4.3.5. HILIC and RP Chromatography

The used UHPLC system was an Ultimate 3000™ liquid chromatographic system (Thermo Scientific™, MA, USA) coupled to an Orbitrap Q Exactive™ mass spectrometer (Thermo Scientific™, MA, USA) equipped with a HESI source operating in the positive and negative ion modes. HILIC chromatographic separation was accomplished using a BEH-HILIC column, 130 Å, 1.7 μm, and 2.1 × 100 mm (Waters, Milford, MA, USA). The used mobile phases were: 20 mM ammonium formate along with 0.1% FA at pH 3.7 (mobile phase A) and ACN (mobile phase B). The gradient consisted of a linear increase of mobile phase B from 5% to 35% over 8.5 min, followed by an additional increase to 50% in 1 min. Phase B was kept constant for 1.5 min and then decreased to 5% in 0.5 min and kept stable for 3.5 min for column re-equilibration (total run time of 15 min). The used flow rate was 0.300 mL/min, the injection volume was 2 μL, and the column was kept at 35 ◦C.

RP chromatographic separation was achieved using an HSS-T3 column, 100 Å, 1.7 μm, and 2.1 × 100 mm (Waters, Milford, MA, USA). The mobile phases were: 0.1% FA in H2O (mobile phase A) and 0.1% FA in ACN (mobile phase B). The gradient ramp consisted of a linear increase to 10% of mobile phase B over 6 min and to 35% in 2 min. Mobile phase

B was further increased to 98% in 2 min, kept constant for 0.5 min, and finally decreased to 0% in 0.5 min and kept stable for 3 min for column re-equilibration (total run time of 15 min). The flow rate was 0.300 mL/min from 0 to 8.0 min, increased to 0.4 mL/min from 8.0 to 12.0 min for column washing, and brought back to 0.3 mL/min from 12.0 to 15.0 min. The injection volume was 2 μL, and the column was kept at 35 ◦C. During LC–MS analysis, samples were kept in the autosampler at 8 ◦C.

## 4.3.6. High-Resolution Mass Spectrometry

Mass spectra were acquired on an Orbitrap QExactive™ mass spectrometer (Thermo Scientific™, MA, USA) operating in both the positive and negative ion modes. The HESI parameters were: 3.20 kV (pos)/–3.20 kV (neg) electrospray voltage, 280 ◦C heated capillary temperature, 50 (pos)/–50 (neg) S-lens RF level, sheath gas (N2) flow of 50 a.u., auxiliary gas (N2) flow of 10 a.u., and gas temperature of 300 ◦C. The acquisition range was set from *m/z* 60 to 900 at a resolution of 70,000 FWHM at *m/z* 200. All data were acquired in profile mode using Xcalibur™ 3.1.66.10. The QExactive™ mass spectrometer was calibrated for the positive and negative modes before sample analysis using the calibration solution provided by the manufacturer (Pierce LTQ ESI Positive Calibration Solution and Pierce LTQ ESI Negative Calibration Solution). For the mass calibration of the instrument, a custom list that included lower masses than the default calibration provided with the instrument was used to ensure that accurate masses were detected at low molecular weights.
