MRM-driven metabolomic workflow for early detection of antibiotic-triggered sub-lethal toxicity using Q-TRAP and zebrafish model

) embryos exposed to environmentally relevant concentrations of the antibiotic's amoxicillin and clarithromycin.Zebrafish embryos were exposed to the lowest concentrations (1 µg/L) and the highest concentration (1 mg/L) of amoxicillin and clarithromycin. A library-assisted multiple reaction monitoring-enhanced product ion (MRM-EPI) approach was applied using a QTRAP LC-MS/MS system, enabling the detection and structural confirmation of 108 endogenous metabolites. A targeted MRM-EPI method was subsequently optimised for sensitivity, reproducibility, and specificity. Multivariate statistical analyses, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), were performed to assess metabolic differences between exposed and control groups.Exposure to both antibiotics resulted in significant alterations in amino acid, purine, lipid, and energy metabolism, indicating that even sub-lethal concentrations can disrupt vital physiological processes in zebrafish embryos. These findings highlight the sensitivity of metabolomics for detecting early biochemical perturbations and support the use of zebrafish embryos as a practical and ethically suitable model for environmental toxicity assessment. The developed MRM-driven workflow provides a reproducible platform for predictive toxicology and ecological risk evaluation.