HPLC analysis of P450 probes Separations were performed on a Zorbax SB-C8 analytical column (5 μm, 4.6 × 75 mm; Agilent Technologies) using linear gradients, 10 mM ammonium acetate in water as mobile phase A, and 10 mM ammonium acetate in 9:1 acetonitrile/methanol (v/v) as mobile phase B. Analytes were eluted at a flow rate of 2 ml/min.
Isoform | Probe | Gradient (% B) | Detection |
|---|---|---|---|
| 1A2 | Phenacetin | 10-50 | UV, 250 nm |
| 2D6 | Bufuralol | 15-45 | Fluorescence, λex = 252 nm, λem = 302 nm |
| 2C9 | Warfarin | 35-45 | Fluorescence, λex = 320 nm, λem = 415 nm |
| 2E1 | Chlorzoxazone | 8-65 | UV, 287 nm |
| 3A4 | Testosterone | 15-45 | UV, 230 nm |
ex, excitation; em, emission.