Uplc beh c18 column 2.1 mm 100 mm

The TGS phenolic profile was assessed by UHPLC-MS/MS analysis (10 mg/mL solution in H₂O:MeOH (50:50, v/v)) as described by Fernández-Fernández et al. [11 (link)]. The identification and quantification of phenolic compounds was carried out using a Thermo Ultimate™ 3000 HPLC (Thermo Scientific, Sunnyvale, CA, USA) coupled to a hybrid quadrupole-orbitrap mass spectrometer (Q-ExactiveTM; Thermo Scientific, Bremen, Germany) equipped with heated electrospray ionization (HESI-II). Chromatographic separation was performed as follows: UPLC BEH C18 column 2.1 mm × 100 mm, 1.7 μm particle size (Waters, Milford, MA, USA); mobile phase, water-acetonitrile gradient at a flow rate of 0.3 mL/min. Thermo Scientific™ Dionex™ Chromeleon™ 7.2 Chromatography Data System (CDS) software was used for data acquisition and processing. The peak area was used to define the relative concentration of the phenolic compounds.

A 50 μL aliquot of each sample extract was diluted 1:3 in 50% methanol containing 2 μM phenol red standard and subjected to LC-MS/MS analysis on a Thermo^TM QExactive^TM mass spectrometer coupled to a Vanquish HPLC system. The mobile phase was an increasing gradient of solution A (100% LC-MS grade water) to solution B (100% LC-MS grade acetonitrile) both containing 0.1% formic acid. The stationary phase column was a Waters^® Acquity^®(Wood Dale, IL, USA) UPLC BEH C18 column, 2.1 mm × 100 mm. The chromatographic program was a 12-min-long run that began with a hold at 5% solution B for 30 s then an increasing linear gradient of solution B from 5–99% over 8.5 min. This 99% B solution was then held for 1.5 min followed by a switch to 0% B for the remaining 1.5 min. The full MS¹ mass spectra were collected in positive mode at a resolution of 17,500, an automatic gain control (AGC) target of 5e5, scan range of 150 to 1500 m/z for the full MS mode (minutes 2–10 of run). The MS/MS spectra were collected at the same resolution and AGC target with an isolation window of 1.0 m/z, a loop count of 5 and stepping of energies 20, 30 and 40 NCE. The data dependent settings included a dynamic exclusion of 1.0 s.