Journal of pharmaceutical and biomedical analysis vol:48 issue:2 pages:347-355
Out of three methods for the analysis of josamycin, the best one was selected and used as starting point for further development. A central composite design was applied to find the most influencing parameters and to optimize the chromatographic conditions and a full factorial design was used to perform a robustness study. The final method uses a Hypersil ODS column 5 µm, 250 × 4.6 mm I.D. maintained at 45 °C. The mobile phase is composed of acetonitrile – phosphate buffer (pH 3, 0.2 mol.l-¹) – tetrabutylammonium hydrogen sulphate 0.2 mol.l-¹ – water (21 : 5 : 3 : 71, v/v/v/v). Strongly retained impurities after the main peak require gradient elution, which is obtained by increasing linearly the acetonitrile concentration (from 21 % to 50 %, v/v) and decreasing the TBA concentration (from 3 % to 0 %, v/v) in the mobile phase. The total run time was 65 min. UV detection is performed at 232 nm and the flow rate is 1 ml/min. The method shows good selectivity, precision, linearity and sensitivity. Five commercial bulk samples were analyzed.