Measurement of Total and Free Cefiderocol Concentrations in Human Plasma by UHPLC‐MS/MS: Application to Patients With Difficult‐To‐Treat Gram‐Negative Bacterial Infections

Cefiderocol is an innovative siderophore cephalosporin against some of the most difficult-to-treat Gram-negative bacterial infections. As with other β-lactam antibiotics, therapeutic drug monitoring of cefiderocol in specific clinical scenarios could improve patient outcomes, minimize toxicity, and maximize treatment effectiveness. Thus, we aimed to develop and validate an ultra-high-performance liquid chromatography-tandem mass spectrometry procedure for measuring total and free cefiderocol concentrations in human plasma. Protein precipitation was used to extract patient samples. Equilibrium dialysis preceded sample preparation for measuring free cefiderocol concentrations. An Acquity-UPLC-BEH (2.1 mm × 100 mm id, 1.7 µm) column was selected for analyte separation, using 0.1% (v/v) formic acid in water and acetonitrile as mobile phases and working in a gradient containing. The cefiderocol and its internal standard ([²H₈-cefiderocol) were detected by electrospray ionization mass spectrometry in positive and multiple reaction monitoring modes, using transitions of 752.2→285.3/468.2 and 760.2→293.3/468.2, respectively. Analysis time was 3.5 min per run. Precisions, absolute relative biases, normalized-matrix effect, and normalized recoveries were ≤13.9%, ≤14.2%, 95.7%-104.1%, and 95.9%-104.8%, respectively. Linearity was observed between 0.50 and 65.0 mg/L with linear regression coefficients greater than 0.9984. No significant interferences and carry-over were observed. The UHPLC-MS/MS procedures we present here could be suitable for clinical research purposes and hold promise for routine analysis. Their versatility would allow for application in the therapeutic drug monitoring of cefiderocol in subjects with difficult-to-treat Gram-negative bacterial infections to guide the treatment and adjust the daily dose of this new β-lactam antibiotic.