Identification of metabolites of honokiol in rat urine using 13C stable isotope labeling and liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry

• The first research focused on the biotransformation of honokiol in rats.
• Combined stable isotope labeling with LC/MS technology in metabolites analysis.
• A reliable, rapid and sensitive method was established for metabolites.
• 51 metabolites of honokiol were detected, 37 of which were tentatively identified.

A general approach based on stable isotope labeling and UPLC/Q-TOF-MS analysis of in vivo novel metabolites of honokiol has been developed in our study. In this method, urine samples were collected after intravenous administration of mixture of regular and [13C6]-labeled honokiol at 1:1 ratio to healthy rats. The metabolites could be easily recognized by the determination of a chromatographically co-eluted pair of isotopomers (MS doublet peaks) with similar peak intensities and mass difference corresponding to that between isotope-labeled and non-isotope-labeled honokiol. A total of 51 metabolites were detected, 37 of which were tentatively identified based on mass accuracy (<5 ppm). Among them, 33 of honokiol metabolites were first reported with 5 metabolites belonging to phase I and other 32 metabolites belonging to phase II metabolites. Our results highlighted that the main phase I metabolic pathways of honokiol in rats were oxidation, and the phase II metabolic pathways were sulfation, glucuronidation, acetylation as well as amino acids conjugation. This was the first research focused on the biotransformation of honokiol in rats, and the identification of these metabolites might provide us essential information for further pharmacological and clinical studies of honokiol.