Abstract
Tandem mass spectrometry methods were used to study the sites of protonation and for identification of 3-amino-1,2,4-benzotriazine 1,4-dioxide (1, tirapazamine), and its metabolites (3-amino-1,2,4-benzotriazine 1-oxide (3), 3-amino-1,2,4-benzotriazine 4-oxide (4), 3-amino-1,2,4-benzotriazine (5), and a related isomer 3-amino-1,2,4-benzotriazine 2-oxide (6). Fragmentation pathways of 3 and 5 indicated the 4-N-atom as the most likely site of protonation. Among the N-oxides studied, the 4-oxide (4) showed the highest degree of protonation at the oxygen atom. The differences in collision-induced dissociation of isomeric protonated 1-, 2- and 4-oxides allowed for their identification by LC/MS/MS. Gas phase and liquid phase protonation of tirapazamine occurred exclusively at the oxygen in the 4-position. A loss of OH radical from these ions (2 +) resulted in ionized 3. Neutralization-reionization mass spectrometry (NR MS) experiments demonstrated the stability of the neutral analogue of protonated tirapazamine in the gas phase in the μs time-frame. A significant portion of the neutral tirapazamine radicals (2) dissociated by loss of hydroxyl radical during the NR MS event, which indicates that previously proposed mechanisms for redox-activated DNA damage are reasonable. The activation energy for loss of hydroxyl radical from activated tirapazamine (2) was estimated to be ∼14 kcal mol−1. Stable neutral analogues of [3+H]+ and [5+H]+ ions were also generated in the course of NR MS experiments. Structures of these radicals were assigned to the molecules having an extra hydrogen atom at one of the ring N-atoms. Quantum chemical calculations of protonated 1, 3, 4 and 5 and the corresponding neutrals were performed to assist in the interpretation of experimental results and to help identify their structures.
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Published online June 18, 2003
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Zagorevskii, D., Song, M., Breneman, C. et al. A mass spectrometry study of tirapazamine and its metabolites: Insights into the mechanism of metabolic transformations and the characterization of reaction intermediates. J Am Soc Mass Spectrom 14, 881–892 (2003). https://doi.org/10.1016/S1044-0305(03)00334-9
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DOI: https://doi.org/10.1016/S1044-0305(03)00334-9