Luna hilic 200a

The extraction procedure and SG profile followed Zimmermann et al. (2012) [91 (link)]. SGs were estimated using a Jasco PU980 HPLC system (JASCO Benelux B.V., Utrecht, the Netherlands) coupled with a UV-visible wavelength detector. A hydrophilic column (Luna HILIC 200A, Phenomenex Inc., Torrance, CA, USA), 5 mm, 250 mm × 4.6 mm (Phenomenex Inc., Torrance, CA, USA), and the corresponding pre-column (4 × 3.0 mm) were used. The UV detection was performed at 205 nm at room temperature, with a flow rate of 0.68 mL/min and a run time of 20 min. Separation was carried out in acetonitrile/water (80:20) as an isocratic mobile phase at pH 3.6 regulated with acetic acid. Chromatograms were gathered online, and data were obtained using a Jasco interface (Hercules 2000 Interface Chromatography). The Common Steviol Glycosides Standards Kit was purchased from Chromadex (LGC Standards S.r.L., Milan, Italy). The calibration curves (0.005–1.00 g L⁻¹) were achieved from standard mixtures containing rubusoside, dulcoside A, stevioside, and rebaudioside A, C, D, E, and M (Reb A, C, D, E, and M), and SGs were quantified using authentic standards. Total SGs were determined as the sum of single SGs [44 (link)].

The level of amino acid uptake was evaluated using HPLC-ESI-QTOF. For HILIC separation, the chromatographic column Luna HILIC 200A (150 × 4.6; 3.5 µm particles, Phenomenex, Torrance, CA, USA) was used according to our previous study [39 (link)]. 0.1% of formic acid (v/v) was added in to the polar part of the mobile phase to enhance the ionization. For detection, a Bruker Maxis Impact mass spectrometer with electrospray by ionization quadrupole time-of-flight (Bruker, Munich, Germany) was utilized. The electrospray ionization source was operated in a positive mode. The voltage of the electrospray capillary was set to 3500 V with a nebulizing gas (N₂) flow rate of 4 L·min⁻¹ and a drying gas temperature set to 350 °C. Scanning was carried out within the range from 50 to 1000 m/z. The samples were diluted 1000 times in isopropanol prior to analysis.