Kinetex 1.7 μm hilic 100

Chromatographic separation was achieved by gradient elution on the LC system Accela 1200 from Thermo Fisher Scientific (Waltham, MA, USA). The following three different chromatographic columns were tested in our study: Hypercarb 3 μm 100 × 2.1 mm from Thermo Fisher Scientific (Waltham, MA, USA), Luna Omega 1.6 μm Polar C18 100 × 2.1 mm, and Kinetex 1.7 μm HILIC 100 × 2.1 mm from Phenomenex (Torrance, CA, USA). Chromatographic conditions in the test (mobile phases, flow rate, and gradient) were the same for each column except for the composition of the mobile phases, which was for Hypercarb and Luna Omega (the reverse-phase columns) opposite to the HILIC column (normal-phase column). After evaluating the chromatographic separation properties of the tested columns, chromatographic column Kinetex 1.7 μm HILIC 100 × 2.1 mm from Phenomenex (Torrance, CA, USA) was used. The gradient LC system was operated using 0.1% formic acid in water: acetonitrile (95:5, v/v, mobile phase A) and 0.1% formic acid in water: acetonitrile (5:95, v/v mobile phase B) at a flow of 200 μL·min⁻¹. A gradient elution for separation on HILIC column was performed: 0–4 min (0% A, 100% B), 8 min (linear gradient to 30% A), 8–12 min (linear gradient to 100% B), and 12–14 min (0% A, 100% B).