An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure...

The quantification of linezolid in serum and plasma matrices is crucial for therapeutic drug monitoring and pharmacokinetic studies. A reference measurement procedure (RMP) was developed and validated to provide accurate, precise, and reliable measurements, fit for its intended use while ensuring suitability for clinical and laboratory settings.

Linezolid quantification was achieved using liquid chromatography-tandem mass spectrometry (LC-MS/MS), with specific ion transitions monitored for the analyte and its stable isotope-labeled internal standard (ISTD). The RMP validation process encompassed key performance parameters including selectivity, matrix effects (MEs), linearity, precision, accuracy, and stability. MEs were assessed by comparing slopes and correlation coefficients (r) across neat solution, analyte-free serum, native serum, and various plasma matrices. Precision and accuracy were quantified through multiday experiments and by analyzing spiked samples across multiple concentrations. Measurement uncertainties were calculated by determining standard uncertainties as well as expanded uncertainties for the entire concentration range.

The described method provides robust quantification of linezolid in human serum and plasma across a concentration range of 0.0800-48.0 μg/mL, demonstrating high specificity and no significant matrix effects. A comprehensive five-day validation study (n=60) demonstrated the method's strong performance, with intermediate precision coefficients of variation (CVs) ≤2.1 % and repeatability CVs≤2.0 %. At the lower limit of the measuring interval (LLMI), the method maintained reliability, achieving an intermediate precision CV of 3.7 % and a repeatability of 1.7 % (n=20). Mean relative biases ranged from -2.0 to 2.9 %. Measurement uncertainty (MU) analysis further supported these results, with standard MUs (k=1) determined to be 1.6-3.8 % for single measurements and 0.8-1.4 % for target value assignments (n=6). Specific assessment at the LLMI yielded an expanded uncertainty (k=2) of 7.6 and 2.8 % for single measurements and target value assignments (n=6), respectively. These results confirm the method's suitability for accurate and precise quantification across the entire measuring range.

This LC-MS/MS-based candidate RMP accurately quantifies linezolid in human serum and plasma. Its robust performance makes it suitable for standardization of routine analytical assays and for analysis of individual patient samples, ensuring analytical reliability and traceability.