NEW SOLID-PHASE EXTRACTION FOR AN IMPROVED HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC PROCEDURE FOR THE QUANTITATION OF HALOFANTRINE AND MONODESBUTYLHALOFANTRINE IN BLOOD OR PLASMA

A rapid, accurate, and sensitive high-performance liquid chromatograph ic (HPLC) method, with fluorimetric detection, for the simultaneous me asurement of halofantrine and desbutylhalofantrine in human plasma or whole blood is described. Sample preparation involved protein precipit ation, followed by an efficient solid-phase extraction on a C-8 cartri dge. Analytes were isolated from 1 ml of the biological fluids and rec overed by a 2% acetic acid in ethyl acetate solution. Chromatographic separation was carried out on a LiChrospher 60 RP select B, C-8 bonded phase (5 mu m particle size, 25 cm x 4 mm I.D.) using a mobile phase of water-acetonitrile (35:65, v/v) containing triethylamine (1%) and a djusted to pH 4 with orthophosphoric acid. The total run time was 14 m in. Relative standard deviations of the intra- and inter-assay precisi ons were less than 5.9%. Assumption of linearity was investigated by s tudying the y-residuals and by ANOVA (analysis of variance). Because o f the wide range of calibration (0.1 to 2.0 mu g/ml) variances were no n-homogeneous (Hartley's test) and the weighted regression line was co mputed in order to allow pharmacokinetic studies. Accuracy was tested using a t-statistic. Limits of decision, detection and quantification were realized from an analysis of the blanks. Application of the metho d to clinical specimens was demonstrated.