In this study, we developed an in-line methodology that combines analytical with pharmacological techniques to characterize metabolites of human histamine H4 receptor (hH4R) ligands. Liquid chromatographic separation of metabolic mixtures is coupled to high-resolution fractionation into 96- or 384-well plates and directly followed by a cell-based reporter gene assay to measure receptor signaling. The complete methodology was designed, optimized, validated, and ultimately miniaturized into a high-density well plate format. Finally, the methodology was demonstrated in a metabolic profiling setting for three hH4R lead compounds and the drug clozapine. This new methodology comprises integrated analytical separations, mass spectrometry, and a cell-based signal transduction-driven reporter gene assay that enables the implementation of comprehensive metabolic profiling earlier in the drug discovery process. © 2012 Society for Laboratory Automation and Screening.
Nijmeijer, S., Vischer, H. F., Rudebeck, A. F., Fleurbaaij, F., Falck, D., Leurs, R., ... Kool, J. (2012). Development of a profiling strategy for metabolic mixtures by combining chromatography and mass spectrometry with cell-based GPCR signaling. Journal of Biomolecular Screening, 17, 1329-1338. https://doi.org/10.1177/1087057112451922