Simultaneous quantification and aerodynamic particle size distribution analysis of tiotropium bromide and olodaterol...

Fixed-dose combination (FDC) inhaled aerosols require precise characterization of both active pharmaceutical ingredients (APIs) and aerodynamic properties to ensure therapeutic efficacy and safety. This study established and validated a sensitive, high-throughput ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous quantification of tiotropium bromide (TTP, a long-acting muscarinic antagonist) and olodaterol (ODT, a long-acting β₂-adrenergic agonist) in nebulized aerosols, coupled with comprehensive aerodynamic particle size distribution (APSD) analysis. The optimized method employed a 50% methanol-water solution as the extraction solvent, with isocratic elution using 0.1% formic acid in water-methanol (50:50, v/v) and multiple reaction monitoring (MRM) in positive ionization mode. The total analysis time was only 3 min, enabling high-throughput analysis. Method validation confirmed excellent linearity for both analytes over the range of 0.5-16 ng/mL (r > 0.998), with a lower limit of quantification (LLOQ) of 0.5 ng/mL. Intra- and inter-batch precision (relative standard deviation, RSD) were < 7.5%, and extraction recoveries ranged from 98.1% to 98.8% (RSD<3%), with good matrix effect tolerance, solution stability (24 h at room temperature/4°C), and dilution integrity. A combination of syringe aspiration and Andersen Cascade Impactor (ACI) sampling was used to characterize aerosol properties at 2 and 8 min of nebulization. Both TTP and ODT exhibited uniform and stable aerosol concentrations (RSD<3.5%), with mass median aerodynamic diameters (MMAD) of 1.87-2.35 μm, geometric standard deviations (GSD) of 2.13-2.44, and fine particle fractions (FPF) exceeding 90%. The proportion of ultrafine particles (<0.41 μm) captured by the micro-orifice collector (MOC) was < 4%, confirming optimal particle size distribution for deep lung deposition. Application of this method to the evaluation of theoretical delivered doses in animal safety assessment tests showed that the doses were 4.7-8.6 times the rat equivalent dose, meeting the design requirements for toxicological studies. This method provides a robust framework for quality control, generic drug consistency evaluation, and preclinical toxicology of TTP/ODT nebulized aerosols, supporting the development of FDC inhaled therapies for chronic obstructive pulmonary disease (COPD).