Accela 1200

Plasma samples were injected directly into a Thermo Fisher Scientific, (Waltham, MA, USA) LC system Accela 1200 equipped with an autosampler with a temperature controlled tray and column. Chromatographic separation was performed on Waters C18 XTerra MS analytical columns (150 × 2.1 mm, size 3.5 μm) with a Waters C18 XTerra MS guard column (10 × 2.1 mm, size 3.5 μm). The column and autosampler tray temperatures were set at 35 °C. The mobile phase consists of 0.1% formic acid in water: methanol (95:5, v/v) A and 0.1% formic acid in water: methanol (5:95, v/v) B, the flow rate was constant 300 µLmin⁻¹. Gradient elution of 0–2 min with mobile phase 95% A and 5% B was started, 2.1–20 min linear gradient from 5% to 90% B, 20.1–25 min 10% A and 90% B, 25.1–30 min linear gradient from 5% to 95% A and 30.1–35 min 95% A and 5% B. The runtime of the method was 35 min.

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).