LAUSR

Abstract • Voriconazole (VCZ) is highly efficient in the treatment of invasive fungal infections. However, the therapy requires the monitoring of VCZ plasma concentration, due to a narrow therapeutic index and large inter-individual variability of pharmacokinetics of VCZ, which often exhibits nonlinearity. Total plasma concentration of VCZ does not always provide sufficient information, especially in hemato-oncology patients, who often show significant fluctuations in plasma protein concentration. Therefore, the monitoring of free drug seems crucial for a complete understanding of the ongoing antifungal therapy. • The study aimed to develop an HPLC-FLD method for the determination of total and free VCZ in clinical samples. • The analysis of total VCZ comprised the one-step samples preparation by plasma protein precipitation. The free drug was isolated from plasma by ultrafiltration. The non-specific bindings (NSB) between the drug and filter membranes were comprehensively evaluated. • The elaborated method fulfilled the validation requirements for bioanalytical methods. The ultrafiltration technique is recommended for the analysis of free VCZ, but requires a preconditioning step. Ultrafiltration was impacted with adsorption of the drug on the filter membrane, which was as high as 61–82% and was found to decrease after pretreatment of the membranes with 5% Tween 80. The method was applied for the quantification of plasma VCZ in patients undergoing antifungal therapy. Total and free VCZ concentrations ranged from 0.21 to 4.92 μg/mL and from 0.07 to 1.28 μg/mL, respectively; the mean protein binding was 47%.