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 (15)N-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 5micro SCX 100A) 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 microM, and limits of detection were estimated to be 0.1-3.0 microM. The mass-spectrometric technique was employed to study the regulation of amino acid levels in barley plants grown at 15 degrees C uniform root temperature (RT) and 20-10 degrees C vertical RT gradient (RTG). The LC-MS-MS results demonstrated enhanced concentration of free amino acids in shoots at 20-10 degrees C RTG, while total free amino acid concentration in roots was similarly low for both RT treatments. (15)NO(3) (-) labeling experiments showed lower (15)N/(14)N 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.
Thiele
Björn
B
Institute for Chemistry and Dynamics of the Geosphere, Institute-3: Phytosphere/BioSpec, Forschungszentrum Jülich GmbH, 52425, Juelich, Germany. b.thiele@fz-juelich.de
Füllner
Kerstin
K
Stein
Nadine
N
Oldiges
Marco
M
Kuhn
Arnd J
AJ
Hofmann
Diana
D
D000596:Amino Acids / Q000032:analysis*; D002138:Calibration; D002412:Cations / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000379:methods*; D001467:Hordeum / Q000737:chemistry* / Q000378:metabolism; D009587:Nitrogen Isotopes; D010936:Plant Extracts / Q000032:analysis; D018515:Plant Leaves / Q000737:chemistry / Q000378:metabolism; D018517:Plant Roots / Q000737:chemistry / Q000378:metabolism; D018520:Plant Shoots / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000596:Amino Acids; D002412:Cations; D009587:Nitrogen Isotopes; D010936:Plant Extracts