Torus diol column

The UHPLC experiments were performed on a Waters H-Class UHPLC system using a modified method described by Krithika et al. [37 (link)]. The UHPLC system was equipped with a Waters BEH C18 column (100 × 2.1 mm, 1.7 µm) and run at 50 °C. Detection was performed using a PDA detector at 280 nm, with the baseline-corrected by 350–400 nm. The injection volume varied between 3 and 5 µL, and the flow rate was 0.6 mL/min. The mobile phase was composed of: (A) Milli-Q water with 0.5% acetic acid or 10 mM formic acid, and (B) acetonitrile with 0.5% acetic acid or 10 mM formic acid.
All UHPSFC/HRMS experiments were performed using a Waters Ultra Performance Convergence Chromatography System (Waters Milford, MA, USA) connected via a flow splitter (ACQUITY UPC² splitter, Waters) to an LTQ Orbitrap Velos Pro mass spectrometer (Thermo Scientific, Waltham, MA, USA), which was equipped with a heated electrospray ionization source (HESI, Thermo Scientific). Chromatographic separation of the samples was performed using a Torus DIOL column (3 mm × 100 mm, 1.7 µm, Waters) protected with a Torus DIOL VanGuard pre-column (2.1 mm × 5 mm, 1.7 µm, Waters). The samples were centrifuged in a 5424R Eppendorf centrifuge (Eppendorf, Hamburg, Germany).

An Acquity UPC² hyphenated to a G2‐Si Synapt HRMS from Waters (Milford, MA, USA) was used. The separation was performed on a Torus DIOL column (3.00×100mm, 1.7μm) with a VanGuard pre‐column (diol, 2.1×5.0mm, 1.7μm) from Waters. The choice of the column was based on literature [12 (link), 13 (link)]. The post‐column splitter interface from Waters was used to connect the UPC² and MS, with a make‐up solvent flow via a quaternary solvent manager (Waters). Methanol as a modifier (B‐solvent), column temperature (40°C), automated backpressure (140bar), flow rate (1.5ml/min), and injection volume (3μl) were kept constant. Strong and weak washing solvents were methanol:water [4:1] and isopropanol, respectively. Both positive (ESI⁺) and negative (ESI⁻) ionization modes were applied. If not stated otherwise, the MS parameters were set as follows: source temperature 120°C, desolvation temperature 600°C, desolvation gas flow 1000L/h, cone gas flow 100L/h, scan rate 0.2scans/s, scan type ‘centroid’, and mass range 50–1200Da. Lockspray calibration with leucine‐enkephalin (0.2ng/ml, Waters) was performed every 30s. The data was acquired with MassLynx; TargetLynx (v4.1; Waters) and R software (RStudio 2022.02) were used to process the data.