A validated high-performance thin-layer chromatography method for the quantification of genistein in the hydroalcoholic extract of Flemingia procumbens Roxb.

Abstract

In the current investigation, a simple, rapid and reproducible high-performance thin-layer chromatography (HPTLC) method was established for the quantitative estimation of genistein in the hydroalcoholic extracts of Flemingia procumbens Roxb. tuber. HPTLC plates (20 cm × 10 cm) precoated with silica gel 60F254 as the stationary phase and a mobile phase consisting of toluene‒ethyl acetate‒acetone‒formic acid (10:2:1:0.5, V/V) were utilized for achieving good separation. Densitometric estimation for fingerprinting and quantification was achieved at a scanning speed of 50 mm/s, 5 × 0.45 nm micro-slit dimension at 271 nm in absorption mode with a deuterium lamp. The developed method was validated for linearity, limits of detection and quantification, precision, accuracy, and robustness parameters as per the International Council for Harmonisation (ICH) guidelines. The retention factor (R F) of genistein was 0.49 ± 0.01. The polynomial regression analysis data for the calibration plots of genistein showed a good linearity relationship with r 2 = 0.999723 ± 0.20%, in the concentration range of 50‒300 ng/spot. The quantification and detection limits were 71.21 and 23.56 ng/band, respectively. The quantity of genistein estimated using the HPTLC‒densitometric method was found to be 7.63 ± 0.02, 27.91 ± 0.05, and 50.41 ± 0.02 µg/mg, respectively, in the hydroalcoholic extract of F. procumbens tuber without peel (i), with only peel (ii), and with whole tubers (iii). This validated HPTLC method of genistein can be applied for the suitable standardization of the plant extract/formulation. Graphical abstract: [Figure not available: see fulltext.]