Exionlctmad system

Metabolites were analyzed using the UPLC-Triple-TOF-MS-based platform (AB SCIEX, USA). The chromatographic separation of metabolites was performed using an ExionLCTMAD system (AB Sciex, USA) equipped with ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm; Waters, Milford, USA). Mobile phase A is water (containing 0.1% formic acid), mobile phase B is acetonitrile/isopropanol (1/1) (containing 0.1% formic acid); the flow rate is 0.40 mL/min, the injection volume is 20 μL, and the column temperature is 40 °C.
As part of the system adjustment and quality control process, a combined quality control sample (QC) was prepared by mixing all samples of equal volume. QC samples were injected at regular intervals (every 9 samples) to monitor the stability of the analysis. QC samples were treated and tested in the same way as analytical samples. It was preferable to represent the entire sample set, and to monitor the stability of the analysis.

Chromatographic separation of the metabolites was performed on a ExionLCTMAD system (AB Sciex, Milford, MA, USA) equipped with an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm; Waters, Milford, MA, USA). The experimental conditions were set up in accordance with Yang [53 (link)]. The mobile phases consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile:isopropanol (1:1, v/v) (solvent B). The solvent gradient changed according to Table 3. The sample injection volume was 20 μL, and the flow rate was set to 0.4 mL/min. The column temperature was maintained at 40 °C. During the period of analysis, all of these samples were stored at 4 °C.
The UPLC system was coupled to a quadrupole time-of-flight mass spectrometer (Triple TOFTM5600+, AB Sciex, Milford, MA, USA) equipped with an electrospray ionization (ESI) source operating in positive mode and negative mode. The following parameters were set in accordance with Yang [53 (link)]: Source temperature, 500 °C; curtain gas (CUR), 30 psi; both Ion Source GS1 and GS2, 50 psi; ion-spray voltage floating (ISVF), −4000 V in negative mode and 5000 V in positive mode, respectively; declustering potential, 80 V; collision energy (CE), 20–60 V rolling for MS/MS. Data acquisition was performed with the Data-Dependent Acquisition (DDA) mode. The detection was carried out over a mass range of 50–1000 m/z.

The metabolites were separated through chromatography using an ExionL CTMAD system (AB Sciex, USA) equipped with an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm; Waters, Milford, USA). The volume and flow rate of sample injection were 20 μL and 0.4 mL/min, respectively. The temperature of the column was set at 40 °C. All the analyzed samples were stored at 4 °C.
The UPLC was attached with a quadrupole-time-of-flight mass spectrometer (Triple TOFTM 5600+, AB Sciex, USA) and an electrospray ionization (ESI) source. The ESI was operated in positive mode and negative mode. The optimal conditions followed in the process of analysis were as stated: source temperature, 500 °C; curtain gas (CUR), 30 psi; both Ion Source GS1 and GS2, 50 psi; ion-spray voltage floating (ISVF), −4000V in negative mode and 5000V in positive mode, respectively; declustering potential, 80V; a collision energy (CE), 20–60V rolling for MS/MS. Finally, data were acquired with Data Dependent Acquisition (DDA) mode. The sample detection was performed over a mass range of 50–1000 m/z.

Chromatographic separation of the metabolites was performed on an ExionLC^TM AD system (AB Sciex, United States) equipped with an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm; Waters, Milford, United States). The mobile phases consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile:isopropanol (1:1, v/v) (solvent B). The solvent gradient changed according to the following conditions: from 0 to 3 min, 95% (A):5% (B) to 80% (A):20% (B); from 3 to 9 min, 80% (A):20% (B) to 5% (A):95% (B); from 9 to 13 min, 5% (A):95% (B) to 5% (A):95% (B); from 13 to 13.1 min, 5% (A):95% (B) to 95% (A):5% (B); and from 13.1 to 16 min, 95% (A):5% (B) to 95% (A):5% (B) to equilibrate the systems. The column temperature was maintained at 40°C. The sample injection volume was 20 μl, and the flow rate was set to 0.4 ml/min. The UPLC system was coupled to a quadrupole-time-of-flight mass spectrometer (Triple TOF^TM 5600⁺, AB Sciex, United States) equipped with an electrospray ionization (ESI) source operating in positive and negative modes. Data acquisition was performed with the data-dependent acquisition (DDA) mode. The detection was carried out over a mass range of 50–1,000 m/z.