Dionex ultimate 3000 hplc instrument

Amino acids were quantified by HPLC after an acidic hydrolysis according to Dhillon, Kumar, and Gujar [66 (link)] and using an AccQ-Tag reagent kit from Waters (Milford, MA, USA) for derivatization of amino acids.
Each RAs sample (ca. 30 mg) was hydrolyzed with 4 mL of HCl and 15 μL of phenol at 110 °C for 24 h, and then entrapped in N₂ atmosphere. The hydrolyzate was filtered through syringe filters (0.45 μm), and then dried with N₂. Next, 10 μL of the sample was mixed with 70 μL of borate buffer (pH 8.2–9.0), then 20 μL of 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate acetonitrile solution (3 mg/mL) were added to the mixture (AccQ-Tag reagent kit, Waters, Milford, USA). Analogous procedures were used in the case of standards.
The amino acid profile (AAs) was identified on a Dionex Ultimate 3000 HPLC instrument (Thermo Scientific, Germering, Germany). Separation was provided on a reverse-phase Nova-Pak C18 column (4 μm, 150 × 3.9 mm) (Waters, Milford, MA, USA) at 37 °C. Elution was run in a two-component gradient at 1 mL/min; eluent A: acetic-phosphate buffer, eluent B: acetonitrile-water (60:40). The detector (VWD-3400RS) was set at 240 nm wavelength. The AAs peaks were computed from AAs standards (Sigma-Aldrich, Poznan, Poland) run at five concentrations. Individual AA values were expressed as mg/100 g of fresh weight of RAs.

All HPLC–MS
experiments were performed using a Dionex UltiMate 3000 HPLC instrument
(Thermo Fisher Scientific, Massachusetts, USA) coupled to a Q Exactive
HF-X Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen,
Germany). Analytes were separated on a Kinetex C18 reversed-phase
column (2.6 μm, 100 × 2.1 mm, Phenomenex, Torrance, USA).
Detailed experimental parameters for HPLC–MS/MS analysis are
provided in Supplementary Note 3.

After 24 and 48 h of growth, supernatants were filtered through Mini-Uniprep syringless filter devices (0.2 µm, Whatman) and analyzed on an Atlantis Dc18 100 A (5 µm 4.6 mm × 150 mm) column (temperature 31 °C) using a Dionex Ultimate 3000 HPLC instrument (Thermo Scientific). Samples were 5-fold diluted in a solution of 20 µM cyclo(Trp-Trp). This latter was used for sample normalization since its retention time of 24.8 min is outside the window at which the detectable metabolite peaks produced by S. ambofaciens emerge. Samples were separated using the following method: 0 to 7 min, isocratic 0.1% HCOOH in water (solvent A)/0.1% HCOOH in CH₃CN (solvent B) (95:5) at 1 ml min⁻¹, 7–30 min, 5–60 % solvent B, 30–35 min, 60–100% solvent B, 35–45 min, 100% solvent B and 50–60 min, 5% solvent B. Metabolite production was detected at 297 nm.