Sample Preparation

The method to simulate glucuronic acid conjugation by microsomes using phase I metabolites was conducted in accordance with WU et al., 2007 [25]. In each reaction mixture protein concentration was normalized to 1 mg/mL, independent of the microsome species. The reaction mixture consisted of 0.3 mM UDPGA, 0.4 mM MgCl2 and 0.1 mM 3- OH or bAE, respectively. The volume of each reaction mixture was filled with 83.4 mM Tris buffer to 200 μL. Microsomes were directly added before the reaction was started. Each tube was gently vortexed and incubated at 37 ◦C for 4 h while gently shaking. To stop the reaction, 400 μL acetonitrile were added to each tube and the samples were centrifugated at 4 ◦C for 5 min and 14,000× *g*. 150 μL of supernatant were diluted with 850 μL of water achieving a final concentration of acetonitrile of 12%, noting the starting conditions of the following HPLC-qTOF-MS method. A blank sample without analyte was used to distinguish analyte signals from matrix signals. Moreover, a second blank sample without liver microsomes was implemented to distinguish enzymatic from chemical reactions. Efficiency of the used microsomal systems was verified with the control 7- -hydroxycoumarin (100 μM).
