Semi-preparative isolation of dihydroresveratrol-3-O-β-d-glucuronide and four resveratrol conjugates from human urine after oral intake of a resveratrol-containing dietary supplement

A method for semi-preparative isolation of major resveratrol metabolites from human urine after oral intake of a trans-resveratrol-containing dietary supplement was developed. Pretreatment of the urine (6 L) by using solid-phase extraction gave a brown oily residue (9.3 g), which was separated using a combination of normal phase column chromatography and reversed-phase flash column chromatography resulting in fractions containing 1.1 g crude trans-resveratrol-3-O-sulfate (M1), 86 mg of a crude mixture of trans-resveratrol-3,5-O-disulfate (M2) and trans-resveratrol-3,4′-O-disulfate (M3), and 568 mg of a crude mixture of trans-resveratrol-3-O-β-d-glucuronide (M4) and dihydroresveratrol-3-O-β-d-glucuronide (M5). Purification of the crude metabolites was performed by semi-preparative reversed-phase HPLC using a gradient of aqueous ammonium acetate (2.5 mmol/L, pH 6.7)/acetonitrile for purification of M1, M2 and M3 or trifluoroacetic acid in water (pH 2.5)/acetonitrile for purification of M4 and M5. From a part of the crude metabolites (50–75 mg), 47 mg M1 (purity 98.7%), 14 mg M2 (purity 96.1%), 10 mg M3 (purity 96.3%), 38 mg M4 (purity 98.2%) and 18 mg M5 (purity 97.8%) were obtained. The structures of all isolated resveratrol metabolites were elucidated by spectroscopic and spectrometric methods such as 1D and 2D NMR, UV, and LC–MS. This method represents a novel approach to obtain resveratrol metabolites being the first method describing the direct isolation of pure resveratrol metabolites from urine samples in quantities sufficient for full chemical characterization and testing in vitro and in preclinical trials.