Separation and characterization of impurity P in azithromycin product

Azithromycin is a macrolide antibiotic which is used to treat a wide variety of bacterial infections. Impurity P is one of the impurities in azithromycin product, which is registered in Pharmacopoeias of Europe and USA. However, to date, the structure of this impurity has still not been elucidated. In this work, we separated impurity P from azithromycin product using preparative chromatography and successfully identified its chemical structure using multiple analytical techniques. First, high-resolution ion trap-time-of-flight mass spectrometry (IT-TOF MS) was used to determine the accurate molecular mass ([M+H]⁺m/z 777.5121) and the chemical formula (C₃₉H₇₂N₂O₁₃) of the impurity. Second, Fourier transforming infrared spectroscopy (FT-IR), ultraviolet-visible absorption spectroscopy (UV-vis) and tandem mass spectrometry (MS/MS) analyses were performed to probe into the key functional groups of the impurity to aid the NMR analysis. Finally, the structure of the impurity was successfully resolved using multidimensional NMR. In addition, a mechanism for the formation of this impurity was proposed.