Evaluation of liquid chromatography methods for the separation of ampicillin and its related substances

Two laboratories collaborated to examine the selectivity of four isocratic liquid chromatography (LC) methods for the separation of ampicillin and its related substances. The United States Pharmacopeia (USP) assay method gave the best selectivity. Similar selectivity was obtained on C18 columns as well as on C8 and poly(styrene-divinylbenzene) copolymer columns. A resolution test using cefradine was proposed to replace the test with caffeine prescribed by the USP. Based on the USP method, a gradient LC method was developed for the analysis of related substances in ampicillin. This LC method has been proposed for assay and purity control in the ampicillin monographs of the European Pharmacopeia.