Several medical plants belonging to the genera Passiflora, Viola, and Crataegus accumulate flavonoid C-glycosides, which likely contribute to their efficacy. Information regarding their phase I and II metabolism in the liver are lacking. Thus, in vitro liver metabolism of orientin, isoorientin, schaftoside, isoschaftoside, vitexin, and isovitexin, all of which accumulated in Passiflora incarnata L., was investigated by incubation in subcellular systems with human liver microsomes and human liver S9 fraction. All metabolite profiles were comprehensively characterized using HPLC-DAD and UHPLC-MS/MS analysis. Mono-glycosylic flavones of the luteolin-type orientin and isoorientin showed a broad range of mono-glucuronidated and mono-sulfated metabolites, whereas for mono-glycosylic flavones of the apigenin-type vitexin and isovitexin, only mono-glucuronidates could be detected. For di-glycosylic flavones of the apigenin-type schaftosid and isoschaftosid, no phase I or II metabolites were identified. The main metabolite of isoorientin was isolated using solid-phase extraction and prep. HPLC-DAD and identified as isoorientin-3 & PRIME;-O-alpha-glucuronide by NMR analysis. A second isolated glucuronide was assigned as isoorientin 4 & PRIME;-O-alpha-glucuronide. These findings indicate that vitexin and isovitexin are metabolized preferentially by uridine 5 & PRIME;-diphospho glucuronosyltransferases (UGTs) in the liver. As only orientin and isoorientin showed mono-sulfated and mono-glucuronidated metabolites, the dihydroxy group in 3 & PRIME;,4 & PRIME;-position may be essential for additional sulfation by sulfotransferases (SULTs) in the liver. The diglycosylic flavones schaftoside and isoschaftoside are likely not accepted as substrates of the used liver enzymes under the chosen conditions.</p>