High-performance liquid chromatographic assay to determine the plasma levels of HIV-protease inhibitors (amprenavir, indinavir, nelfinavir, ritonavir and saquinavir) and the non-nucleoside reverse transcriptase inhibitor (nevirapine) after liquid-liquid extraction.

A single HPLC assay was developed for therapeutic drug monitoring of 5 HIV protease inhibitors (indinavir, amprenavir, saquinavir, ritonavir, nelfinavir) and a non-nucleoside reverse transcriptase inhibitor (nevirapine) in human plasma. After liquid-liquid extraction in a mixture ethyl acetate-hexane, compounds are separated on a C18 column with a gradient elution of solvent A [acetonitrile and 0.025 M tetramethylammonium perchlorate in 0.2% aqueous trifluoroacetic acid (55:45 (v/v))] and solvent B [methanol and 0.025 M tetramethylammonium perchlorate in 0.2% aqueous trifluoroacetic acid (55:45 (v/v))]. The compounds are detected at various wavelengths: 320 nm (nevirapine), 259 nm (indinavir), 254 nm (amprenavir, nelfinavir, saquinavir) and 239 nm (ritonavir). The intra-day and inter-day precision and accuracy are lower than 15%. The limits of quantitation are 0.05 mg/l (amprenavir), 0.2 mg/l (indinavir, saquinavir, nelfinavir) and 0.4 mg/l (ritonavir, nevirapine). This method which allows to estimate simultaneously plasma levels of protease inhibitors and nevirapine can be used for therapeutic drug monitoring.