Development and validation of a sensitive liquid chromatography-tandem mass spectrometry method for the determination of paeoniflorin in rat brain and its application to pharmacokinetic study.

A sensitive and specific method was developed and validated for the determination of paeoniflorin in rat brain with liquid chromatography-tandem mass spectrometry. Sample pretreatment involved protein precipitation following solid-phase extraction. Paeoniflorin and geniposide (internal standard) were separated isocratically on a Waters Symmetry C18 column (150 mm x 2.1 mm i.d., 5 microm), using a mobile phase of methanol/water with 0.1% formic acid (50:50, v/v) at a flow-rate of 200-300 microL/min in 4min. A Finngan LTQ tandem mass spectrometer equipped with electrospray ionization source was operated in the positive ion mode. Selective reaction monitoring was performed to quantify paeoniflorin and the internal standard at m/z transitions of 503-->381 and 411-->231, respectively. A good linearity was found in the range of 2-500 ng/mL (R(2)=0.9939). The intra- and inter-batch assay precisions (coefficient of variation, CV) at 5, 50 and 400 ng/mL (n=5) ranged from 6.3% to 9.7% and 1.2% to 7.2%, respectively, and the accuracies were from 95.9% to 101.6% and 99.4% to 102.9%, respectively. The mean recoveries of paeoniflorin were 81.2%, 80.9% and 82.3% at 5, 50 and 400 ng/mL (n=5), respectively, and the mean recovery of the internal standard was 76.7% with a concentration of 50 ng/mL (n=5). Stability studies showed that paeoniflorin was stable in different conditions. Finally, the method was successfully applied to the pharmacokinetic study of paeoniflorin in rat brain following a single subcutaneous administration (10 mg/kg) to rats.