Debrisoquine

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.

Acetonitrile (ACN), methanol, and water (all LC-MS grade) were purchased from Fisher Scientific (NJ, USA). Debrisoquine, 4-nitrobenzoic acid (4-NBA), and heparin were purchased from Sigma (USA). All standard compounds used for tandem mass spectrometry based validation of metabolite identification were bought from Sigma. Lipid standards were ordered from Avanti Polar Inc. Other reagents for preparation and analyses of samples are described in the respective sections.