Stock solutions were prepared in HPLC grade water (dextrorphan) or methanol (4-hydroxytolbutamide) at 1 mg/ mL. All diluted samples were prepared in 50:50 methanol: water (HPLC grade).
Testing of drug metabolizing enzymes (such as cytochrome P450s) is becoming increasingly important for the evaluation of new chemical entities (NCEs) before evaluation of the new drug in the clinic. Newly synthesized compounds that display proper biological effects on the target of choice in vitro, can and should be evaluated for their effects on drug metabolizing enzymes before an expensive scale-up of the new drug takes place for clinical testing. CYP3A4, 2D6 and 2C9 were chosen for our evaluation because 45-60% of the drugs currently on todays market are metabolized by CYP3A isoforms alone6, and CYP2D6 and CYP2C family members are subject to polymorphisms leading to populations of people that are poor metabolizers7. Having a combined method for all three would, therefore, be useful for testing samples from all of these enzymes at one time, eliminating the need to switch solvents and/or whole methods to evaluate these enzymes.
Good separation of the metabolites is obtained, in less than 5 minutes, on a Varian MonoChrom MS column with 0.1% formic acid (Solvent A) and acetonitrile (Solvent B) with the gradient mentioned (see LC Method, above). Figure 2A C, shows the individual peaks as detected by single reaction mode (SRM) in the 1200L. Limit of detection (LOD) for each of these metabolites was determined to be: 0.5, 0.25 and 0.25 pg (4-hydroxytolbutamide, -hydroxymidazolam and dextrorphan, respectively) and the limit