Pyridyl ethyl diazoacetate derivatization enables LC-MS annotation of cholesterol and its metabolites

Cholesterol and its metabolites are fundamental to membrane organization and cellular signaling, but their poor ionization efficiency and structural similarity complicate mass-spectrometric (MS) analysis. In this work, we developed pyridyl ethyl diazoacetate (PED) as a stable, efficient derivatization reagent that introduces a permanent positive charge on hydroxyl-containing sterols, thereby enhancing ionization and detection sensitivity. Systematic MS² characterization established diagnostic fragmentation motifs of PED-tagged sterols, which we encoded into a query-based spectral-mining strategy for reproducible, rule-based annotation of sterol features in complex matrices. Application of the PED workflow to human liver tissues enabled sterol profiling across cirrhotic, hepatocellular carcinoma, and intrahepatic cholangiocarcinoma specimens, suggesting disease-context-dependent differences in sterol profiles. The workflow effectively integrates chemical derivatization and programmable data interrogation, providing a robust analytical framework for sterol profiling in biological samples and a potential basis for broader lipidomic applications.