Validated and green UPLC-MS/MS method for belumosudil quantification in human liver microsomes: Application to in vitro metabolic stability assessment

Abstract The first targeted Rho-associated protein kinase 2 inhibitor was Belumosudil (Rezurock). After at least two systemic treatments failed for chronic graft-versus-host disease in adults and children aged 12 and older, belumosudil (BLS) was approved on July 16, 2021. This study established a validated Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) system to estimate BLS in HLMs and assess metabolic stability. Agilent C18 column was used to separate BLS and encorafenib (internal standard) in HLMs using an isocratic mobile phase. The electrospray ionization (ESI) source produced BLS parent ions. Multiple reaction monitoring identified and measured BLS daughter ions. The methodology was validated following FDA standards that assessed linearity, selectivity, accuracy, sensitivity, precision, extraction recovery, matrix effects, and stability. The accuracy and precision ranged from −1.42 to 12.50% between days and from −0.89 to 10.5% within a day. The created BLS calibration curve displayed a linearity in the range from 1 ng mL −1 to 3,000 ng mL −1 . The LLOQ of 0.64 ng mL −1 showed the method's sensitivity. The AGEE program displayed that the approach was eco-friendly. In vitro half-life and intrinsic clearance of BLS were 17.55 min and 46.21 mL min −1 kg −1 , demonstrating a high extraction ratio drug. These features can improve the metabolic stability of new derivatives compared to BLS, making them important in medication design.