Rapid, robust LC-MS/MS quantification of cefazolin in whole blood microsamples, plasma, and plasma ultrafiltrate:...

Optimal dosing of cephalosporins in pediatric ICU patients is difficult due to high inter-individual variability in β-lactam pharmacokinetics, limited blood volume, and the lack of validated microsampling-compatible clinical assays. We developed and validated a rapid and reliable liquid chromatography-tandem mass spectrometry method to measure cefazolin concentrations in lithium-heparin plasma, plasma ultrafiltrate (unbound fraction), and capillary whole blood collected with volumetric absorptive microsampling (VAMS). This method was applied in a pilot cohort of pediatric patients. Samples were protein precipitated with cryo-cooled organic solvents, separated by reversed-phase liquid chromatography (reversed-phase HPLC) using a polar C18 column, and detected by multiple reaction monitoring (MRM) on a hybrid triple quadrupole-linear ion trap (QTRAP) mass spectrometer. Cefotaxime-D₃ was used as the internal standard, and calibration curves (0.5-200 mg/L) were established for each matrix. The method was validated in accordance with ICH M10 bioanalytical guidelines for selectivity, linearity, accuracy, precision, carry-over, matrix effect, recovery, process efficiency, stability, and VAMS-specific factors (hematocrit and drying time). The total run time was 3 min, including the internal standard, with an LLOQ of 0.5 mg/L across all matrices. The coefficients of determination (R²) were > 0.995, IS-normalized matrix factors were close to 1.0, and accuracy and precision were within ±15 % at all quality control levels. Cefazolin remained stable under all preanalytical conditions studied. VAMS values were unaffected by changes in hematocrit within clinically relevant ranges. The feasibility of the multi-matrix analytical method in routine practice has been demonstrated. The multi-matrix, microsampling-compatible LC-MS/MS assay described is sensitive and specific for TDM and PK/PD studies, aimed at defining precise dosing regimens for cefazolin in neonatal and pediatric critical care settings.