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

Superoxide anion (O), as the first generated reactive oxygen species (ROS), has been considered to be highly deleterious to cell functions. The measurement of intracellular O level is of great importance to uncover its roles in a variety of oxidative damage diseases. Hydroethidium (HE) fluorescence-based method is dominating intracellular O assay by monitoring the unique product 2-OH-E of HE/O reaction. However, the avoid-less cross-interference of red fluorescence limited its ability to provide trustworthy information on intracellular O formation. By the detection of 2-OH-E, we herein developed and validated an improved LC/MS-based method for the measurement of intracellular O. Firstly, we demonstrated the proportionality of HE/O 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 O. Finally, the developed method has been successfully utilized to evaluate the inhibitory effects of natural compounds on O generation, the result of which was validated by the HE-based fluorescent measurement. Compared with the fluorescent measurement, the LC/MS-based intracellular O assay method is more sensitive, selective and accurate.