Wide-Range Quantification of Belumosudil in Patients with Chronic Graft-Versus-Host Disease by High-Performance Liquid Chromatography.

Abstract

Belumosudil is an oral, selective inhibitor of Rho-associated coiled-coil-containing protein kinase 2, approved for the treatment of chronic graft-versus-host disease (cGVHD). Clinical trials have shown that higher doses of belumosudil are associated with a greater incidence of adverse events. The pharmacokinetics of belumosudil are markedly affected by food intake and concurrent medications, and substantial interindividual variability in plasma concentrations has also been reported. Therefore, monitoring belumosudil blood concentrations may facilitate more effective drug therapy. Belumosudil and luliconazole (internal standard, IS) were extracted from 50 µL of human plasma using methanol. Chromatographic analysis was performed on a reversed-phase column (250 mm × 4.6 mm, 5 µm) under isocratic conditions, with a mobile phase of 0.5% KH2PO4 (pH 4.5) and acetonitrile (40:60, v/v) at a flow rate of 1 mL/min. Ultraviolet detection was performed at 337 nm. The calibration curve for belumosudil demonstrated linearity over the concentration range of 250 to 15,000 ng/mL, with a correlation coefficient (r2) of 0.9998. The accuracy and precision of all validation experiments met the criteria established by the US Food and Drug Administration. In this study, 44 plasma samples from 5 patients with cGVHD were analyzed to determine belumosudil concentrations. The chromatographic peaks of both the IS and belumosudil showed no interference from the biological matrix or from concurrent medications and their metabolites, confirming adequate selectivity and specificity. This study presents the first development and validation of a simple, novel, and broad-range high-performance liquid chromatography-ultraviolet approach for quantifying plasma belumosudil, supporting its suitability for therapeutic drug monitoring in clinical practice.