Acquity uplc hss t3

Metabolomic analysis was performed on the samples described above by LC-MS according to previously described methods (Xue et al., 2017 (link)) with modifications. The freeze-dried samples were extracted with methanol and an internal standard at a ratio of 500:8:0.1. All samples were ground to a fine powder using a grinding mill at 65 Hz for 90 s. Then, the samples were ultrasonicated for 30 min at 40 KHz and allowed to stand for 1 h at −20°C. The samples were centrifuged at 12000 rpm at 4°C for 15 min, and 200 μL of supernatant was transferred to vials for LC-MS analysis.
The analysis platform was LC-Q/TOF-MS (Agilent, 1290 Infinity LC, 6530 UHD and Accurate-Mass Q-TOF/MS, Agilent Technologies, Santa Clara, CA, United States) with a Waters column (ACQUITY UPLC@HSS T3, 2.5 μm 100^∗2.1 mm). Water with 0.1% formic acid and acetonitrile with 0.1% formic acid were used for mobile phase A and B, respectively; the flow rate was 0.4 mL/min, the injection volume was 4 μL, the automatic injector temperature was 4°C, and the column temperature was 40°C. The capillary voltage was 4 and 3.5 kV in positive and negative mode, respectively; the drying gas flow was 11 L/min, and the gas temperature was 350°C. Centroid data were collected from 100 to 1000 m/z.

Chromatographic separation was accomplished in an Thermo Ultimate 3000 system equipped with an ACQUITY UPLC^® HSS T3 (150×2.1 mm, 1.8 μm, Waters) column maintained at 40°C. The temperature of the autosampler was 8°C. Gradient elution was carried out with 0.1% formic acid in water (C) and 0.1% formic acid in acetonitrile (D) or 5 mM ammonium formate in water (A) and acetonitrile (B) at a flow rate of 0.25 mL/min. Injection of 2 μL of each sample was done after equilibration. An increasing linear gradient of solvent B (v/v) was used as follows: 0~1 min, 2% B/D; 1~9 min, 2%~50% B/D; 9~12 min, 50%~98% B/D; 12~13.5 min, 98% B/D; 13.5~14 min, 98%~2% B/D; 14~20 min, 2% D-positive model (14~17 min, 2% B-negative model).
The ESI-MSn experiments were executed on the Thermo Q Exactive Focus mass spectrometer with a spray voltage of 3.5 kV and -2.5 kV in positive and negative modes, respectively. Sheath gas and auxiliary gas were set at 30 and 10 arbitrary units, respectively. The capillary temperature was 325°C. The orbitrap analyzer scanned over a mass range of m/z 81-1 000 for a full scan at a mass resolution of 70,000. Data-dependent acquisition (DDA) MS/MS experiments were performed with a HCD scan. The normalized collision energy was 30 eV. Dynamic exclusion was implemented to remove some unnecessary information in MS/MS spectra.

The quantitative detection of VOCs in human urine was performed using ultra performance liquid chromatography coupled with electrospray tandem mass spectrometry (UPLC-ESI/MSMS) as described in previous studies (20 (link)). The Acquity UPLC^® HSS T3 (Part no. 186003540, 1.8 µm x 2.1 mm x 150 mm, Waters Inc.) column, with 15 mM ammonium acetate and acetonitrile as the mobile phases, was applied for chromatographic separation. In brief, the eluent from the column is firstly ionized using an electrospray interface to generate and transmit negative ions into the mass spectrometer. Then the comparison of relative response factors (ratio of native analyte to stable isotope labeled internal standard) with known standard concentrations yields individual analyte concentrations (https://wwwn.cdc.gov/nchs/data/nhanes/2015-2016/labmethods/UVOC_UVOCS_I_MET.pdf). More details of the laboratory method detecting urine VOCs could be found in the website (https://wwwn.cdc.gov/nchs/data/nhanes/2015-2016/labmethods/UVOC_UVOCS_I_MET.pdf).

Chromatographic separation was accomplished in a Thermo Vanquish system equipped with an ACQUITY UPLC^® HSS T3 (150×2.1 mm, 1.8 µm, Waters) column maintained at 40 °C. The temperature of the autosampler was 8 °C. Gradient elution of analysis was carried out with 0.1% formic acid in water (A2) and 0.1% formic acid in acetonitrile (B2) or 5 mM ammonium formate in water (A3) and acetonitrile (B3) at a flow rate of 0.25 mL/min. Injection of 2 μL of each sample was done after equilibration. An increasing linear gradient of solvent B2/B3 (v/v) was used as follows: 0~1 min, 2% B2/B3; 1~9 min, 2%~50% B2/B3; 9~12 min, 50%~98% B2/B3; 12~13.5 min, 98% B2/B3; 13.5~14 min, 98%~2% B2/B3; 14~20 min, 2% B2-positive model (14~17 min, 2% B3-negative model).

A wide-targeted analysis was performed using an ACQUITY UPLC system (Waters) coupled with a triple quadrupole MS (QTRAP 6500, Sciex). The LC separation was per- formed using a reverse-phase column [ACQUITY UPLC HSS T3 (50 mm × 2.1 mm inner diameter, 1.8 μm particle size; Waters)] with a gradient elution consisting of mobile phase A (methanol/acetonitrile/water = 1:1:3 v/v/v containing 50 mM ammonium acetate and 10 nM EDTA) and mobile phase B (100% isopropanol containing 50 mM ammonium acetate and 10 nM EDTA). The LC gradient consisted of holding solvent (A/B:100/0) for 1 min, which was linearly converted into solvent A/B:50/50 for 4 min, linearly converted to solvent A/B:36/64 for 7 min, linearly converted to solvent A/B:5/95 for 1 min, and then held for 1 min. It was then returned to solvent A/B:100/0 and held for 5 min for re-equilibration. The injection volume was 3.5 μl, the flow rate was 0.350 mL/min, and the column temperature was 50°C. The multiple reaction monitoring (MRM) mode was used to quantify oxidized phospholipids in biological samples.

The purified RPEFs were quantitatively analyzed by using ACQUITY UPLC HSS T₃ (50 mm × 3.0 mm, 1.8 μm, Waters Technologies Co., Massachusetts, USA) coupled with Triple-TOF 5600⁺ Mass Spectrometry (AB SCIEX CO., Framingham, USA). 0.1 % formic acid solution (A) and acetonitrile (B) were adopted as the mobile phase. The extract was filtered through 0.45 μm membrane prior to 4 μL of injection into the system. The flow rate was maintained at 0.5 mL/min and detected at 280 nm UV length. The elution procedure was presented as following: 0–10 min, 5 % to 40 % B; 10–17 min, 40 %-95 % B; 17–25 min, 95 %-5% B. The MS conditions was set as positive/negative ion sweeping mode: scan range 100–1500 m/z, ion source gas 1 and 2 (air) 55 psi, curtain gas (N₂) 35 psi, ion source temperature 600℃ (positive) and 550℃ (negative), source voltage −5.5 kv (positive) and −4.5 kv (negative), collision energy spread 40 ± 20 eV. Data was processed using Analyst software version 4.1 (MassLynx).