PHOTOCHEMICAL CHARACTERIZATION AND OPTIMIZATION OF ARGON ION LASER-INDUCED FLUORESCENCE DETECTION OF O-PHTHALALDEHYDE BETA-MERCAPTOETHANOL-LABELED AMINO-ACIDS AND GAMMA-GLUTAMYL PEPTIDES IN LIQUID-CHROMATOGRAPHY - ULTRATRACE ANALYSIS WITH NEUROBIOLOGICAL SAMPLES

Optimization of argon ion laser-induced (UV multiline) fluorescence detection in liquid chromatography (1 mm i.d. columns) was performed aiming for ultratrace determination of o-phthalaldehyde/beta-mercaptoethanol-labeled neuroactive aminoacids and peptides. The derivatives are shown to undergo facile photochemical decomposition with photodestruction quantum yields, Phi(D), in the range 0.009-0.14. In agreement with theoretical predictions, the optimal signal-to-noise (S/N) ratio was obtained at laser irradiances much lower than are required for prompt fluorescence saturation, Nd/YAG laser flash photolysis experiments yielded a broad absorption band, between <550 and >800 mn, characteristic for solvated electrons. Transient absorptions at shorter wavelengths, also observed, were presumably due to a cation radical species. Based on the differences noted in the photochemical stability of certain derivatives, a new method for peak characterization and enhanced detection selectivity in trace analysis is proposed. The concentration limit of detection was in the range (35-380) x 10(-12) M (S/N = 3, 10 mu L injection volume). Picomolar concentrations of amino acids, gamma-glutamyl, and beta-aspartyl peptides could be determined in brain microdialysates sampled in vivo. Glutathione could be detected in single mammalian neurons, and in a new approach based on fluorophore decay kinetics for peak characterization, gamma-glutamylcysteine could be identified in rat cerebral cortex extracts.