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 glutamate, 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 established with a non-porous ODS-II HPLC column, late-eluting substances were washed out with a one-step low-pressure gradient. Concentrations of the amino acids were determined with a fixed-wavelength fluorescence detector. The detection limit for GABA was 80 fmol in a 15 microl sample, detection limits for glutamate, glycine and taurine were not determined because their concentrations in striatal perfusates were far above their detection limits. Total analysis time was less than 12 min, including the wash-out step. The methods described are relatively simple, sensitive, inexpensive, and fast enough to keep up with the microdialysis sampling.
Piepponen
T P
TP
Department of Pharmacy, University of Helsinki, Finland. petteri.piepponen@helsinki.fi
Skujins
A
A
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005973:Glutamine / Q000032:analysis*; D005998:Glycine / Q000032:analysis*; D007202:Indicators and Reagents / Q000737:chemistry; D008623:Mercaptoethanol / Q000737:chemistry; D017551:Microdialysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013654:Taurine / Q000032:analysis*; D005680:gamma-Aminobutyric Acid / Q000032:analysis*; D009764:o-Phthalaldehyde / Q000737:chemistry
D007202:Indicators and Reagents; D005973:Glutamine; D013654:Taurine; D005680:gamma-Aminobutyric Acid; D008623:Mercaptoethanol; D009764:o-Phthalaldehyde; D005998:Glycine