Abstract
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 ODS~2~ C~18~ 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 in 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. Copyright © 2007 John Wiley & Sons, Ltd.