Sustainable quantification of glycopyrronium, indacaterol, and mometasone along with two genotoxic impurities in a recently approved fixed-dose breezhaler formulations and biological fluids: A machine learning-augmented UV-spectroscopic approach

Abstract

This study presents an innovative, sustainable approach for the simultaneous quantification of glycopyrronium (2–14 μg/mL), indacaterol (6–18 μg/mL), and mometasone (4–20 μg/mL) in a recently approved fixed-dose breezhaler formulations and biological fluids, along with two genotoxic impurities: methyl -toluene sulfonate (2–10 μg/mL) and 4-dimethylamino pyridine (2–10 μg/mL). We developed robust UV spectrophotometric machine-learning chemometric models to address the limitations of existing chromatographic methods. The calibration set was carefully selected at five concentration levels using the multilevel-multifactor experimental design, resulting in 25 calibration mixtures. The Kennard-Stone Clustering Algorithm was employed to construct a representative 13-mixture validation set, overcoming biases associated with random data splitting. Five chemometric models (CLS, PCR, PLS, GA-PLS, and MCR-ALS) were rigorously evaluated, with MCR-ALS demonstrating superior performance. This model achieved 98–102 % recovery percentages for all analytes, with low root mean square error of calibration and prediction of (RMSEC: 0.0225 to 0.5246) and (RMSEP: 0.0039 to 0.4226). The method exhibited excellent relative root mean square error of prediction (RRMSEP: 0.1306 to 0.8517 %), a negligible bias-corrected mean square error of prediction (BCMSEP: −0.0073 to 0.0025), and good sensitivity (LOD: 0.022 to 0.893 μg/mL) across all analytes. Green solvents were selected using the Green Solvents Selection Tool and Greenness Index Spider Charts. The method’s sustainability was comprehensively evaluated using seven state-of-the-art assessment tools. This approach not only offers a green alternative to traditional chromatographic methods but also ensures high accuracy in quantifying both active ingredients and genotoxic impurities, thereby enhancing pharmaceutical quality control and patient safety.