1. INTRODUCTION:

Metformin HCl (MF) is chemically, 1, 1-dimethyl biguanide hydrochloride. It is the first line drug of choice for the treatment of type2 diabetes^8-11. Bio analytical, HPLC, HPTLC and UV-visible spectrophotometry methods have been reported for its individual determination of Metformin and in combination with other drugs^1-4. Glimepiride (GL) is chemically 2-(3-ethyl-4-methyl-2-oxo-3 pyrroline-1-carboxamido) ethyl-phenylsulfonyl-3-(trans-4-methylcyclohexyl) urea^8-11. It is a medium to long acting sulphonyl urea anti-diabetic drug. Several spectrophotometric method, HPLC, HPTLC have been reported for estimation of Glimepiride^5-7. Rosiglitazone (RG) is chemically RS-5-[4-(2-[methyl (pyridin-2-yl) amino] ethoxy) benzyl] thiazolidine-2, 4-dione^8-11. It is an oral antidiabetic agent which acts primarily by increasing insulin sensitivity. Bio-analytical and UV-visible spectrophotometry methods have been reported for its individual determination of Rosiglitazone and in combination with other drugs^12-14.

The present manuscript describes a novel LC method which is simple, rapid, precise, sensitive, selective and accurate isocratic reverse phase HPLC-UV method for simultaneous determination of metformin, rosiglitazone and glimepiride in tablet dosage form.

2. EXPERIMENTAL:

2.1 Reagents and Materials:

A tablet Rosinorm-GM (Zydus Cadila Health Care Limited) contains 500mg of MF, 2 mg of GL and 2mg of RG. The combination of metformin, glimepiride and rosiglitazone is not official in any Pharmacopoeia. Methanol HPLC grade was procured from Merck Ltd, Mumbai. Ortho phosphoric acid and potassium dihydorgen ortho phosphate AR grade were procured from Qualigens Fine Chemicals, Mumbai. Water HPLC grade was obtained from a Milli-QRO water purification system. Reference standards metformin, rosiglitazone and glimepiride were gift samples from Dr. Reddy’s Laboratory Limited (Hyderabad, India)

2.2 Apparatus and Chromatographic conditions:

The HPLC system consisted of a separation module (Water Alliance 2695) and Photo Diode Array (PDA) detector. (Water’s Corporation, (USA). An isocratic elution was performed on XTerra C18column (100 x 4.6 mm, 5µ)

The mobile phase was degassed and filtered (0.45 µ, Millipore) mixture of methanol and phosphate buffer (p^H4.0) adjusted with ortho phosphoric acid (75:25 v/v). Injection volume was 20µl and run time was 14min and flow rate was 1.0ml/min. The column was maintained ambient temperature and the eluents were detected at 239nm.Quantification was achieved by peak area-ratio method with reference to the standards.

2.3 Preparation of stock and standard solutions of Metformin, Glimepiride and Rosiglitazone:

Standard stock solution (1000µg/ml) of metformin, glimepiride and rosiglitazone were prepared separately in methanol. The working standard solutions were prepared and further diluted in mobile phase to contain a mixture of metformin, glimeprirde and rosiglitazone in over the linearity range from 300-700µg/ml, 1.4-2.8µg/ml and 1.4-2.8µg/ml respectively.

2.4Estimation of drugs from marketed formulations:

Twenty tablets, each containing 500mg of MF, 2 mg of GL and 2mg of RG were weighed and tablet contents are finely powdered. A quantity of powder equivalent to 500mg of MF, 2 mg of RG and 2mg of GL were weighed and transferred into 100ml of standard volumetric flask containing 50 ml of methanol. The sample was kept in an ultrasonic bath for 20 min and further diluted to 100ml by using mobile phase. Then it is filtered through 0.2µ membrane filter paper. 10 ml of this solution was further diluted 100ml to get a concentration of 500 µg/ml of MF, 2µg/ml of GL and 2µg/ml of RG.20µl of this solution was injected into HPLC system and chromatograms were recorded. A duplicate injection of the standard solution was also injected into the HPLC system and the chromatograms were recorded. The amount of metformin, glimepiride and rosiglitazone present in each tablet was calculated by comparing the peak area of the standard solution and sample. The amount of the drugs were calculated and tabulated in [Table 1].

3. RESULTS AND DISCUSSION:

The HPLC procedure was optimized with a view to develop precise and accurate assay method. Various mobile phase systems were prepared and used to provide an appropriate chromatographic separation, but the proposed mobile phase comprising of methanol, phosphate buffer (p^H4) (75:25 v/v) gave a better resolution. Using UV-visible PDA detector at 239nm carried out the detection. Amongst the several flow rates tested (0.5 - 2.0 ml/min), the flow rate of 1.00 ml/min was the best for all the drugs with respect to location and resolution of peaks. The retention time of metformin, rosiglitazone and glimepiride was found to be 2.552, 7.472 and 9.195 min respectively. The chromatograms of standard and sample solution of MF, GL and RG were shown in [Figure 1] and [Figure 2] .The asymmetry factor of metformin, rosiglitazone and glimepiride was found to be 0.512, 0.294 and 0.397 respectively, which indicates symmetrical nature of the peak. The percentage label claim of individual drugs found in formulations were calculated and presented in [Table 1] .The results of analysis shows that the amounts of drugs estimated were in good agreement with the label claim of the formulations.

4. METHOD VALIDATION:

The proposed method was validated with respect to accuracy, precision, linearity and range, limit of detection, limit of quantification, robustness and stability of analytical solutions following the guidelines of International Conference on Hormonisation.

Table 1: Table for Assay

S. No	Tablet sample	Label claim (mg/tablet)	Peak Area	^*Amount Present (mg/tablet)	^*Percentage Label claim (%w/w)
1	Metformin	500	32674401	500.98±0.02	100.19±0.04
2	Glimepiride	2	34310	2.05±0.04	102.5±0.08
3	Rosiglitazone	2	59750	1.98±0.05	99.37±0.12

*Average of six determinations, mean ± Standard Deviation

Table 2: Accuracy of the method

Amount(%) of drug added	Theoretical content(µg/ml)	Conc. found (µg/ml)±SD*	Recovery (%)	SEM	RE (%)	RSD (%)
Metformin
0	500	501.12±0.382	100.36	0.215	0.42	1.34
75	875	874.16±0.482	99.84	0.334	0.36	1.14
100	1000	1001.21±0.261	100.16	0.125	0.98	0.67
125	1125	1124.19±0.167	99.75	0.128	1.11	0.96
Glimepiride
0	2	2.39±0.216	101.95	0.129	0.95	0.96
75	3.5	3.54±0.314	101.21	0.249	0.59	0.26
100	4	3.98±0.169	98.77	0.218	1.24	0.62
125	4.5	4.51±0.145	100.41	0.412	1.12	0.37
Rosiglitazone
0	2	2.03±0.231	101.58	0.675	1.56	0.51
75	3.5	3.46±0.452	98.85	0.429	1.42	0.96
100	4	4.14±0.156	102.36	0.498	0.83	0.52
125	4.5	4.55±0.264	101.18	0.614	0.75	0.66

*SD= standard deviation (n=3), SEM= Standard Error of Mean,*RSD=SD/Mean×100,

RE (%)=%Relative Error =(Mean assayed concentration-Added Concentration/ Added Conentration×100).

Figure.1. Chromatogram of standard

Figure.2. Chromatogram of sample

4.1 Accuracy and Precision:

The accuracy of the method was determined by recovery experiments. It was confirmed by studying the recovery at three different concentrations 75%, 100%, and 125 % of those expected by spiking a previously analyzed test solution with additional standard drug solutions, the analysis being done in replicate. The %RSD and % relative error in all cases were within the acceptable limit (≤ 2%). It is evident from the results of accuracy study, reported in [Table 2], that the proposed method enables very accurate quantitative simultaneous estimation of MF, GL, and RG.

The Precision of the method was demonstrated by system precision and method precision studies. In the system precision studies, six replicate injections of the working standard solution prepared as per the proposed method and chromatograms were recorded. Standard deviation and relative standard deviation for the area was calculated and presented in [Table 3]. In the method precision studies, six replicate injections of the analyte solution prepared as per the proposed method and chromatograms were recorded. Standard deviation and relative standard deviation for the area was calculated and presented in [Table 4]. From the data obtained, the developed RP-HPLC method was found to be precise.

Table3: System precision Report

Parameters	Area of Metformin	Area of Glimepiride	Area of Rosiglitazone
Trial 1	32674401	34312	59750
Trial 2	32675789	34516	59486
Trial 3	32676545	34217	59681
Trial 4	32674867	34568	59543
Trial 5	32676941	34691	59648
Trial 6	32674892	34587	59854
Average	32675573	34481	59660
Standard deviation	1019.671	180.216	134.37
% Relative standard deviation	0.0031	0.0054	0.0025

Table 4: Method precision Report

Parameters	Area of Metformin	Area of Glimepiride	Area of Rosiglitazone
Trial 1	32685896	34687	59631
Trial 2	32648956	34568	59684
Trial 3	32678867	34456	59842
Trial 4	32654895	34689	59631
Trial 5	32685494	34784	59758
Trial 6	32548972	34674	59753
Average	32650275	34643	59716.5
Standard deviation	52176.23	114.45	83.04
% Relative standard deviation	0.0015	0.003	0.0013

3.2Linearity and Range:

A linear relationship was observed between the absorbance and concentration over the range from 300-700µg/ml for metformin, 1.4-2.8 µg/ml for glimepiride and 1.4-2.8µg/ml for rosiglitazone [Table 5]. The linearity was expressed as R², which was 0.9992 for metformin, 0.9986 for glimepiride and 0.9995 for rosiglitazone. Values of R2 y-intercept and slope of the regression line are shown in [Figures 3, 4 and 5].

Figure 3: Linearity curve for Metformin

Figure 4: Linearity curve for Glimepiride

Figure 5: Linearity curve for Rosiglitazone

4.3 Limit of Detection (LOD) and Limit of Quantification (LOQ):

Limit of detection (LOD) and limit of quantification (LOQ) were calculated as 3.3 ∂/S and 10 ∂/S, respectively as per ICH guidelines, where ∂ is the standard deviation of the response (y-intercept) and S is the slope of the calibration plot. The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3).The LOD for MF, GL and RG was found to be 2.67 µg/ml,0.02 µg/ml and 0.01µg/ml, respectively. The LOQ is the smallest concentration of the analyte which gives response that can be accurately quantified (signal to noise ratio of 10).The LOQ of MF, GL and RG was found to be 8.1 µg/ml, 0.06 µg/ml and 0.07 µg/ml respectively [Table 5].

Table5.Linearity Report

Parameter	Results
Parameter	Metformin	Glimepiride	Rosiglitazone
Linearity(R²)	0.9992	0.9986	0.9995
RSD,%	≤ 2	≤ 2	≤ 2
Mean Recovery,%	100.14	99.54	101.87
LOD,µg/ml	2.6	0.02	0.01
LOQ,µg/ml	8.1	0.06	0.07
Range, µg/ml	300-700	1.4-2.8	1.4-2.8

4.4 Ruggedness and Robustness:

The ruggedness of the method was determined by carrying out the experiment on different instruments like Shimadzu HPLC (LC 10 AT), Water Alliance 2695 by different operators using different columns. Robustness of the method was determined by making slight changes in the chromatographic conditions. It was observed that there were no marked changes in the chromatograms, which demonstrated that the RP- HPLC method developed is rugged and robust.

4.5 Solution Stability:

In order to demonstrate the stability of both standard and sample solutions during analysis, both solutions were analyzed over a period of 6 h at room temperature. The results show that for both solutions, the retention time and peak area of metformin, glimepiride and rosiglitazone remained unchanged (percentage RSD less than 2.0), thus indicated that both solutions were stable for 24h, which was sufficient to complete the whole analytical process.

4.6 System suitability studies:

The column efficiency, resolution and peak asymmetry were calculated for the standard solutions (Table No.6). The values obtained, demonstrated the suitability of the system for the analysis of this drug combinations.

Table 6: System suitability Report

Parameters	Metformin	Glimepiride	Rosiglitazone
Retention time, min	2.552	9.195	7.472
Tailing factor	0.56	0.98	0.83
Number of theoretical plates	73455	4162	5569
Resolution		12.36	6.37

5. CONCLUSION:

The proposed RP-HPLC method is precise, rugged, robust, simple and rapid. Hence the present RP-HPLC method is suitable for the simultaneous estimation of metformin, glimepiride and rosiglitazone in tablet dosage form.

6. REFERENCE:

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Received on 06.07.2011 Modified on 30.07.2011

Research J. Pharm. and Tech. 4(10): Oct. 2011; Page 1576-1580