| Assay Method Information | |
| | Inhibition Assay |
| Description: | Procedure: 1. A 10 mM solution of a test drug in DMSO was serially diluted with DMSO at four 5-fold dilutions to prepare 10, 2, 0.4 and 0.08 mM DMSO solutions. 2. Each test drug solution of step 1 or DMSO was diluted 160-fold with the human liver microsome solution and dispensed at a concentration of 80 uL/well to a microplate. 3. 10 uL of the substrate solution and 10 uL of the beta-NADPH solution were added to each well of step 2, and the mixture was incubated at 37C. for 10 min. 4. The reaction was terminated by the addition of 300 uL of methanol. 5. The reaction mixture was filtered and analyzed by LC-MS/MS. 3-4 Quantification and CalculationThe amount of 1'-hydroxybufuralol formed was determined by LC-MS/MS, and this value was used as the metabolic activity value of CYP2D6 in each well. The residual activity of each sample-supplemented group was determined by comparison with the activity in the well containing DMSO used as a test drug. |
| Affinity data for this assay | |
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