Identification of BVT.2938 metabolites by LC/MS and LC/MS/MS after in vitro incubations with liver microsomes and hepatocytes

The metabolism of the 5HT2c agonist BVT.2938, 1-(3-{2-[(2-ethoxy-3-pyridinyl)oxy]ethoxy}-2-pyrazinyl)-2(R)-methylpiperazine, was studied in vitro by incubation with rat, monkey and human liver microsomes as well as cryopreserved hepatocytes, followed by liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS analysis on a quadrupole-time of flight mass spectrometer for structural elucidation. Deuterium exchange on column was used to differentiate between hydroxylation and N-oxidation. Liver microsomes were incubated in two different buffer systems with optimum conditions for cytochrome P450 activity or UDP-glucuronosyltransferase activity. The major phase I metabolites of BVT.2938 originated from O-deethylation of the pyridine ring, O-dealkylation of the ethylene bridge, pyrazine ring hydroxylation, hydroxylation of pyridine ring and piperazine ring N-hydroxylation. When a hydrogen carbonate buffer system was supplemented with UDPGA, the piperazine carbamoyl-glucuronide from the parent compound was identified together with several glucuronides of the phase I metabolites. The metabolite pattern in hepatocytes was similar to microsomes except that the sulphate at the N-position of the piperazine ring of BVT.2938 was identified, while the carbamoyl-glucuronide was missing. Excellent correlation was obtained between radioactivity detection and the chemiluminescent nitrogen detector when the nitrogen content of the analytes was taken into account. (C) 2003 Elsevier B.V. All rights reserved.