Biotransformation of flavonols and taxifolin in hepatocyte in vitro systems as determined by liquid chromatography with various stationary phases and electrospray ionization-quadrupole time-of-flight mass spectrometry

Abstract

Liquid chromatography (LC) on various stationary phases was used for the metabolite profile analysis of quercetin, rutin, isoquercitrin and taxifolin. The metabolites were obtained using an in vitro model system of human and rat hepatocytes in the form of cell suspensions and the primary cultures. For separations of the parent compounds and their metabolites, stationary phases based on C₁₈, C₈, cyanopropyl (CNP) or phenyl (PHE) modifications of silica were tested. CNP and PHE stationary phases operating in reversed-phase mode have been shown to be efficient for separation of parent flavonoids and their polar metabolites. Individual metabolites were identified on the basis of an elemental composition determination using electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI-QqTOF MS) on-line connected with an LC system. Detailed analytical parameters such as retention times, selectivity, resolution of chromatographic peaks, MS fragmentation and UV-vis absorption maxima were determined for individual metabolites, namely for phase II biotransformation products. The predominant metabolites were methylated flavonols and flavonol glucuronides. The highest biotransformation rate was found with taxifolin, which was mainly converted to sulfates. The HPLC/ESI-QqTOF MS analyses revealed that quercetin and taxifolin were metabolized more extensively than the studied glycosides, rutin and isoquercitrin.