TABLE 1

Incubation conditions and analytical parameters for CYP1A2, 2A6, 2B6, and 2C8 assays




Phenacetin O-Deethylase (CYP1A2)

Coumarin 7-Hydroxylase (CYP2A6)

Bupropion Hydroxylase (CYP2B6)

Amodiaquine N-Deethylase (CYP2C8)a
Incubation conditions
   Pooled human liver microsomes
      Protein concentration (mg/ml) 0.03 0.03 0.05 0.025
      Incubation time (min) 30 6 20 10
Recombinant P450
      P450 concentration (pmol/ml) 1.2 2.1 1.8 1.7
      Incubation time (min) 20 6 20 10
Analytical conditions
   Analyte Acetaminophen 7-Hydroxycoumarin Hydroxybupropion Desethylamodiaquine
   Internal standard: identity [2H7]Acetaminophen [2H3]7-Hydroxycoumarin [2H6]Hydroxybupropion [2H5]Desethylamodiaquine
   Internal standard: final cone. (μM) 2.0 0.40 0.80 0.75
   Injection volume (μl) 20 20 10 10
   Mobile phase
      System 1 1 1 2
      Gradient program, %B(min) 2(0)→2 (0.5)→98(2.5) 2(0)→2(0.5)→71(2.6) 2(0)→2(0.5)→71(2.7) 2(0)→8(0.5)→18(2.7)
   Mass spectrometer conditions
      Mode Positive Negative Positive Positive
      Capillary voltage 1.25 kV -2.00 kV 2.00 kV 2.75 kV
      Cone voltage 50 V -50 V 30 V 40 V
      Desolvation temperature 325°C 325°C 325°C 325°C
      Source temperature 110°C 110°C 110°C 110°C
      Collision energy 15 eV 22 eV 26 eV 20 eV
      Analyte m/z transition 152→110 161→133 256→139 328→283
      Internal standard m/z transition 159→115 164→136 262→139 333→283
      Rt (min) 1.6 2.2 2.0 1.8
   Standard curve range (nM)
25-10000
10.0-2000
5.00-2000
10.0-1500
  • a The amodiaquine method required injection syringe washes of 50/50 dimethyl sulfoxide/water to prevent assay carryover