Abstract
ABSTRACT
Rationale
Ketoconazole (KCZ) is a clinically essential antifungal and potent CYP3A4 inhibitor. Its gas‐phase fragmentation under positive electrospray ionization tandem mass spectrometry (ESI‐MS/MS) remains poorly characterized, undermining metabolite profiling and structural identification of related azoles.
Methods
We systematically mapped the fragmentation pathways of protonated KCZ using a dual‐platform strategy combining quadrupole time‐of‐flight collision‐induced dissociation (QTOF‐CID) and Orbitrap higher‐energy collisional dissociation (HCD) across a 10–60‐eV collision energy range. Mechanistic assignments and protonation sites were supported by density functional theory (DFT) calculations at B3LYP/6‐311++G(d,p) level in order to quantify thermodynamic barriers and dissociation energies.
Results
Our study identified three distinct dissociation pathways: (1) piperazine ring opening driven by ketene loss (A1,
m/z
489.1453) and alkylamine eliminations; (2) 1,3‐dioxolane ring cleavages (e.g., C10,
m/z
277.1547); and (3) radical‐driven dissociation at the ether linker, yielding an imidazole radical cation (A28,
m/z
82.0531, C
4
H
6
N
2
•+
). DFT calculations established a clear thermodynamic hierarchy, where even‐electron ions (e.g., A1, ΔG ≈ 19 kcal mol
−1
) prevail at low energies, whereas the radical cation (A28, ΔG ≈ 86 kcal mol
−1
) is only accessible at ≥ 40 eV. HCD conditions selectively promoted odd‐electron ion formation, revealing platform‐dependent fragmentation behavior.
Conclusions
This work delivers the first comprehensive, computationally validated fragmentation map for KCZ, directly linking MS/MS observations to thermodynamic predictions and providing a robust reference for high‐confidence identification of synthetic imidazole antifungals.