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 nonprotein 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 mu M, 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.