Ion-pair reversed-phase high-performance liquid chromatographic analysis of halofantrine and desbutylhalofantrine in human plasma

A new ion-pair reversed-phase high-performance liquid chromatographic (HPLC) method for the simultaneous measurements of halofantrine (HF) and its major metabolite, desbutylhalofantrine (Hfm), in human plasma is described. Sample treatment involved protein precipitation with acetonitrile followed by extraction with hexane-diethylether (ratio, 1:1; vol/vol) under alkaline condition. Chromatographic separation was achieved on a 10-microm particle size C-18 column (200 x 4.6 mm internal diameter) using a mobile phase consisting of methanol-0.05 M potassium dihydrogen phosphate (70:30, vol/vol) with 55 mmol/l perchloric acid (pH 3.1). Retention times for Hfm, Hf, and the internal standard were 5.3, 7.5, and 11.5 minutes, respectively. Detection limits of Hf and Hfm were 2.5 and 2.0 ng/ml, respectively (1 ng/ml = 2 nmol/l for Hf; 1 ng/ml = 2.25 nmol/l for Hfm). Intraassay and interassay coefficients of variation for both compounds were less than 7%, with an accuracy of no greater than 8% at concentrations of 40 and 400 ng/ml, respectively. The new HPLC method is sensitive, selective, and rapid. Relative to previous HPLC methods, it is simple and cost-effective. In addition, the internal standard is readily accessible. Application of this method in pharmacokinetic studies was demonstrated.