Aza-Paternò-Büchi derivatization enables annotation of triacylglycerols by negative-ion-mode mass spectrometry at the double bond level.

Triacylglycerols (TGs) represent one of the most abundant lipid classes in biological and food matrices; however, their structural characterization by mass spectrometry remains challenging because conventional workflows often provide only sum composition information. In the present study, an aza-Paternò-Büchi (aPB) derivatization strategy based on 6-azauracil was optimized and applied for negative-ion-mode, isomer-resolved analysis of TGs by high-resolution mass spectrometry. Reaction conditions were systematically investigated through solvent optimization, mixture design modeling, and kinetic studies. Moreover, the ionization and fragmentation behavior of aPB-derivatized TGs was comprehensively investigated, revealing distinct fragmentation pathways associated with charge localization effects in neutral lipids. Application to complex matrices demonstrated the feasibility of the approach, providing direct access to TG regioisomer distributions in human plasma and vegetable seed oils. Overall, the proposed strategy enables untargeted and double-bond-resolved annotation of TGs in complex matrices while maintaining straightforward sample preparation and compatibility with routine lipidomics workflows. For the first time, negative-ion mode was employed for TG annotation, expanding the molecular information accessible in TG lipidomics and food analysis.