[Determination of gamma-aminobutyric acid and glutamic acid in human cerebrospinal fluid by high performance liquid chromatography].

Gamma-aminobutyric acid (Gaba) and glutamic acid (Glu) are believed to be the major neurotransmitter. Levels in cerebrospinal fluid (CSF) may reflect their metabolism in various neurotic and psychiatric diseases. Measurements of Gaba and Glu in body fluids will help to elucidate their metabolic role and diagnostic value. In the present work, the concentrations of glutamic acid and gamma-aminobutyric acid in human cerebrospinal fluid were determined by reversed-phase high performance liquid chromatography using pre-column derivatized with dansyl chloride and UV detection at 254 nm. The mobile phase was A:CH3OH and B: THF/CH3OH/0.05 mol/L NaAc (pH 6.2) (5/75/420, V/V) with gradient elution. The flow rate was 1 mL/min. CSF samples were deproteinizated with methanol. After centrifugation at 15000 r/min for 10 min, the supernatant was introduced into a screw-capped vial and evaporated to near dryness at 80 degrees C. Derivatization was carried out by the addition of 250 microL of bicarbonate solution (pH 9.8) and 250 microL of dansyl chloride solution (4 g/L) followed by heating at 40 degrees C water bath for 30 min. Extraction was performed with 300 microL of ethyl acetate and the organic layer separated was dried at room temperature under nitrogen. The dry residue was dissolved and injected into the HPLC system. The linear range of the method was 5-1000 micromol/L (r = 0.9988) for Glu and 1-600 micromol/L (r = 0.9984) for Gaba. The lower limit of detection were 0.002 micromol/L and 0.001 micromol/L respectively. The recoveries were 96.9%-98.8% and 97.5%-98.8% and the coefficients of variation of peak height measurements were 2.2%-3.4% and 3.8%-5.6% respectivery. The method enables a simple, rapid and reproducible quantification of Glu and Gaba neurotransmitter.