Metabolic Stability Assay with Microsomes

The human and murine pooled microsomes employed were purchased from Tebu-Xenotech (Barcelona, Spain). The compound was incubated at 37 °C with the microsomes in a 50 mM phosphate buffer (pH = 7.4) containing 3 mM MgCl₂, 1 mM NADP, 10 mM glucose-6-phosphate and 1 U/mL glucose-6-phosphate-dehydrogenase. Samples (75 µL) were taken from each well at 0, 10, 20, 40 and 60 min and transferred to a plate containing 4 °C 75 µL acetonitrile and 30 µL of 0.5% formic acid in water were added for improving the chromatographic conditions. The plate was centrifuged (46000 g, 30 min) and supernatants were taken and analyzed in a UPLC-MS/MS (Xevo-TQD, Waters) by employing a BEH C18 column and an isocratic gradient of 0.1% formic acid in water: 0.1% formic acid acetonitrile (60:40). The metabolic stability of the compounds was calculated from the logarithm of the remaining compounds at each of the time points studied [19 (link)].