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  • Stability Indicating RP-HPLC Method For The Simultaneous Estimation of Pyrimethamine and Sulphadoxine in Bulk and Tablet - Taj Pharmaceuticals LTD

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    Page 1 © 2016 Veeragoni Anil Kumar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial- ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). Journal of Applied Pharmaceutical Science Vol. 6 (03), pp. 071-076, March, 2016 Available online at http://www.japsonline.com DOI: 10.7324/JAPS.2016.60312 ISSN 2231-3354 Stability Indicating RP-HPLC Method for the Simultaneous Estimation of Pyrimethamine and Sulphadoxine in Bulk and Tablet Dosage Form Veeragoni Anil Kumar1*, Vasudeva Murthy Sindgi2, Shoba Rani Satla3, Manish Kumar Thimmaraju4 1Department of Pharmaceutical Analysis, Pathfinder institute of Pharmacy Education and Research, Warangal-Telangana. 2Jayamukhi College of Pharmacy, Narsampet, Warangal, Telangana. 3Center for Pharmaceutical Sciences, Institute of Science and Technology, JNTUH, Hyderabad, Telangana. 4Central Analytical Laboratory, Balaji Institute of Pharmaceutical Sciences, Narsampet, Warangal-Telangana. ARTICLE INFO ABSTRACT Article history: Received on: 24/12/2015 Revised on: 15/01/2016 Accepted on: 08/02/2016 Available online: 30/03/2016 A stability indicating simple, selective, accurate high Performance Liquid Chromatographic (HPLC) method was developed and validated for the combined tablet formulation of pyrimethamine & sulphadoxine. Chromatographic separation was optimized by gradient HPLC on a C18 column [Inertsil Silica, 250 x 4.6 mm, 5µ] utilizing a mobile phase of potassium dihydrogen phosphate and acetonitrile taken in the ratio 70:30 at a flow rate of 1.0 ml/min with UV detection at 221nm. The retention time of pyrimethamine and sulphadoxine was 2.77 and 6.57 min respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation, robustness and stress degradation studies. Validation of the method was done in accordance with ICH guidelines for the assay of active ingredients. Thus validated method can be recommended for the routine laboratory analysis. Key words: Pyrimethamine, sulphadoxine, ICH guidelines and stress degradation studies. INTRODUCTION Pyrimethamine is an anti-malarial drug which inhibits the dihydrofolatereductase of plasmodia and there by blocks the biosynthesis of purines and pyrimidines, which are essential for DNA synthesis and cell multiplication. Sulfadoxine is a sulfa drug, often used in combination with pyrimethamine to treat malaria. Review of literature for pyrimethamine & sulphadoxine gave information regarding the various studies conducted and analytical methods established for the drugs alone, in combination and in combination with other drugs in pharmaceutical dosage forms and in biological fluids (Minzi et al., 2013, Sinnaeve et al., 2005). There are few methods reported in the literature for analysis of pyrimethamine & sulphadoxine alone or in combination with other drugs in the pure form, pharmaceuticals formulations and biological fluids by UV- spectrophotometer (Onah and Odeiani, 2002, Meena and . * Corresponding Author Veeragoni Anil Kumar, Department of Pharmaceutical Analysis Pathfinder Institute of Pharmacy Education And Research, Warangal- Telangana. Email: v_aneel@yahoo.co.in Sandhya, 2013), HPTLC (Meena and Sandhya, 2013), Capillary zone electrophoresis (Amin et al., 2012), RP-HPLC (Green et al., 2002, Bergqvist et al., 1991; Astier et al., 1997; Bergqvist et al., 1985; SaeedArayne et al., 2010), LC-MS (Sinnaeve et al., 2005). There was no stability indicating HPLC methods established for the simultaneous estimation of pyrimethamine & sulphadoxine in formulation. The aim of the present work is to develop a stability indicating analytical method for the combined tablet formulation of pyrimethamine & sulphadoxine. Validation of the method was done in accordance with ICH guidelines for the assay of active ingredients. Thus validated metho

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    Page 1 © 2016 Veeragoni Anil Kumar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial- ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). Journal of Applied Pharmaceutical Science Vol. 6 (03), pp. 071-076, March, 2016 Available online at http://www.japsonline.com DOI: 10.7324/JAPS.2016.60312 ISSN 2231-3354 Stability Indicating RP-HPLC Method for the Simultaneous Estimation of Pyrimethamine and Sulphadoxine in Bulk and Tablet Dosage Form Veeragoni Anil Kumar1*, Vasudeva Murthy Sindgi2, Shoba Rani Satla3, Manish Kumar Thimmaraju4 1Department of Pharmaceutical Analysis, Pathfinder institute of Pharmacy Education and Research, Warangal-Telangana. 2Jayamukhi College of Pharmacy, Narsampet, Warangal, Telangana. 3Center for Pharmaceutical Sciences, Institute of Science and Technology, JNTUH, Hyderabad, Telangana. 4Central Analytical Laboratory, Balaji Institute of Pharmaceutical Sciences, Narsampet, Warangal-Telangana. ARTICLE INFO ABSTRACT Article history: Received on: 24/12/2015 Revised on: 15/01/2016 Accepted on: 08/02/2016 Available online: 30/03/2016 A stability indicating simple, selective, accurate high Performance Liquid Chromatographic (HPLC) method was developed and validated for the combined tablet formulation of pyrimethamine & sulphadoxine. Chromatographic separation was optimized by gradient HPLC on a C18 column [Inertsil Silica, 250 x 4.6 mm, 5µ] utilizing a mobile phase of potassium dihydrogen phosphate and acetonitrile taken in the ratio 70:30 at a flow rate of 1.0 ml/min with UV detection at 221nm. The retention time of pyrimethamine and sulphadoxine was 2.77 and 6.57 min respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation, robustness and stress degradation studies. Validation of the method was done in accordance with ICH guidelines for the assay of active ingredients. Thus validated method can be recommended for the routine laboratory analysis. Key words: Pyrimethamine, sulphadoxine, ICH guidelines and stress degradation studies. INTRODUCTION Pyrimethamine is an anti-malarial drug which inhibits the dihydrofolatereductase of plasmodia and there by blocks the biosynthesis of purines and pyrimidines, which are essential for DNA synthesis and cell multiplication. Sulfadoxine is a sulfa drug, often used in combination with pyrimethamine to treat malaria. Review of literature for pyrimethamine & sulphadoxine gave information regarding the various studies conducted and analytical methods established for the drugs alone, in combination and in combination with other drugs in pharmaceutical dosage forms and in biological fluids (Minzi et al., 2013, Sinnaeve et al., 2005). There are few methods reported in the literature for analysis of pyrimethamine & sulphadoxine alone or in combination with other drugs in the pure form, pharmaceuticals formulations and biological fluids by UV- spectrophotometer (Onah and Odeiani, 2002, Meena and . * Corresponding Author Veeragoni Anil Kumar, Department of Pharmaceutical Analysis Pathfinder Institute of Pharmacy Education And Research, Warangal- Telangana. Email: v_aneel@yahoo.co.in Sandhya, 2013), HPTLC (Meena and Sandhya, 2013), Capillary zone electrophoresis (Amin et al., 2012), RP-HPLC (Green et al., 2002, Bergqvist et al., 1991; Astier et al., 1997; Bergqvist et al., 1985; SaeedArayne et al., 2010), LC-MS (Sinnaeve et al., 2005). There was no stability indicating HPLC methods established for the simultaneous estimation of pyrimethamine & sulphadoxine in formulation. The aim of the present work is to develop a stability indicating analytical method for the combined tablet formulation of pyrimethamine & sulphadoxine. Validation of the method was done in accordance with ICH guidelines for the assay of active ingredients. Thus validated metho

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    Stability Indicating RP-HPLC Method For The Simultaneous Estimation of Pyrimethamine and Sulphadoxine in Bulk and Tablet - Taj Pharmaceuticals LTD

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    Page 1 © 2016 Veeragoni Anil Kumar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial- ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). Journal of Applied Pharmaceutical Science Vol. 6 (03), pp. 071-076, March, 2016 Available online at http://www.japsonline.com DOI: 10.7324/JAPS.2016.60312 ISSN 2231-3354 Stability Indicating RP-HPLC Method for the Simultaneous Estimation of Pyrimethamine and Sulphadoxine in Bulk and Tablet Dosage Form Veeragoni Anil Kumar1*, Vasudeva Murthy Sindgi2, Shoba Rani Satla3, Manish Kumar Thimmaraju4 1Department of Pharmaceutical Analysis, Pathfinder institute of Pharmacy Education and Research, Warangal-Telangana. 2Jayamukhi College of Pharmacy, Narsampet, Warangal, Telangana. 3Center for Pharmaceutical Sciences, Institute of Science and Technology, JNTUH, Hyderabad, Telangana. 4Central Analytical Laboratory, Balaji Institute of Pharmaceutical Sciences, Narsampet, Warangal-Telangana. ARTICLE INFO ABSTRACT Article history: Received on: 24/12/2015 Revised on: 15/01/2016 Accepted on: 08/02/2016 Available online: 30/03/2016 A stability indicating simple, selective, accurate high Performance Liquid Chromatographic (HPLC) method was developed and validated for the combined tablet formulation of pyrimethamine & sulphadoxine. Chromatographic separation was optimized by gradient HPLC on a C18 column [Inertsil Silica, 250 x 4.6 mm, 5µ] utilizing a mobile phase of potassium dihydrogen phosphate and acetonitrile taken in the ratio 70:30 at a flow rate of 1.0 ml/min with UV detection at 221nm. The retention time of pyrimethamine and sulphadoxine was 2.77 and 6.57 min respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation, robustness and stress degradation studies. Validation of the method was done in accordance with ICH guidelines for the assay of active ingredients. Thus validated method can be recommended for the routine laboratory analysis. Key words: Pyrimethamine, sulphadoxine, ICH guidelines and stress degradation studies. INTRODUCTION Pyrimethamine is an anti-malarial drug which inhibits the dihydrofolatereductase of plasmodia and there by blocks the biosynthesis of purines and pyrimidines, which are essential for DNA synthesis and cell multiplication. Sulfadoxine is a sulfa drug, often used in combination with pyrimethamine to treat malaria. Review of literature for pyrimethamine & sulphadoxine gave information regarding the various studies conducted and analytical methods established for the drugs alone, in combination and in combination with other drugs in pharmaceutical dosage forms and in biological fluids (Minzi et al., 2013, Sinnaeve et al., 2005). There are few methods reported in the literature for analysis of pyrimethamine & sulphadoxine alone or in combination with other drugs in the pure form, pharmaceuticals formulations and biological fluids by UV- spectrophotometer (Onah and Odeiani, 2002, Meena and . * Corresponding Author Veeragoni Anil Kumar, Department of Pharmaceutical Analysis Pathfinder Institute of Pharmacy Education And Research, Warangal- Telangana. Email: v_aneel@yahoo.co.in Sandhya, 2013), HPTLC (Meena and Sandhya, 2013), Capillary zone electrophoresis (Amin et al., 2012), RP-HPLC (Green et al., 2002, Bergqvist et al., 1991; Astier et al., 1997; Bergqvist et al., 1985; SaeedArayne et al., 2010), LC-MS (Sinnaeve et al., 2005). There was no stability indicating HPLC methods established for the simultaneous estimation of pyrimethamine & sulphadoxine in formulation. The aim of the present work is to develop a stability indicating analytical method for the combined tablet formulation of pyrimethamine & sulphadoxine. Validation of the method was done in accordance with ICH guidelines for the assay of active ingredients. Thus validated metho

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    2016VeeragoniAnilKumaretal.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicenseNonCommercial
    ShareAlikeUnportedLicense(http://creativecommons.org/licenses/byncsa/3.0/).

    JournalofAppliedPharmaceuticalScienceVol.6(03),pp.071076,March,2016
    Availableonlineathttp://www.japsonline.com
    DOI:10.7324/JAPS.2016.60312
    ISSN22313354

    StabilityIndicatingRPHPLCMethodfortheSimultaneousEstimation
    ofPyrimethamineandSulphadoxineinBulkandTabletDosageForm
    VeeragoniAnilKumar1*,VasudevaMurthySindgi2,ShobaRaniSatla3,ManishKumarThimmaraju4
    1DepartmentofPharmaceuticalAnalysis,PathfinderinstituteofPharmacyEducationandResearch,WarangalTelangana.2JayamukhiCollegeofPharmacy,

    Narsampet,Warangal,Telangana.3CenterforPharmaceuticalSciences,InstituteofScienceandTechnology,JNTUH,Hyderabad,Telangana.4Central
    AnalyticalLaboratory,BalajiInstituteofPharmaceuticalSciences,Narsampet,WarangalTelangana.

    ARTICLEINFO
    ABSTRACT

    Articlehistory:
    Receivedon:24/12/2015
    Revisedon:15/01/2016
    Acceptedon:08/02/2016
    Availableonline:30/03/2016
    Astabilityindicatingsimple,selective,accuratehighPerformanceLiquidChromatographic(HPLC)method
    wasdevelopedandvalidatedforthecombinedtabletformulationofpyrimethamine&sulphadoxine.
    ChromatographicseparationwasoptimizedbygradientHPLConaC18column[InertsilSilica,250x4.6mm,
    5]utilizingamobilephaseofpotassiumdihydrogenphosphateandacetonitriletakenintheratio70:30ata
    flowrateof1.0ml/minwithUVdetectionat221nm.Theretentiontimeofpyrimethamineandsulphadoxine
    was2.77and6.57minrespectively.Thedevelopedmethodwasvalidatedintermsofaccuracy,precision,
    linearity,limitofdetection,limitofquantitation,robustnessandstressdegradationstudies.Validationofthe
    methodwasdoneinaccordancewithICHguidelinesfortheassayofactiveingredients.Thusvalidatedmethod
    canberecommendedfortheroutinelaboratoryanalysis.
    Keywords:
    Pyrimethamine,
    sulphadoxine,ICHguidelines
    andstressdegradation
    studies.

    INTRODUCTION
    Pyrimethamineisanantimalarialdrugwhichinhibits
    thedihydrofolatereductaseofplasmodiaandtherebyblocksthe
    biosynthesisofpurinesandpyrimidines,whichareessentialfor
    DNAsynthesisandcellmultiplication.Sulfadoxineisasulfa
    drug,oftenusedincombinationwithpyrimethaminetotreat
    malaria.Reviewofliteratureforpyrimethamine&sulphadoxine
    gaveinformationregardingthevariousstudiesconductedand
    analyticalmethodsestablishedforthedrugsalone,in
    combinationandincombinationwithotherdrugsin
    pharmaceuticaldosageformsandinbiologicalfluids(Minziet
    al.,2013,Sinnaeveetal.,2005).Therearefewmethodsreported
    intheliteratureforanalysisofpyrimethamine&sulphadoxine
    aloneorincombinationwithotherdrugsinthepureform,
    pharmaceuticalsformulationsandbiologicalfluidsbyUV
    spectrophotometer(OnahandOdeiani,2002,Meenaand
    .

    *CorrespondingAuthor
    VeeragoniAnilKumar,DepartmentofPharmaceuticalAnalysis
    PathfinderInstituteofPharmacyEducationAndResearch,Warangal
    Telangana.Email:v_aneel@yahoo.co.in

    Sandhya,2013),HPTLC(MeenaandSandhya,2013),Capillary
    zoneelectrophoresis(Aminetal.,2012),RPHPLC(Greenetal.,
    2002,Bergqvistetal.,1991;Astieretal.,1997;Bergqvistetal.,
    1985;SaeedArayneetal.,2010),LCMS(Sinnaeveetal.,2005).
    TherewasnostabilityindicatingHPLCmethods
    establishedforthesimultaneousestimationofpyrimethamine&
    sulphadoxineinformulation.Theaimofthepresentworkisto
    developastabilityindicatinganalyticalmethodforthecombined
    tabletformulationofpyrimethamine&sulphadoxine.Validationof
    themethodwasdoneinaccordancewithICHguidelinesforthe
    assayofactiveingredients.Thusvalidatedmethodcanbe
    recommendedfortheroutinelaboratoryanalysis.
    MATERIALSANDMETHODS
    Pyrimethamine(PYR)andSulphadoxine(SUL)were
    procuredasgiftsamplesfromTajpharmaceuticals,Mumbai.
    REZIZ(Pyrimethamine25mgandSulphadoxine500mg)tablets
    manufacturedbyShreyalifesciencespvt.Ltd.Indiawasprocured
    fromalocalpharmacy.Acetonitrile(HPLCgrade),ortho
    phosphoricacid,Potassiumdihydrogenorthophosphate,Methanol
    (HPLCgrade),TriethylamineandTDW(TripleDistilledWater).

    Page 2
    072
    AnilKumaretal./JournalofAppliedPharmaceuticalScience6(03);2016:071076

    Instrumentation
    ShimadzugradientHPLCMODELNO(JAPAN),HPLC
    columnInertsil(250x4.6mm,5m),Mobilephasefiltrationunit
    (PallLifesciences,Mumbai,India),LABINDIAU.VwithUV
    Winsoftware,Sonicator,PHmeter(LABINDIA),digitalbalance
    (Denver).
    METHODDEVELOPMENT
    Preparationofstandardsolutions
    Accuratelyweighedandtransferred10mgof
    Pyrimethamineand10mgofSulphadoxineworkingStandards
    intotwoseparate100mlcleandryvolumetricflasks,add30mlof
    diluent,sonicatedfor5minutesandmakeuptothefinalvolume
    withdiluent.
    ChromatographicConditions
    TheHPLCsystemconsistedofShimadzugradientHPLC
    (JAPAN)withdualAbsorbanceUVdetector.Thewavelengthof
    detectionassetat221nm.Separationwascarriedoutingradient
    modeoninertsilC18column(4.6x250mmx5m)andtheretention

    timeofpyrimethamineandsulphadoximewasfoundtobe2.952
    and6.832respectively(figure1),using70:30v/vdihydrogen
    orthophosphate:acetonitrileasmobilephaseataflowrateof1
    ml/min.Themobilephasefilteredthroughnylonmillipore
    (0.2m)membranefilter,purchasedfrompalllifesciences,
    MumbaianddegassedwithUltrasonicatorpriortouse.
    Chromatographywascarriedoutatroomtemperature250cand
    maintainsthecolumntemperatureat320c.
    Fig.1:Chromatogramofpyrimethamineandsulphadoxime.

    PreparationofStandardSolutions
    Stocksolutionsofpyrimethamine(0.5mg/ml)and
    sulphadoxime(1mg/ml)werepreparedinmethanol.Further
    dilutionswerecarriedoutin60%acetonitrileandcalibration
    standardswerepreparedfreshlywithpyrimethamineand
    sulphodoximestocksolutionstogivetheconcentrationsof5,10,
    15,20,25and30g/ml.
    SamplePreparation(Assay)
    5tabletswereweighedandcalculatetheaverageweight
    ofeachtabletthentheweightequivalenttransferredintoa100mL
    volumetricflask,60mLofdiluentaddedandsonicatedfor25min,
    furtherthevolumemadeupwithdiluentandfiltered.Fromthe
    filteredsolution0.2mlwaspipettedoutintoa10mlvolumetric
    flaskandmadeupto10mlwithdiluent.
    RESULTS&DISCUSSIONS
    MethodValidation
    MethodvalidationwasperformedaspertheICH
    guidelinesQ2(R1)ValidationofAnalyticalProcedure.The
    developedmethodwasvalidatedforthefollowingparameters.
    Linearity
    Linearconcentrationsofbothdrugswerepreparedand
    thebestfitlinewascalculated.Widerangecalibrationwas
    determinedbysolutionscontaining5g/mlto30g/ml(table1).
    Correlationcoefficientwasfoundtobe0.999&0.997for
    Pyrimethamine&Sulphadoxinerespectively(showninfig2&3).
    Table1:Linearityresultsofpyrimethamine&sulphadoxine.
    Sno
    Pyrimethamine
    Sulphadoxine
    Concentration
    (g/ml)
    Peakarea
    Concentration
    (g/ml)
    Peakarea
    1
    5
    329102
    5
    2279923
    2
    10
    876684
    10
    5805764
    3
    15
    1225460
    15
    8254245
    4
    20
    1753561
    20
    11870562
    5
    25
    2109537
    25
    14481530
    6
    30
    2614420
    30
    18076428

    Precision
    Theintradayprecisionwasdemonstratedbyinjectingsix
    testsolutionsat25g/mlconcentrationasperthetestprocedure
    (shownintableno2&3)&recordingthechromatogramsofsix
    testsolutions.The%RSDofpyrimethamineandsulphodixime
    wasfoundtobe0.207and0.324respectively.
    Table2:MethodPrecisionofPyrimethamine.
    Sno
    Pyrimethamine(25g/ml)
    Retentiontime(Rt)
    Peakarea
    %Assay
    1
    2.90
    2109429
    100.01
    2
    2.930
    2109837
    99.99
    3
    2.839
    2109941
    99.98
    4
    2.914
    2107535
    100.09
    5
    2.845
    2108530
    100.05
    6
    2.872
    2119528
    99.53
    Mean
    2110800
    99.94
    SD
    4371.18
    0.206
    RSD

    0.207
    0.206
    Table3:MethodPrecisionofSulphadoxine
    Sno
    Sulphadoxine(25g/ml)
    Retentiontime(Rt)
    Peakarea
    %Assay
    1
    6.828
    14482536
    99.99
    2
    6.823
    14481829
    100.00
    3
    6.729
    14599259
    99.19
    4
    6.799
    14499743
    99.87
    5
    6.712
    14481539
    100.00
    6
    6.722
    14481645
    100.00
    Mean
    14504425
    99.84
    SD
    47005.99
    0.3220
    RSD
    0.324
    0.3225

    Page 3
    AnilKumaretal./JournalofAppliedPharmaceuticalScience6(03);2016:071076
    073
    Fig.2:Linearityofpyrimethamine.
    Fig.3:Linearityofsulphadoxine.

    IntermediatePrecision
    Intermediateprecisionoftheanalyticalmethodwas
    determinedbyperformingmethodprecisiononinthreesuccessive
    daysbydifferentanalystsundersameexperimentalcondition.
    Assayofallsixreplicatesamplepreparationswasdeterminedand
    themean%RSDofpyrimethamineandsulphodiximewasfound
    tobe0.259and0.353respectively(shownintableno4).
    Table4:IntermediatePrecisionofpyrimethamine&sulphadoxine.
    S.No
    Parameter
    %RSD
    Mean
    RSD
    Day1
    Day2
    Day3
    Pyrimethamine
    (25g/ml)
    PeakArea
    0.199
    0.264
    0.318
    0.260
    %Assay
    0.198
    0.263
    0.317
    0.259
    sulphadoxine
    (25g/ml)
    PeakArea
    0.317
    0.288
    0.462
    0.356
    %Assay
    0.315
    0.287
    0.4584
    0.353

    Accuracy
    Accuracyofthemethodwasestablishedbyperforming
    recoverystudiesaccordingtotheICHguidelines.Spikedsamples
    werepreparedbyspikingpreanalyzedsamplesolutionswithpure
    drugatthreedifferentconcentrationlevelseachintriplicate.Mean
    percentagerecoveryvaluesatthreedifferentconcentrationsofthe
    twodrugswascalculated.The%recoveryofPyrimethamine
    (98.02100.45%)&Sulphadoxine(99.79100.20%)ateachlevel
    waswithinthelimitsof98%and102%(shownintableno5&6).
    Hence,accuracywasestablishedforthepresentworkandthe
    methodwassaidtobeaccurate.
    Table5:%recoveryofPyrimethamine.
    S.no
    Conc(g/
    ml)
    Conc(g/ml)
    found
    %
    recovery
    Mean
    accuracy
    %RSD
    1
    15
    14.8
    101.35
    100.45
    1.024
    2
    15
    15.1
    99.34

    3
    15
    14.9
    100.67
    4
    20
    20.3
    98.52
    98.02
    1.530
    5
    20
    19.7
    101.52
    6
    20
    20.1
    99.50
    7
    25
    25.2
    99.21
    100.14
    0.832
    8
    25
    24.9
    100.40
    9
    25
    24.8
    100.81
    Table6:%RecoveryofSulphadoxine.
    Sno
    Conc
    (g/ml)
    Conc(g/ml)
    found
    %
    recovery
    Mean
    accuracy
    %RSD
    1
    15
    14.91
    100.60
    100.20
    0.811
    2
    15
    15.11
    99.27
    3
    15
    14.89
    100.74
    4
    20
    20.12
    99.40
    99.79
    0.664
    5
    20
    19.89
    100.55
    6
    20
    20.12
    99.40
    7
    25
    25.09
    99.64
    99.87
    0.428
    8
    25
    25.1
    99.60
    9
    25
    24.91
    100.36

    LimitofDetection&Quantification
    Inthepresentstudy,theLODandLOQwerecalculated
    accordingtothestandarddeviationoftheresponseandtheslope
    ofthecalibrationcurvei.e.,3.3/Sand10/Scriterions,
    respectively;whereisthestandarddeviationofyinterceptsof
    regressionlinesandSistheslopeofthecalibrationcurve.The
    lowestpossibleconcentrationofPyrimethaminethatcanbe
    detectedandquantifiedbythepresentmethodwasfoundtobe
    0.264g/mland0.800g/mlrespectivelyandthatof
    Sulphadoxinewasfoundtobe0.53g/mland1.62g/ml
    respectively.
    Robustness
    Robustnessoftheproposedmethodwasdeterminedby
    varyingvariousparameters,the%RSDreportedwasfoundtobe
    lessthan2%.Asthesystemsuitabilityparametersforthestandard
    andtestchromatogramsofPyrimethamine&Sulphadoxinewere
    withinlimitsforvariationinflowrate(0.1ml)andmobilephase
    composition,theallowablevariationinflowrate,organicsolvent
    ratioinmobilephasecompositionandcolumntemperatureshould
    be10.1ml/min,652mland3050crespectively(shownintable
    no7&8).
    STRESSDEGRADATIONSTUDIES
    StressdegradationstudieswereperformedaspertheICH
    guidelinesQ1A(R2)StabilityTestingofNewDrugSubstances
    andProducts,usingtheproposedvalidatedanalyticalmethodand
    theresultswereshownintableno9&10.
    y=70181x5618
    R=0.999
    0
    500000
    1000000
    1500000
    2000000
    2500000
    3000000
    0
    5

    10
    15
    20
    25
    30
    35
    y=24199x13401
    R=0.997
    0
    2000000
    4000000
    6000000
    8000000
    10000000
    12000000
    14000000
    16000000
    18000000
    20000000
    0
    5
    10
    15
    20
    25
    30
    35

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    AnilKumaretal./JournalofAppliedPharmaceuticalScience6(03);2016:071076

    Aciddegradationstudies
    To1mlofstocksolutionpyrimethamineand
    sulphadoxine,1mlof2NHClwasaddedandrefluxedfor30minat
    600c.Fromtheabovesolution10lwasinjectedintothesystem
    andthechromatogramswererecordedtodetectthestabilityof
    sample(figure4).
    AlkaliDegradationStudies
    To1mlofstocksolutionofofstandarddrugandsample
    pyrimethamineandsulphadoxine,1mlof2NNaOHwasadded
    andrefluxedfor30minat60C.Fromtheabovesolution10l
    wasinjectedintothesystemandthechromatogramswererecorded
    todetectthestabilityofsample(figure5).
    Oxidation
    To1mlofstocksolutionofstandarddrugandsampleof
    pyrimethamineandsulphadoxine,1mlof20%H2O2wasaddedand
    refluxedfor30minat600c.Fromtheabovesolution10lwas
    injectedintothesystemandthechromatogramswererecordedto
    detectthestabilityofsample(figure6).
    PhotoStabilityStudies
    Thephotochemicalstabilityofthedrugwasalsostudied
    byexposingthe25g/mlsolutiontoUVLightbykeepingthe
    beakerinUVChamberfor7daysor200Watthours/m 2inphoto
    stabilitychamber..ForHPLCstudy,fromtheabovesolution10l
    wasinjectedintothesystemandthechromatogramswererecorded
    todetectthestabilityofsample(figure7).
    PyrimethamineandSulphadoxineundergoesdegradation
    inacidic,oxidation,alkaline,andUV.Moredegradationwas
    foundforoxidation.AsperICHguidelinespeakpurityangle
    shouldbelessthanpeakpuritythreshold.Hence,methodofthe
    analysisofPYRandSULintabletdosageformshowsthatthe
    degradationproductdoesntinterferewiththeanalytical
    determination.Thestressdegradationstudiesshowedthatthedrug
    formulationcontainingpyrimethamineandsulphadoxine
    undergoesdegradationinacidic,oxidation,alkaline,andUV
    (7.29%,15.01%,16.13%,7.88%,5.51%and12.92%,10.72%,
    7.13%,13.32%,6.01%).hencetheproposedanalyticalmethodis
    alsousefulforthedeterminationofpyrimethamineand
    sulphadoxinestabilityinsampleofpharmaceuticaldosageform.
    Table7:RobustnessofSulphadoxine.
    Parameter
    Variationinflow
    VariationinMobilephase
    Variationincolumntemp
    flowrate
    flowrate
    Buffer:Acetonitrile(75:25)
    Buffer:Acetonitrile(80:20)
    3050
    30+50
    Stds
    (0.9ml/mim)
    (1.1ml/mim)
    1
    9709676
    8894030
    8990708
    9020771
    8962774
    8921543
    2
    9721151
    8862782
    8982909
    9025663
    8965300
    8925743
    Mean
    9715414
    8878406
    8986809
    9023217
    8965537
    8922143
    SD
    8114.7
    22095.7
    5515
    2044.6
    3163.4
    2262.4
    %RSD
    0.1

    0.2
    0.1
    0.1
    0.1
    0.1
    Retentiontime
    7.09
    2.715
    6.03
    7.07
    6.47
    6.37
    Tailingfactor
    1.33
    1.33
    1.32
    1.3
    1.32
    1.43
    Theoreticalplates
    5217
    5082
    5089
    5306
    5253
    5346
    Table8:RobustnessofPyrimethamine.
    Parameter
    Variationinflow
    VariationinMobilephase
    variationincolumntemp
    flowrate
    flowrate
    Buffer:Acetonitrile(75:25)
    Buffer:Acetonitrile(80:20)
    3050
    30+50
    Stds
    (0.9ml/mim)
    (1.1ml/mim)
    1
    1588766
    1485375
    1435845
    1539898
    1454227
    1434907
    2
    1603920
    1484112
    1425867
    1549198
    1480862
    1440355
    Mean
    1596343
    1484743
    1430856
    1544548
    1467545
    1437631
    SD
    10715
    892.8
    7055.5
    6576.4
    18833.7
    3852.6
    %RSD
    0.7
    0.1
    0.4
    0.4
    1.3
    0.3
    Retentiontime
    2.95
    2.715
    2.43
    3.076
    2.71
    2.66
    Tailingfactor
    1.47
    1.44
    1.42
    1.39
    1.44
    1.43
    Theoreticalplates
    3679
    3665
    3797
    3824
    3746
    3981
    Table9:ResultsofstressdegradationstudiesofSulphadoxine.
    Sno
    Stressconditions
    Time
    %Assay
    %Degradation
    1
    AcidDegradation
    30min
    88.082
    11.918
    2
    BaseDegradation
    30min
    87.281
    12.719
    3
    PeroxideDegradation
    30min
    93.214
    6.786
    4
    UVDegradation
    7days
    92.887
    7.113
    Table10:ResultsofstressdegradationstudiesofPyrimethamine.
    Sno
    Stressconditions
    Time
    %Assay
    %Degradation
    1
    AcidDegradation

    30min
    92.713
    7.287
    2
    BaseDegradation
    30min
    85.985
    14.015
    3
    PeroxideDegradation
    30min
    82.798
    17.202
    4
    UVDegradation
    7days
    93.502
    6.498

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    AnilKumaretal./JournalofAppliedPharmaceuticalScience6(03);2016:071076
    075
    Fig.4:ChromatogramforSampleaciddegradation.
    Fig.5:ChromatogramforSampleAlkalidegradation.
    Fig.6:ChromatogramforSamplePeroxidedegradation.

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    AnilKumaretal./JournalofAppliedPharmaceuticalScience6(03);2016:071076

    CONCLUSION
    TheproposedHPLCmethodwasfoundtobesimple,
    specific,precise,accurate,rapidandeconomicalforsimultaneous
    estimationofpyrimethamine&sulphadoxineinbulkandtablet
    dosageformandwasfoundtobesuitablefortheroutineanalysis
    andqualitycontrolandpercentagedegradationofpharmaceutical
    preparationscontainingthesedrugseitherindividuallyorin
    combination.
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    Howtocitethisarticle:
    AnilKumarV,SindgiVM,SatlaSR,ThimmarajuMK.Stability
    IndicatingRpHplcMethodfortheSimultaneousEstimationof
    PyrimethamineandSulphadoxineinBulkandTabletDosageForm.
    JAppPharmSci,2016;6(03):071076.

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