Seite - (000120) - in Biomedical Chemistry: Current Trends and Developments

simultaneously quantify CYP probe substrates/metabolites. At this level, LC-MS and LC-MS/MS are undeniably involved in the characterization and quantification of the metabolites and have been most frequently used to support these in vitro studies (Table 2.1.6). To increase throughput, many probes have been applied to assess multiple CYP activities simultaneously within a single experiment. This strategy, named the cocktail approach, is starting to be employed in vitro to predict the clinical impact of genetic polymorphism to CYP-based DDI (Rhodes, 2011; Kozakai 2012; Pillai 2013). Traditional analytical methods using LC coupled with UV, radiometric, fluorescence and luminescence detections have some limitations including a lack of substrate specificity, solvent assay sensitivity and analytical interferences. Thus, LC-MS and LC-MS/MS offer advantages over these classical methods such as high specificity and sensitivity to investigate several enzyme- specific substrate probes and selectively quantify each specific metabolite. The current methodologies employing the cocktail approach to assess in vitro the activity of human CYPs can be found in the recent review published by Spaggiari et al. (2014). According to Table 2.1.6, ion trap mass spectrometers have been used to a large extent to support in vitro metabolic studies as a qualitative tool, but in comparison with classical quadrupole MS/MS, the quantitative features of ion trap mass spectrometers remain less used. In fact, due to its small footprint and high sensitivity, the ion trap was considered somewhat of an alternative to the triple quadrupole MS for quantitative high-throughput analysis. However, the generation of MS/MS data requires a relatively long duty cycle, limiting the numbers of analytes that can be quantified simultaneously. Furthermore, the LC- Q-TOF MS technique has been widely applied to obtain high-resolution mass spectral data of precursor and product ions due to its high- throughput analysis, specificity, selectivity and resolution power. The enhancement of its selectivity is attractive to reduce the problems such as matrix suppression and metabolite interferences. It is also of great value in the interpretation and elucidation of the chemical structure of the metabolites of each NCE. As a result, the best strategy uses an ion trap to obtain the structural information by sequential MSn experiments and MRM screening could be subsequently used to perform quantitation