Mass Spectrometry-Based Analysis of the Pharmacokinetics and Intracellular Metabolism of Nucleoside Reverse Transcriptase Inhibitors
Mon 4:00 PM - Track 1: Small Molecule Analysis II
Leon Coulier
TNO Quality of Life
Leon Coulier, TNO Quality of Life, Zeist, the Netherlands (presenting author)
Henk Gerritsen, TNO Quality of Life, Zeist, the Netherlands
Ivana Bobeldijk, TNO Quality of Life, Zeist, the Netherlands
Jeroen van Kampen, Erasmus MC, Rotterdam, the Netherlands
Marleen Reedijk, Erasmus MC, Rotterdam, the Netherlands
Theo Luider, Erasmus MC, Rotterdam, the Netherlands
Rob Gruters, Erasmus MC, Rotterdam, the Netherlands
Highly active antiretroviral therapy (HAART) has resulted in a significant reduction of the mortality and morbidity of Human Immunodeficiency Virus 1 (HIV-1) infected patients. However, the emerging of viral resistance and drug toxicity remain serious problems. Antiretroviral drugs must penetrate into HIV-1 susceptible cells to exert its action. In many cases, the plasma pharmacokinetics correlates poorly to the intracellular pharmacokinetics. This is especially true for the nucleoside reverse transcriptase inhibitors (NRTIs), since NRTIs are intracellularly phosphorylated to their active triphosphate form (NRTI-TP) and compete with endogenous deoxynucleoside-triphosphates (dNTPs) as substrate for incorporation in the DNA copy of the viral RNA.

Within the Top Institute Pharma-project ‘’Multi-disciplinary approach to monitor and select effective therapy in HIV-infection’’ (T4-212) a comprehensive analytical platform was set-up for NRTIs and their phosphorylated metabolites and endogenous nucleotides in plasma and peripheral blood mononuclear cells (PBMCs). NRTIs in plasma and PBMCs are analyzed using a relatively straightforward reversed phase (UP)LC-MS/MS method. However, the chromatographic separation of phosphorylated compounds, like phosphorylated NRTIs and endogenous nucleotides like ATP, require special attention due to the strong polarity of these metabolites resulting in irreversible adsorption or peak tailing. Previously, it was shown that ion-pair LC-ESI-MS is able to analyse a variety of polar metabolites in extracts of microbial samples, such as nucleotides, coenzyme A esters and sugar nucleotides1.

This method was adapted successfully to analyze multiple endogenous nucleotides and phosphorylated NRTIs in PBMCs. With this method approx. 25 exogenous and endogenous mono, di- and tri-phosphorylated compounds all can be distinguished from each other by independent parameters, e.g. MS/MS transition and/or retention time2. The optimized ion-pair LC-MS/MS method was validated successfully with respect to linearity, sensitivity, accuracy and precision as described in the FDA guidelines. An important factor in the method development was the sensitivity needed for analysis of clinical samples. The intracellular concentrations of these antiretroviral drugs in one million PBMCs are approximately 10-15 to 10-12 mole. It is a major technical challenge to measure these low drug concentrations, in a clinical setting, in HIV-1 infected patient, especially in children, without drawing unacceptably large volumes of blood. Our ion-pair LC-MS/MS method is capable to detect and separate the different compounds at clinically relevant concentrations. Some first clinical applications will be shown.

1 Coulier et al., Anal. Chem. 78 (2006), 6573-65823
2 Coulier et al., Proteomics Clin. Appl. 2 (2008), 1557-1562.

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