Summary of incubation conditions and analytical methods for in vitro metabolism assays
| Index Reaction | Reaction Time | Microsomal Protein Concentration | Internal Standard | Analytical Method | ||||
|---|---|---|---|---|---|---|---|---|
| Liver | Lymphoblast | Column2-a | Mobile Phase | Flow Rate | Detection System | |||
| min | μg/ml | ml/min | ||||||
| Ethoxyresorufin O-deethylation (CYP1A2) | 5 | 62.5 | 160 | Phenacetin | HPLC; A | 65:35 25 mM KH2PO4 (pH 7):CH3OH | 0.8 | Resorufin: fluorescence (excitation 530 nm, emission 580 nm); phenacetin: UV 230 nm |
| Methoxyresorufin O-deethylation (CYP1A2) | 4 | 62.5 | 80 | Phenacetin | HPLC; A | 65:35 25 mM KH2PO4 (pH 7):CH3OH | 0.8 | Resorufin: fluorescence (excitation 530 nm, emission 580 nm); phenacetin: UV 230 nm |
| Phenacetin O-deethylation (CYP1A2) | 20 | 250 | 1000 | 2-Acetamido-phenol | HPLC; B | 87:13 50 mM KH2PO4:CH3CN | 1.5 | UV 254 nm |
| Bupropion hydroxylation (CYP2B6) | 20 | 250 | 250 | Trazodone | HPLC; B | 79:21 50 mM KH2PO4 (pH 3):CH3CN | 2.0 | UV 214 nm |
| S-mephenytoin 4-hydroxylation (CYP2C19) | 60 | 500 | 1000 | Phenacetin | HPLC; B | 80:20 50 mM KH2PO4:CH3CN | 1.5 | UV 204 nm |
| Conversion of trazodone to m-CPP (CYP3A) | 6 | 200 | 500 | Dextrorphan | HPLC; B | 80:20 50 mM KH2PO4:CH3CN | 1.4 | UV 249 nm |
| Triazolam 1-hydroxylation (CYP3A) | 20 | 200 | 500 | Phenacetin | HPLC; A | 67.5:22.5:10 50 mM KH2PO4:CH3CN:CH3OH | 1.3 | UV 220 nm |
| Diazepam 3-hydroxylation (CYP3A) | 20 | 500 | 1000 | U-31485 (Pharmacia & Upjohn) | GC; 3% SP-2250 packed 6-ft coiled glass, 2 mm i.d. (Supelco); 275°C | Argon:methane (95:5) | 30 | 63Ni electron-capture detector at 310°C (Hewlett-Packard) |
| Bufuralol 1′-hydroxylation (CYP2D6) | 20 | 125 | 80 | Trazodone | HPLC; B | 70:30 50 mM KH2PO4:CH3CN | 1.4 | Hydroxybufuralol: fluorescence (excitation 530 nm, emission 580 nm); trazodone: UV 230 nm |
| Desipramine 2-hydroxylation (CYP2D6) | 10–20 | 250 | 100 | Trazodone | HPLC; B | 67:33 50 mM KH2PO4:CH3CN | 1.3 | UV 254 nm |
| Nortriptyline E-10 hydroxylation (CYP2D6) | 10–20 | 250 | 200 | Chlorzoxazone | HPLC; B | 70:30 50 mM KH2PO4:CH3CN | 1.4 | UV 240 nm |
↵2-a A and B refer to C1815-cm × 3.9-mm NovaPak and C18 30-cm × 3.9-mm μBondapak reversed-phase steel columns (Waters), respectively.
GC, gas chromatography; m-CPP,meta-chlorophenylpiperazine.