Stability Indicating RP-HPLC Method For The Estimation Of .

Available online on www.ijpqa.comInternational Journal of Pharmaceutical Quality Assurance 2016; 7(3); 46-50ISSN 0975 9506Research ArticleStability Indicating RP-HPLC Method for the Estimation of MetforminHydrochloride and Repaglinide as API and Estimation in TabletDosage FormSonia K1*, Nappinnai M2, Manikandan K11Department of Pharmaceutical Analysis, SRM College of Pharmacy, Kattankulathur.Department of Pharmaceutics, Surya College of Pharmacy, Vikravandi, Villupuram2Available Online:15th September, 2016ABSTRACTA new simple, precise, sensitive, economical RP-HPLC method was developed for the estimation of Metforminhydrochloride and Repaglinide in bulk and tablet dosage form. The λmax for Metformin hydrochloride and Repaglinidewas found to be nm and nm respectively. The linearity in the concentration range of 25µg/ml to 150 µg/ml (r2 0.999) and0.1 to 0.6 µg/ml (r2 0.999) for Metformin hydrochloride and Repaglinide respectively. The chromatographic values forMetformin hydrochloride and Repaglinide were found to be satisfactory on BDS Hypersil 18, 250 x 4.6mm, 5µm, columnusing mobile phase of ACN: Buffer in the ratio of 58: 42 v/v with the flow rate of 1.0ml/min. The developed method wasvalidated according to ICH guidelines and found to be accurate, precise, rugged and robust and found to be in goodaccordance with the prescribed values. The newly developed method can be used for routine analysis estimation ofMetformin hydrochloride and Repaglinide in bulk and tablet dosage form in pharmaceutical industry.Keywords: Metformin hydrochloride, Repaglinide, Stability studies, method development and validation.INTRODUCTIONGlucose is the obligatory source of energy for the brain andphysiological control of blood glucose reflects the need tomaintain adequate fuel supplies in the face of intermittentfood intake and variable metabolic demands. Increasedblood sugar causes increased insulin secretion, whereasreduced blood sugar reduces insulin secretion. Insulin isthe main hormone controlling intermediary metabolism.It’s most obvious acute effect is to lower blood glucose1-2.Metformin hydrochloride is the drug of choice inBiguanide classification. It is chemically 1, 1-dimethylbiguanide hydrochloride3 (Figure 1). Metformin lowersblood glucose additionally reduces low density and verylow density lipoprotein LDL and VLDL respectively4.Repaglinide is chemically(S)-( )-2 –ethoxy-4-[2-(3methyl-1-[2-(piperidin-1-yl) phenyl] butylamino)-2oxoethyl] benzoic acid5 (Figure 2). It acts by blocking thesulfonyl urea receptor on K ATP channels in pancreatic Bcell membranes6. From literature review shows that thereis developed method including UV7-8, Fluorometric9,HPTLC10, LC-MS11-12 methods. The review of theliterature reveals that the present study is to develop andvalidate a stability indicating HPLC method for metforminhydrochloride and Repaglinide according to ICHguidelines13.MATERIALS AND METHODSThe reagents were used are of Analytical grade. HPLCModel Shimadzu equipped with BDS Hypersil C18 (4.6 x*Author for Correspondence: soniapharm68@yahoo.com250mm, 5 m) Column and pump of LC-20 AT VP serieswith injector of Rheodyne.Optimization of Chromatographic ConditionsThe chromatographic conditions were optimized toAcetonitrile: NaH2PO4 ratio 58:42, Flow rate: 1.0 ml/minand Wave length: 231 nm.Preparation of standard solution of MetforminHydrochloride and Repaglinide250 mg Metformin hydrochloride and 1 mg Repaglinidewas taken and made up the volume to 100 ml.Preparation of standard solution of RepaglinideAccurately weighed 10mg Repaglinide of transferred toFigure 1: Metformin hydrochlorideFigure 2: Repaglinide

Sonia et al. / Stability Indicating RP-HPLC Figure 3: the chromatogram of both the standard and sample.Figure 4: the chromatogram of both the standard and sample.Table 1: Standard stock solution of Repaglinide /Metformin nideHydrochloride1.Water 2.Acetonitrile 3.0.1N NaOH 4.0.1N HCl 5.Methanol 10ml volumetric flask volume made up to 10ml usingmobile phase. Pipetted 0.1ml and made up to the markusing mobile phase.SolubilitySolubility of drugs was observed by dissolving it indifferent solvents and it wasfound that drugshaving good solubility in following solvents. The table 1shows the different solubility of Metformin hydrochlorideand Repaglinide.Standard stock solution of Repaglinide / MetforminHydrochlorideAccurately 10 mg of Repaglinide / Metformin HCl wasweighed into a clean and dry 10 ml volumetric flask,dissolved with sufficient volume of mobile phase and thenmade up to the volume with mobile phase. The maximumwavelength was found to be 231 nm. The chromatogramof both the standard and sample in Figure 3.AssayWeigh about 20 tablets and powdered. Powder equivalentto 500mg and 2mg of Metformin HCl and Repaglinidewere taken into 50 ml volumetric flask. Add about 10 mlof mobile phase and sonicated until the contents weredissolved. Filter the contents by using 0.45µ membranefilter under vacuum. Make up to the mark with mobilephase. Inject 20µl of sample solution into thechromatographic system. Measure the area of MetforminHCl and Repaglinide and calculate the percentage of assay.StandardMarket sample analysisLinearityTo 250 mg Metformin hydrochloride and 1 mgRepaglinide was taken and made up the volume to 100 ml.pipette out 1, 2, 3, 4, 5, and 6ml from the stock solutionand made up the volume using 100 ml respectively. Theresults were tabulated in Table 2 and Chart showing thelinearity of Repaglinide and Metformin hydrochloride inFigureAccuracyFrom the Standard stock solution pipette out 5 ml andmake upto 100 ml (Spiking Standard Solution). From thestandard stock solution pipette out 1, 2, 3, 4 and 5mlIJPQA, Volume 7, Issue 3, July 2016 - September 2016Page 47

Sonia et al. / Stability Indicating RP-HPLC Table 2:Sample No.1.2.3.4.5.6.MeanLabel Claim (mg)Metformin HClRepaglinide5002500250025002500250025002Amount present (mg)Metformin 1.97499.871.99499.831.99499.871.99Standard DeviationMetformin HCl .0122310.0186630.012235Figure 5: The Linearity chromatogram of both Metformin hydrochloride and Repaglinide6(a)6(b)Figure 6: The chart showing the Linearity of both Metformin hydrochloride and RepaglinideTable 3: Linearity data of Repaglinide / MetforminHydrochlorideS.Concentration Peak Area StatisticalNO µg/mlAnalysis1.0.124.4592.0.249.334Slope (m): 247.293.0.370.5564.0.497.251Intercept icient (R2):0.999respectively and made up the volume using 100ml(Standard Stock Solution). The average % Recovery ofRepaglinide and Metformin HCl was found to be 99.80 and99.70 respectively. The results were tabulated in table 4.PrecisionThe closeness of agreement (degree of scatter) between aseries of measurements obtained from multiple samplingsof the same homogeneous sample. Should be investigatedusing homogeneous, authentic samplesRepeatabilityEstablished the repeatability of the analytical method byestimating the assay for 5 sample proportion of the samebatch under normal operating conditions. Calculated theassay for all 5 sample preparation and reported the %RSDfor the sample. The results were tabulated in table 4.RuggednessIntermediate precision (ruggedness)IJPQA, Volume 7, Issue 3, July 2016 - September 2016Page 48

Sonia et al. / Stability Indicating RP-HPLC Table 4: Accuracy studies for Repaglinide and Metformin HClS.Mixture of pure andCon. of pure drug,NoDrugformulationµg/ml1.Repaglinide0.4 0.050.052.0.5 0.050.053.0.6 0.050.051.Metformin100 12.512.52.HCl125 12.512.53.150 12.512.5Table 5: Repeatability of Repaglinide and Metformin HClMetformin HClS. No.Retention 74AverageStd. Deviation 0.0186634.517515Std. Deviation%RSD0.890.88%RSDTable 6: Ruggedness for Repaglinide and MetforminHClAnalystRetention time ofRetention time ofRepaglinide (min)Metformin Hcl(min)Analyst 15.5832.193Analyst 25.5832.193Table 7: Robustness of Repaglinide and MetforminHClWave lengthRT ofRT of MetforminRepaglinideHcl228 nm5.4372.057232 nm5.3372.050Flow rateRT ofRT of MetforminRepaglinideHcl0.9 mL/ min4.9035.6401.1 mL/ min2.4201.907Table 8: Limit of Detection and Limit of Quantizationof Repaglinide and Metformin 6169.000HydrochloridePlates (N)2LOD, µg/ml3.732203LOQ, s (N)2LOD, µg/ml0.012913LOQ, µg/ml0.04030Intermediate precision study was carried out by repeatingthe complete experiment with different analysts, ondifferent days in same laboratory as per the followingpreparation. To 250 mg of Metformin hydrochloride and 1Conc. of 2RepaglinideRetention time6.0075.9875.996.0075.985.99420.0122350.20% Recovery ofpure 187127.642127.376126.796126.6750.9677130.76mg of Repaglinide made up the volume to 100 ml. thesampleMethod PrecisionAbility to reproduce data within the predefined precisionis called method precision. The standard and sample wasprepared and readings were taken.RobustnessCapacity to remain unaffected by small but deliberatevariations in method parameters. Variations may include:stability of analytical solution, variation of pH in a mobilephase, different column (lot/supplier), temperature, andflow rate. From the standard stock solution and sample, therobustness can be measured with different flow rate andwavelength. The method remains unaffected by the smalldeliberate changes that were introduced. the results weretabulated in table 7.SpecificityTreating with AcidsTo 1 ml from the stock solution into a 10 ml volumetricflask. To that 1 ml of 0.1M hydrochloric acid added.Observed for any change took place in the retention of thepeak.Treating with BaseTo 1 ml from stocks solution into a 10 ml volumetric flask.To this 1 ml of 0.1 M sodium hydroxide were added andObserved for any degradedness.HeatingFor the specificity study 1 ml from the stock solutionshould be taken in a 10 ml flask, make up to the volumewith the mobile phase. The solution should be heated at40oC for a period of 30 min. Observed for any degradationoccurs or not. From the specificity performed, variousdegradation products are formed and there is no change inthe detection of the analyte in the presence of othercomponents.Limit of Detection and Limit of QuantizationIJPQA, Volume 7, Issue 3, July 2016 - September 2016Page 49