4 years ago

Development and validation of a LC/MS-based method for the measurement of intracellular superoxide anion

Development and validation of a LC/MS-based method for the measurement of intracellular superoxide anion
Superoxide anion (O2 .-), as the first generated reactive oxygen species (ROS), has been considered to be highly deleterious to cell functions. The measurement of intracellular O2 .- level is of great importance to uncover its roles in a variety of oxidative damage diseases. Hydroethidium (HE) fluorescence-based method is dominating intracellular O2 .- assay by monitoring the unique product 2-OH-E+ of HE/O2 .- reaction. However, the avoid-less cross-interference of red fluorescence limited its ability to provide trustworthy information on intracellular O2 .- formation. By the detection of 2-OH-E+, we herein developed and validated an improved LC/MS-based method for the measurement of intracellular O2 .-. Firstly, we demonstrated the proportionality of HE/O2 .- reaction. Secondly, ungerimine was used as internal standard to eliminate daily basis and matrix effect in the LC/MS-based detection of 2-OH-E+. Afterward, the total protein concentration was utilized for cell number normalization. Accordingly, an equation was further proposed to calculate the relative abundance (RA) of intracellular O2 .-. Finally, the developed method has been successfully utilized to evaluate the inhibitory effects of natural compounds on O2 .- generation, the result of which was validated by the HE-based fluorescent measurement. Compared with the fluorescent measurement, the LC/MS-based intracellular O2 .- assay method is more sensitive, selective and accurate.

Publisher URL: www.sciencedirect.com/science

DOI: S0003267017312515

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