Development and Validation of a Novel High-Performance Thin-Layer Chromatography Method for the Simultaneous Determination of Apigenin and Luteolin in Hygrophila spinosa T. Anders

The present study was undertaken to develop and validate a novel high-performance thin-layer chromatography (HPTLC) method for the quantification of apigenin and luteolin in Hygrophila spinosa. Separation was carried out on HPTLC plates using the mobile phase toluene—ethyl acetate—formic acid (6.0:4.0:1.0, v/v), and detection was achieved at 349 nm. The method was validated by the International Conference on Harmonization (ICH) guidelines. The calibration range was 80–560 ng per band (R2 = 0.997) for apigenin and 40–280 ng per band (R2 = 0.998) for luteolin. The method was accurate in triplicate results at different standard addition levels with average recoveries of 99.94 and 100.01% for apigenin and luteolin, respectively. The limits of detection and quantification were 6.25 and 18.95 ng, respectively, for apigenin, and 2.36 and 7.55 ng, respectively, for luteolin. The quantity of apigenin was 8.84 and 15.67 mg g−1 in the alcoholic extract and its ethyl acetate fraction, respectively. The luteolin contents in the alcoholic extract and ethyl acetate fraction were 0.172 and 0.464 mg g−1, respectively. Further, luteolin was found to be present only in the flower (6.87 mg g−1) extract, but apigenin was detected in the husk (2.38 mg g−1), flower (7.97 mg g−1), spine (1.01 mg g−1), fruits (0.60 mg g−1), and leaf (4.28 mg g−1). The developed method could be used for the quality control analysis and quantification of apigenin and luteolin in herbal preparations containing H. spinosa.