Determination of Protocatechuic Acid in Rat Plasma by High Performance Liquid Chromatography

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 Diamondsil (TM) C-18 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. 997 8. 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: C-max of (3. 16 +/- 0. 03) mu g/mL, t(max) of (0. 50 +/- 0. 00) h, AUC(0 -> t) of (39. 9 +/- 5. 9) mu g . mL(-1) . h, AUC(0 ->infinity) of (54. 8 +/- 8. 1) mu g . mL(-1) x h and t(1/2) 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.