A simple, efficient, and sensitive method for simultaneous detection of anti-HIV drugs atazanavir, ritonavir, and tenofovir by use of liquid chromatography-tandem mass spectrometry

Abstract

In the treatment of HIV infection, a combination of anti-HIV drugs is commonly used in highly active antiretroviral therapy (HAART). One such combination recommended for clinical therapy consists of the two HIV protease inhibitors atazanavir and ritonavir and the HIV nucleotide reverse transcriptase inhibitor tenofovir. The detection of plasma and cell drug concentrations provides an assessment of actual drug exposure and patient compliance. We thus developed a simple, efficient, and sensitive method to simultaneously extract and detect these three drugs in plasma and peripheral blood mononuclear cells. The use of a liquid-liquid extraction followed by protein precipitation provided a simple process, yielding a high recovery rate for all three drugs in plasma (>92%) and in cells (>86%). The liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was able to detect 0.01, 0.25, and 2.5 pg (2, 50, and 500 pg/ml, respectively) in 5 μl for atazanavir, ritonavir, and tenofovir, respectively. Validation of the method exhibited high precision and accuracy. This method was subsequently applied to a primate study to determine the concentrations of all three drugs in both plasma and cell samples. This validated method can be useful for an evaluation of drug concentrations in biological samples in an efficient and sensitive manner.

FIG 1

Chromatograms of the drugs atazanavir (A), ritonavir (B), tenofovir (C), and IS (cyheptamide) (D). RT, retention time.

FIG 2

Extracted plasma and cell samples from primates compared to neat (undiluted) samples of atazanavir (A), ritonavir (B), and tenofovir (C). The data are presented as mean values from 6 repetitions with linear square regression lines. The main curve in each panel represents plasma-extracted samples versus neat samples, while the inset represents cell-extracted samples versus neat samples.

FIG 3

Time course plasma concentrations of atazanavir (top), ritonavir (middle), and tenofovir (bottom) in two primates (M. nemestrina). The two primates were subcutaneously administered atazanavir (20 mg/kg), ritonavir (10.2 mg/kg), and tenofovir (12.2 mg/kg). Plasma samples were extracted with the validated assay, as described in the text, and were analyzed using LC-MS/MS. The data were analyzed for each drug at specific time points and are presented as the means ± SD (n = 6). The symbols represent the samples collected from two different primates.