Incubation with Human Liver Microsomes in the Presence of Glutathione

(R)-ND-336 (20 μM final concentration) was incubated with human liver microsomes (Xenotech, 1 mg/mL) in 1 mL volume of 100 mM potassium phosphate buffer (pH 7.4) containing 1 mM NADPH, 5 mM l-glutathione (Sigma–Aldrich), and 10 mM MgCl₂ at 37 °C. Aliquots were drawn at several time points and protein was precipitated with one volume of acetonitrile. Samples were analyzed by UPLC with UV and mass spectrometry detection. Samples were screened for the glutathione adduct at m/z 643 in positive ESI mode.