Readout: % Binding, % Remaining at time point, % Recovery
Controls: Phenacetine, Quinidine, Warfarin
Working solutions (1 mM in DMSO) are prepared for each test and control compound. The dosing solutions are prepared by diluting the working solutions to 5 µM in plasma.
The dialysis plate (RED device)⁵ is prepared by adding buffer to one chamber of the RED device and dosing solution to the other chamber. The plate is sealed with an adhesive film and incubated at 37 °C while shaking for 5 h. Equal volumes of post dialysis samples are removed from both the plasma and the buffer chambers in separate microcentrifuge tubes and equal volumes (50 µL) of fresh phosphate buffer and plasma are added to the tubes, respectively.
Plasma samples are diluted 5-fold and then all samples are treated with quenching solution (terfenadine/tolbutamide in methanol/ACN). Sample mixtures are then centrifuged and the supernatant is removed for LC-MS/MS analysis.
To assess plasma stability, aliquots of dosing solution are stored at 4 °C (t = 0 h sample) and at 37 °C for 5 h (t = 5 h sample). Following incubation, aliquots are removed for LC-MS/MS analysis.
The MS detection is performed by using a Sciex API 4000 Q trap instrument. Each compound is analyzed by reversed phase HPLC using a Kinetex 2.6µ C18 100Å column (3.0 mm X 30 mm, Phenomenex). Mobile phase – Solvent A: water with 0.1% formic acid, solvent B: ACN with 0.1% formic acid. The amount of parent compound is determined on the basis of the peak area ratio (compound area to IS area) for each time point.
The calculation of percentage binding is determined using the following equations:
Binding % = (Cpe – Cb) / Cpe x 100
% Stability of test compound = (Conc. of stability sample / Conc. of time zero sample) x 100
% Recovery = (Cpe + Cb) / Conc. of stability sample x 100
Cpe = Concentration of test compound in plasma at equilibrium
Cb = Concentration of test compound in buffer at equilibrium
HPLC High-performance liquid chromatography
LC Liquid chromatography
MS Mass spectrometry
PPB Plasma Protein Binding
RED Rapid Equilibrium Dialysis