Incubation and analysis conditions to determine IC50 values in vitro in human liver microsomes for trans-(±)-2-phenylcyclopropylamine (tranylcypromine) and cyclopropylbenzene in seven CYP-specific activities
| CYP | Substrate, Conc. in Incubation | Protein | Cofactor System | Incubation Time | Determination, λ | HPLC Conditions |
|---|---|---|---|---|---|---|
| mg/ml | min | nm | ||||
| CYP1A2 | Ethoxyresorufin, 1 μM | 0.1 | NADPH-regenerating | 5 | Fluorometric, ex 530, em 585 | |
| CYP2A6 | Coumarin, 10 μM | 0.2 | NADPH-regenerating | 10 | Fluorometric, ex 395, em 454 | |
| CYP2C9 | Tolbutamide, 200 μM | 0.75 | NADPH-regenerating | 20 | UV-HPLC, 236 | Column: Symmetry C18 3.9 × 150 mm, 5-μm (Waters Co.) |
| CYP2C19 | rac-Mephenytoin, 100 μM | 0.5 | NADPH | 20 | UV-HPLC, 204/240 | Flow: 1.0 ml/min |
| CYP2D6 | Dextromethorphan, 100 μM | 0.5 | NADPH-regenerating | 20 | UV-HPLC, 204/280 | Eluent: CYP2C19: 50 mM H3PO4, pH 7.0 + acetonitrile (75:25); CYP2C9, CYP2E1: 50 mM |
| CYP2E1 | Chlorzoxazone, 100 μM | 0.5 | NADPH-regenerating | 20 | UV-HPLC, 282 | H3PO4, pH 3.0 + acetonitrile (70:30); CYP2D6: 50 mM H3PO4, pH 3.0 + acetonitrile (75:25); CYP3A4: 40% acetonitrile |
| CYP3A4/5 | Midazolam, 10 μM | 0.5 | NADPH | 10 | UV-HPLC, 245 |
ex, excitation; em, emission.