A validated high-performance thin-layer chromatography method for the quantification of chlorogenic acid in the hydroalcoholic extract of Gynura cusimbua leaves

Abstract

A simple, reliable, and reproducible high-performance thin-layer chromatography (HPTLC) method was developed for the quantitative determination of chlorogenic acid in the hydroalcoholic extract of Gynura cusimbua (D. Don) S. Moore leaves (HAGC). HPTLC plates (10 cm × 10 cm) pre-coated with silica gel 60F254 as the stationary phase and a mobile phase consisting of ethyl acetate‒formic acid‒acetic acid‒water (100:11:11:2.6, V/V) were used for achieving good separation. Densitometric evaluation for fingerprinting and quantification was performed at a scanning speed of 100 mm/s and 6 × 0.45-nm micro-slit dimension at 366 nm in absorption mode with a deuterium lamp. The method was validated in terms of linearity, limits of detection and quantification, precision, accuracy, and robustness parameters. The RF of chlorogenic acid was 0.43 ± 0.01. The linear regression analysis data for the calibration plots of chlorogenic acid showed a good linearity relationship with r2 = 0.9996, in the concentration range of 50‒250 ng/spot. The limits of quantification and detection were 43.12 and 14.36 ng/band, respectively. The amount of chlorogenic acid in HAGC was found to be 3.37% w/w. The quantification and the method validation of chlorogenic acid have not yet been reported for G. cusimbua which can be employed for the appropriate standardization of the plant extract/formulation. Graphical Abstract: [Figure not available: see fulltext.].