Development and Validation of the First Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Quantification of Multiple Antiretrovirals in Meconium

Abstract

A novel method for the simultaneous quantification of 16 antiretroviral (ARV) drugs and 4 metabolites in meconium was developed and validated. Quantification of 6 nucleoside/nucleotide reverse transcriptase inhibitors, 2 non-nucleoside reverse transcriptase inhibitors, 7 protease inhibitors, and 1 integrase inhibitor was achieved in 0.25 g of meconium. Specimen preparation included methanol homogenization and solid-phase extraction. Separate positive and negative polarity multiple reaction monitoring mode injections were required to achieve sufficient sensitivity. Linearity ranged from 10 to 75 ng/g up to 2500 ng/g for most analytes and 100–500 ng/g up to 25 000 ng/g for some; all correlation coefficients were ≥0.99. Extraction efficiencies from meconium were 32.8–119.5% with analytical recovery of 80.3–108.3% and total imprecision of 2.2–11.0% for all quantitative analytes. Two analytes with analytical recovery (70.0–138.5%) falling outside the 80–120% criteria range were considered semiquantitative. Matrix effects were −98.3–47.0% and −98.0–67.2% for analytes and internal standards, respectively. Analytes were stable (>75%) at room temperature for 24 h, 4 °C for 3 days, −20 °C for 3 freeze–thaw cycles over 3 days, and on the autosampler. Method applicability was demonstrated by analyzing meconium from HIV-uninfected infants born to HIV-positive mothers on ARV therapy. This method can be used as a tool to investigate the potential effects of in utero ARV exposure on childhood health and neurodevelopmental outcomes