A targeted lipidomics workflow for high-throughput phenotyping and mechanism-based in vitro hepatotoxicity studies.

Animal-based toxicity testing is limited in throughput and mechanistic characterization, underscoring the need for cell-based approaches. Incorporating molecular readouts, such as lipidomics, can enhance the translational relevance of cell-based assays. Because untargeted lipidomic analyses are time-consuming and can require large sample volumes, a targeted workflow was used in this study to characterize drug-induced liver injury-related lipid responses in primary hepatocytes from multiple species. Multiple reaction monitoring was applied in a targeted lipidomics workflow by coupling liquid-chromatography with triple quadrupole mass spectrometry (LC-MS/MS) for 148 lipid targets across 3 categories and 11 classes. We used primary hepatocytes from human, rat, monkey, and dog cultured in 96-well plates and exposed to chlorpromazine (1-30 μM), bosentan (1-200 μM), and fialuridine (1-100 μM) from culture day 4 to 8. Chlorpromazine induced the most pronounced lipid alterations in human, monkey, and rat hepatocytes, while responses in dog hepatocytes were minimal, with human cells showing a dose-dependent trend up to 10 μM. Bosentan induced dose-dependent lipid changes across species, strongest in human hepatocytes, whereas fialuridine caused limited lipid alterations. Overall, lipidomic effects closely aligned with conventional toxicity markers, reflecting compound and species-specific hepatotoxic sensitivity. The targeted lipidomics workflow provided mechanism-informed phenotyping of chemical-induced lipid responses in cultured hepatocytes. Moreover, it provided a framework for mechanistic evaluation, supported cross-species comparison, and complemented in vitro hepatotoxicity assessments.