| Assay Detail |
Compounds were quantified using an LC/MS assay. Low resolution mass spectra (ESI) were collected on a Shimadzu HPLC, CTC autosampler & API 4000 (AB SCIEX, USA). Compounds were resolved on a Phenomenex Luna C18 (2.0 × 50 mm^2, 5 μm) column at room temperature with a flow of 0.5 mL/min. The gradient consisted of eluents A (0.1% formic acid in double distilled water) and B (0.1% formic acid in HPLC-grade acetonitrile). The gradient method started at 5% of B and followed a linear gradient to 95% B in 2.0 minutes. The column was then washed with 95% B for 0.2 minutes, brought back down to 5% B in 0.2 minutes, and equilibrated at 5% B for an additional 0.6 minutes. Samples (50 μL) from the Caco-2 compartments were mixed with methanol (μL) containing internal standards (200 nM Imipramine,200 nM Labetalol and 200 nM Diclofenac) and injected into the LC/MS. Papp was quantified according to the following equation: Papp = {V_a / (Area * time)} * {[drug]acceptor / [drug]initial_donor}.
Human, epithelial Caco-2 cells were seeded onto a 24-well transwell plate at a density of 3.90 х 10^4 cells/cm^2. The medium was changed every other day and cells were utilized after 21 days. Sample stock solutions in DMSO were diluted to 5 μM with HBSS (10 mM HEPES, pH 7.4). Compounds were analyzed in the apical to basolateral (A-B) direction by adding 100 μL compound solution to apical compartment, 600 μL HBSS to basolateral compartment, incubating for 2 hours at 37°C, and then taking sample from both compartments as per the LC/MS method. Compounds were analyzed in the basolateral to apical (B-A) direction by adding 100 μL HBSS to apical compartment, 600 μL compound solution to basolateral compartment, incubating for 2 hours at 37°C, and then taking samples from both compartments as per the LC/MS method.
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