Stability Indicating Assay Method for Quantification of Hypoxia‐Inducible Factor Inhibitor Daprodustat: Characterization of Novel Degradation Products by Quadrupole Time‐of‐Flight (Q‐TOF) Mass Spectrometry and In Silico Toxicity Thereof

Abstract A selective stability‐indicating HPLC method was developed for daprodustat. Among the stress conditions involving acid hydrolysis, base hydrolysis, and oxidation, the drug was found to degrade under acid and base hydrolysis. Whereas it was found to be stable under oxidative stress. With an injection volume of 10 µL, the drug and its degradation products (DPs) were separated on Luna C‐18 (150 × 4.6 mm, 5 µm) column using a mobile phase combination of 0.1% formic acid buffer and acetonitrile at a flow rate of 1 mL/min by gradient elution. Diode array detection was performed at 265 nm. Meanwhile, the developed HPLC method was validated in compliance with ICH guideline Q2(R2) with LOD of 14.7 ng/mL and LOQ of 44.7 ng/mL. Overall, two DPs were formed under accelerated stress conditions. Furthermore, the DPs were characterized by LC‐ESI‐Q‐TOF‐MS/MS. The mechanism via which daprodustat forms all the DPs has been discussed. In addition, DEREK software was used to predict the in silico toxicity of the drug and its two degradation products.