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TurkJChem26(2002),521{528.
cTUB_ITAK
HPLCMethodfortheAnalysisofParacetamol,CaeineandDipyrone
M.LeventALTUNDepartmentofPharmacognosy,FacultyofPharmacy,AnkaraUniversity,06100
Ankara-TURKEY
Received27.06.2001
Anaccurate,simple,reproducibleandsensitivemethodforthedeterminationofparacetamoanddipyronewasdevelopedandvalidated.Paracetamol,caeineanddipyronewereseparated
-BondapackC8columnbyisocraticelutionwithaflowrateof1.0ml/min.Themobilephasecompo
sitionwas0.01MKH2PO4--methanol-acetonitrile-isopropylalcohol(420:20:30:30)(v/v/v
spectrophotometricdetectionwascarriedoutat215nm.Thelinearrangeofdeterminationcaeineanddipyronewere0.409-400g/ml,0.151-200g/mland0.233-600g/ml,respectively.Themethodwasshowntobelinear,reproducible,speci
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c,sensitiveandrugged.
KeyWords:Paracetamol,Acetaminophen,Caeine,Dipyrone,HighperformanceliquidchromatogValidation.
Introduction
Paracetamol(acetaminophen)isoneofthemostpopularover-the-counteranalgesicandantiParacetamolisavailableindierentdosageforms:tablet,capsules,drops,elixirs,suspentories.Dosageformsofparacetamolanditscombinationswithotherdrugshavebeenlistedinvarpharmacopoeias1;2.
Thecombinationofparacetamolwithdipyroneisusedasanantipyretic,analgesicandantidrug.
Numerousmethodshavebeenreportedfortheanalysisofparacetamolanditscombinationsi
orinbiologicalfluids.Paracetamolhasbeendeterminedincombinationwithotherdrugstitrimetry3;4,voltammetry5,fluorimetry6,colorimetry6,UV-spectrophotometry7-9,quantitativethin-layerchromatography(TLC)10,high-performanceliquidchromatography(HPLC)11-16andgaschromatography
(GC)17inpharmaceuticalpreparations.
Dipyroneisananalgesic,antipyretic,andantiinflammatorydrug.Afteroraladministratioedto4-methylaminoantipyrineandfurthermetabolizedto4-aminoantipyrine,4-formylaminoantine,
and4-acetylaminoantipyrine18.
Dipyrone(noramidopyrinemethanesulfonatesodium,metamizole)isawater-solublepyrazolontivedrugavailableinoralandparenteralforms.Formorethan60yearsithasbeenusedasa
521
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HPLCMethodfortheAnalysisofParacetamol,CaeineandDipyrone,M.L.ALTUN
antipyretic,antispasmodic,andantiinflammatorydrug.Althoughthedrugiswidelyusedin
inothersithasbeenrestrictedorbannedbecauseoftheallegedrisksofadversereactiagranulocytosis18.
Dipyronehasbeendeterminedbytitrimetric19,UV-spectrophotometric8;9;20,colorimetric21rographic22,TLC,HPTLC20,gasliquidchromatography(GLC)23andHPLC24methods.
Thesimultaneousdeterminationofparacetamolanddipyroneintabletsbyderivativespectrwasreportedintheliterature25.
AlthoughthereareUV9andPLS(PartialLeast-squaresSpectrophotometric)26methodsforthedeterminationofparacetamol,dipyroneandcaeineintheirternarymixture.AsuitableHPLCmethodtodeterminetheternarymixtureofparacetamol,dipyroneandcaeinewasnotlocatedinthel
Theobjectiveofthisstudywastodevelopandvalidateaspeci
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c,accurate,preciseandreproduciblequalitycontrolmethodforparacetamol,caeineanddipyroneintheirternarycombination.
Experimental
Chemicals
Paracetamol(acetaminophen)wasusedfromUSPreferencestandard(103-90-2),caeinewasobMerckChemicals(Merck-2584)anddipyronewasreceivedfromUnitedPharmaceuticalWorks.Cicgradedouble-distilledwater,analyticalgradeKH2PO4(Merck-104871),HPLC-gradeacetonitrile(Merck-100030),methanol(Riedel-deHaen-34860)andisopropylalcohol(CarloErbareagentiereused.
Apparatus
ThemethoddevelopmentwasperformedwithaLCsystemconsistingofaWatersmodel515solsystem,aWatersmodel996Photodiode-arraydetector(MILFORD,MA,USA)andaWaters717pautosamplerusinga20-lsampleloop.ThesystemwascontrolledanddataanalyseswerepertheMillennium2010software.TheassayswereperformedwithanotherLCsystemconsistingmodelPU-980pumpandJASCOUV-975UV/VISdetector.Sampleswereinjectedwitha7725Rheoinjectorsystemwitha20lsampleloop.Thedetectorwassetat215nm(0.02a.u.f.s)andintegratedautomaticallybycomputerusingtheBorwinsoftwareprogram.
Separationwascarriedoutatambienttemperatureusinga-BondapakC8column(5m,250mmx
4.6mmI.D.;Waters,Milford,MA,USA).Aguardcolumn(10mBondapakC18indisposableplasticinsertsandWatersGuard-Pakholder)wasusedtosafeguardtheanalyticalcolumn.Alltheconcerningthequantitativeanalysiswereperformedwithexternalstandardizationbythempeakareas.StockandStandardSolutions
Paracetamol(20.00mg),caeine(10.00mg)anddipyrone(15.00mg)wereaccuratelyweighedvolumetricflaskanddissolvedinthemobilephaseand
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lleduptovolumewiththemobilephase.
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HPLCMethodfortheAnalysisofParacetamol,CaeineandDipyrone,M.L.ALTUN
Standard
WorkingSolution
Standardworkingsolutionswerepreparedindividuallyinmobilephaseforparacetamol,caeAliquotsfromeachworkingsolutionwerecombinedanddilutedwithmobilephasetoyieldwith
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nalconcentrationsof100g/ml,50g/mland150g/ml.Studiesonthestabilityoftheanalinstandardworkingsolutionshowedthattherewerenodecompositionproductsinthechromdierenceinareasduringanalyticalprocedure,evenafterstorageforfourdaysat+4C.
Procedure
ChromatographicConditions
HPLCanalysiswasperformedbyisocraticelutionwithaflowrateof1.0ml/min.Themobilcompositionwas0.01MKH2PO4-methanol-acetonitrile-isopropylalcohol(420:20:30:30)(vsolventswere
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lteredthrougha0.45mmillipore
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lterbeforeuseanddegassedinanultrasonicbath.Volumesof10lpreparedsolutionsandsampleswereinjectedintothecolumn.Quanti
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cationwaseectedbymeasuringat215nmasestablishedfromthethree-dimensionalchromatogram.Thechromat
runtimewas10minandthecolumnvoidvolumewas1.735min.
Throughoutthestudy,thesuitabilityofthechromatographicsystemwasmonitoredbycalcuthecapacityfactor(k0),theresolution(R),theselectivity()andpeakasymmetry(T).
Calibration
Mixedstandardsolutionscontainingparacetamol(25-400g/ml),caeine(12.5-200g/ml),and(37.5-600g/ml)werepreparedinthemobilephase.
Triplicate10linjectionsweremadeforeachstandardsolutiontoseethereproducibilityresponseateachconcentrationlevel.Thepeakareaofeachdrugwasplottedagainstthetoobtainthecalibrationgraph.The
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veconcentrationsofeachcompoundweresubjectedtoregressionanalysistocalculatethecalibrationequationandcorrelationcoecients.
ResultsandDiscussion
MethodDevelopment
Themobilephasewaschosenafterseveraltrialswithmethanol,isopropylalcohol,acetoniandbuersolutionsinvariousproportionsandatdierentpHvalues.Amobilephaseconsist
0.01MKH2PO4-methanol-acetonitrile-isopropylalcohol(420:20:30:30)(v/v/v/v)wasseleemaximumseparationandsensitivity.Flowratesbetween0.5and1.5/minwerestudied.Aflowrateof1.0ml/mingaveanoptimal
tonoiseratiowithareasonableseparationtime.Usingareversed-phaseC8column,theretentiontimesforparacetamol,caeineanddipyronewereobservedtobe4.880min,5.845minand8.093minreTotaltimeofanalysiswaslessthan9min.
Themaximumabsorptionofparacetamol,caeineanddipyronetogetherasdetectedat215nmthiswavelengthwaschosenfortheanalysis.Thechromatogramat215nmshowedacompleteresolutionofallpeaks(Figure).
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HPLCMethodfortheAnalysisofParacetamol,CaeineandDipyrone,M.L.ALTUN
AU
0.500
0.400
0.300
0.200
0.100
0.000
(I) (II)(III)4.8185.7527.8852.00 4.00 6.00 8.00minutesFigureChromatogramofthemixtureofparacetamol(I),caeine(II)anddipyrone(III)develmethod
Linearity
Table1presentstheequationoftheregressionline,correlationcoecient(r2),relative(RSD)valuesoftheslopeandinterceptforeachcompound.Excellentlinearitywasobtainebetweenthepeakareasandconcentrationsof25-400g/mlwithr2=0.9987,12.5-200g/mlwithr2=0.9999and37.5-600g/mlwithr2=0.9993forparacetamol,caeineanddipyrone,respectively.
Table1.LinearityResults,LimitofDetection(LOD)andLimitofQuanti
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cation(LOQ)
Compound.Equationr2Slope(RSD%)
Intercept(RSD%)LOQ
g/mLLOD
g/mLParacetamol215Y=20491.74X+150586.10.99870.1922.0311.2260.409Caeine215Y=
53717.01X+197602.20.99990.1501.8680.5020.151Dipyrone215Y=19043.82X+111528.50.99930.0582.9140.7760.233
X=Concentration(g/mL);Y=Area
LimitsofDetectionand
Quanti
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cation
Limitsofdetection(LOD)wereestablishedatasignal-to-noiseratio(S/N)of3.Limitso
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cation(LOQ)wereestablishedatasignal-to-noiseratio(S/N)of9.LODandLOQwereexperimenta
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edbysixinjectionsofparacetamol,caeineanddipyroneattheLODandLOQconcentrations.Tcalculatedtobe0.409,0.151and0.233g/mlandtheLOQwascalculatedtobe1.226,0.502
g/mlforparacetamol,caeineanddipyrone,respectively(Table1).
Suitabilityof
theMethod
Thechromatographicparameterssuchasresolution,selectivityandpeakasymmetryweresatthesecompounds(Table2).Thecalculatedresolutionvaluesbetweeneachpeak-pairwerenoandtheselectivitywasnotlessthan1.30.
k.valueswerefoundtobe1.81,2.37and3.66forparacetamol,caeineanddipyrone,respecti
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HPLCMethodfortheAnalysisofParacetamol,CaeineandDipyrone,M.L.ALTUN
Table2.SystemPerformanceParametersofParacetamol,CaeineandDipyrone
Compoundtr(n=9,mean)Area(n=9,mean)k.R.TParacetamol4.880(0.33)*2308090.67(0.56)*
1.813.628(0.64)*1.307(0.24)*1.179Cafeine5.8452853026.332.371.154(0.47)*(0.47)*6.4331.547Dipyrone8.093(0.47)*3062225.89(0.46)*3.66(0.77)*(0.16)*1.505
*RSD%valuesaregiveninparenthesesRSD%=(StandardDeviation/Mean)x100
Precision
Theprecisionofthemethod(within-dayvariationsofreplicatedeterminations)wascheckeparacetamol,caeine,dipyrone9timesattheLOQlevel.Theprecisionofthemethod,expreRSD%attheLOQlevel,was2.02,2.51and5.54%forparacetamol,caeineanddipyrone,res(Table3).
Table3.PrecisionoftheDevelopedMethodattheLOQlevel(n=9)
Compound.PeakArea(mean)RSD%Paracetamol21524179.442.02Caeine21525541.222.51Dipyrone21518617.505.54
Accuracy
Astandardworkingsolutioncontainingparacetamol,caeineanddipyrone,yielding
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nalconcentrationsof100g/ml,50g/ml,and150g/mlrespectivelywasprepared.Thepreparedmixtureofstandarinjected9timesasatestsample.Fromtherespectiveareacounts,theconcentrationsofcaeineanddipyronewerecalculatedusingthedetectorresponses.Theaccuracy,de
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nedintermsof%deviationofthecalculatedconcentrationsfromtheactualconcentrations,islistedinTa
Table4.AccuracyoftheDevelopedMethod(n=9)
CompoundSpiked
Concentrationg/mLMeasuredConcentration
g/mLMeanSDRSD%Deviation%Paracetamol100105.28
0.6300.6005.28Caeine5049.430.2490.5041.14Dipyrone150154.940.7420.4793.29
(SpikedConcentration-MeanMeasuredConcentration)
%Deviation=100
SpikedConcentration
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HPLCMethodfortheAnalysisofParacetamol,CaeineandDipyrone,M.L.ALTUN
Ruggedness
TheruggednessoftheHPLCmethodwasevaluatedbycarryingouttheanalysisusingastandsolution,thesamechromatographicsystemandthesamecolumnondierentdays.Thepreparestandardswasinjected9timesasatestsample.Smalldierencesinareasandgoodconstantimeswereobservedafter96hours.AnRSDoflessthan0.560%forareasand0.472%forrewereobtained(Tables5and6).Thecomparabledetectorresponsesobtainedondierentdaysthemethodiscapableofproducingresultswithhighprecisionondierentdays.
Similarly,theruggednessofthemethodwastestedbyinjectingthestandardworkingsolutdierentHPLCunit.Thehighdegreesofreproducibilityofdetectorresponsesandretentionthatthemethodisfairlyrugged.
Table5.DaytoDayVariabilityAccordingtoAreaa
June16,2000June20,2000ParacetamolCaeineDipyroneParacetamolCaeineDipyroneAreaS.D.2308090.6712921.172853026.3313356.873062225.89
14124.23238184811432.762949559.889166.103020215.897476.78RSD%0.5600.4680.4610.4800.3110.248
aMeanvaluesofninedeterminationsTable6.DaytoDayVariabilityAccordingtoRetentionTimea
June16,2000June20,2000ParacetamolCaeineDipyroneParacetamolCaeineDipyroneRtS.D.4.8800.0165.8450.0278.0930.0385.1220.007
6.4560.0118.934
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0.020RSD%0.3290.4680.4720.1390.1710.221
aMeanvaluesofninedeterminations
Analysisof
SyntheticMixtures
Recoverystudiesinthismethodwereperformedonthesyntheticmixturespreparedbyaddinweighedamountsofthedrugs(Table7).MeanrecoveriesandRSDwerefoundtobe101.62anparacetamol,100.11and3.38%forcaeine100.86and2.88%fordipyrone,respectively.
Table7.RecoveryResultsforParacetamol,CaeineandDipyroneinSyntheticMixturesbyHP
ParacetamolCaeineDipyroneMixtureAddedFoundRecoveryAddedFoundRecoveryAddedFoundRecovery
Nogg%gg%gg%1400400.97100.24200194.2097.10600600.02100.002300302.77100.92150155.87103.91450455.34101.193200190.8495.42100103.55103.55300289.0796.364100105.29105.295049.4398.87150154.94103.2955053.12106.252524.2897.127577.60103.46X=101.62X=100.11X=100.86RSD%=4.28RSD%=3.38RSD%2.88
X=Mean
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HPLCMethodfortheAnalysisofParacetamol,CaeineandDipyrone,M.L.ALTUN
Conclusion
Thedevelopedmethodissuitablefortheidenti
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cationandquanti
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cationoftheternarycombinationof
paracetamol,caeineanddipyrone.AhighpercentageofrecoveryshowsthatthemethodcanbesuccessfullyusedonaroutineTheproposedmethodissimple,sensitive,rapid,speci
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candcouldbeappliedforqualityandstability
monitoringofparacetamol,caeineanddipyronecombinations.
References
1.J.E.F.Reynolds,\MartindaleTheExtraPharmacopoeia",31sted.,pp.27-28,PharmaceuticalPress,London,1996.2.TheUnitedStatesPharmacopoeia,24threvision,pp.17-39,U.S.PharmacopeialConvention,Rockville,MD,2000.3.BritishPharmacopoeiaCD1998,version2,TheStationeryOceLtd.,Norwich,1998.4.EuropeanPharmacopoeia1997,thirdedition,pp.748-749,ConventionontheElaborationof
Parmacopoeia(EuropeanTreatySeriesNo.50),Strasbourg,1996.5.O.V.Lau,S.F.LukandY.M.Cheung,Analyst,114,1047-1051(1989).6.H.A.El-ObeidandA.AAl-Badr,\AnalyticalPro
8/8/2019 HPLC for Analysis Paracetamol
26/29
lesofDrugSubstances",Vol.14,pp.551-596,AmericanPharmaceuticalAssociation,1985.7.H-K.ChanandD.J.W.Grant,IntJ.Pharm.,57,117-124(1989).8.N.ErkandF.Onur,Anal.Lett.,30(6),1201-1210(1997).9.
H.N.Dogan,Pharmazie,51(10),773-774(1996).10.P.Kahela,E.LaineandM.Anttila,DrugDev.Ind.Pharm.,13(2),213-224(1987).Univ.,27(2),93-100(1998).
11.S.Suzen,C.Akay,S.Tartlms,R.S.Erdol,A.OnalandS.Cevheroglu,J.FacultyofPhar12.W.R.Sisco,C.T.Rittenhuse,L.A.EverhartandA.M.McLaughlin,J.Chromatogr.,354,355-313.
M.HossainandJ.W.Ayres,Int.J.Pharm.,133,223-235(1996).14.H.M.Ali,M.M.A.Homeida,J.Ford,C.A.Truman,C.J.C.RobertsandA.A.Badwan,Int.J.P155-159(1988).15.K.H.Yuen,K.K.Peh,Y.L.QuahandK.L.Chan,DrugDev.Ind.Pharm.,23(2),225-228(1997)16.D.D.Orsi,L.Gagliardi,A.BolascoandD.Tonelli,Chromatographia,43(9/10),496-500(1917.J.J.BerghandA.P.Lotter,DrugDev.Ind.Pharm.,10(1),127-136(1984).18.E.Z.Katz,Y.Caraco,L.GranitandM.Levy,Clin.Pharmacol.Ther.,58(2),198-209(199519.
M.K.Srivastava,S.Ahmad,D.SinghandI.C.Shukla,Analyst,110,735-737(1985).20.T.Aburjai,B.I.Amro,K.Aiedeh,M.AbuirjeieandS.Al-Khalil,Pharmazie,55,751-754(221.A.M.HapetteandS.A.Poulet,J.Liq.Chromatogr.,13(2),357-370(1990)
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HPLCMethodfortheAnalysisofParacetamol,CaeineandDipyrone,M.L.ALTUN
22.F.Belal,Electroanalysis,4,589(1992).
23.A.Siou
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andD.Colussi,J.Chromatogr.,146,503-507(1978).24.G.Geisslinger,R.BockerandM.Levy,PharmaceuticalResearch,13(8),1272-1275(1996).25.M.UstunOzgurandS.Sungur,ChimicaActaTurcica,23,119-123(1995).26.A.Bozdogan,A.M.Acar,G.KuntandH.Caglar,Pharmazie,49,457-458(1994).
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