title: Evaluation of evaporative light-scattering detection for metabolite quantification without authentic analytical standards or radiolabel.
abstract: Development of a sensitive and specific technique for the quantitation of drug metabolites without the use of synthetic analytical standards or radiolabel would represent a major advance in preliminary route of metabolism screening in drug discovery. In this study, the ability of evaporative light-scattering detection (ELSD) to quantify metabolites of 7-ethoxycoumarin (EC) was evaluated. Because ELSD operates as a mass detector, the complex nature of in vitro-derived samples from hepatocyte incubations resulted in an inability to detect the analytes of interest in this matrix using ELSD. Additionally, the gradient nature of the analysis required to temporally separate ethoxycoumarin from its metabolites and matrix components interfered with the ELSD response. Furthermore, using less-complex contrived mixtures, ELSD demonstrated insufficient sensitivity (limit of detection of 1000-10,000 ng/mL) and an inconsistent inter-analyte response. Together, the limitations outlined in these experiments demonstrate that ELSD is at present an inadequate technique for generating semi-quantitative data on metabolites in drug discovery.
authors.affiliation: Preclinical Drug Discovery, Cardiovascular and Urogenital Center of Excellence in Drug Discovery, GlaxoSmithKline, King of Prussia, PA 19406, USA. harvey.e.fries@gsk.com
authors.forename: Harvey E
authors.initials: HE
authors.lastname: Fries
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Evans
authors.forename: Keith W
authors.initials: KW
authors.lastname: Ward
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003374:Coumarins / Q000378:metabolism*; D004285:Dogs; D022781:Hepatocytes / Q000378:metabolism; D008027:Light; D012542:Scattering, Radiation; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
chemical_list: D003374:Coumarins; C017299:7-ethoxycoumarin