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

Abstract

A sensitive and selective liquid chromatographic assay has been developed for 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 sample spiked with internal standard was made by a solid-phase extraction procedure using a polymeric reversed-phase sorbent. Liquid chromatography was performed using a narrowbore C18 reversed-phase column and gradient elution. A double ultraviolet detection at 265 nm (amprenavir) and at 210 nm (indinavir, nelfinavir, ritonavir, saquinavir and internal standard) was used. Calibration curves were linear in the range 25–10000 ng/mL and the assay has been validated over the range 25–5000 ng/mL. Average accuracy at four concentrations 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 than 10%. Mean absolute recoveries varied from 85.4% (ritonavir) to 98.8% (saquinavir). No metabolite of the protease inhibitors was found to coelute with the drugs of interest or with the internal standard. At this time, among the tested drugs, especially all the presently licensed nucleoside and nonnucleoside reverse transcriptase inhibitors that can be used in combination with the protease inhibitors, none was found to interfere with the assay. This method is now in use in the authors' laboratory for the therapeutic monitoring of the HIV-protease inhibitors.