TABLE 1

Incubation and analytical conditions used for cytochrome P450 probe substrates


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.