An improved high-performance liquid chromatographic method for the simultaneous measurement of halofantrine and desbutylhalofantrine in human serum
Keeratithakul, D.; Teja-Isavadharm, P.; Shanks, G.D.; Webster, H.K.; Edstein, M.D.
Therapeutic Drug Monitoring 13(1): 64-68
A rapid, accurate, and sensitive high-performance liquid chromatographic (HPLC) method, with ultraviolet detection, for simultaneous measurement of halofantrine (HAL) and desbutylhalofantrine (BHAL) in human serum is described. Sample preparation involved protein precipitation, followed by a solid-phase clean-up and a liquid-liquid extraction. Chromatographic separation was carried out on an Ultrasphere C8 column (25 cm .times. 4.6 mm I.D., 5 .mu.m particle size) using a mobile phase of acetonitrile:water, 75:25, (vol/vol), with 0.2% (w/vol) sodium dodecyl sulfate and 0.2% (vol/vol) glacial acetic acid. The serum sample volume used was 0.5 ml, with concentrations normalized to 1 ml. Retention times of BHAL, HAL, and the intermal standard were 5.5, 8.3, and 11.5 min, respectively, with a total run time of 13.5 min. The average extraction recovery for HAL was 85.6% and 100.5% for BHAL. Inter- and intra-assay precisions of HAL and BHAL were .ltoreq. 7.5%, with an accuracy of .ltoreq. 10.8% over three concentrations (0.02, 0.5, 2.0 .mu.g/ml). Detection limits of HAL and BHAL were 5 and 3 ng/ml, respectively. The new HPLC method resulted in cleaner chromatograms and a 1.7-fold faster run time than previous HPLC methods. Application of the method with clinical specimens was demonstrated.