In Vitro Measurement of Plasma Protein Binding

The unbound fraction of compound in plasma from rhesus macaques was measured by equilibrium dialysis of 2.5 μM compound in 100% plasma against 100 mM PBS buffer using HT Dialysis plates (Model HTD96b) with 12–14 MWCO dialysis membranes. The plate was equilibrated for 4 h at 37°C in a humidified incubator with a 5% CO₂ environment. The study samples were prepared for a protein precipitation extraction method by addition of acetonitrile containing a cocktail of internal standards (labetalol, imipramine, and diclofenac). The peak area ratio of test compound to internal standard in plasma and buffer were determined by ultra-high performance liquid chromatography (UPLC) coupled with a SCIEX API triple quadruple mass spectrometer. Chromatographic separation was performed using reverse phase LC gradient methods. The fraction unbound in plasma is the ratio of test compound/internal standard peak area ratio in buffer to that of test compound/internal standard peak area ratio in plasma.