Identification of a major metabolite of danshensu in rat urine and simultaneous determination of danshensu and its metabolite in plasma: application to a pharmacokinetic study in rats.

Danshensu, as the effective component of Salvia miltiorrhiza (Danshen), has been widely used in clinical studies for treatment of cardiovascular diseases in China. A new metabolite, 4-hydroxy-3-methoxyphenyllactic acid was isolated from the urine of rats, and its chemical structure was identified by ultraviolet (UV), Infrared Spectroscopy (IR), mass spectrometry (MS) and nuclear magnetic resonance (NMR). Furthermore, a selective and sensitive high performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method was developed for the simultaneous quantification of danshensu and its major metabolite, 4-hydroxy-3-methoxyphenyllactic acid, in rat plasma after oral and intravenous administration of danshensu. The separation was performed on a Hypersil Gold C18 column (150 × 2.1 mm i.d., 3.0 µm, Thermo, San jose CA, USA) with gradient elution using a mobile phase composed of methanol and water (containing 0.1% formic acid) at a flow rate of 0.2 mL/min. Linear detection responses were obtained for danshensu and 4-hydroxy-3-methoxyphenyllactic acid ranging from 5 to 10000 ng/mL and 5 to 4000 ng/mL, respectively. The lower limits of quantification (LLOQs) for the two compounds were both 5 ng/mL. The intra-and inter-day precision (R.S.D %) were within 5.61% for the two analytes. The average recoveries of the analytes were greater than 72.43%. The method was proved to be stable during all sample storage, preparation and analytic procedures. This method was successfully applied to the pharmacokinetic studies of danshensu and 4-hydroxy-3-methoxyphenyllactic acid after oral and intravenous administration of danshensu in rats.