title: 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.
authors.affiliation: Bioanalytical Science, Drug Innovation Pharmacokinetics, Vertex Pharmaceuticals Incorporated, Cambridge, MA, USA. hongying_gao@vrtx.com
authors.forename: Hongying
authors.initials: H
authors.lastname: Gao
authors.forename: Olivier L
authors.initials: OL
authors.lastname: Materne
authors.forename: David L
authors.initials: DL
authors.lastname: Howe
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Brummel
mesh_terms: D000818:Animals; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005260:Female; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008252:Macaca fascicularis; D008297:Male; D008660:Metabolism; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism; D051381:Rats; D013045:Species Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D015262:Xenobiotics / Q000145:classification / Q000493:pharmacokinetics*
chemical_list: D015262:Xenobiotics