Dear Editor,High-throughput analysis of in vitro cytochrome p450 inhibition samples using mass spectrometry coupled with an integrated liquid chromatography/autosampler system A significant amount of effort in drug discovery is devoted to the study and understanding of drug-drug interactions (DDI), because the effectiveness and/or toxicity of pharmaceutical drugs can be profoundly influenced by the coadministration of other agents. DDI is preferentially assessed by means of an in vitro assay whereby the inhibitory potential of a test drug is measured with human liver microsomes as the CYP450 enzyme source, along with a known probe enzyme substrate, e.g., midazolam with CYP3A4/5. 1 The production of the specific probe substrate metabolite is monitored and compared to the production of metabolite in the absence of the test drug. These assays can be readily performed in multi-well format, and automated using the latest generation of sample preparation robotics. Consequently, the timeliness of results delivery concerning DDI is predominantly dictated by the analysis of samples and data processing, and for this reason breakthroughs in analysis technology have been of recent interest. Some notable examples in the area of mass spectrometry include ultra-performance liquid chromatography, 2 laser diode thermal desorption (LDTD), 3 matrix-assisted laser desorption/ionization (MALDI), 4 direct analysis in real time (DART), 5 and column switching liquid chromatography/ mass spectrometry (LC/MS). 6 All of these approaches take advantage of the relatively simple nature of the sample matrix to completely eliminate or drastically reduce the need for chromatography, thereby dramatically improving sample turnaround time compared to traditional LC/MS, while preserving data quality. In this work, an integrated chromatography/autosampler system 7,8 is evaluated that achieves a 7 s cycle time, and is evaluated for DDI sample analysis with respect to precision, carryover, matrix effects and correlation of results to existing LC/MS methodology.Experimental details consisting of the reagents used, sample preparation procedures and experiments performed can be found in the Supporting Information. An Applied Biosystems/MDS Sciex API 4000 triple-quadrupole mass spectrometer (Concord, ON, Canada) operated in positive ion, multi-reaction monitoring (MRM) mode was used for all experiments. The declustering potentials and collision energies were individually optimized for all three sets of analytes and internal standards, which corresponded to the CYP P450 isoforms used in the DDI assay. 1 0 -Hydroxybufuralol (m/z 278.3!186.2) was monitored to evaluate the
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