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 chromatographic (HPLC) method, with fluorimetric detection, for the simultaneous measurement of halofantrine and desbutylhalofantrine in human plasma or whole blood is described. Sample preparation involved protein precipitation, followed by an efficient solid-phase extraction on a C-8 cartridge. Analytes were isolated from 1 ml of the biological fluids and recovered 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 adjusted to pH 4 with orthophosphoric acid. The total run time was 14 min. Relative standard deviations of the intra- and inter-assay precisions were less than 5.9%. Assumption of linearity was investigated by studying the y-residuals and by ANOVA (analysis of variance). Because of the wide range of calibration (0.1 to 2.0 mu g/ml) variances were non-homogeneous (Hartley's test) and the weighted regression line was computed 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 method to clinical specimens was demonstrated.