Measurement of drug concentration in brain and blood

Measurement of tissue drug concentration was performed at Pharmaron (Bejing, China). DO264 and (S)-DO271 were administered as described above and incubated for 4 h. The brain tissues were homogenized with 4x volume of water (mL) by brain weight (g). Blood was collected via cardiac puncture and immediately quenched with acetonitrile at 1:4 blood:acetonitrile ratio. The brain homogenate and blood samples from drug-treated mice were diluted by 1000 to 2000-fold with 50% ACN in H₂O. 5 μL of the diluted sample solutions were added to 50 μL of the blank C57BL/6J mice brain homogenate and blood respectively to make dose samples. For making calibration standards, the stock solutions of DO264 and (S)-DO271 (1 mg/mL in DMSO) were diluted with 50% ACN in H₂O to generate serial concentrations of working solution. 5 μL of the working solutions (5, 10, 20, 50, 100, 500, 1000, 5000, 10000 ng/mL) were added to 50 μL of the blank C57BL/6J mice blood to achieve calibration standards of 0.5-1000 ng/mL (0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng/mL). 55 μL of calibration standards and 55 μL dose samples were added to 200 μL of ACN for precipitating protein respectively. Then the samples were vortexed for 30 s. After centrifugation at 4 °C, 4000 rpm for 15 min, the supernatant was diluted 5 times with water. 5 μL of the diluted supernatant was injected into the LC/MS/MS system for quantitative analysis (LCMS-8060, SHIMADZU).