Simultaneous Quantitation of HIV-Protease Inhibitors Ritonavir, Lopinavir and Indinavir in Human Plasma by UPLC-ESI-MS-MS

A selective, sensitive and high-throughput ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method has been developed and validated for the quantification of HIV-protease inhibitors ritonavir (RN), lopinavir (LPV) and indinavir (IDV) in human plasma. Sample clean-up involved protein precipitation of both drugs and fluconazole used as internal standard from 100 mu L human plasma. All the analytes were chromatographically separated on a Waters Acquity UPLC BEN C18 (2.1 x 50 mm, 1.7 tin particle size) analytical column using 0.1% formic acid and methanol (40:60, v/v) as the mobile phase. The parent -> product ion transitions for ritonavir (m/z 721.40 -> 296.10), lopinavir (m/z 629.40 -> 447.40) and indinavir (m/z 614.4 -> 421.0) IS (m/z 307.10 -> 220.10) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion mode. The method was validated over the concentration range of 30-15,000 ng/mL for LPV and IDV and 3-1500 ng/mL for RTV. The method was successfully applied to a pilot bioequivalence study in 36 healthy human subjects after oral administration of lopinavir 200 mg and ritonavir 50 mg tablet formulation under fasting conditions.