MET is the first-line drug of choice for the treatment of type 2 diabetes, particular in overweight and obese people and those with normal kidney function. It is used in the treatment of polycystic ovary syndrome and has been investigated for other diseases where insulin resistance may be an important factor. Literature survey reveals that some methods have been reported for determination of MET by UV Spectroscopy [8], HPLC [9], HPTLC [10] and LC/MS [11]. This present work describes the development and validation of a new RP HPLC method for simultaneous estimation of VIDA and MET in bulk and in tablet dosage formulation.

2 EXPERIMENTAL:

2.1 Materials

Reference standard of VIDA and MET gifted by Fortune Health Care Pvt. Ltd., Vadodara (Gujarat), India and Novartis Pharmaceuticals Pvt. Ltd., Hyderabad, (A.P.) India respectively and they were used as such without further purification. Water HPLC grade was obtained from a Milli-QRO water purification system. All the chemicals and reagents used were of analytical reagent grade.

2.2 Instrumentation

Chromatographic separation was performed on HPLC system consisted of a waters pump, UV-Visible detector, Lichrocart C18 column (250 x 4.60 x 5μm) was used for separation. Mobile Phase 0.05 M KH₂PO₄: Acetonitrile (70:30 v/v, pH 3.5 with Ortho Phosphoric Acid) was filtered through 0.45 m filter paper. Then it was degassed by sonicator. The employed flow rate for analysis was 1.0 ml/min. After making suitable dilutions sample was scanned under UV range and maximum absorbance was found at λ _max215 nm.

2.3 Sample Preparation

2.3.1 Preparation of Standard Stock Solution

10 mg of VIDA and 10 mg of MET were weighed accurately and transferred to separate 10 ml volumetric flask, and the volume was adjusted to the mark with the mobile phase to give a stock solution of 1000 μg/ml.

2.3.2 Preparation of Working Standard Solution

From stock solution of VIDA 05-25 µg/ml and from stock solution of MET 10-50 µg/ml concentrations were prepared.

2.4 Calibration

The contents of the drug were determined using a calibration curve established with six dilutions of each standard at concentrations ranging from 5 to 25 mg/mL for VIDA and 10 to 50 mg/mL for MET. Each concentration was measured in triplicate. The corresponding peak areas were plotted against the concentrations of the drug sample injected. Peak identification was achieved by comparison of both the retention time and UV absorption spectrum with those obtained for standards.

2.5 System Suitability [13]

According to United State Pharmacopoeia 2007¹⁴system suitability tests are an integral part of LC method in the course of optimizing the conditions of the proposed method¹⁵.

Figure No .1 Chromatogram of VIDA and Metformin

The system suitability test solution was injected and chromatographic parameters like resolution, tailing factor and theoretical plates for VIDA and MET were evaluated for proving the system suitability.

Table No.1 System Suitability Parameters

System Suitability Parameter	VIDA*	MET*
Retention Time	6.645 + 0.3 min	5.187 + 0.3 min
Area Under Curve	5233.62	6570.23
Theoretical Plates	1.97	1.15

(* Results are average of 3 readings)

Table No. 2 Summary of Validation Parameters of Proposed method

PARAMETERS	VIDA	MET
Linearity
Range	5 – 25 mg/ml	10 – 50 mg/ml
Intercept	- 0.0855	- 0.9885
Slope	0.0009	0.0032
Correlation Coefficient	0.999	0.999
Equation of Line	y=5056x + 114.3	y=3034x + 306.7
Accuracy
Mean % Recovery	99.53	99.71
Precision (%RSD)
Intraday (n=3)	0.37	0.46
Interday (n=3)	0.88	0.65
Repeatability (n=6)	0.159	0.156
Ruggedness (%RSD)
Analyst 1 (n=3)	0.110	0.115
Analyst 2 (n=3)	0.225	0.220

2.6 Validation parameter [13-19]

The method was validated according to ICH guideline for linearity, precision, accuracy, selectivity. Selectivity was checked using VIDA and MET and a mixture of standards in order to optimize separation and detection. Linearity of the method was performed by analyzing a standard solution of drugs by the method in the concentration ranging 5-25 mg/mL for VIDA and 10-50 mg/mL for MET. The accuracy of the proposed method was determined by a recovery study carried out by addition method. The samples were spiked with three different amounts of standard compounds. The spiked samples were extracted in triplicate and analyzed under the previously established optimal conditions. The obtained average contents of the target compounds were used to calculate the spike recoveries. Precision was determined by repeatability and inter-day and inter-day reproducibility experiments. Standard solution containing VIDA and MET were injected six times. Drugs were also extracted six times to evaluate the repeatability of the extraction process. The mean amount and standard deviation (SD) value of each constitute were calculated. All the calculated data for analytical parameters summarized in Table No. 2.

2.7 Tablet studies

The proposed method was successfully applied to the analysis of marketed products and the results obtained are given in Table 3.

Table No. 3 Analysis for VIDA and MET in Tablet Formulation

Std. Conc. µg/ml	VIDAGLIPTIN		METFORMIN
Std. Conc. µg/ml	5 µg/ml	10 µg/ml	30 µg/ml	40 µg/ml
1	4.97	9.98	29.93	39.98
2	4.98	9.98	29.99	39.91
3	4.95	9.99	29.96	39.99
Mean	4.96	9.98	29.96	39.96
%found*	99.33	99.83	99.86	99.9
SD	0.015	0.005	0.03	0.043
%RSD	0.307	0.057	0.100	0.109

*Each reading is mean reading of three batch of formulation

3 CONCLUSION:

The results showed that the method provided adequate accuracy, precision, sensitivity, reproducibility with better resolution for the simultaneous analysis of VIDA and MET. The advantages of proposed method are its short analysis time and a simple procedure for sample preparation. The RP-HPLC method developed for simultaneous analysis of VIDA and MET can be used for routine quality control of their bulk drug mixture and their combined dosage form.

4 ACKNOWLEDGEMENT:

The authors wish to thanks Principal and Management of Shivnagar Vidya Prasarak Mandals College of Pharmacy, Malegaon (Bk), Tal- Baramati, Dist-Pune and SCAN Research Laboratories, Bhopal for providing required lab facilities with enthusiastic environment.

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Received on 22.12.2012

Modified on 28.02.2013

Accepted on 03.04.2013

Research Journal of Pharmaceutical Dosage Forms and Technology. 5(2): March- April, 2013, 95-98