Pro asn

Liquid chromatography-mass spectrometry (LC-MS)-grade isopropanol, acetonitrile, methanol and water were purchased from Fisher Scientific (New Jersey, USA). Debrisoquine, Pro-Asn, glycoursodeoxycholic acid andmalic acid, 4-nitrobenzoic acid (4-NBA) were products of Sigma (St. Louis, MO, USA). High purity formic acid (99%) was provided by Thermo-Scientific (Rockford, IL). The serum lipid extraction was conducted as described previously [37 (link)]. Briefly, the plasma samples were thawed on ice before being vortexed. For metabolite extraction, 25 μL of plasma sample was mixed with 175 μL of extraction buffer (25% acetonitrile in 40% methanol and 35% water). The sample was then incubated on ice for 10 min before centrifuged again at 14,000 rpm at 4 °C for 20 min. Subsequently, the produced supernatant was transferred to a fresh tube and dried under vacuum. After drying, the dried samples were reconstituted in 200 μL of buffer containing 5% methanol, 1% acetonitrile and 94% water. Fine particles were removed by centrifuge at 13,000 rpm for 20 min at 4 °C. Finally, the supernatant was transferred to a glass vial for ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) analysis.