Determination of paeoniflorin, calycosin-7-O-β-d-glucoside, ononin, calycosin and formononetin in rat plasma after oral administration of Buyang Huanwu decoction for their pharmacokinetic study by liquid chromatography-mass spectrometry

The purpose of this study was to simultaneously investigate the pharmacokinetics of five bioactive compounds in rat plasma after oral administration of Buyang Huanwu decoction (BYHWD) using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). The separations were performed on a Thermo Hypersil Gold C₁₈ analytical column (50 × 2.1mm, 3μm) with the column temperature kept at 30°C. The quantitative analysis was performed using a quadrupole mass spectrometer detector operated under selected ion monitoring mode. A linear gradient elution of A (0.1% formic acid solution) and B (100% acetonitrile) was used at a flow rate of 0.2mL/min. The method was validated within the concentration ranges 1.8-450, 6.0-1500, 2.0-500, 1.2-300 and 1.2-150ng/mL for paeoniflorin, calycosin-7-O-β-d-glucoside, ononin, calycosin and formononetin, respectively. The calibration curves were linear with correlation coefficients > 0.99. The lower limits of quantitations were < 6.0ng/mL. The method was further applied to assess the pharmacokinetics of the five bioactive constituents of BYHWD in rat plasma.