Highly selective fluorimetric determination of acidic amino acids by high-performance liquid chromatography following intramolecular excimer-forming derivatization with a pyrene-labeling reagent

Abstract

A highly selective fluorimetric determination method for acidic amino acids, glutamic acid, aspartic acid and N-methyl-D-aspartic acid (NMDA), has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid hydrazide (PBH), followed by reversed-phase high-performance liquid chromatography (HPLC). Aspartic acid and NMDA, containing two carboxyl moieties in a molecule, were converted to the corresponding dipyrene-labeled derivatives by a reaction with PBH in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and pyridine. Glutamic acid was PBH-derivatized after the amine-protection with orthophthalaldehyde. These derivatives afforded intramolecular excimer fluorescence (440-540 nm), which could clearly be discriminated from the normal fluorescence (360-420 nm) emitted from PBH and monopyrene-labeled derivatives of monocarboxylic acids. The PBH derivatives of acidic amino acids could be separated by reversed-phase HPLC on an ODS column with isocratic elution using aqueous 67%(v/v) acetonitrile as the mobile phase. The detection limits (signal-to-noise ratio = 3) for glutamic acid, aspartic acid and NMDA were 21, 32 and 460 fmol, respectively, for a 20 μl injection.