3.2. In Vitro Simulation of Metabolism by HLM

Metabolism reactions were performed in vitro with the use of the human liver microsome fraction. The incubation system consisted of 50 μM substrate, 55 mM phosphate buffer (pH 7.4) and 0.5 mg·mL⁻¹ HLM. Following the 2-min preincubation period at 37 °C, metabolic reactions were initiated by the addition of 10 μL NADPH (20 mM). The total volume of the reaction suspension was equal to 200 μL. The reaction was terminated after 0, 30, 60, 90 and 120 min of incubation with the use of 200 μL ice-cold acetonitrile–methanol mixture (1:1). Next, the precipitated samples were centrifuged at 15,000 rpm for 10 min at 4 °C and the supernatants (40 μL) were transferred into the vials for the LC–MS analysis. The negative control samples were prepared in the same manner without adding the NADPH solution.