Analysis of amino acids without derivatization in barley extracts by LC-MS-MS

A method has been developed for quantification of 20 amino acids as well as 13 N-15-labeled amino acids in barley plants. The amino acids were extracted from plant tissues using aqueous HCl-ethanol and directly analyzed without further purification. Analysis of the underivatized amino acids was performed by liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS-MS) in the positive ESI mode. Separation was achieved on a strong cation exchange column (Luna 5 mu SCX 100 angstrom) with 30 mM ammonium acetate in water ( solvent A) and 5% acetic acid in water ( solvent B). Quantification was accomplished using d(2)-Phe as an internal standard. Calibration curves were linear over the range 0.5-50 mu M, and limits of detection were estimated to be 0.1-3.0 mu M. The mass-spectrometric technique was employed to study the regulation of amino acid levels in barley plants grown at 15 C uniform root temperature (RT) and 20 - 10 C vertical RT gradient (RTG). The LC-MS-MS results demonstrated enhanced concentration of free amino acids in shoots at 20 - 10 C RTG, while total free amino acid concentration in roots was similarly low for both RT treatments. (NO3-)-N-15 labeling experiments showed lower N-15/N-14 ratios for Glu, Ser, Ala and Val in plants grown at 20-10 degrees C RTG compared with those grown at 15 degrees C RT.