EVALUATION OF LIQUID-CHROMATOGRAPHY METHODS FOR THE ANALYSIS OF BENZYLPENICILLIN AND ITS RELATED SUBSTANCES

Seven isocratic liquid chromatography (LC) methods have been examined for the separation of benzylpenicillin and its related substances on C -18 or C-8, phenyl and poly(styrene-divinylbenzene) columns. The metho d proposed by a manufacturer, using a C-8 column and a mobile phase co nsisting of 0.05 M phosphate buffer, pH 3.5-methanol (62:38, v/v) gave the better selectivity. The amount of organic modifier was slightly a dapted when it was used with different columns. Similar selectivity wa s obtained not only on C-8 materials but also on C-18 packings. The se lectivity on poly(styrenedivinylbenzene) was worse. A resolution test with phenylacetic acid was used as the method performance test. The ro bustness of this method was examined by applying a full factorial desi gn to test the influence of content of organic modifier in the mobile phase, the pH of the mobile phase, the concentration of buffer in the mobile phase and the column temperature. The results show that the met hod is robust.