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

Abstract

A method has been developed for quantification of 20 amino acids as well as 13 ¹⁵N-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 5µ SCX 100Å) 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 µM, and limits of detection were estimated to be 0.1–3.0 µM. 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. ¹⁵NO₃ ⁻ labeling experiments showed lower ¹⁵N/¹⁴N ratios for Glu, Ser, Ala and Val in plants grown at 20–10 °C RTG compared with those grown at 15 °C RT.