HPLC, Densitometric and Spectrophotometric Methods for the Simultaneous Determination of Colchicine and Probenecid in Their Binary Mixture

Abstract

Aim: To develop methods with  complete validation according to ICH guidelines and to be  applied for the determination of both drugs in laboratory prepared mixtures and in pharmaceutical formulation Study Design: High performance liquid chromatography (HPLC), densitometric and different spectrophotometric methods (zero order, derivative ratio, ratio difference and mean centering) are developed for simultaneous determination of colchicine and probenecid in their combined pharmaceutical formulation.  Methodology: High performance liquid chromatography separation is developed using C18 column and methanol: ammonia (100: 1.5 v/v) as a mobile phase. The densitometric method based on the separation of both drugs using chloroform: methanol: ethyl acetate: water: ammonia (7: 5:2.5:0.5:0.5 by volume) as mobile phase and scanning λ at 254 nm. Zero order determination is based on measurement of colchicine absorbance at 349 nm.  The first derivative ratio of peak amplitudes at 367 nm& at 290.4 nm and the ratio difference with the amplitude difference between (385 nm and 362.4 nm) and ( 270 nm and 255 nm)  for colchicine and probenecid, respectively are developed for the determination of both drugs. Mean centering determination of probenecid is developed by measurement at 279 nm using 3.6 µg/mL of colchicine as a divisor. Results: HPLC method was applied over the  concentration ranges of 1.0-45.0 µg/mL & 0.5-30.0, while densitometric method was  linear over the concentration 0.15. 0-0.6 & 0.15-0.45 µg / band  and spectrophotometric methods were linear over the concentration ranges 10.00-55.0 & 3.6-20.0 µg/mL  for colchicine and probenecid, respectively. Conclusion: Novel, simple and accurate method for the determination of colchicine and probenecid simultaneously in their binary mixture.