Development of a novel stability-indicating UHPLC method for revefenacin quality control and impurity identification using 2D-LC-HRMS.

Revefenacin (Rev) inhalation solution is the only once-daily nebulized bronchodilator approved in the USA for chronic obstructive pulmonary disease treatment. Despite the critical role of quality control in pharmaceutical manufacturing, no established methods exist for Rev, and its degradation pathways remain unreported. This study performed forced degradation to generate impurity-containing samples, which were used to develop a novel ultrahigh-performance liquid chromatography (UHPLC) method. Qualitative analysis of 12 impurities was conducted using two-dimensional liquid chromatography-high-resolution mass spectrometry. Characteristic fragment ions and mass fragmentation processes were examined, leading to the identification and verification of four specific impurities using reference standards. Impurities A (C₂₂H₃₃N₃O₄), B (C₃₅H₄₂N₄O₅), and C (C₁₂H₁₁N) were identified definitively by comparison to reference materials, while DP-9 and DP-11 were tentatively identified based on MS studies. The remaining seven impurities were not identified due to concentrations below the identification threshold. The primary degradation pathways of Rev were oxidation and hydrolysis. The UHPLC method achieved baseline separation of the 12 degradation products (DPs), and its effectiveness was confirmed by assessing sample stability under the proposed chromatographic conditions. The results demonstrate that this method is suitable for Rev quality control. The novel stability-indicating UHPLC method developed for quantifying Rev DPs offers a reliable approach for quality assurance.