Abstract
We have recently reported on the effect of the environmental conditions on the quantity of diosgenin. Attempts for the simultaneous quantification of trigonelline and diosgenin using normal-phase silica gel plates were not successful. A high-performance thin-layer chromatography (HPTLC) method was developed using glass-backed plates coated with RP-18 silica gel 60 F-254S and acetonitrile-water (7.5:2.5, V/V) as the mobile phase. Trigonelline and diosgenin peaks were well separated with R-F values 0.29 +/- 0.02 and 0.17 +/- 0.01, respectively. The TLC plates were directly scanned at 267 nm for trigonelline and at 430 nm after derivatization with vanillin-sulfuric acid for diosgenin. Linear regression analysis revealed a good linear relationship between the peak area and the amounts of trigonelline and diosgenin in the range of 200-1400 and 50-300 ng per band, respectively. The method was validated in accordance with the International Conference on Harmonization (ICH) guidelines for precision, accuracy, and robustness.