Investigation of aclidinium bromide degradation by stability-indicating HPLC methods, characterization of impurities by NMR, and identification of degradation products by LC-MS

Abstract

Aclidinium bromide (ACL) is a long-acting muscarinic receptor antagonist used for the long-term treatment of chronic obstructive pulmonary disease (COPD). The aim of this study was to investigate the degradation of aclidinium bromide under stress and stability testing conditions, for which we developed and validated the first stability-indicating, specific, precise, accurate, and robust assay and related substances HPLC methods. Nine of the compounds used as reference standards were synthesized and fully characterized by 1H and 13C NMR, MS, and FTIR techniques. Two of these molecules, namely ACL-dimer and ACL-desphenyl, are novel compounds and reported herein for the first time. Hydrolysis of aclidinium resulted in major degradation via the formation of ACL-desDTG and DTGA metabolites. ACL-desphenyl and phenol were observed only under oxidative conditions at very low levels (< 0.10%), while ACL-hydroxy, known as a metabolite of aclidinium and confirmed by LC/QDa and LC/Q-TOF m/z data, formed under oxidative stress-testing conditions, UV light, and daylight. The identification of two impurities formed only when aclidinium bromide was treated with hydrogen peroxide was done by LC/QDa and LC/Q-TOF studies and the novel structures were proposed as ACL-bromophenoxy and ACL-bromothiophenyl formed via bromination of the phenyl and thiophene ring on the aclidinium, respectively. The stress and photostability testing studies showed that the aclidinium bromide drug substance is not sensitive to elevated temperature (105 °C, 10 days), slightly sensitive to daylight and UV-radiation, and it showed significant degradation under all hydrolysis and oxidizing conditions. The related substances HPLC method reported herein is also capable to monitor the potential genotoxic impurity 3-bromopropoxybenzene (3-BPB), a class 2 impurity according to ICH M7, and ensures that the impurity remains below the threshold of toxicological concern (TTC) limit, making it safe for patients.