A reversed-phase liquid chromatography/tandem mass spectrometry method is described for the investigation of spiramycin and related substances. The method uses an XTerra C18 column (250 x 4.6 mm i.d.), 5 microm, and a mobile phase consisting of acetonitrile, methanol, water and ammonium acetate solution, pH 6.5. Mass spectral data were acquired on an LCQ ion trap mass spectrometer equipped with atmospheric pressure chemical ionization (APCI) operated in the positive ion mode. Using this method, the fragmentation behavior of spiramycin and its related substances was studied and the unknown impurities occurring in commercial samples were investigated. In total 17 compounds were identified, among which three reported as specified impurities in the European Pharmacopoeia. The other impurities showed mainly a modification in the forosamine sugar or in the substituent at C-3 and C-6 positions. In one impurity, the mycarose sugar is absent.
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