Determination of submicromolar concentrations of neurotransmitter amino acids by fluorescence detection using a modification of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate method for amino acid analysis

A sensitive method for quantitatively determining submicromolar levels of n eurotransmitter amino acids (e.g. Asp, Glu and gamma-aminobutyric acid) in microdialysates from brain and cerebrospinal fluids is reported. 6-Aminoqui nolyl-N-hydroxysuccinimidyl carbamate (AQC) was employed as the derivatizat ion reagent, followed by HPLC separation and fluorescence detection of the derivatives. The derivatization was conducted simply by mixing the AQC dire ctly with the microdialysis samples. The reaction was complete within secon ds after mixing at room temperature. Separation development optimizing the gradient profile, eluent pH and column temperature resulted in an excellent separation of the required amino acids in less than 30 min. Other resolved amino acids in the same profile include Gly, taurine, and Pro. Recoveries for the amino acids of interest spiked into high salt containing perfusion buffers were greater than 97%. The sensitivity of the method was increased by employing a 16-mu l flow cell in the detector and analyzing 20-mu l aliq uots of the derivatization mixtures. With the optimized conditions, the det ection limits were 3-7 nM (fmol/mu l). Typical reproducibility (%R.S.D.) fo r quantitation of these amino acids at submicromolar levels was approximate ly 2%. Excellent linearity (r(2)>0.999) was achieved over the range 0.2-20 mu M. The low detection limits permitted the analysis of a number of differ ent microdialysate samples including those from cerebrospinal fluid, as wel l as substantia nigra and hypothalamus from brain samples, even at basal le vels when gamma-aminobutyric acid concentration may be <50 nM. The excellen t sensitivity made it easy to distinguish basal from stimulated levels of n eurotransmitter amino acids, even from sample sizes as small as 10 mu l. (C ) 1998 Published by Elsevier Science B.V. All rights reserved.