An improved high-performance liquid chromatographic method for the simultaneous measurement of halofantrine and desbutylhalofantrine in human serum.
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 x 4.6 mm I.D., 5 microns 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 for BHAL, HAL, and the internal 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 less than or equal to 7.5%, with an accuracy of less than or equal to 10.8% over three concentrations (0.02, 0.5, 2.0 micrograms/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.
Keeratithakul
D
D
Department of Immunology and Biochemistry, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.
Teja-Isavadharm
P
P
Shanks
G D
GD
Webster
H K
HK
Edstein
M D
MD
D000962:Antimalarials / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008288:Malaria / Q000188:drug therapy*; D010616:Phenanthrenes / Q000097:blood* / Q000627:therapeutic use; D014466:Ultraviolet Rays
D000962:Antimalarials; D010616:Phenanthrenes; C044961:1,3-dichloro-6-trifluoromethyl-9-phenanthryl-3-(n-butyl)aminopropanol; C023768:halofantrine