P450 Isoform | Substrate | Enzyme (pmol P450/inc) | Metabolite | Internal Standard (200 μl of Acetonitrile) | Mobile Phase Acetonitrile/10 mM Potassium Phosphate Buffer (pH 8.0) | Excitation | Emission |
---|---|---|---|---|---|---|---|
nm | |||||||
CYP1A2 | 2.5 μM CEC | 0.5 | CHC | 0.5 μM HMC | 0–10 min; 20:80–70:30 linear gradient | 408 | 450 |
CYP2A6 | 0.5 μM Coumarin | 0.4 | HC | 0.1 μM HMC | 0–5 min; 20:80–30:70 linear gradient, 5–10 min; 30:70 | 368 | 456 |
CYP2C8 | 0.5 μM DBF | 2.5 | FL | 40 nM DCFL | 0–5 min; 15:85–32:68 linear gradient, 5–12 min 70:30 | 490 | 525 |
CYP2C9 | 35 μM MFL | 2.5 | FL | 40 nM DCFL | 0–10 min; 15:85–35:65 linear gradient | 490 | 525 |
CYP2C19 | 1.5 μM MFL | 2.5 | FL | 40 nM DCFL | 0–10 min; 15:85–35:65 linear gradient | 490 | 525 |
CYP2D6 | 5.0 μM MAMC | 2.5 | HAMC | 0.1 μM HC | 0–10 min; 10:90–30:70 linear gradient | 370 | 470 |
CYP2E1 | 100 μM MFC | 5.0 | HFC | 0.25 μM HC | 0–10 min; 20:80–70:30 linear gradient | 385 | 502 |
CYP3A4 | 0.5 μM DBF | 0.5 | FL | 40 nM DCFL | 0–5 min; 15:85–32:68 linear gradient, 5–12 min 70:30 | 490 | 525 |
CHC, 3-cyano-7-hydroxycoumarin; HC, 7-hydroxycoumarin; FL, fluorescein; HAMC, 7-hydroxy-4-(aminomethyl)coumarin; HFC, 7-hydroxy-4-(trifluoromethyl)coumarin; HMC, 7-hydroxy-4-methylcoumarin; DCFL, 2,7-dichlorofluorescein.