Separation and Characterization of New Components and Impurities in Leucomycin by Multiple Heart-Cutting Two-Dimensional Liquid Chromatography Combined with Ion Trap/Time-of-Flight Mass Spectrometry

Abstract

In this study, nine new components and six impurities in leucomycin were discovered. A method was developed for the separation and characterization of new components and impurities in leucomycin by multiple heart-cutting two-dimensional liquid chromatography combined with ion trap/time-of-flight mass spectrometry in both positive and negative electrospray ionization modes. With this method, a non-volatile buffer solution was used as mobile phase in the first-dimensional system for good separation. Eluent of each peak from the first-dimensional system was trapped by a switching valve and sent to the liquid chromatography-mass spectrometry system using a volatile mobile phase. The complete fragmentation patterns of the new components and degradation impurities were deduced based on MSn data. The structures of nine new components in leucomycin were deduced as unsaturated ketone in the 16-membered ring of leucomycin. The structures of six impurities were characterized for the first time, four of which were acid degradation products, and the other two were process impurities. The correlation between impurities and the purification process of leucomycin was also studied. The degradation impurities were produced during purification of leucomycin fermentation broth, which requires a low-pH environment. Based on the characterization of impurities, this study not only revealed the mechanism of impurity production, thus providing guidance to pharmaceutical companies for manufacturing process improvement and impurity reduction, but also provided a scientific basis for further improvement of official monographs in pharmacopoeias.