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

• Published in: Analytical and bioanalytical chemistry 2008-08, Vol.391 (7), p.2663-2672
• Main Authors: Thiele, Björn, Füllner, Kerstin, Stein, Nadine, Oldiges, Marco, Kuhn, Arnd J, Hofmann, Diana
• Format: Article
• Language: English
• Subjects: amino acids; Amino Acids - analysis; Analytical Chemistry; Biochemistry; Calibration; Cations - chemistry; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chromatography, Ion Exchange - methods; Food Science; Hordeum - chemistry; Hordeum - metabolism; Laboratory Medicine; Liquid chromatography-mass spectrometry; Monitoring/Environmental Analysis; Nitrogen Isotopes; Original Paper; Plant Extracts - analysis; Plant Leaves - chemistry; Plant Leaves - metabolism; Plant metabolism; Plant Roots - chemistry; Plant Roots - metabolism; Plant Shoots - chemistry; Plant Shoots - metabolism; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization - methods; Strong cation exchange column; Tandem Mass Spectrometry - methods
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-008-2167-9

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.