Novel Approach to Performing Metabolite Identification in Drug Metabolism

Abstract

A novel online method is developed, using liquid chromatography (LC)-accurate radioisotope counting dynamic-flow (ARC) coupled with a radioactivity detector and mass spectrometer, for metabolite identification in drug discovery and development. This method offers the advantages of improved sensitivity for detecting radiolabeled drugs as well as streamlining the process of identifying and characterizing metabolites. For the purposes of evaluating this method, in vitro human liver microsomal incubations with [(14)C]dextromethorphan are conducted. Online separation and identification of [(14)C]dextromethorphan metabolites are achieved without intensive sample preparation, concentration, or fraction collection. Mass spectrometric analysis identified and characterized the metabolites of dextromethorphan formed by N - and O -dealkylation, correlated well with previously published results. Chromatographic peaks for [(14)C]dextromethorphan and its metabolites are collected online, then infused for extended periods of time at a flow rate of 10 microL/min while maintaining the column pressure. The continuous analytical signal input allowed acquisition of a higher order of multistage fragmentation for both major and minor metabolites. The multistage MS fragmentation pattern obtained for the metabolites allowed defining the sites of metabolism for dextromethorphan. Further evaluations of this method are also conducted using a [(14)C]compound A to check the linearity and sensitivity of the dynamic-flow method. The R(2) value is 0.996 for the dynamic-flow method between 50 and 600 disintegrations per minute (dpm); the limit of detection for LC-ARC is 20 dpm, which is approximately 10 times more sensitive than conventional continuous-flow radioactivity detection techniques. The overall results suggest that the combination of LC-ARC with radioactivity detection and mass spectrometry has great potential as a powerful tool for enhancing the sensitivity of radioisotope measurement in metabolite identification studies during drug discovery and development.

Identification of Novel Metabolites of Colchicine in Rat Bile Facilitated by Enhanced Online Radiometric Detection

“…In a comparative sensitivity analysis of radiolabeled colchicine, the PSNR of the colchicine peak using the XFlow LC-ARC system was approximately 20 times higher than that using conventional LC-FSA. The observed sensitivity improvement was consistent with previous published results (Nassar and Lee, 2007). The enhanced sensitivity and chromatographic resolution of this system enabled detection of three novel colchicine metabolites in rat bile samples.…”

“…Their applications cover identifying binding sites of target enzyme or receptors (Weeks et al, 2005), localizing target tissues (Jang et al, 2007), obtaining drug metabolism and disposition information in absorption, distribution, metabolism, and excretion (ADME) studies (Dalvie, 2000;Marathe et al, 2004), and imaging pharmacological response and drug localization in animals and humans through positron emission tomography (Aloj and Morelli, 2004). In drug metabolism and pharmacokinetics, 3 H-and 14 C-labeled drug candidates are commonly used for in vitro studies measuring absorption (Jigorel et al, 2005), metabolism (Nassar and Lee, 2007), and covalent binding (Evans et al, 2004) and in vivo experiments assessing metabolism and excretion routes in preclinical and clinical settings (James et al, 2005;Burkey et al, 2006).…”

“…The XFlow LC-ARC system is a novel analytical device for enhanced on-line radiometric detection (Nassar et al, 2003;Nassar and Lee, 2007). It controls the scintillation liquid flow as well as oncolumn and postcolumn LC flows.…”

“…It controls the scintillation liquid flow as well as oncolumn and postcolumn LC flows. It has been demonstrated that dynamic liquid flow optimization, in conjunction with an improved flow cell design and a high-efficiency liquid scintillation cocktail, enhanced radiometric detection sensitivity and resolution without prolonging LC run times (Nassar and Lee, 2007). Integration of the XFlow LC-ARC system with mass spectrometric detection allows parallel acquisition of high-sensitivity radiometric and MS data and thus facilitates direct and effective metabolite identification in ADME studies.…”

ADMEProfiling in Drug Discovery and Development: An Overview

“…In majority cases, this difference is mostly quantitative rather than qualitative-in other words, (1) even at artificially higher NCE concentrations, commonly used for met ID studies, the profile of metabolites would not alter significantly (in most cases), although their relative amounts and the enzymes involved in their formation may vary significantly. Very sensitive analytical LC/MS/MS-or LC/NMR-based methods have revolutionized for metabolite identification, quantitation, and characterization [89][90][91][92][93][94][95][96][97].…”