Simultaneous determination of the five HIV-protease inhibitors: Amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir in human plasma by solid-phase extraction and column liquid chromatography

A sensitive and selective liquid chromatographic assay has been developed f or the determination of the five protease inhibitors currently approved by the Food and Drug Administration (FDA) (amprenavir, indinavir; nelfinavir, ritonavir, and saquinavir) in a single run. Pretreatment of a 1-mL plasma s ample spiked with internal standard was made by a solid-phase extraction pr ocedure using a polymeric reversed-phase sorbent. Liquid chromatography was performed using a narrowbore C18 reversed-phase column and gradient elutio n. A double ultraviolet detection at 265 nm (amprenavir) and at 210 nm (ind inavir, nelfinavir, ritonavir, saquinavir and internal standard) was used. Calibration curves were linear in the range 25-10000 ng/mL and the assay ha s been validated over the range 25-5000 ng/mL. Average accuracy at four con centrations was in the range of 100.5-104.2% and 96.9-100.5% for within-day and between-day. respectively. The coefficients of variation were less tha n 10%. Mean absolute recoveries varied from 85.4% (ritonavir) to 98.8% (saq uinavir). No metabolite of the protease inhibitors was found to coelute wit h the drugs of interest or with the internal standard. At this time, among the tested drugs, especially all the presently licensed nucleoside and nonn ucleoside reverse transcriptase inhibitors that can be used a combination w ith the protease inhibitors, none was found to interfere with the assay. Th is method is now in use in the authors' laboratory for the therapeutic moni toring of the HIV-protease inhibitors.