Identification and Reaction Analysis of Antibiotic Transformation Products in an Urban River via Suspect and Nontarget Screening with a Self-Curated Library

Antibiotics enter urban river basins mainly through sewage effluent. They undergo transformation during treatment and in river systems, generating transformation products (TPs) that may still have ecological effects. Yet, identifying these TPs by high-resolution mass spectrometry is hindered by the scarcity of reference standards and comprehensive MS/MS spectral libraries. To overcome this, we constructed a dedicated MS/MS library containing over 6,500 MS/MS spectra for 244 parent antibiotics and 362 TPs, thereby enabling rapid suspect screening through spectral matching. For novel TPs beyond library coverage, we integrated diagnostic fragment searching and seed-driven molecular networking to propagate annotations to structurally related unknowns. Applying this workflow to water, sediment, and effluent samples from an urban river system in Beijing, we identified 39 parent antibiotics and 158 TPs, of which 20 TPs are not previously documented in environmental samples. The major transformation reactions were cleavage, dealkylation, and hydrolysis, alongside compound-specific pathways like hemiacetal formation for erythromycin. These reactions differentially affect the environmental properties of antibiotics, and some reactions (e.g., dehydrogenation and dehydration) showed potential to enhance ecotoxicity. These results confirm that transformation substantially diversifies antibiotic signatures in urban aquatic environments and underscore the necessity of including TPs in environmental monitoring and risk assessment.