A high-performance liquid chromatographic assay for the determination of desbutylhalofantrine enantiomers in rat plasma.

PURPOSE To develop a stereoselective high performance liquid chromatographic assay for determination of desbutylhalofantrine (DHF) enantiomers in rat plasma. METHODS After protein precipitation of 100 microL of rat plasma, racemic DHF and internal standard (quinidine sulfate) were extracted into hexane in the presence of pH 8 phosphate buffer. After transfer and evaporation of the hexane, the residue was derivatized using 0.25 M (+)-di-O-acetyl-L-tartaric acid anhydride at 4 degrees C. After 5 min the reaction was stopped by addition of methanol in water, and the tube contents were dried, reconstituted in the mobile phase, and injected into a C(18) analytical column under reverse phase conditions. RESULTS The derivatized enantiomers were baseline resolved and free of interference from endogenous components in plasma. Standard curves were linear (r(2)>0.99) over the range of enantiomer concentrations from 25-1000 ng/mL. The assay was validated to concentrations as low as 25 ng/mL, based on 100 microL of rat plasma. The nature of diastereomers formed was found to be dependent on the temperature used during the derivatization step. In a preliminary experiment in the rat, stereoselectivity in the plasma concentrations of DHF were observed, indicating stereoselectivity in the pharmacokinetics of the metabolite. CONCLUSIONS The assay was sensitive and appropriate for use in pharmacokinetic studies of DHF in the rat.