Aromatic nitro compounds including nitrazepam, chloramphenicol, and p-nitrophenyl phosphate are often found in pharmaceuticals and as common reagents 1) for the assay of enzymes such as phosphatase and peptidase. The nitration of tyrosine residues in proteins has been recognized as an important process for regulating pathological conditions of various diseases induced by oxidative stress.2) Thus, general and sensitive methods for the determination of aromatic nitro compounds in biological fluids are needed.Spectrophotometric methods are currently used for the analysis of aromatic nitro compounds. As an alternative, cathodic detection can be considered, since aromatic nitro groups are known to undergo electrochemical reduction at a relatively positive potential. [3][4][5] Yet, few studies on the reductive detection of aromatic nitro compounds are to be found in the literature. [6][7][8] This is mainly because deoxygenation of the medium must be carried out for electrochemical detection in a cathodic mode.3,9-12) Otherwise, dissolved oxygen, whose potential for reduction is quite low at common electrodes such as carbons, exhibits a cathodic response, preventing the sensitive and reproducible determination of analytes. Notably in high performance liquid chromatography with an electrochemical detector (ECD/HPLC) in the cathodic mode, a rather tedious procedure must be applied continuously for a mobile phase. However, a cathodic ECD/HPLC system has the following advantages over other HPLC systems: (1) in general, downsizing of an electrochemical HPLC system is more feasible than that of a spectrophotometric one; (2) cathodically active compounds are rare in biological fluids, so there are fewer problems from interference or separation in comparison to anodic ECD/HPLC. To develop a useful bioanalytical method based on these advantages, especially the second, we tested a cathodic ECD/HPLC system as a sensitive and precise tool for the determination of p-nitrophenol (NP). Recently, ECD/HPLC systems with a dual mode have been developed for the determination of a nitrophenol analogue, nitrotyrosine. [13][14][15] The detection of nitrotyrosine using such systems comprises two electrochemical processes for transformation and detection as follows: cathodic reduction to the corresponding aminophenol at an upstream electrode and oxidation of the product at a downstream (detection) electrode. Although dual ECD/HPLC was demonstrated to provide a highly sensitive tool for the analysis of nitrotyrosine, a sophisticated electrochemical device is required and anodic oxidation-based detection will encounter problems with separation since biological fluids contain a large number of anodically active compounds. The purpose of this study is to take full advantage of the potential utility of cathodic detection, to develop a simple and general ECD/HPLC system for NP in biological samples. Herein, we report that utilizing not only a commercially available helium-purging device but also PEEK tubes for all tubing makes the ECD/HPLC system a ...