Non-Target Screening of Antibiotic Fermentation Residues: Unveiling Biosynthetic Intermediates and Transformation Products by FT-ICR-MS

Antibiotic fermentation residues (AFRs) represent a complex source of emerging contaminants, yet their molecular composition remains poorly characterized, hindering evidence-based risk assessment. Here, we employed dual-mode electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) combined with suspect screening and paired mass distance (PMD) network analysis to systematically characterize molecular inventories and their chemical relationships in penicillin G (PFRs), cephalosporin C (CFRs), and erythromycin (EFRs) fermentation residues. Molecular formula assignment yielded 2,162 (PFRs), 990 (CFRs), and 1,674 (EFRs) molecules, with putatively annotated 71 penicillin-related, 4 cephalosporin-related, and 51 macrolide compounds, respectively. Dual-mode analysisd apparent ionization selectivity, with EFRs showing 12-fold higher formula counts in ESI(−) mode. Key high-intensity compounds included ring-opened β -lactam hydrolysis products in PFRs, biosynthetic intermediates in CFRs, and acid-catalyzed dehydration products in EFRs. PMD network analysis differentiated biosynthetic/transformation paths: PFRs were dominated by redox reactions (56.3%) and hydration/dehydration (30.4%); CFRs by methylation/demethylation and redox reactions (70.6%); and EFRs by methylation/demethylation combined with redox reactions (88.1%). Critically, while PFRs consisted largely of deactivated hydrolysis products, CFRs and EFRs contained high levels of bioactive intermediates and derivatives that retain their core pharmacophores. This study provides a molecular baseline for prioritizing high-risk compounds and guiding sustainable pharmaceutical waste management.