Determination and assay validation of pinosylvin in rat serum: application to drug metabolism and pharmacokinetics

A method of analysis of pinosylvin in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and determine its concentration in natural products. A novel and simple high-performance liquid chromatographic method was developed for simultaneous determination of pinosylvin and products of its metabolism in rat serum and liver microsomes. Serum, or microsomes (0.1 mL) were precipitated with acetonitrile after addition of the internal standard, 7-ethoxycoumarin. Separation was achieved on an amylose tris 3,5 dimethylphenylcarbamate column (150 mm × 4.6 mm, ID, 5 μm) with UV detection at 308 nm. The calibration curves were linear ranging from 0.5 to 100 μg/mL. The mean extraction efficiency was >99%. Precision of the assay (coefficient of variation) was <10%, including the limit of quantitation (0.5 μg/mL). Bias of the assay was lower than 15%. The limit of detection was 100 ng/mL for a 0.1 mL sample. The assay was successfully applied to both the in vitro and in vivo metabolic kinetic study of pinosylvin. Three metabolites of pinosylvin, two oxidative and one glucuronidated, have been identified. The two oxidative metabolites of pinosylvin have been identified as E- and Z-resveratrol.