Reverse Phase-HPLC

A simple, economic and precise RP-HPLC method has been developed and validated for simultaneous estimation of thiocolchicoside (THC) and diclofenac (DCF) both in bulk drug and in capsule formulation. Reversed-phase chromatography was performed on a C18 Phenomenex-Gemini column with mobile phase acetonitrile: water (70:30 % v/v, adjusted at pH 3.0) at a flow rate of 1.0 ml/min. Detection was performed at 258 nm and sharp peaks were obtained for THC and DCF at a retention time of 1.537 min and 4.010 min respectively. The method was validated for accuracy, precision, detection and quantification limits, and system suitability in accordance with ICH guidelines. Linear regression analysis data for the calibration plot showed there was a linear relationship between response and concentration for THC in the range 4 - 24 μg/ml with the correlation coefficient 0.9998 and the linear regression equation y = 20.64x + 26.08. Linearity was observed in the range of 25 - 150 μg/ml with the correlation coefficient 0.9998 and the linear regression equation y = 11.13x + 65.27 for DCF. The detection (LOD) and quantification (LOQ) limits for THC were found to be 1.052 and 3.187 μg/ml and for DCF it was found to be 1.475 and 4.470 μg/ml. Statistical analysis proved that the method was precise, reproducible and accurate for simultaneous estimation of THC and DCF. The wide linearity range, sensitivity, accuracy, short retention time, and simple mobile phase imply that the method is suitable for routine quantification of THC and DCF with high precision and accuracy.