Stereospecific determination of cis- and trans-resveratrol in rat plasma by HPLC: Application to pharmacokinetic studies

A simple, accurate, precise, specific and reproducible high-performance liquid chromatography (HPLC) method was developed for simultaneous determination of resveratrol isomers in rat plasma. Cis-resveratrol was made by exposure of a trans-resveratrol solution to sunlight for 5 days followed by separation by HPLC and identification by mass spectrometry (MS). The assay procedure involved simple liquid-liquid extraction of resveratrol isomers and internal standard (IS, caffeine) from a small plasma volume directly into acetonitrile. The supernatant liquid was added an equal volume of water and injected onto a Hypersil ODS₂ C₁₈ column (5 μm, 4.6 × 250 mm). Mobile phase consisting of methanol and distilled water was used at a flow rate of 1.0 mL/min for the effective separation of cis-, trans-resveratrol and caffeine (IS). The detection of the analyte peak was achieved by monitoring the eluate using a UV detector set at 303 nm. The ratio of peak area of analyte to IS was used for quantification of plasma samples. Nominal retention times of cis-, trans-resveratrol and IS were 3.2, 4.3 and 6.1 min, respectively. The calibration curve was linear ranging from 0.066 to 6.64 and 0.134 to 13.4 μg/mL with correlation coefficients of 0.9998 and 0.9997 for trans and cis isomers, respectively. The absolute recovery of both isomers was more than 85%. The inter- and intra-day precisions m the measurement of quality control (QC) samples, 0.066, 0.664 and 6.64 μg/mL of trans-resveratrol, were in the range 2.37-6.95% relative standard deviation (RSD) and 0.77-6.97% RSD, respectively. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 0.134, 1.34 and 13.4 μg/mL of cis-resveratrol, were in the range 1.93-3.72% relative standard deviation (RSD) and 1.13-6.57% RSD, respectively. Both analytes and IS were stable in the battery of stability studies and freeze-thaw cycles. Resveratrol isomers were found to be stable for a period of 30 days on storage at -20°C. The application of the assay to determine the pharmacokinetic disposition after a single oral dose to rats is described.