Determination of resveratrol and its sulfate and glucuronide metabolites in plasma by LC–MS/MS and their pharmacokinetics in dogs

An analytical approach for the determination of trans-resveratrol (3,5,4′-trihydroxy-trans-stilbene) and its glucuronide and sulfate conjugates in dog plasma by LC–MS/MS (without enzymatic hydrolysis of the conjugates) was validated to support pre-clinical toxicological and pharmacological studies. The approach required two independent sample extractions and consequent instrument runs. Samples for resveratrol determination were prepared by protein precipitation with acetonitrile; acetonitrile–methanol was used instead for resveratrol metabolites. Chromatographic separation was performed using a C18 column (30 mm × 2.0 mm) at a flow rate of 0.25 mL/min. For resveratrol the mobile phase consisted of A: 5 mM ammonium acetate in water–isopropanol (98:2, v/v) and B: methanol–isopropanol (98:2, v/v) and for metabolites the mobile phase was modified as follows: A: 0.1% (v/v) formic acid in water and B: 0.1% (v/v) formic acid in acetonitrile. Total run time was 12 min for each run with retention times of about 4–5 min for all analytes. A turbo ion spray source was used operating in negative mode for resveratrol and resveratrol sulfate and in positive mode for resveratrol glucuronide. Calibration curves were linear from 5 to 1000 ng/mL for resveratrol and its glucuronide, and 10–2000 ng/mL for resveratrol sulfate. Linearity was assessed using the internal standard method for resveratrol and the external standard method for the metabolites. Method accuracy was 90–112% of the true value for all analytes with precision of 9% RSD or less for all validation experiments. The validated method was applied to a preclinical toxicology study in dogs after oral administration (200–1200 mg/kg) of the agent. Peak plasma resveratrol concentration (Cmax) for most animals was observed within 1–5 h of dosing, with group mean values in the 1.7–9.9 μg/mL (7.5–43 μM) range. Area under the plasma concentration–time curve (AUC) mean values for resveratrol ranged from 3.6 to 44 h μg/mL for all study groups and were generally proportional to the dose, with no consistent statistically significant changes observed for gender or number of doses. Mean molecular-weight adjusted ratios of resveratrol metabolites to resveratrol for AUC ranged from 1 to 9 for resveratrol glucuronide and from 2 to 11 for resveratrol sulfate.

► Plasma method validated for resveratrol metabolites without enzymatic hydrolysis. ► Peak resveratrol plasma concentration in dogs observed within 1–5 h after oral dosing. ► Resveratrol was rapidly metabolized to glucuronide and sulfate. ► AUC ratios of resveratrol metabolites to resveratrol ranged from 1 to 11.