Metabolite profiling in human urine by LC–MS/MS: Method optimization and application for glucuronides from dextromethorphan metabolism

Analysis of human urine for specific compounds or metabolites is an established method for biomonitoring occupational or environmental exposures. Modern liquid chromatography–tandem mass spectrometry is not limited to single compounds but can simultaneously analyze whole classes of urine constituents with both high sensitivity and specificity. Individual differences in the composition of urine are very large in humans, which raises a number of problems that are not encountered in animal experimentation. In this report, we investigated whether analysis of glucuronides as a class could reflect differences between human individuals regarding the polymorphic activity of the cytochrome P450 enzyme CYP2D6. From a group of 152 students that had been classified for CYP2D6 activity, urine of 12 “poor metabolizers” and 35 “extensive metabolizers” was collected 90 min after ingestion of 10 mg of the antitussive drug dextromethorphan (DEX) and analyzed for glucuronides. Methods development included the following aspects: adjustment of urine samples to equal creatinine concentration to avoid differences between samples in retention times and ion suppression; on-line enrichment of low-level analytes by column switching; precursor ion scan vs. theoretical multiple reaction monitoring; use of quality control samples to check for reproducibility in large sample series; peak extraction and handling of null entries to build the data matrix; logarithmic data transformation and different scaling procedures; principal component analysis (PCA) vs. discriminant analysis. Our results show that an optimized procedure not only identified the known DEX metabolites as predictors of CYP2D6-specific metabolic pathways but also indicated the presence of additional, so far unknown path-specific glucuronide metabolites. We conclude that metabolite profiling of urine and other biofluids by modern mass spectrometric methodology may help characterize individual differences and become useful in drug development and personalized pharmacotherapy.