Quantification of Amino Acids by UPLC-MS/MS

The concentrations of all 20 proteinogenic amino acids as well as those of 4-aminobutyric acid, citrulline, and ornithine were quantified with external calibration, in triplicate, by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS), using an Acquity system equipped with an HSS T3 column (Waters, Milford, MA, United States), as described previously (Van der Veken et al., 2022 (link)). Minor modifications included a constant flow rate of 0.35 mL/min for the mobile phase and the following gradient: 0.0 to 1.0 min, 99% A and 1% B; 1.0 to 8.0 min, 30% A and 70% B; 8.1 min to 10.0 min, 100% B; and 10.1 to 25.0 min, 99% A and 1% B. Samples were prepared by the addition of 300 μL of acetonitrile (Merck) to 100 μL of aqueous extract, followed by microcentrifugation at 18,000 × g for 15 min. Then, 900 μL of ultrapure water with 0.10% formic acid and 8.0 mg/L of 2-aminobutyric acid (IS; Sigma-Aldrich) was added to 100 μL of supernatant, and the mixture was filtered with a 0.2-μm LG H-PTFE filter (Millex; Merck) before injection (10 μL) into the column.