Validated thin-layer chromatographic method for alternative and simultaneous determination of two anti-gout agents in their fixed dose combinations

A rapid, simple and sensitive thin-layer chromatography (TLC) spectrodensitometric method was developed for the simultaneous determination of colchicine and probenecid in their binary mixtures. The two drugs were quantitatively separated using silica gel 60 F-254 as stationary phase and toluene-ethyl acetate-methanol-ammonia (30:20:20:0.1, v/v/v/v) as mobile phase with UV detection at 248 nm for both drugs and at 354 nm for colchicine alone. Both drugs were efficiently separated with R-f values of 0.33 +/- 0.03 and 0.60 +/- 0.03 for probenecid and colchicine, respectively. The linearity was found to be 16-320 and 120-2400 (ng/band) with quantitation limits of 17.59 and 225.82 ng/band for colchicine and probenecid, respectively, at 248 nm. At 354 nm, the linearity range of colchicine was found 16-240 ng/band with a quantitation limit of 54.03 ng/band. The experimental determination ranges were greatly extended with lower quantitation limits (15.60 and 116.13 ng/band for colchicine and probenecid, respectively at 248 nm, and 13.20 for colchicine at 354 nm) and correlation coefficients were improved when polynomial regression analysis was used. The quadratic model was found to be the best fit for all responses. The method has been validated according to the International Conference on Harmonization (ICH) guidelines providing good correlation coefficients (0.9997-0.9999) for both drugs, and it has been successfully applied in the determination of both drugs in their commercial dosage form without interference from excipients.