Novel liquid chromatography-tandem mass spectrometry method for simultaneous detection of anti-HIV drugs Lopinavir, Ritonavir, and Tenofovir in plasma.

For HIV infection, anti-HIV drug combinations are typically used as highly active antiretroviral therapy (HAART), intended to maximize viral suppression. Three drugs used frequently in combination are the protease inhibitors lopinavir and ritonavir and the nucleotide reverse transcriptase inhibitor tenofovir. We have successfully developed a simple, efficient, and sensitive method to simultaneously extract and determine the concentrations of lopinavir, ritonavir, and tenofovir in plasma samples. The plasma extractions were performed using a liquid-liquid extraction followed by protein precipitation of the remaining aqueous layer. The collected fractions were combined, dried, and reconstituted in the mobile phase. The drugs were quantified using liquid chromatography coupled with tandem mass spectrometry. The assay was applied to a study of plasma drug levels in two primates (Macaca nemestrina). The bioanalytical assay was optimized and validated to exhibit a high extraction efficiency and good sensitivity and reproducibility. When the assay was applied in a primate study, all three drugs could be detected in plasma within minutes of subcutaneous dosing. This validated assay will be useful for evaluation of drug concentrations in an efficient, selective, and sensitive manner.