Journal of separation science vol:25 issue:7 pages:399-404
A linear gradient elution method is described for the separation of tetracycline, its fermentation impurities, and its degradation products. This method uses XTerra RP-18, 5 mum (25 cm x 4.6 mm ID), a silica-based stationary phase with reduced silanol activity. Gradient elution with mobile phases containing acetonitrile, 0.3 M tetrabutylammonium hydrogen sulfate pH 7.5, 0.3 M ethylenediaminetetraacetic acid pH 7.5, water was used at a flow rate of 1.0 mL/min. The column temperature was set at 40degreesC. The UV detection was performed at 280 nm. A central composite experimental design was performed to optimize the separation. The method permits a good separation of TC and its impurities. Some of these were not reported before and have now been identified for the first time. Some unidentified impurities were also separated.