Revisiting the Fragmentation of Ketoconazole by Positive Electrospray Ionization Tandem Mass Spectrometry and Density...

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.

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.

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, C₄H₆N₂ ^•+). 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.

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.