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
Tp. Piepponen et A. Skujins, 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, J CHROMAT B, 757(2), 2001, pp. 277-283
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.