Determination ofβ-Cyano-L-alanine,γ-Glutamyl-β-cyano-L-alanine, and Common Free Amino Acids inVicia sativa(Fabaceae) Seeds by Reversed-Phase High-Performance Liquid Chromatography
A method for determination ofβ-cyano-L-alanine,γ-glutamyl-β-cyano-L-alanine and other free amino acids inVicia sativais presented. Seed extracts were derivatized by reaction with diethyl ethoxymethylenemalonate and analyzed by reverse-phase high-performance liquid chromatography. Calibration curves showed very good linearity of the response. The limit of detection and quantification was 0.15 and 0.50 μM, respectively. The method has high intra- (RSD = 0.28–0.31%) and interrepeatability (RSD = 2.76–3.08%) and remarkable accuracy with a 99% recovery in spiked samples. The method is very easy to carry out and allows for ready analysis of large number of samples using very basic HPLC equipment because the derivatized samples are very stable and have very good chromatographic properties. The method has been applied to the determination ofγ-glutamyl-β-cyano-L-alanine,β-cyano-L-alanine, and common free amino acids in eight wild populations ofV. sativafrom southwestern Spain.