Development for the analysis of reactive oxygen species using capillary electrophoresis with laser-induced fluorescence detection

Abstract

Reactive oxygen species (ROS) have been identified as important chemical mediators that regulate signal transduction pathways. Today, the importance of ROS in pathological events has become increasingly apparent. To monitor the ROS in biological systems, various techniques such as cytochrome c peroxidase assay and malonaldialdehyde detection were developed and used clinically. However, these methods are very laborious and time-consuming. Therefore, in this study, we developed a capillary electrophoresis (CE)-based ROS monitoring technique using dihydrorhodamine 123 (DHR-123). DHR-123 is a nonfluorescent compound, and can be oxidized irreversibly and rapidly by ROS to rhodamine 123 (Rho-123), which is a fluorescent dye. Upon detecting the amount of the fluorescence from Rho-123, we could indirectly quantify the amount of ROS generation in biological systems. Determination of Rho-123 was performed in an uncoated silica capillary (27 cm×75 μm i.d.) using a CE system with laser-induced fluorescence detection. Optimum conditions for the Rho-123 detection were 50 mM borate buffer (pH 10.0), running at 20°C, and 5 s injection. This method allowed the detection of Rho-123 with a detection limit around 50 pM. We also applied this developed method to the analysis of intracellular ROS level in methamphetamine (MA) treated PC12 cells. © 2001 John Wiley & Sons, Inc. J Micro Sep 13: 327–331, 2001