Monitoring of amino acids in plasma by LC with fluorescence detection

Amino acids are a very interesting class of biologically active compounds, being the principal component of proteins and precursors of important neurotransmitters and neuromodulators, such as catecholamines (phenylalanine and tyrosine), histamine (histidine), nitric oxide (arginine), and serotonin (tryptophan). Plasma amino acid concentration may be a regulating factor of neurotransmitter biosynthesis in the brain. As cognitive disorders increase exponentially with age, few data are available about plasma amino acid concentrations in elderly persons in relation to their cognitive status. Various dietary supplements containing amino acids are proposed to influence ageing, memory loss and cognitive function, despite sparse scientific data for the efficiency of these supplements. Plasma analysis is hampered by the presence of a variety of amino acids at considerable different concentrations and their direct determination is difficult for the lack of significant chromophore and fluorophore groups. The reversed-phase liquid chromatography (RP-LC) combined with pre-column derivatization is a reliable way to overcome the problem. The purpose of the present work was to optimize and apply an HPLC method, which involves o-phthaldialdehyde (OPA) as fluorogenic derivatization reagent, to the selective and sensitive determination of amino acids in plasma of elders before and after oral treatment with poly amino acid formulations. Blood samples were subjected to centrifugation and the obtained plasma was deproteinized with 5-sulfosalicylic acid. The derivatization reaction was carried out at room temperature for 1 min in presence of borate buffer (pH 9.5; 0.4 M). The RP-HPLC separations with fluorescence detection were performed on a Phenomenex Gemini 5mm C18 (250 x 3 mm i.d.) column using two different mobile phases under gradient elution conditions. The combination of both chromatographic methods, employing mobile phases at different pH, provided a system with orthogonal selectivity, which allows a sure identification of a variety of amino acids in a complex matrix as plasma. Studies are in progress to extend the application to a large number of samples in order to evaluate both the correlation of plasma amino acid profile with the cognitive status and the effect of dietary supplement administration.