Single use red

In vitro plasma protein binding was performed by the ADME Center at USAMRICD using Thermo Scientific’s protocol and their Single-Use RED (rapid equilibrium dialysis) Plates. Samples (100–500μL) were prepared by spiking test compound with plasma at the appropriate concentrations and places into the sample chamber. Dialysis buffer (300–750 μL) was added to the buffer chamber. The unit was covered with sealing tape and incubated at 37°C on an orbital shaker at approximately 250 rpm or 20 rpm on an up and down shaker for 4h. 50 μL from both the buffer and the plasma chambers were placed in separate microcentrifuge tubes or into a deep well plate for analysis. 50 μL of plasma was added to the buffer sample and an equal volume of buffer to the collected plasma sample. 300 μL of Internal Standard containing precipitation buffer (such as cold 90/10 acetonitrile/water with 0.1% formic acid) was added to precipitate protein and release compound. Vortexed and incubated 30 minutes on ice, centrifuged for 10 minutes at 13,000–15,000 × g. The supernatant was analyzed with LC-MS/MS. The test compound concentration in the buffer and plasma chambers were determined from peak areas relative to the internal standard. The percentage of the test compound bound was calculated as follows: % Free = (Concentration buffer chamber/Concentration plasma chamber) × 100%. % Bound = 100% - % Free.