Characterization of Prednisone Degradants via Forced Degradation Studies: A UHPLC‐Based Approach With HRMS/MS, NMR, and Toxicity Predictions Using Quantitative Structure–Activity Relationship Models

ABSTRACT Prednisone, a steroid therapeutically vital for its anti‐inflammatory and immunosuppressive activity, often necessitates thorough impurity profiling. Impurity profiling involves monitoring process impurities during drug substance screening and examining degradation products (DPs) in drug formulations, thereby enabling future stability studies that are critical to ensuring product quality. Prednisone—as reported in the European Pharmacopoeia 11.4—is associated with 10 impurities, primarily process‐related impurities; however, the method used to detect these impurities has some limitations. In this study, a single reproducible ultrahigh‐performance liquid chromatography method has been developed to quantify 12 impurities. The limitations of the European Pharmacopoeia method are also discussed. Forced degradation studies were performed using the new method, and the results demonstrated an adequate mass balance. This method also enables the identification and quantification of two novel degradation products, detected at relative retention time (RRT) of 0.24 and 0.93. The impurity at RRT of 0.24 forms rapidly under mild alkaline conditions at room temperature, with significant formation occurring within a short period. The impurity at RRT of 0.93 is generated via an oxidative degradation pathway. The presence of these impurities was first noted during the initial stages of drug product development and pre‐formulation studies. Using this insight, optimized approaches were implemented to effectively minimize the impurity levels in the final formulation. The new DPs were identified and characterized by nuclear magnetic resonance spectroscopy and liquid chromatography–high resolution mass spectrometry. The impurities were established as 17‐formyloxy prednisone (DP1) and 1,2‐epoxy prednisone (DP2), respectively, and a preliminary toxicity evaluation was undertaken using quantitative structure–activity relationship models from CASE Ultra software. A plausible degradation mechanism was proposed to guide its control strategies. The new analytical method was validated as per the principles of International Council for Harmonization guidances.