Empower v 3

The chromatographic analysis was carried out using a hydrophilic interaction liquid chromatography method (HILIC), using the Waters e2695 Alliance chromatographic system (Waters, Milford, MA, USA). Chromatographic separation was managed, chromatograms were recorded and data were processed with the software Empower^® v.3.0 (Waters, Milford, MA, USA). Chromatographic separations were carried out by using a Shodex SUGAR SZ5532 (150 × 6.0 mm, particle size 6 μm) column equipped with a Shodex SUGAR SZ-G (10 × 4.6 mm, particle size 6 μm) pre-column (Shodex Group, Tokyo, Japan). The volume of the injection was 10 µL. The flow rate was 1 mL/min, and gradient elution was used. The mobile phase consisted of water (solvent A) acetonitrile (solvent B). The following conditions of elution were applied: 0–5 min, 19% A; 5–20 min, 19–30% A; 20–22 min, 30% A; and 22–25 min, 30–19% A. The column was operated at a constant temperature of 60 °C. Detection was made with the evaporative light scattering detector (ELSD) Waters ACQUITY UPLC^® ELS Detector (Waters, Milford, MA, USA), with sprayer temperature—40 °C, nitrogen gas flow pressure—25 psi and evaporator temperature—60 °C. The identification of the chromatographic peaks was achieved by comparing the retention times and spectral characteristics of the eluting peaks with those of the reference compounds.

HCEA separation was performed using a Waters ACQUITY™ ultraperformance liquid chromatography (UPLC) system (Waters Corporation, Milford, MA, USA) equipped with a quaternary solvent manager system, a column compartment, an autosampler, and a photodiode array (PDA) detector. A 1 μL aliquot of the sample solution was injected into a CORTECS UPLC T3 column (2.1 × 100, 1.6 μm) maintained at 45°C. The autosampler temperature was maintained at 25°C. The mobile phase consisted of acetonitrile (A) and 0.1% (v/v) formic acid solution (B). It was delivered using the following optimized gradient program: 15%–25% A (0–4 min), 25%–35% A (4–5 min), 35%–15% A (5–5.01 min), and 15% A (5.01–8 min). The flow rate was 0.4 mL min⁻¹ with a detection wavelength of 308 nm.
The Waters ACQUITY™ UPLC system was equipped with electrospray ionization (ESI) in the negative ion mode. The MS was operated using a capillary voltage of 0.8 kV, sample cone voltage of 15 V, desolvation temperature of 600°C, source temperature of 120°C, and desolvation gas flow of 240 L h⁻¹. The data were collected from 150 Da to 700 Da using a 0.2 s scan time and uploaded to Empower v. 3.0 (Waters Corporation, Milford, MA, USA).