Ion Mobility-Enhanced Liquid Chromatography Coupled with Mass Spectrometry (LC-MS) Enables Reliable Detection of OXA-48-Like Carbapenemases Beyond Conventional Activity-Based Assays

Carbapenemases are major drivers of carbapenem resistance in Gram-negative bacteria and pose a critical threat to last-line antibiotic therapy. Rapid identification of carbapenemase classes is essential for appropriate treatment and epidemiological surveillance; however, current functional methods lack class-level resolution and may yield false-negative results for OXA-48-like enzymes. In this study, we developed and validated an assay based on liquid chromatography-mass spectrometry with trapped ion mobility spectrometry-time-of-flight [LC-MS (timsTOF)] for simultaneous detection and class-level differentiation of five clinically relevant carbapenemases (KPC, NDM, VIM, IMP, and OXA-48-like). The method employs three carbapenem substrates (meropenem, imipenem, and ertapenem). A total of 55 clinical isolates were analyzed using a standardized 2-hour incubation protocol, with a total analysis time of 7 min per sample. Ion mobility enabled unambiguous identification of the OXA-48-specific meropenem-derived {beta}-lactone based on its distinct collisional cross-section (185 [A]{superscript 2} vs. 191 [A]{superscript 2} for intact meropenem), despite identical mass and nearly identical retention time. This marker was detected in all OXA-48-like producers and was absent in all other groups. In contrast, imipenem and ertapenem did not provide comparable discrimination, highlighting the central role of meropenem. Distinct hydrolysis profiles enabled class-level differentiation supported by multivariate analysis. LC-MS (timsTOF) thus enables rapid, sensitive, and specific functional detection of carbapenemases within a single workflow. The ion mobility dimension is critical for accurate identification of OXA-48-like enzymes and supports the potential implementation of this approach in routine clinical microbiology laboratories. ImportanceThis study introduces an ion mobility-enabled LC-MS (timsTOF) approach for functional detection and class-level differentiation of clinically relevant carbapenemases within a single analytical workflow. By leveraging collisional cross-section measurements, the method enables reliable identification of OXA-48-like carbapenemase through detection of a meropenem-derived {beta}-lactone that is indistinguishable by mass alone. This directly addresses a major diagnostic limitation of conventional activity-based assays, which may yield false-negative results for OXA-48-like enzymes. The approach further demonstrates the potential of integrating ion mobility into routine clinical mass spectrometry to enhance specificity beyond traditional mass and retention time measurements. These findings support the development of next-generation diagnostic strategies capable of detecting both known and emerging resistance mechanisms without reliance on predefined targets.