title: A generic method to detect electrophilic intermediates using isotopic pattern triggered data-dependent high-resolution accurate mass spectrometry.
abstract: A need still exists for a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method that can detect broad classes of glutathione (GSH) conjugates and provide characterization of their structures. We now describe the development of a method that multiplexes high-resolution accurate mass analysis with isotope pattern triggered data-dependent product ion scans, for simultaneous detection and structural elucidation of GSH conjugates within a single analysis using a LTQ/Orbitrap. This method was initially developed to detect GSH conjugates generated from incubating 10 microM test compound with pooled human liver microsomes fortified with NADPH-regenerating system and a 2:1 ratio of 5 mM glutathione and [(13)C(2) (15)N-Gly]glutathione. The GSH conjugates were detected by isotope search of mass defect filtered and control subtracted full scan accurate MS data using MetWorks software. This was followed by elucidation of reactive intermediate structures using chemical formulae for both protonated molecules and their product ions from accurate masses in a single analysis. The mass accuracies measured for the precursor and product ions by the Orbitrap were <2 ppm in external mass calibration mode. Successful detection and characterization of GSH conjugates of acetaminophen, tienilic acid, clozapine, ticlopidine and mifepristone validated this method. In each case, the detected GSH conjugates were within the top five hits by isotope search. This method also has a broader detection capability since it is independent of the collision-induced dissociation behavior of the GSH conjugates. Furthermore, this method is amenable to a broad class of reactive intermediate trapping agents as exemplified by the simultaneous detection and structural elucidation of the cyano-N-methylene iminium ion conjugates of verapamil and its O-desmethyl metabolites, which we report for the first time. In addition to the chemically tagged reactive intermediates, this method also provides information on stable metabolites from the full scan accurate MS data.
authors.affiliation: Drug Metabolism and Pharmacokinetics, Global Preclinical Development, Johnson and Johnson Pharmaceutical Research Institute, Raritan, NJ 08869, USA. hlim5@prdus.jnj.com
authors.forename: Heng-Keang
authors.initials: HK
authors.lastname: Lim
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.forename: Kevin
authors.initials: K
authors.lastname: Cook
authors.forename: Carlo
authors.initials: C
authors.lastname: Sensenhauser
authors.forename: Jose
authors.initials: J
authors.lastname: Silva
authors.forename: David C
authors.initials: DC
authors.lastname: Evans
mesh_terms: D002851:Chromatography, High Pressure Liquid; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling; D007554:Isotopes; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D007554:Isotopes; D004364:Pharmaceutical Preparations; D005978:Glutathione