Acquity uplc hss t3 column

The LC-MS/MS analysis was run on an Acquity UPLCTM system. The analytes were separated on an Acquity UPLC TM HSST3 column (2.1×100 mm, 1.7 m, Waters) that was run at a constant temperature of 30°C. The mobile phase, consisting of 0.1% formic acid in water (Solvent A) and acetonitrile (Solvent B), was used under conditions of gradient elution: 0-0.5 min, 10% B; 0.5-1.5 min, 60% B; 1.5-1.75 min, 10% B; 1.75-2 min, and 10% B; at a flow rate of 0.5 mL/min. ESI-MS/MS conditions were set as follows: the instrument was operated using an electrospray ionization source (ESI) in positive mode. ESI parameters were a capillary voltage of 3.0 kV, an extractor voltage of 30 V, a source temperature of 120°C, a desolvation temperature of 350°C, a cone gas flow of 80 L/h, and a desolvation flow of 650 L/h (both gases were nitrogen). Multiple reaction monitoring (MRM) transitions, cone voltages of 28 V, and collision energies of 25 V were applied during the analysis. Data acquisition was performed using Mass Lynx 4.0 software with the Quan Lynx program (obtained from Waters).

Inorganic polysulfides were identified using liquid chromatography electrospray ionization high resolution mass spectrometry (LC-ESI-HRMS) using a micrOTOF-Q II Mass Spectrometer (Bruker Daltronics, Billerica, MA, USA) coupled to an UltiMate 3000 (Thermo Fisher) UHPLC system, as described previously [18 (link)]. A Waters Acquity UPLC HSS T3 column (1.8 µm, 150 mm × 2.1 mm inner diameter) was used with mobile phases A (water containing 0.1% formic acid) and B (acetonitrile containing 0.1% formic acid). Samples were diluted 10-fold in water and 20 µL was injected with a linear gradient (0–90% B, 30 min) at a flow rate of 0.4 mL/min. The mass spectrometer was used in the positive ion mode with the capillary voltage set to 2200 V and drying gas set to 8.0 L/min at 180 °C. The IAM polysulfide adducts were detected as the [M + Na]⁺ ion using their exact masses ±0.002 m/z: S₁ (149.038, 171.020), S₂ (181.010, 202.992), S₃ (212.982, 234.964), S₄ (244.954, 266.936), S₅ (276.926, 298.908), S₆ (308.898, 330.880). In these experiments, H₂S (1 mM, as Na₂S) was incubated in 10 mM phosphate buffer with 1 mM 1,4-NQ for 10 min, derivatized with 5 mM IAM for 30 min, and subjected to LC-ESI-HRMS within 1–2 h.

The LC-MS system utilized for metabolomic analysis was composed of a Waters Acquity I-Class PLUS ultraperformance liquid chromatography system coupled to a Waters Xevo G2-XS QTof high-resolution mass spectrometer. The chromatographic column used was an Acquity UPLC HSS T3 column (1.8 µm, 2.1 mm × 100 mm) purchased from Waters. The mobile phase consisted of aqueous formic acid solution (A) and acetonitrile (B), and the gradient elution program was as follows: 0 min, 98%; 0.25 min, 98%; 10 min, 2%; 13 min, 2%; 13.1 min, 98%; 15 min, 98% and 0 min, 2%; 0.25 min, 2%; 10 min, 98%; 13 min, 98%; 13.1 min, 2%; and 15 min, 2%. The Xevo G2-XS QTof high-resolution mass spectrometer can collect primary and secondary mass spectral data in MSE mode under the control of acquisition software (MassLynx V4.2, Waters). In each data acquisition cycle, dual-channel data acquisition with low collision energy and high collision energy can be carried out simultaneously. The low collision energy was 2 V, the high collision energy was 10–40 V, and the scanning frequency was 0.2 s. The parameters of the electrospray ionization (ESI) source were as follows: capillary voltage, 2000 V (positive ion mode) or −1500 V (negative ion mode); taper hole voltage, 30 V; ion source temperature, 150°C; desolvation gas temperature, 500°C; back blowing flow rate, 50 L/h; and flow rate of desolvation gas, 800 L/h.

Quercetin concentration in plasma of in vivo pharmacokinetic studies was analyzed by UPLC-MS method (Waters Acquity I CLASS/ABSCIEX QTRAT-4500, Milford, MA). Chromatographic separation was carried out on a ACQUITY UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 µm) with a ACQUITY UPLC HSS T3 guard pre-column (5.0 mm × 2.1 mm, 1.8 µL, 100 A, 3/pkg, Waters, Milford, MA). The mobile phase consisted of a gradient mobile phase system consisting of methanol containing 0.1% formic acid (phase A) and water containing 0.1% formic acid (phase B) at a flow rate of 0.2 mL /min. The pump was programed as follows: 0-0.5 min, 70% phase B; 0.5-2.5 min, 70-35% phase B; 2.5-4.5 min, 35-15% phase B; 4.5-6 min, 15-0% phase B; 6-7 min, 0% phase B; 7-7.1 min, 70% phase B; 7.1-10 min, 70% phase B. A 10 µl sample was injected into the system and column temperature maintained at 30 °C.
The mass spectrometer worked in the negative ion mode. The MS parameters were as follows: the ion spray voltage was set at −4.5 kV, and the source temperature was set at 500 °C. The curtain gas was 30 psi. The ion source gas1 and ion source gas 2 were both set at 50 psi respectively. The multiple reaction monitoring transitions were performed at m/z 3 0 1→151 for quercetin, m/z 3 1 5→300.1 for isorhamnetin, m/z 477 → 301 for quercetin 3-O-β-D-Glucuronide (Que-glu) and m/z 4 1 7→122 for nimodipine (internal standard).

For High Performance Liquid Chromatography analysis of tryptophan metabolites, feacal samples were collected and froze in liquid nitrogen before metabolites measurement. The metabolites were extracted from feacal samples using high throughput tissue lyser according to the manufacture’s protocol. Then extracted metabolites were resolved using ACQUITY UPLC® HSS T3 Column (2.1×150 mm, 1.8 μm, Waters, Watertown, MA, USA) on a ACQUITY analysis system from Waters and identified using Lipid Search software.