Simultaneous Determination of Total and Unbound Amphotericin B by LC–MS/MS: Application to Protein Binding Analysis

Amphotericin B (AmB), a first‐line antifungal agent, is highly protein‐bound, and its efficacy and toxicity are primarily determined by the unbound fraction. However, current therapeutic drug monitoring mainly relies on total plasma concentrations, and clinical data on unbound AmB remain limited. In this study, a rapid and sensitive LC–MS/MS method was developed and validated in accordance with ICH M10 guidelines for the simultaneous quantification of total and unbound AmB concentrations. The method was successfully applied to 52 clinical samples from 26 patients. Correlation analysis combined with locally estimated scatterplot smoothing demonstrated that the protein binding rate (PBR) of AmB was associated with multiple biochemical parameters as well as total drug concentration. Subgroup analyses further revealed significant differences in PBR stratified by total concentration, albumin levels, and eGFR. The method requires only a small plasma volume and shows good applicability in clinical samples, providing a practical tool for investigating unbound pharmacokinetics and supporting individualized therapeutic drug monitoring, particularly in patients with altered protein‐binding capacity.