Elaboration and Verification of the HPTLC Methodology for the Simultaneous Estimation of Glycyrrhizic Acid (GLY), Ellagic Acid, and Kaempferolin in Herbal Formulations

Abstract

Glycyrrhizic acid (GLY), Ellagic acid (EA), and kaempferol (KAE) can be detected simultaneously using the suggested herbal formulation using a simple, precise, and proven high performance thin layer chromatography (HPTLC) method. Using Merck TLC aluminum sheets of silica gel G60 F254 with a 200 µm thickness, the separation was carried out using ethyl acetate: toluene: formic acid (4.5:4.5:1 v/v/v) as the mobile phase. The chemical was densitometrically measured in absorbance mode at 265 nm. For ellagic acid, glycyrrhizic acid, and kaempferol, the aforementioned mobile phase yielded discrete peaks with Rf values of 0.41±0.03, 0.28±0.03, and 0.76±0.03, respectively. The linear regression analysis results for the calibration plots for EA, GLY, and KA showed a reasonable linear association in the concentration range of 300-900 ng/spot for all the chemicals, with regression coefficients (r2) of 0.9936, 0.9923, and 0.9919, respectively. For kaempferol, the limits of quantitation and detection were 44.9678 and 136.2661 ng/spot; for GLY, they were 126.6847 and 383.8930 ng/spot; and for EA, they were 62.5197 and 189.4539 ng/spot, respectively. The established HPTLC approach that has been proposed can be used to identify and quantify measurement of EA, GLY, and KAE in their extracts and herbal formulations. In the end, the statistical analysis of the data showed that the approach is both selective and repeatable for estimating every molecule.