Isolation and identification of four major impurities in capreomycin sulfate

Capreomycin has good clinical utility to treat multidrug resistant tuberculosis, but it is only used as a second line drug due to its adverse reactions. Literature has demonstrated that the toxicity of capreomycin product is significantly influenced by the impurities in it. Unfortunately, so far, no one impurity in capreomycin has ever been isolated and definitely identified due to its extremely strong basic character and high polarity. An ion-pair method reported in literature can provide separation of capreomycin and its impurities, but it is hard to be used for the preparative purpose. In this study, this ion-pair method was further improved to detect more impurities in capreomycin sulfate substance. Besides the four main components (IA, IB, IIA and IIB), four impurities (impurity A-D) with their contents much higher than the identification threshold were observed. Furthermore, a two dimensional (2D) LC quadrupole-time of flight (Q-TOF) MS method was established to realize high resolution MS analysis of these impurities. For the purpose of preparative isolation, a hydrophilic interaction chromatography (HILIC) method was established. The four main components were well isolated, but unfortunately, the four impurities were co-eluted with each other or with IB by the HILIC method. Fortunately, the degradation experiments revealed that IA and IB could yield clean impurity A and B respectively in acidic medium, and can yield clean impurity D and C respectively in alkaline medium. Therefore, IA and IB were first isolated by the preparative HILIC method, then pure IA and IB underwent acid degradation and base degradation separately and followed by re-isolation by the HILIC method to obtain pure impurity A-D respectively. Based on Q-TOF MS and NMR analysis, the structures (including absolute configuration) of the four isolated impurities were definitely identified.