A Validated High-Performance Thin-Layer Chromatographic Method for the Quantitation of Protodioscin in Trigonella foenum-graecum and Its Herbal Formulations

Summary

A simple, rapid, and sensitive high-performance thin-layer chro-matographic (HPTLC) method was developed for the estimation of protodioscin from Trigonella foenum-graecum. Chromatographic separation was achieved on aluminum plates precoated with silica gel G60 F₂₅₄ as the stationary phase and chloroform—methanol—glacial acetic acid (6.5:3.0:0.5 v/v) as the mobile phase. Densitometric evaluation was carried out at 515 nm after derivatizing the spots with p-dimethyl aminobenzaldehyde. The R_F value of the protodi-oscin was found to be 0.31 ± 0.02. The response in terms of peak area was found to be linear over the concentration range from 400 to 1400 ng spot⁻¹ with a regression coefcient of 0.9981 and mean recovery of 100.90 ± 3.00%. The developed method was validated as per the International Conference on Harmonization (ICH) guidelines. The limit of detection and limit of quantitation were found to be 100 ng spot⁻¹ and 400 ng spot⁻¹, respectively. Robustness of the developed method was assessed using experimental design, and results were analyzed statistically and graphically. The method was found to be accurate, precise, and robust to carry out the estimation of protodioscin from the plant matrix. The developed method was successfully applied for the estimation of protodioscin from marketed herbal formulations and the seeds, leaves, and roots of T. foenum-graecum.