Development and validation of an online extraction HPLC-HRMS method for the quantification of carbapenems in...

Quantifying carbapenems in fecal samples is analytically challenging due to their instability and the complexity of intestinal matrices. We developed and validated a novel HPLC-HRMS method enabling direct quantification of imipenem and meropenem in human fecal material collected with the FecalSwab™ system. Sample processing consisted of minimal handling, involving clarification and direct online extraction using TurboFlow™ prior to chromatographic separation on a reversed-phase column. Detection was performed using Orbitrap high-resolution mass spectrometry in full-scan mode. Because only a small fraction of parenterally administered carbapenems reaches the intestinal lumen, but even low concentrations can exert a strong ecological pressure on gut microbiota and promote selection of resistant Enterobacterales, sensitive measurement of residual fecal levels is essential to understand antibiotic-driven dysbiosis and resistance emergence. The method was validated according to ICH M10 guidelines. Linearity was achieved over 1.5-750 ng/L for imipenem and 2.9-1500 ng/L for meropenem (R² > 0.995). Trueness and precision were within ±15% at all QC levels (±20% at LLOQ). Matrix effects were controlled using isotopically labeled internal standards, yielding normalized matrix factors with CV < 12%. Stability studies confirmed rapid degradation of imipenem and moderate stability of meropenem in fecal medium, prompting the identification and semi-quantification of their hydrolysis products. The method was successfully applied to 40 clinical samples, detecting carbapenems or their degradation products in 17/20 imipenem-exposed and 18/20 meropenem-exposed patients. This validated workflow provides a robust and clinically applicable approach for assessing intestinal exposure to carbapenems and supports ecological investigations of antibiotic-microbiota interactions.