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The European Pharmacopoeia, the U.S. Pharmacopoeia, and the Pharmacopoeia of the People's Republic of China all prescribe a high-performance liquid chromatography-ultraviolet method within a C18 column system for the separation of Azithromycin (AZT) and its related substances. The identification of impurities in the AZT complex was performed according to the relative retention time (RRT) between each impurity and AZT. However, the RRT values of impurities often vary on different types of C18 packing materials and at different column temperatures, which could affect the accurate and fast identification of impurities. In our study, five different commonly used C18 columns as well as nine different column temperature set points were assessed for the analysis of AZT and its related substances. A factorial design was applied to analysis the relationships between column types/column temperatures and RRT value of each impurity. The results showed that the change rates of the RRT values of impurities were different on different columns and at different column temperature set points. Therefore, the current method adopted by the three Pharmacopeias, in which the RRT values were used to identify the related substances, is not suitable to identify the ones in the AZT chromatographic system.

Factors Influencing the HPLC Determination for Related Substances of Azithromycin