| Adenosine (Ado) and adenine ribonucleotides are essential in cell metabolism and energy production, cellular signaling, and DNA and RNA synthesis. The biosynthesis of these molecules takes place in both the intracellular and extracellular space via transphosphorylation reactions catalyzed by several distinct kinase enzymes like adenylate kinase. Several analytical detection methodologies have been developed to monitor these molecules in biological tissue, including both liquid chromatography (LC) and capillary electrophoresis (CE) techniques. However, many of these methodologies are limited by separation resolution and sample injection volume requirements. This thesis presents a novel capillary electrophoresis-laser induced fluorescence detection (CE-LIF) method with high separation power to analyze Ado and Inosine (Ino), a metabolite of Ado, by derivatization with 2,4,6-trinitrobenzenesulfonic acid to form fluorescent trinitrophenylated complexes of Ado (TNP-Ado) and Ino (TNP-Ino). The development and validation of the CE-LIF method, optimization of the trinitrophenylation reaction, and fluorescence enhancement of TNP-Ado and TNP-Ino with gamma-cyclodextrin will be discussed. Detection limits were 1.6 muM for Ado and 4 muM for Ino in rat brain tissue. Large-volume sample stacking (LVSS) was employed to further enhance the sensitivity of the CE-LIF method, with detections limits of 310 nM and 159 nm for Ado and Ino, respectively. The CE-LIF method offers promise for the analysis of Ado, Ino and potentially other adenine ribonucleotides in small volume generating biological experiments like in vivo microdialysis and single cell metabolomics. |
