Identification of 2-Aminothiazolobenzazepine Metabolites in Human, Rat, Dog, and Monkey Microsomes by Ion-Molecule Reactions in Linear Quadrupole Ion Trap Mass Spectrometry

2-Aminothiazolobenzazepine (2-ATBA), 7-\[(1-methyl-1H-pyrazol-4-yl)methyl]-6,7,8,9-tetrahydro-5H-[1,3]thiazolo[4,5-h\]\[3\]benzazepin-2-amine, is a D2 partial agonist that demonstrated antipsychotic effects in a rodent in vivo efficacy model. Metabolite profile showed that 2-ATBA is mainly metabolized by oxidation. However, identification of the oxidation site(s) in the 2-aminothiazole group presents a challenge for the traditional metabolite identification methods such as the LC/MS and NMR due to the lack of unique MS/MS fragmentation patterns for ions with the 2-aminothiazole group oxidized at different sites and the lack of stability for purification or reference standard synthesis. This paper describes the characterization of the oxidized heteroatoms of the 2-aminothiazole group via Gas-Phase Ion-molecule Reactions (GPIMR) in a modified linear quadrupole ion trap (LTQ) mass spectrometer. The GPIMR reagents used were dimethyl disulfide (DMDS), tert-butyl peroxide (TBP) and tri(dimethylamino)borane (TDMAB). Each reagent was introduced into the ion trap through the helium line and was allowed to react with the protonated metabolites. The ionic ion-molecule reaction products and their fragmentation profiles were compared to the profiles of the ionic ion-molecule reaction products of protonated reference compounds that had specific hetero atom functionalities. The oxidized 2-aminothiazole metabolite of 2-ATBA showed a similar GPIMR profile to that of the reference compounds with a tertiary N-oxide functionality and distinct from the profiles of the reference compounds with N-aryl hydroxylamine, nitroso or pyridine N-oxide functionalities. This study demonstrates the feasibility of fingerprinting the chemical nature of oxidized nitrogen functional groups via GPIMR profiling for metabolite structure elucidation.