Incubation conditions and analytical parameters for CYP3A and CYP2E1 assays
Chlorzoxazone 6-Hydroxylase (CYP2E1) | Felodipine Dehydrogenase (CYP3A) | Midazolam 1′-Hydroxylase (CYP3A) | Testosterone 6β-Hydroxylase (CYP3A) | |
|---|---|---|---|---|
| Incubation conditions | ||||
| Pooled human liver microsomes | ||||
| Protein concentration (mg/ml) | 0.05 | 0.01 | 0.03 | 0.03 |
| Incubation time (min) | 20 | 10 | 4 | 10 |
| Recombinant P450 | ||||
| P450 concentration (pmol/ml) | 6.3 | 0.095/0.61 | 0.95/1.2a | 2.4/6.1a |
| Incubation time (min) | 20 | 10/10 | 4/4a | 10/10a |
| Analytical conditions | ||||
| Analyte | 6-Hydroxychlorzoxazone | Dehydrofelodipine | 1′-Hydroxymidazolam | 6β-Hydroxytestosterone |
| Internal standard: identity | [2H215N]6-Hydroxychlorzoxazone | [2H3]Dehydrofelodipine | [2H4]1′-Hydroxymidazolam | [2H3]6β-Hydroxytestosterone |
| Internal standard: final conc. (μM) | 5.0 | 0.25 | 0.10 | 2.5 |
| Injection volume (μl) | 20 | 10 | 20 | 30 |
| Mobile phase | ||||
| System | 1 | 1 | 1 | 1 |
| Gradient program, %B(min) | 2(0)→15(0.5)→40(2.5) | 2(0)→40(0.5)→98(2.8) | 2(0)→28(0.5)→58(3) | 2(0)→24(0.5)→36(2.6) |
| Mass spectrometer conditions | ||||
| Mode | Negative | Positive | Positive | Positive |
| Capillary voltage | -2.00 kV | 3.25 kV | 3.25 kV | 3.65 kV |
| Cone voltage | -60 V | 60 V | 50 V | 50 V |
| Desolvation temperature | 300°C | 325°C | 275°C | 350°C |
| Source temperature | 110°C | 110°C | 110°C | 110°C |
| Collision energy | 40 eV | 25 eV | 21 eV | 16 eV |
| Analyte m/z transition | 184→120 | 382→354 | 342→324 | 305→269 |
| Internal standard m/z transition | 187→123 | 387→359 | 346→328 | 308→272 |
| Retention time (min) | 2.0 | 1.9 | 2.0 | 2.0 |
| Standard curve range (nM) | 100-4000 | 5.00-2000 | 1.00-1000 | 30.0-10,000 |
↵ a Values represent those for recombinant CYP3A4/CYP3A5