Summary of incubation conditions and analytical methods for different CYP3A substrates
Substrate | Concentration Range | Incubation Time | Protein Concentration | Extraction Solvent | Internal Standard | Analytical Method | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| cDNA | Liver | cDNA | Liver | cDNA | Liver | Column | Mobile Phase | Detection | ||||||||
| μM | min | pmol/ml | mg/ml | |||||||||||||
| TST | 5-200a | 20 | 15 | 15 | 10a | 0.05 | Ethyl acetate | 11 α-Progesterone | Develosil ODS-UG-5 column (4.6 × 100 mm, 5 μm) | Gradient of methanol/water/acetonitrile | UV, 240 nm | |||||
| 5-400b | 2b | |||||||||||||||
| ERM | 1.25-235 | 5 | 15 | 15 | 100 | 0.25 | Hexane | None | Monitoring [14C]formaldehyde formation from the [N-methyl-14C]erythromycin as described by Zhang et al. (1996) | |||||||
| MDZ | 0.5-200 | 0.5 | 2 | 4 | 10 | 0.05 | Ethyl acetate | 15N3-1′-OH/4-OH midazolam | GC/MS method as described (Paine et al., 1997) | |||||||
| FLZ | 10-500 | 30 | 30 | 30 | 60 | 0.3 | Methanol | Clonazepam | C18 Inertisil ODS-2 column (4.6 × 150 mm, 5 μm) | Water/methanol/acetonitrile (55:40:5) | UV, 234 nm | |||||
| CBZ | 50-2000 | 50 | 30 | 30 | 80 | 0.5 | t-Butyl methyl ether | 2-Methyl carbamazepine | Zorbax C8 column (4.6 × 250 mm, 5 μm) | Water/methanol/acetonitrile (50:40:10) | UV, 210 nm | |||||
| DXM | 10-1500 | 10 | 15 | 15 | 100 | 0.25 | Acetonitrile | None | Zorbax C8 column (4.6 × 150 mm, 5 μm) | 70% 15 mM ammonium acetate (pH 4) and 30% acetonitrile | Fluorescence (excitation, 235 nm; emission, 310 nm) | |||||
| TFN | 0.05-200 | 0.2 | 2 | 2 | 5 | 0.01 | Acetonitrile | Ethoxy-oxo-terfenadine | Zorbax C8 column (2.1 × 50 mm, 5 μm) | 5 mM ammonia acetate/methanol gradient | LC/MS; the monitored ions were m/z 472, 488, 502, and 528, which correspond to the [M + H+] of TFN, TFN alcohol, TFN acid, and internal standard | |||||
| LDC | 25-3200 | 10 | 15 | 45 | 20 | 0.2 | t-Butyl methyl ether | Procaine | Phenomenex C8 guard column and Microsorb-MV C18 column (24 cm) | Gradient of acetonitrile and 0.1 M KPi/0.01% triethylamine (pH 3.0) | UV, 214 nm | |||||
| ITZ |
| 0.5 |
| 2 |
| 0.05 | acetonitrile | None | Zorbax C8 column (2.1 × 50 mm, 5 μm) | 5 mM ammonium acetate (pH 7)/acetonitrile gradient | LC/MS; the monitored ions were m/z 721 and 705, which correspond to the [M + H+] of ITZ-OH and ITZ | |||||
GC/MS, gas chromatography/mass spectrometry; LC/MS, liquid chromatography/mass spectrometry.
↵ a Kinetic comparison of CYP3A4 and CYP3A5 (coexpressed with reductase and supplemented with b5).
↵ b Kinetic comparison of CYP3A4 coexpressed with reductase and b5 and CYP3A4 coexpressed with reductase and supplemented with reductase and b5.