Determination of γ-aminobutyric acid in serum by reverse-phase high performance liquid chromatography

Abstract

A new highly sensitive HPLC method for determination γ-aminobutyric acid (GABA) in serum was developed by precolumn derivatiza-tion of GABA with 9-fluorenylmethyl chlorormate (FMOC-Cl) using ε-amino-n-caproic acid as an internal standard. The column used was Shim-Pack CLC-ODS (M), 4.6 mm × 150 mm, 5 μm. Mobile phase A consisted of sodium acetate buffer (0.05 mol/L, pH3.65) /water/ tetranhydrofunan/glacial acetic acid (250/100/ 15/2.2). Mobile phase B was acetonitrile/ methanol (4/1). The conditions of derivatization and chromatography were studied to find a optimal procedure, which included pH value, reaction time, ionic strength and amount of derivatizing reagent. The coefficient of variance of the method was less than 4.6% for within runs, 6.1% for between runs, respectively. The minimal detection limit (S/N = 2) was 3.1 nmol/L. The linearity was observed from 10 to 1 000 nmol/L, and the coefficient of determination was 0.9992. The average recovery was 97.1%.