4.8.1. Chromatography

Chromatographic experiments were performed on a Thermo Syncronis C18 column (2.1 mm i.d. × 100 mm, 1.7 <sup>μ</sup>m) using an Acquity UPLCTM System (Waters Corp., Milford, MA, USA). The column was maintained at 35 ◦C, and the mobile phase, at a flow rate of 0.4 mL/min, consisting of solvent A (0.1% formic acid in water) and mobile phase B (acetonitrile). 0–12 min, 5%–20% B; 12–14 min, 20%–25% B; 14–19 min, 25%–55% B; 19–21 min, 55%–70% B; 21–25 min, 70%–95% B.

### 4.8.2. Mass Spectrometry

MS spectrometry was performed with a Waters SynaptTM Q-TOF/MS (Waters Corp., Milford, CT, USA). The conditions used for the electrospray ion (ESI) source were as follows: capillary voltage of 3.0 kV, sample cone voltage of 30.0 V; extraction cone voltage of 2.0 V, source temperature of 120 ◦C, desolvation temperature of 350 ◦C. Nitrogen was used as desolvation and cone gas with a flow rate of 600 and 50 L/h, respectively. Leucine-enkephalin was used as the lock mass generating an [M + H]<sup>+</sup> ion (*m*/*z* 556.2771) and [M-H]− ion (*m*/*z* 554.2615) at a concentration of 200 pg/mL and flow rate of 100 μL/min to ensure accuracy during the MS analysis via a syringe pump.
