Characterization of 28 unknown impurities in 16-membered macrolides by liquid chromatography coupled with ion trap/time-of-flight mass spectrometry.

In terms of risk assessment, the study of the impurity profile is important to ensure the safety and effectiveness of drugs in clinical application. Sixteen-membered macrolides are produced by microbial fermentation, and many closely related substances in the product make the components and impurities complicated. In this study, methods were developed to separate and identify the impurities in three representative 16-membered macrolides (josamycin, midecamycin and meleumycin) using a high-performance liquid chromatography coupled to high-resolution ion trap/time-of-flight mass spectrometry (IT-TOF MS). In total, 53 impurities were characterized in the positive mode of electrospray ionization, among which 28 novel impurities were found. The proposed structures of impurities were deduced based on MS/MS data, and the ultraviolet (UV) absorption behaviors of impurities were discussed. In addition to the impurities with maximum absorption wavelengths (λmax) of 231 nm and 280 nm, there was a new group of impurities with λmax of 205 nm in meleumycin, midecamycin and josamycin.

International standards and international reference preparations: amphotericin B, vancomycin, capreomycin, cefalotin, demethylchlortetracycline, gentamycin, gramicidin S, kanamycin and kanamycin B, lincomycin, lymecycline, methacycline, paromomycin, rifamycin SV, ristocetin and ristocetin B, spiramycin, and triacetyloleandomycin.

Each of the preparations described here was obtained and evaluated at the request of a WHO Expert Committee on Biological Standardization. Unless otherwise stated, a standard procedure was used to distribute the material into individual ampoules. The procedure was as follows. Upon receipt by the National Institute for Medical Research (NIMR), London, materials were stored temporarily in the dark at a temperature of -10 degrees C or lower, and protected from moisture. At a convenient time they were brought back to room temperature, mixed, and distributed into individual neutral glass ampoules so that each ampoule contained 50-100 mg of powder. If it was known that the material was light-sensitive non-actinic glass ampoules were used. After exhaustive drying in vacuum over phosphorus(V) oxide, the ampoules were either constricted (up to 1963) or fitted with capillary leak plugs, dried for a further period under the same conditions, filled with dry nitrogen, and sealed by fusion of the glass. The total drying period varied from 8 to 38 days according to the nature of the material. After they had been tested for leaks, the ampoules were stored in the dark at -20 degrees C.