Isolation and identification of a major impurity in a new bulk drug candidate by preparative LC, ESI-MSn, LC-MS-MS, and NMR

Abstract

A simple preparative liquid chromatography (LC) method is developed to isolate a major impurity in a new bulk drug candidate, 6-bromo-4- (carbamidinemethyl)-5-hydroxy-1-methyl-2-(phenylthiomethyl)-1H-indole-3- carboxylic acid ethyl ester hydrochloride monohydrate (carmidole). The carmidole solution for preparation is exposed to daylight before isolation. Based on the electrospray ionization (ESI)-mass spectroscopy (MSn) spectral data of the impurity fraction and carmidole, the impurity is preliminarily characterized as 6-bromo-4-(carbamidinemethyl)-5-hydroxy-1-methyl-2-methyl-1H- indole-3-carboxylic acid ethyl ester. LC-MS-MS is used to analyze a carmidole sample. The impurity, lyophilate, is obtained from the fraction of preparative LC, and the impurity standard is synthesized. By comparison of the retention times of high-performance liquid chromatography, ESI-MSn, and 1H-nuclear magnetic resonance of the impurity lyophilate with impurity standard and carmidole itself, the structure of the impurity is confirmed and its formation is discussed.