Time Dependent Inhibition, LC-MS/MS Detection (IC50 Shift)
Assay System:
- Human liver microsomes, pooled from 50 donors.
- Inhibition potential against CYP isoforms (IC50) measured after pre-incubation with and without NADPH in the presence of different concentrations of test compound by monitoring activity of individual CYP enzymes using isoform-specific probe substrates with LC-MS/MS detection.
- Time-dependent inhibition (IC50 shift) is determined by comparing IC50 values determined after pre-incubation with and without NADPH.
Assay Conditions:
| Test Compound Concentration |
Eight concentrations ranging from 0.05 µM to 100 µM |
| Microsome Concentration |
0.1 mg/mL |
| Percent Organic Solvent in Incubation |
2% acetonitrile |
| CYP Isoforms Available for Testing |
CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP 3A4 |
| Pre-Incubation Time |
30 minutes |
| Incubation Time |
10-20 minutes (depending on CYP isoform) |
| pH |
7.4 |
| Temperature |
37 °C |
| Analysis |
LC-MS/MS |
| Number of Repeats |
2 |
Deliverable:
- IC50 of test compound and positive control compound both with and without NADPH pre-incubation.
- IC50 shift (IC50 -NADPH/ IC50 +NADPH)
Test Compound Requirement: 1-2 mg dry powder or 100-200 µL of 10 mM acetonitrile stock solution.
| CYP Isoform |
Probe Substrate |
Monitored Metabolite |
Positive Control Inhibitor |
| 1A2 |
Phenacetin |
Acetaminophen |
Furafylline |
| 2B6 |
Bupropion |
OH Bupropion |
Ticlopidine |
| 2C8 |
Amodiaquine |
Desethylamodiaquine |
Gemfibrozil Glucuronide |
| 2C9 |
Tolbutamide |
4-OH Tolbutamide |
Tienilic Acid |
| 2C19 |
S-Mephenytoin |
4’-OH Mephenytoin |
(S)-Fluoxetine |
| 2D6 |
Dextromethorphan |
Dextrorphan |
Paroxetine |
| 3A4 |
Testosterone |
6β-OH Testosterone |
Mibefradil |
| 3A4 |
Midazolam |
1’-OH Midazolam |
Mibefradil |
