Incubation Conditions | Probe Substrate | ||||||
---|---|---|---|---|---|---|---|
Midazolam | (±)-Bufuralol | Diclofenac | Phenacetin | ||||
HLMs | |||||||
Substrate concentration (μM) | 1.0 | 0.2 | 1.0 | 1.0 | |||
Protein concentration (mg/ml) | 0.2 | 0.2 | 0.5 | 1.0 | |||
Incubation time (min) | 30 | 60 | 30 | 60 | |||
Recombinant P450 | |||||||
Substrate concentration (μM) | 0.2 | 0.1 | 0.5 | 1.0 | |||
Protein concentration (mg/ml) | 20 | 20 | 20 | 50 | |||
Incubation time (min) | 30 | 30 | 30 | 15 | |||
Analytical conditions | |||||||
IS identity | Triazolam | Propranolol | Gemfibrozil | Acetanilide | |||
IS concentration (μM) | 2 | 2 | 10 | 2 | |||
Injection volume (μL) | 40 | 20 | 50 | 40 | |||
Mobile phase system | 1 | 1 | 2 | 1 | |||
LC-MS/MS conditions | |||||||
Mode | Positive | Positive | Negative | Positive | |||
Capillary voltage (V) | 3.5 | 3.5 | 2.5 | 3.5 | |||
Cone voltage (kV) | 50 | 30 | -25 | 40 | |||
Desolvation temperature (°C) | 250 | 250 | 150 | 250 | |||
Source temperature (°C) | 100 | 100 | 80 | 100 | |||
Collision energy (eV) | 25 | 20 | 10 | 20 | |||
Analyte MRM transition | 326 → 292 | 262 → 188 | 294 → 250 | 180 → 110 | |||
IS MRM transition | 343 → 309 | 260 → 116 | 249 → 121 | 136 → 94 | |||
Retention time (min) | 1.3 | 1.2 | 1.6 | 1.1 |
IS, internal standard; MRM, multiple reaction monitoring.