A White-green Stability-indicating HPLC Method for Targeted Determination of Four Specified Process-related Impurities in Revumenib with LC-MS/MS-based Characterization and In Silico Toxicity Assessment of Degradation Products

A green and white stability-indicating HPLC method was proposed for the targeted determination of four specified process-related impurities of revumenib and for monitoring degradation products (DPs) formed under forced degradation conditions. The proposed method displays excellent linearity within 25–150 µg/mL for revumenib and 0.25–1.50 µg/mL for impurities ( R ² > 0.999) with high reproducibility (% Relative Standard Deviation [RSD] < 1.0) and accuracy. The Design of Experiments-based robustness evaluation demonstrates statistically significant models (p < .01), satisfactory model performance (R² up to 0.95), and acceptable resolution between critical peak pairs across the experimentally investigated conditions. Forced degradation studies showed controlled degradation (≤10.25%) with complete resolution between revumenib and all DPs. Stress-degradation produced four DPs, and the major DPs detected under the investigated stress conditions were structurally characterized by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) with mass errors of less than 5 ppm and well-defined fragmentation pathways. The sustainability assessment using Analytical GREEnness (AGREE) (0.89), AGREE-Prep (0.75), Green Analytical Procedure Index, and the Red Green Blue (RGB)-12 model yielded an overall whiteness score of 80.1%. These findings demonstrate that the proposed method provides a suitable balance between analytical performance, environmental sustainability, and practical efficiency for the targeted determination of the four specified process-related impurities and stability assessment of revumenib.