Rapid and sensitive step gradient assays of glutamate, glycine, taurine and gamma-aminobutyric acid by high-performance liquid chromatography-fluorescence detection with o-phthalaldehyde-mercaptoethanol derivatization with an emphasis on microdialysis samples

We developed a rapid step-gradient HPLC method for determination of glutama te, glycine and taurine, and a separate method for determination of gamma - aminobutyric acid (GABA) in striatal microdialysates. The amino acids were pre-column derivatized with o-phthalaldehyde-2-mercaptoethanol by using an automated refrigerated autoinjector. Separation of the amino acids was esta blished with a non-porous ODS-II HPLC column, late-eluting substances were washed out with a one-step low-pressure gradient. Concentrations of the ami no acids were determined with a fixed-wavelength fluorescence detector. The detection limit for GABA was 80 fmol in a 15 mul sample. detection limits for glutamate, glycine and taurine were not determined because their concen trations in striatal perfusates were far above their detection limits. Tota l analysis time was less than 12 min, including the wash-out step. The meth ods described are relatively simple, sensitive, inexpensive, and fast enoug h to keep up with the microdialysis sampling. (C) 2001 Elsevier Science B.V . All rights reserved.