UV Spectrophotometric Simultaneous Determination of Metformine Hydrochloride and Pioglitazone Hydrochloride in Combined Dosage Form

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1 ISSN- (Print) ISSN- (Online) RESEARCH ARTICLE UV Spectrophotometric Simultaneous Determination of Metformine Hydrochloride and Pioglitazone Hydrochloride in Combined Dosage Form Sonali D. Rathod*, P.M. Patil, S. B. Jadhav and P.D. Chaudhari. Department of Pharmaceutical Chemistry. Modern Collage of Pharmacy, Nigdi, Pune. *Corresponding Author ABSTRACT: Simple, sensitive, specific and economic spectrophotometric method was developed and validated for simultaneous quantitation of Metformine Hydrochloride and Pioglitazone Hydrochloride in tablet dosage form. New method based on the simultaneous estimation of drugs in a binary mixture without previous separation was developed. In simultaneous equation method, Metformine Hydrochloride and Pioglitazone Hydrochloride were quantified using their absorptivity values of at selected wavelengths, viz., nm and. nm respectively. The accuracy and reproducibility of the proposed method was statistically validated by studies. The simultaneous equation method permits simple, rapid and direct determination of Metformine Hydrochloride and Pioglitazone Hydrochloride in commercially available tablet dosage form without previous separations and can therefore be used for routine analysis of both drugs in quality control laboratories. KEYWORDS: Metformine Hydrochloride and Pioglitazone Hydrochloride, Vierodt s method.. INTRODUCTION: Diabetes is one of the costliest health problems in the world. Globally, diabetes is likely to be the fourth leading cause of death []. Approximately 0% of people with diabetes have type diabetes. It usually begins as insulin resistance, a disorder in which the cells do not use insulin properly. As the need for insulin rises; the pancreas gradually loses its ability to produce insulin. Type II diabetes is associated with older age, obesity, family history of gestational diabetes, impaired glucose metabolism, physical inactivity and race/ ethnicity []. If the glycemic target level is not achieved with one oral agent alone, combination oral and/or insulin therapy is recommended [, ]. Combination oral therapy becomes an obvious choice when glycemic control is not achieved with conventional monotherapy []. The advantages of oral dose combinations as compared to their components which are taken alone are lower cost and better patient compliance [, ].Combination therapy has been shown to achieve greater blood glucose lowering than monotherapy because different classes have different and complimentary mechanisms of action. Therefore, it is more logical to add another drug than replace the existing drug. The rapid introduction of combination therapy with two or three complementary oral anti diabetics help in targeting the dual effect and also reduced adverse effects []. Pioglitazone is a thiazolidine dione derivative and it is used in patient with NIDDM type of antidiabetic, chemically it is -(-[-(-ethypyridin--yl] benzyl) thiazolidine-, -dione. The molecular formula is CH0NOS. Chemically, metformin is,-dimethyl biguanide hydrochloride, pioglitazone is (± )--[p- [-(- ethyl--pyridyl)-ethoxy] benzyl]-,-thiazolidinedione. Metformin improves hepatic and peripheral tissue sensitivity to insulin without the problem of serious lactic acidosis, pioglitazone has been shown to affect abnormal glucose and lipid metabolism associated with insulin resistance by enhancing insulin action on peripheral tissues.[] Fig shows that the structures of Pioglitazone [a] and Metformine[b] Received on 0..0 Accepted on.0.0 Asian Pharma Press All Right Reserved Asian J. Pharm. Ana. (): Jan.-Mar. 0; Page 0-0 [a] Pioglitazone

2 [b] Metformine Fig : Structures of Pioglitazone [a] and Metformine[b]. MATERIAL AND METHODS:.. Apparatus A double beam UV/Vis spectrophotometer, Shimadzu UV- 00 Pharmaspec, was employed with a pair of cm quartz cells for all analytical work. Graph : Pioglitazone Hydrochloride.. Reagents and chemicals Metformine Hydrochloride and Pioglitazone Hydrochloride were obtained from Gen Pharmaceuticals Ltd.Pune, Maharastra, India as a gift sample and were used as working standards. Sodium hydroxide of analytical grade and double distilled water were used throughout the analysis... Commercial formulation A commercial pharmaceutical preparation, Glime P tablet (Metformine Hydrochloride 00 mg and mg Pioglitazone Hydrochloride) was procured from the local market... Preparation of standard solution Standard stock solution of Metformine Hydrochloride and Pioglitazone Hydrochloride was prepared by dissolving 0 mg of each drug separately in 00mL volumetric flask using 0.N sodium hydroxide as solvent up to 0 ml and volume make up with distilled water and both sample sonicate upto 0 min. Stock solutions of 00 g/ml were obtained in this manner. From these stock solutions, working standard solutions of concentration were prepared by appropriate dilutions. Working standard solutions were scanned in the entire UV range to determine the max. The max of Metformine Hydrochloride and Pioglitazone Hydrochloride were found to be nm and. nm respectively... Calibration curves Seven standard dilutions of each drug were prepared separately having concentrations of -0 g/ml. The absorbances of these standard solutions were measured at nm and. nm and calibration curve was plotted. The absorptivity coefficients of the two drugs were determined using calibration curve (graph and ) Graph :Metformine Hydrochloride.. Preparation of sample solution Sample solution containing both the drugs was prepared by dissolving 0 mg of each drug in 00mL volumetric flask using 0 ml of 0.N sodium hydroxide to give stock solutions then both sonicate for 0 min and further with distilled water and made 00 g/ml stock solution, working standard solution of 0 g/ml concentration was prepared by appropriate dilution. Seven standard dilutions of concentrations of,,, 0,, and 0 g/ml was prepared from working standard solution. The absorbance of this sample solution was measured at nm and. nm and their concentrations were determined using proposed analytical methods... Simultaneous equations method: Method was based on simultaneous equation method of Vierodt. The method is applicable in the case of sample containing two drugs, each of which absorbs at the max of the other (Beckett et al, ). Two equations were constructed based upon the fact that the absorbance of the mixture of Metformine and Pioglitazone at nm and.nm is the sum of the absorbances at respective wavelengths. Two equations were developed using absorptivity coefficient values.

3 CM = A0.0 - A 0.0 / () CP = A A 0.0 / () Where CM and C P are concentrations of Metformine Hydrochloride and Pioglitazone Hydrochloride in g/00ml respectively in the sample solution. A and A are absorbances of the mixture at nm and. nm, respectively. Solving these two equations, the concentrations C H and C T can be readily determined... Precision and accuracy Journal of Applied Pharmaceutical Science 0 (0); 0: - The precision of the method was evaluated by inter day and intraday variation studies. In intraday studies, working solutions of standard and sample were analysed thrice in a day and percentage relative standard deviation (% RSD) was calculated. In the inter day variation studies, working solution of standard and sample were analysed on three consecutive days and percentage relative standard deviation (% RSD) was calculated. The data is shown in table -. The accuracy of the method was determined by studies. The studies were performed by the standard addition method at 0%, 00% and 0% level and the percentage recoveries were calculated and are shown in Table -. Fig: Spectra of Mixture (Metformine Hcl and Pioglitazone Hcl).. Estimation in the marked formulation Twenty tablets were weighed and crushed to a fine powder. An accurately weighed powder sample equivalent to 0 mg of Pioglitazone Hydrochloride and Metformine Hydrochloride was transferred to a 0 ml volumetric flask, dissolved in ml 0.N sodium hydroxide, shaken for 0 min and the volume was made up to the mark with 0.N sodium hydroxide. The solution was then filtered through Whatman filter paper no.. The solution was further diluted to get different concentrations in the range of -0 g/ml of both the drugs. For this method the absorbances of the sample solution, i.e., A and A, were recorded at nm and. nm respectively, and concentration of two drugs in the sample were determined using the equations() and (). The analysis procedure was repeated three times with the formulation. The result of analysis of the formulation is shown in Table... Method validation The method validation parameters like linearity, precision, accuracy, repeatability, limit of detection and limit of quantitation were checked as per ICH guidelines..0. Linearity and range The linearity for Metformine and Pioglitazone were determined at seven concentration levels, ranging from - 0/mL using working standards shown in table -.. Limit of detection and limit of quantitation The Limit of Detection (LOD) is the smallest concentration of the analyte that gives the measurable response. LOD was calculated using the following formula and shown in Table -. LOD =. ( / S) Where, S = slope of calibration curve, = standard deviation of the response. The Limit of Quantification (LOQ) is the smallest concentration of the analyte, which gives a response that can be accurately quantified. LOQ was calculated using the following formula and shown in Table -. LOQ = 0 ( / S) Where, S = slope of calibration curve, = standard deviation of the response.. RESULTS AND DISCUSSION: In the present work, new method, namely, simultaneous equation method (Vierordt's method) was used for the simultaneous spectroscopic estimation of Metformine HCl and Pioglitazone HCl in commercially available tablet dosage form. The concentrations in the range of -0 g/ml of mixed working standard and two sampling wavelengths of nm ( max of Metformine Hcl), and. nm ( max of Pioglitazone HCl) gave optimum accuracy, precision, time, economy, and sensitivity for this method. The proposed procedure was successfully applied to the determination of Metformine Hcl and Pioglitazone Hcl in the commercially available tablets dosage form. The studies were carried out at different concentrations by spiking a known concentration of standard drug to the preanalysed sample and contents were reanalysed by proposed methods. The results of marketed formulation analysis and Recovery studies are depicted in Table -. The method was validated Statistically for range, linearity, precision, accuracy, repeatability, LOD, and LOQ Table -. Accuracy was ascertained on the basis of Recovery studies. Precision was calculated as inter and intraday Variation for both the drugs. The percentage

4 recoveries for of Metformine HCl and Pioglitazone HCl were found to be.±.0-.±0. and.±0.-.±0. for this method respectively. The contents estimated using the proposed method was found in agreement with the labeled amount Table -.The relative standard deviations was found to be within the limit, indicating good accuracy, precision, and repeatability of the proposed method. Table : Determination of Accuracy by percentage method for Pioglitazone HCl Ingredients Tablet amount Amount added Level of addition Amount recovered Pioglitazone 0 0% % 0 0%. Percentage.%.% 00.% Average %.±0. Table : Determination of Accuracy by percentage method for Metformine HCl Ingredients Tablet amount Amount added Level of addition Amount recovered Metformine 0 0% % 0 0%. Percentage.%.% 00.0% Average % 00.%±. Table :Validation parameters for Pioglitazone Sr. No Parameters Results Absorption (nm) Linearity range Standard regression equation Correlation coefficient (r ) A (%, cm) Accuracy (% ± SD) Precision (% CV) Specificity.nm - µg/ml y=0.0x r =0..±0. 0. %,00.% A 0 µg/ml solution of candidate drug in solvent (0. N NaOH and distilled water 0 LOD LOQ mixture in the ratio of 0:0 respectively ) at UV detection show an absorbance value of of. nm will Table :Validation parameters for Metformin Sr. No Parameters Results Absorption (nm) Linearity range Standard regression equation Correlation coefficient (r ) A (%, cm) Accuracy (% ± SD) Precision (% CV) Specificity nm -0 µg/ml y= r = 00.%± %,0.% A µg/ml solution of candidate drug in solvent (0. N NaOH and distilled water 0 LOD LOQ mixture in the ratio of 0:0 respectively ) at UV detection an absorbance value of of nm will show Table : Precision data for the developed method Assays of Pioglitazone as % of labeled amount Sample number Analyst I(Intra- day precision) Analyst II (Inter- day precision) Average ±S.D. 0. ± ± 0.

5 Table : Precision data for the developed method Assays of Metformine as % of labeled amount Sample number Analyst I (Intra- day precision) Analyst II (Inter- day precision) Average± S.D. 00.± 0. 0.± 0.. CONCLUSION: The Vierordt s method permits simple, rapid and direct determination of of Metformine HCl and Pioglitazone HCl in commercially available tablet dosage form without previous separation. The results of analysis of two drugs from tablet formulation using method was found close to 00%, Standard deviation was satisfactorily low indicating accuracy and reproducibility of the method. Recovery studies were satisfactory which showed that there is no interference of excipients.. ACKNOWLEDGEMENT: The authors wish to thank Gen Pharmaceuticals Ltd Pune, Maharashtra for providing the gift samples of of Metformine HCl and Pioglitazone HCl.. REFERENCES:. Wild S., Roglic G., Green A., Diabetes Care, 00, :0-0.. Fujimoto W.Y., American Journal of Medicine, 000,0:-.. Inzucchi S.E., Journal of American Medical Association,000, :0-.. Lebovitz H.E., Oral Antidiabetic Agents, Medicine Clinics of North America, 000, :-.. G. Charpentier,F. Fluery, M. Kabir, S. Kaur, S. Halimit ;Improved glycaemic control by addition of glimepiride to metformin monotherapy in Type diabetic patients;00 Diabetic UK,Diabetic Medicine,,-. Van Gaal L.F., De Leeuw I.H., Rationale and Options for Combination Therapy in the Treatment of Type II Diabetologia, 000, : -0.. Reassner C.A., Promising New Approaches, Diabetes, Obesity and Metabolism,, : -.. Cox S.L., Diabetes Today,00, 0:-.. R.S. Satoskar, S.D. Bhandarkar, N. Nirmala, Pharmacology & Pharmacotherapeutics, Vol II, 0. th Ed, Popular Prakashan, Mumbai, India.. Validation of analytical procedures, Proceedings of the International Conference on. Harmonization (ICH). Commission of the Japan (). B. Praveenkumar reddy, D. Boopathy et.al. ; Method development and validation of simultaneous determination of pioglitazone and glimepiride in pharmaceutical dosage form by RP-HPLC. International journal of chemical tech. research vol., No. ;0-. K.S. Lakshmi, T. Rajesh et.al; Development and validation of liquid chromatography and uv derivative spectrophotometric methods for the determination of metformine, pioglitazone and glimepiride in pharmaceutical formulations. Der Pharma Chemica, 00,() -. Rekha Gangola, Sunil Kaushik et.al; Spectrophotometric simultaneous determination of hydrochlorothiazide and telmisartan in combined dosage form. Journal of applied pharmaceutical science (),0;-. Sagar Tatane, development of UV Spectrophotometric method of telmisartan in tablet formulation JAPHR 0;issue.