INTRODUCTION:

Metformin was first approved in Canada in 1972, followed by 1995 in the USA Metformin is an antihyperglycemic agent of the biguanide class, used for the management of type II diabetes¹. Chemically Metformin hydrochloride is identified as 3-(diaminomethylidene)-1,1-dimethylguanidine; hydrochloride with molecular weight of 165.62g/mol². It is currently the drug of first choice for the treatment of type II diabetes; it is prescribed to at least 120 million people worldwide³. Metformin hydrochloride is freely soluble in water and is practically insoluble in acetone, ether, and chloroform. The p^Ka of metformin is 12.4. The pH of a 1% aqueous solution of metformin hydrochloride is 6.68⁴. Metformin is BCS class III agent i.e. with high solubility and low permeability⁵.

USP recommends pH 6.8 phosphate buffer for dissolution testing of Metformin hydrochloride tablets and adopted UV absorption method at the wavelength of maximum absorbance at about 233 nm⁶. Slowly absorbed after oral administration, about 30 to 50 % of an oral dose is excreted in the urine as unchanged drug in 24 h, and about 30% of the dose is eliminated unchanged in the faeces⁷. Maximum plasma concentration (C_max) is reached in approximately 2.5 hours (t_max). After oral administration, metformin absorption is saturable and incomplete. It is assumed that the pharmacokinetics of metformin absorption is non-linear⁸.

Glimepiride is an oral antidiabetic drug which belongs to the sulfonylurea group and usually given as an oral antidiabetic therapy for patients with type 2 diabetes mellitus. Glimepiride acts to lower blood glucose by stimulating the release of insulin from pancreatic β-cells⁹. Glimepiride, which is sometimes referred to as a third-generation agent, was released in 1995¹⁰. Chemically itis identified as 1-[[p-[2-(3-ethyl-4-methyl-2-oxo-3-pyrroline-1carboxamido) ethyl] phenyl] sulfonyl]-3-(trans-4-methylcyclohexyl) urea with molecular weight of 490.62. Glimepiride is practically insoluble in water¹¹. Glimepiride belongs to BCS II drug with low solubility and high permeability¹². USP recommends pH 7.8 phosphate buffer for dissolution testing of Glimepiride tablets and provided HPLC method for analysis at wavelength of 228 nm¹³. The bioavailability of Glimepiride after oral administration is complete. Maximum serum concentrations (C_max) are reached approx. 2.5 hours after oral intake (mean 0.3 µg/ml during multiple dosing of 4 mg daily) and there is a linear relationship between dose and both C_max and AUC (area under the time/concentration curve)¹⁴. Less soluble over Physiological pH range¹⁵.

Mali et al has reported simultaneous estimation of Metformin hydrochloride and Glimepiride by UV Spectrophometer using methanol as solvent¹⁶. Present study involves development of spectrophotometric method for simultaneous estimation of Metformin and Glimepiride using physiological buffer i.e. pH 6.8 buffer media.

As per ICH various parameters that are need to be estimated for validation of analytical method are as Specificity, Solution, Linearity and Range, Precision, Accuracy, Ruggedness and Robustness and system suitability¹⁷.

MATERIAL AND METHODS:

Chemicals and reagents:

Metformin Hydrochloride and Glimepiride were procured. Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride and water used were of analytical grade (Loba Chemie Pvt. Ltd., Mumbai). All other chemicals and reagents used were analytical grade unless otherwise indicated.

Instrumentation:

The proposed work was carried out on a Jasco, Japan UV-visible spectrophotometer (model V-630), which possesses a double beam double detector configuration with a 1 cm quartz matched cell. All weighing was done on electronic balance (Shimadzu, Japan). A Labhosp, India was used for degassing the physiological media and pH meter of Chemline, India was used to adjust pH. USP dissolution apparatus Type II of Electrolab, India was used to carry dissolution activity.

Selection of Physiological media:

Physiological media is selected based on fact that it will suit as per Pharmacopoeial requirement of dissolution for various solid oral dosage forms of combination drugs.

USP monograph recommends pH 6.8 buffer for immediate release tablets of Metformin Hydrochloride and pH 7.8 phosphate buffer with surfactant for immediate release tablets. Hence based recommendation for selection of dissolution media pH 6.8 phosphate buffer selected in this study.

Diluent - pH 6.8 phosphate buffer

Phosphate buffered saline pH 6.8:

Dissolve 1.0g of potassium dihydrogen phosphate, 2.0g of dipotassium hydrogen phosphate and 8.5g of sodium chloride in 900ml of water, adjust the pH if necessary and dilute to 1000.0ml with the same solvent.

Development of analytical method:

Determination of λ_max of Metformin Hydrochloride:

50 mg of Metformin hydrochloride is dissolved in 30 ml diluent in 50ml volumetric flask sonicate to dissolve. Allow to cool at room temperature and make up the volume with diluents (Metformin 1000ppm). Further pipette out 5ml of Stock solution into a 50ml volumetric flask containing 10ml of diluent, mixed well and make up to the mark with the diluent (100 ppm of Metformin). Further take 5ml of the above solution into a 50 ml volumetric flask containing 10ml of diluent, mixed well and make up to the mark with diluent (10 ppm Metformin).

Take absorbance spectrum of solution was taken on the UV spectrophotometer in range from 200 – 400nm against the blank as distilled water.

Fig. 1: Standard solution determination of λ_max of Metformin Hydrochloride

Determination of λ_max of Glimepiride:

10mg Glimepiride is dissolved in 30ml diluent in 50ml volumetric flask sonicate to dissolve. Allow to cool at room temperature and make up the volume with diluent (Stock Solution) (Glimepiride 200ppm).

Further pipette out 10ml of Stock solution into a 50ml volumetric flask containing 10ml of diluent, mixed well and make up to the mark with the diluents (40ppm of Glimepiride).

Further take 10ml of the above solution into a 50ml volumetric flask containing 10ml of diluent, mixed well and make upto the mark with diluents (4ppm Glimepiride).

Take absorbance spectrum of solution was taken on the UV spectrophotometer in range from 200 – 400nm against the blank as distilled water.

Fig. 2: Standard solution for determination of λ_max of Glimepiride

Simultaneous estimation of Metformin hydrochloride and Glimepiride:

Standard preparation:

50mg of Metformin hydrochloride and 20mg Glimepiride is dissolved in 30ml diluent in 50ml volumetric flask sonicate to dissolve. Allow to cool at room temperature and make up the volume with diluent (Stock Solution) (Metformin 1000ppm and Glimepiride 400 ppm).

Further pipette out 5ml of Stock solution into a 50ml volumetric flask containing 10ml of diluent, mixed well and make up to the mark with the diluents (100ppm of Metformin and 40ppm of Glimepiride).

Further take 5ml of the above solution into a 50ml volumetric flask containing 10ml of diluent, mixed well and make upto the mark with diluent (10ppm Metformin and 4 ppm Glimepiride).

Fig. 3: Standard solution of Metformin and Glimepiride

Sample solution preparation:

Take 900ml of dissolution media dissolution vessel (at 37⁰C±0.5⁰C) immerse paddle and adjust 50rpm speed. Place one tablet in each vessel taking care to exclude air bubbles. At the end of specified time withdraw 20ml aliquot from zone midway between surface of dissolution media and top of paddle. Immediately filter with whatman filter paper discard first 5ml. For Metformin dilute 2ml of filtrate solution into a 100ml volumetric flask with dissolution media. For Glimepiride use filtrate directly. (Solution is stable upto 24 hr at 25⁰C)

Placebo solution preparation:

Place placebo of 1 dosage unit in dissolution vessel and prepare placebo preparation as per the same solution preparation.

Procedure- Measure absorbance of five replicate at above 226nm and 233nm using 1cm cell in dissolution media as a blank.

Relative standard deviation of 5 replicate injections should not be more than two.

For Metformin hydrochloride:

% dissolved	At	X	Ws	X	5	x	5	x	900	x	100	x	P
	As		50		50		50		LC		2

For Glimepiride:

% Dissolved	At	X	Ws	X	5	x	5	x	900	x	P
	As		50		50		50		LC

At = Absorbance of test solution As = Absorbance of standard solution Ws = weight of working standard in mg

LC= label claim P = potency (in %)

Validation of analytical procedure:

1. Specificity (interference):

Method: Specificity of method is done by taking absorbance of blank, placebo and standard preparation. Calculate interference due to placebo, blank and standard preparation.

Acceptance criteria:

Interference due to blank solution should not be more than 1% and placebo solution not be more than 2% with respect to that of standard absorbance.

2. Solution stability:

Method: Standard solution and sample solution were prepared by spiking drug substance and placebo powder equivalent to one dosage unit. Standard and sample solutions were stored at room temperature (25⁰C) and analyzed at initial and intermediate time intervals.

Acceptance criteria:

Absolute difference in % Assay/Release at time intervals with respect to initial should not be more than 2.

3. Filter compatibility:

Method: Spike the API into placebo powder equivalent to one dosage unit, one aliquot was centrifuged and other was filtered.

Acceptance criteria:

Absolute difference should not be more than 2 in results obtained by centrifuged solution and filtered solution.

4. Filter saturation:

Method: Spike the API into placebo powder equivalent to one dosage unit, at filtration stage 1ml, 3ml and 5ml of sample solution filtered and discarded using separate filters followed by filtration of 5ml aliquot and collect filtrate in separate test tubes.

Acceptance criteria:

Absolute difference should not be more than 2 in result obtained by two consecutive aliquots.

5. Linearity and Range:

Linearity:

Method: Linearity solution was prepared by quantitative dilution of the stock solution of standard to obtain solutions at 30% to 150% of target concentration. Each solution were analyzed and the absorbance was recorded.

Slope, Y-intercept, correlation coefficient of the regression line and residual sum square was calculated.

For range, analyzed six replicates of lower and higher concentration level and calculate the mean and relative standard deviation and also recorded the concentration levels over which the results are linear.

Range:

Method: Dilute standard 30–150 % of working concentration. Inject six replicate of lower and higher concentration.

Acceptance criteria: Relative standard deviation should not be more than 2.0 %.

6. Precision (Repeatability):

Method: Spike API to placebo powder in dissolution media. Dissolution carried out as described in methodology on six units. % dissolved was calculated.

Acceptance criteria: Relative standard deviation should not be more than 5.0 %.

7. Accuracy:

Method :

Spike API to placebo in dissolution vessel containing placebo powder equivalent to one dosage unit to obtain solution from 50 % lower concentration, 100 % and 120 % higher concentration with respect to sample in triplicate. % recovery calculated.

Acceptance criteria:

Absolute difference NMT 5 between the mean results obtained in Method precision and Intermediate precision.

RESULTS:

Sr. No.	Parameters	Experiment	Acceptance Criteria		Results of Metformin Hydrochloride		Results of Glimepiride
1.	Specificity	Interference with blank and Placebo	Interference due to blank solution should not be more than 1 % and placebo solution not be more than 2 % with respect to that of standard absorbance		Complies		Complies
2.	Solution Stability	Standard and sample solutions were stored at 25^ᵒC and analyzed at initial and intermediate time intervals.	Absolute difference in % Assay/ Release at time intervals with respect to initial should not be more than 2.		Absolute Difference
					Standard	Sample	Standard	Sample
					1.6	0.9	1.8	1.4
					Standard and sample solution is stable up to 24 hours at 25^ᵒC.
3.	Filter Compatibility	Sample solution was filtered through whatman filter paper and results compared with centrifuged sample	Absolute difference NMT 2 in results obtained by centrifuged solution and filtered solution		Absolute difference
					0.7		0.2
					Whatman filter is found to be suitable
4.	Filter Saturation	Sample solution was prepared after discarding 1.0 ml and 5.0 ml of sample solution and analyzed.	Absolute difference NMT 2 in result obtained by two consecutive aliquots.		Absolute difference
					0.4		0.1
					5 ml volume to be discarded to saturate the filters.
5.	Linearity and Range	Correlation Coefficient (R)	NLT 0.995		0.9972		0.9985
		Slope	Record Results		0.0402		0.0214
		Y-Intercept	Record Results		0.0002		0.0007
		Residual sum of squares	Record Results		0.002735		0.0011
		Range	RSD NMT 2.0 %	Lower % level	1.26		1.28
				Higher % level	0.17		0.52
				% level	30 % to 150 %
6.	Precision	Repeatability	RSD NMT 5.0 %		1.14		0.51
7.	Accuracy	Individual Recoveries	95.0 % to 105.0 %		96.19 % to 99.23 %		97.85 % - 99.44 %

DISCUSSION AND CONCLUSION:

UV spectrophotometric methods for is developed and is suitable for estimation of Metformin and Glimepiride simultaneously in various Pharmaceutical dosage form. Method is validated as per ICH Q2 requirement and study reveals method is Precise, accurate, specific and linear over specified range.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of Department of Pharmaceutics, Shri. Vivekanand Nursing Home Trust’s College of B. Pharmacy, Shri Shivajinagar, Rahuri Factory, Dist. Ahmednagar for providing facilities to carry our research activities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 23.01.2020 Modified on 12.03.2020

Research J. Pharm. and Tech. 2020; 13(11):5360-5364.

DOI: 10.5958/0974-360X.2020.00937.3