1 methylnicotinamide

Reagents for sample preparation and LC mobile phases (Optima^TM grade) were obtained from Thermo Fisher Scientific^TM 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.

Rifampicin (RIF), verapamil (Ver), ritonavir (RTV), pravastatin (PV), 5-fluorouracil and fexofenadine (FEX) were purchased from Shanghai Macklin Co., Ltd (Shanghai, China), Tetradecanedioate (TDA), hexadecanedioate (HAD), 1-methylnicotinamide, pyridoxine, N-acetyl-L-phenylalanine, N-acetylvaline and azelaic acid (AZA) were purchased from Sigma-Aldrich (St Louis, MO, USA). L-serine-O-sulfate was purchased from Toronto Research Chemicals (Toronto, Canada). All primers for the real-time quantitative polymerase chain reaction (PCR) detecting system (qPCR) or quantitative real-time polymerase chain reaction (qRT-PCR) were synthesized by Sunbiotech Co., Ltd. (Beijing, China). Anti-P-gp was purchased from Abcam (ab170904, rat, USA). Anti-Oatp1b2 was purchased from Santa Cruz Biotechnology (sc-376904, rat, USA). Anti-β-actin was purchased from Affinity (AF7018, rat, China). Methanol and acetonitrile were high-performance liquid chromatography (HPLC)-grade (Fisher Scientific, NJ, USA). All other reagents and solvents were of analytical grade and were commercially available.