The development and validation of an LC-MS/MS method for the quantitation of ceftobiprole in plasma and urine in older patients

A liquid chromatography–tandem mass spectrometry method for quantifying ceftobiprole levels in plasma and urine from older patients was developed and validated. Protein precipitation with ceftobiprole-D 4 (internal standard) was used for sample preparation. Chromatographic separation was performed on a Shim-pack GIST Amide column (2.1 × 100 mm, 3 μm) with a gradient mobile phase of acetonitrile/water containing 2.5 mM ammonium formate and 0.1 % formic acid. Detection utilized positive electrospray ionization and multiple reaction monitoring, achieving lower limits of quantification of 0.3 (plasma) and 2 μg/mL (urine). The method demonstrated linearity over the ranges of 0.3–30 (plasma, correlation coefficient [r 2 ] > 0.99) and 2–100 μg/mL (urine, r 2 > 0.99). The results for precision (relative standard deviation [RSD], ≤15 %), accuracy, specificity, carryover, reinjection reproducibility, and hemolysis effects complied with International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use criteria. Matrix effects ranged 77.1 %–126.1 % (RSD, ≤9.2 %), whereas the extraction recoveries were > 90.5 % (plasma) and > 95.4 % (urine). Stability studies under clinical conditions revealed accuracy within 85 %–115 % (RSD, ≤8.4 %) for 4 h at room temperature, 12 h at 4 °C, 7 days at −40 °C, and three freeze–thaw cycles. Dilution integrity (10-fold) yielded accuracies of 90.7 %–105.9 %. Pharmacokinetic profiling in six older patients receiving 250 mg every 12 h revealed significant inter-individual variability (area under the curve from 0 to 24 h = 124.2–281.9 h·μg/mL). This method provides a reliable approach for therapeutic monitoring of ceftobiprole in older populations. • First dual-matrix validated LC-MS/MS method for Ceftobiprole in elderly patients. • Full validation per ICH M10 guidelines (precision RSD ≤15 %, accuracy 85–115 %). • Clinically proven sample stability protocols. • Revealed geriatric-specific PK variability. • AUC 0–24h differences (124.2–281.9 h·μg/mL) in older patients.