Detection and identification of 14C-labelled flavonol metabolites by high-performance liquid chromatography–radiocounting and tandem mass spectrometry

One hour after the ingestion of feed containing [2-14C]quercetin-4′-O-β-d-glucoside, an extract from the gastrointestinal tracts of three rats was purified by partitioning and solid-phase extraction techniques. The resultant ethyl acetate and aqueous fractions were then analysed by reversed-phase HPLC with on-line radioactivity detection and ion trap mass spectrometry capable of performing data dependent MS–MS analysis. The presence of the 14C-labelled metabolites was determined with the radioactivity monitor and the mass spectra produced were used to identify 16 of the 17 metabolites detected. These included methylation of the aglycone and the formation of mono and diglucuronides and sulfate conjugates. In the light of these findings, the use of mass spectrometry, operating in various modes, to identify flavonols and their metabolites in body tissues and fluids is discussed.