Research Article Development and Validation of HPTLC Method for Simultaneous Estimation of Diosgenin and Quercetin in Fenugreek Seeds (Trigonella foenum-graceum) Omi Laila, 1,2 Imtiyaz Murtaza, 1 M. Z. Abdin, 2 S. Ahmad, 3 Nisar Ahmad Ganai, 4 and Majid Jehangir 1 1 Biochemistry and Molecular Biotechnology Laboratory, Biochemistry Section, Division of Post Harvest Technology, SKUAST-K, Shalimar Campus, Jammu and Kashmir 191121, India 2 Centre for Transgenic Plant Development, Department of Biotechnology, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India 3 Bioactive Natural Product Laboratory, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India 4 TSRI, Mirgund, SKUAST-K, Kashmir, India Correspondence should be addressed to Imtiyaz Murtaza; imz murtaza@hotmail.com and M. Z. Abdin; mzabdin@rediffmail.com Received 17 November 2013; Accepted 22 December 2013; Published 10 April 2014 Academic Editors: M. A. Lacaille-Dubois, F. J. Se˜ nor´ ans, and X. Wu Copyright © 2014 Omi Laila et al. is 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 aſter spraying with anisaldehyde-sulphuric acid reagent. e retardation factorvalue of diosgenin and quercetin was found to be 0.69 ± 0.02 and 0.57 ± 0.02, respectively. e 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). e 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. 1. Introduction Fenugreek (Trigonella foenum-graceum Linn., Fam. Legumi- nosae), highly esteemed by both east and west, has a long history of being used as a medicinal herb and has been regarded as a treatment for just about every ailment known to man [1]. Right from early times, it has been extensively used in both Indian Ayurveda and Unani systems of medicines as well as traditional Chinese medicines for treatment of epilepsy, paralysis, gout, dropsy, chronic cough, diabetes, piles sinus, and lung congestion, inflammation, infection mitigation, hair treatment, breast enhancement, and aphrodisiac effects [2– 4]. e crop species has also long been used as a galactagogue to promote lactation in weaning mothers as well as for its ability to treat wounds and sore muscles [2, 5]. With the growing need for safer drugs, such type of traditional herbal medicines have been extensively preferred to prevent and cure human diseases because of easy accessibility by the local people and most importantly low toxicity [6]. erefore, in view of this, human use of fenugreek is expected to increase day by day. Research literature survey suggests pharmacolog- ical properties of fenugreek seed are attributed to presence of specific bioactive compounds like steroidal diosgenin, alka- loid trigonelline, flavonoid quercetin, galactomannan, and unusual amino acid 4-hydroxyisoleucine [7]. Among these above mentioned key bioactive compounds of fenugreek, diosgenin, and quercetin (Figure 1), have been reported to be of utmost importance and are known to produce desired Hindawi Publishing Corporation ISRN Chromatography Volume 2014, Article ID 583047, 8 pages http://dx.doi.org/10.1155/2014/583047