Atmospheric pressure photoionization (APPI) was developed as an

alternative to electrospray ionization (ESI) for the generation of protonated

molecules using liquid chromatography and optimized using dopants such as

toluene, which predominantly forms protonated molecules, and chlorobenzene,

which favors the formation of radical cations, although the latter has not been fully

exploited. Based on 40 diverse low-molecular-weight compounds and micro liquid

chromatography (μLC) coupled with APPI tandem mass spectrometry (APPI-

MS/MS), the potential of radical cations was investigated. Chromatographic and

ionization conditions were decoupled by post-column addition of methanol,

allowing separate study and optimization. Due to the mass flow sensitive behavior

of APPI, sensitivity is not affected by post-column dilution, and for 8 of 35 analytes, the radical cation response with μLC-APPI is

better than for protonated molecules using μLC-ESI. Collision-induced fragmentation (CID) of radical cations produced within a

collision energy range from 10−115 eV have, in the median, 65% of the fragments found in electron ionization (EI) spectra. This

similarity allowed identification of 86% of the analytes using data-dependent acquisition (DDA) of radical cations and NIST EI

library searches. We propose a workflow that uses multimodal DDA of protonated precursor molecules using ESI or APPI with

toluene as a dopant, and radical cations produced by chlorobenzene-assisted μLC-APPI with post-column addition of methanol.

This increases the confidence of molecular identification by allowing orthogonal library searches using MS/MS libraries for

protonated precursor CID spectra and EI libraries for radical cation CID spectra.