Comprehensive analysis of the intracellular metabolism of antiretroviral nucleosides and nucleotides using liquid chromatography–tandem mass spectrometry and method improvement by using ultra performance liquid chromatography

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2011-09, Vol.879 (26), p.2772-2782
• Main Authors: Coulier, Leon, Gerritsen, Henk, van Kampen, Jeroen J.A., Reedijk, Mariska L., Luider, Theo M., Osterhaus, Albert D.M.E., Gruters, Rob A., Brüll, Lars
• Format: Article
• Language: English
• Subjects: adenosine triphosphate; Analysis; Analytical, structural and metabolic biochemistry; antiretroviral agents; Antiretroviral drugs; Biological and medical sciences; Chromatography; Chromatography, High Pressure Liquid - methods; detection limit; Drugs; Fundamental and applied biological sciences. Psychology; General pharmacology; HIV; HIV infections; HIV Infections - blood; Human immunodeficiency virus; Humans; Inhibitors; Ion-pair LC–MS/MS; ionization; Leukocytes, Mononuclear - metabolism; Liquid chromatography; Liquids; Mass spectrometry; Medical sciences; metabolism; metabolites; mononuclear leukocytes; NRTI; Nucleosides; Nucleosides - blood; Nucleosides - chemistry; Nucleosides - metabolism; Nucleotides - blood; Nucleotides - chemistry; Nucleotides - metabolism; Pharmacology. Drug treatments; Phosphorylated metabolites; Platforms; Reproducibility of Results; Reverse Transcriptase Inhibitors - blood; Reverse Transcriptase Inhibitors - chemistry; Reverse Transcriptase Inhibitors - metabolism; RNA-directed DNA polymerase; Sensitivity and Specificity; tandem mass spectrometry; Tandem Mass Spectrometry - methods; toxicity; ultra-performance liquid chromatography; UPLC; Zidovudine - blood; Zidovudine - chemistry; Zidovudine - metabolism
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2011.07.045

Nucleoside reverse transcriptase inhibitors (NRTIs) are a key class of drugs for the treatment of HIV infection. NRTIs are intracellularly phosphorylated to their active triphosphate metabolites and compete with endogenous deoxynucleotides (dNTP) for substrate binding. It is therefore important to analyze the intracellular concentrations of these compounds to understand drug efficacy and toxicity. To that purpose an analytical platform was developed that is capable of analyzing 8 NRTIs, 12 phosphorylated NRTIs and 4 dNTPs in small numbers of peripheral blood mononuclear cells, i.e. 1 × 10 6 cells. The platform consists of two liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods: a reversed-phase method for NRTIs using positive electrospray ionization (ESI) and an ion-pair LC–MS/MS method for the phosphorylated compounds using negative ESI. The methods use the same LC–MS system and column and changing from one method to the other only includes changing the mobile phase. The methods were partially validated, focussing on sensitivity, accuracy and precision. Successful transfer of the methods to ultra performance liquid chromatography (UPLC) led to a significant improvement of speed for the analysis of NRTIs and sensitivity for both NRTIs and phosphorylated NRTIs. The latter was demonstrated by the improved separation by UHPLC of dGTP vs. AZT-TP and ATP which made direct analysis of dGTP possible using the optimal MS/MS transition thereby significantly improving the detection limit of dGTP. Typically LLOQs observed for both the NRTIs and phosphorylated NRTIs were 1 nM, while the mean accuracy varied between 82 and 120% and inter- and intra-assay precision was generally