known mechanism-based inhibitors ranged from 0.04 (ritonavir) to 108mM
(rutaecarpine), and from 0.01 (diltiazem) to 1.62 min^1 (L-754394), respectively
(Atkinson et al., 2005; Chiba et al., 1995; Iwata et al., 2005; Luo et al., 2003).
16.3.3 General Incubation Procedure and Sample Preparation
A general experimental procedure for an enzyme inactivation reaction has been
described previously (Lu et al., 2003). The incubation mixtures contained liver
microsomal proteins (0.5–2 mg/mL final concentration), 1 mM NADPH and
100 mM potassium phosphate buffer (pH 7.4). The samples were incubated at
37 C (up to 30 min) in the presence of solvent alone, or an inhibitor at varying
concentrations (eight concentration points assigned between 0.25 and 4KI)ina
shaking water bath (final volume of 0.2 mL). All chemical inhibitors and
substrates were dissolved in 50% (v/v) acetonitrile/water. The final volume of
acetonitrile in the incubation was less than 1% (v/v). At the appropriate times (0,
5, 10, 20, and 30 min), aliquots (25mL) of the incubate were removed and added
to separate vials containing 225mL (10-fold dilution) of fresh buffer preheated at
37 C. A marker substrate (Table 16.1) and NADPH (1.0 mM) were added and
the reaction was allowed to proceed for additional 10 min. The reaction was
terminated with acetonitrile (two volumes) containing an appropriate amount of
internal standard. The samples were vortexed, centrifuged (3500 rpm for 10 min)
and the supernatant was separated and diluted with an equal volume of 0.05%
formic acid in water. Aliquots (20mL) were subjected to LC/MS/MS analysis.
Incubations carried out in the presence of solvent alone (time zero) were
designated controls (100%). HPLC flow was diverted from the mass
FIGURE 16.6 (Continued)IRREVERSIBLE INHIBITION 531