Three-dimensional mass spectral libraries for improved metabolites identification using collision energy profiles of protonated and radical cation precursors

Electron impact ionization (EI) is routinely applied in industrial and national laboratories for structural identification using standardized libraries but is restricted to volatile and thermostable analytes separated by gas chromatography. In contrast, collision-induced dissociation (CID) of radical cations generated by atmospheric pressure photoionization (APPI) makes EI-library searches applicable to liquid chromatography and supercritical fluid chromatography, enabling analysis of thermolabile and involatile analytes. Within this study, we extend the utility of radical cation CID beyond EI-Library searches, by systematically investigating collision energy profiles of radical cations and protonated precursors from 40 low-molecular-weight compounds. Across collision energies from 10-115 eV, 79% of the 10 most abundant elemental formula losses differ between radical cation and protonated precursor CID. Most frequent elemental formula losses for radical cations are methyl and hydrogen radical losses, and for protonated precursors water and carboxyl group losses. Common fragment ions from radical cation and protonated precursor CID often show distinct collision energy profiles with shifted intensity maxima. While most fragments from radical cation CID resemble those produced by EI, additional structurally informative fragments become accessible at lower collision energies and are likely missed under 70 eV EI conditions due to excessive internal energy deposition. Collectively, these results establish collision-energy-resolved fragmentation as a basis for structurally informative 3-dimensional spectral libraries incorporating collision energy, fragment intensity, and m/z values for compound identification. The applicability of this approach is demonstrated for 9 exogenous substances spiked into human plasma using SWATH data acquisition and collision-energy profile matching with an emphasis on radical cations.