Simultaneous Quantification of Novel Antiretroviral Drug Combination by Stability-indicating High Performance Liquid Chromatography Method

Research in the many areas of human immunodeficiency virus treatment, eradication and prevention has necessitated measurement of antiretroviral concentrations in nontraditional specimen types. For HIV infection, drug combinations are typically used as highly active antiretroviral therapy, intended to maximize viral suppression. A novel four drugs combination was used, which contains two nucleoside analog reverse transcriptase inhibitors (lamivudine and tenofovir) and two protease inhibitors (darunavir and ritonavir). A new simple, efficient, and sensitive reverse phase high performance liquid chromatographic method has been developed for simultaneous extraction and determination of the concentrations of lamivudine, tenofovir, darunavir and ritonavir in bulk and in their tablets. Four compounds were separated on a reversed-phase C18 column at 30 +/- 2.0 degrees using a gradient mobile phase combination containing potassium dihydrogen phosphate, acetonitrile and methanol. The pH was adjusted to 3.5 +/- 0.05 by the addition of orthophosphoric acid. The samples were detected using a UV detector, 260 nm for lamivudine, tenofovir and darunavir and 240 nm for ritonavir. The procedure separated analytes and its potential degradation products such as lamivudine, tenofovir, darunavir and ritonavir eluting at about 2.385, 4.055, 11.353 and 14.010 mins, respectively. The linear range of lamivudine, tenofovir, darunavir and ritonavir was 58.32-174.96 mu g/ml, 58.32-174.96 mu g/ml, 72.00-216.00 mu g/ml and 112.50-337.5 mu g/ml, respectively. The relative standard deviation for precision was less than 2.0%. The drug was subjected to acid, alkaline peroxide and photolytic stress conditions and the performance of the method was validated according to the International Conference on Harmonization guidelines for specificity, linearity, accuracy, precision and robustness.