Next-generation Derivatization Reagents Optimized for Enhanced Product Ion Formation in Photodissociation-mass Spectrometry of Fatty Acids

Ultraviolet-photodissociation (UVPD) mass spectrometry is an emerging analytical tool for structural elucidation of biomolecules including lipids. Gas phase UVPD of ionised fatty acids (FAs) can promote fragmentation that is diagnostic for molecular structure including the regiochemistry of carbon-carbon double bonds and methyl branching position(s). Typically, however, lipids exhibit poor conversion to photoproducts under UVPD and thus require longer integration times to achieve the signal-to-noise required for structural assignments. Consequently, the integration of UVPD into liquid-chromatography mass spectrometry (LC-MS) workflows for FAs has been limited. To enhance photofragmentation efficiency, an alternative strategy has been devised using wet-chemical derivatization of FAs to explicitly incorporate photolabile groups. FA derivatives that include an aryl-iodide motif have photodissociation conversions of up to 28% when activated by a single 266 nm photon. The radical-directed dissociation product ions resulting from UVPD of these derivatives provide key details of molecular structure and discriminate between lipid isomers. Herein, we describe the structure-activity guided development of new FA derivatives capable of photoproduct yields of up to 97%. UVPD-action spectroscopy demonstrates that photodissociation for FAs derivatized with N-(2-aminoethyl)-4-iodobenzamide (NIBA) is maximised near 266 nm and highlights the key role of the 4-iodobenzamide motif in the efficient formation of [M - I]˙+ radical cations (and diagnostic secondary product ions). The high photodissociation yield of NIBA-derivatized lipids is maintained across 37 commonly observed FAs with the resulting UVPD mass spectra shown to be effective in the discrimination of isomeric FAs that differ in the position(s) of carbon-carbon double bonds. Integration of this strategy with reversed-phase LC-MS workflows is confirmed with high-quality UVPD mass spectra acquired across each chromatographic peak.