Plant Amino Acid Quantification by UPLC

Amino acids were extracted by homogenizing plant shoots (200 mg FW) in 1 ml of 80% (v/v) aqueous ethanol (MagNALyser; Roche, Vilvoorde, Belgium; 1 min, 7000 rpm), spiked with norvaline to estimate the loss of amino acids during extraction, and centrifugation at 20 000 g for 20 min. The supernatant was vacuum‐evaporated, and the pellet resuspended in 1 ml of chloroform. The plant residue was re‐extracted with 1 ml HPLC grade water using MagNALyser and the supernatant after centrifugation (20 000 g for 20 min) was mixed with the pellet suspended in chloroform. Then the extracts were centrifuged for 10 min at 20 000 g and the aqueous phase was filtered by Millipore microfilters (0.2‐μm pore size) before assaying amino acid concentrations. Amino acids were measured by using a Waters Acquity UPLC‐tqd system (Milford, MA, USA) equipped with a BEH amide 2.1 × 50 column (Sinha et al., 2013).