Acquity uplc beh hilic column

Targeted analysis of TMAO and its derivatives (choline, betaine, TMAO, creatinine, and L‐carnitine) in serum samples using the UPLC‐MS method. 20 μl of serum sample mixed with add 10 μl of internal standard solution, and then add 750 μl of 1% formic acid‐acetonitrile solution, vortex for 30 s, centrifuge at 12,000 rpm for 5 min at 4°C, take 500 μl of supernatant, filter through 0.22 μm of membrane, and filter add the liquid to the test bottle. Samples were performed using an ACQUITY UPLC BEH HILIC column (2.1 × 100 m, 1.7 μm; Waters) with an ESI source in positive ionization mode, injection volume 5 μl, column temperature 40℃, mobile phase A‐acetonitrile, B‐water (containing 0.1% formic acid), and 10 mM of ammonium formate at a flow rate of 0.4 ml/min. The solvent gradient was set as follows: 0–1 min, 80% A; 1–2 min, 80%–70% A; 2–2.5 min, 70% A; 2.5–3 min, 70%–50% A; 3–3.5 min, 50% A; 3.5–4 min, 50%–80% A; and 4–6 min, 80% A. The ion source temperature was 500℃, the ion source voltage was 5000 V, the collision gas was 6 psi, the curtain gas was 30 psi, and the atomization gas and auxiliary gas were both 50 psi. Multiple reaction monitoring (MRM) was used for scanning.

In the ¹⁵N incorporation assay, cells were inoculated into 5 mL KDC medium in ϕ25 test tubes, which were stoppered with butyl-rubber, and the gas phase was replaced with a mixture of ¹⁵N₂:O₂ (82:18) (Shoko Science, Kanagawa, Japan) for the ¹⁵N₂ sample or air for the ¹⁴N₂ sample. Test tubes were statically incubated at 25 °C for 6 d. In the ¹³C incorporation assay, cells were inoculated in KDC medium in which glucose or citrate was replaced with ¹³C₆-glucose (>99 atom % ¹³C) or 1,5-¹³C₂-citrate (98 atom % ¹³C), respectively. Culturing was performed in 5 mL of medium in non-hermetic ϕ25 test tubes at 25 °C for 6 d. To analyze glutamate in the supernatant, the culture supernatant was diluted 10-fold with methanol and transferred to a UFLC Nexera system (Shimadzu) connected to a Triple TOF 5600 system (SCIEX, Tokyo, Japan). Samples were separated using an ACQUITY UPLC BEH HILIC column, 130 Å, 1.7 µm, 2.1 × 50 mm (Waters, Milford, MA, USA). LC conditions were as follows: mobile phase A, H₂O + 0.1% formic acid; mobile phase B, acetonitrile + 0.1% formic acid; 95% B for 1 min, 95%–40% B over 5 min, 40% B for 3 min, and 95% B for 3 min, at a flow rate of 0.4 mL min⁻¹. Ions of 2.7 ± 0.2 min (retention time of standard glutamate) were analyzed. MS analyses were simultaneously performed using electrospray ionization in the positive mode.

BABA was quantified in material from plants treated with chemical inducers as well as after infection with PstDC3000. Plant material was harvested, flash-frozen and ground to fine powder in liquid nitrogen. The extraction protocol was conducted as described by Thevenet et al. (2017) (link); in brief 100 mg of ground tissue was extracted in 500 μL of 0.1% HCOOH/H₂O (v/v) containing the deuterium labeled internal standard (BABA-d₃) using a Retsch mixer mill. After centrifugation of the extract at 18400 g during 4 min, the supernatant was purified by solid phase extraction on an Isolute SCX-2 cartridge (1 mL, 100 mg). The eluate was concentrated to dryness in a centrifugal evaporator (Speedvac) at 35°C. Samples were finally resuspended in 300 μL (1: 3) organic mobile phase B/EtOH 80% (v/v) leading to a final concentration of internal standards of 50 ng mL⁻¹. Extracted BABA was quantified using an Ultimate 3000 RSLC (Dionex, Thermo Fisher Scientific) interfaced with a 4000 QTRAP (AB Sciex) by injecting 3.5 μL of extract on an Acquity UPLC BEH HILIC column (100 mm × 2.1 mm, 1.7 μm, Waters).

Analysis was performed using a UPLC/Q-TOF (Bruker Impact II). An Acquity UPLC BEH HILIC column (2.1 × 50 mm, 1.7 μm) from Waters™ was used to analyze amino acids and other metabolites in the standard solution and fish embryo extracted samples according to the protocol proposed by WATERS [28 ] with few modifications (Table S1 in Supporting Information). Acquisition (FSR > 60,000) was performed in full scan mass over 60 to 1000 m/z (Supporting Information Table S2). The ionization source operated in positive (4 eV of ion energy, 20–30 eV switching normalized collision energy) and negative mode (6 eV of ion energy, 24–36 eV switching normalized collision energy) alternating MS scans of the precursor ions (MS1) and all-ion fragmentation (MS2). Acquisitions in positive and negative ESI were performed in different chromatographic runs, as Bruker’s Q-TOF technology does not allow simultaneous acquisition in both modes. All solvents were analytical grade or higher purity.