Optimization of the separation of erythromycin and related substances by high-performance liquid chromatography
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An improved high-performance liquid chromatographic method for analysis of erythromycin is described. The separation can be performed under mild conditions of pH (6.5) and temperature (35T) on C, and CIS silica-based reversed-phase ma¬terials of different origins. The mobile phase, with a flow-rate of 1.5 ml/min, con¬tained various amounts of acetonitrile (25-40%, v/v), 5% (v/v) 0.2 M ammonium phosphate buffer pH 6.5, 20% (v/v) 0.2 M tetramethylammonium phosphate and water. UV detection at 215 nm allows quantitation of erythromycins A, Band C, N-demethylerythromycin A, erythromycin A enol ether and anhvdroerythromycin A. The column history plays a major role, older columns often giving better separations.