Agilent 1290 infinity uhplc system

Serum samples were thawed at 4°C, and diluted with methanol at a ratio of 1:3. After 3-min vortex, the samples were centrifuged at 12,000 g for 10 min at 4°C. The supernatant was separated with chromatography using an Agilent 1290 Infinity UHPLC system (Agilent Technologies, Wilmington, DE). The column oven was set at 40°C. An acquity UPLC^® HSS T3 C₁₈ column (1.8 µm 100 × 2.1 mm, Waters, Milford, MA) was used for the reverse phase separation. The mobile phase consisted of 0.1% formic acid (A) and acetonitrile modified with 0.1% formic acid (B), using a gradient elution of 2% B at 0–2 min, 2–95% B at 2–17 min, 95% B at 17–19 min. The total run time was 25 min including 6-min equilibration. The flow rate was 400 µl/min and the injection volume was 4 µl. For HILIC analysis, an acquity UPLC^® BEH HILIC C₁₈ column (1.7 µm 100 × 2.1 mm, Waters) was used on the same LC system. The mobile phase consisted of 10 mM ammonium formate modified with 0.1% formic acid (A) and ACN modified with 0.1% formic acid (B), using a gradient elution of 95% B at 0–10 min, 95–90% B at 10–14 min, 90% B at 14–20 min, 90–60% B at 20–23 min, 60% B at 23–25 min and post time was set to 10 min for equilibrating the system. The total run time was 35 min. The flow rate was 350 µl/min and the injection volume was 4 µl.