Catecholamines derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole: characterization of chemical structure and fluorescence properties

4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was evaluated as a fluorogenic derivatization reagent for the analysis of the catecholamines, dopamine, epinephrine, norepinephrine, and their naturally occurring metabolites, metanephrine and normetanephrine, homovanillic acid, 3,4-dihydroxyphenyl acetic acid. These compounds reacted rapidly with NBD-F under mild conditions to form stable derivatives. The optimal reaction conditions were found to be 12.5 mM borate buffer pH 8.0 in water:acetonitrile (1:1) at 50 deg. C for 5 min. New NBD derivatives of all the catecholamines and metabolites were prepared and purified and were shown by electrospray mass spectrometry to be fully reacted at all available catechol and amine sites, resulting in di- or tri-substituted derivatives. Homovanillic acid and 3,4-dihydroxyphenyl acetic acid reacted with NBD-F but gave non-fluorescent derivatives. The fluorescence excitation wavelength maximum demonstrated a red shift for the derivatives with increasing polarity of the solvent and the fluorescence intensity increased linearly with increasing organic ratio in the solvent-aqueous buffer complex. The presence of electrolyte in the solvent and the electrolyte concentration in the solvent-electrolyte complex had little effect on the fluorescent intensity. The fluorescence quantum yields in acetonitrile were also obtained. The separation behavior of the NBD-catecholamines was determined by high-performance liquid chromatography (HPLC). The studies demonstrated good potential for the application of NBD-F derivatization to the quantitative analysis of catecholamines and related compounds in biological matrices