DEVELOPMENT AND VALIDATION OF SPECTROPHOTOMETRIC METHOD FOR SIMULTANEOUS ESTIMATION OF GLICLAZIDE AND METFORMIN HYDROCHLORIDE IN BULK AND TABLET DOSAGE FORM BY SIMULTANEOUS EQUATION METHOD

DEVELOPMENT AND VALIDATION OF SPECTROPHOTOMETRIC METHOD FOR SIMULTANEOUS ESTIMATION OF GLICLAZIDE AND METFORMIN HYDROCHLORIDE IN BULK AND TABLET DOSAGE FORM BY SIMULTANEOUS EQUATION METHOD

Ketan P. Dadhania*¹, Parthika A. Nadpara ², Yadvendra K. Agrawal ³

Smt. R. D. Gardi B. Pharmacy College ¹, Opp. Garden Dinner Club, Rajkot-Jamnagar Highway, Nyara, Rajkot, Gujarat, India

Department of Pharmaceutical Sciences, Saurashtra University ², Rajkot, Gujarat, India

Institute of Research & Development, Forensic Sciences University ³, Ganhinagar, Gujarat, India

ABSTRACT

A simple, rapid, accurate, precise, specific and economical spectrophotometric method for simultaneous estimation of Gliclazide (GLC) and Metformin hydrochloride (MET) in combined tablet dosage form has been developed. It employs formation and solving of simultaneous equation using two wavelengths 227.0 nm and 237.5 nm. This method obeys Beer’s law in the employed concentration ranges of 5-25 μg/ml and 2.5-12.5 μg/ml for Gliclazide and Metformin hydrochloride, respectively. Results of analysis were validated statistically and by recovery studies.

Metformin hydrochloride, Spectrophotometric method, Simultaneous Equation

INTRODUCTION: Metformin hydrochloride (N, N-dimethylimidodicarbonimidic diamide hydrochloride or 1, 1-dimethyl biguanide hydrochloride) is oral antihyperglycemic drugs used in the management of type 2 diabetes ^1,2. It is an antihyperglycemic agent, not hypoglycemic which improves glucose tolerance in patients with type 2 diabetes, lowering both basal and postprandial plasma glucose. It does not cause insulin release from the pancreas and does not cause hypoglycemia, even in large doses. It decreases hepatic glucose production, decreases intestinal absorption of glucose, and improves insulin sensitivity by increasing peripheral glucose uptake and utilization. It has no significant effects on the secretion of glucagon, cortisol, growth hormone, or somatostatin ^3,4.

Gliclazide (1- (3- azabicyclo [3.3.0] oct- 3- yl) - 3- p-tolylsulfonylurea or 1-(hexahydrocyclopenta [c]pyrrol-2 (1H)- yl)- 3- [(4-methylphenyl) sulfonyl] urea) is hypoglycemic agent used in the treatment of type-II diabetes mellitus ^{2, 5}. It reduces blood glucose levels by correcting both defective insulin secretion and peripheral insulin resistance, increasing the sensitivity of ß-cells to glucose, decreasing hepatic glucose production, and increasing glucose clearance. It also has anti-platelet adhesive activity and reduces levels of free radicals, thereby preventing vascular complications. It also has been reported to reduce plasma cholesterol and triglyceride levels after repeated administration ^{3, 6}. Gliclazide and Metformin hydrochloride are now widely used in combination for the effective treatment of type-II diabetes mellitus.

Literature survey reveled that several methods like UV-Visible spectrophotometry, HPLC are available for the drug identification, impurity analysis, metabolite identification and assay of Gliclazide and Metformin hydrochloride as an individual drug and in combination with other drug/s in bulk drug, formulations and biological fluids ^7-26.

The HPLC method has been reported for the simultaneous estimation of Gliclazide and Metformin hydrochloride in tablet dosage form. Thus the present study was undertaken to develop and validate a simple, sensitive, accurate, precise and reproducible U.V method for simultaneous estimation of Gliclazide and Metformin hydrochloride in tablet dosage form.

MATERIALS AND METHODS:

Apparatus: A double beam UV-visible Spectro- photometer (Elico Ltd, SL 164, India), attached to a computer software UV-VIS Spectrasoft, with a spectral width of 2 nm, wavelength accuracy of ±0.5 nm and pair of 1 cm matched quartz cells, Analytical balance (Keroy (Balance) Pvt. Ltd, KEROY, Varanasi, India), Ultrasonicator (Toshniwal Process Instruments Pvt. Ltd., TOSHCON SW 4, Ajmer, India), Corning volumetric flasks and pipettes of borosilicate glass were used for the development of proposed method.

Reagents and Materials: Gliclazide (GLC) and Metformin Hydrochloride (MET) were provided by RPG Life Sciences Ltd (Ankleshwar, India) and Troikaa Pharmaceuticals Ltd (Ahmedabad, India) respectively. Methanol, acetonitrile were of analytical reagent grade and purchased from S. D. Fine Chemicals Ltd., India. HPLC grade methanol and acetonitrile were purchased from S. D. Fine Chemicals Ltd., India. The marketed formulations, tablet(s), were procured from the local market which had a label claim of Gliclazide 80 mg and Metformin hydrochloride 500 mg.

Preparation of Standard Stock Solution: Stock solutions (100μg/ml) of Gliclazide and Metformin hydrochloride were prepared by dissolving separately 10 mg of drug in methanol and making up the volume with methanol. The stock solution was suitably diluted to produce solution of concentration 10 μg/ml. These working solutions were scanned in the entire UV range (200-400 nm) to determine the λ_max.

Procedure for Calibration Curve: Standard solutions of Gliclazide in the concentration range of 5 μg/ml to 25 μg/ml obtained by transferring (0.5, 1, 1.5, 2 and 2.5 ml) of Gliclazide stock solution (100 μg/ml) to the series of 10 ml volumetric flasks and standard solutions of Metformin hydrochloride in the concentration range of 2.5 μg/ml to 15 μg/ml were obtained by transferring (0.25, 0.5, 0.75, 1, 1.25 and 1.5 ml) of Metformin hydrochloride stock solution (100 μg/ml) to the series of 10 ml volumetric flasks. Then methanol was added to each volumetric flask up to 10 ml. All dilutions were scanned in wavelength range of 200 nm to 400 nm. The absorbances were plotted against the respective concentrations to obtain the calibration curves.

Estimation of Gliclazide and Metformin hydrochloride in Pharmaceutical tablet Dosage form: Twenty tablets were weighed and finely powdered. The powder equivalent to 16 mg GLC and 100 mg MET was accurately weighed and transferred to volumetric flask of 100 ml capacity contains 25 ml of the methanol and sonicated it for 5 min. The flask was shaken and volume was made up to the mark with methanol. This solution was carefully filtered through Whatman filter paper (No. 41). Aliquot (0.06 ml) was pipette out and transferred to volumetric flask of 10 ml capacity. Volume was made up to the mark with HPLC grade methanol to give a solution containing 6.0 µg/ml MET and 0.96 µg/ml GLC.  The absorbance of sample solution was measured at 227.0 nm and 237.5.0 nm against blank. The content of Gliclazide and Metformin hydrochloride in tablet was calculated using two framed simultaneous equations and results of analysis are shown in Table 1.

TABLE 1: RESULTS OF ANALYSIS OF TABLET FORMULATIONS

Recovery Studies: Recovery studies were done so as to check the accuracy of the method. The accuracy of the method was assessed by taking known amounts of Gliclazide and Metformin hydrochloride in standard mixture solution and absorbance were determined at 227.0 nm and 237.5 nm. Concentration of the drugs in the mixture was calculated using the equations. The analysis was done in a set of 3 replicates and results of analysis are shown in Table 2.

TABLE 2: RECOVERY STUDIES

RESULTS AND DISCUSSION:

Absorption Maxima: Absorption maxima of Gliclazide and Metformin hydrochloride were detected at 227 nm (λ₁) and 237.5 nm (λ₂), respectively and overlain spectra was recorded Fig. 1.

Absorptivity Coefficients: The absorptivity coefficients of the two drugs were determined by using Beer’s law: A = E (1%, 1cm) CL. The absorptivity coefficients of Gliclazide at 227.0 nm and 237.5 nm were 29.4689±1.0844 and 16.7762±0.7069 and for Metformin hydrochloride were 52.5711±2.6003 and 83.2329±1.9087.

FIG. 1: OVERLAIN SPECTRUM OF GLICLAZIDE AND METFORMIN HYDROCHLORIDE

Formation of Simultaneous Equation: A set of two simultaneous equations were framed using the mean absorptivity.

Where, A₁ and A₂ are the absorbance of sample solutions at 227.0 nm and 237.5 nm respectively. C_x and C_y are concentration of Gliclazide and Metformin hydrochloride in mg/mL in sample solution. By substituting the values of A₁ and A₂ the values of C_x and C_y can be calculated by solving the two equations simultaneously. Here, a_x1 and a_x2 are the absorptivity coefficient of Gliclazide at 227.0 nm and 237.5 nm respectively; a_y1 and a_y2 are the absorptivity coefficient of Metformin hydrochloride at 227.0 nm and 237.5 nm respectively. The optical parameters & regression characteristics for Gliclazide and Metformin hydrochloride are shown in Table 3.

TABLE 3: OPTICAL AND REGRESSION CHARACTERISTICS

Method Validation: The linearity range for Gliclazide and Metformin hydrochloride were 5-25 μg/mL and 2.5-12.5 μg/ mL respectively. Recovery studies was carried out by addition of standard drug solution to pre-analyzed tablet sample solution at three different concentration levels taking into consideration percentage purity of added bulk drug sample. The results of the recovery studies are found to be satisfactory and shown in Table 2. The results obtained from recovery study (accuracy study) indicated that mean percentage recovery were 99.68±1.24 and 100.06±0.22 for Gliclazide and Metformin hydrochloride, respectively. Other validation parameters were found to be satisfactory and are shown in Table 4.

TABLE 4: VALIDATION PARAMETERS

* t- Test was performed for comparison of results

CONCLUSION: Based on the results obtained, it was concluded that the proposed method of analysis is accurate, precise, reproducible & economical and can be employed for routine quality control of Gliclazide and Metformin Hydrochloride in tablet formulations.  

 ACKNOWLEDGEMENT: The authors are thankful to Nirma University of Science and Technology, Ahmedabad Gujarat, India for providing necessary facilities to carry out this work. We are also thankful to RPG Life Sciences Ltd., Ankleshwar, Gujarat, India and Troikaa Pharmaceuticals Ltd, Ahmedabad, Gujarat, India for providing us the free gift samples of Gliclazide and Metformin Hydrochloride, respectively which were required for our research work.

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