Oxysterol fingerprinting via UPLC-APCI-MS/MS for monitoring developmental stages in the zebrafish model

Oxysterols, oxidized derivatives of cholesterol, play multifaceted roles in cholesterol homeostasis and cellular signalling. Despite their recognized biological relevance, the temporal dynamics of oxysterol profiles during vertebrate development remain poorly understood. In this study, we developed and validated a robust UHPLC- MS/MS method for the quantification of ten oxysterols in zebrafish (Danio rerio) embryos across five develop mental stages (3-, 24-, 48-, 72-, and 96-hours post-fertilization). The method exhibited excellent performance in terms of sensitivity, recovery, and reproducibility, following EMA guidelines. A combination of hierarchical clustering analysis (HCA) and Random Forest classification revealed distinct stage-specific oxysterol profiles. Notably, 6 α-hydroxycholesterol emerged as the most discriminative marker, followed by 24- and 27-hydroxycho lesterol, indicating their potential involvement in neurodevelopmental and metabolic regulation processes. These findings provide new insights into the role of oxysterols during embryogenesis and establish a reliable platform for evaluating oxysterol dynamics in developmental, toxicological, and pharmacological studies using the zebrafish model.