column using polar organic mode tris(3-chloro-4-methylphenylcarbamate)chiral a cellulose on Simultaneous determination chiral and achiral of impurities ofivabradine Journal of Pharmaceutical and Biomedical Analysis

ContentslistsavailableatScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

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

Simultaneous determination of chiral and achiral impurities of ivabradine on a cellulose tris(3-chloro-4-methylphenylcarbamate) chiral column using polar organic mode

Elek Ferencz

, Béla Kovács

, Francisc Boda

,

Mohammadhassan Foroughbakhshfasaei

, Éva Katalin Kelemen

, Gerg ˝o Tóth

, Zoltán-István Szabó

aFacultyofPharmacy,UniversityofMedicine,Pharmacy,SciencesandTechnologyofTarguMures,Gh.Marinescu38,RO-540139,TîrguMures¸,Romania

bGedeonRichterRomaniaS.A.,RO-540306,TîrguMures¸,Romania

cDepartmentofPharmaceuticalChemistry,SemmelweisUniversity,H ˝ogyesE.u.9,Budapest,Hungary

a r t i c l e i n f o

Articlehistory:

Received14June2019

Receivedinrevisedform29August2019 Accepted30August2019

Availableonline30August2019

Keywords:

Chemoselectivity Polarorganicmode Chiralseparation Relatedsubstance HPLC

a b s t r a c t

A high performance liquid chromatographic method was developed for the simultaneous deter- mination of the related substances (R-ivabradine, dehydro-S-ivabradine, N-demethyl-S-ivabradine, ((S)-3,4-dimethoxy-bicyclo[4.2.0]octa-1,3,5-triene-7-yl-methyl)-methyl-amine)and1-(7,8-dimethoxy- 1,3,4,5-tetrahydro-2H-3-benzazepine-2-on-3-yl)-3-chloro-propane) ofthe heart-rate lowering drug, ivabradine. The separation capability of seven different polysaccharide-type chiral columns (Lux Amylose-1,Luxi-Amylose-1,LuxAmylose-2,LuxCellulose-1,LuxCellulose-2,LuxCellulose-3andLux Cellulose-4)wasinvestigatedwithamobilephaseconsistingof0.1%diethylamineinmethanol,2- propanolandacetonitrile.DuringthescrenningexperimentsthebestresultswereobtainedonLux Cellulose-2(basedoncellulosetris(3-chloro-4-methylphenylcarbamate)columnwithmethanolwith anidealcase,wherealltheimpuritieselutedbeforetheS-ivabradinepeak.Chromatographicparameters (flowrate, temperatureandmobilephase constituents)wereoptimized byafull factorialscreen- ingdesign. Using optimizedparameters(LuxCellulose-2columnwith 0.06%(v/v)diethylaminein methanol/acetonitrile98/2(v/v)with0.45mL/minflowrateat12^◦C)baselineseparationswereachieved betweenallcompounds.TheoptimizedmethodwasvalidatedaccordingtotheInternationalCouncilon HarmonizationQ2(R1)guidelineandprovedtobereliable,linear,preciseandaccuratefordetermination ofatleast0.05%forallimpuritiesinS-ivabradinesamples.Methodapplicationwastestedonacommer- cialtabletformulationandprovedtobesuitableforroutinequalitycontrolofbothchiralandachiral relatedsubstancesofS-ivabradine.

©2019TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Ivabradine 3-{3-[((S)-3,4-dimethoxy-bicyclo[4.2.0]octa-1,3,5- triene-7-ylmethyl)-methyl-amino]-propyl}-7,8-dimethoxy- 1,3,4,5-tetrahydro-2H-3-benzazepine-2-one; IVA) is an orally bioavailable,hyperpolarization-activated,cyclicnucleotide-gated

∗ Correspondingauthorat:DepartmentofPharmaceuticalChemistry,Semmel- weisUniversity,H-1092Budapest,H ˝ogyesE.u.9,Hungary.

∗∗ Correspondingauthorat:FacultyofPharmacy,UniversityofMedicine,Phar- macy,SciencesandTechnologyofTarguMures,Gh.Marinescu38,GedeonRichter RomaniaS.A.,RO-540306,TîrguMures¸,Romania.

E-mail addresses: toth.gergo@pharma.semmelweis-univ.hu (G. Tóth), zoltan.szabo@gedeon-richter.ro,zoltan.szabo@umfst.ro(Z.-I.Szabó).

channel inhibitor. It is used for chronic heart failure not fully managedby␤-blockers[1,2].IVAhasasingleasymmetriccarbon atom,resultingin twoopticalisomers.Thedrugismarketedas a single enantiomeric agent; the pharmaceutical formulations containonlytheS-enantiomer,becauseofitsimprovedelectro- physiological selectivity [3,4]. Nevertheless, to date, only one applicationnotefromDaicel Chiraltechnologiesisavailablefor chiral separation of IVA using Chiralpak IG(based on amylose tris(3-chloro-5-methylphenylcarbamate) column [5]. Moreover, thenumberofthedescribedmethodfordeterminationofachiral relatedsubstancesinIVAisalsolimited[6–9].Highperformance liquid chromatography (HPLC) using a chiral stationary phase (CSP)isthegoldenstandardinanalyticalenantioseparationdueto theseveraladvantagesitoffers,suchaseaseofuse,robustnessand

https://doi.org/10.1016/j.jpba.2019.112851

0731-7085/©2019TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.

0/).

CSPsavailableonthemarket,polysaccharide-type CSPsarethe most frequently applied, due to their high enantiorecognition capability for most of the compounds and multimodal nature [10–12].Thesecolumnscanbeoperatedinnormalphase,reversed phase and polar organicmobile phase modes. In polarorganic modeonlypolarorganicsolvents,neatalcohols(methanol,ethanol and2-propanol),neatacetonitrile(ACN)ortheircombinationsare usedasmobilephase.Polarorganicmodehasseveraladvantages, suchasshorterruntimes,highefficiency,andusuallyhighersolu- bilityoftheanalytesinthemobilephase[12–15].Apartfromtheir abilitytodiscriminatebetweenenantiomers,polysaccharide-type CSPsmayalsopresentexcellentchemoselectivityforstructurally relatedcompounds,thereforetheycanbeusedforsimultaneous determinationofchiralandchemicalimpurities[14,16,17].

Allspecifiedimpurities, includingR-enantiomerareofinter- estin the analysis of S-IVA. Therefore, the aim of the present studywastodevelopamethodforthesimultaneousdetermination ofR-IVA,dehydro-S-ivabradine(DHIVA),N-demethyl-S-ivabradine (DMIVA),((S)-3,4-dimethoxy-bicyclo[4.2.0]octa-1,3,5-triene-7-yl- methyl)-methyl-amine (IMP-1) and 1-(7,8-dimethoxy-1,3,4,5- tetrahydro-2H-3-benzazepine-2-on-3-yl)-3-chloro-propane(IMP- 2)(Fig.1)inS-IVAsampleswithalimitofquantificationofatleast 0.05%orbelow.

2. Materialsandmethods

2.1. Materials

S-IVA (as a hydrobromide salt), racemic IVA, N-demethyl-S-ivabradine (DMIVA, 3-{3-[((S)-3,4-dimethoxy- bicyclo[4.2.0]octa-1,3,5-triene-7-yl)-methyl-amino]-propyl}- 7,8-dimethoxy-1,3,4,5-tetrahydro-2H-3-benzazepine-2-one), dehydro-S-ivabradine (DHIVA, 3-{3-[((S)-3,4-dimethoxy- bicyclo[4.2.0]octa-1,3,5-triene-7-ylmethyl)-methyl-amino]- propyl}-7,8-dimethoxy-1,3-dihydro-2H-3-benzazepine-2-one), ((S)-3,4-dimethoxy-bicyclo[4.2.0]octa-1,3,5-

triene-7-yl-methyl)-methyl-amine (IMP-1) and 1-(7,8-dimethoxy-1,3,4,5-tetrahydro-2H-3-benzazepine-2-on- 3-yl)-3-chloro-propane (IMP-2) (Fig. 1) were obtained from a pharmaceutical company in Târgu Mures¸. Diethylamine (DEA)

≥99.5%waspurchasedfromSigma-Aldrich(Budapest,Hungary).

Gradientgrademethanol(MeOH),2-propanol(IPA)andacetoni- trile(ACN)werepurchased fromThomaskerFinechemicals Ltd.

(Budapest,Hungary).

Ultrapure,deionizedwater waspreparedbyaMilli-QDirect 8 Millipore system (Milford, MA, USA). S-IVA 5mgfilm-coated tabletswereobtainedfromCentralPharmacyofSemmelweisUni- versity (Budapest, Hungary). All chiral columns with identical dimensions(4.6x150mm,5␮mparticles)wereorderedfromPhe- nomenex(Torrance,CA,USA):LuxCellulose-1[cellulosetris(3,5- dimethylphenylcarbamate)], Lux Cellulose-2 [cellulose tris(3- chloro-4-methylphenylcarbamate)], Lux Cellulose-3 [cellulose tris(4-methylbenzoate)],LuxCellulose-4[cellulosetris(4-chloro- 3-methylphenylcarbamate)], Lux Amylose-1 [amylose tris(3,5- dimethylphenylcarbamate)],Lux i-Amylose-1 [amylose tris(3,5- dimethylphenylcarbamate)], immobilized and Lux Amylose-2 [amylosetris(5-chloro-2-methylphenylcarbamate)] (Supplemen- taryInformationFigureS1).

2.2. LC-UVanalysis

LC-UVexperimentswascarriedoutonaJASCOHPLCsystem (JASCOPU-2089Plusbinarygradientpump,AS-4050autosampler, MD-2010PlusdiodearraydetectorandCO2065Pluscolumnoven).

wasChromNAV.UVdetectionwasperformedat286nm.MeOHwas usedasasamplesolventforthepreparationofsolutionsthrough- outthestudy.Forthepreliminaryexperiments,stocksolutionsof 200␮g/mLS-IVAwerepreparedinMeOHandwerespikedwith impuritiesataround2%(around4␮g/mL).Thefinaltestsolution ofS-IVAusedforvalidationandmethodapplicabilitytestingwas about8000␮g/ml.Allimpuritylevelpercentagesarereportedrel- ativetothisconcentration.Aninjectionvolumeof5␮Lwasused andthreeparallelmeasurementswereperformedinallcases.

Forpreparationofsamplesolutions,tentabletswereweighted, thengroundand mixedina mortar.In a5mLvolumetric flask, MeOH was added to an accurately weighted portion of the tablet powder corresponding to about 40mg S-IVA. Then the suspensionwas sonicatedfor 30min and centrifugedfor 2min applying4000rpm(Sartorius2–16Pbenchtopcentrifuge,Goettin- gen,Germany).Theclearsupernatantwasfilteredthrough0.22␮m poresizesyringecontainingPVDFfilter(FilterBiomembraneCo., LTD,NantongCity,China).

Theexperimentaldesign andmultivariatemethodoptimiza- tionwasperformedwiththeaidofModde11software(Umetrics, Sweden).

3. Resultsanddiscussion

3.1. Methodscoutingphase

Inroutinequalitycontrolofsingleenantiomericpharmaceutical substances,the“classicalapproach”wouldbetodevelopseparate methodsforthequantificationofachiralandchiralrelatedsub- stances.Methodsthatenablesimultaneousquantificationofboth chiral and achiral impurities arethus highly welcome,because theycansignificantlyshortentheanalysistime.However,inthese cases,thedevelopedmethodsneedtodisplaybothenantioselec- tivity(chiralseparation)andchemoselectivity(achiralseparation) towardstheanalytes.Althoughthiscanalsobeachievedbytandem couplingofchiralandachiralcolumns,usingasinglechiralcolumn ismoredesirable.Thus,forthepresentmethod,basedonregula- toryconsiderations,theanalyticaltargetprofilewasdefinedasa methodthatwouldallowthepreciseandaccuratedetermination ofallselectedimpurities(chiralandchemical)atalevelofatleast 0.05%orbelow,inshortanalysistime(t≤30min).

Recently, wehave successfully appliedpolysaccharide-based CSPsinpolarorganicmodeforthechiralseparationofalargevari- etyofanalytes[18–21],howeverpolysaccharide-typeCSPshave alsoprovedtheircapabilityforsimultaneouschiral-achiralanaly- ses[14,16,17].

Polysaccharide-basedCSPscomeinawidevarietyofcommer- cially available columns and usually display a great variety of enantio-andchemoselectivity.Inthepresentstudy,sevendifferent polysaccharide-basedCSPs,includingamylose-basedLuxAmylose- 1,Luxi-Amylose-1andLuxAmylose-2,aswellascellulose-based LuxCellulose-1,LuxCellulose-2,LuxCellulose-3andLuxCellulose- 4weretestedinpolarorganicmodeusingamobilephaseconsisting of0.1%DEAinMeOH,IPAorACN,with0.5mL/minflow rateat 25^◦.Inourscreeningthecombinationofpolarorganicsolvents wasnotused,howeveritshouldbenotedthatsolventmixturecan changetheenantiorecognitionmechanismandalsocanimprove theresolution.

Duringthescreeningstepinallcases,IMP-1andIMP-2were wellresolvedfromeachotherandthemainpeak,however,dif- ficultieswereobservedduringtheseparationofthestructurally verysimilarDHIVA,DMIVA,R-IVAfromthemainpeakforwhich co-elutionswereoftenobserved.Particularlychallengingwasthe resolutionofIVAenantiomers,whichwasidentifiedasthecritical

Fig.1.Name,chemicalstructureandabbreviationsofthecompoundsusedinthisstudy.

Table1

Summaryofthechromatographicdataobtainedduringthepreliminaryscreeningphase.

Column Mobilephase^* Rs Enantiomerelution

order

tr,min Lastelutedcompound

LuxAmylose-1

MeOH 0.25 R<S 23.1 S-IVA

IPA 1.34 R<S 16.1 S-IVA

ACN 0.24 S<R 19.0 DMIVA

Luxi-Amylose-1

MeOH – – 11.2 IVA

IPA 0.86 R<S 20.3 S-IVA

ACN – – 10.8 IVA

LuxAmylose-2

MeOH 1.06 S<R 18.2 R-IVA

IPA – – 49.3 DMIVA

ACN 1.80 S<R 49.9 DMIVA

LuxCellulose-1

MeOH 2.15 S<R 10.6 R-IVA

IPA 1.64 S<R 19.6 DMIVA

ACN 2.60 S<R 8.9 DMIVA

LuxCellulose-2

MeOH 1.78 R<S 19.9 S-IVA

IPA – – 40.9 DMIVA

ACN 0.20 R<S 39.3 DMIVA

LuxCellulose-3

MeOH 2.73 S<R 9.3 R-IVA

IPA – – 11.7 DMIVA

ACN – – 5.0 DMIVA

LuxCellulose-4

MeOH 0.25 R<S 14.1 S-IVA

IPA – – 35.8 DMIVA

ACN 1.11 R<S 26.4 DMIVA

Rs,resolutionbetweenR-andS-IVA.

tr,retentiontimeofthelastelutedpeak.

*allmobilephasescontained0.1%DEAasbasicmodifier.

-noenantiorecognitioncouldbeobserved.

parameter.Thus,thescreeningwasfocusedmainlyonselectingthe adequatecolumnforbaselineseparationoftheenantiomerswith thedesiredelutionorder(chiralimpurityandR-IVAelutingbefore

theeutomer).Someofthechromatogramsobtainedaredepicted inFig.2,whiletherelevantchromatographicdataarepresentedin Table1.Asitcanbeobserved,baselinechiralseparationbetween

Fig.2.Representativechromatogramsobtainedduringthepreliminarystudy.Chromatographicconditions:(A)LuxCellulose-2with0.1%DEAinMeOH;(B)LuxCellulose-3 with0.1%DEAinMeOH;(C)LuxCellulose-2with0.1%DEAinACN;(D)LuxCellulose-4with0.1%DEAinMeOH(flowrate:0.5mL/min,temperature25^◦C,detectionat 286nm.)1.IMP-1,2.IMP-2,3.DHIVA,4.DMIVA,5.R-IVA,6.S-IVA.

enantiomerswasachieved ontheLuxCellulose-1 columnwith highresolutionvaluesforallthreemobilephases,buttheelution orderoftheenantiomerswasunfavorable.Distomer-firstelution orderwasobservedonLux-i-Amylose-1,LuxCellulose-2andLux Cellulose-4,butthebaselineseparationwasachievedonlyinthe caseofLuxCellulose-2using0.1%DEAinMeOHasmobilephase.

Mobilephasedependentenantiomerelutionorderwasrecorded onLuxAmylose-1column,whenchangingthemaincomponentof themobilephasefromalcoholstoACN.

Sincethe final goalwasto develop a singlemethodfor the separation of all related substances, not only enantioselectiv- ity, but chemoselectivity wasalso important. Thus, for further methodoptimizationthefollowingstarting-point wasselected:

LuxCellulose-2column,with0.1%DEAinMeOHwith0.5mL/min flowrateat25^◦C.Byapplyingthismethod,allinvestigatedrelated substancescouldberesolved.Moreover,notonlytheenantiomeric impurity R-IVA, but allinvestigated impurities eluted beforeS- IVA(Fig.2A).InanattempttogainhigherresolutionMeOHwas changedtoEtOH,howeverusingtheethanolicmobilephaseDMIVA andS-IVAco-eluted,thereforeitwasnotsuitable(Supplementary InformationFigureS2).AdditionofsmallamountsofACNtoMEOH asasecondarysolvent,however,leadtoreducedretentiontimes withoutmodifyingtheresolutions significantly (Supplementary InformationFigureS3).Thus,thisoptionwasalsoevaluatedduring furthermethodoptimization.

3.2. Methodoptimizationbyexperimentaldesign

Conventionalmethodoptimizationisusuallyperformedbythe so-called OFATapproach (one factorat a time),which leadsto amultitudeofexperiments,whilstgainingonlylittleknowledge

abouttheprocessitself.Duetotheseshortcomings,experimental design-based,multivariateoptimizationofanalyticaltechniques havebecomewidespreadinrecentyears.Themainadvantageof thisapproachisthatitoffersmaximumprocessunderstandingwith aminimalnumberofexperiments[22,23].

Thus,amultivariatemethodologywasundertakenforarapid optimizationofnumerousparametersaffectingthesimultaneous separationoftheanalytes.Basedonthepreliminaryexperimental runs,fourcriticalqualityattributeswereselectedandmonitored asresponsesintheexperimentalruns.Theseincluded:R_sbetween DHIVAandDMIVA(abbreviatedR_s1),R_s betweenDMIVAandR- IVA (R_s2), R_s betweenR-IVA and S-IVA (R_s3)and total analysis time(measuredasretentiontimeofS-IVA,t_r,S-IVA).Criticalprocess parameterswereidentifiedbasedonourpreviousexperiencewith polarorganicmodeonpolysaccharide-typeCSPsandariskassess- mentofdifferentfactorsthatcouldinfluencetheperformanceof themethod.Thecriticalprocessparametersselectedasexperimen- talvariablesduringtheDoErunsweredefinedasDEAvolumeratio addedtothemobilephase(DEA%,range0.05%–0.15%),ACN/MeOH volumeratioofthemobilephase(ACN%,0–10%),flowrate(0.3– 0.7mL/min)and columntemperature(T,10–40^◦C).Inourwork DEAwasusedasmodifierhoweverthenatureofthebasicand/or acidicmodifiermaydeeplyinfluencetheseparation[15].

Inordertoobtainthoroughinformationabouttheinfluenceof definedfactorsonselectedresponsesandtherebytoachievethe optimalchromatographicconditions,aimingtofulfilallresponse targetsettings,afullfactorialexperimentaldesignwiththreecen- terpointswasselected.

Thedesignreturnedatotalof19experimentalruns,including threereplicatecenterpointedexperiments.Thesampleconsisted of 8000␮g/mL S-IVA and 80␮g/mL of each impurity (impurity

Table2

Optimizedmethodconditionswithpredictedandexperimentallyobtainedresponsevalues.

Optimizedmethodconditions Rs1 Rs2 Rs3 tr,S-IVA

DEA% ACN% Flowrate

(ml/min)

T(^oC) Predicted values

2.02 (1.85–2.19)

1.58 (1.54–1.62)

2.00 (1.95–2.05)

23.71 (21.98–25.57)

0.06 2 0.45 12 Experimental

obtainedvalues

2.13 1.55 1.98 23.20

levelsat1.0%relative toS-IVA)inMeOH. Allexperimentalruns wereperformedintriplicateandtheaveragevalueswereintro- ducedintheworksheet.Thedesignmatrixandtherecordedvalues forallresponsesaresummarizedinSupplementaryInformation TableS1.Theobtaineddatawasfittedbypartialleastsquares(PLS) regressionandthemodelpassedthereplicativeplotanalysisforall responses(SupplementaryInformationFigureS4),meaningthat replicate errors didnot interferewithdata analysis.Histogram plotsofresponsesindicatednormaldistributionforreponsesR_s1, R_s3,butnotforR_s2andt_r,S-IVA.Forthelattertwo,thehistograms showedstrongpositiveskewness,implyingthatthefrequencyof lowervaluesinthedatasetwasmuchhigher.Inordertoachieve approximatelynormal distribution,logarithmic transformations wereemployed,whichresultedinasubstantialincreaseinmodel predictability (Q²) for both responses. The model was further adjustedbydeletingnon-significantfactorsandaddingdetected squarevaluestoincreasetheQ²values.Thefinalcoefficientplots obtained,withthesignificantfactorsaffectingeachresponseare presentedinSupplementaryInformationFigureS4.Forallofmon- itoredresponses,theexperimentalvariablesACN%,flowrateand Tpresentedanegativecorrelation,indicatingthatanincreasein anyofthesefactorsresultedinadecreaseinallR_svalues,butalso adecreaseint_r,S-IVA.DEA%inmostcaseshadnosignificanteffect upontheresponses,apartfromR_s2where,incomparisontoACN%, flowrateandTalsopresentsasignificantinverseproportionality withtheselectedresponse,althoughbeingtheleastinfluentialof thedefinedfactors.

Furthermodelrefinementimpliedtheappraisalof quadratic termsinsidethemodel.Thepresenceofquadratictermsindicatesa non-linearrelationshipin-betweenfactorsandresponsesandsug- geststheexistenceofcurvatureinsidethemodel.Quadraticterms werenotidentifiedincaseofR_s2andR_s3.RegardingR_s1andt_r,_S-IVA

significantquadratictermswereobservedandincludedintheanal- ysisofmodelperformanceindicators.Astherefinedmodelallows onlyonequadratictermtobeincludedintheregressionequation, thiswasfulfilledbyselectingtheoneforwhichthecorresponding singularfactorhasthegreatestimpactontheselectedresponse.

Hence,incaseofR_s1 andt_r,_S-IVA,ACN%xACN%andFlowxFlow wereretained,respectively.

Inallcases,significantregressionmodelswereobtained(p<

0.05)andapartfromthecaseoft_r,S-IVA,nolackoffitwasdetected (p> 0.05).In thelattercase,thelackof fitwasalsoevidentby thenegativemodelvalidityvalue.Incontrasttothis,modelrepro- ducibilityshowedextremelyhighvalues( ˜0.99),sincethereplicate runsresultedinalmostidenticalretentiontimesofthemainpeak (seeSupplementaryInformationTableS1).Althoughretentiontime reproducibility is desirable and mostly expected in chromato- graphictechniques,thisalsomeans,thatthepureerrorinsidethe modeltendstozeroandthus,thelowmodelvalidityincaseof t_r,_S-IVAcanbeexplainedbyhighreproducibility.Themethodwas subsequentlyoptimizedusingthepredictionspreadsheetfunction ofthesoftware,applyingthefollowingcriteria:allR_svaluesmaxi- mized,withacriticalminimalvalue≤1.5,andt_r,S-IVAminimized.

Theoptimizersetpoint resultsobtainedaftertheperformed experimentsduring screeningis basedona Monte Carlosimu- lation,where parameter settingswere chosen accordingtothe

lowest log(D) value, with DEA=0.058%, ACN=1.68%, flow rate

=0.46mL/minandcolumntemperature=12.0^◦C.Designspacewas evaluatedaroundtheaforementionedparametersettings.The4D designspaceexplorermap(SupplementaryInformationFigureS5) indicatesthatthechromatographicsystemreturnsoptimalresults onlyatlowcolumntemperatureandflowratesettings.Atthelow- estsetpointsofboththeflowrateandcolumntemperature,the proportionofACNisallowedtobeusedinawiderrange,showinga shifttowardstighterspecificationlimitsashighersettingsforboth flowrateandcolumntemperaturearerequired.Thesetpointanaly- sisfunctionofthesoftwarerevealedthatthefrequencyhistograms oftheselectedfactorsanddefinedresponsesfollowanormaldis- tribution(SupplementaryInformationFigureS6andS7),andthe individualprobabilityoffailureforthedefinedresponsesissitu- atedbelow1%,withanoverallprobabilityoffailureof1.7%.Forease ofuse,thefinalproposedmethodusesslightlydifferentparame- tersettings(mostparametersettingsarerounded),whichstilllie inside thedesign spaceand returnsatisfactory resultsin terms ofselectedresponserequirements.Table2showstheoptimized parametersofthechromatographicsystemandtheclosenessof thepredictedandexperimentallyobtainedvaluesforallresponses, usingtherecommendedsettings.Sincetheanalyticaltargetprofile ofthemethodwasfulfilled,furthermethodoptimizationwasnot undertaken.Theparametersofthefinalmethodwerethefollow- ingLuxCellulose-2columnwith0.06%(v/v)DEAinMeOH/ACN98/2 (v/v)with0.45mL/minflowrateat12^◦C.

3.3. Methodvalidationandapplication

Validationoftheoptimizedmethodwasperformedaccording toInternationalCouncilforHarmonizationguidelineQ2(R1)forall relatedsubstancesandforR-IVAaschiralimpurity,withrespect ofsensitivity,linearity,accuracy,andprecision[24].Thelimitof detection(LOD) and thelimitof quantification(LOQ) werecal- culated based onsignal-to-noise ratiosof 3:1and 10:1 for the LOD and LOQ,respectively. (Baseline noise was measuredcon- sideringa peaktopeakwithin3minselectedinthree different partsofthechromatogramofthestandardsolution.)Theobtained valuesaresummarizedinTable3.Basedontheresults,thelin- earityofthemethodwasevaluatedateightconcentrationlevels forallimpuritiesandcalibrationplotswererepresentedbyplot- tingpeakareasagainstcorrespondingconcentrations(expressed in␮g/mL).Thecorrelationcoefficientwasdeterminedbylinear leastsquaresregressionanalysisanditishigherthan0.9972inall cases.Moreover,forallimpurities95%confidenceintervalsofthe y-interceptsincludedzeroandrandomdistributionoftheresiduals wasobserved.

Theaccuracyandprecisionwereanalyzedbyperformingintra- (repeatability)andinter-dayevaluation(twoconsecutivedays)of threeconcentrationlevelsforallimpurities,coveringthelinear- ity range,each solutionbeing injectedfive times.Theaccuracy (expressedinmeanrecovery%)rangedfrom97.52%to104.32%.The repeatability(expressedasRSD%)determinedbyfiveparallelinjec- tionsofthesolutionsonthesamedaywasbetween0.41%and2.29%.

Intermediate precisionof themethod(expressed in RSD%) was investigatedontwoconsecutivedaysandwaslowerthan3.56%.

Assayvalidationdata.

Parameter Level IMP-1 IMP-2 DHIVA DMIVA R-IVA

Range(␮g/mL) Range(%)

2–80 0.025%-1%

2–80 0.025%-1%

2.7–64 0.034%-0.8%

2.7–64 0.034%-0.8%

4–80 0.05%-1%

r² 0.9992 0.9989 0.9980 0.9997 0.9972

LOD(␮g/mL) 0.12 0.12 0.81 0.60 1.20

LOQ(␮g/mL) 0.40 0.40 2.70 2.0 4.0

Accuracy(%)

I^* 98.90±0.90 104.32±1.01 99.69±1.03 103.92±1.45 103.45±1.19

II^** 97.52±1.12 99.55±2.21 102.65±0.99 97.91±0.75 98.94±0.67

III^*** 98.29±0.22 101.81±0.88 103.51±0.75 99.81±0.19 98.62±0.34

Content repeatability(RSD)

I^* 1.95 1.53 2.29 1.50 0.63

II^** 0.68 0.55 1.19 1.02 0.97

III^*** 0.41 0.56 0.55 0.49 0.66

Contentprecision (RSD)

I^* 2.12 1.74 3.56 1.69 1.11

II^** 0.69 0.38 1.26 1.09 1.01

III^*** 0.82 0.49 0.81 1.52 2.27

* LevelI:IMP-1=4␮g/ml,IMP-2=4␮g/ml,DHIVA=4␮g/ml,DMIVA=5.4␮g/ml,R-IVA=8␮g/ml.

** LevelII:IMP-1=16␮g/ml,IMP-2=16␮g/ml,DHIVA=12␮g/ml,DMIVA=10.8␮g/ml,R-IVA=16␮g/ml.

***LevelIII:IMP-1=56␮g/ml,IMP-2=56␮g/ml,DHIVA=40␮g/ml,DMIVA=54␮g/ml,R-IVA=56␮g/ml.

Fig.3.Chromatogramsof(A)Solution ofIvabradine5mgtablet.(B) Solution ofIvabradine 5mgfilm-coated tabletspiked with all impuritiesat the0.05%

level.Experimentalconditions:LuxCellulose-2columnwith0.06%(v/v)DEAin MeOH/ACN98/2(v/v)with0.45mL/minflowrateat12^◦C.1.IMP-1,2.IMP-2,3.

DHIVA,4.DMIVA,5.R-IVA,6.S-IVA.

Basedontheobtainedresultstheoptimizedmethodprovedto besensitive,linear,accurateandpreciseforthedeterminationof fivedifferentimpurities,includingR-IVAaschiralimpurity.

Theoptimizedandvalidatedmethodwasappliedtotheanalysis ofrealsamples,intheformoffilm-coatedtabletswithanomi- nalcontentof5mgS-IVAbase.Therepresentativechromatograms recordedforthesamplesolutionandthesamplesolutionspiked withimpuritiesareshowninFig.3AandB,respectively.OnlyR-IVA ofthemonitoredimpuritiescouldbequantifiedinthecommercial tablet,theotherimpuritieswerebelow(orclose)theLOD.Thecon- tentofR-IVAwas0.032±0.001%.Inadditiontotheknownrelated substances,minorunidentifiedpeakscouldalsobeobservedinthe samplechromatogram,whichcouldbeindicatingunknownimpu- ritiesormaybetheresultsofdifferentexicipientsusedinthetablet formulation.Usingpeaknormalizationat286nm,thesumofthe totalimpuritieswascalculatedas0.14±0.01%.

4. Concludingremarks

Anovel,singleHPLCmethodusingLuxCellulose-2columnin polarorganicmodewasdevelopedusingDoEforthesimultane- ousdeterminationofDHIVA,DMIVA,IMP-1andIMP-2asrelated substances aswellas R-IVA asenantiomeric impurity in S-IVA.

ThemethodwasvalidatedaccordingtotheInternationalCoun- cilforHarmonizationguidelineQ2(R1)andprovedtobeprecise andaccuratefordeterminationofatleast0.05%orbelowforall impuritiesinS-IVAsamples.Applicationofthemethodwastested ona commercialtablet and showedthatthetablet contains R- IVAimpurity,butbelow0.05%.Ourmethodcouldbeappliedinan industrialenvironmentforsimultaneousquantificationofchemi- calandchiralrelatedsubstancesofS-IVAinoneruntosavetime andmoney.Furthermore,thepresentmethodisanotherexample thatrelatedsubstancesandenantiomericimpuritiescanbedeter- minedbyasinglemethodusingpolysaccharide-typeCSPinpolar organicmode.

Acknowledgements

ThisworkwassupportedbytheJánosBolyaiResearchScholar- shipoftheHungarianAcademyofSciencesandbytheSemmelweis Innovation Found STIA-M-17 and STIA-18-KF (to G.Tóth). The financialsupportfromBolyai+NewNationalExcellenceProgram (grant number: UNKP-19-4-SE-28) of the Ministry of Human Capacitiesishighlyappreciated(G.Tóth).

AppendixA. Supplementarydata

Supplementarymaterial relatedto this articlecanbe found, in theonline version, at doi:https://doi.org/10.1016/j.jpba.2019.

112851.

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