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

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