Development of a Quantitative Method for Hydroxychavicol in the Ethyl Acetate Fraction of Piper betle L. Ethanol Extract Using High-Performance Liquid Chromatography

In this study, we developed a quantitative analysis method for hydroxychavicol (HC) in the ethyl acetate fraction of the total ethanol extract of Piper betle using high-performance liquid chromatography. The stationary phase utilized a reversed-phase silica gel C18 column (5 μm x 4.6 mm x 250 mm), while the mobile phase consisted of acetonitrile and 0.05% orthophosphoric acid in water (30:70, v/v). The flow rate was set to 1 ml/min, with a chromatography run time of 20 minutes and a column temperature of 40 °C. Detection was carried out at a wavelength of 210 nm, corresponding to the maximum absorption wavelength of HC. The injection volume was 20 µl. The retention time for HC was about 14.03 minutes. Within the concentration range of HC from 7.68 to 276.50 µg/ml, there was a strong linear correlation between peak area and solution concentration. The method ensured specificity for HC, with high accuracy and precision, evidenced by a recovery rate from 98% to 102% and a relative standard deviation of repeatability ≤ 2%. The limits of detection and quantification were 0.87 µg/ml and 2.87 µg/ml, respectively. Hydroxychavicol, quantification, high-performance liquid chromatography, Piper betle.