Automated precolumn fluorescence labelling by carbodiimide activation of N-acetylaspartate and N-acetylaspartylglutamate applied to an HPLC brain tissue analysis

Abstract

An automated method is described to couple carboxyl-containing metabolites to the fluorophore 2-aminoanthracene in aqueous solution (containing 75% methanol) in the presence of N,N-dicyclohexylcarbodiimide. The reaction was optimized for N-acetylaspartate ( N-Ac-Asp) and N-acetylaspartylglutamate ( N-Ac-Asp-Glu). The reactions occurred within 5 min at room temperature in the presence of 0.5–2 m m HCl. At concentrations of electrolytes exceeding 10 m m the coupling reaction became suboptimal. Derivatization was performed in a commercial precolumn derivatization unit. Additional tubing was needed to provide the reagents prior to reversed-phase HPLC and fluorescence detection. The assay is linear over at least three orders of magnitude; as little as 1 pmol could reproducibly be assayed in 100 μg wet weight brain tissue extracted with a mixture of methanol and 4 m m HCL (9:1, v/v). N-Ac-Asp and N-Ac-Asp-Glu levels in several brain regions and spinal cord were similar to those so far reported. The compounds could not be detected in peripheral tissue. The advantages, prospects and limitations of the present approach over existing methods to estimate water-soluble carboxylic acids is discussed.