Caffeine Biotransformation in Zebrafish Larvae: Integrated LC-MS/MS Quantification and FTIR Spatial Mapping

Caffeine (CAF) is one of the most widely consumed psychoactive substances worldwide. It is primarily metabolized to paraxanthine (PAR), theobromine (THR), and theophylline (THY). While CAF metabolism has been extensively characterized in humans and rodent models, corresponding data in zebrafish (Danio rerio) larvae remain limited. This study provides a comprehensive characterization of CAF biotransformation in zebrafish larvae using validated LC-QQQ-MS for quantitative metabolite profiling and FTIR imaging for spatially resolved tissue distribution mapping. Zebrafish larvae at 4 days post fertilization (dpf) were exposed to CAF at concentrations of 15, 25, and 50 mg/L for 18 h. The LC–MS/MS analysis demonstrated dose-dependent conversion of CAF to PAR (up to 4.54%), THR (up to 1.32%), and THY (up to 0.27%). The PAR/CAF and THR/CAF ratios increased as CAF concentration rose, while the THY/CAF ratio declined. In parallel, FTIR imaging was applied to visualize the spatial distribution of CAF and selected metabolites in larval tissue sections, confirming the presence of CAF, PAR, and THR and revealing their localization within the tissue. However, THY was not detected by this method. Metabolite localization differed across larval tissues, indicating compartmentalized metabolic processes. This study provides the first comprehensive characterization of CAF metabolism in zebrafish larvae, revealing low but detectable CYP450-mediated metabolic activity prior to full hepatic maturation. The findings support the utility of zebrafish larvae as a model for developmental pharmacokinetic studies.