HPLC For Analysis Paracetamol

TurkJChem
26(2002),521{528.
¨
cTUB_
ITAK
HPLC
Method
for
the
Analysis
of
Paracetamol,
Caeine
and
Dipyrone
M.
Levent
ALTUN
Department
of
Pharmacognosy,
Faculty
of
Pharmacy,
Ankara
University,
06100
Ankara-TURKEY
Received27.06.2001
Anaccurate,simple,reproducibleandsensitivemethodforthedeterminationofparacetamol
anddipyronewasdevelopedandvalidated.Paracetamol,caeineanddipyronewereseparatedu
-BondapackC8
columnbyisocraticelutionwithaflowrateof1.0ml/min.Themobilephasecompo
sitionwas0.01MKH2PO4--methanol-acetonitrile-isopropylalcohol(420:20:30:30)(v/v/v/
spectrophotometricdetectionwascarriedoutat215nm.Thelinearrangeofdeterminationf
caeineanddipyronewere0.409-400g/ml,0.151-200g/mland0.233-600g/ml,respectively.
Themethodwasshowntobelinear,reproducible,speci
c,sensitiveandrugged.
KeyWords:Paracetamol,Acetaminophen,Caeine,Dipyrone,Highperformanceliquidchromatogr
Validation.
Introduction
Paracetamol(acetaminophen)isoneofthemostpopularover-the-counteranalgesicandantip
Paracetamolisavailableindierentdosageforms:tablet,capsules,drops,elixirs,suspens
tories.
Dosageformsofparacetamolanditscombinationswithotherdrugshavebeenlistedinvari
pharmacopoeias1;2
.
Thecombinationofparacetamolwithdipyroneisusedasanantipyretic,analgesicandantii
drug.
Numerousmethodshavebeenreportedfortheanalysisofparacetamolanditscombinationsin
orinbiologicalfluids.Paracetamolhasbeendeterminedincombinationwithotherdrugsu
titrimetry3;4,voltammetry5,fluorimetry6,colorimetry6,UV-spectrophotometry7-9,quantita
tivethin-layer
chromatography(TLC)10,high-performanceliquidchromatography(HPLC)11-16
andgaschromatography
(GC)17
inpharmaceuticalpreparations.
Dipyroneisananalgesic,antipyretic,andantiinflammatorydrug.Afteroraladministration
ed
to4-methylaminoantipyrineandfurthermetabolizedto4-aminoantipyrine,4-formylaminoantip
ne,
and4-acetylaminoantipyrine18
.
Dipyrone(noramidopyrinemethanesulfonatesodium,metamizole)isawater-solublepyrazolone
tive
drugavailableinoralandparenteralforms.Formorethan60yearsithasbeenusedasan
521
HPLCMethodfortheAnalysisofParacetamol,CaeineandDipyrone,M.
L.
ALTUN
antipyretic,antispasmodic,andantiinflammatorydrug.Althoughthedrugiswidelyusedin
inothersithasbeenrestrictedorbannedbecauseoftheallegedrisksofadversereactio
agranulocytosis18
.
Dipyronehasbeendeterminedbytitrimetric19,UV-spectrophotometric8;9;20,colorimetric21,
rographic22,
TLC,HPTLC20,gasliquidchromatography(GLC)23andHPLC24
methods.
Thesimultaneousdeterminationofparacetamolanddipyroneintabletsbyderivativespectro
wasreportedintheliterature25
.
AlthoughthereareUV9andPLS(PartialLeast-squaresSpectrophotometric)26
methodsforthedetermination
ofparacetamol,dipyroneandcaeineintheirternarymixture.AsuitableHPLCmethodto
determinetheternarymixtureofparacetamol,dipyroneandcaeinewasnotlocatedintheli
Theobjectiveofthisstudywastodevelopandvalidateaspeci
c,accurate,preciseandreproducible
qualitycontrolmethodforparacetamol,caeineanddipyroneintheirternarycombination.
Experimental
Chemicals
Paracetamol(acetaminophen)wasusedfromUSPreferencestandard(103-90-2),caeinewasobt
MerckChemicals(Merck-2584)anddipyronewasreceivedfromUnitedPharmaceuticalWorks.Ch
ic
gradedouble-distilledwater,analyticalgradeKH2PO4
(Merck-104871),HPLC-gradeacetonitrile
(Merck-100030),methanol(Riedel-deHaen-34860)andisopropylalcohol(CarloErbareagenti-
ere
used.
Apparatus
ThemethoddevelopmentwasperformedwithaLCsystemconsistingofaWatersmodel515solv
system,aWatersmodel996Photodiode-arraydetector(MILFORD,MA,USA)andaWaters717pl
autosamplerusinga20-lsampleloop.Thesystemwascontrolledanddataanalyseswereperf
theMillennium2010software.TheassayswereperformedwithanotherLCsystemconsistingo
modelPU-980pumpandJASCOUV-975UV/VISdetector.Sampleswereinjectedwitha7725Rheod
injectorsystemwitha20lsampleloop.Thedetectorwassetat215nm(0.02a.u.f.s)and
integratedautomaticallybycomputerusingtheBorwinsoftwareprogram.
Separationwascarriedoutatambienttemperatureusinga-BondapakC8
column(5m,250mmx
4.6mmI.D.;Waters,Milford,MA,USA).Aguardcolumn(10mBondapakC18
indisposableplastic
insertsandWatersGuard-Pakholder)wasusedtosafeguardtheanalyticalcolumn.Allthec
concerningthequantitativeanalysiswereperformedwithexternalstandardizationbytheme
peakareas.
Stock
and
Standard
Solutions
Paracetamol(20.00mg),caeine(10.00mg)anddipyrone(15.00mg)wereaccuratelyweighedi
volumetricflaskanddissolvedinthemobilephaseand
lleduptovolumewiththemobilephase.
L.
ALTUN
Standard
Working
Solution
Standardworkingsolutionswerepreparedindividuallyinmobilephaseforparacetamol,caei
Aliquotsfromeachworkingsolutionwerecombinedanddilutedwithmobilephasetoyielda
with
nalconcentrationsof100g/ml,50g/mland150g/ml.Studiesonthestabilityoftheanaly
instandardworkingsolutionshowedthattherewerenodecompositionproductsinthechroma
dierenceinareasduringanalyticalprocedure,evenafterstorageforfourdaysat+4C.
Procedure
Chromatographic
Conditions
HPLCanalysiswasperformedbyisocraticelutionwithaflowrateof1.0ml/min.Themobile
compositionwas0.01MKH2PO4-methanol-acetonitrile-isopropylalcohol(420:20:30:30)(v/
solventswere
lteredthrougha0.45mmillipore
lterbeforeuseanddegassedinanultrasonicbath.
Volumesof10lpreparedsolutionsandsampleswereinjectedintothecolumn.Quanti
cationwaseected
bymeasuringat215nmasestablishedfromthethree-dimensionalchromatogram.Thechromato
runtimewas10minandthecolumnvoidvolumewas1.735min.
Throughoutthestudy,thesuitabilityofthechromatographicsystemwasmonitoredbycalcul
thecapacityfactor(k0),theresolution(R),theselectivity()andpeakasymmetry(T).
Calibration
Mixedstandardsolutionscontainingparacetamol(25-400g/ml),caeine(12.5-200g/ml),and
(37.5-600g/ml)werepreparedinthemobilephase.
Triplicate10linjectionsweremadeforeachstandardsolutiontoseethereproducibility
responseateachconcentrationlevel.Thepeakareaofeachdrugwasplottedagainstthec
toobtainthecalibrationgraph.The
veconcentrationsofeachcompoundweresubjectedtoregression
analysistocalculatethecalibrationequationandcorrelationcoecients.
Results
and
Discussion
Method
Development
Themobilephasewaschosenafterseveraltrialswithmethanol,isopropylalcohol,acetonit
andbuersolutionsinvariousproportionsandatdierentpHvalues.Amobilephaseconsisti
0.01MKH2PO4-methanol-acetonitrile-isopropylalcohol(420:20:30:30)(v/v/v/v)wasselec
e
maximumseparationandsensitivity.
Flowratesbetween0.5and1.5/minwerestudied.Aflowrateof1.0ml/mingaveanoptimal
tonoiseratiowithareasonableseparationtime.Usingareversed-phaseC8
column,theretentiontimesfor
paracetamol,caeineanddipyronewereobservedtobe4.880min,5.845minand8.093minres
Totaltimeofanalysiswaslessthan9min.
Themaximumabsorptionofparacetamol,caeineanddipyronetogetherasdetectedat215nma
thiswavelengthwaschosenfortheanalysis.
Thechromatogramat215nmshowedacompleteresolutionofallpeaks(Figure).
L.
ALTUN
AU
0.500
0.400
0.300
0.200
0.100
0.000
(I) (II)
(III)
4.818
5.7527.885
2.00 4.00 6.00 8.00
minutes
FigureChromatogramofthemixtureofparacetamol(I),caeine(II)anddipyrone(III)develo
method
Linearity
Table1presentstheequationoftheregressionline,correlationcoecient(r2),relatives
(RSD)valuesoftheslopeandinterceptforeachcompound.Excellentlinearitywasobtained
betweenthepeakareasandconcentrationsof25-400g/mlwithr2
=0.9987,12.5-200g/mlwith
r2
=0.9999and37.5-600g/mlwithr2
=0.9993forparacetamol,caeineanddipyrone,respectively.
Table1.LinearityResults,LimitofDetection(LOD)andLimitofQuanti
cation(LOQ)
Compound.
Equationr
2
Slope
(RSD%)
Intercept
(RSD%)
LOQ
g/mL
LOD
g/mL
Paracetamol
215Y=
20491.74X+
150586.10.99870.1922.0311.2260.409
Caeine
215Y=
53717.01X+
197602.20.99990.1501.8680.5020.151
Dipyrone
215Y=
19043.82X+
111528.50.99930.0582.9140.7760.233
X=Concentration(g/mL);Y=Area
Limits
of
Detection
and
Quanti
cation
Limitsofdetection(LOD)wereestablishedatasignal-to-noiseratio(S/N)of3.Limitsof
cation
(LOQ)wereestablishedatasignal-to-noiseratio(S/N)of9.LODandLOQwereexperimental
ed
bysixinjectionsofparacetamol,caeineanddipyroneattheLODandLOQconcentrations.Th
calculatedtobe0.409,0.151and0.233g/mlandtheLOQwascalculatedtobe1.226,0.502
g/mlforparacetamol,caeineanddipyrone,respectively(Table1).
Suitability
of
the
Method
Thechromatographicparameterssuchasresolution,selectivityandpeakasymmetryweresati
thesecompounds(Table2).Thecalculatedresolutionvaluesbetweeneachpeak-pairwereno
andtheselectivitywasnotlessthan1.30.
k.
valueswerefoundtobe1.81,2.37and3.66forparacetamol,caeineanddipyrone,respectiv
L.
ALTUN
Table2.SystemPerformanceParametersofParacetamol,CaeineandDipyrone
Compoundtr
(n=9,mean)
Area
(n=9,mean)
k.
R.
T
Paracetamol4.880
(0.33)*
2308090.67
(0.56)*
1.813.628
(0.64)*
1.307
(0.24)*
1.179
Cafeine5.8452853026.332.371.154
(0.47)*(0.47)*6.4331.547
Dipyrone8.093
(0.47)*
3062225.89
(0.46)*
3.66(0.77)*(0.16)*1.505
*RSD%valuesaregiveninparentheses
RSD%=(StandardDeviation/Mean)x100
Precision
Theprecisionofthemethod(within-dayvariationsofreplicatedeterminations)waschecked
paracetamol,caeine,dipyrone9timesattheLOQlevel.Theprecisionofthemethod,expres
RSD%attheLOQlevel,was2.02,2.51and5.54%forparacetamol,caeineanddipyrone,resp
(Table3).
Table3.PrecisionoftheDevelopedMethodattheLOQlevel(n=9)
Compound.
PeakArea
(mean)
RSD%
Paracetamol21524179.442.02
Caeine21525541.222.51
Dipyrone21518617.505.54
Accuracy
Astandardworkingsolutioncontainingparacetamol,caeineanddipyrone,yielding
nalconcentrationsof
100g/ml,50g/ml,and150g/mlrespectivelywasprepared.Thepreparedmixtureofstandard
injected9timesasatestsample.Fromtherespectiveareacounts,theconcentrationsoft
caeineanddipyronewerecalculatedusingthedetectorresponses.Theaccuracy,de
nedintermsof%
deviationofthecalculatedconcentrationsfromtheactualconcentrations,islistedinTab
Table4.AccuracyoftheDevelopedMethod(n=9)
Compound
Spiked
Concentration
g/mL
Measured
Concentration
g/mL
Mean±
SD
RSD
%
Deviation
%
Paracetamol100105.28±
0.6300.6005.28
Caeine5049.43±
0.2490.5041.14
Dipyrone150154.94±
0.7420.4793.29
(SpikedConcentration-MeanMeasuredConcentration)
%Deviation=×
100
SpikedConcentration
L.
ALTUN
Ruggedness
TheruggednessoftheHPLCmethodwasevaluatedbycarryingouttheanalysisusingastanda
solution,thesamechromatographicsystemandthesamecolumnondierentdays.Theprepared
standardswasinjected9timesasatestsample.Smalldierencesinareasandgoodconstanc
timeswereobservedafter96hours.AnRSDoflessthan0.560%forareasand0.472%forret
wereobtained(Tables5and6).Thecomparabledetectorresponsesobtainedondierentdays
themethodiscapableofproducingresultswithhighprecisionondierentdays.
Similarly,theruggednessofthemethodwastestedbyinjectingthestandardworkingsoluti
dierentHPLCunit.Thehighdegreesofreproducibilityofdetectorresponsesandretention
thatthemethodisfairlyrugged.
Table5.DaytoDayVariabilityAccordingtoAreaa
June16,2000June20,2000
ParacetamolCaeineDipyroneParacetamolCaeineDipyrone
Area
S.D.
2308090.67
12921.17
2853026.33
13356.87
3062225.89
14124.23
2381848
11432.76
2949559.88
9166.10
3020215.89
7476.78
RSD%0.5600.4680.4610.4800.3110.248
aMeanvaluesofninedeterminations
Table6.DaytoDayVariabilityAccordingtoRetentionTimea
June16,2000June20,2000
ParacetamolCaeineDipyroneParacetamolCaeineDipyrone
Rt
S.D.
4.880
0.016
5.845
0.027
8.093
0.038
5.122
0.007
6.456
0.011
8.934
0.020
RSD%0.3290.4680.4720.1390.1710.221
aMeanvaluesofninedeterminations
Analysis
of
Synthetic
Mixtures
Recoverystudiesinthismethodwereperformedonthesyntheticmixturespreparedbyadding
weighedamountsofthedrugs(Table7).MeanrecoveriesandRSDwerefoundtobe101.62and
paracetamol,100.11and3.38%forcaeine100.86and2.88%fordipyrone,respectively.
Table7.RecoveryResultsforParacetamol,CaeineandDipyroneinSyntheticMixturesbyHPL
ParacetamolCaeineDipyrone
MixtureAddedFoundRecoveryAddedFoundRecoveryAddedFoundRecovery
Nogg%gg%gg%
1400400.97100.24200194.2097.10600600.02100.00
2300302.77100.92150155.87103.91450455.34101.19
3200190.8495.42100103.55103.55300289.0796.36
4100105.29105.295049.4398.87150154.94103.29
55053.12106.252524.2897.127577.60103.46
X=101.62X=100.11X=100.86
RSD%=4.28RSD%=3.38RSD%2.88
X=Mean
L.
ALTUN
Conclusion
Thedevelopedmethodissuitablefortheidenti
cationandquanti
cationoftheternarycombinationof
paracetamol,caeineanddipyrone.
Ahighpercentageofrecoveryshowsthatthemethodcanbesuccessfullyusedonaroutineb
Theproposedmethodissimple,sensitive,rapid,speci
candcouldbeappliedforqualityandstability
monitoringofparacetamol,caeineanddipyronecombinations.
References
1.
J.E.F.Reynolds,\MartindaleTheExtraPharmacopoeia",31st
ed.,pp.27-28,PharmaceuticalPress,
London,1996.
2.
TheUnitedStatesPharmacopoeia,24th
revision,pp.17-39,U.S.PharmacopeialConvention,Rockville,
MD,2000.
3.
BritishPharmacopoeiaCD1998,version2,TheStationeryOceLtd.,Norwich,1998.
4.
EuropeanPharmacopoeia1997,thirdedition,pp.748-749,ConventionontheElaborationofa
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
lesofDrugSubstances",Vol.14,pp.551-596,American
PharmaceuticalAssociation,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.Süzen,C.Akay,S.Tartlms,R.S.Erdöl,A.OnalandS.Cevheroglu,J.FacultyofPharm
12.
W.R.Sisco,C.T.Rittenhuse,L.A.EverhartandA.M.McLaughlin,J.Chromatogr.,354,355-36
13.
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.Ph
155-159(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(199
17.
J.J.BerghandA.P.Lötter,DrugDev.Ind.Pharm.,10(1),127-136(1984).
18.
E.Z.Katz,Y.Caraco,L.GranitandM.Levy,Clin.Pharmacol.Ther.,58(2),198-209(1995)
19.
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(20
21.
A.M.HapetteandS.A.Poulet,J.Liq.Chromatogr.,13(2),357-370(1990)
L.
ALTUN
22.F.Belal,Electroanalysis,4,589(1992).
23.A.Siou
andD.Colussi,J.Chromatogr.,146,503-507(1978).
24.
G.Geisslinger,R.BöckerandM.Levy,PharmaceuticalResearch,13(8),1272-1275(1996).
¨¨
25.M.UstünOzgürandS.Sungur,ChimicaActaTurcica,23,119-123(1995).
26.A.Bozdogan,A.M.Acar,G.KuntandH.Caglar,Pharmazie,49,457-458(1994).

HPLC For Analysis Paracetamol

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

HPLC For Analysis Paracetamol

Uploaded by

Copyright:

Available Formats

TurkJChem

You might also like

HPLC For Analysis Paracetamol

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

HPLC For Analysis Paracetamol

Uploaded by

Copyright:

Available Formats

TurkJChem

You might also like

TurkJChem