Determination of the Neurotoxin 3-N-Oxalyl-2,3-Diaminopropionic Acid and Other Free Amino Acids in Lathyrus cicera and L. sativus Seeds by Reversed-Phase High-Performance Liquid Chromatography
A method for determination of the neurotoxic non-protein amino acid 3-N-oxalyl-2,3-diaminopropionic acid (ODAP) and other free amino acids in Lathyrus cicera and Lahtyrus sativus is presented. Seed extracts were derivatised by reaction with diethyl ethoxymethylenemalonate (DEEMM) and analysed by reversed-phase high-performance liquid chromatography (RP-HPLC). Calibration curves showed very good linearity of the response. The limits of detection (LOD) and quantification (LOQ) were 0.15 and 0.50 μM, respectively. The method has a high intra- [relative standard deviation (RSD) < 0.42 %] and inter-repeatability (RSD = 2.01–2.33 %) and a remarkable accuracy with a 99 % recovery in spiked samples. The method yielded similar results in comparison with a previously established colorimetric method. 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 derivatised samples are very stable and have very good chromatographic properties.
KeywordsLathyrus cicera Lathyrus sativus 3-N-Oxalyl-2,3-diaminopropionic acid Free amino acids Diethyl ethoxymethylenemalonate
This work was carried out with the financial support of Junta de Andalucía (Spain) to the Laboratory of Bioactive and Functional Components of Plant Products (Instituto de la Grasa, C.S.I.C.) and the project PAI09-0015-7008 (Consejería de Educación y Ciencia, Junta de Comunidades de Castilla—La Mancha). Cristina Megias and Isabel Cortés-Giraldo are, respectively, recipients of a JAE-Doc (C.S.I.C.) contract and a JAE-Pre (C.S.I.C) fellowship from the “Junta para la Ampliación de Estudios” program (cofinanced by the European Social Fund). Thanks are due to María Dolores García Contreras for technical assistance. The authors are indebted to the Banco de Germoplasma Vegetal de Cuenca, BGV-CU (Cuenca, Spain) for supplying the plant material. We are grateful to the European Social Fund and to “Fundación Parque Científico y Tecnológico de Albacete” for additional financing.
Compliance with Ethics Requirements
This article does not contain any studies with human or animal subjects.
Conflict of Interest
Cristina Megías declares that she has no conflict of interest. Isabel Cortés Giraldo declares that she has no conflict of interest. Manuel Alaiz declares that he has no conflict of interest. Julio Girón Calle declares that he has no conflict of interest. Javier Vioque declares that he has no conflict of interest. Omar Santana Méridas declares that he has no conflict of interest. David Herraiz Peñalver declares that he has no conflict of interest. Raul Sánchez Vioque declares that she has no conflict of interest.
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