Method for rapid metabolite profiling of drug candidates in fresh hepatocytes using liquid chromatography coupled with a hybrid quadrupole linear ion trap

Abstract

Rapid information on metabolic profiling is required to evaluate the structural liabilities of drug candidates in early drug discovery. In this study, a sensitive and rapid semi-quantitative method was developed to simultaneously monitor the drug candidate and metabolites as well as collect tandem mass (MS/MS) spectra for subsequent metabolite identification. The simultaneous semi-quantitation and identification of metabolites in fresh hepatocytes is achieved using high-performance liquid chromatography (HPLC) coupled with a hybrid quadrupole linear ion trap. The survey experiment consists of monitoring multiple-reaction monitoring (MRM) transitions for the internal standard, the parent, and 48 MRM transitions designed to cover the most common phase I and II biotransformations. An information-dependent acquisition (IDA) method was employed to trigger product ion scans above the MRM signal threshold. Three biotransformations of a lead compound have been identified through enhanced product ion scans and the respective MRM transitions of those metabolites were selected for semi-quantitation. Parent disappearance and formation of the metabolites as a function of incubation time in five different species were monitored by their respective MRM responses. The method provides the necessary sensitivity to detect minor metabolites in a relevant therapeutic concentration range. Enzymatic turnover of the parent and the metabolites in different species are revealed based on the different initial concentrations of the parent. This methodology integrates the parent disappearance, metabolite identification, and the formation of the metabolites along the time course using a single rapid LC/MS/MS analysis. This method can be used as a complementary tool to the conventional method of metabolic profiling. It provides a rapid and sensitive initial profile of the metabolism of potential structural series at the lead selection stage. The method can also be incorporated into the overall metabolite profiling scheme to evaluate the drug candidates in drug discovery.

A Simple Liquid Chromatography-Tandem Mass Spectrometry Method to Determine Relative Plasma Exposures of Drug Metabolites across Species for Metabolite Safety Assessments

“…The list of metabolites can be selected as Ն10% of total parentrelated material based on the radiolabeled ADME study or the estimation from LC-UV-MS responses from a multiple-dose human study. In the latter case, the accuracy of the percentage of the total parent-related materials is not critical; rather, the list of metabolites should be comprehensive to cover all metabolites suspected to be important because the mass spectrometric method has the capability of simultaneously monitoring 50 mass transitions (Gao et al, 2007). The list of metabolites should be generated during routine metabolite identification studies and transferred to bioanalytical scientist.…”

“…Multiple ion monitoring (MIM) can be used to trigger an enhanced product ion (EPI) scan to identify a large number of metabolites in a single chromatographic run (Yao et al, 2008). Likewise, multiple reaction monitoring (MRM) can be used to trigger EPI scans to profile predicted drug metabolites in in vitro samples Gao et al, 2007).…”

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].…”

“…A strong expertise in sample preparation and novel sample introduction into the LC/MS/MS [e.g., Ultra Performance Liquid Chromatography (UPLC) and nano-flow techniques] tremendously improves success in detection and identification of in vivo metabolites. There are a multitude of very good articles discussing several of these modern analytical strategies involved in metabolite identification and are highly recommended for further reading [89][90][91][92][93][94][95][96][97].…”

Driving efficiency in a high-throughput metabolic stability assay through a generic high-resolution accurate mass method and automated data mining

“…Several laboratories have established 'anticipated SRM channels' based on the fragmentation patterns of parent compounds in order to monitor putatively predicted metabolites alongside the parents using quadruple-based instruments (Poon et al, 1999;Tiller and Romanyshyn, 2002;Gao et al, 2007). The unit mass accuracy of quadruple analyzers, however, often leads to ambiguous metabolite identification in complex biological matrixes where many isobaric substances are present.…”