HPTLC METHOD DEVELOPMENT AND VALIDATION FOR QUANTITATIVE ESTIMATION OF ELLAGIC ACID, GLYCYRRHIZIC ACID AND HESPERIDIN FROM PUNICA GRANATUM PEEL, GLYCYRRHIZA GLABRA ROOT AND CITRUS SINENSIS PEEL EXTRACT

The present study focuses on developing and validating a high-performance thin-layer chromatography (HPTLC) densitometry method for identifying and quantifying ellagic acid, glycyrrhizic acid and hesperidin in the ethanolic extract of Punica granatum, Glycyrrhizic glabra and Citrus sinensis. The separation was achieved by mobile phase for ellagic acid was Methanol: Water: Formic acid (3.8:4.7:0.8) and spotting was done on RP TLC Al plate silica gel 60, for glycyrrhizic acid was ethyl acetate: Formic acid: Glacial acetic acid: Water (14.7:0.8:0.5:1) and mobile phase for hesperidin was n – butanol: Acetic acid: Water (2.8:0.6:0.9) on TLC aluminium plate pre-coated with silica gel 60 F254. Densitometric scanning was carried out at a wavelength of 254nm and 287nm in absorbance mode. The peak corresponding to reference standard and that of the extract had the same RF of 0.19 for ellagic acid, 0.14 for glycyrrhizic acid and 0.58 for hesperidin. Following the International Conference of Harmonization (ICH) requirements for precision, accuracy, and reproducibility, the method was verified. The regression equation showed a linear function with a correlation coefficient of 0.999, 0.997 and 0.999, respectively. The limit of detection was 39, 48.26 and 22.39ng/spot respectively and limit of quantification was 120, 146.2 and 67.87 ng/spot for Ellagic acid, Glycyrrhizic acid and Hesperidin, respectively. The content of ellagic acid from the Punica granatun extract was found to be 0.78% w/w, glycyrrhizic acid from the Glycyrrhiza glabra extract was found to be 0.35% w/w and hesperidin from the Citrus sinensis extract was found to be 0.134% w/w. The validated HPTLC method can be routinely used for the quality control and standardization of the marker Ellagic acid, Glycyrrhizic acid and Hesperidin and formulations containing Punica granatun, Glycyrrhiza glabra and Citrus sinensis.