Hyphenated chromatographic analysis of bioactive gallic acid and quercetin in Hygrophila auriculata (K. Schum) Heine growing wildly in marshy places in India by validated HPTLC method

Abstract

ObjectiveA simple, accurate, and rapid high-performance thin-layer chromatographic (HPTLC) method for simultaneous quantification of the two biologically active flavonoidal compounds, gallic acid and quercetin, in Hygrophila auriculata (K. Schum) Heine (HA) has been established and validated. MethodsChromatography was performed on aluminium foil-backed silica gel 60 F254 HPTLC plates with the binary mobile phase toluene: ethyl acetate: formic acid (5:4:1, v/v/v). Ultraviolet detection was performed densitometrically at the maximum absorbance wavelength, 270nm. The method was validated for precision, recovery, robustness, specificity, and detection and quantification limits, in accordance with ICH guidelines. ResultsThe system was found to give compact spots for gallic acid (GA) and quercetin (QE) (Rf value of 0.31 and 0.50, respectively). The limit of detection (23 and 41 ng band−1) limit of quantification (69 and 123 ng band−1), recovery (99.4–99.9 and 98.7–99.4%), and precision (i.e ≤1.98 and 1.97) were satisfactory for gallic acid and quercetin respectively. Linearity range for GA and QE were 100–1000 (r2= 0.9991) and 150–900 ng band−1 (r2= 0.9956) and the contents estimated as 0.28±0.01% and 0.41±0.01% w/w respectively. ConclusionsThis simple, precise and accurate method gave good resolution from other constituents present in the extract. The method has been successfully applied in the analysis and routine quality control of herbal material and formulations containing Hygrophila auriculata (K. Schum) Heine.