TABLE 2

Incubation conditions and analytical parameters for CYP2C9, 2C19, and 2D6 assays

Diclofenac 4′-Hydroxylase (CYP2C9)
Tolbutamide Methylhydroxylase (CYP2C9)
(S)-Mephenytoin 4′-Hydroxylase (CYP2C19)
Dextromethorphan O-Demethylase (CYP2D6)
Incubation conditions
   Pooled human liver microsomes
      Protein concentration (mg/ml) 0.03 0.1 0.2 0.03
      Incubation time (min) 10 20 40 10
   Recombinant P450
      P450 concentration (pmol/ml) 1.6 7.7 3.7 1.2
      Incubation time (min) 10 10 20 14
Analytical conditions
   Analyte 4′-Hydroxydiclofenac 4-Hydroxytolbutamide 4′-Hydroxymephenytoin Dextrophan
   Internal standard: identity [13C6]4′-Hydroxydiclofenac [2H9]4-Hydroxytolbutamide [2H3]4′-Hydroxymephenytoin [2H3]Dextrorphan
   Internal standard: final conc. (μM) 0.50 1.0 2.0 0.20
   Injection volume (μl) 20 20 20 20
   Mobile phase
      System 1 1 1 1
      Gradient program, %B(min) 2(0)→40(0.5)→78(2.5) 2(0)→20(0.5)→60(2.5) 2(0)→20(0.5)→72(2.5) 2(0)→10(0.5)→38(2.5)
   Mass spectrometer conditions
      Mode Negative Positive Negative Positive
      Capillary voltage -3.00 kV 3.00 kV -3.25 kV 2.75 kV
      Cone voltage -45 V 50 V -50 V 60 V
      Desolvation temperature 300°C 325°C 300°C 300°C
      Source temperature 110°C 110°C 110°C 110°C
      Collision energy 12 eV 18 eV 13 eV 40 eV
      Analyte m/z transition 310→266 287→171 233→190 258→157
      Internal standard m/z transition 316→272 296→171 236→193 261→157
      Retention time (min) 2.0 2.0 1.7 2.0
   Standard curve range (nM)
10.0-2000
10.0-5000
50.0-10,000
1.00-1000