*2.7. LC-ESI-LTQ-Orbitrap-MS Analyses*

Liquid chromatography (LC) analysis was conducted using an Accela chromatograph (Thermo Scientific, Hemel Hempstead, UK) equipped with a quaternary pump, photodiode array detector, and thermostated autosampler. Chromatographic separation was performed in an Atlantis T3 column 2.1 × 100 mm, 3μm (Waters, Milford, MA, USA). Gradient elution

of analytes was performed utilizing H2O/0.1% HCOOH (solvent A) and CH3CN (solvent B) at a continuous flow rate of 0.350 mL/min, and an injection volume of 5 μL. The following gradient was applied: 0 min, 2% B; 0–2 min, 8% B; 2–12 min, 20% B; 12–13 min, 30% B; 13–14 min, 100% B; 14–17 min, 100% B; 17–18 min, 2% B and the column was equilibrated to the initial conditions for 5 min [24].

The LC equipment was coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific, Hemel Hempstead, UK) employed for accurate mass measurements and equipped with an electrospray ionization (ESI) source operating in negative mode. The working parameters were as follows: source voltage, 4 kV; sheath gas, 20 a.u. (arbitrary units); auxiliary gas, 10 a.u.; sweep gas, 2 a.u.; and capillary temperature, 275 ◦C. Default values were used for most of the other acquisition parameters (FT Automatic gain control target <sup>5</sup>·10<sup>5</sup> for MS mode and 5·10<sup>4</sup> for MSn mode). Samples were analyzed in FTMS mode with a resolving power of 30,000 (FWHM at *m*/*z* 400) and data-dependent MS/MS events were acquired with a resolving power of 15,000. The most intense ions detected in the FTMS spectrum were chosen for the data-dependent scan. The parent ions were fragmented by high-energy C-trap dissociation by normalized collision energy of 35 V and an activation time of 10 ms. The mass range in FTMS mode was from *m*/*z* 100 to 1500. Instrument control and data recovery were conducted using Xcalibur 3.0 software (Thermo Fisher Scientific). The tentative identification of analytes was performed by comparing MS/MS spectra with fragments found in databases and the literature when no standard compound was available [25].

Individual compounds were semi-quantified using pure standards or the most similar compounds. Some analytes, such as glycosylated forms, dimers, or oligomers, were semi-quantified using the aglycone form of the monomer [2].
