Comprehensive Impurity and Degradation Profiling of Iptacopan Using a White-Green Stability-Indicating HPLC Method...

A comprehensive, stability-indicating, and environmentally sustainable analytical strategy was developed for the impurity and degradation profiling of iptacopan. Optimum separation was achieved on a Hypersil BDS C18 (250 × 4.6 mm, 5 μm) column using 0.8-mL/min isocratic flow of 10-mM ammonium acetate buffer (pH 4.5) and ethanol (60:40, v/v) with 234-nm UV detection. The method produced excellent linearity (r² ≥ 0.9995), precision (%RSD ≤ 0.75), accuracy (98.32%-100.26%), and robustness. Forced degradation studies generated four distinct degradation products (DPs) under acidic and oxidative stress, characterized by LC-MS/MS. DP 3 showed a +30-Da mass increase (m/z 453.4996), consistent with an indole-carboxylic acid analogue, whereas DP 1 (m/z 439.4730) exhibited a -14-Da shift, indicating demethylation. DP 2 displayed a -42-Da difference (m/z 381.4369) due to dealkylation, and DP 4 showed a +14-Da shift (m/z 437.5002), corresponding to a linker ketone analogue. In silico toxicity assessment suggested moderate toxicity for most DPs, with DP 4 showing a comparatively favorable profile. Greenness and whiteness evaluation using AGREE (0.82), AGREE-Prep (0.81), GAPI (E-factor = 10), and RGB-12 (78.0%) confirmed excellent environmental and practical performance. The method provides a robust and sustainable platform for quality control and stability assessment of iptacopan.