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

Abstract

A sensitive method for quantitatively determining submicromolar levels of neurotransmitter amino acids (e.g. Asp, Glu and γ-aminobutyric acid) in microdialysates from brain and cerebrospinal fluids is reported. 6-Aminoquinolyl- N-hydroxysuccinimidyl carbamate (AQC) was employed as the derivatization reagent, followed by HPLC separation and fluorescence detection of the derivatives. The derivatization was conducted simply by mixing the AQC directly with the microdialysis samples. The reaction was complete within seconds 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-μl flow cell in the detector and analyzing 20-μl aliquots of the derivatization mixtures. With the optimized conditions, the detection limits were 3–7 n M (fmol/μl). Typical reproducibility (%R.S.D.) for quantitation of these amino acids at submicromolar levels was approximately 2%. Excellent linearity ( r 2>0.999) was achieved over the range 0.2–20 μ M. The low detection limits permitted the analysis of a number of different microdialysate samples including those from cerebrospinal fluid, as well as substantia nigra and hypothalamus from brain samples, even at basal levels where γ-aminobutyric acid concentration may be <50 n M. The excellent sensitivity made it easy to distinguish basal from stimulated levels of neurotransmitter amino acids, even from sample sizes as small as 10 μl.