Simulation of oxidative stress of guanosine and 8-oxo-7,8-dihydroguanosine by electrochemically assisted injection–capillary electrophoresis–mass spectrometry
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Oxidative stress plays a crucial role in DNA and RNA damage within biological cells. As a consequence, mutations of DNA can occur, leading to disorders like cancer and neurodegenerative and cardiovascular diseases. The oxidative attack of guanosine and 8-oxo-7,8-dihydroguanosine is simulated by electrochemistry coupled to capillary electrophoresis–mass spectrometry. The electrochemical conversion of the compound of interest is implemented in the injection protocol termed electrochemically assisted injection (EAI). In this way, oxidation products of guanosine can be generated electrochemically, separated by capillary electrophoresis, and detected by electrospray ionization time-of-flight mass spectrometry (EAI–CE–MS). A fully automated laboratory-made EAI cell with an integrated buffer reservoir and a compartment holding screen-printed electrodes is used for the injection. In this study, parameters like pH of the sample solution and the redox potential applied during the injection were investigated in terms of corresponding formation of well-known markers of DNA damage. The important product species, 8-oxo-7,8-dihydroguanosine, was investigated in a separate study to distinguish between primary and secondary oxidation products. A comparison of product species formed under alkaline, neutral, and acidic conditions is presented. To compare real biological systems with an analytical approach for simulation of oxidative stress, it is desirable to have a well-defined control over the redox potential and to use solutions, which are close to physiological conditions. In contrast to typical HPLC–MS protocols, the hyphenation of EAI, CE, and MS enables the generation and separation of species involved without the use of organic solvents. Thus, information of the electrochemical behavior of the nucleoside guanosine as well as the primary oxidation product 8-oxo-7,8-dihydroguanosine can be characterized under conditions close to the physiological situation. In addition, the migration behavior found in CE separations of product species can be used to identify compounds if several possible species have the same mass-to-charge values determined by MS detection.
KeywordsNucleoside oxidation Electrochemistry Capillary electrophoresis Mass spectrometry Electrochemically assisted injection Guanosine Oxidative stress
Electrochemically assisted injection
Electrochemistry–μ-liquid chromatography–mass spectrometry
Electrospray ionization time-of-flight mass spectrometry
Liquid chromatography–mass spectrometry
Quadrupole time-of-flight mass spectrometry
Reactive oxygen species
Screen-printed carbon electrode
This research was supported by the Research Executive Agency (REA) of the European Union under grant agreement number PITN-GA-2010-264772 (ITN CHEBANA). We thank J. Kiermaier for the measurements concerning high-resolution mass spectrometry.
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