Characterization of two unknown impurities in roxithromycin by 2D LC–QTOF/MS/MS and NMR

•Two unknown impurities in roxithromycin were separated and characterized by 2D LC–QTOF MS/MS.•The structures of the impurities were confirmed by NMR after purifying by preparative HPLC.•The mechanisms for formation of the impurities were proposed. Two unknown impurities in roxithromycin were discovered and preliminarily characterized by two-dimensional liquid chromatography coupled with QTOF mass analyzer (2D LC–QTOF MS/MS). The column-switching technique of 2D LC made the chromatographic conditions in official standard of roxithromycin compatible with mass spectrometric detector. The complete MS/MS fragmentation patterns of the impurities were studied to obtain structural information of these impurities. Furthermore, these two impurities were separated and purified by preparative HPLC, and their structures were confirmed by 1D and 2D nuclear magnetic resonance (NMR). Structural elucidation of two impurities by 1H NMR, 13C NMR, the 1H–1H COSY, HSQC and HMBC NMR spectra has been discussed. Based on high resolution MS/MS and NMR data, the structures of these two impurities were elucidated respectively as 11-O-[(2-Methoxyethoxy) methyl] roxithromycin and de(N-methyl)-N-formyl roxithromycin. In addition, the mechanisms for formation of the impurities were also proposed.