Determination of Free and Total Underivatized Amino Acids Including L-Canavanine in Bitter Vetch Seeds Using Hydrophilic Interaction Liquid Chromatography

摘要

A new hydrophilic interaction liquid chromatography method coupled with diode-array detector was developed for the determination of 17 underivatized amino acids including L-canavanine in bitter vetch [Vicia ervilia (L.) Willd.] seeds. Amino acids were extracted as free as well as total extracts after acid hydrolysis, followed by chromatographic separation on a Zorbax Rx-SIL column with a mobile phase of acetonitrile/potassium phosphate buffer (12.5mM; pH 3.0) using gradient elution and detection at 190nm. The method is characterized by a wide linear range (0.01-200 mu g/mL, r0.9987), sufficient accuracy (relative error 86.3-109.1%), and suitable precision for the results (relative standard deviation 4.9% in the case of intra-day and 9.8% in the case of inter-day precision). The limits of detection and quantification for free amino acids ranged from 0.01 to 0.24mg/g and 0.03 to 0.72mg/g, respectively, whereas the total amino acids ranged from 0.02 to 0.47mg/g and 0.07 to 1.43mg/g, respectively. The mean recoveries of free and total amino acids in spiked samples exceeded 70.3% for most amino acids. The mean total content of free and total amino acids in bitter vetch seeds was 1.71 and 14.88g/100g seed, whereas the corresponding values for canavanine were 0.07 and 0.19g/100g seed, respectively.