Development of a reversed-phase high-performance thin-layer chromatography method for the simultaneous determination of trigonelline and diosgenin biomarkers in trigonella foenum-graecum L. Seeds grown in variable environment

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.