Methyl tert butyl ether (mtbe)

25-OHD₃-S, 25-OHD₃-G and d₆-25-OHD₃ were purchased from Toronto Research Chemicals (Toronto, Canada). The purity of d₆-25-OHD₃, used to prepare the deuterated internal standards, was 96.6%. Iodobenzene I,I-diacetate, 3’-phosphoadenosine-5’-phosphosulfate (PAPS), uridine 5’-diphosphoglucuronic acid ammonium salt (UDPGA), alamethacin and 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
4-(4’-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (precursor of DAPTAD) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). HPLC-grade acetonitrile, methanol, ethyl acetate, methyl tert-butyl ether, formic acid, acetic acid, and ammonium hydroxide were obtained from Fisher Chemicals (Waltham, MA, USA). Vitamin D₃-free human serum was purchased from Golden West Biologicals (Temecula, CA, USA). Human plasma was obtained from the Puget Sound Blood Bank (Seattle, WA). Pooled human liver cytosol and microsomes were prepared in-house using liver tissue provided by the University of Washington School of Pharmacy Human Tissue Bank.

1, 2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) pre-dissolved in chloroform was obtained from Avanti Polar Lipids (Alabaster, AL). Fmoc-Arg (Pbf)-Nova Syn TGA resin (0.23 mmol/g), 9-Fluorenylmethyl-oxycarbonyl-O-succinimide (Fmoc-OSu) and all amino acids were purchased from Novabiochem, San Diego, CA. Triisopropylsilane (TIS), anisole, hexafluro-2-propanol (HFIP), 30% ammonium hydroxide [NH₃ (aq.)], piperidine were purchased from Sigma-Aldrich, St. Louis, MO. 2,2,6,6-Tetramethylpiperidine-1-oxyl-4-carboxylic acid (TOAC) was purchased from Acros Organics, Belgium. Trifluoroacetic acid (TFA), 1-hydroxybenzo-triazole (HOBt), (2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), diisopropylethylamine (DIEA), M-methyl-pyrrolidone (NMP), and dichloromethane (DCM) were purchased from Applied Biosystems, Foster City, CA. Acetic anhydride (Ac₂O) and methyl-tert-butyl ether were purchased from Fisher Scientific, Pittsburgh, PA.

MPA and progesterone were measured by Wisconsin National Primate Center Assay Services. Briefly, plasma, standards and quality control samples were diluted in 500 μL of ultrapurified water (Fisher Scientific). Methyl tert butyl ether (Fisher Scientific) was then added, vortexed vigorously, and incubated at room temperature for 5 min. The organic phase containing steroids was transferred into a new tube, evaporated to dryness by air stream and heated water bath (60 °C), and then resuspended in 50 μL of 20% acetonitrile in water and analyzed via LC-MS/MS. The calibration curve ranged from 0.195 to 25 ng/mL and the linearity was r > 0.9990. The intraassay CV was 2.76% and the interassay CV was 4.7%.

Progesterone, 6β-OHP, and 16α-OHP were obtained from Steraloids, Inc. (Newport, RI). 6β-hydroxytestosterone, β-nicotinamide adenine dinucleotide phosphate tetrasodium salt (NADPH), and ketoconazole were purchased from Sigma-Aldrich, Co. (St. Louis, MO). Letrozole, α-naphthoflavone, omeprazole, pilocarpine, quecertin, quinidine, and sulfaphenazole were obtained from Sigma-Aldrich, Santa Cruz Biotechnology (Dallas, TX), or Cayman Chemical (Ann Arbor, MI). Pooled human liver microsomes (HLMs) from 150 individual donors and baculovirus–expressed CYP3A4+b5, CYP3A5+b5 and CYP3A7+b5, CYP2A6, CYP19A1 were purchased from Corning Gentest (Woburn, MA). Acetonitrile (ACN) and methyltert-butyl ether were obtained from Fisher Scientific (Fair Lawn, NJ). All other chemicals were of high performance liquid chromatography (HPLC) grade and purchased from reputable manufacturers.