In vitro Blood-Brain-Barrier Model. Permeation of test compounds through a human brain endothelial cell barrier was evaluated using the Corning Costar 3470 Transwell Plate system (0.4 µm pore diameter with 6.5 mm inserts) and HBEC-5i cells (ATCC, CRL-3245). Filter inserts were coated one day before seeding with 50 µL 0.01% rat-tail collagen type I (C7661, Sigma-Aldrich) in PBS. 60,000 HBEC-5i cells in 100 µL HBEC-5i-medium (DMEM/F12 with 10% FCS and 40 µg/mL ECGS) were seeded after aspiration of the coating-supernatant in each insert with 600 µL HBEC-5i-medium in each receiver well. After 24 h incubation at 37 °C with 5% CO2, medium was exchanged to 600 µL T98G cell supernatant in receiver wells and 100 µL fresh HBEC-5i-medium in inserts. The medium exchange was repeated every second day. On day 7 after seeding, medium in the receiver wells was replaced by 600 µL Hank's balanced salt solution (HBSS) containing 10 mM HEPES and 0.1% DMSO and 100 µL test compound mixtures (antipyrine, propranolol and test compound; each at 10 µM) in HBSS containing 10 mM HEPES and 0.1% DMSO were added to inserts. After 60 min incubation. 100 µL samples were taken from the receiver wells and diluted in 400 µL (75%/25% A/B; A=Formic acid 0.1% in water; B=MeCN). Test compound concentrations in the samples were determined by LC-MS/MS on an API-3200-QTrap (Sciex) equipped with an Agilent Technologies 1100 series setup including a binary pump (G1311A), a degasser (G1322A), and a Shimadzu SIL 20A HT autosampler under the control of Analyst 1.6 (Sciex). An XBridge BEH C18 (3.5 µm, 150 mm x 3 mm, Waters, protected with a 0.5 µm frit) served as stationary phase in combination with a gradient method starting with 0.1% formic acid in water (A) and MeCN (B) as mobile phase (80%:20% = A:B) for 6 min going to (50%:50%) after 8 min. 5 µL of aspirated supernatant diluted in mobile phase starting conditions was loaded onto the column, separated at a flow rate of 400 µL/min, and quantified per Area of MRM (multiple reaction monitoring).