Trace-Level Amino Acid Analysis by Capillary Liquid Chromatography and Application to in Vivo Microdialysis Sampling with 10-s Temporal Resolution

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Abstract
A sensitive method was developed to determine 16 amino acids, including all the neurotransmitter amino acids and neuromodulators, in physiological samples. Samples were derivatized with o-phthalaldehyde/tert-butyl thiol followed by two scavenging reactions that reduced the chemical background caused by excess derivatization reagent by ∼90%. A total of 250 nL of the derivatized sample was injected and concentrated onto a 50-μm-inner diameter capillary column packed with 5-μm reversed-phase particles and separated using gradient elution. Analytes were detected amperometrically at a cylindrical 9-μm carbon fiber microelectrode. The combination of on-column concentration, scavenging reactions after derivatization, high sensitivity electrochemical detection, and protocols to minimize amine contamination allowed detection limits of 90−350 pM (20−80 amol) for all the amino acids tested. This method was used to analyze in vivo microdialysate samples from probes implanted in the striatum of anesthetized rats. Probes were perfused at 1.2 μL/min and fractions collected every 10 s. The 200-nL fractions were diluted to 2 μL to facilitate sample handling for off-line analysis. The suitability of this method for simultaneous monitoring of all the major amino acid neurotransmitters with 10-s temporal resolution under basal conditions, during potassium stimulation, and during selective uptake inhibition of γ-aminobutyric acid is demonstrated.

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