Determination of nelfinavir,a potent HIV protease inhibitor,and its active metabolite M8 in human plasma by high-performance liquid chromatography with photodiode-array detection

摘要

We developed and characterized a high-performance liquid chromatographic assay for the determination of nelfinavir (NFV),a potent HIV protease inhibitor,and its active metabolite M8 in human plasma.Extraction of the internal standard,M8 and NFV from the plasma buffered at pH 9.5 was achieved by a liquid-liquid extraction with a mixtfure of methyl-tert.-butyl ether and hexane.Following two washes of the reconstituted sample with hexane,separation was achieved on an octadecylsilyl analytical column with a mobile phase containing 0.1% trifluoroacetic acid-acetonitrile-methanol (51:46:5,v/v).Detection was performed using an ultraviolet photodiode-array detector.The signal was monitored at a wavelength of 220 nm. The assay was found to be linear and has been validated over the concentration range of 25 to 3000 #mu#g/l for M8 and 25 to 6000 #mu#g/l for NFV,from 500 #mu#l of plasma.Recoveries were 98.9% (SD 8.9%),and 100.2% (SD 11.7%) for M8 and NFV,respectively.Concentrations that gave a signal-to-noise ratio of three (15 #mug/l for both M8 and NFV) were selected to determine the limit of detection. The lower limit of quantification (25 #mu#g/l for both M8 and NFV) was defined as the concentration for which the relative standard deviation and the percent deviation from the nominal concentration were lower than 20%.