Analytical methods for the separation and detection of metabolites of the CYP-marker reactions
| Reaction | Analytical Method | Wavelength | Mobile Phase and Run Procedure | Retention Times |
|---|---|---|---|---|
| nm | min | |||
| 7-Ethoxyresorufin- | Fluorometric | excitation: 544 | ||
| O-dealkylation | emission: 590 | |||
| Chlorzoxazone | HPLC/UV | 287 | A: 20 mM NaClO4 (pH 2.5) | 6-OH-chlorzoxazone: 4.2 |
| 6-hydroxylation | B: acetonitrile. Gradient: 0–5 min (20%) B, 5–5.1 min (20– | chlorzoxazone: 12.8 | ||
| 30%) B, 5.1–14 min (30%) B, 14–14.1 min (30–20%) B, | ||||
| 14.1–16 (20%) B. Flow rate: 1.0 ml/min. | ||||
| Testosterone | HPLC/UV | 254 | 60% methanol in 50 mM KH2PO4 | 6β-OH-testosterone: 3.6 |
| 6β-hydroxylation | Flow rate: 1 ml/min | testosterone: 16 | ||
| Bufuralol | HPLC/Fluoro. | excitation: 252 | A: 20 mM perchlorate B: acetonitrile. Isocratic run of | 1′-OH-bufuralol: 3.3 |
| 1′-hydroxylation | emission: 302 | 70%A and 30%B. Flow rate: 1.0 ml/min. | bufuralol: 16.2 | |
| Paclitaxel | HPLC/UV | 230 | 40% acetonitrile, 10% methanol in 0.1 M ammonium | 6α-OH-paclitaxel: 9.0 |
| 6α-hydroxylation | acetate. Flow rate: 1 ml/min | paclitaxel: 14.7 | ||
| S-Mephenytoin | HPLC/UV | 222 | A: acetonitrile B: 20 mM NaClO4, 36 mM K2HPO4, | 4-OH-mephenytoin: 6.2 |
| 4-hydroxylation | 14mM KH2PO4 and 17% acetonitrile. Gradient: 0– | S-mephenytoin: 8.8 | ||
| 6 min (100%) B, 6–7.5 min (100–30%) B 10.5–10.6 min (30– | ||||
| 100%) B, 10.6–13 min (100%) B. Flow rate: 1.5 ml/min | ||||
| Diclofenac | HPLC/UV | 280 | A: 20% acetonitrile in 1 mM perchloric acid B: methanol. | 4-OH-diclofenac: 8.2 |
| 4-hydroxylation | Gradient: 0–3 (40%) B, 3–8 (40–100%) B, 8–10 (100–40%) B. | diclofenac: 9.4 | ||
| Flow rate: 1 ml/min |