Simultaneous Determination of Lamivudine, Lopinavir, Ritonavir and Zidovudine Concentration in Plasma of HIV-Infected Patients by HPLC-MS/MS

The nucleoside reverse transcriptase inhibitors lamivudineand zidovudine and the protease inhibitors lopinavir and ritonavirare currently used in human immunodeficiency virusAQ4 (HIV) therapy. Here, a HPLC-MS/MS method, using a hybridquadrupole time-of-flight mass analyzer, is reported for thesimultaneous quantization of lamivudine, lopinavir, ritonavir,and zidovudine in plasma of HIV-infected patients. The volumeof plasma sample was 600 lL. Plasma samples were extractedby solid-phase using 1 cc Oasis HLB Cartridge (divinylbenzeneand N-vinylpyrrolidone) and evaporated in a water bath undernitrogen stream. The extracted samples were reconstituted with100-lL methanol. Five microliters of the reconstituted sampleswere injected into a HPLC-MS/MS apparatus, and the analyteswere eluted on a Vydac column (1 3 250 mm2 i.d.) filled with3-lm C18 particles. The mobile phase was delivered at 70 lL/min with a linear gradient elution, both acetonitrile andultrapure water solvents contained 0.2% formic acid. Thecalibration curves were linear from 0.47 to 20 ng/mL. Theabsolute recovery ranged between 91 and 107%. The minimalconcentration of lamivudine, lopinavir, ritonavir, and zidovudinedetectable by HPLC-MS/MS is 0.47, 0.28, 0.30, and0.66 ng/mL, respectively. The great advantage of the newHPLC-MS/MS method here reported is the possibility toachieve a very high specificity toward the selected anti-HIVdrugs, despite a simple and rapid sample preparation.[...