Revisiting the Fragmentation of Ketoconazole by Positive Electrospray Ionization Tandem Mass Spectrometry and Density Functional Theory Calculations

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.