Abstract:

An improved high performance liquid chromatography (HPLC) method for the determination of γ-aminobutyric
acid (GABA) and L-glutamic acid (L-Glu) in microbial fermentation broth was developed. Amino acids in samples were
derivatized with 4 mmol/L 9-fluorenylmethyl chloroformate (FMOC-Cl) after being pretreated with 10% trichloroacetic
acid. The chromatographic separation was performed on a Phenomenex C18 column (4.6 mm × 250 mm, 5 μm) using A
[50 mmol/L sodium acetate (pH 4.8):methanol:acetonitrile:diethylene oxide = 82:8.5:8.5:1, V/V] and B [50 mmol/L sodium
acetate (pH 4.8):methanol:acetonitrile:diethylene oxide = 22:38.5:38.5:1, V/V] (30:70, V/V) as mobile phase at a flow rate of
1.0 mL/min. The chromatogram was detected at a wavelength of 265 nm with the column temperature being maintained at
40 ℃. This method was proved to be of good stability, high sensitivity, good repeatability and short determination period.
The linear ranges for the analysis of γ-aminobutyric acid and L-glutamic acid were 20?400 μg/mL. Compared with those
from the common derivatizing agent, o-phthalaldehyde (OPA), the derivatives in the present method, without gradient
elution, were more stable.

Key words: high performance liquid chromatography (HPLC), microbial fermentation broth, γ-aminobutyric acid (GABA), L-glutamic acid (L-Glu), 9-fluorenylmethyl chloroformate (FMOC-Cl)