INTRODUCTION:

Metformin HCl is a biguanide derivative, chemically 1,1-dimethylbiguanide hydrochloride and oral hypoglycemic agent official in IP, BP and USP. Whereas, Pioglitazone HCl is a thiazolidine dione derivative, chemically 5-(4-[2-(5-ethypyridin-2-yl)ethoxy] benzyl) thiazolidine-2, 4-dione hydrochloride and official in IP and USP^1-3. Pioglitazone is a potent agonist for peroxisome proliferator activated receptor-gamma (PPARγ), activation of which modulates the transcription of a number of insulin responsive genes involved in the control of glucose and lipid metabolism. Whereas Metformin decreases hepatic glucose production, decreases intestinal absorption of glucose and improves insulin sensitivity by increasing peripheral glucose uptake and utilization⁴.

Literature survey reveals that few methods are reported for estimation of Metformin HCl^5-6 and PIO^7-8 alone. Some chromatographic methods are also reported for simultaneous estimation of MET and PIO^9-13.

There are also some UV-Visible spectrophotometric methods, reported for simultaneous estimation of MET and PIO^4,
14-16. The methods reported are mostly Simultaneous Equation using methanol as solvent. Only single reporting is there for Q-analysis and First order Derivative spectroscopy using N,N-dimethyl formamide as a solvent¹⁷. Since both the drugs are available as hydrochloride salt, are soluble in NaOH. The absorbance of PIO is more in NaOH than in Methanol. Therefore we have developed a cost effective, simple, accurate and precise method using 0.1 N NaOH as a solvent. Since the two drugs are available in very vast differential concentration, the method is based on the standard addition technique.

MATERIALS AND METHODS:

Instrumentation:

A Double beam UV‐Visible spectrophotometer (Shimadzu UV-1800) with 10mm matched quartz cells was used. All weighing were done on single pan balance (Shimadzu).

Reagents and chemicals:

MET and PIO reference standards were kindly provided by Aarti Drugs Pvt. Ltd, Mumbai. All the reagents were of analytical grade. Glass double distilled water was used throughout the experiment. Tablets were purchased from local market each containing 500 mg of MET and 30 mg of PIO (PIOZ MF 30, USV Ltd).

Determination of λmax values:

Standard stock solutions of MET (100 μg/ml) and PIO (100 μg/ml) were prepared by dissolving 10 mg of each drug separately in 100 ml of 0.1N NaOH prepared in Glass Double Distilled water. For the selection of analytical wavelengths, standard solutions of MET (10 μg/ml) and PIO (10 μg/ml) were prepared by appropriate dilutions of Standard stock solutions with 0.1 N NaOH and scanned in the spectrum mode from 200 to 400 nm. From the overlain spectra of these drugs [Figure 1], wavelengths 232.5 nm (λmax of MET) and 267 nm (λmax of PIO) were determined.

Figure 1: Overlay spectra of MET and PIO

Second order derivative spectrophotometric method:

Standard solutions of 5-30 µg/ml of MET and 2.5 -15 µg/ml of PIO were prepared and scanned separately in the range of 200-400 nm. These spectrums were converted to second order derivative spectra (Figure 2) by using derivative mode with 21 data point. From the overlain spectra wavelengths 247.5 nm (Zero crossing for PIO) and 279.5 nm (Zero crossing for MET) were selected for determination of MET and PIO respectively. There was proportionate increase in amplitude at both the wavelengths. Calibration curves (Figure 3 and 4) were plotted for both the drugs to obtain regression equation and used for determination of the drugs in tablet formulation.

Fig. 2: Overlay of second order derivative spectra of MET and PIO

Figure 3: Calibration curve for MET at 247.5 nm

Figure 4: Calibration curve for PIO at 279.5nm

Formulation Analysis:

For estimating MET and PIO in tablet formulation, twenty tablets (PIOZ* MF 30 by USV Ltd.) containing 500 mg of MET and 30 mg of PIO were weighed and average weight was calculated. The tablets were crushed to obtain fine powder. Powder equivalent to 100 mg of MET and 6 mg of PIO was weighed, 44 mg of standard PIO was added externally by standard addition method and transferred to 100 ml volumetric flask. Mixture was dissolved in 50 ml of 0.1 N NaOH by sonication for 20 min and volume was made up to the mark with same solvent. The solution was then filtered though Whatmann filter paper No. 41. Appropriate aliquots were taken for further analysis.

Method Validation:

The proposed method was validated for the parameters like Linearity, Precision, Accuracy, Limit of Detection and Limit of Quantification as per ICH guidelines.

RESULT AND DISCUSSION:

In the present work a second order derivative UV-Visible spectrophotometric method was developed for simultaneous estimation of Metformin HCl and Pioglitazone HCl and applied for commercially available bilayer tablet formulation. The assay result (% label claim) [Table 1] was found to be 98.68 with S. D. ± 1.51for MET and 99.098 with SD ± 1.22 for PIO. It shows that method have been successfully applied for marketed formulation for routine analysis.

Table 1: Analysis of Marketed formulation

Drug	Label claim (mg)	Amount of drug found (mg)	% Drug found ± SD
Metformin HCl	500	493.4	98.68±1.51
Pioglitazone HCl	30	29.729	99.098±1.22

The method was validated for the parameters like Linearity, Precision, Accuracy, Limit of Detection and Limit of quantification as per ICH guidelines. Linearity [Table 2] of the method was observed by linear regression equation method in different concentration range 5-30 μg/ ml and 2.5-15 μg/ ml of MET and PIO respectively. The Correlation coefficient was found to be close to 1.00, indicating good linearity. LOD and LOQ were calculated by equations 3.3 σ/SD and 10 σ/SD respectively. Low values indicate sensitivity of method.

Table 2: Summary of Validation parameters

Parameters	Metformin HCl	Pioglitazone HCl
Wavelength of measurement (nm)	247.5	279.5
Linearity (µg/ml)	5-30	2.5-15
Correlation coefficient	0.999	0.999
LOD (µg/ml)	1.628	0.27
LOQ (µg/ml)	4.884	0.71

Accuracy studies [Table 3] were done by % recovery analysis at three levels, which was found in a range of 96.89-101.197 for MET and 97.38-101.166 for PIO, shows that method is accurate. Precision [Table 4] of the method is done by interday and intraday analysis where the % R.S.D. found to be less than 2, which indicates the validity of method.

Table 3: Recovery data

Drug	Level of addition	Amount added (mg)	Amount Recovered (mg)	% Recovery
Met formin	50%	4	3.8756	96.89
	100%	8	7.9184	98.68
	150%	12	12.2366	101.197
Pio glitazone	50%	2	1.9476	97.38
	100%	4	4.036	100.9
	150%	6	6.07	101.166

Table 4: Intraday and interday precision data

Drug	Intra day		Inter day
Drug	SD	% RSD	SD	% RSD
Metformin HCl	1.55	1.5707	1.49	1.5099
Pioglitazone HCl	1.32	1.3320	1.40	1.4127

CONCLUSION:

A simple, accurate and sensitive second order derivative UV-Visible spectrophotometric method was developed and validated for simultaneous estimation of Metformin HCl and Pioglitazone HCl in a bilayer tablet formulation using UV-Visible Spectrophotometer without prior separation by using 0.1 N NaOH as solvent. Statistical analysis proves that, the method is specific, accurate and repeatable. It can therefore be concluded that use of this method can save much time and money with accuracy.

ACKNOWLEDGEMENT:

The authors are thankful to Aarti Drugs Pvt. Ltd., Mumbai, for providing gift samples of Metformin HCl and Pioglitazone HCl. The authors are thankful to Management of PES Modern College of Pharmacy (for Ladies), Moshi, Pune for providing necessary facility for the work.

REFERENCES:

1. Indian Pharmacopoeia, Government of India, Ministry of Health and Family Welfare, The Indian Pharmacopoeia Commission, Ghaziabad, Vol-I, 1657-1659 and Vol-II, 1916-1918, 2010.

2. British Pharmacopoeia, British Pharmacopoeia Commission Office, London, Vol-II, 1410-1411, 2008.

3. United States Pharmacopeia-34/National Formulary-29, United States Pharmacopeial Convention, MD, Vol-III, 3442-3444 and 3933-3935, 2011.

4. Sujana K, Swathi Rani G, Bhanu Prasad M, Saheethi Reddy M, Simultaneous Estimation of Pioglitazone Hydrochloride and Metformin Hydrochloride using UV Spectroscopic Method. Journal of Biomedical Sciences and Research.2(2); 2010: 110-115.

5. Al-Rimawi F, Development and Validation of an Analytical Method for Metformin Hydrochloride and Its Related Compound (1-Cyanoguanidine) in Tablet Formulations By HPLC-UV. Talanta.79; 2009: 1368-1371.

6. Arayne MS, Sultana N, Zuberi MH. Development and Validation of RP-HPLC Method for the Analysis of Metformin. Pakistani Journal of Pharmaceutical Sciences. 19(3); 2006: 231-5.

7. Radhakrishna T, Sreenivas Rao D, Om Reddy G. Determination of Pioglitazone Hydrochloride in Bulk and Pharmaceutical Formulations by HPLC and MEKC Methods. Journal of Pharmaceutical and Biomedical Analysis. 29(4); 2002: 593-607.

8. Jedlicka A, Klimes J, Grafnetterová T, Reversed-Phase HPLC Methods for Purity Test and Assay of Pioglitazone Hydrochloride in Tablets. Pharmazie. 59(3); 2004: 178-82.

9. Lakshmi KS, Rajesh T, Shrinivas Sharma. Simultaneous Determination of Metformin and Pioglitazone by Reversed Phase HPLC in Pharmaceutical Dosage Forms. Indian Journal of Pharmacy and Pharmaceutical Sciences. 1(2); 2009: 162-166.

10. Shankar MB, Modi VD,Shah DA, Bhatt KK, Mehta RS, Geetha M et al., Estimation of Pioglitazone Hydrochloride and Metformin Hydrochloride in Tablets by Derivative Spectrophotometry and Liquid Chromatographic Methods. J J AOAC International. 88; 2005: 1167-1172.

11. Sahoo PK, Sharma R, Chaturvedi SC. Simultaneous Estimation of Metformin Hydrochloride and Pioglitazone Hydrochloride by RP-HPLC Method From Combined Tablet Dosage Form. Indian Journal Pharmceutical Sciences. 70; 2008: 383-386.

12. Kolte BL, Raut BB, Deo AA, Bagool MA, Shinde DB. Simultaneous High-Performance Liquid Chromatographic Determination of Pioglitazone and Metformin in Pharmaceutical-Dosage Form. Journal of Chromatography Science.42; 2004: 27-31.

13. Srinivas P, Venkataramana K, Srinivasa Rao J, Srinivasa Rao N. Simultaneous Determination of Metformin and Pioglitazone Tablets in Pharmaceutical Dosage Form by RP-HPLC Method. International Journal of Pharmaceutical, Chemical and Biological Sciences. 2(1); 2012: 104-109.

14. Bodar J, Kumar S, Yadav Y et. al. Development of the Spectrophotometric Method for the Simultaneous Estimation of Pioglitazone and Metformin. Pharma Science Monitor, 2011; 957-964.

15. Rathod SD, Patil PM, Jadhav SB, Chaudhari PD. UV Spectrophotometric Simultaneous Determination of Metformin Hydrochloride and Pioglitazone Hydrochloride in Combined Dosage Form. Asian Journal of Pharmaceutical Analysis. 2(1); 2012:05-09.

16. Sayed S, Thomas A. Simultaneous Estimation of Pioglitazone and Metformin Hydrochloride in Tablet Dosage Form along with in vitro Studies. Journal of Pharmacy Research. 2(9); 2009: 1485-1488.

17. Goswami L, Mukhopadhyay S, Durgapal S. Simultaneous Estimation of Metformin and Pioglitazone by Ultraviolet Spectrophotometry. Indian Journal of Pharmaceutical Sciences. 72(4); 2010: 508-510.

Received on 24.07.2014 Accepted on 27.08.2014

Asian J. Pharm. Ana. 4(3): July-Sept. 2014; Page 121-124