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

Abstract

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 has demonstrated antipsychotic effects in a rodent in vivo efficacy model. The 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 liquid chromatography/mass spectrometry and NMR due to the lack of unique tandem mass spectrometry fragmentation patterns for ions with the 2-aminothiazole group oxidized at different sites and the lack of stability for purification or reference standard synthesis. We describe 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 mass spectrometer. The GPIMR reagents used were dimethyl disulfide, tert-butyl peroxide, and tri(dimethylamino)borane. 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 with the profiles of the ionic ion-molecule reaction products of protonated reference compounds that had specific heteroatom 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.

Graph-based machine learning interprets and predicts diagnostic isomer-selective ion–molecule reactions in tandem mass spectrometry

“…Over the years, more than 20 different reagents have been developed, with more developed every year, for the identication of different functionalities in protonated analytes. 4,[7][8][9]11,13,43,44 Chemical diversity in the reagents results in increased reliability of the identication. At this time, nine different reagents can be tested simultaneously by using a nine-pulsed valve system developed for rapid introduction of nine reagents while each analyte in a mixture elutes from an HPLC column.…”

“…This approach is limited by the fact that isomeric ions oen generate identical fragmentation patterns, making identication of compounds via CAD mass spectra unreliable. 4,7 To address this issue, a MS/MS approach based on diagnostic, reliable and predictable gas-phase ion-molecule reactions has been developed. [7][8][9][10][11] This approach can be used to identify specic functional groups or their combinations in ionized and isolated mixture components to thereby facilitate the differentiation of isomeric ions, oen without the need for reference compounds.…”

“…The only modication to any commercial ion trap or multiquadrupole instrument is the addition of an inlet system for the neutral reagents, which is straightforward. 7,8,12,13 One of the neutral reagents used previously to differentiate two isomeric drug metabolites is 2-methoxypropene (MOP). 7 In these experiments, protonation of the analytes was achieved through atmospheric pressure chemical ionization (APCI) in a linear quadrupole ion trap mass spectrometer.…”

“…7,8,12,13 One of the neutral reagents used previously to differentiate two isomeric drug metabolites is 2-methoxypropene (MOP). 7 In these experiments, protonation of the analytes was achieved through atmospheric pressure chemical ionization (APCI) in a linear quadrupole ion trap mass spectrometer. The protonated analytes were transferred into the ion trap, isolated and allowed to react with MOP that was continuously introduced into the ion trap ( Fig.…”

Pharmacokinetic and Metabolism Studies of Monomethyl Auristatin F via Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry

“…However, M7 was eluted after parent. It is usually N-oxide-type metabolites that are eluted at a later time than the parent when oxidation occurs [38,39,40]. Therefore, M5 was presumed to be oxidized to dimethyl groups within the range suggested, whereas M7 was presumed to be oxidized to the nitrogen or more sterically hindered location in the structure.…”