INTRODUCTION:

Metformin (MET):

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 action ³. 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-diabetic⁴ patients^5,6,7. Metformin is found official in Merck index⁸. There are various spectrophotometric methods developed⁹ for estimation of metformin ^10,11.

GLIMEPIRIDE (GLIM):

Glimepiride is 1-{(p-[2-(3-ethyl-4-methyl-2-oxo-3- pyrroline-1-carboxamide) ethyl] phenyl) sulfonyl}-3- (trans-4-methylcyclohexyl) urea, is 3rd generation sulfonylurea derivative used for the treatment of type II diabetes mellitus ¹². It appears to lower blood glucose by stimulating insulin release from beta cells in the pancreatic islets possibly due to increased intracellular cAMP ¹³.

Literature survey reveals that some method was reported for the Multicomponent mode determination of these drugs in combined dosage form ¹⁴. The Aim of present work was to developed simple, accurate and rapid UV spectrophotometric methods for Multicomponent mode of Metformin and Glimepiride in combined dosage form.

Fig. 1 Chemical structure of Metformin

Fig. 2 Chemical structure of Metformin HCl

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 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 MET. 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 GLIM. Absorbances of the above solutions were measured at 228 nm and a calibration curve of absorbance against concentration was plotted.

Multi Component mode method:

Calibration curves were plotted at 233nm for Metformin and 228nm for Glimepiride. 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 Metformin and glimepiride 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 standard solution of 10 μg/ml concentration was prepares with appropriate dilutions. The absorbance of sample solution was measured at 233 nm and 228 nm. The absorbance of each solution was substituted in the Multi component mode equation to calculate the amount of the drug present.

Validation of spectrophotometric method¹⁵

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.

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).

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 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 nm for estimation of MET and GLIM respectively (Fig 3 & 4).The calibration curves were constructed by plotting absorbance versus concentration and the regration equation were generated (Fig 5 and 6).

Fig. 3 Overlain spectra of Mixed Standards of Metformin HCl and

Glimepiride.

Fig. 4 Overlain spectra of the Optimized standard solutions of Metformin HCl, Glimepiride and mixture.

Fig. 5 Calibration curve for MET at 233 nm.

Fig.6 Calibration curve for GLIM at 228 nm

Stability in standard solutions:

Standard solutions of MET and GLIM were found to be stable in methanol up to 6 hours of preparations when stored at room temperature

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 99-101% justifies the accuracy of the method.

Table 1. Statistical data of MET and GLIM at 233 nm and 228 nm respectively by multicomponent mode method

Parameter	MET at 233 nm	GLIM at 228 nm
Linear Range (μg/ml)	5-10	3-18
Slope	0.078	0.051
Intercept	0.030	0.001
Correlation coefficient (r²)	0.999	0.999
Limit of Detection (μg/ml)	0.0823	0.04213
Limit of Quantitation (μg/ml)	0.2561	0.13902

Table 2. Results of recovery studies

Level	% Recovery
Level	MET	GLIM
80%(n=3)	99.95	99.94
100%(n=3)	99.95	99.87
120%(n=3)	99.96	99.89

CONCLUSION:

The result shows that the developed spectrophotometric method is sensitive, precise and specific for Multi-Component Mode UV Spectrophotometric of MET and GLIM. Proposed study describes method for the estimation of MET and GLIM combination in mixture. 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 HCl and Glimepiride. The authors are very thankful to Director, HOD and Management of IIMT College of Medical Sciences for providing necessary facilities to carry out research work.

REFERENCES:

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Received on 08.10.2013 Modified on 02.11.2013

Asian J. Research Chem. 7(1): January 2014; Page 07-10