During the enzyme reaction of a substrate, multiple metabolite isomers may
be formed. Thus, a baseline LC separation of the metabolites is desired. For
example, more than five hydroxylated testosterone isomers (2-, 6-, 15-, and 16-
positions) were seen after an incubation of testosterone with HLM. BDS
Hypersil C8 was successfuly used for a separation of the five isomers
(Table 16.1). Majority of the metabolites (>82%) was due to the 6b-OH-
testosterone. Similarly, two hydroxylated isomers of midazolam at 1 and 4^0 -
positions were also isolated with the given HPLC condition.
Mass to charge value for each metabolite formed is shown in Table 16.1.
For quantitation of the metabolites, the mass spectrometer was operated in the
multiple reaction-monitoring (MRM) modes to monitor the metabolite(s) and
internal standard with dwell time set to 150 ms for each reaction. After
optimization, heated nebulizer parameters were set as follows (arbitrary units):
NEB: 10, CUR: 8, TEMP: 300C, and NC: 5. The flow rate of heated gas (gas
2) was operated at 5 L/min.
16.2.5 Automated Sample Preparation and Incubation
An automation procedure for sample preparation and incubation has been
described previously (Yao et al., 2007). A working solution of HLM was
prepared with phosphate buffer to form 0.1 to 0.25 mg/mL for the assays.
Metabolite standards were manually prepared with 50% acetonitrile in water
as stock solution, and further diluted with HLM for quantitation of the
metabolites formed. Individual probe substrates were prepared at designated
concentrations around theirKmvalues. Positive controls (inhibitors) or test
compounds were prepared in DMSO (or 50% acetonitrile) as stock solution.
TABLE 16.2 Kmvalues for individual CYPs in pooled human liver microsomes.
Enzyme
involved Substrate Metabolite Internal standard Km(mM)
CYP1A2 Phenacetin Acetaminophen 40 -OH-Butyranilide 87.7
CYP2A6 Coumarin 7-OH-Coumarin None 97.8
CYP2B6 Bupropion OH-Bupropion DL-Propranolol 98.0
CYP2C8 Taxol 6 a-OH-Taxol Baccatin III 14.4
CYP2C9 Diclofenac 40 -OH-Diclofenac Flufenamic acid 11.2
CYP2C19 (S)-Mephenytoin 4^0 -Mephenytoin Phenytoin 77.9
CYP2D6 Bufuralol 10 -OH-Bufuralol DL-Propranolol 14.0
CYP2D6 Dextromethorphan Dextrorphan Levallorphan 11.7
CYP2E1 Chlorzoxazone 6-OH-Chlorzoxazone Cpd A 274
CYP3A4 Testosterone 6 b-OH-Testosterone Cortisone 47.2
CYP3A4 Nifedipine Oxidized nifedipine Nimodipine 13.0
CYP3A4 Midazolam 10 -OH-Midazolam 10 -OH-Triazolam 2.98
CYP3A4 Midazolam 4-OH-Midazolam 4-OH-Triazolam 24.4
REVERSIBLE INHIBITION 521