RATIONALE A simple and sensitive method was developed for the separation and characterization of four unknown impurities in azithromycin and erythromycin imino ether using two-dimensional liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry (2D LC/QTOFMS) with positive and negative electrospray ionization. METHODS The chromatographic separation in the first dimension was performed with a Waters Xbridge RP18 column in gradient mode using binary mobile phase: (A) phosphate buffer (pH 8.2)-acetonitrile (47:53, v/v) and (B) water-acetonitrile (90:10, v/v). In the second dimension, the chromatographic separation was performed using a Shimadzu Shim-pack GISS C18 column with volatile mobile phases: (A) ammonium formate solution (10 mM) and (B) methanol. RESULTS The molecular formulae and structures of the four impurities were deduced based on the LC/MS/MS data, and further confirmed using 1 H NMR, 13 C NMR, 1 H-1 H COSY, HSQC and HMBC NMR spectra after semi-preparative isolation of impurities. In addition, the mechanism for the formation of the impurities was also proposed. CONCLUSIONS The contradiction between the non-volatile salt mobile phase and mass spectrometry was solved by means of a multiple heart-cutting 2D LC approach and on-line desalination technology. Four impurities were separated and characterized. These results could further improve the method of official monographs in pharmacopoeias and guides to improve the process of reducing impurity content.

中文翻译：

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使用二维液相色谱结合高分辨率四极杆飞行时间质谱和核磁共振，对阿奇霉素和红霉素亚氨基醚中的四种未知杂质进行表征。

原理开发了一种简单而灵敏的方法，用于分离和表征阿奇霉素和红霉素亚氨基醚中的四种未知杂质，方法是将二维液相色谱与高分辨四极杆飞行时间质谱（2D LC / QTOFMS）联用和负电喷雾电离。方法使用二元流动相，使用Waters Xbridge RP18色谱柱以梯度模式进行一维色谱分离：（A）磷酸盐缓冲液（pH 8.2）-乙腈（47:53，v / v）和（B）水-乙腈（90:10，v / v）。在第二维中，色谱分离是使用具有挥发性流动相的Shimadzu Shim-pack GISS C18色谱柱进行的：（A）甲酸铵溶液（10 mM）和（B）甲醇。结果根据LC / MS / MS数据推导了四种杂质的分子式和结构，并通过半制备后的1 H NMR，13 C NMR，1 H-1 H COSY，HSQC和HMBC NMR光谱进一步证实分离杂质。另外，还提出了形成杂质的机理。结论非挥发性盐流动相与质谱法之间的矛盾通过多中心切割二维液相色谱方法和在线脱盐技术得以解决。分离并表征了四种杂质。这些结果可进一步改善药典中官方专着的方法，并指导改进减少杂质含量的过程。半制备性分离杂质后的HSQC和HMBC NMR光谱。另外，还提出了形成杂质的机理。结论非挥发性盐流动相与质谱法之间的矛盾通过多中心切割二维液相色谱方法和在线脱盐技术得以解决。分离并表征了四种杂质。这些结果可进一步改善药典中官方专着的方法，并指导改进减少杂质含量的过程。半制备性分离杂质后的HSQC和HMBC NMR光谱。另外，还提出了形成杂质的机理。结论非挥发性盐流动相与质谱法之间的矛盾通过多中心切割二维液相色谱方法和在线脱盐技术得以解决。分离并表征了四种杂质。这些结果可进一步改善药典中官方专着的方法，并指导改进减少杂质含量的过程。结论非挥发性盐流动相与质谱法之间的矛盾通过多中心切割二维液相色谱方法和在线脱盐技术得以解决。分离并表征了四种杂质。这些结果可进一步改善药典中官方专着的方法，并指导改进减少杂质含量的过程。结论非挥发性盐流动相与质谱法之间的矛盾通过多中心切割二维液相色谱方法和在线脱盐技术得以解决。分离并表征了四种杂质。这些结果可进一步改善药典中官方专着的方法，并指导改进减少杂质含量的过程。