Evaluation of analytical and clinical performance of a dual-probe phenotyping method for CYP2D6 polymorphism and CYP3A4 activity screening

A bioanalytical method for the determination of dextromethorphan (DEX) and its metabolites dextrorphan (DTX), 3-methoxymorphinan (3MM), and 3-hydroxym orphinan (3HM) in human urine was developed for CYP2D6 phenotyping and CYP3 A4 activity measurements in clinical pharmacology studies using dextrometho rphan administered in a drinking solution as substrate. The method was eval uated by thorough conventional validation and by a cross-validation of the method with a previously applied method for dextromethorphan and dextrorpha n only (CYP2D6 phenotyping). Cross-validation with the former method showed no significant differences in measured concentrations of volunteer samples . This guaranteed the consistency of epidemiologic data in the database col lected from two methods. For the CYP2D6 and CYP3A4 evaluations, the clinica l parameters are ratios of concentrations. It appeared that severe variance in individual concentrations generally did not infltrence the variance of ratios significantly, because experimental errors in concentrations of two analytes proved to correlate considerably. For CYP2D6 values around the ant imodes, the chance of a misclassification is very small. The chance of clas sifying an extensive metabolizer as a poor metabolizer or vice versa is neg ligible. For CYP3A4 activity determinations it was concluded that in genera l a change in dextromethorphan/3-methoxymorphinan (DEX/3MM) ratios of 10% o r more as detected with the current method, is a significant increase or de crease in the activity of CYP3A4. The authors concluded that they had obtai ned an analytically valid and clinically reliable bioanalytical method for the determination of dextromethorphan and its metabolites dextrorphan, 3-me thoxymorphinan, and 3-hydroxymorphinan in human urine for CYP2D6 phenotypin g and CYP3A4 activity measurements for clinical pharmacology studies.