Characterization of the in vivo and in vitro metabolic profile of PAC-1 using liquid chromatography–mass spectrometry

In the present study, the metabolic profile of PAC-1, a potential anticancer drug, was investigated using liquid chromatography–mass spectrometric (LC/MS) techniques. Two different types of mass spectrometers – a quadrupole time-of-flight (Q-TOF) mass spectrometer and an ion trap (IT) mass spectrometer – were employed to acquire structural information on PAC-1 metabolites. A gradient liquid chromatographic system composed of 0.2% formic acid in methanol and 0.2% formic acid in water was used for metabolite separation on an Agilent TC-C18 column. A total of 16 metabolites were detected. The corresponding product ion spectra were acquired and interpreted, and structures were proposed. Accurate mass measurement using LC-Q-TOF was used to determine the elemental composition of metabolites thereby confirming the proposed structures of these metabolites. Phase I metabolic changes were predominantly observed, including debenzylation, dihydrodiol formation, hydroxylation, and dihydroxylation. The detected phase II metabolites included PAC-1 and hydroxylated PAC-1 glucuronide conjugates. Based on metabolite analysis, several PAC-1 metabolic pathways in rat were proposed.