INTRODUCTION:

Metformin Hydrochloride is 1,1-Dimethylbiguanide hydrochloride¹ and is used in the treatment of diabetes. It is completely different from the hypoglycemic sulfonamides² both in its structure and its mode of action³. It possibly interferes with mitochondrial respiratory chains and promote peripheral glucose utilization by enhancing anaerobic glycolysis or it enhances binding of insulin to its receptors and potentiates its action. Other explanation is that it suppresses hepatic gluconeogenesis and inhibits intestinal absorption of glucose. It causes little or no hypoglycemia in non-diabetic patients⁴. Metformin is found official in Merck index⁵. There are various spectrophotometric methods developed⁶ for estimation of metformin.^7,8

Glimepiride i.e. 1-[[4-[2-(3-Ethyl-4-methyl-2-oxo-3-pyrroline-1-carboxamido)-ethyl]phenyl] sulphonyl]-3- trans-(4-methylcyclohexyl) urea is a hypoglycemic agent belonging to the second generation sulfonylureas. It appears to lower blood glucose by stimulating insulin release from beta cells in⁹ the pancreatic islets possibly due to increased intracellular Camp ¹⁰.

Fig.1.1. Structure of Metformin

Fig.1.1. Structure of Glimepiride

MATERIALS AND METHODS:

Apparatus:

A Shimadzu model 1800 double beam UV-Visible spectrophotometer with spectral width of 1 nm, wavelength accuracy of ± 0.1 nm and a pair of 10 mm matched quartz cell was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV-Probe system software (Ver.2.34).

Solvent used:

In the present investigation acetonitrile was selected as solvent for Glimepiride and 0.1 N HCl: Distilled water (25:75) was selected as a solvent for Metformin.

Preparation of standard stock solution:

Standard stock solution of Metformin HCl and Glimepiride, Each having concentration of 1000 μg/ml, were prepared in 0.1N NaOH. From these solutions, 100 μg/ml of Metformin HCl and Glimepiride respectively standard solution were prepared by dilution with 0.1N NaOH.

Selection of analytical wavelengths:

Appropriate dilutions were prepared for each drug from the standard stock ‘B’ solution and scanned in the spectrum mode from 200 nm to 400 nm. The absorption maximum was found at 233 nm for metformin and the absorption maximum was found at 228.4 for glimepiride respectively.

Preparation of calibration curve for Metformin HCl and Glimepiride:

· Calibration curve for the Metformin HCl –

Appropriate volume of aliquots was pipetted out from the standard stock solution in to a series of 10 ml volumetric flasks. The volume was made up to the mark with 0.1N NaOH to obtain concentrations of 5, 6, 7, 8, 9, 10 μg/ml of TELM. Absorbances of the above solutions were measured at 233 nm.

· Calibration curve for the Glimepiride–

Appropriate volume of aliquots was pipetted out from the standard stock solution in to a series of 10 ml volumetric flasks. The volume was made up to the mark with 0.1N NaOH to obtain concentrations of 3, 6, 9, 12, 15, 18 μg/ml of AML. Absorbances of the above solutions were measured at 228 nm and a calibration curve of absorbance against concentration was plotted.

Simultaneous Equation method

Calibration curves were plotted at 228nm for telmisartan and 233nm for Amlodipine. The regression equations of the two drugs were determined using calibration curves equations. The concentration of test solution was determined from the respective regression equations.

Stability in standard solutions

The standard solutions of Telmisartan and amlodipine were analysed after storing at 0, 1,2,4,6 hours at room temperature after preparation by the developed methods.

Analysis of tablet formulation:

Twenty tablets were weighed and their average weight was determined and finely powdered. The powder equivalent to 500 mg of MET and 2 mg of GLIM was accurately weighed and transferred to 100 ml volumetric flask and dissolved in 50 ml 0.1N NaOH and the content was kept in Sonicator for 20 min. The flask was shaken and volume was made up to the mark with distilled water and made 100 μg/ml stock solution,working stamdard solution of 10 μg/ml concentration was prepares with appropriate dillutions. The absorbance of sample solutions were measured at 233 nm and 228.4 nm and their concentrations were determined using proposed analytical methods.

Validation of spectrophotometric method¹¹

Accuracy

To study the accuracy, 20 tablets were weighed and powered and analysis of the same was carried out. To ascertain the accuracy of proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% and 120%).

Precision

The precision is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogenous samples. It is usually expressed as the standard deviation or relative standard deviation (coefficient of variation).

Intra-day precision

Variation of results within the same day was analyzed. Intraday precision was determined by analyzing Metformin HCL and Glimepiride individually for three times in the same day at their selected analytical wavelengths.

Inter-day precision

Variation of results between the days was analysed. Inter-day precision was determined by analyzing Metformin HCL and Glimepiride individually daily once for three days at their selected analytical wavelengths.

Repeatability

Standard solutions of Metformin (5, 6, 7, 8, 9, 10 μg/ml) were prepared and absorbance was measured at 233 nm using 0.1N NaOH as the blank. The absorbance of the same concentration solution was measured six times and standard deviation was calculated.

Similarly, standard solutions of Glimepiride (3, 6, 9, 12, 15, 18 μg/ml) were prepared and absorbance was measured at 228.4 nm and 233 nm taking the 0.1N NaOH as the blank. The absorbance of the same concentration solution was measured six times and standard deviation was calculated.

Reproducibility

The absorbances were measured by another analyst and the values obtained were evaluated using t - test to verify their reproducibility.

Linearity and Range

The linearity of analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in sample within a given range. The range of analytical method is the interval between the upper and lower levels of analyte that have been demonstrated to be determined within a suitable level of precision, accuracy and linearity.

Limit of detection and limit of quantitation

Detection limit is the lowest amount of analyte in a sample that can be detected, but not necessarily quantitated. The quantitation limit is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. Detection limit and quantitation limit were determined based on the standard deviation of response and slope of calibration curve.

RESULT AND DISCUSSION:

The absorbance of solution was measured at 233 nm and 228.4 nm for estimation of MET and GLIM, respectively (Fig 1 and 2).The calibration curves were constructed by plotting absorbance versus concentration and the regration equation were generated (Fig 3 and 4).

Validation of the method:

Results of validation studies are summarized in Table 1. The accuracy of the method was confirmed by recovery studies from tablet at 80%, 100% and 120% levels of standard addition and the results are depicted in Table 2. Recovery in the range of 100-101% justifies the accuracy of the method.

Analysis of Marketed Formulation:

The method was applied to marketed tablet (AMRYL) containing MET 500 mg and GLIM 2 mg. The results are depicted in Table 3.Results shows that this method can be successfully applied to marketed formulations.

Table 1 Statistical data of MET and GLIM at 233 nm and 228 nm respectively by simultaneous equation method

Parameter	MET at 233 nm	GLIM at 228 nm
Linear Range (μg/ml)	5-10	3-18
Slope	0.0778	0.0512
Intercept	0.0054	-0.0013
Correlation coefficient (r²)	0.9992	0.9999
Limit of Detection (μg/ml)	0.08408	0.0429
Limit of Quantitation (μg/ml)	0.2531	0.130

Table 2 Determination of Accuracy of MET and GLIM by simultaneous equation method

Level of % Recovery	% Recovery*
Level of % Recovery	MET	GLIM
80%( n=3)	99.76	99.53
100%( n=3)	99.87	99.71
120%( n=3)	98.98	99.83

CONCLUSION:

Proposed study describes method for the estimation of MET and GLIM combination in mixture. The method was validated and found to be simple, sensitive, accurate and precise as per ICH guidelines .The method was successfully used for determination of drugs in their pharmaceutical formulation.

ACKNOWLEDGMENTS:

The authors are thankful to USV Pharmaceutical Ltd. Baddi, India for providing gift sample of Metformin and Glimepiride. The authors are very thankful to Principal and Management of IIMT College of Medical Sciences for providing necessary facilities to carry out research work.

REFERENCES:

1. Klepser T.B., Kelly M.W.; Metformin hydrochloride: An antihyperglycemic agent. Am J Health System Pharm. 1997; 54:893–903.

2. Hitt E.;Uses of metformin may extend beyond patients with type 2 diabetes. Drugs. 1995; 63:1879–94.

3. Maria C.R., Ranetti A.;The pharmacokinetics of some oral antidiabetics –A pharmaceutical approach. Farmacia, 2006; LIV, 6, 3- 13.

4. Hermann L. S.; Metformin:A review of its pharmacological properties and therapeutic use. Diabete metab. 1979; 5(3): 233-45.

5. Budavari S, in. The Merck Index., ed. Merck and Co Inc., 13thed., 1997; Whitehouse Station (NJ), 5792.

6. Arayne MS, Sultana N, Zuberi MH, Siddiqui FA. Spectrophotometric quantitation of Metformin in bulk drug and pharmaceutical formulations using multivariate technique. Indian J Pharm Sci 2009; 71 (3): 331-335.

7. Chaturvedi PK, Sharma R. Simultaneous spectrophotometric estimation and validation of three component tablet formulation containing pioglitazone hydrochloride, Metformin hydrochloride and Glibenclamide. Analytical Letters 2008; 41(12):2133-2142.

8. Rohokale BS, Jadhav VM, Kadam VJ. Studies in prospective process validation of Metformin HCl tablet Dosage formulation. Int J PharmaTech Res 2010; 2(3):1673-1678.

9. Satoskar RG, Bhandarkar SD, Nirmala N, pharmacology and pharmacotherapeutics, Vol II,17^th ed, Popular publication, Mumbai, Maharastra, India.

10. Muller G, Hartz D, Punter J, Okonomopulos R, Kramer W; Differential interaction of glimepiride and glibenclamide with the beta-cell sulfonylurea receptor. I. Binding characteristics. Biochim Biophys Acta. 1994; 1191(2):267-77.

11. ICH, Q2 (R1) Validation of analytical procedure: Text and methodology, International Conference on Harmonization, Geneva, Switzerland, 2005.

Received on 11.07.2013 Modified on 25.07.2013

Asian J. Research Chem. 6(9): September 2013; Page 845-848