A simple LC-MS/MS method for the simultaneous quantification of resveratrol and its major phase II metabolites: Assessment of their urinary and biliary excretions in rats

- SALLE was applied to extract Res, its metabolites in bile, urine and feces.
- LC-MS/MS method was validated with acceptable recovery and good accuracy.
- SALLE generated cleaner extracts with low endogenous substance interferences.
- Biliary excretion only took a minor proportion of Res and its metabolites.
- Urine excreted the majority of Res with R3G and R3S as two dominated forms.

Trans-resveratrol (Res) is rapidly metabolized, extensively distributed into various tissues and mainly excreted by urine. The present study aimed to establish a simple LC-MS/MS method to simultaneously quantify Res and its major phase II metabolites (Res-3-O-β-d-glucuronide, R3G; Res-4′-O-β-d-glucuronide, R4′G; Res-resveratrol 3-sulfate, R3S; and Res-4′-sulfate, R4′S), and apply this method to assess their urinary and biliary excretions in rats. A simplified salting-out assisted liquid-liquid extraction (SALLE) strategy was developed to prepare samples with acetonitrile-methanol mixture (8:2, v/v) as extractant and ammonium acetate solution (10 M) as salting-out reagent. The method validation demonstrated an acceptable recovery (>80%), good accuracy (85-115%), low deviation of detection (<15%) and no obvious matrix effect (<20%). Then the validated method was successfully applied to analyze the excretion of Res and its metabolites after intragastric administration of Res at 50 mg/kg in rats. Only a minor proportion of Res (0.51 nmol) and its metabolites (R3S, 35.8 nmol; R4S, 0.25 nmol; R3G, 142.3 nmol; R4′G, 0.19 nmol) were eliminated via bile, while the majority of Res (1670.2 nmol), R3G (14,089.0 nmol) and R3S (2975.6 nmol) were excreted through urine. The major forms found in feces were Res and R3S, which were accumulated up to 241.8 and 250.8 nmol, respectively. In summary, the SALLE technique simplified the samples preparation and could be well popularized, especially for those highly polar compounds in biosamples like urine, bile and feces, where various endogenous substances could significantly affect the extraction recovery and detection response.