Development and Validation of an LC-MS/MS Method for Ceftriaxone Therapeutic Drug Monitoring in Pediatric Patients

Background: Ceftriaxone is a widely used third-generation cephalosporin, yet its pharmacokinetics in pediatric populations remain incompletely characterized. Accurate measurement of plasma concentrations is essential to guide therapeutic drug monitoring (TDM) and optimize dosing in vulnerable patients. We developed and validated a sensitive LC–MS/MS method for quantifying total ceftriaxone in plasma, employing an isotopically labeled internal standard ([13C,2H3]-ceftriaxone). Methods: Sample preparation involved protein precipitation with 200 µL of methanol, while chromatographic separation was performed on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) using water with 0.1% formic acid and 2 mM ammonium acetate (Phase A) and methanol with 0.1% formic acid and 2 mM ammonium acetate (Phase B). The method was validated according to ICH M10 guidelines, assessing linearity, precision, accuracy, matrix effects, extraction recovery, carryover, and stability. Results: The assay demonstrated excellent calibration performance across the range of 0.27–200 mg/L (R2 = 0.99), with IS-normalized matrix effects of 87–89%, extraction recoveries of 95–106%, intra- and inter-run accuracies ranging from 92% to 114%, and intra- and inter-run precision (CV%) of 2–8% and 3–11%, respectively. Peak shapes for both ceftriaxone and the internal standard showed good symmetry, and matrix effects were minimal. Application to 18 pediatric plasma samples confirmed the method’s suitability for clinical use, with a median ceftriaxone concentration of 77.89 mg/L (IQR 91.63 mg/L). Conclusions: The described LC–MS/MS method is rapid, robust, and reliable, enabling accurate quantification of total ceftriaxone in plasma. Its application supports TDM, pharmacokinetic studies, and individualized dosing in pediatric patients, providing a practical tool to optimize therapy in populations with variable pharmacokinetics.