Trace-level amino acid analysis by capillary liquid chromatography and application to in vivo microdialysis sampling with 10-s temporal resolution

A sensitive method was developed to determine 16 amino acids, including all the neurotransmitter amino acids and neuromodulators, in physiological sam ples. Samples were derivatized with o-phthalaldehyde/tert-butyl thiol follo wed by two scavenging reactions that reduced the chemical background caused by excess derivatization reagent by similar to 90%. A total of 250 nL of t he derivatized sample was injected and concentrated onto a 50-mu m-inner di ameter capillary column packed with 5-mu m reversed-phase particles and sep arated using gradient elution. Analytes were detected amperometrically at a cylindrical 9-mu m carbon fiber microelectrode. The combination of on-colu mn concentration, scavenging reactions after derivatization, high sensitivi ty 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 fro m probes implanted in the striatum of anesthetized rats. Probes were perfus ed at 1.2 mu L/min and fractions collected every 10 s. The 200-nL fractions were diluted to 2 mu L to facilitate sample handling for offline analysis. The suitability of this method for simultaneous monitoring of all the majo r amino acid neurotransmitters with 10-s temporal resolution under basal co nditions, during potassium stimulation, and during selective uptake inhibit ion of gamma-aminobutyric acid is demonstrated.