Method for (S)-mephenytoin 4′-hydroxylase, dextromethorphan O-demethylase, terfenadine hydroxylase, midazolam 1′-hydroxylase, and testosterone 6β-hydroxylase assays
Parameter(s) | (S)-Mephenytoin 4′-Hydroxylase | Dextromethorphan O-Demethylase | Terfenadine Hydroxylase | Midazolam 1′-Hydroxylase | Testosterone 6β-Hydroxylase |
|---|---|---|---|---|---|
| (CYP2C19) | (CYP2D6) | (CYP2J2) | (CYP3A) | (CYP3A) | |
| Microsomal protein concentration (mg/ml) | 0.25 | 0.15 | Not conducted | 0.1 | 0.15 |
| Recombinant P450 concentration (pmol/ml) | Not conducted | Not conducted | 2 | 5 | Not conducted |
| Incubation time (min) | 5 | 5 | 5 | 5 | 5 |
| Substrate (final concentration, μM) | (S)-Mephenytoin (55) | Dextromethorphan (10) | Terfenadine (1) | Midazolam (5) | Testosterone (75) |
| Analyte | 4′-Hydroxymephenytoin | Dextrorphan | Terfenadine alcohol | 1′-Hydroxymidazolam | 6β-Hydroxytestosterone |
| Internal standard | 6-Hydroxychlozoxazone | Propranolol | Trazodone | α-Hydroxymidazolam-D4 | 6β-Hydroxytestosterone-D3 |
| Column | Luna Pheny-hexyl 2 × 150 mm, 5 μm | Luna Pheny-hexyl 2 × 150 mm, 5 μm | Luna Pheny-hexyl 2 × 150 mm, 5 μm | Zorbax SB-C18 150 × 2.1 mm, 5 μm | Zorbax SB-C18 150 × 2.1 mm, 5 μm |
| Flow rate | 0.3 ml/min | 0.3 ml/min | 0.3 ml/min | 0.6 ml/min | 0.6 ml/min |
| Mobile phase A | 0.1% Formic acid | 0.1% Formic acid | 0.1% Formic acid | 0.1% Formic acid | 0.1% Formic acid |
| Mobile phase B | ACN containing 0.1% formic acid | ACN containing 0.1% formic acid | ACN containing 0.1% formic acid | ACN containing 0.1% formic acid | Methanol containing 0.1% formic acid |
| Mass spectrometer conditions | |||||
| Mode | Negative | Positive | Positive | Positive | Positive |
| Declustering potential (V) | -55 | 81 | 106 | 76 | 71 |
| Collision energy (V) | -22 | 53 | 39 | 31 | 21 |
| Turbo-V source temperature (°C) | 400 | 400 | 350 | 400 | 350 |
| Analyte m/z transition | 233 → 190 | 258 → 157 | 488 → 452 | 342 → 324 | 305 → 269 |
| Internal standard m/z transition | 184 → 120 | 260 → 183 | 372 → 148 | 346 → 328 | 308 → 272 |
| Standard curve range (nM) | 2-100 | 25-10,000 | 0.1-500 | 2-1000 | 5-5000 |