Quantification of Belzutifan in Biological Samples: LC–MS/MS Method Validation and Pharmacokinetic Study in Rats

Belzutifan, an inhibitor of hypoxia inducible factor-2α, is used to treat cancer associated with von Hippel Lindau disease. The quality control and pharmacokinetic study of this drug is crucial for effective chemotherapy. Since no bio-analytical method has been reported, this work aimed to develop an LC–MS/MS technique for the determination of belzutifan and its application for pharmacokinetic profiling in rat plasma. Belzutifan and apalutamide (IS) were quantified on a symmetry shield (150 × 4.6 mm, 3.5 μm) column using acetonitrile and buffer (30:70% v/v) as the mobile phase, with a run time of 7 min. The spiked samples and quality controls were extracted with an optimized protein precipitation technique. Belzutifan and IS were quantified in MRM mode. The validation of the method in compliance with the US FDA's guidelines was performed. The analyte and IS were quantified at m/z 384.3422 → 311.4205 and 478.4154 → 341.1629, respectively. The results indicate linearity between 5 and 100 ng/mL concentration with r² = 0.9997, which proved to be accurate with % recovery between 95.0% and 97.98%, along with other essential metrics within the accepted limits. The pharmacokinetic study demonstrates that the established LC–MS/MS method accurately quantifies the drugs in rat plasma and might be useful for routine quantification of belzutifan in biological matrices.