A simple and specific high performance liquid chromatographic (HPLC) method with UV detection using picroside II as the internal standard was developed and validated to determine the concentration of paeoniflorin in rat plasma and study its pharmacokinetics after an single intravenous administration of 40 mg kg−1 paeoniflorin to Wistar rats. The analytes of interest were extracted from rat plasma samples by ethyl acetate after acidification with 0.05 mol L−1 NaH2PO4 solution (pH 5.0). Chromatographic separation was achieved on an Agilent XDB C18 column (250 × 4.6 mm I.D., 5 μm) with a Shim-pack GVP-ODS C18 guard column (10 × 4.6 mm I.D., 5 μm) using a mobile phase consisting of acetonitrile–water–acetic acid (18:82:0.4, v/v/v) at a flow rate of 1.0 mL min−1. The UV detection was performed at a wavelength of 230 nm. The linear calibration curves were obtained in the concentration range of 0.05–200.0 μg mL−1 in rat plasma with the lower limit of quantification (LLOQ) of 0.05 μg mL−1. The intra- and inter-day precisions in terms of % relative standard deviation (RSD) were lower than 5.7 and 8.2% in rat plasma, respectively. The accuracy in terms of % relative error (RE) ranged from −1.9 to 2.6% in rat plasma. The extraction recoveries of paeoniflorin and picroside II were calculated to be 69.7 and 56.9%, respectively. This validated method was successfully applied to the pharmacokinetic study of a new paeoniflorin frozen dry power formulation. After single intravenous administration, the main pharmacokinetic parameters t1/2, AUC0-∞, CLTOT, VZ, MRT0-∞ and Vss were 0.739 ± 0.232 h, 43.75 ± 6.90 μg h mL−1, 15.50 ± 2.46 L kg−1 h−1, 1.003 ± 0.401 L kg−1, 0.480 ± 0.055 h and 0.444 ± 0.060 L kg−1, respectively.
