Liquid Chromatography Combined With Tandem Mass Spectrometry for the Pharmacokinetic and Metabolism Studies of PROTAC ARV‐471 in Rats

ABSTRACT Proteolysis targeting chimera (PROTAC) is emerging as a promising medicinal modality, which has aroused widespread interest among the field of pharmaceutical manufacturing in the recent years. ARV‐471 is an orally active PROTAC estrogen receptor degrader against breast cancer, which leads to the ubiquitylation and subsequent degradation of estrogen receptors via the proteasome. In this study, we developed a highly sensitive liquid chromatography tandem mass spectrometry method (LLOQ = 0.5 ng/mL) for the measurement of ARV‐471 in rat plasma. The acetonitrile precipitated sample was separated on ACQUITY BEH C 18 column using acetonitrile‐0.1% formic acid as mobile phased with gradient elution. Multiple reactions monitoring in positive ESI mode was employed for the quantification of ARV‐471 ( m/z 724.4 → 396.2). The assay showed good linearity over the concentration range of 0.5–1000 ng/mL with correlation coefficient > 0.996. The assay was validated according to FDA guidance, and all the validation parameters were within the predefined acceptance criteria. After validation, the assay was applied to the pharmacokinetic study of ARV‐471 in rats. Additionally, the metabolites in rat plasma were identified using liquid chromatography–high resolution mass spectrometry. Four metabolites were identified and characterized. Hydrolysis, glucuronidation and deamination were the main metabolic pathways of ARV‐471 in rats.