|Huo, Cheng-Jun - UNIVERSITY OF KENTUCKY|
|Dougherty, Charles - UNIVERSITY OF KENTUCKY|
Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 2, 2008
Publication Date: June 18, 2008
Citation: Kagan, I., Coe, B.L., Smith, L.L., Huo, C., Dougherty, C.T., Strickland, J.R. 2008. A validated method for gas chromatographic analysis of gamma-aminobutyric acid in tall fescue herbage. Journal of Agricultural and Food Chemistry. 56:5538-5543. Interpretive Summary: Gamma-aminobutyric acid (GABA) is an amino acid that is not incorporated into proteins but has been isolated from bacterial cultures and many plant and animal tissues. GABA is a neurotransmitter in animals and has been associated with stress responses in plants. A method for quantifying GABA in forages was developed, with the long-term goals of (1) understanding the role of GABA in forage plants and (2) evaluating the relationship between GABA content of tall fescue and symptoms of tall fescue toxicosis in grazing animals. GABA in flash-frozen, lyophilized herbage was quantified by gas chromatography (GC) coupled to flame ionization detection (FID). Herbage samples were extracted with 80% ethanol/20% water, and amino acids in the extract were chemically modified (derivatized) to make them volatile, a requirement for GC analysis. The derivatization and GC method, including the column for separation, were initially tried with a commercial kit intended for derivatizing and separating amino acids in serum. In its original form, the kit was not fully satisfactory for analyzing GABA in herbage extracts. Consequently, the derivatization procedure was modified, and the column and separation parameters were changed. This adapted method was validated by verifying the identity of the putative GABA peak, evaluating the extent of matrix effects (interference of sample extract with quantification), determining the limits of GABA detection and quantitation, and determining percent recovery in tall fescue extracts. The method is rapid, easy to use, and generates little solvent waste.
Technical Abstract: Gamma-Aminobutyric acid (GABA) is an inhibitory neurotransmitter in animals that is also found in plants and has been associated with plant responses to stress. A simple and relatively rapid method of GABA separation and quantification was developed from a commercially available kit for serum amino acids (Phenomenex EZ:faast) and validated for tall fescue (Festuca arundinacea). Extraction in ethanol-water (80:20, v/v) at room temperature yielded detectable amounts of GABA. Clean separation from plant components in 28 min was achieved by gas chromatography (GC) with flame ionization detection (FID), using a 30-meter, 5% phenyl/95% dimethylpolysiloxane column. The identity of the putative GABA peak was confirmed by GC with mass spectrometric detection (MS). The relatively small effects of sample matrix on GABA measurement were verified by demonstrating slope parallelism of GABA curves prepared in the presence and absence of fescue extracts. Limits of quantification and detection were 2.00 and 1.00 nmol per 100 µL, respectively. Average method recovery and interassay coefficient of variation were 93.4 and 6.4 %, respectively.