Characterization of a new component and impurities in josamycin by trap-free two-dimensional liquid chromatography coupled to ion trap time-of-flight mass spectrometry

Guijun Liu; Yu Xu; Jing Sang; Bingqi Zhu; Jian Wang

doi:10.1002/rcm.8439

Characterization of a new component and impurities in josamycin by trap-free two-dimensional liquid chromatography coupled to ion trap time-of-flight mass spectrometry

Rapid Commun Mass Spectrom. 2019 Jun 30;33(12):1058-1066. doi: 10.1002/rcm.8439.

Authors

Guijun Liu¹, Yu Xu¹, Jing Sang², Bingqi Zhu², Jian Wang^{1

2}

Affiliations

¹ Zhejiang University of Technology, Hangzhou, 310014, China.
² Zhejiang Institute for Food and Drug Control, Hangzhou, 310052, China.

PMID: 30907019
DOI: 10.1002/rcm.8439

Abstract

Rationale: The toxicities of the impurities of a drug will affect the clinical effects and cause potential health risk; therefore, it is essential to study profiles of the impurities. In this study, a new structural type of component and two acid degradation impurities in josamycin were discovered and characterized for the further improvement of official monographs in pharmacopoeias.

Methods: The component and acid degradation impurities in josamycin were separated and preliminary characterized by trap-free two-dimensional liquid chromatography coupled to high-resolution ion trap time-of-flight mass spectrometry (2D LC/IT-TOF MS) in both positive and negative electrospray ionization mode. The eluent of each peak from the first dimensional chromatographic system was trapped by a switching valve and subsequently transferred to the second dimensional chromatographic system, which was connected to the mass spectrometer. Full scan MS was firstly conducted to obtain the exact m/z values of the molecules. Then LC/MS/MS and LC/MS/MS/MS experiments were performed on the compounds of interest.

Results: A new structural type of component, which was named as josamycin A, and two acid degradation impuritiess, which were identified as impurity I and impurity II, were discovered in josamycin. Their structures and fragmentation pattern were deduced according to MSⁿ data. Furthermore, josamycin A was synthesized and impurity I was separated by preparative HPLC. The structures of josamycin A and the impurities were confirmed by ¹ H NMR and ¹³ C NMR data.

Conclusions: Josamycin A was produced when the hydroxyl group on the macrolide of josamycin was oxidized into a carbonyl group. Impurity I and impurity II were produced by the loss of one molecule of acetyl mycaminose from josamycin and josamycin A, respectively. Compared with josamycin, the experimental results showed that josamycin A had a higher antibacterial activity with similar cytotoxicity, while impurity I had no antibacterial activity but a higher cytotoxicity. As a result, the control of impurity I is significant.