Comprehensive impurity profiling and stability assessment of indacaterol maleate integrating LC-MS and in silico...

This study describes the development and validation of stability-indicating analytical methods for indacaterol maleate, an ultra-long-acting β₂-adrenoceptor agonist used in asthma and chronic obstructive pulmonary disease (COPD). RP-HPLC-UV methods were established for quantifying indacaterol and its related substances, while LC-QDa-MS and high-resolution LC-QTOF-MS were employed for impurity identification and structural elucidation. All methods were developed and validated in accordance with ICH guidelines to support comprehensive assessment of process-related impurities and degradation behavior. Eight potential impurities were identified, synthesized, and fully characterized. The validated RP-HPLC-UV assay and related substances methods were applied to forced degradation studies under thermal, photolytic, oxidative, neutral, acidic, and alkaline conditions, as well as to accelerated and long-term stability samples. Trace-level impurities were structurally elucidated using LC-QDa-MS and LC-QTOF-MS. In silico mutagenicity assessment of thirteen impurities using Nexus software classified IND-B ((R)-8-(benzyloxy)-5-(2-bromo-1-hydroxyethyl)-quinolin-2(1H)-one) as a Class 3 potential genotoxic impurity due to its alkyl bromide moiety. Although the TTC-based limit for IND-B (based on the ICH M7 threshold of 1.5 µg/day for a maximum daily dose of 300 µg) corresponds to ≤ 0.5%, all specified impurities were conservatively controlled at ≤ 0.15% and unspecified impurities at ≤ 0.10%, in line with ICH M7 and Q3A guidelines. The RP-HPLC-UV assay and related substances methods demonstrated specificity, precision, accuracy, linearity, and robustness, confirming their suitability as stability-indicating methods. Overall, this integrated approach provides a robust framework for impurity profiling, stability assessment, and genotoxic risk evaluation of indacaterol maleate, ensuring regulatory compliance and product quality throughout shelf life.