Determination of twelve antiretroviral agents in human plasma sample usingreversed-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 (P Is), nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs, NNRTIs) using a single 1-ml plasma samples. A solid-liquid extraction proc edure without internal standard was coupled with two separate reversed-phas e HPLC systems; one for the determination of amprenavir, efavirenz, indinav ir, nelfinavir, ritonavir, saquinavir (run time=32 min) and one for the det ermination of abacavir, didanosine, lamivudine, stavudine, nevirapine, zido vudine (run time=40 min). The first requires a mobile phase containing sodi um phosphate buffer+ion pair-acetonitrile (50:50, v/v) through a C-18 Symme try column (250x4.6 mm I.D., 5 mu m particle size), using variable waveleng ths (241, 254 and 261 nm). The second system requires three mobile phases ( potassium phosphate buffer+ion pair-acetonitrile) for different elution thr ough a C-18 Symmetry Shield column (250x4.6 mm I.D., 5 mu m), using a singl e wavelength (260 nm). Peak-areas are linear; correlation coefficients are better than 0.998 for all compounds, with both inter- and intra-day relativ e standard deviations lower than 12%. Extraction recoveries are higher than 93% for PIs and NNRTIs and higher than 70% for NRTIs. The method is specif ic and sensitive and was used to determine trough and peak levels of antire troviral drugs in HIV infected patients under various combinations of RTIs and PIs. (C) 2000 Elsevier Science B.V. All rights reserved.