Analysis of Trinitrophenylated Adenosine and Inosine by Capillary Electrophoresis and gamma-Cyclodextrin-Enhanced Fluorescence Detection

作者：Stephen Terilyn K L; Guillemette Katherine L; Green Thomas K^*

来源：Analytical Chemistry, 2016, 88(15): 7777-7785.

DOI：10.1021/acs.analchem.6b01796

Monitoring molecules such as adenosine (Ado) and inosine (Ino) in the central nervous system has enabled the field of neuroscience to correlate molecular concentrations dynamics to neurological function, behavior, and disease. In vivo sampling techniques are commonly used to monitor these dynamics; however, many techniques are limited by the sensitivity and sample volume requirements of currently available detection methods. Here, we present a novel capillary electrophoresis-laser induced fluorescence detection (CE-LIF) method that analyzes Ado and Ino by derivatization with 2,4,6-trinitrobenzenesulfonic acid to form fluorescent trinitrophenylated complexes of Ado (TNP-Ado) and Ino (TNP-Ino). These complexes exhibit similar to 25-fold fluorescence enhancement upon the formation of inclusion complexes with gamma-cyclodextrin (gamma-CD). Association constants were determined as 4600 M-1 for Ado and 1000 M-1 for Ino by CE-LIF. The structure of the TNP-Ado:gamma-CD complex was determined by 2D nuclear magnetic resonance (NMR) spectroscopy. Optimal trinitrophenylation reaction conditions and CE-LIF parameters were determined and resulted in the limit of detection of 1.6 mu M for Ado and 4 mu M for Ino. Ado and Ino were simultaneously quantified in homogenized rat forebrain samples to illustrate application of the technique. Simulated biological samples, desalted by ultrafiltration in the presence gamma-CD, were concentrated on-capillary by large-volume sample stacking (LVSS) to achieve detection limits of 32 and 38 nM for TNP-Ado and TNP-Ino, respectively.

• 出版日期2016-8-2