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JournalofPharmaceuticalandBiomedicalAnalysis52(2010)645–651ContentslistsavailableatScienceDirectJournalofPharmaceuticalandBiomedicalAnalysisjournalhomepage:ficationofinternalstandardsforquantificationby1HNMRspectroscopyTorgnyRundlöfa,∗,MarieMathiassona,SomerBekiroglub,1,BirgitHakkarainena,b,TimBowdenc,TorbjörnArvidssona,baMedicalProductsAgency,P.O.Box26,SE-75103Uppsala,SwedenbDivisionofAnalyticalPharmaceuticalChemistry,DepartmentofMedicinalChemistry,UppsalaUniversity,BiomedicalCentre,Box574,SE-75123,Uppsala,SwedencDivisionofPolymerChemistry,DepartmentofMaterialsChemistry,UppsalaUniversity,ÅngströmLaboratory,Box538,SE-75121Uppsala,SwedenarticleinfoArticlehistory:Received23November2009Receivedinrevisedform29January2010Accepted4February2010Availableonline11February2010Keywords:NuclearmagneticresonancespectroscopyQuantificationInternalreferencestandardAbsolutepurityabstractInquantitativeNMR(qNMR)selectionofanappropriateinternalstandardprovestobecrucial.Inthisstudy,25candidatecompoundsconsideredtobepotentinternalstandardswereinvestigatedwithrespecttotheabilityofprovidinguniquesignalchemicalshifts,purity,solubility,andeaseofuse.The1Hchemicalshift(ı)values,assignments,multiplicitiesandnumberofprotons(foreachsignal),appropri-ateness(astobeusedasinternalstandards)infourdifferentdeuteratedsolvents(D2O,DMSO-d6,CD3OD,CDCl3)werestudied.Takingintoaccountthepropertiesofthese25internalstandards,themostversatileeightcompounds(2,4,6-triiodophenol,1,3,5-trichloro-2-nitrobenzene,3,4,5-trichloropyridine,dimethylterephthalate,1,4-dinitrobenzene,2,3,5-triiodobenzoicacid,maleicacidandfumaricacid)werequali-fiedusingbothdifferentialscanningcalorimetry(DSC)andNMRspectroscopyemployinghighlypureacetanilideasthereferencestandard.Thedatafromthesetwomethodswerecomparedaswellasutilizedinthequalityassessmentofthecompoundsasinternalstandards.Finally,theselectedinternalstandardsweretestedandevaluatedinarealcaseofquantitativeNMRanalysisofaparacetamolpharmaceuticalproduct.©2010ElsevierB.V.Allrightsreserved.1.IntroductionQuantitativeNMR(qNMR)wasintroducedalreadymorethan50yearsagoandthefirststudyonpharmaceuticalswaspublishedin1963[1,2].ThestateoftheartofqNMRinthefieldofpharmaceu-ticalsandrelatedareaswasreviewed[3–6]andduringtherecentyearsscientificpublicationsemergedatanescalatingrate.AnadvantageofqNMRcomparedtootherinstrumentalanalyt-icalmethodsisthatitisaprimaryratiomethodofmeasurement,sincethepeakareasareproportionaltothenumberofcorre-spondingnucleigivingrisetothesignals[7–9].TheuncertaintyinquantificationmeasurementbyNMRspectroscopyislow[10,11]andtheperformanceofqNMRissimilartothatofthealternativeanalyticaltechniques[12].Comparedtothetraditionalchromato-graphicmethodsthatarestillfavoredinroutinequantitativeanalyses,NMRspectroscopyhavecertainadvantages[10]suchas∗Correspondingauthorat:MedicalProductsAgency,P.O.Box26,SE-75103Upp-sala,Sweden.Tel.:[phone];fax:[phone].E-mailaddress:[e-mail](T.Rundlöf).1Presentaddress:TUBITAKMarmaraResearchCenter,FoodInstitute,P.O.Box21,41470Gebze/Kocaeli,Turkey.(i)simpleandeasysamplepreparation,(ii)possibilitytosimulta-neouslydeterminemolecularstructures,(iii)noneedforindividualexperimentalsetup,e.g.,referenceofthesamecompoundandcal-ibrations,(iv)relativelyshortmeasuringtimes,(v)non-invasiveandnon-destructivecharacterofthemethod,(vi)noneedforpriorisolationoftheanalytepresentinamixture,(vii)possibilityofsimultaneousquantitativeanalysisofmultipletargetanalytesinamixture.TheapplicabilityofqNMRhasincreasedduetoavailabilityofhighsensitivity/homogeneityNMRsystemstogetherwithmodernsoftwarepackageswhichallowaccurateandprecisedataprocess-ing.InqNMRareferencestandardoftheanalyteisnotneeded,i.e.,thequantificationmaybeperformedusinganinternalstan-dard[1,5].VariousinternalstandardshavebeenusedinqNMR[6],usuallyco-dissolvedwiththeanalyte,butalsointroducedinaseparatecoaxialinserttube[13].Inrecenttimealsoquantifica-tiontowardsanelectronicallygeneratedsignal(peak)intheNMRspectrum,so-calledEretic,wasevolved[14].However,adescrip-tiveandcomparativestudyoninternalstandardsisnon-existentintheliterature.Thescopeofthepresentworkwastostudycompoundsthatcanbeusedasinternalstandardsusing1HNMRexperiments,since1HnucleiareatthecoreofqNMRapplicationsduetohighsensitivity0731-7085/$–seefrontmatter©2010ElsevierB.V.Allrightsreserved.doi:10.1016/j.jpba.2010.02.007646T.Rundlöfetal./JournalofPharmaceuticalandBiomedicalAnalysis52(2010)645–651Chart1.andwidespreadpresenceinorganicmolecules,eventhoughotherNMRactivenucleilike13C,19F,and31Pcanbeemployed[1].InqNMRthesignalfromtheinternalstandardandthesignalfromtheanalytemustnotoverlap,andintheselectionofcompoundsinthisstudyattentionwasattemptedsoastoavoidnormallycrowdedspectralregions.Inthepresentwork25candidateqNMRinternalstandardswerethoroughlyinvestigated.Chemicalshift(ı)values,assignments,multiplicitiesandnumberofprotons(foreachsignal),appropri-ateness(astobeusedasinternalstandards)of25compoundsinfourdifferentdeuteratedsolvents(D2O,DMSO-d6,CD3OD,CDCl3)wer