Ab tripletof 5600plus system

HPLC and MS/MS analyses were performed to analyse metabolites in enzyme assays and plant materials. For the latter, samples were prepared as described previously [28 (link)].
HPLC analysis was performed on a Waters 2695 HPLC system (Waters Corp, Milford, MA, USA) equipped with a Sunfire C18 ODS column (4.6 × 250 mm, 5 μm), quaternary solvent manager and a 2998 PDA detector. Separation was conducted using water (A) and acetonitrile (B) with the following gradient: 0–5 min, 20% B; 5–10 min, 20%–27% B; 10–15 min, 27% B; 15–25 min, 27%–40% B; 25–35 min, 40%–60% B; 35–40 min, 60%–80% B; 40–42 min, 80%–100% B; 42–45 min, 100%–20% B; 45–50 min, 20% B. The flow rate was set as 1 mL·min-1 and the injection volume was 10 μL. The temperature of the column and samples were maintained at 25°C and 8°C, respectively. Metabolites were detected at the wavelength of 200–400 nm.
MS/MS was conducted on an AB TripleTOF 5600plus System (AB SCIEX, Framingham, MA, USA). MS2 spectra were obtained in positive ion mode (ESI) or negative ion mode and the exact mass was measured.

For this, an AB TripleTOF 5600^plus system (AB SCIEX, Framingham, USA) was used, with these optimal MS conditions applied: scan range = 100–2000 m/z; for negative ion mode: source voltage = –4.5 kV and source temperature = 550°C; for positive ion mode: source voltage = +5.5 kV and source temperature = 600°C. The pressure of gas 1 (air) and gas 2 (air) was set to 50 psi; the curtain gas (N₂) pressure was set to 35 psi. The maximum tolerable error was ± 5 ppm, with a declustering potential (DP) = 100 V and collision energy (CE) = 10 V used. The IDA-based auto-MS2 was performed on the eight most intense metabolite ions in a full scan cycle (1 s). The m/z scan range of the precursor ion and product ion were set to 100–2000 Da and 50−2000 Da, respectively. The CE voltage was set to 20 V, 40 V, and 60 V in the positive ESI mode and, conversely, to −20 V, −40 V, and −60 V in the negative ESI mode. Finally, the ion release delay (IRD) used was 67, and the ion release width (IRW) was 25.
The exact mass calibration was performed automatically before each analysis, by using the Automated Calibration Delivery System. Spearman correlations (non-parametric) of the data were carried out in SPSS 20.0 (IBM Co., Armonk, NY, USA).

The identification of individual phenolic compounds was analyzed on a Waters UPLC (Waters Corp., Milford, MA, USA) coupled with Mass Spectrometry on a AB Triple TOF 5600 plus System (AB SCIEX, Framingham, MA, USA). The LC separation parameters and column were the same as showed in 3.7. The optimal MS conditions: scan range m/z 100–2000. Negative ion mode: source voltage was −4.5 kV and the source temperature was 550 °C. The pressure of Gas 1 (Air) and Gas 2 (Air) were set to 50 psi, the pressure of Curtain Gas (N2) was set to 30 psi. The Injection volume was set at 10 μL. The flow rate was 0.2 mL·min⁻¹. The Maximum allowed error was set to ±5 ppm. Declustering potential (DP), 100 V; collision energy (CE), 10 V. For MS/MS acquisition mode, the IDA-based auto-MS2 was performed on the 8 most intense metabolite ions, the parameters were almost the same except that the collision energy (CE) was set at −40 ± 20 V, ion release delay (IRD) at 67, ion release width (IRW) at 25. In a cycle of full scan (1 s). The scan range of m/z of precursor ion and product ion were set as 100–2000 Da and 50–1500 Da, respectively. The exact mass calibration was performed automatically before each analysis employing the Automated Calibration Delivery System.