Table of Contents
ISRN Chromatography
Volume 2014, Article ID 583047, 8 pages
Research Article

Development and Validation of HPTLC Method for Simultaneous Estimation of Diosgenin and Quercetin in Fenugreek Seeds (Trigonella foenum-graceum)

1Biochemistry and Molecular Biotechnology Laboratory, Biochemistry Section, Division of Post Harvest Technology, SKUAST-K, Shalimar Campus, Jammu and Kashmir 191121, India
2Centre for Transgenic Plant Development, Department of Biotechnology, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India
3Bioactive Natural Product Laboratory, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India
4TSRI, Mirgund, SKUAST-K, Kashmir, India

Received 17 November 2013; Accepted 22 December 2013; Published 10 April 2014

Academic Editors: M. A. Lacaille-Dubois, F. J. Señoráns, and X. Wu

Copyright © 2014 Omi Laila et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


A sensitive, fast, and reproducible high performance thin-layer chromatographic method has been developed for simultaneous analysis of diosgenin and quercetin from fenugreek seeds, using TLC aluminium plates precoated with silica gel G60F254. Among the different combinations of mobile phases used, best separation was achieved in Toluene-ethyl acetate-formic acid (5 : 4 : 1, v/v/v). Densitometric scanning of the plates directly at 275nm was used for analysis of quercetin. While as for analysis of diosgenin, plates were scanned at 450nm after spraying with anisaldehyde-sulphuric acid reagent. The retardation factorvalue of diosgenin and quercetin was found to be 0.69 ± 0.02 and 0.57 ± 0.02, respectively. The method was validated for specificity, precision (intraday and interday), accuracy, and robustness. Accuracy of the method was checked by recovery study of three different levels with the average percentage recovery of 99.13 ± 0.26 for diosgenin and 99.63 ± 0.34 for quercetin, respectively. Dried fenugreek seed samples were found to contain diosgenin in the range of 0.113–0.135% (w/w) and quercetin in the range of 0.009–0.012% (w/w). The present method is being reported for the first time and can be used for routine quality control and quantification of these marker compounds in various plant samples, extracts, and market formulations.