A liquid chromatographic assay for the stereospecific quantitative analysis of halofantrine in human plasma

A stereospecific liquid chromatographic (LC) assay was developed for the quantification of the antimalarial drug, halofantrine, in human plasma. Following protein precipitation with acetonitrile, the enantiomers of halofantrine were extracted from human plasma using ammonium hydroxide and tert-butyl methyl ether-hexane. A precolumn derivatization step was employed using (+)-di-O-acetyl-L-tartaric acid anhydride to form diastereomeric derivatives of the halofantrine enantiomers. Chromatographic resolution of the diastereomers was performed using reversed-phase LC with UV detection at 254 nm. The recovery of (+/-)-halofantrine from human plasma at 25 and 2000 ng ml-1 was 68.2 and 61.4%, respectively. The derivatization yield following extraction and derivatization of 2000 ng ml-1 of (+/-)-halofantrine was 95.6%. Using 0.5 ml of plasma, the limit of quantification for each halofantrine enantiomer was 12.5 ng ml-1. Linear responses in analyte/internal standard peak height ratios were observed for analyte concentrations ranging from 12.5 to 1000 ng ml-1. Chromatograms of drug-free plasma showed no interfering peaks with retention times similar to those for (+)- and (-)-halofantrine or internal standard. Based on the validation data, the assay performed well over the enantiomer concentration range of 12.5-500 ng ml-1.