Pooled human liver cytosol

The incubation mixtures (final volume, 0.2 mL) consisted of 50 mM KH₂PO₄ (pH 7.4), compound 1 (final concentration, 10 or 50 µM), 0.5 mg of protein/mL of pooled human liver microsomes (Xenotech LLC, Lenexa, KS, USA) or pooled human liver cytosol (Xenotech LLC), and 0.5 mM NADPH (mixtures containing liver microsomes) or oxidized nicotinamide adenine dinucleotide (NAD; mixtures containing liver cytosol). The reactions were started following a 3 min thermal equilibration at 37 °C by adding the microsomes or the cytosolic fraction, conducted at 37 °C under aerobic conditions for different time periods, and terminated by adding 0.1 mL of ice-cold acetonitrile. After protein removal by centrifugation at 20,000× g for 10 min (4 °C), an aliquot of the supernatant was analyzed by HPLC with diode array detection, and metabolite peaks were collected manually and subjected to mass spectrometry as described below.

EXE was purchased from Sigma-Aldrich (St. Louis, MO). 17β-DHE, 17β-DHE-Gluc, 17β-OH-EXE-d3 (D₃-17β-DHE), 17β-OH-EXE-d3-17-O-β-d-glucuronide, and EXE-19-d3 (D₃-EXE) were purchased from Toronto Research Chemicals (North York, ON, Canada). Ammonium formate was obtained from Sigma-Aldrich, whereas ammonium acetate and formic acid were purchased from Thermo Fisher Scientific (Waltham, MA). Acetonitrile was purchased from Merck (Kenilworth, NJ). Solvent and buffer modifiers for ultra-pressure liquid chromatography–mass spectrometry (UPLC-MS) analysis including Acetonitrile, Ammonium formate, ammonium acetate, and formic acid were all liquid chromatography–mass spectrometry (LC-MS) grade. Milli-Q water (Sigma, St. Louis, MO) was used for the preparation of all solutions. Pooled human liver cytosol (HLC; mixed gender, pool of 50 subjects) was purchased from XENOTECH (Kansas City, KS). Reduced l-glutathione (GSH) and glutamyltranspeptidase (γ-GT) from equine kidney were purchased from Sigma-Aldrich. l-Cysteine was purchased from Alfa Aesar (Heysham, Lancashire, UK). Preparative C-18 thin-layer chromatography (TLC) plates (20 cm × 20 cm × 1 mm) were obtained from Sigma-Aldrich. Hexane, diethyl ether, Acetonitrile, potassium hydroxide, and methanol used for chemical synthesis were of American Chemical Society reagent grade or higher quality and were purchased from VWR (Radnor, PA).