Development and validation of new tools for the study of drug metabolism

In this research, chemometric tools coupled with LC-MS methods were used to study the dextromethorphan metabolism catalyzed by CYP2D6 and CYP3A4. A new constraint was implemented into the Multivariate Curve Resolution - Alternating Least Squares (MCR-ALS) algorithm to obtain kinetic constants from these enzymatic systems at steady-state. This work also compared the validity of the analytical results obtained by traditional quantification methods such as chromatographic area integration vs. MCR-ALS. Using direct infusion of mixtures containing levallorphan and 3-methoxymorphinan, the preferential ionization LV over MM was found to level off at approximately 2 muM. The ionization suppression between analytes and the unretained species was identified in a chromatographic gradient method and resolved by the use of chemometrics. The effect of the resolution on the quantification of the dextromethorphan metabolites was studied using two isocratic methods containing NH4COOH as the mobile phase additive. Using MCR-ALS with a quadratic correction internal standard method for an in-vitro fast metabolizer, the high resolution method gave a value (0.0055 +/- 0.0017) muL/pmol P450/min for the intrinsic clearance for CYP3A4, and for CYP2D6 (2.82 +/- 0.81) muL/pmol P450/min. The result of the low resolution method for CYP3A4 gave (0.0074 +/- 0.0031) muL/pmol P450/min, and for CYP2D6 (2.45 +/- 0.74) muL/pmol P450/min.; Evidence for the N-demethylation of dextromethorphan by CYP2D6 was found, but the variability in the data did not allow the fitting of a kinetic model to obtain the clearance for this reaction.