Method Development And Validation Of Forced …

1 International Journal of PharmTech ResearchCODEN (USA): IJPRIF ISSN : , , pp1750-1757, Oct-Dec2012 Method Development And Validation Of ForcedDegradation Studies Of Pioglitazone HydrochlorideBy Using UV SpectroscopyNarsimha rao. Doredla*, ,Raju. Bojjagani, Syam vardhan. MadasuDepartment of Pharmaceutical Analysis Sarada College of PharmaceuticalSciences, Kondakavuru, Narasaraopet, Guntur No: +91-9985361467 Abstract:Pioglitazone hydrochloride is an oral antidiabetic agent used in the treatment of type 2 diabetesmellitus and also known as non-insulin dependent diabetes mellitus (NIDDM) or adult onset , an accurate and economic, precise and reproducible UV Spectroscopy Method has been developedfor the estimation ofPioglitazone hydrochloride tabletdosage form and validated by ICH guidelines. Thestandard (10 g/ml) was scanned between 200-400 nm and maximumabsorption was recorded at 270 assay results are found to be The linearity range of 10-50 g/ml proved that it obeyedBeer s Law and the correlation coefficient (r2) was found to be at 270 nm with an intercept a slope of with RSD complied pH degradation studies of tablet formulationwere found to be less at pH 6-8.

2 The force degradation studies of pioglitazone tablet formulation was doneonStress degradation by hydrolysisunder alkaline condition by using NaOH was found to be 60min, for 90min. Stress degradation by hydrolysis under acidic conditionby using 3N HCl andproduct degradation was found to be for 60min and for 90min. Dry heat induceddegradation was done by using 700c temperature was found to for 48 degradationwas done by using hydrogen peroxide and product degradationwas found to be 15 degradationwas found to for3hrs and : Pioglitazone Hydrochloride, UV Spectroscopy, PH degradation, is an oral anti diabetic agentbelonging to the class of thiazolidinediones thatacts primarily by decreasing insulin resistance. Itis used in the management of type 2 diabetesmellitus. It improves sensitivity to insulin inmuscle and adipose tissue and inhibits hepaticgluconeogenesis also improves glycemic controlwhile reducing circulating insulin [( )-5-[[4-[2-(5-ethyl-2- pyridinyl)ethoxy] phenyl] methyl]-2,4-thiazolidinedionemonohydroch loride belongs to a differentchemical class and has a different pharmacologicalaction than the sulfonylureas, metformin, or the-glycosidase inhibitors(1).]

3 The chemical structurefor Pioglitazone Hydrochloride was show in 1:Chemical structure of PioglitazoneHClNarsimha rao. Doredla et ,4(4)1751 Determination of pioglitazone by variousanalytical methods like Spectrophotometricmethod(2) and HPLC and MECK Method (3) intablet dosage form, HPLC and solid phaseextraction Method in human serum(4) and in dogserum(5), HPLC and LC MS in human plasma(6), (7)have been reported. Pioglitazone is not official inany pharmacopoeia. There is a need for a simple,rapid, cost effective and reproducible Method forassay of pioglitazone in its dosage formsBut therewas no reported Method for the Forceddegradation studies of pioglitazone hydrochlorideby using UV spectroscopy. So the present work isto carry out the force degradation studies alongwith itspH degradationstudies. The Method wasvalidated according to the ICH (Q2A1995)guidelines (8) Forced degradation studies may helpfacilitate pharmaceutical Development as well inareas such as formulation Development ,manufacturing,and packaging, in whichknowledge of chemical behaviour can be used toimprove a drug product.

4 The available regulatoryguidance provides useful definitions and generalcomments about degradation studies(9). TheInternational Conference on Harmonization (ICH)guidelines(10-11)indicates that stress testing isdesigned to determine the intrinsic stability of themolecule by establishing degradation pathway inorder to identify the likely degradation productsand to validate the stability indicating power oftheanalytical procedure used. ICH guidelines stabilitytesting of new drug substances and products Q1A(R2)(10)and (Q1B)(11)requires that stresstesting should be carried out to elucidate thesubstance. It suggests that the degradationproducts that areformed under the variety ofcondition should include the effect of temperature,appropriate oxidation, photolysis and susceptibilityto hydrolysis across a wide range of pH value.

5 Inthe guideline, the study of effect of temperature issuggested to be done in 100C increment above theaccelerated temperature (500C, 600C etc.) andthat of humidity at a level of 75 % or details are however provided for the studyof oxidation, photolysis and hydrolysis at differentpH values(12)EXPERIMENTALI nstrumentAbsorption spectral measurements were carriedout with a UV Visible spectrophotometer(Shimadzu Model 1700) using UV Probe softwareversion 2 was employed with spectral bandwidthof 1 nm and wavelength accuracy of nm (withautomatic wavelength correction with a pair of 5cm matched quartz used for dilution was distilled in thelaboratory. A double beam UV spectrophotometer(Shimadzu UV-1800) was used with 1 cm matchedquartz cell. Tablet formulation [Pioz 15, USV Ltd.,Baddi, Solan- Dist, Himachal pradesh] wereprocured from a local pharmacy with labeledamount 15 mg per solubility study of drugSolubility of the drug was determined at 28 1 small quantity of standard drugs were dissolvedin different solvents like distilled water, methanol,ethanol, acetonitrile, isopropyl alcohol, dimethylsulfoxide, dimethyl formamide, N HCl,chloroform, acetonitrile and pH4, 7, The results are reported in table 1: Solubility data for Pioglitazone water (HPLC and spectroscopicgrade)In soluble3 MethanolFreely methyl sulfoxideFreely methyl propyl PH 4 solutionIn PH 7 solutionIn solubleNarsimha rao.)

6 Doredla et ,4(4)1752 Solvent Selection:Various solvents were selected for the solubilitystudies and it was found that Pioglitazone wassoluble in the following solvents; dimethylsulfoxide, dimethyl formamide, methanol, NHCl, chloroform, acetonitrile, the presentinvestigation methanol was selected as a of analytical wavelength andabsorption maxima:Appropriate 10 g/ml dilutions were prepared fordrug from the standard stock solution and thesolutions were scanned in the wavelength range of200-400 nm. The absorption spectra thus obtainedwas derivatized for zero order spectroscopy. Thiszero order spectrum was selected for the analysisof the drugs. The absorption maximum was foundat 270 nm which can be further used for analysisas shown of stock solutions:Standard Pioglitazone 100mg was weighed andtransformed to a 100 ml volumetric flask anddissolved in 25 ml of methanol.

7 The flask wasshaken and volume was made up to the mark withmethanol to give a solution containing 1000 g/ml(Stock solution A). From this stock solution A,pipette out 5 ml and place into 50 ml volumetricflask. The volume was made up to the mark withmethanol to give a solution containing 100 g/ml(Stock solution B)Selection of analytical concentration range:From the standard stock solution B ofPioglitazone, appropriate aliquots 1, 2, 3, 4 and 5ml were pipetted out in 10 ml volumetric flasksand dilutions weremade with methanol to obtainworking standard solutions of concentrations from1-50 g/ml. Absorbance for these solutions weremeasured at 270 nm. For standard solutionanalytical concentration range was found to be 1-50 g/ml and overlain spectra was obtained andoptical characteristic and linearity data wasreported in table curve for thePioglitazone:Appropriate volumes of aliquots from standardPioglitazone stock solution B were transferred todifferent volumetric flasks of 10 ml capacity.

8 Thevolume was adjusted to the mark with methanol toobtain concentrations of 10, 20, 30 ,40 and50 g/ml. Absorbance value of each solutionagainst methanol as a blank were measured at 270nm. From that absorbance value, regressionequation and correlation coefficient (r2) aredetermined and 2: UV Spectra of Pioglitazone StandardNarsimha rao. Doredla et ,4(4)1753 Table 2: UV Optical characteristic and linearity dataParametersPioglitazone max (nm)270 Beer s law limits in equationY=mx+cY= + (c) 3: UV Linearity graph of PioglitazoneAnalysisof Pioglitazone fromTabletDosageform:Twenty tablets of formulation were weighed andfinely powdered. The powder equivalent to 100mg of Pioglitazone was accurately weighed. It wasthen transferred to volumetric flask of 100 mlcapacity containing 25 ml of methanol andsonicated for 30 min.

9 The flask was shaken andthe solution was filtered through Whatmann filterpaper (No. 41) into 100 ml volumetric was made up to the mark with methanolto give a solution of 1000 g/ml (Stock solutionA). From this solution 5 ml was takenand placedin 50 ml volumetric flask. The volume was madeup to the mark using methanol to give a solution of100 g/ml(Stock solution B). From the stocksolution B, ml was taken and diluted to 10 mlto give 20 g/ml and it was further used for theestimation of Pioglitazone. The result was reportedin Table 3: Analysis data of Tablet formulation by UVDrugLabelclaimmg/tabAmountfoundmg/tabL abel claim(%) *% * : Pioglitazone, :Standard deviation, COV: Coefficient of variation, : Standard errorNarsimha rao. Doredla et ,4(4)1754 Method VALIDATIONV alidation parameters:The Method was validated with reference toaccuracy, precision, and accuracy of the proposed methods wasassessed by recovery studies at three differentlevels 80%, 100%, 120%.

10 The recovery studieswere carried out by adding known amount ofstandard solution of the drug to preanalysed tabletsolutions. The resulting solutions were then re-analysed by proposed methods; the results arereported in Table of the methods was studied as intra-day,interday and repeatability. Intra-day study wasperformed by analyzing, the three differentconcentration of drug for three times in the sameday. Inter-day precision was performed byanalyzing three different concentration of the drugfor three days in a week. Repeatability wasperformed by analyzing same concentration ofdrugs for six times. The results are reported inTable of the proposed Method is determinedby analysis of aliquots from homogenous slot bydifferent analysts using similar operational andenvironmental conditions. The results arereportedin Table 4: Results of Accuracy studies by UV spectroscopyLevel ofrecoveryAmount ofsample ( g/ml)Amount ofdrug added( g/ml)**Amount ofdrug recovered( g/ml)**%Recovery **80% ** is average of six determinationsTable 5: Precision study data of Pioglitazone by UV spectroscopyConcentration( g/ml)Inter-dayAbsorbancemean SD**% RSDI ntra-dayAbsorbancemean SD**% ** is average of six 6: Ruggedness study data of Pioglitazone by UVAnalyst 1 Analyst 2 Sample Labelclaim(mg)Amountfound(mg)% Recovery SD**Amountfound(mg)% Recovery SD** ** is average of six determinationsNarsimha rao.