Optimization of dansyl derivatization and chromatographic conditions in the determination of neuroactive amino acids of biological samples.
BACKGROUND: Amino acid neurotransmitters represent a major class of compounds that are involved in neuronal communication at CNS synapses. METHODS: Twelve amino acids were separated after precolumn derivatization with dansyl chloride. The biologically important amino acids, taurine, aspartate, glutamate, glycine, alanine and gamma-aminobutyric acid were determined in rat brain tissue and rabbit plasma. RESULTS: The major modifications to previous methods included isocratic elution instead of gradient elution to reduce time and cost of serial analyses; optimization of mobile phase to improve the separation of free dansyl with derivatives, so as to elide complex steps for the clean of the chromatogram; and selection of an internal standard (Trazodone) to improve the reproducibility and the reliability of procedures. Twelve amino acids were assayed within 35 min. Other alpha-amino acids that are relevant to the make-up of mammalian proteins did not interfere with the determination. The standard curve, recovery, analytical precision and detection limits for each neuroactive amino acid were determined. CONCLUSION: This assay separated 12 amino acids in a single run. Six neuroactive amino acids were also simultaneous measured by isocratic HPLC with UV detection. The method is applicable in determination of Tau, Glu, Asp, Gly, Ala and GABA in biological samples.