A quantitative method for the analysis of total and unbound concentrations of amoxicillin, ampicillin, ceftazidime, ceftriaxone, ertapenem, fosfomycin and penicillin G in human plasma with liquid chromatography–tandem mass spectrometry assay

Monitoring antibiotic plasma levels is critical in populations with altered pharmacokinetics, such as critically ill patients in neonatal or adult intensive care units. This study aimed to develop and validate a rapid, reproducible and sensitive liquid chromatography–tandem mass spectrometry assay (LC–MS/MS) for measuring total and unbound concentrations of amoxicillin, ampicillin, ceftazidime, ceftriaxone, ertapenem, fosfomycin and penicillin G in human plasma. The method required 20 and 250 μl sample volumes for measuring total and unbound concentrations, respectively. Sample preparation involved protein precipitation and the addition of an internal standard. Ultrafiltration separated unbound drugs. Method validation covered selectivity, carryover, linearity, accuracy, precision, dilution effects, matrix effects and stability. The LC–MS/MS was performed within a run time of 7.5 min. Calibration curves were linear for ceftazidime and ertapenem (ranges 0.1–50 and 0.05–100 mg/l, respectively) and quadratic for other analytes (0.1–50 mg/l, except for ampicillin: 0.1–20 mg/l; R² > 0.990). Accuracy was within ±15% of the nominal concentration, and precision did not exceed ±15% (relative standard deviation). Samples showed no significant degradation at the tested temperatures and time points. Clinical applicability was demonstrated in a critically ill neonate. This method with minimal sample volume and short analysis time enables the measurement of total and unbound concentrations of selected antibiotics, and is suitable for routine clinical care and studies.