HPLC for Analysis Paracetamol

8/8/2019 HPLC for Analysis Paracetamol

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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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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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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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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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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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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.

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HPLC for Analysis Paracetamol

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