Development and Validation of High-performance Thin layer Chromatography Method for Simultaneous Determination of Gallic Acid and Quercetin from Annona squamosa L. leaf Extract

Abstract

The objective of this research was to develop a validated high performance thin layer chromatography (HPTLC) procedure for identification, estimation and standardization of two biomarker compound Quercetin and gallic acid from Annona squamosa L. Leaf hydro alcoholic Extract. The chromatographic quantification were conducted on normal HPTLC (20 cm x 10 cm glass backed silica gel 60 F 254) with Toluene: Ethyl acetate: Formic Acid (5.4:3.8:0.8 v/v/v) used as a mobile phase . The detection and evaluation were performed by densitometric scanning at 254nm. The system was found to give a well resolved, compact and intense peaks for quercetin (Rf 0.45±0.05) and gallic acid ( Rf 0.28±0.05) at 360nm and 280nm respectively from other constituents present in the extract of Annona squamosa L .The proposed analytical method for both the biomarker compound was found to be handy, simple, precise, linear (%RSD =0.673808 and 0.896642 for GA and Quercetin respectively), accurate, reliable and sensitive for analysis of both the biomarkers. The LOD/ LOQ (ng) for Gallic acid and Quercetin were recorded as 15/45 and 12/36, respectively in the linearity range of 200-1200ng per spot. The obtained result showed quantities of Gallic acid and Quercetin in leaf hydroalcoholic extract as 0.023% w/w and 0.048 w/w, respectively. The developed HPTLC was found to be suitable for the routine analysis of both of these biomarkers in the hydroalcoholic extract of Annona squamosa L. and can be further employed in the process quality control of herbal formulations containing the mentioned biomarkers.