Simultaneous Estimation of Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride in Pharmaceutical Dosage Forms by RP-HPLC Method

Chetan Ramolia^3*, Zarna Dedania¹, Ronak Dedania¹, Sheth NR², G Vidya sagar¹, Bhavna Patel⁴ and Bhatt KK⁵

¹Department of Pharmaceutical Analysis, Veerayatan Institute of Pharmacy, Kutch

²Department of Pharmaceutical Sciences, Saurastra University, Rajkot, Gujarat,

³CRO, Veeda, Ahmedabad

⁴CVM Institute for Degree Course in Pharmacy, New Vallabh Vidhyanagar

⁵Ipcowala Indukaka College of Pharmacy, Vallabh Vidhyanagar

*Corresponding Author E-mail: zaroo229@yahoo.co.in

ABSTRACT:

A simple, specific, accurate and stability indicating reversed phase high performance liquid chromatographic method was developed for the simultaneous determination of and Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride in tablet dosage forms. Detector consists of photodiode array detector; the reversed phase column used was RP-C18 (2.27µm size, 250 mm´4.6 mm i.d.) at ambient temperature, in isocratic mode, with mobile phase containing Methnol: Acetonitrile: Phosphate Buffer, pH 5.39 (20: 40: 40 % v/v/v) adjusted to pH 5.39 using ortho phosphoric acid was used. The flow rate was 1.0 ml/min and effluents were monitored at 238 nm. The retention times of Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride were 3.69 min, 8.18 min and 12.5 min respectively.

The calibration curves were linear in the concentration range of 4-24 μg/ml for Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride. The proposed method was validated and successfully applied to the estimation of Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride in combined tablet dosage forms. Linearity was obtained in the concentration range of 4 to 24 µg/ml of Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride with a correlation coefficient of 0.9963, 0.9992 and 0.9957 respectively. Detector consists of photodiode array detector; the reversed phase column used was RP-C18 (2.27µm size, 250 mm´4.6 mm i.d.) at ambient temperature. The developed method was validated according to ICH guidelines and values of accuracy, precision and other statistical analysis were found to be in good accordance with the prescribed values. Thus the proposed method was successfully applied for simultaneous determination of Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride in routine analysis.

KEYWORDS: Metformin Hydrochloride, Rosiglitazone Maleate And Glimepiride, RP-HPLC

INTRODUCTION:

Metformin Hydrochloride 1-(diaminomethylidene)-3,3-dimethyl-guanidine-hydrochloride is an anti-diabetic drug from the biguanide class. It decreases intestinal absorption of glucose and improves insulin sensitivity by increasing peripheral glucose uptake and utilization. Rosiglitazone maleate 5-((4-(2-(methyl-2-pyridinylamino) ethoxy) phenyl)methyl)- 2,4-thiazolidinedione is an anti-diabetic drug from the thiazolidinedione class.

Its mechanism of action is by activation of the intracellular receptor class of the peroxisome proliferator-activated receptors (PPARs), specifically PPARγ. The cell will then take more glucose in, and therefore lower the amount of sugar in the bloodstream. Glimepiride 3-ethyl-N,N-bis (3-ethyl-4-methyl-2-oxo-5H-pyrrol-2-yl)- 4-methyl-2-oxo-5H-pyrrole-1- carboxamide. Glimepiride distinctly lower the blood glucose level by both defects of NIDDM, by stimulating pancreatic beta cells to produce more insulin and induced increased activity of peripheral insulin intra cellular receptor.

Literature survey reveals that RP HPLC – MS in plasma ¹, spectrophotometry ², ion pair LC ³HPLC⁴are available for the determination of Metformin Hydrochloride ; LC in formulation^5,6,7 and plasma^8,9,10for determination of Rosiglitazone maleate; Spectrometry ¹¹, LC-MS ¹²RP-HPLC in formulation ^{13, 14}are available for Glimepiride. The review of the literature revealed that no RP-HPLC method has so far been reported for the combination of Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride. So an attempt has been made to develop a simple, precise, accurate reverse phase high performance liquid chromatographic method for the simultaneous estimation of Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride in combined dosage forms.

Fig 1: Chromatogram containing 12µg/ml standard Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride in Methnol: Acetonitrile: Phosphate Buffer, pH 5.39 (20:40:40), Flow rate - 1.0ml/min.

Table 1: System Suitability Test Parameters

System Suitability Parameters	Proposed Method
System Suitability Parameters	Metformin Hydrochloride	Rosiglitazone Maleate	Glimepiride
Retention times (R_T)	3.69	8.18	12.5
Theoretical plates (N)	576.0	2973.8	3906.2
Tailing factor (A_S)	1.00	1.16	1.13
Resolution (R_S)	7.50	6.28	–

MATERIAL AND METHODS:

Chemicals and Reagents:

Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride were obtained as gift samples from Panacea Biotec Limited, Baddi (H.P.), India. Methanol, Acetonitrile, Water were of HPLC grade and Orthophosphoric acid were used were of Analytical grade obtained from E. Merck (India) Ltd., Mumbai. The commercial formulation contains 500mg Metformin Hydrochloride, 2mg Rosiglitazone Maleate and 2mg Glimepiride as tablet.

Instrumentation:

The present work was carried out on isocratic high pressure liquid chromatograph, LC system used consist of pump (Perkin Elmer, USA) with universal loop injector (Rheodyne) of injection capacity 20 µl. Detector consists of photodiode array detector; the reversed phase column used was RP-C₁₈ (2.27µm size, 250 mm´4.6 mm i.d.) at ambient temperature.

Chromatographic Condition:

The mobile phase containing Methanol: Acetonitrile: Phosphate Buffer, pH 5.39 (20:40:40 % v/v/v) adjusted to pH 5.39 using ortho phosphoric acid was used. The mobile phase was filtered on a 0.45 micron membrane filter and ultrasonicated for 10 min. The flow rate was 1.0 ml/min for 20 min and effluents were monitored at 238 nm.

Preparation of standard Stock Solutions:

The standard stock solution of Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride was prepared by dissolving 20 mg of each drug separately in 100.0 ml mobile phase. The flask was shaken to dissolve the solid and volume was made up to the mark with mobile phase to give a solution containing 200µg/ml of Metformin Hydrochoride, Rosiglitazone Maleate and Glimepiride.

Sample Preparation:

A total of 20 tablets were accurately weighed and powdered. An amount equivalent to one tablet (500mg MET, 2mg ROS and 2mg GLI) was transferred to a 100ml volumetric flask; 10 ml of mobile phase was added and the flask was kept in an ultrasonic bath for 10 min. The solution was filtered through 0.2 micron nylon membrane filter. Pure drug solution of Rosiglitazone Maleate and Glimepiride was added and volume was made up to the mark with mobile phase to give a solution containing 15µg/ml Metformin Hydrochoride, 14.9µg/ml Rosiglitazone Maleate and 14.9µg/ml Glimepiride used for the estimation of Metformin Hydrochoride, Rosiglitazone Maleate and Glimepiride.

RESULTS AND DISCUSSION:

Chromatographic Method:

Initially Methanol: Water, Methanol: Acetonitrile were tried as mobile phase, in which satisfactory peak was not obtained. Then different ratio of Methanol: Acetonotrile: Phosphate buffer (40:20:20, 20:30:50, 20:40:40, 30:35:35) at flow rate of 1.0 or 1.3ml/min. were tried but resolution was not satisfactory. Finally the system containing Methnol: Acetonitrile: Phosphate Buffer, pH 5.39 (20:40:40) was found to be satisfactory and gave well resolved peaks for Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride with retention time 3.6 minutes, 8.18 minutes and 12.5 minutes respectively. Peak with retention time 1.9 min was of maleic acid and confirmed with single chromatogram of rosiglitazole maleate.

System Suitability:

The system suitability test was applied to a representative chromatogram to check the various parameters such as column efficiency, resolution, precision and peak tailing. The result obtained is shown in Table 1. All these parameters were evaluated with the background of regulatory requirements, which also suggests good chromatographic condition.

Table 2: Recovery Study for proposed method:

Amt.of sample(μg/ml)

Amount added %

Total amount added (μg/ml)

Amount recovered *

(μg/ml) ± SD

% Recovery ± SD

Metformin Hydrochloride

12 μg/ml

100

150

5.98 ± 0.11

12.07 ± 0.07

18.03 ± 0.08

99.77 ± 1.82

101.36 ± 0.70

100.16 ± 0.44

Rosiglitazone Maleate

12 μg/ml

100

150

5.99 ± 0.09

12.01 ± 0.04

17.98 ± 0.09

99.83 ± 1.6

100.58 ± 0.33

99.92 ± 0.51

Glimepiride

12 μg/ml

100

150

6.03 ± 0.06

11.98 ± 0.11

18.04 ± 0.08

100.61 ± 1.10

99.86 ± 0.94

100.24 ± 0.46

· Each value indicate mean of three determination

Table 3: Precision data for proposed method

Drug

Concentration µg/ml

Intra day (n=3) % RSD

Inter day (n=3) % RSD

Metformin Hydrochloride

100.28 ± 0.79

100.10 ± 0.49

101.04 ± 0.09

100.45 ± 0.76

99.72 ± 0.81

101.23 ± 1.09

Rosiglitazone Maleate

100.32 ± 0.26

101.28 ± 0.11

100.14 ± 0.32

101.68 ± 0.15

101.42 ± 0.45

99.75 ± 0.86

Glimepiride

101.30 ± 0.56

100.38 ± 0.86

100.15 ± 0.57

99.62 ± 0.16

100.23 ± 0.76

100.61 ± 1.05

Table 4: Summary of Validation Parameters for Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride by RP-HPLC method

Parameter	Metformin Hydrochloride	Rosiglitazone Maleate	Glimepiride
Linear Range(µg/ml)	4 – 24	4 – 24	4 – 24
Slope	151381	81454	99324
Intercept	33271	29846	149452
Standard deviation of slope	4629.8	1169.3	3270.4
Standard deviation of intercept	72122.0	18214.8	50945.8
Limit of Detection (μg/ml)	0.02914	0.01436	0.05384
Limit of Quantitation (μg/ml)	0.09715	0.0478	0.17946
Repeatability (RSD, n=6)	0.000733	0.000672	0.001145
Specificity	Specific	Specific	Specific
Solvent suitability	Solvent suitable for 24 hrs	Solvent suitable for 24 hrs	Solvent suitable for 24 hrs

Table 5: Assay Results of Marketed Formulation

Label claim (mg/tablet)	% of Drug found *	% RSD
Metformin Hydrochloride 500 mg	100.58 ± 0.18	0.18
Rosiglitazone Maleate 2 mg	99.45 ± 0.45	0.45
Glimepiride 2 mg	99.75 ± 0.56	0.56

Accuracy and Precision:

The recovery experiment was carried out by spiking the already analyzed sample of the tablets with their different known concentration of standard Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride. The percent recovery for Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride is summarized in Table 2. Intra day and inter day precision data is summarized in Table 3.The summary of other validation parameter shown in Table 4.

Assay:

The content of Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride found in the tablets by the proposed method are shown in Table 5. The low R.S.D indicates that the method is precise and accurate.

CONCLUSION:

The proposed RP-HPLC method allows for accurate, precise and reliable measurement of Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride simultaneously in combined dosage form. The developed RP-HPLC method was found to be simple, rapid, selective, accurate and precise for the concurrent estimation of drug component tablet dosage form of Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride. The method was evaluated in a mass of facets, such as best condition, linear relation including coefficient of correlation, robustness, accuracy, reproducibility and precision. The RSD for all parameters was found to be less which indicates the validity of method and assay results obtained by this method are in fair agreement. The developed method can be used for routine quantitative simultaneous estimation of Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride in pharmaceutical preparation.

ACKNOWLEDGMENTS:

The authors are grateful to Institute of Science and Technology for Advanced Studies and Research (ISTAR) and A. R. college of Pharmacy, Vallabh Vidhyanagar, India, for providing the facilities to carry the experiment and Panacea Biotec Limited, Baddi(H.P.) for providing gift samples of Metformin Hydrochloride, Rosiglitazone Maleate and Glimepiride.

REFERENCES:

1. Bonfigli AR, Manfrini S, Gregorio F, Testa R, Testa I,De sio G, Coppa G. Determination of plasma metformin by a new cation-exchange HPLC technique. Department of Gerontological Research, Center of Biochemistry, INRCA, ancona, Italy

2. Ajithasdas.A and Nancey.K Simultaneous estimation of metformin and glipizide by UV spectrophotometry. Indian Drugs 2000.

3. Vasaderan M, Rami J, Ravisankar S, Suresh B. Ion-pair liquid chromatography technique for the estimation of metformin in its multicomponent dosage forms. Journal Pharmaceutical and Biomedical Analysis 2001;84: 77-84.

4. Lad NR, Bhoir SI, Bhoir IC and Sundaresan M, Concurrent assay of metformin and glimepiride in tablet using RP-HPLC with wavelength programming. Indian Journal of Pharmaceutical Sciences 2003;65(11-12): 650.

5. Kolte BL, Raut BB, Deo AA, Bagool MA, Shinde DB. Liquid chromatographic method for the determination of rosiglitazone in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2003;88: 37-44.

6. Radhakrishna T, Satyanarayana J and Satyanarayana A. LC Determination of rosiglitazone in bulk and pharmaceutical formulation. J Pharm Biomed Anal 2002;29: 873-80.

7. Gomes P, Sippel J, Jablonski A and Steppe M. Determination of rosiglitazone in coated tablets by MEKC and HPLC methods. J Pharm Biomed Anal 2004;36: 909-13.

8. Mamidi RN, Benjamin B, Ramesh M and Srinivas NR. Simple method for the determination of rosiglitazone in human plasma using a commercially available internal standard. Biomed Chromatogr 2003;17: 417-20.

9. Muxlow AM, Fowles S and Russell P. Automated HPLC method for the determination of rosiglitazone in human plasma. J Chromatogr B Biomed Sci Appl 2001;752: 77-84

10. Kim KA, Park JY. Simple and extractionless HPLC determination of rosiglitazone in human plasma and application to pharmacokinetics in human plasma. Biomed Chromatogr 2004;18: 613-5.

11. Altinoz S and Tekeli D. Analysis of glimepiride by using derivative UV spectrophotometric method. J Pharm Biomed Anal 2001;24: 507-15.

12. Pistos C and Koutsopoulou MI. Improved liquid chromatographic tandem mass spectrometric determination and pharmacokinetic study of glimepiride in human plasma. Biomed Chromatogr 2005;19: 394-401.

13. Pistos C, Astraka C, Kalovidouris M, Vassilopoulos E and Koutsopoulou M. Bioequivalence evaluation of two brands of glimepiride 4 mg tablets in healthy subjects. Int J Clin Pharmacol Ther 2005;43: 203-8.

14. Wanjari DB and Gaikwad NJ. Reversed phase HPLC method for determination of glimepiride in tablet dosage forms. Indian J Pharm Sci 2005;67: 253-5.

Received on 14.09.2009 Modified on 17.11.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 83-86