Shekhar M. Bhavsar et al _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ J. Chem. Pharm. Res., 2010, 2(2): 563-572 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
few ml of the filtrate. Dilute 10ml of this solution to 25.0mL with mobile phase and mix to obtain final concentration of 80ppm of both the drugs.
Results and Discussion
Literature review reveals that the published analytical methods for estimation of Lornoxicam and Thiocolchicoside in their formulation were not reported for the estimation in their combined dosage form.
Pure drugs chromatogram was run in different mobile phases containing methanol and different buffers in different ratios. Different columns (e.g. C8, C18, phenyle) with different dimensions were used. The retention time and tailing factor was calculated for each drugs and for each chromatogram. Finally Buffer(5.7606 gm Ammonium Dihydrogen Phosphate in 2000mL of milli- Q water, adjust pH 7.3 with Tri Ethyl Amine): Methanol as a mobile phase in the volume of ratio 45:55 v/v and Inertsil ODS 3V C-18 (250 x 4.6 mm), 5 µ analytical column was selected which gave a sharp and symmetrical peak with minimum tailing. Inertsil ODS 3V C-18 (250 x 4.6 mm), 5 µ column has an embedded polar groups. It has high carbon loads, which provide high peak purity and more retention to polar drugs.
Calibration graph was found to be linear at range 0.24 – 120 g mL-1and 0.235 – 120 g mL-1 for Lornoxicam and Thiocolchicoside respectively. Six different concentrations of a mixture of two drugs in the range given above were prepared and 10 µL of each solution injected in HPLC. Regression analysis of the calibration data for Lornoxicam and Thiocolchicoside showed that the dependent variable (peak area) and the independent variable (concentration) were represented by the equations: y = m x + c was found to y = 29738.77 x + 2700.34 and y = 18227.53 x -3713.71 for Lornoxicam and Thiocolchicoside respectively.
The correlation of coefficient (r2) obtained was found to be 0.9999 for both the drugs. It was observed that the concentration range showed a good relationship. The limit of detection for both the drugs was found to be 0.8 g mL-1 and the limit of quantification was found to be 0.24 g mL- and 0.235 g mL-1 for Lornoxicam and Thiocolchicoside respectively. It proves the sensitivity of method for the drugs. The % assay or average amount of Lornoxicam and Thiocolchicoside was found to be 96.3% and 100.2% respectively in each tablet. The average % recovery for Lornoxicam and Thiocolchicoside was found to be 100.83% and 100.53% respectively which shows that method is free from interference from excipients present in the formulation. The low values of standard deviation and coefficient of variation at each level of the recovery experiment indicate high precision of the method. 1
The proposed method was applied for routine analysis of solid dosage form. The method was found linear, robust, specific and reproducible.
Method Validation Validation was done as per ICH guideline Q2 (R1) . The developed method was validated with respect to parameters such as linearity, precision, accuracy, specificity, ruggedness, robustness and solution stability.