Triple-column ion-exchange physiological amino acid analysis with fluorescent detection: baseline characterization of...

A highly resolving triple-column amino acid analyzer with fluorometric detection is described. The reliability of this technique was evaluated and it was used in a baseline investigation of amino acids and related compounds in human cerebrospinal fluid (CSF). The procedure employs three distinct ion-exchange columns to elute the acidic, neutral, and basic amino acids, respectively. Each column is run isocratically with lithium citrate buffers designed to provide overlapping elution profiles. Studies using CSF collected under strictly controlled conditions documented nanomolar concentrations of aspartate, gamma-aminobutyric acid (GABA), beta-alanine, 1-methylhistidine, and 3-methylhistidine, as well as low levels of glutamate, methyllysine, and ammonia. In addition, other common amino acids were also quantified. Chromatograms of CSF from all three systems (acidic, neutral, and basic) exhibited numerous uncharacterized compounds emphasizing the resolution and sensitivity of the analytical procedure. In vitro stability studies revealed that levels of aspartate, glutamate, GABA, homocarnosine, and ammonia are subject to significant change when CSF is maintained at room temperature for various periods of time up to 24 h. It is concluded that the valid and accurate measurement of CSF amino compounds, especially the neurotransmitter amino acids, requires a highly specific and sensitive assay procedure as well as strict control of CSF manipulation in vitro.