Simultaneous quantification of four antiretroviral drugs in breast milk samples from HIV-positive women by an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method

<div><p>The primary strategy to avoid mother-to-child transmission of human immunodeficiency virus (HIV) through breastfeeding is administration of highly active antiretroviral therapy (HAART) to HIV-positive pregnant women. Because significant changes in the pharmacokinetics of antiretroviral (ARV) drugs occur during pregnancy, quantifying HAART and the viral load in breast milk in this population is essential. Here, we developed an analytical assay for the simultaneous quantification of four ARV drugs in breast milk using ultra-performance liquid chromatography coupled to tandem mass spectrometry. We validated this method following Mexican and international guidelines. ARV drugs. We extracted the ARV drugs from 200 μL samples of breast milk and detected these drugs in a triple quadrupole mass spectrometer with positive electrospray ionization. The validated concentration ranges (ng/mL) for zidovudine, lamivudine, lopinavir, and ritonavir were 12.5–750, 50–2500, 100–5000 and 5 to 250, respectively. Additionally, the absolute recovery percentages (and matrix effects) were 91.4 (8.39), 88.78 (28.75), 91.38 (11.77) and 89.78 (12.37), respectively. We determined that ARV drugs are stable for 24 h at 8°C and 24°C for 15 days at –80°C. This methodology had the capacity for simultaneous detection; separation; and accurate, precise quantification of ARV drugs in human breast milk samples according to Mexican standard laws and United States Food and Drug Administration guidelines.</p></div

Clinical manifestations in the ILI and PH1N1 women.

<p>ILI: Pregnant women with influenza-like illness.</p><p>PH1N1: Confirmed H1N1pdm2009 virus-infected pregnant women.</p><p>*Fisher’s exact test.</p><p>Clinical manifestations in the ILI and PH1N1 women.</p

Correlation between nominal concentration <i>vs</i> measured concentration (ng/mL) for intra-day (n = 3) and inter-day (n = 3) validation of each ARV.

<p>Correlation between nominal concentration <i>vs</i> measured concentration (ng/mL) for intra-day (n = 3) and inter-day (n = 3) validation of each ARV.</p

Demographic and obstetric characteristics of the study population.

<p>HW. Healthy women.</p><p>HP. Healthy pregnant women.</p><p>ILI. Pregnant women with influenza-like illness.</p><p>PH1N1. H1N1pdm2009 virus-infected pregnant women.</p><p>Gw. Gestational weeks.</p><p>n/a not applicable.</p><p>*Fisher’s exact test.</p><p>Demographic and obstetric characteristics of the study population.</p

Accuracy and precision for the quantification of antiretroviral in breast milk.

<p>Accuracy and precision for the quantification of antiretroviral in breast milk.</p

Nominal concentrations of each calibration curve and quality control standards in breast milk samples.

<p>Nominal concentrations of each calibration curve and quality control standards in breast milk samples.</p

Representative ion chromatograms of separation and retention time (minutes) of each ARV drug and IS: 0.29, 0.62, 1.61, 1.71 and 2.11 for LMV, ZDV, RTV, LPV, and SMV, respectively, corresponding only to LQC (ng/mL) of each ARV.

<p>Total run time was 2.5 minutes, with an ACQUITY UPLC BEH C 18 column, formic acid and ACN gradient as mobile phase.</p

Chromatographic conditions: Mobile phase gradient program.

<p>Chromatographic conditions: Mobile phase gradient program.</p

Higher percentage of CD69+ lymphocytes in H1N1pdm2009 virus-infected pregnant women.

<p>The peripheral blood leukocytes from the HW, HP, ILI and PH1N1 women were immunostained with CD3-, CD19-, CD14- and CD69-specific antibodies and analyzed by flow cytometry. Representative data of each group are shown in the dot plots for CD69+CD3+ cells (a). The distribution of CD69 expression on CD3− (b), CD19− (c) or CD14− (d) gated cells. The Kruskal-Wallis test with Dunn’s multiple comparison post-test was performed using the GraphPad Software. The significance values were *p<0.05, **p<0.01, ***p<0.001.</p

Transition ions and optimal conditions to obtain relative abundance of product ions.

<p>Transition ions and optimal conditions to obtain relative abundance of product ions.</p