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EurasianJournalofAnalyticalChemistry,2016,11(2),101-113

Copyright©2014byiSER,InternationalSocietyofEducationalResearchISSN:1306-3057

OptimizationofStabilityIndicatingRP-HPLCmethodforTheEstimationofanAntidepressantAgentsAlprazolamandImipramineinPure&PharmaceuticalDosageFormPayalP.Chauhan,DivyaY.Patel,DepartmentofQualityAssurance,SardarPatelCollegeofPharmacy,INDIASamirK.ShahDepartmentofPharmacology,SardarPatelCollegeofPharmacy,INDIA�Received18August2015�Revised25December2015�Accepted09January2016

ASimple,SpecificandPrecisestabilityindicatingReversePhasehighperformanceliquidChromatography method has been developed and validated for the estimation ofAlprazolam and Imipramine in tablet dosage form using ODS-BP Hyperchrome C18column(250mm×4.6mmid,5μmparticlesize)asastationaryphase,Water(pH-6):Methanol:Triethylamine(70:30:0.1%v/v/v)asmobilephase, flowrateof1.0mL/minanddetectionwascarriedoutat216nm.Theretention timeofAlprazolamwas3.181minute and Imipramine was 5.045 minute. RP-HPLC method was developed withlinearityrangeof0.5–1.5μg/mLAlprazolamand50–150μg/mLImipramine.Theco-relationcoefficientwasfoundtobe0.9999forAlprazolamand0.9998forImipramine.Theassayresultsobtained ingoodagreementwith thecorresponding labeledamountby developed method within range of 99.58% – 101.45% and 98.84% – 99.14% forAlprazolam and Imipramine respectively. Accuracy, Precision, LOD, LOQ, Specificity,Robustnessweremetalltheacceptancecriteriaforthevalidationofanalyticalmethodas per ICH Q2 (R1) guideline. This method can be conveniently used to detect thepossible degradation product in the combined dosage form of Alprazolam andImipramineduringstabilitystudies(acidic,alkaline,oxidative,photolyticandthermal).The method proved to be affective on application to a stressed marketed tabletformulation.

Keywords: analytical method development, validation, simultaneous estimation,alprazolam,imipramine,stabilityindicating

Correspondence:PayalP.Chauhan

DepartmentofQualityAssurance,SardarPatelCollegeofPharmacy,Bakrol,Gujarat,INDIA

E-mail:[email protected]:10.12973/ejac.2016.125a

P.P.Chauhan,D.Y.Patel&S.K.Shah

102 ©2016iSER,EurasianJAnalChem,11(2),101-113

INTRODUCTION

Alprazolamisananxiolyticdrug.Itisbelongingtoclassofabenzodiazepine.Itischemically 8-Chloro- 1-methyl-6-phenyl-4H - [1,2,4]-triazol [4,3-a] [1,4] benzo-diazepine.Itisusedinthetreatmentofanxietydisorder,ortheshort-termreliefofsymptoms of anxiety and for the treatment of panic disorder, with or withoutagoraphobia and panic disorder. It is binding with inhibitory neurotransmitterGABA to the site opens the chloride channel, resulting in a hyperpolarized cellmembranethatpreventsfurtherexcitationofthecell.SomespectrophotometricandHPLCmethodsarereportedinliteratureforestimationofAlprazolamaloneandincombination with other drugs. Imipramine is a tricyclic antidepressant. It ischemically10,14,Dihydro-5H-Dibenz(b,f)azepine-5-9dimethylaminopropyl.It isused in the treatment of depression and nocturnal enuresis in children. TCAs arepotent inhibitors of serotonin and nor epinephrine reuptake. TCAs also blockhistamine H1 receptors, α1-adrenergic receptors and muscarinic receptors, whichaccounts for their sedative, hypotensive and anticholinergic effects [1,2]. In theliterature,therearemethodsdescribedfortheindividualestimationofAlprazolamand Imipramineby spectroscopyand liquid chromatography.A fewmethodshavealso been described for the simultaneous determination of Alprazolamwith otherdrugs such as Propranolol, Fluoxetine HCl, Sertraline, Melatonin [3-14]. A fewmethodswere also described for simultaneous determination of Imipraminewithotherdrugs suchasChlordiazepoxide,Diazepam [15-25]. So,need todevelop fast,Accurate,Precise,SpecificstabilityindicatingRP-HPLCMethodforthesimultaneousestimation of both the drugs and to validate the developed method as per ICHGuidelineQ2(R1)[26-28].

Figure1.AlprazolamFigure2.Imipramine

EXPERIMENTAL

Instrument&Apparatus

A high Performance Liquid Chromatography system S 1122 HPLC (AnalyticalTechnologies) comprised of S 1122 Pump, 2203 UV -Visible detector, ODS-BPhyperchromeC18 (250mm×4.6mm,5µm),Rheodyne injector(20µl)wasused foranalysis.Table1.OptimizedchromatographicconditionParameter OptimizedConditionStationaryphase ODS-BPHyperchromeC18column(250mm×4.6mm,5µm).

Mobilephase Water(pH6adjustedwith1%Ortho-PhosphoricAcid):Methanol:Triethylamine(70:30:0.1%v/v/v)

Diluent SameasMobilePhaseFlowrate 1.0mL/minWavelength 216nm.Injectionvolume 20μl.

OptimizationofStabilityIndicatingRP-HPLCmethod

©2016iSER,EurasianJAnalChem,11(2),101-113 103

Chemicals&Reagents

StandardAlprazolamwasprocured as a gift sample fromAril Pharmaceuticals,Mumbai,Gujarat, India&StandardImipramineprocuredasagiftsamplefromSatiChemicals, Ahmedabad, India. The reagents utilised for analysis are HPLC gradeMethanol,HPLCgradewater,HPLCgradeAcetonitrile,ARgradeOrthoPhosphoricacidandARgradeTriethylamine.

SolubilityDetermination

Solubility of Alprazolam and Imipramine was determined in different solventslikeWater,MethanolandAcetone.

Wavelengthselection

Standard stock solutions of Alprazolam 1 μg/mL and Imipramine 100 μg/mLwere prepared for the selection of wavelength and it was found that both drugsshowed reasonably good response at 216 nm. So, 216 nm was selected as awavelengthforestimation.(Figure3).

PreparationofStockSolution

PreparationofALPStandardStockSolution(10μg/mL)

Accuratelyweighed10mgofAlprazolamwastransferredto100mLvolumetricflask, dissolved and diluted up to mark with mobile phase to obtain finalconcentrationof100μg/mLAlprazolam.Solutionwas furtherdilutedwithmobilephase toobtainstandardstocksolutionsof10μg/mLofAlprazolam.Thissolutionwasfilteredthrough0.45μmmembranefilterpaperandsonicatedfor10minandusedasstandardstocksolution.

PreparationofIMIStandardStockSolution(1000μg/mL)

Accuratelyweighed100mgofImipraminewastransferredto100mLvolumetricflask, dissolved and diluted up to mark with mobile Phase to obtain finalconcentrationof 1000μg/mL Imipramine.This solutionwas filtered through0.45μm membrane filter paper, sonicated for 10 min and used as standard stocksolution.

PreparationofALPandIMI.WorkingStandardSolutions

From the Standard Stock Solution of Alprazolam (10 μg/mL) and Imipramine(1000μg/mL).take1mLsolutionin10mLofvolumetricflaskseparatelyforeachandmakeupwithmobilephase.Thissolutionwascontaining1μg/mLAlprazolamand100μg/mLofImipramine.

Preparationofcombinedstandardsolution

AccuratelyweighedquantitiesofAlprazolam(10mg)andImipramine(1gm)weretransferredinto100mLvolumetricflask.Theyweredissolvedanddiluteduptothemark with mobile phase to give a combined stock solution (100 𝜇g/mL) ofAlprazolam and (10,000𝜇g/mL) of Imipramine. Stock solution (10 mL) wastransferredin100mLvolumetricflaskanddiluteduptomarkwithmobilephasetoobtain combined working standard solution of Alprazolam 10 𝜇g/mL and



Imipramine1000𝜇g/mL.This solutionwasused toprepare standard solution forlinearity.

CalibrationCurveforALPandIMI.

Toestablish the linearityofanalyticalmethod,aseriesofdilutionranging from0.5–1.5μg/mLforAlprazolamand50–150μg/mLforImipraminewereprepared.The combined solution of Alprazolam and Imipramine ranging from 0.5 to 1.5𝜇g/mLand50to150𝜇g/mLwerepreparedbypipettingout0.5,0.75,1,1.25and1.5mLfromthecombinedworkingstandardsolutionofAlprazolam(10𝜇g/mL)andImipramine(1000𝜇g/mL)intoseriesof10mLvolumetricflasksandthevolumewasadjustedtomarkwithmobilephase.Allofthesesolutionswerefilteredandinjected,Chromatogramofeachsolutionwasrecordedanditwasrepeatedforfivetimes.Acalibration graph was plotted between the mean peak area vs. respectiveconcentrationandtheregressionequationwasderived.

METHODVALIDATION

Linearity

Linearitystudywascarriedatfivedifferentconcentrationlevels.Linearityrangeof Alprazolam and Imipraminewas found to be 0.5-1.5 μg/mL and 50-150μg/mL,respectively.

Accuracy

Accuracywascalculatedbyadditionofstandarddrugstopreanalyzedsampleat3 different concentration levels and computing percentage recoveries. Standardlimitof%recoverystudyis98-102%asperICHguideline.Fromthestudiesitwasconcluded that % recovery study of Alprazolam and Imipramine complies withstandardlimitofICHguideline.

Precision

Repeatability

Solutioncontainingmixture1μg/mLAlprazolamand100μg/mLImipraminewasprepared.Preparedsolutionwasanalyzedsixtimesinsamedayaspertheproposedmethod.

Intermediateprecision

Intraday Precision: Solution containing mixture of 0.5, 1, 1.5 μg/mL Alprazolamand50,100,150μg/mL Imipraminewasprepared from their respective standardstocksolution.Analysiswasreplicatedfor3differenttimeswithinsameday.Intraday Precision: Solution containing mixture of 0.5, 1, 1.5 μg/mL Alprazolamand50,100,150μg/mL Imipraminewasprepared from their respective standardstocksolution.Analysiswasreplicatedfor3differentdays.

LimitofDetection(LOD)andLimitofQuantitation(LOQ)

The limits of Detection and Quantification of the developed method werecalculated from the standard deviation of they-intercepts and slope of thecalibrationcurvesofAlprazolamandImipramineusingtheformulaeasgivenbelow.



LimitofDetection=3.3α/SLimitofQuantitation=10α/SWhereαis the standard deviation of they-intercepts andSis the slope of the

calibrationcurve.

Robustness

As per ICH, the prepared solutionwas analyzed as per proposedmethodwithsmallbutdeliberatechangeinchromatographicconditionsaslistedbelow:

• Changeinflowrate:0.8mL/min;1.2mL/min;• Change in mobile phase composition: Water : Methanol (± 2% of Mobile

phasecomposition)• ChangeinmobilephasepH:6.2,5.8.

SystemSuitabilityParameters

System suitability tests were carried out on standard stock solution of ALP (1μg/mL) and IMI (100 μg/mL) and these solutionswere injected under optimizedchromatographic condition. Various parameters like Resolution, Selectivity,AsymmetryandTheoreticalplateswerechecked.

AnalysisofPharmaceuticalFormulation

Twenty tablets were weighed accurately and their average weight wasdetermined.Thetabletswerecrushedtofinepowderandfromthetriturate;tabletpowder equivalent to 100mgof Imipraminewasweighed and transferred to 100mLvolumetricflask.Tothisflask,50mLmobilephasewasaddedandtheflaskwassonicatedfor15min.Thevolumewasadjusteduptothemarkwithmobilephase.The solution was then filtered through 0.45 μm membrane filter paper. Filtratecontained mixture of 10 μg/mL Alprazolam and 1000 μg/mL Imipramine. Thefiltratedsolutionwassuitablydilutedwithmobilephasetogetafinalconcentrationof1μg/mLofAlprazolamand100μg/mLof Imipramineandsonicated for5min.Prepared solution was injected to system. The chromatogram was stopped afterseparationachievedcompletely.ConcentrationofAlprazolamandImipraminewerecomputed by putting value of their peak areas in respective standard regressionequationobtainedfromcalibrationcurve.

ForcedDegradationstudy

Forced degradation studieswere performed to evaluate the stability indicatingproperties (Specificity) of the proposed method. ALP (API) and IMI (API)individually and its standard mixtures were subjected to Acid, Base, Oxidation,Thermal & Photo Degradation to ensure the effective separation of degradationpeaksandmainpeaks.

ForceddegradationStandardstockpreparation

Alprazolam (15 mg) and Imipramine (150 mg) were accurately weighed andtransferredtoa100mLvolumetric flask,dissolvedinsufficientquantityofMobilePhase(Water:Methanol :Triethylamine)(70:30:0.1%v/v/v)andthendiluteduptothemarkwithMobilephase.(ThesolutioncontainsALP150μg/mL&IMI1500μg/mL)AndFurtherDilutethe10mLofAlprazolamSolutionin100mLVolumetricFlaskandmakeuptothemark.(Thesolutioncontains15μg/mLofALP).ThefinalsolutionwaslabeledasStandardStockSolution.



Forceddegradationworkingstandardpreparation

From Standard Stock Solution take 1 mL of ALP and 1 mL of IMI in 10 mLVolumetricFlaskandmakeuptothemarkwithMobilePhase.(1.5μg/mLALPand150μg/mLIMI)ThissolutionwaslabeledaWorkingStandardSolutionwhichisuseforforcedegradationstudyandchromatographed.

Aciddegradation

1mLofstandardstocksolutionwastransferredinto10mLofvolumetricflask.2mLof0.1MHClsolutionwasaddedandmixedwell.Thevolumetricflaskwasheatinwaterbathat70°Cfor2hrs.Aftertimeperiodthecontentwascooledtoambienttemperature.Theabovesolutionwasneutralizedwith2mLof0.1MNaOHsolutionandthendilutedthevolumewithMobilePhaseandchromatographed.

Basedegradation

1mLofstandardstocksolutionwastransferredinto10mLofvolumetricflask.2mLof0.1MNaOHsolutionwasaddedandmixedwell.Thevolumetricflaskwasheatinwaterbathat70°Cfor2hrs.Aftertimeperiodthecontentwascooledtoambienttemperature.Theabove solutionwasneutralizedwith2mLof0.1MHCl solutionandthendilutedthevolumewithMobilePhaseandchromatographed.

OxidationDegradation

1mLofstandardstocksolutionwastransferredinto10mLofvolumetricflask.2mL3%H2O2wasaddedandheat thevolumetric flask for2hrsat70°C,after timeperiod content was cooled to ambient temperature and diluted to volume withMobilePhaseandchromatographed.

PhotoDegradation

1mLofstandardstocksolutionwastransferredinto10mLofvolumetricflask.Andplacethevolumetricflaskintothesunlightfor4hrs,aftertimeperiodcontentwascooled to ambient temperature and diluted to volume with Mobile Phase andchromatographed.

ThermalDegradation

200mgpowderofAlprazolamandImipraminewereheatedinovenat105ºCfor4 hrs. After time period the content was cooled to ambient temperature andpreparedthesolutionasperworkingstandardsolutionandchromatographed.All these solutions were filtered through the 0.45 μ membrane filter before

injecting.

RESULTANDDISCUSSION

Methoddevelopment

Fortheselectionofanalyticalwavelengthforthequantificationofthedrugs,thestandardsolutionofAlprazolam1μg/mLandImipramine100μg/mLwerescannedin UV-Visible spectrophotometer and their overlain spectra were shown strongabsorbance at about 216nmwhichwas selected as the analytical wavelength forfurther analysis (Figure 3). For the effective separation of Alprazolam and



Imipramine, attempts were made by using mobile phases containing solvents ofvarying polarity, at different concentration level with implicating ODS-BPHyperchromeC18column(250mm×4.6mmid,5μmparticlesize)asastationaryphase. Various mobile phase systems like water: methanol, water: Acetonitrile,Water: Methanol: 0.1% Triethylamine at different concentration levels withdifferentpHwas tried.Among thedifferentmobilephasecombinationsemployed,bestresolutionwithsharpwelldefinedpeaksobtainedwithmobilephasecomposedof Water (pH:6): Methanol : 0.1% Triethylamine in the ratio of 70:30:0.1 v/v/v(Table1).The typical retention timeofAlprazolamand ImipramineWere found tobeabout3.207minand5.073min.ArepresentativechromatogramofstandardandtestwasshowninFigure4(a)and(b).

Figure3.OverlaidSpectraofAlprazolam(1μg/mL)andImipramine(100μg/mL)inMethanol

Figure 4(a).Chromatogram of Alprazolam (1 µg/mL) and Imipramine (1 µg/mL)standardinOptimizedCondition{Water(pH6):Methanol:Triethylamine:70:30:0.1%v/v/v}

λ : 216 nm



Figure4(b).ChromatogramofSampleAlprazolam(1µg/mL)&Imipramine(100µg/mL){Water(pH6):Methanol:Triethylamine:70:30:0.1%v/v/v}Table1.OptimizedmobilephaseconditionMobilePhase FlowRate RtAlprazolam RtImipramine ResolutionWater(pH-6):Methanol:Triethylamine(70:30:0.1%v/v/v)

1mL/min 3.207 5.073 9.692

MethodValidation

TheproposedRP–HPLCmethodwasvalidatedasperICHGuidelines.

LinearityandRange

ThelinearrelationshipwasexistedbetweenconcentrationandareaforALPandIMIintheconcentrationrangeof0.5-1.5μg/mLand50-150μg/mL,respectively.ThecorrelationcoefficientforAlprazolamwas0.9999andforImipraminewas0.9998.ArepresentativedataoflinearityisshowninTable2.

Table2.ResultofLinearityforRP-HPLCMethod(n=5)Parameter Alprazolam ImipramineRange 0.5–1.5 50–150Linearity Equation y=153.75x-1.9981 y=50.922x-80.285

R2 0.9999 0.9998%RSD 0.23–0.50 0.26–1.84

LOD(μg/mL) 0.010 2.202LOQ(μg/mL) 0.031 6.673

Accuracy(StandardAdditionMethod)

Result obtained reveals that % recovery of ALP and IMI was found to be100.10–100.94and100.39–100.74respectively(Table3).

Table3.ResultofaccuracyforRP-HPLCMethod(n=3)Drug %ofStdAdded %Recovery(Mean±SD) %RSDAlprazolam 80% 100.94±0.82 0.81

100% 100.10±1.05 1.05120% 100.27±1.27 1.26

Imipramine 80% 100.39±1.53 1.53100% 100.66±0.66 0.65120% 100.74±1.06 1.05



Precision

For repeatability, % RSD was found to be 0.41 and 1.40 for ALP and IMI,respectively.Forintradayprecision,%RSDwasfoundtobe0.91–1.29%and0.72–1.07%forALPandIMI,respectively.Forintradayprecision,%RSDwasfoundtobe0.25–0.52%and1.17–0.27%forALPandIMI,respectively(Table4,5,6).

Table4:ResultofRepeatabilityforRP-HPLCMethodDrug Area

(Mean,n=6)SD

(n=6)%RSD

Alprazolam 152.4613 0.62 0.41Imipramine 5072.426 71.17 1.40

Table5:ResultofIntradayPrecisionforRP-HPLCMethod(n=3)Drug Conc.

inμg/mLArea

(Mean,n=3)SD

(n=3)%RSD

Alprazolam 0.5 73.955 0.453 0.613

1 151.7733 0.304 0.2001.5 227.582 0.456 0.200

Imipramine 0.5 2438.4366 34.975 1.4351 5028.7466 7.485 0.1481.5 7547.5916 12.828 0.169

Table6:ResultofInterdayPrecisionforRP-HPLCMethod(n=3)Drug Conc.

inμg/mLAREA

(Mean,n=3)SD

(n=3)%RSD

Alprazolam 0.5 73.9626 0.39 0.52

1 152.069 0.39 0.251.5 228.206 0.81 0.35

Imipramine 0.5 2397.355 6.48 0.271 5003.295 58.69 1.171.5 7554.571 11.99 0.15

Robustness

Variationintheflowrate,mobilephase,andpHhasbeenmadetotheanalyticalmethod in order to evaluate and measure the capacity of the method to remainunaffectedbysuchvariations.The%RSDwasfoundtobelessthan2.(Tables7).

Table7:ResultofRobustnessforRP-HPLCMethod(n=3)VariationandLevel Conc.inμg/mL MeanArea±SD %RSD

ALP IMI ALP IMI ALP IMIChangeinFlowRate

0.8mL/min 1 100 158.35±0.92 5250.79±28.77 0.58 0.541.2mL/min 1 100 149.02±0.91 4935.34±24.96 0.61 0.50

ChangeinpH 5.8pH 1 100 156.61±0.92 519279±27.55 0.58 0.536.2pH 1 100 149.07±1.05 4936.99±31.11 0.72 0.64

ChangeinMobilephaseComposition

+2% 1 100 150.66±1.42 5021.72±98.39 0.94 1.95-2% 1 100 149.59±1.54 4946.80±59.46 1.03 1.20

LODandLOQ

LODwerefoundtobe0.010and2.202𝜇g/mLforALPandIMIrespectively.LOQwerefoundtobe0.031and6.673𝜇g/mLALPandIMIrespectively.

SystemsuitabilityParameters

System suitability was established to determine the adequate resolution andreproducibility of the proposed method. Parameters including Retention time,Tailingfactor,ResolutionandNo.oftheoreticalplateswerecalculated.(Table8).



Table8:ResultsofSystemsuitabilityparametersforRP-HPLCMethodSystemsuitabilityparameters Drug

Alprazolam ImipramineResolution(R

s) 9.7068

RelativeRetentionorSelectivityFactor(α) 3.1812 5.045TailingfactororSymmetryfactor(T) 1.278 1.380ColumnefficiencyorTheoreticalPlates(N) 7450.6±25.55 7193±34.14

AssayofmarketedFormulation

PercentagepurityofALPandIMIwasfoundtobe100.51%and99.%forALPandIMI,respectively(Table9).Table9:AssayofAlprazolamandImipramineinmarketedformulation(ALZOSET)(n=3)Drug %Assay

MeanSD

%RSD

Alprazolam 100.51 0.94 0.93Imipramine 99.00 0.16 0.16

Forceddegradationstudies

From thedegradationof these solutionsunder the stress condition givesus anidea about the origin of degrading products. Degradants did not show anyinterferencewiththeelutionofdrugpeaks.Hence,themethodisstabilityindicating.

Table10.SummaryofforcedegradationstudiesStressCondition

Alprazolam ImipramineArea R.T %Degradation Area R.T %Degradation

Assuch 294.427 3.210 --- 7796.094 5.150 ---Acid 258.821 3.187 12.1 7043.264 5.143 9.65Base 263.779 3.230 10.40 6357.070 5.163 18.46Oxidation 261.202 3.187 11.28 6927.428 5.087 11.14PhotolyticCondition 264.828 3.193 10.06 6601.144 5.073 15.33

ThermalCondition 247.657 3.213 15.89 7042.034 5.083 9.67

Figure5.Degradationpeaksofsamplesolutioninacidcondition



Figure6.Degradationpeaksofsamplesolutioninbasecondition

Figure7.Degradationpeaksofsamplesolutioninoxidationcondition

Figure8.Degradationpeaksofsamplesolutioninphotolyticcondition

Figure9.Degradationpeaksofsamplesolutioninthermalcondition

CONCLUSION

A simple, Accurate and Precise stability indicating HPLC method has beendevelopedforestimationofImipramineandAlprazolamintabletdosageform.TheRP-HPLCMethodforImipramineandAlprazolamisvalidatedforvariousparameterlike specificity, Linearity, Range, LOD, LOQ, Robustness, Precision Accuracy.Linearityofthedevelopedmethodwasnearto1,rangewasfound0.5-1.5μg/mLforAlprazolamand50–150μg/mLforImipramine.%RSDwasfoundtobelessthan2for repeatability, intradayprecisionand intermediateprecision,Robustness. Forcedegradationstudyofdrugs incombineddosage formwascarriedoutaccording toICHguidelineQ1A(R2).IndegradationstudyitwasfoundthattheAlprazolamwas



moresusceptibleunder stress condition in comparisonwith Imipramineexcept inthermal condition. So, the Degradation study by the RP-HPLC method can besuccessfully applied for the simultaneous estimation of these drugs in combineddosageform.Thepeaksofthedegradantsineachconditionwerewellresolvedfrommainpeaks.Thereisnointerferenceofanydegradantsattheretentiontimeofthemain peaks indicates that the developed method is stability indicating. Theproposed method can be used as an alternative method for the analysis ofAlprazolamandImipramineinitsFormulation.

ACKNOWLEDGEMENT

The authors are thankful to the Institute Sardar Patel college of Pharmacy,Bakrol,Indiaforprovidingnecessaryfacilitiestocarryoutresearchwork.

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