A high-resolution accurate mass approach to identification of graveoline metabolites using ultra-high-performance liquid chromatography combined with a photo diode array detector andquadrupole/time-of-flight tandem mass spectrometry.

Abstract

Graveoline is a biologically active ingredient extracted from Ruta graveolens. Current work aimed at investigating in vitro metabolism of graveoline using rat or human liver microsomes and hepatocytes. Graveoline (20 μM) was incubated with nicotinamide adenine dinucleotide phosphate-supplemented rat and human liver microsomes as well as hepatocytes. LC coupled to a photo diode array detector and quadrupole/time-of-flight tandem mass spectrometry was used to detect and identify the metabolites. The structures of the metabolites were identified by accurate mass, elemental composition, and indicative fragment ions. A total of 12 metabolites, comprising 6 phase I and 6 phase II metabolites, were obtained. The metabolic pathways included demethylenation, demethylation, hydroxylation, glucuronidation, and glutathion conjugation. The metabolite (M10) produced by opening the ring of the methylenedioxyphenyl moiety was detected as the most abundant in both liver microsomes and hepatocytes, mainly catalyzed by CYP1A2, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5. This study provides valuable information on the in vitro metabolism of graveoline, which is indispensable for further development and safety evaluation of this compound.