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

PURPOSE: To develop a stereoselective high performance liquid chromatograph ic assay for determination of desbutylhalofantrine (DHF) enantiomers in rat plasma. METHODS: After protein precipitation of 100 muL of rat plasma, rac emic DHF and internal standard (quinidine sulfate) were extracted into hexa ne 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 degreesC. After 5 min the reaction was stopped by addition of methanol in water, and the tube contents were dried, recons tituted in the mobile phase, and injected into a C-18 analytical column und er reverse phase conditions. RESULTS: The derivatized enantiomers were base line resolved and free of interference from endogenous components in plasma . Standard curves were linear (r(2)>0.99) over the range of enantiomer conc entrations from 25-1000 ng/mL. The assay was validated to concentrations as low as 25 ng/mL, based on 100 muL of rat plasma. The nature of diastereome rs formed was found to be dependent on the temperature used during the deri vatization step. In a preliminary experiment in the rat, stereoselectivity in the plasma concentrations of DHF were observed, indicating stereoselecti vity in the pharmacokinetics of the metabolite. CONCLUSIONS: The assay was sensitive and appropriate for use in pharmacokinetic studies of DHF in the rat.