HPLC法定量分析微生物法制备液中产物γ-氨基丁酸和底物L-谷氨酸

建立一种测定微生物法制备液中γ-氨基丁酸(γ-aminobutyric acid,GABA)和L-谷氨酸(L-glutamic,L-Glu)的高效液相色谱法。样品经10%三氯乙酸溶液预处理后,用4 mmol/L氯甲酰芴甲酯进行柱前衍生。采用Phenomenex C18色谱柱(4.6 mm×250 mm,5μm),以A50 mmol/L乙酸钠溶液(p H 4.8)-甲醇-乙腈-四氢呋喃(82∶8.5∶8.5∶1,V/V)]∶B50 mmol/L乙酸钠溶液(p H 4.8)-甲醇-乙腈-四氢呋喃(22∶38.5∶38.5∶1,V/V)]=30∶70为流动相,流速1.0 m L/min,柱温40℃洗脱,检测波长265 nm。该方法稳定、灵敏、测定周期短、重复性好。在GABA和L-Glu质量浓度为20~400μg/m L范围内有良好的线性关系。与常用衍生剂邻苯二甲醛相比,衍生产物的稳定性更好,且无需梯度洗脱。

Quantitative Analysis of γ-Aminobutyric Acid and L-Glutamic Acid in Microbial Fermentation Broth by HPLC

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.