Reagents for sample preparation and LC mobile phases (OptimaTM grade) were obtained from Thermo Fisher ScientificTM Inc. (Waltham, MA, USA). Authentic chemical standards were of the highest purity available: debrisoquine sulfate, 4-nitrobenzoic acid, carnitine, creatine, hippuric acid, 4-pyridoxic acid, phenylacetylglycine, 4-hydroxy-3-methoxyphenylglycol sulfate, 1-methylhistamine, 1-methylnicotinamide (Sigma-Aldrich® LLC, St. Louis, MO, USA), and N1-acetylspermidine (Cayman Chemical Company, Ann Arbor, MI, USA).
Urine samples were prepared as previously described [20 (link)]. Urine (20 μL) was deproteinated with 50% acetonitrile (80 μL) containing internal standards (2 μM debrisoquine sulfate, 30 μM 4-nitrobenzoic acid), incubated on ice for 10 min, vortexed for 30 s, and centrifuged for 10 min (10,000× g, 4 °C). A quality control (QC) sample was prepared by mixing 1 μL of urine from each sample and prepared as above and run every 10 samples.
Samples were injected (2 μL) and analyzed by an ACQUITY UPLC (BEH C18 1.7 μM, 2.1 × 50 mm column) coupled to a Xevo® G2 QTOF-MS (Waters Corp., Milford, MA, USA). Data-independent acquisition was performed in both negative and positive electrospray ionization (ESI) modes as previously described [6 (link)] using leucine enkephalin (556.2771 [M + H]+ or 554.2615 [M − H]−) as Lockspray® to calibrate accurate mass.
Urine samples were prepared as previously described [20 (link)]. Urine (20 μL) was deproteinated with 50% acetonitrile (80 μL) containing internal standards (2 μM debrisoquine sulfate, 30 μM 4-nitrobenzoic acid), incubated on ice for 10 min, vortexed for 30 s, and centrifuged for 10 min (10,000× g, 4 °C). A quality control (QC) sample was prepared by mixing 1 μL of urine from each sample and prepared as above and run every 10 samples.
Samples were injected (2 μL) and analyzed by an ACQUITY UPLC (BEH C18 1.7 μM, 2.1 × 50 mm column) coupled to a Xevo® G2 QTOF-MS (Waters Corp., Milford, MA, USA). Data-independent acquisition was performed in both negative and positive electrospray ionization (ESI) modes as previously described [6 (link)] using leucine enkephalin (556.2771 [M + H]+ or 554.2615 [M − H]−) as Lockspray® to calibrate accurate mass.