Important note: Information in this article was accurate in 2000. The state of the art may have changed since the publication date.
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J Chromatogr B Biomed Sci Appl. 1999 Dec 10;735(2):159-70. Unique Identifier : AIDSLINE MED/20134192
Lamotte C; Peytavin G; Farinotti R; Service de Pharmacie Clinique et des Biomateriaux, Groupe; Hospitalier X.Bichat-C.Bernard, Paris, France.
Abstract: 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%.
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