Incubation conditions and analytical parameters for CYP2C9, CYP2D6, and CYP2E1 metabolism assays
| P450 | CYP2C9 | CYP2D6 | CYP2D6 | CYP2E1 |
|---|---|---|---|---|
| Substrate | Flurbiprofen | Bufuralol | Dextromethorphan | Chlorzoxazone |
| Metabolite | 4′-Hydroxyflurbiprofen | 1′-Hydroxybufuralol | Dextrorphan | 6-Hydrochlorzoxazone |
| Internal standard | 2-Fluoro-4-biphenyl acetic acid | Levallorphan | Levallorphan | 7-Hydroxycoumarin |
| Incubation conditions | ||||
| Substrate concentrations, μM | 2–300 | 0.5–50 | 1–200 | 5–1000 |
| Incubation time, min | 20 | 10 | 20 | 20 |
| Quench solvent | 90% Acetonitrile/10% phosphoric acid, 100 μl | Acetonitrile, 100 μl | 50% Phosphoric acid/50% water, 50 μl | Acetonitrile, 100 μl |
| Analytical parameters | ||||
| Injection volume, μl | 30 | 20 | 30 | 50 |
| Column | Brownlee Spheri-5 C18, 4.6 × 100 mm (PerkinElmer Life and Analytical Sciences, Waltham, MA) | Aqua C18, 2 × 150 mm (Phenomenex, Torrance, CA) | Cyano, 4.6 × 150 mm (Regis Technologies, Morton Grove, IL) | YMC-Pack Pro C8, 4.6 × 150 mm (YMC America, Allentown, PA) |
| Mobile phase A | 90% 5 mM Potassium phosphate (pH 3)/10% acetonitrile | 100 mM Ammonium acetate (pH 3) | 10% Acetonitrile/0.25% triethylamine (pH 3) | 10 mM Ammonium acetate |
| Mobile phase B | Acetonitrile | Acetonitrile | Methanol | Acetonitrile |
| Flow rate, ml/min | 1 | 0.45 | 1 | 1 |
| Program, % B | Isocratic, 33 | 20 (0 min) to 60 (8 min) to 20 (9 min) to 20 (10.5 min) | Isocratic, 10 | Isocratic, 20 |
| λ (nm), excitation/emission | 260/320 | 252/302 | 280/310 | 287 |
| Analyte retention time, min | 2.5 | 2.1 | 3.5 | 5.4 |
| Source | Tracy et al., 2002 | Crespi et al., 2006 | Subramanian et al., 2009 | Chittur and Tracy, 1997 |