Stable isotope labeled internal standards

Both standards of metabolites and stable isotope-labeled internal standards (IS) were obtained from Sigma-Aldrich (St. Louis, MO, United States), Cayman Chemical (Ann Arbor, MI, United States), Bidepharm (Shanghai, China), Steraloids (Newport, RI, United States), Cambridge Isotope Laboratories (Cambridge, MA, United States), and Cayman Chemical or Steroids (Supplementary Table S1). Detailed information was given in our previous study (Hu et al., 2020a (link)). Organic solutions (e.g., acetonitrile, isopropyl alcohol, and methanol) of HPLC grade were purchased from Fisher Scientific (Pittsburgh, PA, United States). The modifier of the mobile phase—formic acid—was obtained from Co., Inc. (Fairfield, OH, United States). Ultrapure Millipore water was prepared by a purification system.

MS grade methanol, acetonitrile, isopropanol, and formic acid were purchased from Sigma-Aldrich (St. Louis, MO). Ultrapure water was prepared by the Milli-Q system (Millipore, Billerica, MA). All the reference standards and stable isotope-labeled internal standards were purchased from Sigma-Aldrich (St. Louis, MO), Steraloids Inc. (Newport, RI), TRC Chemicals (Toronto, ON, Canada), and Nu-Chek Prep (Elysian, MN). The derivatization regents including 3-nitrophenylhydrazine (3-NPH) and N-(30(dimethylamino)propyl)-N'-ethylcarbodiimide (EDC) were purchased from Sigma-Aldrich (St. Louis, MO).