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

This study aimed to develop a targeted metabolomics workflow using low-resolution tandem mass spectrometry (MS/MS) to identify metabolic alterations in zebrafish (Danio rerio) 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. © 2025 Informa UK Limited, trading as Taylor & Francis Group.