Simultaneous Determination of Ritonavir and Atazanavir in Human Plasma by LC-MS/MS and Its Pharmacokinetic Application

A simple and rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and validated for simultaneous quantification of two protease inhibitors ritonavir and atazanavir in human plasma. Saquinavir was used as an internal standard. The analytes were extracted from human plasma samples by solid-phase extraction technique using a Orpheus C18 extraction cartridges. The reconstituted samples were chromatographed on a C18 column by using a 85:15 (v/v) mixture of methanol and 5mM ammonium acetate as the mobile phase at a flow rate of 0.9 mL/min. The calibration curves obtained were linear (r ³ 0.99) over the concentration range of 8.0-1600.0 ng/mL for ritonavir and 50.5-5995.2 ng/mL for atazanavir. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. A run time of 2.0 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was found to be applicable to clinical studies.