Optimization of high-performance liquid chromatographic analysis for isoxazolyl penicillins using factorial design

A 3 X 3 factorial design has been used to study the effects of pH and acetonitrile concentration of the eluents on the retention and resolution of cloxacillin, flucloxacillin and dicloxacillin on a C18 column. The logarithm of the capacity factors of these solutes have been found to vary linearly with the pH and quadratically with the acetonitrile content. The equations generated have been employed to predict experimental conditions necessary for an optimum separation. The chromatographic condition selected has been applied to the quantitation of flucloxacillin in human plasma using dicloxacillin as the interval standard. Sample preparation consists of protein precipitation and solid-phase extraction. The detection limit of the assay at 220 nm for flucloxacillin is in the region of 0.1 microgram/ml. This assay has been employed in a study of the relative bioavailability of two commercial flucloxacillin sodium capsules in ten healthy volunteers.