Evaluation of LC methods for the separation of phenoxymethylpenicillin and its related substances

Five isocratic reversed-phase liquid chromatography (LC) methods have been examined for the separation of phenoxymethylpenicillin and its related substances. A method previously selected for the analysis of benzylpenicillin gave the best selectivity. The mobile phase of this method is composed of 0.5 M phosphate buffer (pH 3.5)-water-methanol (10:50:40), the amount of organic modifier was slightly adapted when it was used with different columns. Similar selectivity is obtained not only on different C18 materials but also on C8 packings. The selectivity on poly(styrenedivinylbenzene) is less good. As method performance test a resolution test with benzylpenicillin was used. The robustness of this method was examined by applying a full factorial design to test the influence of the content of organic modifier in the mobile phase, the pH of the mobile phase, the concentration of buffer in the mobile phase and the temperature of the column. The results show that the method is robust.