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Journal of Analytical Methods in Chemistry
Volume 2014, Article ID 409089, 5 pages
Research Article

Determination of β-Cyano-L-alanine, γ-Glutamyl-β-cyano-L-alanine, and Common Free Amino Acids in Vicia sativa (Fabaceae) Seeds by Reversed-Phase High-Performance Liquid Chromatography

Instituto de la Grasa (C.S.I.C.), Avenida Padre García Tejero 4, 41012 Sevilla, Spain

Received 3 October 2014; Accepted 8 December 2014; Published 22 December 2014

Academic Editor: Karoly Heberger

Copyright © 2014 Cristina Megías et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


A method for determination of β-cyano-L-alanine, γ-glutamyl-β-cyano-L-alanine and other free amino acids in Vicia sativa is 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 of γ-glutamyl-β-cyano-L-alanine, β-cyano-L-alanine, and common free amino acids in eight wild populations of V. sativa from southwestern Spain.