LC analysis of hydrophilic and hydrophobic compounds

For quality control of the metabolomic analysis, we pipetted 10 μL of each of the 30 plasma samples to pool a mixture, which was equally separated into 4 parts. When running sample sets on the UPLC column, 1 part of the control sample was first added, and the remaining 3 parts were sequentially injected after per 10 samples.

The sample extracts of hydrophilic compounds were analyzed using the ultra-performance liquid chromatography (UPLC) of a LC-MS/MS system (Shim-pack UFLC SHIMADZU CBM A system, MS, QTRAP® 6500+ System). The samples were injected onto a Waters HSS T3 column (1.8 µm, 2.1 mm × 100 mm). The column temperature, flow rate and injection volume were set 40 °C, 0.4 mL/min and 2 μL, respectively. Mobile phase A (2% ACN, 0.1% FA) and mobile phase B (90% ACN, 0.1% FA) were used to establish a 12 min separation gradient (0 min - 5% B; 11 min - 90% B; 12 min - 5% B).

The sample extracts of hydrophobic compounds were also analyzed using the same UPLC (Shim-pack UFLC SHIMADZU CBM A system, MS, QTRAP® 6500+ System). The samples were injected onto a Thermo Accucore™ C30 column (2.6 μm, 2.1 mm × 100 mm). The column temperature, flow rate and injection volume were set 45 °C, 0.35 mL/min and 2 μL, respectively. Mobile phase A (2% ACN, 0.1% FA) and mobile phase B (90% ACN, 0.1% FA) were used to establish a 20 min separation gradient (0 min - 20% B; 2 min - 30% B; 4 min - 60% B; 9 min - 85% B; 14 min - 90% B; 15.5 min - 95% B; 17.3 min - 90% B; 20 min - 20% B).