Abstract: A method was developed for the quantitative determination of protocatechuic acid in rat plasma by high performance liquid chromatography (HPLC). With added p-hydroxybenzoic acid as internal standard, the plasma samples were extracted with a solvent mixture of methanol and acetonitrile (1∶5, v/v). The analyte was determined by HPLC with a DiamondsilTM C18 column and a mobile phase of acetonitrile-water (adjusted to pH 2.5 with H3PO4 , 9∶91, v/v) at a flow rate of 1.2 mL/min. The UV detection wavelength was 260 nm. The assay exhibited a good linearity in the concentration range from 0.050 to 3.20 mg/L with a correlation coefficient of 0.9978. The limit of quantification was 0.050 mg/L. The relative standard deviations of  intra-day and inter-day determination  were both less than 7.0% and the accuracy ranged from -1.4% to 2.6%. The extraction recoveries of protocatechuic acid  from rat plasma samples at three concentration levels were 83.4%, 87.3%, and 91.1%, respectively. The developed method was applied to a pharmacokinetic study. The main pharmacokinetic parameters in rats after a single oral dose of 10 mL/kg body weight were as follows: Cmax of (3.16±0.03) μg/mL, tmax of (0.50±0.00) h, AUC0t of (39.9±5.9) μg·mL-1·h, AUC0∞ of (54.8±8.1) μg·mL-1·h and t1/2 of (3.87±0.25) h. The method was proved to be simple, sensitive and accurate, thus suitable for the pharmacokinetic study of protocatechuic acid in rat plasma.

Key words: protocatechuic acid, rat plasma , high performance liquid chromatography (HPLC)