Separation, Identification, and Characterization of Transformation Products Related to Relugolix Upon Subjecting to In Vitro Gastrointestinal Fluids Utilizing Liquid Chromatography, Mass Spectrometry, and Nuclear Magnetic Resonance Spectroscopy

Abstract

This study investigated the gastrointestinal stability of relugolix, a biopharmaceutical classification system Class IV drug. Relugolix was found to be unstable in simulated gastric fluid but stable in simulated intestinal fluid and fed‐state–simulated gastric fluid. The high‐performance liquid chromatography method was developed and validated for separating the drug and its transformation products. The optimized method comprises mobile phase ammonium acetate (pH 4.75) as Solvent A and acetonitrile as Solvent B. A Phenomenex Gemini C18 (250 × 4.6 mm, 5 µm) column was used as the stationary phase. The transformation product, desmethyl relugolix, was identified and characterized using high‐resolution mass spectrometric and nuclear magnetic spectroscopic studies by enriching the compound using forced degradation studies under acidic stress hydrolytic conditions. Sodium alginate microspheres were prepared and evaluated to improve relugolix stability in the stomach. Additionally, gastrointestinal stability studies demonstrated that the microspheres protected relugolix from degradation in simulated gastric fluid. In silico analysis predicted minimal toxicity risks for both relugolix and desmethyl relugolix. These findings suggest that relugolix sodium alginate microspheres could be a promising strategy to mitigate the formation of desmethyl relugolix by protecting it from degradation in the stomach's acidic environment.