Fast Quantitation of Drug Metabolites with a Small Amount. Quantitative Method for Hydroxytestosterone by GC/MS and Its Application for Measurement of P450 Enzyme Activity

Cytochrome P450 (Cyt. P450) is capable of metabolizing a wide range of endogenous and xenobiotic compounds. The metabolism of testosterone has been used to probe into Cyt. P450 isoenzyme activities of rat liver preparations in vitro. Then we studied the quantitation method of hydroxytestosterone by GC/MS for the measurement of Cyt. P450 isoenzyme activities.Testosterone and 4 kinds of hydroxytestosterones (2α, 6β, 16α, and 16β-OH) were derivatized into the ethoxime-dimethylisopropylsilyl ether derivatives and separated completely on fused silica capillary column. Linearity of the method was established over the concentration range of 0.1-20 ng/sample for 2α, 6β, and 16α-hydroxytestosterones.The present method enabled the measurement of testosterone 2α and 6β-hydroxylation in a trace amount of rat liver homogenate. And the method revealed the relationship between the change in actual isoenzyme activity and inducer treatment such as phenobarbital and dexamethasone. The method is applicable to the measurement of wide variety of Cyt. P450 isoenzyme activities.