Identification of Degradation Products and a Stability-Indicating RP-HPLC Method for the Determination of Belzutifan...

Hypoxia-inducible factor-2alpha (HIF-2α) is a protein, the selective inhibitor of which (belzutifan) is used in the treatment of cancers related to von Hippel-Lindau (VHL) disease. No analytical procedure has been so far reported in the identification of forced degradation products of belzutifan. Thus, the current project was conducted to design and approve the liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) technique to perform a full analysis of it.

This research was to develop and confirm a strong high-performance liquid chromatography (HPLC) procedure in the quantification of belzutifan in the tablet dosage form and to utilize LC-MS/MS in the characterization of the forced degradation products of belzutifan. Approach: The separation was done by the chromatographic method utilizing a Zorbax C₁₈ column with the mobile phase comprised of acetonitrile and 0.1% formic acid (50:50, v/v), and the flow rate was 1.0 mL/min with 247nm as a detector wavelength. Belzutifan had a retention time of 2.338 min and a total reaction time of 4 min.

The derived technique showed great linearity (R2 > 0.999) in the range of concentrations (10-60 μg/ml) and good accuracy, precision, and strength. The forced degradation analysis identified specific degradation products, which were further identified using the LC-MS/MS, thus gathering significant information about the stability profile of belzutifan in stressful environments.

This study developed and validated a simple, rapid, cost-effective, and highly sensitive RP-HPLC method for quantitative estimation of belzutifan in tablets. The method complied with ICH guidelines, demonstrating excellent linearity, accuracy, specificity, robustness, and stability. Linearity was achieved over a wide range, with no interference from excipients, confirming its reliability. Relative standard deviations (RSD) were below 2%, indicating precision and reproducibility. Owing to its simplicity and efficiency, the method is suitable for routine quality control in pharmaceutical laboratories.