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

Reactive oxygen species (ROS) have been identified as important chemical me diators that regulate signal transduction pathways. Today, the importance o f ROS in pathological events has become increasingly apparent. To monitor t he ROS in biological systems, various techniques such as cytochrome c perox idase assay and malonaldialdehyde detection were developed and used clinica lly. However, these methods are very laborious and time-consuming. Therefor e, in this study, we developed a capillary electrophoresis (CE)-based ROS m onitoring technique using dihydrorhodamine 123 (DHR-123). DHR-123 is a nonf luorescent compound, and can be oxidized irreversibly and rapidly by ROS to rhodamine 123 (Rho-123), which is a fluorescent dye. Upon detecting the am ount of the fluorescence from Rho-123, we could indirectly, quantify the am ount of ROS generation in biological systems. Determination of Rho-123 was performed in an uncoated silica capillary (27 cm x 75 mum i.d.) using a CE system with laser-induced fluorescence detection. Optimum conditions for th e Rho-123 detection were 50 mM borate buffer (pH 10.0), running at 20 degre esC, 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 c ells. (C) 2001 John Wiley & Sons, Inc.