Simultaneous determination of lamivudine, lopinavir, ritonavir, and zidovudine concentration in plasma of HIV-infected patients by HPLC-MS/MS.

The nucleoside reverse transcriptase inhibitors lamivudine and zidovudine and the protease inhibitors lopinavir and ritonavir are currently used in anti-human immunodeficiency virus (HIV) therapy. Here, a high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method, using a hybrid quadrupole time-of-flight mass analyzer, is reported for the simultaneous quantification of lamivudine, lopinavir, ritonavir, and zidovudine in plasma of HIV-infected patients. The volume of plasma sample was 600 µL. Plasma samples were extracted by solid-phase using 1 cc Oasis HLB Cartridge (divinylbenzene and N-vinylpyrrolidone) and evaporated in a water bath under nitrogen stream. The extracted samples were reconstituted with 100-µL methanol. Five microliters of the reconstituted samples were injected into a HPLC-MS/MS apparatus, and the analytes were eluted on a Vydac column (250 × 1.0 mm i.d.) filled with 3-µm C(18) particles. The mobile phase was delivered at 70 µL/min with a linear gradient elution, both acetonitrile and ultrapure water solvents contained 0.2% formic acid. The calibration curves were linear from 0.47 to 20 ng/mL. The absolute recovery ranged between 91 and 107%. The minimal concentration of lamivudine, lopinavir, ritonavir, and zidovudine detectable by HPLC-MS/MS is 0.47, 0.28, 0.30, and 0.66 ng/mL, respectively. The great advantage of the new HPLC-MS/MS method here reported is the possibility to achieve a very high specificity toward the selected anti-HIV drugs, despite the simple and rapid sample preparation. Moreover, this method is easily extendible to the analysis of co-administrated drugs.

Determination of total and unbound concentrations of lopinavir in plasma using liquid chromatography-tandem mass spectrometry and ultrafiltration methods.

Lopinavir is an HIV protease inhibitor with high protein binding (98-99%) in human plasma. This study was designed to develop an ultrafiltration method to measure the unbound concentrations of lopinavir overcoming the non-specific binding issue. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of total concentrations of lopinavir in plasma was developed and validated, and an adaptation was also optimized and validated for the determination of unbound concentrations. The chromatographic separation was performed with a C18 column (100 mm × 2.1mm i.d., 5 µm particle size) using a mobile phase containing deionized water with formic acid, and acetonitrile, with gradient elution at a flow-rate of 350 µL min(-1). Identification of the compounds was performed by multiple reaction monitoring, using electrospray ionization in positive ion mode. The method was validated over a clinical range of 0.01-1 µg/mL for human plasma ultrafiltrate and 0.1-15 µg/mL in human plasma. The inter and intra-assay accuracies and precisions were between 0.23% and 11.37% for total lopinavir concentrations, and between 3.50% and 13.30% for plasma ultrafiltrate (unbound concentration). The ultrafiltration method described allows an accurate separation of the unbound fraction of lopinavir, circumscribing the loss of drug by nonspecific binding (NSB), and the validated LC-MS/MS methodology proposed is suitable for the determination of total and unbound concentrations of lopinavir in clinical practice.