Simultaneous determination of belzutifan and cabozantinib, oral agents for renal cell carcinoma, in human plasma using high-performance liquid chromatography.

Belzutifan and cabozantinib are oral drugs for advanced renal cell carcinoma, which display exposure-dependent toxicities. No validated method exists for simultaneously quantifying these drugs in human plasma. In this study, we developed and validated a high-performance liquid chromatography-ultraviolet method for simultaneously quantifying belzutifan and cabozantinib in human plasma. Plasma samples (50 μL) were processed via simple protein precipitation. Chromatographic separation on a C18 column was performed isocratically with 0.5% KH2PO4 (pH 4.5)/acetonitrile (47/53, v/v) at 1.0 mL/min, with UV detection at 230 nm. Performance characteristics (calibration, precision, accuracy, recovery, stability, selectivity, and interference by concomitant drugs) were systematically evaluated. Linear calibration ranges were 50-4,000 ng/mL for belzutifan and 25-4,000 ng/mL for cabozantinib with excellent linearity (r2 ≥ 0.9998). Intra- and interday coefficients of variation were ≤ 5.41% for belzutifan and ≤ 12.25% for cabozantinib. Both analytes were stable under bench-top, short-term, long-term, and freeze-thaw conditions. No interference from endogenous plasma components or six commonly co-administered medications was observed. The newly developed assay for the simultaneous quantification of belzutifan and cabozantinib in human plasma is rapid, requires minimal sample volumes, and demonstrates robust analytical performance.