Readout: Blood-to-Plasma ratio (%)
Fresh blood (EDTA-K2) is incubated in a humidified atmosphere of 5% CO2 at 37 °C. 1 mL blood is removed into a 1.5 mL tube and centrifuged.
A stock solution of the test compound and verapamil working solution with DMSO are spiked into 4 mL blood, respectively, and vortexed gently. Then 1 mL of blood is removed and centrifuged. At the 0 min time point, an aliquot of blood is removed and sonicated to lyse cells. Blank plasma and quenching solution are then added.
After incubation for 60 min in a CO2 incubator with shaking, an aliquot of blood (sonicated) is removed with addition of equal volume of blank plasma and centrifuged. The remaining incubation mixture is centrifuged to obtain plasma sample. An aliquot of plasma with equal volume of blank blood is removed and quenching solution is added.
After vortexing and centrifuging, the supernatant is analyzed by LC-MS/MS. 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 blood to plasma ratio is calculated from the following equation:
Blood-to-Plasma ratio (%) = (CB/CP) x 100%
Where CB is the LC-MS/MS response (peak area ratio to an internal standard) for the blood fraction
Where CP is the LC-MS/MS response (peak area ratio to an internal standard) for the plasma fraction
EDTA-K2 Ethylenediaminetetraacetic acid, dipotassium salt
HPLC High-performance liquid chromatography
LC Liquid chromatography
MS Mass spectrometry