Determination of asiatic acid in beagle dog plasma after oral administration of Centella asiatica extract by precolumn derivatization RP-HPLC

A novel precolumn derivatization reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV–vis detection for the quantitative determination of total concentration of asiatic acid (AA) in beagle dog plasma is described. AA was extracted with n-hexane-dichloromethane-2-propanol (20:10:1, v/v/v) from plasma, which had been hydrolyzed by acid and derivatized with p-Toluidine. Chromatographic separation was achieved on a C₁₈ column using gradient elution in a water–methanol system. Detection was set at UV wavelength of 248 nm. A calibration curve ranging from 0.01 to 1.5 μg/mL was shown to be linear, and the lower limit of quantification (LLOQ) was 0.01 μg/mL. The intra- and inter-day precisions which were determined by three different concentrations (0.05, 0.2 and 0.8 μg/mL) ranged from 4.4% to 13.1% and 4.6% to 14.2%, respectively. Mean extraction recoveries were no less than 65% for AA and ursolic acid (IS). Plasma samples containing asiatic acid were stable for 30 days at ?20 °C. The method was successfully applied to a pharmacokinetic study in beagle dogs after oral administration of Centella asiatica extract, and the main pharmacokinetic parameters obtained were: T_1/2, 4.29 h; T_max, 2.70 h; C_max, 0.74 μg/mL; AUC_0–t and AUC_0–∞, 3.74 and 3.82 μg h/mL, respectively.