LC-MS/MS analysis of plasma, kidney, liver, skin, and spleen samples was carried out using a Shimadzu LC-20AD coupled with an AB Sciex API 4000 (Shimadzu, Kyoto, Japan). Standard curve samples (20 µL), quality control samples and rat plasma samples were mixed with 60 µL of acetonitrile containing internal standard (200 ng/mL of tolbutamide or 50 ng/mL of propranolol) in Eppendorf tubes. After vortexing the mixture for 1 minute, the mixture was centrifuged for 10 minutes at 13,000 rpm at 4°C and then the supernatant (50 µL) was transferred to a 96-well plate which was pre-added in 150 µL of water. After shaking the samples for 10 minutes, 10 µL of sample was injected into LC-MS/MS. The separation was carried out on a Thermo Betasil C18 column (2.1×50 mm, 5 µm particle size) with 0.1% trifluoroacetic acid in water (A) and acetonitrile (B) as the mobile phase at a flow rate of 0.5 mL/minute. Method: 10% B (0–0.5 minutes), 10%–95% B (0.5–1.1 minutes), 95% B (1.1–1.4 minutes), 95%–10% B (1.4–1.5 minutes), 10% B (1.5–2.2 minutes). Mass transitions: 260.3/116.2 for propranolol (internal standard); 761.4/289.1 for BMK-20113.
The lung, muscle, and CSF samples were analyzed by LC-MS/MS using a Shimadzu LC-20AD coupled with an AB Sciex API 4000. The other analytical conditions are the same as those used for the plasma, kidney, liver, skin, and spleen samples.
The lung, muscle, and CSF samples were analyzed by LC-MS/MS using a Shimadzu LC-20AD coupled with an AB Sciex API 4000. The other analytical conditions are the same as those used for the plasma, kidney, liver, skin, and spleen samples.