Development and validation of a fully automated LC method for the determination of cloxacillin in human plasma using anion exchange restricted access material for sample clean-up

In the framework of a preliminary investigation on the plasma profile of cloxacillin after oral administration, a simple and rapid LC method was developed for the direct determination of this compound in human plasma. The on-line sample clean-up was carried out using a weak anion exchanger (diethylaminoethyl groups) as restricted access material (RAM). The effects of the washing liquid pH, the ionic strength and the addition of organic modifier to the washing liquid were studied in order to obtain an efficient sample clean-up and a high recovery of cloxacillin. The separation was achieved on octadecylsilica stationary phase using a mobile phase consisting in a mixture of phosphate buffer (pH 4.0; 25 mM) and acetonitrile (72:28, v/v). The UV detection was performed at 215 nm. The most appropriate regression model of the response function as well as the limit of quantitation (LOQ) were first selected during the pre-validation step. These criteria were then assessed during the formal validation step. The LOQ was 50 ng/ml. The method was also validated with respect to analyte recovery, precision, trueness, accuracy and linearity. Finally, it was successfully applied for the analysis of the first plasma samples obtained from patients having taken an oral dose of 500 mg cloxacillin.