QUANTITATION OF PROTEIN-BOUND 3-NITROTYROSINE AND 3,4-DIHYDROXYPHENYLALANINE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH ELECTROCHEMICALARRAY DETECTION

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in myriad disease etiologies and may represent an obl igate pathologic sequelus of inflammation. Unfortunately, few sensitiv e and specific analytical techniques exist for the routine assay of bi omarkers indicative of ROS and RNS elaboration. In this study, high-pe rformance liquid chromatography is used in conjunction with coulometri c electrochemical array (HPLC-EC) detection to allow ultrasensitive de termination of protein-bound 3-nitrotyrosine and 3,4-dihydroxyphenylal anine (3-hydroxytyrosine) as specific in situ biomarkers of protein ex posure to reactive nitrating and oxidizing species. Tyrosine and deriv atives can be analyzed simultaneously with practical detection limits for tyrosine, 3-NT, and 3,4-Dopa being 10, 50, and 2 pmol, respectivel y, in as little as 20 mu L of sample. HPLC-EC array detection allows t wo-dimensional resolution of chromatograms, greatly facilitating peak detection and confidence assignment. A method of sample preparation wh erein tyrosine analogs are enzymatically hydrolyzed from protein witho ut the need for sample extraction, concentration, or derivatization is reported. (C) 1997 Academic Press.