title: High-resolution twin-ion metabolite extraction (HiTIME) mass spectrometry: nontargeted detection of unknown drug metabolites by isotope labeling, liquid chromatography mass spectrometry, and automated high-performance computing.
abstract: The metabolic fate of a compound can often determine the success of a new drug lead. Thus, significant effort is directed toward identifying the metabolites formed from a given molecule. Here, an automated and nontargeted procedure is introduced for detecting drug metabolites without authentic metabolite standards via the use of stable isotope labeling, liquid chromatography mass spectrometry (LC/MS), and high-performance computing. LC/MS of blood plasma extracts from rats that were administered a 1:1 mixture of acetaminophen (APAP) and (13)C6-APAP resulted in mass spectra that contained "twin" ions for drug metabolites that were not detected in control spectra (i.e., no APAP administered). Because of the development of a program (high-resolution twin-ion metabolite extraction; HiTIME) that can identify twin-ions in high-resolution mass spectra without centroiding (i.e., reduction of mass spectral peaks to single data points), 9 doublets corresponding to APAP metabolites were identified. This is nearly twice that obtained by use of existing programs that make use of centroiding to reduce computational cost under these conditions with a quadrupole time-of-flight mass spectrometer. By a manual search for all reported APAP metabolite ions, no additional twin-ion signals were assigned. These data indicate that all the major metabolites of APAP and multiple low-abundance metabolites (e.g., acetaminophen hydroxy- and methoxysulfate) that are rarely reported were detected. This methodology can be used to detect drug metabolites without prior knowledge of their identity. HiTIME is freely available from https://github.com/bjpop/HiTIME .
authors.affiliation: †School of Chemistry and Bio21 Institute of Molecular Science and Biotechnology, University of Melbourne, 30 Flemington Road, Melbourne, Victoria 3010, Australia.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Leeming
authors.affiliation: ‡Victorian Life Sciences Computation Initiative, University of Melbourne, 187 Grattan Street, Carlton, Victoria 3010, Australia.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Isaac
authors.affiliation: ‡Victorian Life Sciences Computation Initiative, University of Melbourne, 187 Grattan Street, Carlton, Victoria 3010, Australia.|§Department of Computing and Information Systems, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Bernard J
authors.initials: BJ
authors.lastname: Pope
authors.affiliation: ∥Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia.|¶Royal Children's Hospital Melbourne, 50 Flemington Road, Victoria 3052, Australia.
authors.forename: Noel
authors.initials: N
authors.lastname: Cranswick
authors.affiliation: ∥Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia.|⊥ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Wright
authors.affiliation: ∥Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia.|⊥ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: James
authors.initials: J
authors.lastname: Ziogas
authors.affiliation: †School of Chemistry and Bio21 Institute of Molecular Science and Biotechnology, University of Melbourne, 30 Flemington Road, Melbourne, Victoria 3010, Australia.|⊥ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Richard A J
authors.initials: RA
authors.lastname: O'Hair
authors.affiliation: #School of Chemistry, University of New South Wales, Sydney, New South Wales 2052, Australia.
authors.forename: William A
authors.initials: WA
authors.lastname: Donald
mesh_terms: D000082:Acetaminophen / Q000008:administration & dosage / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001331:Automation; D002853:Chromatography, Liquid; D003205:Computing Methodologies; D007553:Isotope Labeling; D008297:Male; D013058:Mass Spectrometry; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000082:Acetaminophen