Simultaneous Estimation of Metformin Hydrochloride and Rosiglitazone Maleate from Tablet Dosage Form by Derivative Spectroscopic Method.

Swapnil D. Jadhav*, Akshay A. Magdum, Ramchandra M. Panchal and Pradip S. Koli

Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy,

Near Chitranagari, Kolhapur – 416013.

*Corresponding Author E-mail: swapnil.jadhav@bharatividyapeeth.edu

ABSTRACT:

Metformin HCl (MET) and Rosiglitazone Maleate (ROS) are used in combination for treatment of diabetes as both drugs are oral hypoglycemic gents. The present work deals with simple derivative spectrophotometric method development for simultaneous estimation of MET and ROS in tablet formulation (AVANDAMET). For determination of sampling wavelength, 10 μg/ml of each of MET and ROS were scanned on UV-630 double beam spectrophotometer in 200-400 nm range. The estimation of ROS was carried out at 312 nm in zero order mode where MET showed zero crossing point and MET was estimated at 247 nm in first order mode where ROS showed zero crossing point. The marketed tablet formulation contains 500 mg of MET and 4 mg of ROS. Hence for analysis of ROS, standard addition of 100 mg of pure drug is carried out. For this method, linearity was observed in range of 10-90 μg/ml for MET and 2-18 μg/ml for ROS. The method is validated as per ICH guidelines. The recovery studies confirmed accuracy of proposed method and low values of standard deviation confirmed precision of method. The proposed method can be optimized further for the simultaneous estimation of both drugs from biological fluids, used in pharmacokinetic and bioequivalence studies.

KEYWORDS: Derivative Spectroscopy, First Order, Zero Order, Metformin, Rosiglitazone.

INTRODUCTION:

Rosiglitazone (ROS) [(RS)-5-[4-(2-[methyl(pyridin-2-yl)amino]ethoxy)benzyl]thiazolidine-2,4-dione] is an oral antidiabetic agent, which acts primarily by increasing insulin sensitivity. Rosiglitazone improves glycemic control while reducing circulating insulin levels. Pharmacologic studies in animal models indicate that rosiglitazone improves sensitivity to insulin in muscle and adipose tissue and inhibits hepatic gluconeogenesis¹.Metformin (MET) hydrochloride (Figure 1) (N,N-dimethylimidodicarbonimidic diamide hydrochloride) decreases blood glucose levels by decreasing hepatic glucose production, decreasing intestinal absorption of glucose, and improving insulin sensitivity by increasing peripheral glucose uptake and utilization².

Figure 1: Structure of Metformin (MET) and Rosoglitazone (ROS)

MET and ROS are recently introduced in the market as combined tablet dosage form which is widely used in the treatment of diabetes. Literature survey has revealed that there are methods are reported for analysis and estimation of Metformin^3-7 and Rosiglitazone^8-12 individually and in combination with other drugs. Hence our plan was to develop method that will be simple, accurate, precise, sensitive and selective as well as it will be cheaper and do not requiring prior separation. It has been observed that chromatographic methods have been reported for quantification of MET and ROS from formulations and biological fluids.^13-14 but no UV-Visible spectrophotometric has been reported. Hence, we proposed to develop analytical method for simultaneous estimation of ROS and MET from their tablet dosage form (AVANDAMET).

MATERIALS AND METHODS:

Materials:

Instrument:

Spectrophotometric analysis was carried out on a JASCO UV-630 double beam spectrophotometer using a 1 cm quartz cell. The instrument settings were zero order and first derivative mode and band width of 2.0 nm in the range of 200–400nm.

Reagents and chemicals:

Metformin HCl and Rosiglitazone maleate were supplied by Cipla India Pvt. Ltd., Goa. All solvents were spectrophotometric grade were obtained from SD fine chemicals. Water was purified by glass distillation apparatus in laboratory.

Methods:

Linearity Study:

Stock solutions were prepared separately in water: ethanol (50:50) to obtain 100 μg/ml of both drugs. The nine working mixed standard were prepared by dilution of stock solution in same solvent system in concentration range 2-18 μg/ml of ROS and 10-90 μg/ml for MET. MET and ROS were initially scanned for determining sampling wavelength in range 200-400 nm. Sampling wavelengths were 214 nm for ROS where MET showed zero crossing point and 231 nm for MET where ROS showed zero crossing point in first order derivative mode (Figure 2). The nine solutions of each drug were scanned in wavelength range f 200-400 nm in zero order derivative modes. These spectra were then processed to first order derivative mode by software. The absorbance values of both drugs were recorded at respective wavelengths. The calibration graphs were constructed for MET and ROS. The calibration curve data will be useful for quantitative estimation of both drugs at respective wavelength.

Assay of Tablet Formulation:

Twenty tablets of AVANDAMET (MET-500 mg and ROS-4 mg) were weighted individually and average weight of tablet was calculated. Average weight amount of tablet powder was taken and 100 mg of pure ROS was added and mixed properly. The powder equivalent to 50 mg of ROS taken, dissolved in 60 ml of solvent system. This solution was then filtered through Whatman Filter Paper No. 41. The volume was made up to 100 ml with solvent system and sonicated for 10 minutes. One ml of this stock solution was diluted to 10 ml to get concentration equal to 50μg/ml of ROS. This solution is scanned on UV-630 system in range 200-400 nm using solvent system as blank. The spectra obtained were processed to first order derivative mode and absorbances were noted at respective wavelengths. The concentrations of MET and ROS were calculated from regression equations generated from calibration graph. The concentration of ROS was calculated by subtracting total concentration of ROS from concentration of extra added (100mg) ROS. The results of marketed formulation (AVANDAMET) analysis are reported in Table 1.

Figure 2. Overlain Spectra of MET (10 µg/ml) and ROS (10 µg/ml)

A: Zero Order B: First Order.

RESULT AND DISCUSSION:

After thorough literature survey, it has been observed that RP-HPLC methods have been reported for simultaneous estimation of MET and ROS from formulations and biological fluids^13-14 but no UV-Visible spectrophotometric has been reported for simple, accurate and precise simultaneous estimation of both. Hence, we proposed to develop analytical method for simultaneous estimation of ROS and MET from their tablet dosage form (AVANDAMET). This tablet contains 4 mg of ROS and 500 mg of MET. Hence standard addition of pure drug (ROS) is carried out as on dilution its concentration will not fall down below limit of detection.

The overlain spectra of MET and ROS in zero order mode indicated that estimation of ROS is possible at 312 nm at which MET has zero crossing point means, MET has zero absorbance. In zero order mode, estimation of MET is not possible as no zero crossing point is available for ROS at which it has zero absorbance. Hence estimation of MET is carried out at 247 nm in first order derivative mode where ROS has zero crossing point. The quantification of individual drug is carried out by using calibration curve data i.e. slope and intercept values. The concentrations of drugs calculated using linear regression equation a = A+ B × C. Where, a is absorbance of drug, A is intercept, B is slope and C is concentration of drug.

The developed method was validated by following ICH Q2B (R1) guidelines.¹⁵ The following parameters were studied for validation.

Accuracy:

Recovery studies were performed by standard addition method at three levels i.e., 80%, 100% and 120%. Known amounts of pure MET and ROS were added to pre-analyzed sample of marketed formulation and they were subjected to analysis by the proposed method. Results of recovery studies are shown in Table 1. The results of recovery studies were found to be in range of 98.21 to 101.91 with standard deviation value of 1.712.

Precision:

Precision study was performed to find out intra-day and inter-day variations. The results of precision studies are reported in Table 2 and values of standard deviation less than 2% indicates high degree of precision.

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ were separately determined based on the calibration curves. The standard deviation of the y-intercepts (σ) and slope of the regression lines (S) were used. These values were calculated using following formula

LOD = 3.3 × σ/ S

LOQ = 10 × σ / S

The limit of quantitation and limit of detection were found to be 0.214ng/mL and 0.087ng/mL for ROS and 0.257ng/mL and 0.090ng/mL for MET respectively. These low LOD and LOQ indicate that very small quantities of analyte can be estimated by this method.

Robustness:

The robustness of method was studied by changing composition of solvent system. The results of robustness studies are reported in Table 3

Ruggedness:

The ruggedness study was carried out by using different instruments and analyst. The results are as shown in Table 3.

Table 1. Results of Tablet (AVANDAMET) Analysis and Recovery Study

Drug	Label Claim (mg/tablet)	%Label Claim Estimated Mean^a± SD^b	Amount Added (mg)	% Recovery Estimated Mean^a± SD^b
MET	500	99.76±1.266	400	99.25±1.257
		100.87±1.186	500	101.91±1.169
		99.92±1.601	600	98.78±1.032
ROS	4	99.04±0.9143	3.2	98.21±1.034
		99.84±0.7453	4	98.75±1.289
		99.55±0.9560	4.8	99.31±1.712

a: Average of Three Determinations b: Standard Deviation.

Table 2. Results of Precision Studies

Precision		Amount of Pure Drug Added in mg		% Concentration Found (Mean^a ± SD^b)
		MET	ROS	MET	ROS
Intra-Day	T1	400	3.2	99.04 ±1.242	98.33 ± 1.158
	T2	500	4	99.55 ± 0.515	99.40 ± 1.244
	T3	600	4.8	99.60 ±0.661	98.56± 1.450
Inter-Day	D1	-	-	1001.11±0.673	98.75±1.297
	D2	-	-	100.02±0.963	99.31±1.7158
	D3	-	-	100.04 ± 1.242	99.59 ± 0.2767

a: Average of Three Determinations b: Standard Deviation.

Table 3. Result of Robustness and Ruggedness Study

Parameter	Modification	% Recovery Mean^b± SD^c		Parameter	Modification	% Recovery Mean^b± SD^c
		MET	ROS			MET	ROS
Robustness Study				Ruggedness Study
Solvent System Ratio (Water: Ethanol)	45:55	98.91±1.258	100.11 ± 1.307	Instrument	UV-530	101.19±0.731	99.91±0.374
	48:52	98.78±1.028	100.39 ± 0.344		UV-630	99.15 ± 0.872	99.68 ± 0.914
	50:50^a	99.59 ±0.015	98.21±1.046	Analyst	I	99.42 ± 0.545	98.37 ± 0.352
	52:48	99.72 ± 0.542	98.75±1.297		II	98.45 ± 0.541	99.67 ± 1.190
	55:45	99.87 ± 0.831	99.31±1.758

a: Optimized Parameter for Developed Method b: Mean of Three Readings c: Standard Deviation

CONCLUSION:

The results of analysis and various validation parameters indicated that developed method is simple, accurate, precise and sensitive method simultaneous determination of MET and ROS from their pharmaceutical formulation. The optimization of developed method will lead to estimation of MET and ROS from biological fluids.

ACKNOWLEDGEMENT:

The authors are thankful to Cipla India Pvt. Ltd., Goa for providing gift samples of drugs. The authors are also thankful to Principal, Bharati Vidyapeeth College of Pharmacy, Kolhapur for providing facilities required for this work.

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Received on 26.03.2013 Accepted on 05.06.2013

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Asian J. Pharm. Ana. 3(3): July-Sept. 2013; Page 94-97