Addressing Analytical Challenges to Improve Laboratory Consistency in Unbound Flucloxacillin Measurement

Accurate quantification of flucloxacillin (FLU) in plasma is essential for therapeutic drug monitoring (TDM). Although TDM of FLU traditionally targets total concentrations, only the unbound drug is pharmacologically active. Ultrafiltration (UF) enables the separation of the unbound drug; however, multiple pre-analytical variables can affect results. Furthermore, the lack of standardized protocols currently limits interlaboratory reproducibility. This study aims to develop and validate a robust liquid chromatography-high-resolution mass spectrometric (LC-HRMS) method for quantifying unbound FLU, including an extensive assessment of analyte stability. Cross-validation with an external laboratory was performed to support harmonization of UF protocols.

Ultrafiltrate was obtained after incubation (10 minutes at 37°C) and UF (30 minutes at 1900 g and 37°C), and analyzed using LC-HRMS. Analytical and pre-analytical protocols were validated in accordance with European Medicine Agency guidelines, evaluating calibration curves, selectivity, carryover, accuracy, precision, matrix effect, stability, dilution integrity, as well as nonspecific binding, and membrane protein leakage. Cross-validation was conducted using patient samples from clinical routine.

The method validation was successful. Ultrafiltrate stability was substantially lower than plasma stability, emphasizing the need for immediate postfiltration analysis. Interlaboratory comparison demonstrated strong concordance, with >90% of samples differing by <±25.0%.

A validated LC-HRMS method for quantifying unbound FLU was developed using UF. Cross-validation showed that comparable results can be achieved across laboratories when key UF parameters, such as device type and temperature, are harmonized. Because of the limited stability of FLU in ultrafiltrate, sample storage in plasma matrix is recommended.