Azetidination-based photocycloaddition enables regioisomer-resolved annotation of cholesteryl esters by negative-ion-mode LC-MS/MS

Cholesteryl esters (CE) play central roles in lipid transport and storage, yet their structural characterization remains challenging due to their extreme hydrophobicity and poor ionization efficiency. Conventional CE lipidomics workflows typically rely on positive-ion-mode analysis and report CE at the sum-composition level, without resolving double-bond (C=C) positional isomerism. Chemical derivatization strategies enabling isomer-resolved analysis of CE are therefore highly desirable but remain largely unexplored. In the present study, the use of the aza-Paternò-Büchi (aPB) reaction with 6-azauracil was extended to CE, enabling negative-ion-mode LC–HRMS/MS analysis with annotation of C=C bond regiochemistry. Optimization of the derivatization conditions allowed efficient reaction of highly hydrophobic CE while maintaining compatibility with electrospray ionization. Tandem mass spectrometry revealed a previously unreported set of diagnostic fragment ions that proved particularly suitable for quantitative applications. The workflow enabled both relative and absolute quantitation of CE regioisomers with good linearity, repeatability, and trueness over a wide dynamic range, using a single dominant diagnostic ion to simplify data processing. Application to complex biological matrices of clinical interest demonstrated the feasibility of the approach, providing direct access to CE regioisomer distributions in human plasma and bovine liver. This study establishes aPB-based derivatization as a viable and complementary strategy for structurally resolved CE analysis, expanding the analytical toolbox for lipidomics studies where detailed molecular structure is critical for biological interpretation. • Aza-Paternò-Büchi derivatization enables negative-ion-mode CE analysis • C=C regioisomers of cholesteryl esters resolved by aPB reaction and LC–MS/MS • Distinct fragmentation mechanisms identified for aPB-derivatized CE • Diagnostic product ions allow relative and absolute quantitation of CE regioisomers • CE regioisomer distributions determined in plasma and liver samples