substrate concentration profile using Michaelis–Menten equation (Eq. 16.1).
Figure 16.1 shows the typical M–M kinetics of (S)-mephenytoin 4^0 -hydroxyla-
tion. Table 16.2 showsKmvalues of the various substrates for the individual
human CYP activities in HLM.
y¼Vmax½S
Kmþ½Sð 16 : 1 Þ16.2.4 LC/MS/MS Analysis
For all 16 assays in Table 16.1, HPLC separation of metabolite(s) formed from
each substrate was achieved using LC columns eluted with the following
mobile phase gradient at a flow rate of 1.5 mL/min. The mobile phase solvents
were used for CYP1A2 assay (A: 0.05% formic acid in water; B: 0.05% formic
acid in acetonitrile) and for all other CYP assays (A: 0.05% formic acid in
water:methanol 90:10; B: 0.05% formic acid in acetonitrile) as indicated in
Table 16.1. The columns were re-equilibrated to starting conditions before the
next set of samples was injected. HPLC flow was diverted from the mass
spectrometer to waste for the first and last minute of the gradient.
FIGURE 16.1 Hyperbolic saturation curve of CYP2C19-catalyzed (S)-mephenytoin
40 -hydroxylation in human liver microsomes.KmandVmaxvalues were calculated with
Equation 16.1 (Km= 23.72.15mM andVmax= 0.360.01 nmol/min/mg). Data were
fitted by nonlinear regression (Eq. 16.1, SigmaPlot 9.0).
520 ANALYSIS OFIN VITROCYTOCHROME P450 INHIBITION