Stability studies of glycopyrronium bromide and in vitro toxicity evaluation of its major transformation products by LC-MS/MS and NMR.

Abstract

Characterization of impurities is very important in drug chemistry and process development. Due to these transformation products (TPs), the drug may lose its stability, safety and efficiency. Hence, it is imperative to develop a selective analytical method enabling chromatographic separation of drug and their TPs. Glycopyrronium bromide is a quaternary ammonium drug that acts as an anticholinergic agent and is used to treat chronic obstructive pulmonary disease. Glycopyrronium bromide was forced to degrade under various conditions, according to the International Council for Harmonisation Q1A (R2) guidelines. The resulting TPs were further separated on an Accucore C18 (150 × 4.6 mm, 2.6 μm) column using a gradient program. Glycopyrronium bromide yielded two TPs under acid and base hydrolytic stress conditions. Structural characterization of both TP1 and TP2 was carried out by ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry in combination with HRMS. Further, the TPs were isolated using semi-preparative HPLC, and their structures were confirmed by ¹H and ¹³C NMR spectroscopy. In vitro toxicity studies of isolated TPs were carried out using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay method and they were found non-toxic.