Plasma stability assay

Plasma stability assays were conducted with final concentrations of 99% plasma, 1% DMSO, and 5 μM compound. Incubations occurred in EDTA-treated plasma (BioreclamationIVT) from five species – C57BL/6 mouse, Sprague Dawley rat, Beagle dog, Cynomolgus monkey, and human. Time points up to 4 h were collected and quenched on ice with 6X volume of acetonitrile containing an internal standard, 100 ng mL^-1 glyburide. Samples were vortexed then centrifuged to precipitate protein. A 6:5 ratio of water:supernatant were mixed for injection on a Sciex 4000 with a TSI source in positive mode with a source temperature, spray voltage, gas 1, gas 2, CAD gas, and curtain gas settings of 500, 5500, 60, 60, 10, and 30, respectively. Transitions for 1, 2, and glyburide were 524.2 > 84.1, 496.4 > 127.1, and 494.2 > 168.8, respectively, with declustering potentials of 70, 100, and 70 V, respectively, and collision energies of 45, 65, and 40 V, respectively. An Agilent liquid chromatography system used a ACE C18 30x2.0 mm and 3 μm column (Advanced Chromatography Technologies Ltd.) with solvents A and B containing 0.1% formic acid in water (A) and acetonitrile (B) at a flow rate of 0.7 mL min^-1 with a gradient profile of 2% B from 0 to 0.5 min, an increase from 2% to 98% B from 0.5 to 2 min, 98% B from 2 to 2.5 min, a decrease from 98% to 2% B from 2.5 to 2.6 min, and 2% B from 2.6 to 3 min.