B-418 Simultaneous LC-MS/MS quantification of Cefepime, Meropenem, Piperacillin and Tazobactam in pediatric serum: method validation for therapeutic drug monitoring

Abstract

ß-lactam antibiotics and ß-lactamase inhibitors are cornerstone therapies for serious pediatric infections, yet drug exposure in children is highly variable due to developmental pharmacokinetics, critical illness, and rapidly changing renal function. Underexposure can contribute to treatment failure, while overexposure-particularly with renal dysfunction-may increase adverse effects (e.g., cefepime-associated neurotoxicity). Therapeutic drug monitoring (TDM) can support individualized dosing in high-risk pediatric patients but requires rapid turnaround, small specimen volumes, and robust quantification across a wide concentration range. We developed and validated a rapid LC-MS/MS assay for simultaneous quantification of cefepime, meropenem, piperacillin, and tazobactam in pediatric serum for routine clinical TDM. Residual de-identified serum specimens previously negative/undetectable for the target analytes were used as the validation matrix. Samples were fortified at low, mid, and high QC concentration levels. For preparation, 50 µL of serum was precipitated with an acidic organic solvent, followed by centrifugation and post-crash dilution of the supernatant prior to injection. Chromatographic separation was performed on a C18 column using gradient elution. Quantification was performed by LC-MS/MS with positive electrospray ionization and multiple reaction monitoring (MRM). Calibration used a 6-point curve spanning 0.5-60 µg/mL. Validation assessed calibration model performance, within-run and between-run precision, accuracy (bias), carryover, matrix effects, analyte stability, and method comparison to an external reference laboratory. The method achieved chromatographic separation and MRM detection of cefepime, and tazobactam in a 5.0-min run without clinically significant interferences. Calibration across 0.5-60 µg/mL was linear for meropenem, and tazobactam and quadratic for cefepime, with r = 0.99. Within-run and between-run imprecision were =6% CV for all analytes, and bias was within ±20% at low, and high QC levels. The method met predefined acceptance criteria for carryover, and analyte stability. Bias versus the external reference laboratory was within ±20%. We developed and validated a rapid, low-volume LC-MS/MS method for simultaneous quantification of cefepime, and tazobactam in pediatric serum. The assay demonstrated acceptable analytical performance across clinically relevant ranges and is suitable for routine therapeutic drug monitoring in children.