DETERMINATION OF GLUTAMATE AND ASPARTATE IN MICRODIALYSIS SAMPLES BY REVERSED-PHASE COLUMN LIQUID-CHROMATOGRAPHY WITH FLUORESCENCE AND ELECTROCHEMICAL DETECTION

Five different systems for fast determination of aspartate and glutama te in microdialysis samples are described: (I) a high-speed HPLC using a gradient pump with a sharp elution profile, (II) a column switching technique, (III) an isocratic pump with a low-pressure switching valv e for one-step gradients, (IV) microbore chromatography using injectio ns of acetonitrile as a wash-out step, (V) on-line connection of micro dialysis and HPLC/derivatization. In all cases, automated precolumn de rivatization with o-phthalaldehyde-2-mercaptoethanol reagent were used . Both fluorescence and electrochemical detection techniques were eval uated in terms of reproducibility, sensitivity, interference, maintena nce and troubleshooting. The electrochemical detection method required a second derivatization step with 0.2 M iodoacetamide to remove exces s of a thiol moiety and regular recalibrations after each six to ten i njections. Under these conditions the correlation coefficients for ele ctrochemical vs. fluorescence detectors were 0.918 for Asp and 0.988 f or Glu for 65 microdialysis samples. Coefficients of variation for six analyses between calibrations were below 3% for both detectors. The l imits of detection for both amino acids were about 0.4 pmol for electr ochemical detection with a thiol scavenger step, 50 fmol far fluoresce nce detection using conventional columns and about 20-30 fmol for the microbore system. AU systems are suitable for detecting basal levels o f Asp and Glu in 5-10 mu l microdialysis samples from a rat brain wher e typical concentrations lie around 1-10 pmol or more. It is concluded that a microbore setup with one isocratic pump and an autosampler opt imized for injections of washing solvent between samples is the most p ractical and economical. The system allows analysis of minute sample v olumes down to 1-2 mu l. (C) 1998 Elsevier Science B.V.