Eluent b

Fifteen laboratories returned results. One laboratory did not analyze the two liquid samples and one laboratory only analyzed the SPIFAN powder samples. A survey was conducted among the participants to evaluate the diversity of instrument and reagents used during this study, and thirteen laboratories provided answers (see Supplemental Table 1).
Different instruments (six Waters Acquity, four Waters Acquity H-class, one Waters Acquity I-class, one Thermo 3000 RS, and one Agilent 1200 Infinity II) were used. All laboratories used the column specified in the method. For the mobile phase A, 11 laboratories prepared Eluent A from the AccQ·Tag Ultra Eluent A concentrate according to the protocol, one laboratory only used 120 mL of the concentrate (instead of 150 mL), and one laboratory used the Alternative Eluent A. For the mobile phase B, seven laboratories used the Eluent B from Waters and six laboratories used the Alternative Eluent B. Twelve laboratories used the AccQ·Tag Ultra derivatization kit from Waters and one laboratory used the Alternative Derivatizing Reagent.

The free amino acids and ammonium in the extracts of roots, nodules, stems, and leaves of the plants and xylem sap were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate (AQC) reagent (AccQ·Flour, Waters, Milford, MA, USA), and analyzed using a Waters Aquity UPLC system equipped with a Waters AccQ Tag Ultra column (2.1 mm × 100 mm, particle size 1.7 μm; Waters, Milford, MA, USA). The column temperature was 60 °C and the sample temperature was 20 °C. The wavelength for the detection of AQC-amino acid derivatives was at 260 nm using an Aquity UPLC TUV Detector. Elution was performed using a gradient of Waters AccQTag Ultra Eluent A and Eluent B with a flow rate of 0.7 mL/min [51 (link)]. For the internal standard, 2.5 mM 2-aminoisobutylic acid (AABA) was used. For the calibration of amino acids, 200 μM of a standard amino acid mixture was used (18 amino acids by Amino Acid Standard H, Thermo Fisher Scientific Ltd, Waltham, MA, USA. with added GABA, Trp, Asn, Gln, and AABA).