Determination of anti-HIV drug concentration in human plasma by MALDI-TOF/TOF

The antiretroviral therapeutic drug monitoring is becoming increased in clinical care to determine the best dosage regimen adapted to each patient. Here, the determination of the anti-HIV drugs lamivudine, lopinavir, and ritonavir concentration in the plasma of HIV-infected patients by MALDI-TOF/TOF is reported. The volume of the plasma sample was 600 μL. Plasma samples were extracted by solid-phase (divinylbenzene and N-vinylpyrrolidone) and evaporated in a water bath under a nitrogen stream. The extracted samples were reconstituted with methanol (100 μL), mixed (1:1) with a saturated matrix solution (4-hydroxybenzoic acid in 50% acetonitrile–0.1% trifluoracetic acid), and spotted onto the MALDI-TOF/TOF sample target plate. The lamivudine, lopinavir and ritonavir concentration was determined by standard additions analysis. Regression of standard additions was linear over the anti-HIV drug concentration ranges explored (lamivudine, 0.010–1.0 pmol/μL; lopinavir and ritonavir, 0.0025–0.50 pmol/μL). Moreover, emtricitabine (i.e., the fluorinated analog of lamivudine) was used as the internal standard to determine the lamivudine concentration. The calibration curve was linear on the emtricitabine concentration ranging between 0.050 and 5.0 pmol/μL. The absolute recovery ranged between 80 and 110%. Values of the lamivudine, lopinavir and ritonavir concentration determined by MALDI-TOF/TOF are in excellent agreement with those obtained by HPLC-UV and HPLC-MS/MS. MALDI-TOF/TOF experiments allowed also the detection of the ritonavir metabolite R5. Zidovudine was undetectable by MALDI-TOF/TOF analysis because also the minimal laser intensity may induce the anti-HIV drug photolysis. The MALDI-TOF/TOF technique is useful to determine very low concentrations of anti-HIV drugs (0.0025–0.010 pmol/μL).