3.4.5. Plasma Stability Assay

A DMSO stock solution of each compound was incubated in triplicate in the presence of human plasma and HEPES buffer (25 mM, 140 mM NaCl, pH 7.4) at the final concentration of 100 µM at 37 °C under shaking. At the selected time point of 0 and 24 h, 50 µL of the mixture was collected, treated with 1.0 mL of cold ACN, and centrifuged at 5000 rpm for 10 min. The supernatant was collected, and the amount of unmodified compound was quantified with the HLPC-UV/MS method above reported. Calculations of modified compounds were made using time zero as 100% of the unmodified compound.