Determination of twelve antiretroviral agents in human plasma sample using reversed-phase high-performance liquid chromatography

A new high-performance liquid chromatography (HPLC) with UV detection assay was developed for the simultaneous determination of protease inhibitors (PIs), nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs, NNRTIs) using a single 1-ml plasma samples. A solid–liquid extraction procedure without internal standard was coupled with two separate reversed-phase HPLC systems; one for the determination of amprenavir, efavirenz, indinavir, nelfinavir, ritonavir, saquinavir (run time=32 min) and one for the determination of abacavir, didanosine, lamivudine, stavudine, nevirapine, zidovudine (run time=40 min). The first requires a mobile phase containing sodium phosphate buffer+ion pair–acetonitrile (50:50, v/v) through a C18 Symmetry column (250×4.6 mm I.D., 5 μm particle size), using variable wavelengths (241, 254 and 261 nm). The second system requires three mobile phases (potassium phosphate buffer+ion pair–acetonitrile) for different elution through a C18 Symmetry Shield column (250×4.6 mm I.D., 5 μm), using a single wavelength (260 nm). Peak-areas are linear; correlation coefficients are better than 0.998 for all compounds, with both inter- and intra-day relative standard deviations lower than 12%. Extraction recoveries are higher than 93% for PIs and NNRTIs and higher than 70% for NRTIs. The method is specific and sensitive and was used to determine trough and peak levels of antiretroviral drugs in HIV infected patients under various combinations of RTIs and PIs.