Simultaneous determination of indinavir, ritonavir and saquinavir in plasma by high-performance liquid chromatography

Background and Purpose: Antiretroviral regimens consisting of a combination of protease inhibitors have been shown to be highly potent for the treatment of HIV infection. Therapeutic drug monitoring may ensure optimal drug levels to improve efficacy and minimize side effects. The purpose of this study was to establish a method for simultaneous determination of plasma concentrations of indinavir (IDV), ritonavir (RTV) and saquinavir (SQV) by high-performance liquid chromatography (HPLC) in a single run. Methods. A C-18 column and an ultraviolet detector set at 215 nm were used in the HPLC. The mobile phase consisted of phosphate buffer (50 mM, pH 5.6) and acetonitrile (55:45, v/v), and the flow rate was 1.5 mL/min. Plasma sample (0.5 mL) containing IDV, RTV, and SQV was alkalinized with ammonia water (10%) and propylparaben added as an internal standard (IS), which was then extracted with methyl tert.-butyl ether. The organic layer was taken and evaporated to dryness. The residue was dissolved in the mobile phase, and the solution was washed with n-hexane followed by HPLC analysis. Results: The calibration curves were linear for each of the 3 drugs in the studied concentration range (0.1 to 5 mug/mL). The extraction recoveries were greater than 90%. One HPLC run takes less than 15 minutes. Analysis of plasma samples of patients treated with combinations of these drugs demonstrated that the calibration curves covered the clinical concentration range. Conclusions: This study established a simple and accurate method for simultaneous determination of IDV, RTV, and SQV in plasma by isocratic HPLC. The method is practical for therapeutic drug monitoring.