High-performance liquid chromatographic method for the determination of nelfinavir, a novel HIV-1 protease inhibitor, in human plasma.

Nelfinavir mesylate, a potent and orally bioavailable inhibitor of HIV-1 protease (Ki=2 nM), has undergone Phase III clinical evaluation in a large population of HIV-positive patients. A high-performance liquid chromatography analytical method was developed to determine the pharmacokinetic parameters of the free base, nelfinavir, in these human subjects. The method involved the extraction of nelfinavir and an internal standard, 6,7-dimethyl-2,3-di-(2-pyridyl)quinoxaline, from 250 microl of human plasma with a mixture of ethyl acetate-acetonitrile (90:10, v/v). The analysis was via ultraviolet detection at 220 nm using a reversed-phase C18 analytical column and a mobile phase consisting of 25 mM monobasic sodium phosphate buffer (adjusted to pH 3.4 with phosphoric acid)-acetonitrile (58:42, v/v) that resolved the drug and internal standard peaks from non-specific substances in human plasma. The method was validated under Good Laboratory Practice (GLP) conditions for specificity, inter- and intra-assay precision and accuracy, absolute recovery and stability. The mean recovery ranged from 92.4 to 83.0% for nelfinavir and was 95.7% for the internal standard. The method was linear over a concentration range of 0.0300 microg/ml to 10 microg/ml, with a minimum quantifiable level of 0.0500 microg/ml for nelfinavir.