminationsimultanée dediclofénac(Dic)etses7impuretéspotentielles,ycomprisledimère,dansplusieursformes pharmaceutiques.Laséparationchromatographiqueaétéeffectuéeenmodegradientàl’aide d’unecolonneAcquityHSST3(C18,100×2,1mm,1,8␮m)etd’unephasemobilecomposéed’un mélanged’acidephosphoriqueàpH2,3etdeméthanol.Ledébitetlevolumed’injectionont étéfixésrespectivementà0,35mL·min⁻¹et1␮L.Ladétectionaétéréaliséeparundétecteur à barrettediodes à254nm. Les échantillons de Dicont été soumis aux différentes condi- tionsdedégradationforcéeprescritesparlaConférenceinternationaled’harmonisation(ICH) (hydrolyse,température,oxydationetphotolyse).Laméthodeaétévalidéeconformémentaux directivesdel’ICHQ2R1encequiconcernelaspécificité,leslimitesdedétectionetdequantifi- cation,lafidélité,lalinéarité,l’exactitudeetlarobustesse.Lesproduitsdedégradationsont bienséparés,prouvantlaspécificitédelaméthode.Laméthodemontreunebonnelinéarité, unrecouvrementélevéetunefidélité(répétabilitéetfidélitéintermédiaire)satisfaisanteavec uncoefficientdevariationinférieurà3%pourledosageduDicetsesimpuretés.Lesvaleurs obtenuespourlimitedequantificationsontinférieuresà0,5␮g·mL⁻¹pourtouteslesimpuretés.

Lanouvelleméthodeprésentéeestexacte,ﬁdèle,spéciﬁque,linéaireetrobuste.Enplus,la méthodeaétéappliquéeavecsuccèspourledosageduDicetsesimpuretéspotentiellesdans plusieursformespharmaceutiques.

Introduction

Diclofenac acid (Dic) (Table 1), chemically known as {2- [(2,6-dichlorophenyl)-amino]phenyl}acetateacid,hasbeen used extensively and for a long time as a nonsteroidal anti-inﬂammatorydrug(NSAID).SeveralDicsaltsarecom- monly used in various drug formulations such as tablets, capsules,solutionsfor injection,suppositoriesandgels to alleviate the painandswellingassociated withconditions suchasarthritis,toothache,dysmenorrhoeaandothermus- culoskeletaldisorders[1—3].

Aliteraturesurveyrevealsthatalargenumberofpapers ondeterminationofDicindividuallyorincombinationwith otherdrugsinpharmaceuticaldosageformsbyavarietyof analyticaltechniquesareavailable.Themostcommonbeing High Performance Liquid Chromatography (HPLC) [4—24], UV spectrometry (UV) [25—32], Capillary Electrophoresis (CE) [22,33,34], Thin Layer and High Performance Thin Layer Chromatography (TLC and HPTLC) [35—37]. On the other hand, although the description of several potential

impurities, and the importance and requirement of their controlin drugproductby theregistration authorities for assuringthemaximumsafetyofdrugtherapyeveninsmall amounts[38,39],onlyfewpapershasbeenfocusedondeter- mining the content of Dic with some of its impurities in differentpharmaceuticalforms[23,24,40—50].

Furthermore,although the current global officialPhar- macopoeias including European (EP) [51], United States (USP)[52], British (BP) [53] and Japanese (JP) [54] have dealt with Dic impurities in API (Active Pharmaceutical Ingredient)andfinishproducts,onlytwoknownimpurities couldbequantified.Outofthesixpotentialimpuritieslisted byEPintheAPI,onlytwoimpurities,namelyImp-AandImp- F,couldbequantified.ItshouldbenotedthatonlytheImpA couldbequantifiedbytheBPandUSPinthefinishproducts andnoknownimpuritycouldbequantifiedbyJPintheAPI orinfinishproduct.Attemptshavebeenmaderecentlyby Novakovaetal.[55]andElzayatetal.[56] todevelop an indicatingstabilityUPLCmethodfailtoquantifyallknown potentialimpuritiesinthestudiedmatrixformulations.

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360 M.Azougaghetal.

Table1 ChemicalstructureofDic,itspotentialimpurities,somecommonpreservativesandtheirUVspectrum,aswell astheirretentiontimesandtheoreticalplatesparameters.

StructureschimiquesdeDic,sesimpuretéspotentielles,quelquesconservateurslesplusutilisésdanssaformulation, leursspectresUVainsiqueleurstempsderétentionrelatifsetlenombredeplateauxthéoriques.

Chemicalname Chemicalstructure RT,min Theoretical plates

UVSpectra

Diclofenac:

2-(2-(2,6-dichlorophenyl amino)phenyl)aceticacid

4.568 15558

ImpurityA(PhEur):1-(2,6 dichlorophenyl)-

1,3-dihydro-2H-indol-2-one

2.544 12958

ImpurityB(PhEur):

2-[(2,6-dichlorophenyl) amino]benzaldehyde

5.740 87000

ImpurityC(PhEur):

[2-[(2,6-dichlorophenyl) amino]phenyl]methanol

3.621 15397

ImpurityD(PhEur):

[2-[(2-bromo-6-chlorophenyl) amino]phenyl]aceticacid

4.984 16402

ImpurityE(PhEur):

1,3-dihydro-2H-indol-2-one

0.937 5703

ImpurityF(PhEur):

N-(4-chlorophenyl)-2-(2,6- dichlorophenyl)

acetamide

3.864 15252

Dimer:4,4-bis[(2,6-Dichloro phenyl)amino],biphenyl}

-3,3-bisaceticaciddisodium salt

7.099 237935

Benzoicacid 0.996 —

Benzylalcohol 0.846 5368

Methylparaben 0.997 6027

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Table1 (Continued)

Chemicalname Chemicalstructure RT,min Theoretical plates

UVSpectra

Propylparaben 1.611 10089

Ethylparaben 1.215 7455

To the extent ofour knowledge,none of the currently existingmethodshasrevealedthepresenceofdimerimpu- rityorusedasindicating stabilityforallknownimpurities indifferentpharmaceuticaldosageforms.

The aim of thepresent study wastodevelop an inno- vativefaster,speciﬁc,sensible,accurateandpreciseUPLC method for the estimation of Dic in the presence of its relatedcompoundimpuritiesincludingDimerimpurityand some preservatives commonly formulated in Dic dosage forms.

Experimental

Materialsandreagents

ThechemicalstructureofDicacid,itspotentialimpurities andthecommonpreservatives usedinitsformulationare showninTable1.

The working standards of Dic sodium (99.7%), dimer impurity (99%) and Imp-F (99%) were kindly provided by Maphar, ZenithPharma and Novartis Moroccan drug indus- tries,respectively.TheReferencestandardsoftheotherDic impurities,speciﬁcally:Imp-A(99.9%),Imp-B(99.8%),Imp- C(99.3%),Imp-D(99.7%)andImp-E(99.9%)werepurchased fromLCG(Germany).Allthecompoundswereusedwithout furtherpuriﬁcation.

ThecommonpreservativesforDicformulationsincluding benzoicacid(BcA),benzylalcohol(ByA),methyl(MP),ethyl (EP),andpropyl(PP)parabenswerepurchasedfromSigma- Aldrich(Germany).

Analytical grade reagents including dibasic potassium phosphate,sodiumhydroxide,phosphoricacid, hydrochlo- ride acid, sulphuric acid and hydrogen peroxide were purchasedfromSigma-Aldrich(Germany).Theacetonitrile andmethanolsolventswereHPLCgradeandobtainedfrom Merck(Germany)andusedthroughout.Allaqueoussolutions including the HPLC mobile phase were prepared with in- housewater usingMillipore Milli-QPlus waterpuriﬁcation system(USA).

Commercially dosageformsincludingtablets,capsules, gels,suppositoriesandsolutionsforinjection,fromdiffer- entcountries(Congo;China,India,Morocco,France,Spain, Yemen and Zambia) wereused for the method speciﬁcity veriﬁcationandmethodapplication.

Chromatographicconditions andequipments

LiquidChromatographywascarriedoutonaWatersAquity UPLC-HClass withphotodiode array detector. The output signalwasmonitored andprocessedusing empowerssoft- ware.ThechromatographiccolumnusedwasAcquityHSST3 (C18,100×2.1mm,1.8␮m)columnmaintainedattemper- atureof45^◦C.Theseparation wasachievedonagradient elution by a mobile phase consisting of phosphate buffer (solvent A)and methanol givenin Table 2. The solvent A waspreparedbymixingequalvolumesof0.5g·l⁻¹ofphos- phoricacidand0.75g·l⁻¹ ofmonobasicsodiumphosphate, previouslyadjustedwithadditionalportionsof phosphoric acid,toapHvalueof2.3andﬁlteredthrough0.45␮mnylon membraneanddegassed beforeuse.The sampleinjection volumewassetat 1␮l andmonitored byUVdetection at 254nm.

Preparationofstandardandsamplesolutions

AstandardsolutionofDicof1mg·ml⁻¹waspreparedbydis- solvinganappropriateamountofDicNainadiluentphaseof solventAmixedwithmethanol(35:65,v/v)forcontenttest.

TheworkingstandardimpurityandAPIstocksolutionswere preparedbydissolving10mgofeachproductinto100mlof diluentphaseforimpuritytest.

Samplesequivalenttotheconcentrationof1mg·ml⁻¹of Dicbaseinthediluentphasewerepreparedaccordingtothe dosageformsforDiccontentandimpuritytestsasdescribed below:

Tabletsandcapsules

Anamount of crushedtabletsequivalentto100mgofDic basewastransferredtoa50-mlvolumetricﬂaskcontaining 20mlofmethanol.Thesolutionwassonicatedfor15minutes (min)andthenmadeuptothevolumewithmethanol.

Gels

Aportionofsamplecorrespondingto100mgofDicbasewas accuratelyweighedandwastransferredin30mlofmethanol ina50-mlvolumetricflask.Thismixturewassubjected to vigorousshakingforabout 20minfor complete extraction ofdrugandsonicatedfor5minbeforefillinguptheflaskto 50ml.

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362 M.Azougaghetal.

Table2 Gradientprogramusedinthestudy.

Legradientd’élutionutilisédanscetteétude.

Time(min) Flowrate(ml·min⁻¹) %A %Methanol

Initial 0.35 38.0 62.0

4.00 0.35 38.0 62.0

4.50 0.35 10.0 90.0

6.50 0.35 10.0 90.0

7.00 0.35 38.0 62.0

9.00 0.35 38.0 62.0

Solutionsfor injection

Anaccuratelyweighedportionofthepowder,equivalentto about100mgofDicbasewastakenin50mlvolumetricﬂask.

Volumewasmadetothemarkwithmethanolandsonicated for5min.

Suppositories

Similarly,anamountofhomogenizedsampleequivalentto 100mgofDicbasewasaccuratelyweightedandmixedwith 25mlofmethanolina50-mlvolumetricﬂask.Theﬂaskwas putinanultrasonicbathwherethewatertemperaturewas setat 37^◦C. Aftercomplete disintegration ofthe sample, 25ml ofmethanolwere added;the sample wassonicated for10minandmadeuptothevolumewithmethanol.

Afterwards5mlfromeachof theabovesolutionswere transferredtoa10-mlvolumetricflaskanddilutedto10ml withdiluentphaseandfilteredusing0.22␮mNylonmem- branefilterbeforetobeanalyzedbyUPLC.

Stressdegradationstudies

Forceddegradationstudiesunderdifferentconditionswere carried out to determine if Dic was successfully sepa- ratedfromitspotentialimpuritiesandinterferingproducts.

Stressstudieswereperformedataninitialconcentrationof 1mg·ml⁻¹ofDicaccordingtotheICHQ1Aconditions[57].

Thesestressconditionstudiesincluded photolyticUVlight (254nmfor2days),thermal(60^◦Cfor24h),acidhydrolysis (1NHClat60^◦Cfor1hand1NH2SO4atroomtemperature for 1h), base hydrolysis (1NNaOH at 60^◦C for 24h), and oxidation(3%H2O2atroomtemperaturefor24h).

Method validation

ThedescribedmethodhasbeenvalidatedforDicassaywith itssevenpotentialimpuritiesbyUPLCdeterminationaccord- ingtoICHQ2R1[58]withrespecttospeciﬁcity,precision, accuracy,linearity,limitofdetection(LOD),limitofquan- titation(LOQ)androbustness.

Speciﬁcity

Specificityisdefinedastheabilitytoaccessunequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products,and matrix components. Specificity wasinvesti- gatedbyinjectinga freshlypreparedsolutionofdifferent Dicdosageforms(tablets,capsules,gels,suppositoriesand

solutions for injection)at aconcentrationof 1mg·ml⁻¹, a solutionoftheDicimpuritiesandcommonpreservatives.In addition,samplesolutionssubjectedtostressstudieswere analysedtoprovideanindicationofthestabilityindicating proprietyandspeciﬁcityofthemethod.Thepossibleinter- ferentsfromexcipientsandthepotentialimpurityproducts withAPIwereevaluatedbythepeakpuritytestusingaPDA detector.

Precision

Precision (repeatability and intermediate precision) was veriﬁed by spiking the matrix containing Dic with known amounts of impurity using the sample preparation procedure.

Toevaluatedrepeatability(Intra-Dayvariation) sixsep- arate Dicsolutions withconcentration of 1mg·ml⁻¹,from one homogenous sample, were spiked with each impurity at speciﬁcationlevel,i.e0.5%bythesameanalystonthe samedaywiththesameequipment.The%relativestandard (%RSD)wascalculatedforthepeakareaofeachimpurityand Dic,respectively.

Intheintermediateprecision(inter-dayvariation)study, the same procedure of method precision was carried out using the same equipment by two analysts, on different days.The%RSDof the18resultswascalculatedaccording toISO5725[59]foreachcomponent.

Accuracy

Accuracyofthecontentmethodwasevaluatedbytherecov- eryintriplicateatthreeconcentrationlevels(i.e.50,100 and150%)ofknownamountofDicspikedintoplaceboequiv- alenttonominalDicconcentration(1mg·ml⁻¹.Similarly,the recoveryofallimpuritiesfromspiked samplecontaininga concentration of 1mg·ml⁻¹ of Dic was conducted in trip- licate at threedifferent spikelevels(i.e. 0.1, 1and 2%).

Thecorrespondingpercentagerecoveryagainstareference solutionand%RSDvalueswerecalculated.

Linearity

DetectorresponselinearityforDicanditssevenimpurities wereassessedbyinjectingseparatelysevenpreparedimpu- ritysolutionsoverthecalibrationrangesfromLOQto200%

(i.e. 0.5,1,2.5, 5,10,15, 20␮g·ml⁻¹).Similarly, the lin- earityofDicwastestedintherangesof50to150%atﬁve concentrations(rangingfrom0.5to1.5mg·ml⁻¹).

Thestandard curveswereconstructed byplottingeach peakareaversusitscorrespondingstandardconcentration.

The date wassubjected tostatisticalanalysis usingleast- squares methodtoobtain the slope,y-intercept,residues andcoefﬁcientofcorrelation(r²)values.

Limitofdetectionandquantiﬁcation

Signal-to-noise (S/N) ratiomethod wasadopted todeter- minethelimitofquantiﬁcation(LOQ)andlimitofdetection (LOD)asperICHQ2(R1)guideline[58].TheLODandLOQof themethodweredeterminedbyinjectingstandardsolutions ofprogressivelydecreasingconcentrationuntiltoachievea S/Nratioof3:1and10:1,respectively.Theprecision(n=6)

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wasalsodetermined at theLOQ level, andthe %RSDwas calculatedforthepeakareaforeachimpurity.

Robustness

Therobustnessofananalyticalprocedureisameasureofits capacitytoremainunaffectedbysmall,butdeliberatevari- ationsinmethodparametersandprovidesanindicationof itsreliabilityduringnormalusage.Thevariablesevaluated inthestudywereﬂowrate(±0.05ml·min⁻¹),columntemperature(±5^◦C),pHofthemobilephasebuffer(±0.2)and

%organic inthe mobilephaseat startinggradient (±5%).

Inallthedeliberatevariedchromatographicconditions,the resolution(RS)betweencriticaladjacentpeaks(Imp-E/MP orByA,Imp-C/Imp-FandDIC/Imp-D)andpeakssensibilities werechecked.

Solutionstability

Thestabilityofanalyticalsolutionswasdeterminedbyleav- ingtestandstandardDicspikedwithitsimpuritiessolutions intightlycappedvolumetricﬂasksatroomtemperaturefor 48handtheamountofallimpuritieswasmeasuredatdif- ferenttimeintervals,speciﬁcally0,12,24and48hagainst afreshlypreparedstandardsolution.

Results and discussion

Optimizationof chromatographicconditions

Theoptimisationofchromatographicseparationsisachieved byvaryingtheexperimentalconditionsoftherununtilthe Dic,itssevenpotentialassociatedimpuritiesandthepreser- vativesareseparatedcleanlyinareasonableamountoftime withadequatesensitivity.

The matrix containing Dic, five common preservatives and the seven impurities at a concentration of 1, 0.2 and0.001mg·ml⁻¹,respectively,wasstudied.Initialexperi- mentsconductedbyisocraticelutionusingphosphatebuffer (pH=2.5)andmethanol(30:70,V/V)asmobilephaseflow- ingatavariableratesoverAcquityBEH,(C18,50×2.1mm, 1.7␮m)columnwerenotappropriatetoseparateallprod- ucts. Attempts to improve the separation were made by increasingthesamestationaryphasecolumnto100mm,also fail toprovide satisfactory results. Hence, attempts have beenmadetodevelopagradientelutionusingvariouspro- portionsofdifferentaqueousphasesandorganicmodifiers asmobilephaseatseveralflowrates.Aftersometrialgra- dients,theoptimumchromatographicconditions obtained for the best compromise between reasonable retention timesandresolution,cannotseparateadequatelytheadja- centpeaks(Imp-C/Imp-FandImp-E/MPorByA)whichwere elutedwithapoorresolution of0and1.4values,respectively,(Fig.1a).ReplacingBHSstationaryphasecolumnwith CSH(Phenylethyl100×2.1mm1.7␮m)resultedontheone handinimprovingtheresolutionofgreaterthan2.5between Imp-C/Imp-F peaks but on the other hand in decreasing the resolution between Imp-E/MP or ByA) peaks to 1.1 (Fig. 1b). Using the identical chromatographic conditions withanewtypeHSST3,(C18,100×2.1mm,1.8␮m)col- umnhasresultedinanimprovementinresolutionbetween

allcriticalpeaksbutwithoutasatisfactorylevel.Theimpact of the phosphate buffer wasinvestigated by changing its percentageatstartinggradientfrom25%to50%,itspHover therangeof 2 to3 andthe columntemperature from25 to50^◦C.ThebestclearseparationbetweentheDicaswell astheir seven impurities withinrelatively shortrun time wasobtainedbyinjectingavolumeof1␮lintheHSST3col- umnmaintainedat45^◦Cwithastartinggradientfrom32%of phosphatebuffersetapHvalueof2.3.Thebestcompromise betweenreasonableresolutionandsensitivitywasachieved atthewavelengthof254nm(Fig.1c).

Basedontheabove-mentionedresults,thebestsepara- tionbetweenDicanditsimpurityproductsinthepresenceof interferingexcipientswasachievedbyusingthechromato- graphicconditions.

Stressdegradationstudies

ThedegradationstudyrevealedthatDicwasverysensitive toacidhydrolysis(30%)andUVlightstress(35%)compared to other degradation conditions. The major degradation products formed afteracid hydrolysis were Imp A and an unknownimpurityatretentiontime(RT)of5.8min.Several degradationproductswereobservedwhenthedrugwassub- jectedtotheUVlightstress;themajorpeakswereelutedat RTof1.832and2.987minwiththepresenceofmanyminor peaksat RT 2.654,3.022, 3.230,6.986 and7.272min.No considerabledegradationwasnoticed(<3%)whenthedrug wasstressedinalkalinehydrolysis;onlyoneminorpeakwas elutedatRT2.240min.Intheoxidationconditionsthedrug wasslightly susceptibletodegradation(5%), theobtained chromatogram displayed four minor degradation products appeared at RT 1.378, 1.908, 2.949 and 5.821min. Solid statestressedatelevated temperatureshowedtheforma- tionoftwominordegradationproductselutingatRT2.258 and5.833min.

Validation

Speciﬁcity

Therewerenointerferencesbetweenthepeakcorrespond- ing to the drug substance and other impurities or other componentsoftheformulationspeaks.Underthestresscon- ditions, none of the observed degradation product peaks interfereswiththepeakcorrespondingtoDic.Theobtained purityangleswerewithinthepuritythresholdlimits,which conﬁrmthatDicpeaksarehomogenousandpureinallthe analyzed samples subjected toforced degradation conditions.

Linearity

The results for linearity obtained from the calibration curvesover theconcentrationranges describedpreviously of Dic and its impurities are shown in Table 3. Correla- tion coefﬁcients were found to be more than 0.999 for both drug and each of its impurities. The t-student test onthey-interceptdemonstratednostatisticallysigniﬁcant differencefromtheorigin.Thegraphicexaminationofthe residualsdistributedrandomly aroundhorizontalzeroaxis demonstratedtheabsenceofconstantsystematicerror.All

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364M.Azougaghetal.

Table3 Regression,precision,LOQandaccuracyresults.

Résultatsdelarégression,laﬁdélité,laLDQetl’exactitude.

Parameters Dic Dic Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F Dimer

Linearityrange(␮g·ml⁻¹) 500—1500 0.5—2 0.5—2 0.5—2 0.5—2 0.5—2 0.5—2 0.5—2 0.5—2

Regressionequation(Y=bX+a)

Slope(b) 8211224 7975180 10922911 15637721 7647248 7679175 25569108 25463252 4280266

Intercept(a) −287.4 −36.1 33.7 −245.6 −36.7 −9.9 −209,9 58.2 249.3

r² 1.000 0.9991 0.9995 0.9995 0.9995 0.9995 0.9996 0.9994 0.9997

Teststudent^a−⁰

ıa _0.031 _0.029 _0.026 _0.14 _0.043 _0.011 _0.081 _0.049 _1.077

Precision(1%)

Repeatability(%RSD) 0.45 N.A 0.98 1.51 1.21 1.55 0.96 1.31 2.61

Intermediateprecision(%RSD) 0.51 N.A 1.49 1.82 1.84 2.83 1.51 1.93 2.78

LOQ

LOQ(␮g·ml⁻¹) N.A N.A 0.5 0.5 0.4 0.5 0.5 0.4 0.5

LOQRecovery(%) N.A N.A 96.2 98.5 98.7 104.2 107.4 102.4 90.5

LOQrepeatability(%RSD) N.A N.A 6.69 4.79 6.5 3.6 3.53 4.47 7.02

Accuracy

Level1 500(␮g·ml⁻¹) (0.1%) (0.1%) (0.1%) (0.1%) (0.1%) (0.1%) (0.1%)

Recovery(%) 99.9 N.A 95.68 101.02 96.13 108.31 106.36 96.14 92.02

(%RSD) 0.61 N.A 5.3 4.22 5.93 5.29 2.47 4.28 8.54

Level2 1000(␮g·ml⁻¹) (1%) (1%) (1%) (1%) (1%) (1%) (1%)

Recovery(%) 100.2 N.A 98.02 100.78 97.59 104.48 97.59 99.02 96.16

(%RSD) 0.54 N.A 2.64 2.94 2.39 1.75 1.26 2.64 1.94

Level3 1500(␮g·ml⁻¹) (2%) (2%) (2%) (2%) (2%) (2%) (2%)

Recovery(%) 100.2 N.A 99.66 99.25 99.34 99.49 99.55 101.42 98.13

(%RSD) 0.64 N.A 1.80 3.11 2.71 2.64 2.93 2.34 3.40

␦a:standarddeviationofintercepta;t(5%,5)=2.57with5%and5arecriticalvalueanddegreesoffreedom,respectively;N.A:notapplied.

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Figure1. Typicalchromatogramsobtainedduringmethodoptimisationsteps.

Chromatogrammestypiquesobtenusdurantlesétapesd’optimisationdelaméthode.

these data prove that the method was considered to be linearinthestudiedranges.

Accuracy

Accuracy datafollowing thedeterminationof each of the compoundsofinterestaresummarizedinTable3.Theabso- lute recoveries were calculated by comparing the areas underthepeaksobtainedfromstandardworkingsolutions withthepeakareasfromstandardsamples.Themeanrecov- eryofDicwasrangedfrom99to101%witha%RSDlessthan 1%ateachlevel.Thepercentagerecoveryofimpuritiesin

Dicsamplesvariedfrom92.0to108.5%andthe%RSDwas foundtobelessthan6%.Alldatahaveshowngoodconsis- tentrecoveriesindicatingthatthemethodishighlyaccurate forthedeterminationofDicanditsimpurities

Precision

Data obtained from precision experiments of Dic and its sevenimpuritiesaregiveninTable3.The%RSDvaluesless than2.5%and3%obtainedforrepeatabilityandintermedi- ateprecisionstudies,respectively,conﬁrmthatthemethod wassufﬁcientlyprecise.

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366 M.Azougaghetal.

Figure2. Chromatogramsofdiclofenacspikedwithitssevenimpuritiesandcommonpreservativesobtainedfromrobustnessstudies.

Chromatogrammesdediclofénacdopéavecsesseptimpuretésetlesconservateurslesplusutilisésdanssaformulation,obtenusàpartir desétudesdelarobustesse.

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Limitof detectionandquantiﬁcation

TheLODandLOQvaluesforeachimpurityarereportedin Table3.TheLODandLOQwereﬁndtocorrespondto0.02%

andto0.05%,respectively.

The %RSDfor peakareas ofall impuritiesat LOQlevel arewithin8%.TherecoveriesatLOQlevelareintherange of 92—107.5%.The resultsshow clearlythatthe proposed methodcanquantifysmallquantityofimpuritiesinDicsam- ples.

Robustness

In allthe deliberately variedchromatographicconditions, allanalyteswereadequatelyresolvedandtheelutionorder remainedunchangedasitisshowninFig.2.Theresultsof themethodshowedclearly,robustnessofthemethod.

Solutionstability

All chromatograms showed that the retention times and peakareasofthedrugsremainedalmostunchangedandno signiﬁcant degradation wasobserved duringthe periodof 24h.

Beyondthisperiod,although, theDicandthemajority peakareaswerestillstabletheDimerpeakareadecrease.

Theseresultsindicatedthatthesesolutionswerestablefor at least 24h whenkept at room temperature, which was sufﬁcientforthewholeanalyticalprocess.

Sampleresults

The assay determination of Dic and its impurities in the pharmaceuticaldosagesformsusedtocheckthespeciﬁcity showed that content of Dic in all samples comply with standard limits i.e ±5%. However differences in impurity proﬁleswereobservedinthesesamples(resultsnotshown).

The developedUPLCmethodwassuccessfullyvalidated inaccordancetotheICHguidelinesalongwithitsapplica- tion in the routine quality control assay of marketed Dic formulationsliketablets,capsules,solutionsforinjection, suppositoriesandgels.

Conclusion

TheproposedUPLCmethodpresentedinthispaperisdevel- opedforquantificationDicanditssevenpotentialimpurities in API and five pharmaceutical dosage forms. This is the firststability-indicatingassayfordetectingthedimerimpu- rity.The methodwassuccessfullyvalidatedforspecificity, precision,LODandLOQ,linearity,accuracyandrobustness toensurecompliancewithICHguideline.Themethodwas provedtobestabilityindicatingbyconductingforceddegra- dationstudyandachievingdesiredseparationofallanalytes in the presence of preservatives and impuritypeaks. The resultsofpeakpurityindicatethattheproposedmethodis suitableforestimationofDicinthepresenceofitsimpuri- ties.

The proposed method wassuccessfully appliedfor Dic withitsimpuritiesestimationinmarketedtablets,capsules, gels,suppositoriesandinjectionformulations.Itallowsthe analysis to be used for routine testing in pharmaceutical

industriestomonitortheimpuritiesofDicinvariousphar- maceuticaldosageforms.

Acknowledgements

The authorsthank theLNCM for sponsoring thiswork and alsoallanalyticalscientistsinLNCMfortheirsupportofthis study

Disclosure of interest

Theauthorsdeclarethattheyhavenocompetinginterest.

References

[1]GanTJ.Diclofenac:anupdateonitsmechanismofactionand safetyproﬁle.CurrMedResOpin2010;26:1715—31.

[2]Gostick IN, Khong James G, et al. Controlled-release indomethacin and sustained-release diclofenac sodium in the treatment of osteoarthritis — a comparative controlled clinical-trial ingeneral-practice. Curr Med ResOpin 1990;12:135—42.

[3]Nair B, Taylor-Gjevre R. A review of topical diclofenac use in musculoskeletal disease. Pharmaceuticals (Basel) 2010;3(6):1892—908.

[4]ShahY,JoshiS,JindalKC,KhannaS.Highperformanceliquid chromatographic determination of diclofenac diethylammo- niumingels.DrugDevIndPharm1993;20:1303—7.

[5]GonzalezL,YulnG,VolonteMG.Determinationofcyanocobal- amin,betamethasone,anddiclofenacsodiuminpharmaceuti- calformulationsbyhighperformanceliquidchromatography.

JPharmBiomedAnal1999;20:487—92.

[6]Klimes J, Sochor J, Dolez P, Korner J. HPLC evaluation of diclofenac in transdermal therapeutic preparations. Int J Pharm2001;217:153—60.

[7]Patil D, Raman B. Simultaneous estimation of dextro- propoxyphen HCl, diclofenac sodium and paracetamol in capsulebyRP-HPLC.IndianDrugs2001;38:36—9.

[8]Subramanian G, Musmade P. A Validation of a RP-HPLC method for simultaneous determination of paracetamol methocarbamol and diclofenac potassium in tablets. IJPSR 2000;66:694—6.

[9]HanysováL, MokryM,Kastner P,KlimesrJ.HPLCevaluation ofdiclofenacinthevariousformsoftherapeuticpreparations.

ChemPap2005;59:103—8.

[10]KasperekR.Determinationofdiclofenacsodiumandpapaver- inehydrochlorideintabletsbyHPLCmethod.ActaPolPharm DrugRes2008;65:403—8.

[11]ChaudharyB,GoyalA,KaushikD.Simultaneousestimationof diclofenacsodiumandrabeprazolebyHPLCmethodincom- bineddosageform.IntJPharmTechRes2009;1:43—5.

[12]PhaleMD,HamrapurkarPD.AvalidatedandsimpliﬁedRP-HPLC ofmetoprolol succinatefrom bulkdrugs. AsianJ ResChem 2009;2:119—21.

[13]Elkady E. Simultaneous determination of diclofenac potas- siumandmethocarbamolinternarymixturewithguaifenesinby reversephaseliquidchromatography.Talanta2010;82:1604—7.

[14]RathnamMV,SinghRR.SimultaneousRP-HPLCdetermination ofcamyloﬁndihydrochlorideanddiclofenacpotassiuminphar- maceuticalpreparations.PharmAnalActa2010;1:1—4.

[15]BirajdarAS,MeyyanathanS,SureshB.ARP-HPLCmethodfor determiınatıonofdiclofenacwithrabeprazoleinsoliddosage form.PharmSciMonit2011;2:171—8.

(11)

368 M.Azougaghetal.

[16]Adhikari K, JanaK, Behera A, MoitraSK. Searchfor simple mobilephasesinrapidLCseparations-analysisofdrugsincom- plexmatrix(diclofenacgelandinjections).IntJPharmPharm Sci2011;3:136—8.

[17]BadgujarMA,PingaleSG,MangaonkarKV.Simultaneousdeter- minationofparacetamol,chlorzoxazoneanddiclofenacsodium intabletdosageformbyhighperformanceliquidchromatog- raphy.JChem2011;8:1206—11.

[18]Rele RV, Parab JM, Mhatre VV, Warkar CB. Simultaneous RP-HPLC determination of diclofenac potassium and famotidine inpharmaceutical preparations. ResJPharm Technol 2011;4:638—41.

[19]UmarkarAR,RewatkarNS.RP-HPLCmethoddevelopmentand validationforestimationofthiocolchicosideandpotassiumin bulkandcapsuledosageforms.JPharmRes2011;4:1307—8.

[20]Chaudhary H,AminKKS, AroraS,KumarV,RatheeS,Rathe P.DevelopmentandvalidationofRP-HPLCmethodforsimul- taneousestimationofdiclofenacdiethylamineandcurcumin in transdermal gels. J Liquid Chromatogr Relat Technol 2012;35:174—87.

[21]Charde MS, Wanare M, Welankiwar AS, Kumar J, Chakole RD. Developoment of validated stability indicating assay methodforsimultaneousestimationofdiclofenacsodiumand misoprostol in their combined dosage form. IJAPA 2014;4:

12—7.

[22]Aurora-Prado MS, Steppe M, Tavares MFM, Kedor-Hackmann ERM, Santoro MIRM. Comparison between capillary elec- trophoresisandliquidchromatographyforthedetermination ofdiclofenacsodiumina pharmaceuticaltablet.JAOACInt 2002;85(2):330—40.

[23]Vignaduzzo SE, Castellano PM, KaufmanTS. Experimentally designed, validated HPLC simultaneous determination of pridinolanddiclofenacintheircombinedpharmaceuticalfor- mulations,whichallowslimitingdiclofenacrelatedcompound A.JLiquidChromatogrRelatTechnol2010;33:720—1732.

[24]PandaSS,PatanaikD,KumarR. NewstabilityindicatingRP- HPLCmethodfordeterminationofdiclofenacpotassiumand metaxalone from their combined dosage form. Sci Pharm 2012;80:127—37.

[25]AgrawalYK,ShivramchandraK.Spectrophotometricdetermi- nationofdiclofenacsodiumintablets.JPharmBiomedAnal 1991;9:97—100.

[26]ShakyaAK,JoshiGK, MishraP.Spectrophotometricdetermi- nationofdiclofenacsodiuminpharmaceuticaldosageforms.

IndianJPharmSci1992;54:44—6.

[27]Olakunle SI, Olajire AA, Bolaji AO, Ajibola AO. Colorimet- ric assaymethod fordiclofenac intablets.Pak JPharm Sci 2006;19:134—41.

[28]Kumar RS, Karthikeyan C, Moorthy N, Trivedi P. New spectrophotometric methods applied to the simultaneous determinationofdiclofenacpotassiumandtizanidine.IndianJ PharmSci2006;68:317—22.

[29]ChapleDR,MehtaSA,YeoleMP,TartePS.Simultaneousestima- tionoffamotidineanddiclofenacpotassiumincombinedtablet dosageformbymulticomponentmodeofanalysis.PharmaRev 2009;2:119—22.

[30]Mehta SA, Umarkar AR, Chaple DR,Thote LT. Development ofUVspectrophotometricmethodsfor simultaneousestima- tion of famotidine and diclofenac potassium in combined dosageformusingsimultaneousequationmethod.JPharmRes 2011;4:2045—6.

[31]Vanparia DJ, ShahSA, MaroliaBP, BodiwalaKB, PatadiaRK.

Spectrophotometricmethodsfor simultaneous estimationof thiocolchicosideanddiclofenacpotassiumintheircombined dosageform.AsianJResChem2011;4:123—7.

[32]Revathi G, Rama RN, Venkata SP. Simultaneous UV- spectrophotometricdeterminationandvalidationofdiclofenac

sodiumandrabeprazolesodiumusinghydrotropicagentsinits tabletdosageform.IntJDrugDevRes2012;4:316—24.

[33]Prado MSA, Steppe M, Tavares MM, Kedor-Hackmann EM, SantoroMI.Methodvalidationfordiclofenacsodiuminpharma- ceuticalbycapillaryelectrophoresis.JCapillaryElectrophor 1999;6:125—9.

[34]JinWR,ZhangJ.Determinationofdiclofenacsodiumbycap- illaryzoneelectrophoresiswithelectrochemicaldetection.J ChromatogrA2000;868:101—7.

[35]RathnamMV,SinghMV.DevelopmentandvalidationofaHPTLC methodforsimultaneousestimationofdrotaverinehydrochlo- rideanddiclofenacpotassiumincombineddosageform.Pharm AnalActa2008;1:1—4.

[36]Khatal LD, Kamble AY. Validated HPTLC method for simul- taneousquantitation of paracetamol,diclofenac potassium, and famotidine in tablet formulation. J AOAC Int2010;93:

765—70.

[37]Dhaneshwar SR, Bhusar VK. Validated HPTLC method for simultaneousestimationofdiclofenacsodiumandmisopros- tol in bulk drug and formulation. Asian J Pharm Biol Res 2011;1:15—21.

[38]Gorog S. Drug safety, drug quality, drug analysis. J Pharm BiomedAnal2008;48:247—53.

[39]AhujaS.Assuringqualityofdrugsbymonitoringimpurities.Adv DrugDelivRev2007;59:3—11.

[40]BeaulieuN,LoveringEG,LefrancoisJ,OngH.Determination ofdiclofenacsodiumandrelatedcompoundsinrawmaterials andformulations.JAOAC1990;73:698—701.

[41]KubalaT,GambhirB,Borst SI. Aspeciﬁcstability indicating HPLCmethodtodeterminediclofenacsodiuminrawmaterials andpharmaceuticalsoliddosageforms.DrugDevIndPharm 1993;19:749—57.

[42]Hao Y, Mei Z. RP-HPLC simultaneouly determination of diclofenacsodium,misoprostol and itsrelatedimpurities in compoundmisoprostoltablet.ChinPharmJ2000;35:192—3.

[43]RoyJ,IslamM,KhanAH,DasSC,AkhteruzzamanM,DebAK, etal.Diclofenacsodiuminjectionsterilizedbyautoclaveand theoccurrenceofcyclicreactionproducingasmallamountof impurity.JPharmSci2001;90:541—4.

[44]HájkováR,SolichP,PospísilováM,SíchaJ.Simultaneousdeter- minationofmethylparaben,propylparaben,sodiumdiclofenac anditsdegradationproductinatopicalemugelbyreversed- phaseliquidchromatography.AnalChimActa2002;467:91—6.

[45]TehraniMH,FarniaF,EmamiM.Determinationofsyntheticpre- cursorsasimpuritiesindiclofenacsodiumrawmaterial.Iranian JPharmRes2002;1:51—3.

[46]Shaﬁee A, Amini M, Hajmahmodi M. Improved chromatographic method for determination of sodium diclofenac in injectablesolutionandpredictionofchemicalstability.JSci 2003;14:21—5.

[47]GaudianoMC,ValvoL, BertocchiP,MannaL.RP-HPLCstudy ofthedegradationofdiclofenacand piroxicaminthepres- ence of hydroxyl radicals. J Pharm Biomed Anal 2003;32:

151—8.

[48]Mulgund SV, Phoujdar MS, Londhe SV, Mallade PS, Kulkarni TS, Deshpande AS, et al. Stability indicating HPLC method forsimultaneousdeterminationofmephenesinanddiclofenac diethylamine.IndianJPharmSci2009;71:35—40.

[49]Korodi T, Bukowsk K, Lachmann B. Evaluation of a short stability-indicatingHPLCmethodfordiclofenacsodiumgels.

Pharmazie2012;67:980—3.

[50]KrzekJ,StarekM.Densitometricdeterminationofdiclofenac- (2,6-dichlorophenyl)indolin-2-oneandindolin-2-oneinparma- ceuticalpreparations and modelsolutions. JPharm Biomed Anal2002;28:227—43.

[51]EuropeanPharmacopoeia.8.2Ed;2014.

[52]UnitedStatesPharmacopoeia.38thEd;2014.

(12)

[53] BritishPharmacopoeia;2014.

[54] JapanesePharmacopoeia.XIVthEd;2011.

[55] Novakova L, Solichova D, Solich P. Advantages of ultra performance liquid chromatography over high-performance liquid chromatography: comparison of different analytical approaches during analysis of diclofenac gel. J Sep Sci 2006;29:2433—43.

[56] ElzayatEM,IbrahimMF,Abdel-RahmanAA,AhmedSM,Alanazi FK,HabibWA.Avalidatedstability-indicatingUPLCmethodfor determinationofdiclofenacsodiuminitspureformandmatrix formulations.ArabJChem2014[inpress].

[57]InternationalConferenceonHarmonization(ICH)oftechnical requirements for registration of pharmaceuticalsfor human use.Stability,Q1A.

[58]InternationalConferenceonHarmonization(ICH)oftechnical requirements for registration of pharmaceuticalsfor human use.Validationofanalyticalprocedures:textandmethodology, Q2(R1).

[59]ISO5725-2Accuracy(truenessandprecision)ofmeasurement methodsand results—–Part 2:basicmethod forthe determination of repeatability and reproducibility of a standard measurementmethod;1994.