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.
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 mixture 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 microg/l for M8 and 25 to 6000 microg/l for NFV, from 500 microl 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 microg/l for both M8 and NFV) were selected to determine the limit of detection. The lower limit of quantification (25 microg/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%.
Lamotte
C
C
Service de Pharmacie Clinique et des Biomatériaux, Groupe Hospitalier X.Bichat-C.Bernard, Paris, France.
Peytavin
G
G
Farinotti
R
R
D019380:Anti-HIV Agents / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017320:HIV Protease Inhibitors / Q000097:blood*; D006801:Humans; D019888:Nelfinavir / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D019380:Anti-HIV Agents; D017320:HIV Protease Inhibitors; D019888:Nelfinavir