Detection and Structural Characterization of Glutathione-Trapped Reactive Metabolites Using Liquid Chromatography−High-Resolution Mass Spectrometry and Mass Defect Filtering

Abstract

The present study was designed to apply the mass defect filter (MDF) approach to the screening and identification of reactive metabolites using high-resolution mass spectrometry. Glutathione (GSH)-trapped reactive metabolites of acetaminophen, diclofenac, carbamazepine, clozapine, p-cresol, 4-ethylphenol, and 3-methylindole in human liver microsomes (HLM) were analyzed by HPLC coupled with Orbitrap or Fourier transform ion cyclotron resonance mass spectrometry. Through the selective removal of all ions that fall outside of the GSH adduct MDF template windows, the processed full scan MS chromatograms displayed GSH adducts as major components with no or a few interference peaks. The accurate mass LC-MS data sets were also utilized for the elimination of false positive peaks, detection of stable oxidative metabolites with other MDF templates, and determination of metabolite molecular formulas. Compared to the neutral loss scan by a triple quadrupole instrument, the MDF approach was more sensitive and selective in screening for GSH-trapped reactive metabolites in HLM and rat bile and far more effective in detecting GSH adducts that do not afford the neutral loss of 129 Da as a significant fragmentation pathway. The GSH adduct screening capability of the MDF approach, together with the utility of accurate mass MS/MS information in structural elucidation, makes high-resolution LC-MS a useful tool for analyzing reactive metabolites.

The Pneumotoxin 3-Methylindole Is a Substrate and a Mechanism-Based Inactivator of CYP2A13, a Human Cytochrome P450 Enzyme Preferentially Expressed in the Respiratory Tract

“…GS-A3 was not detected by high-resolution mass analysis. In an earlier report, incubations with human liver microsomes produced at least seven distinct GSH adducts detected at m/z 453 (Yan et al, 2007;Zhu et al, 2007); however, cleavage of the thioether bond in the MS 2 product ion spectrum is only consistent with 3-glutathionyl-S-3-methyloxindole. Therefore, we propose GS-A3 to be 3-glutathionyl-S-3-methyloxindole; its formation could be rationalized in terms of a nucleophilic attack of 2,3-epoxy-3MI by GSH at the C-3 position, followed by loss of water to form an imine and subsequent oxidation to a lactone (Scheme 1), as was suggested recently (Yan et al, 2007).…”

Nucleophile addition of reduced glutathione on 2-methyl-2-nitroso compound: A combined electron paramagnetic resonance spectroscopy and electrospray tandem mass spectrometry study

“…In particular, dehydration of m/z 322.1 was observed to occur from a 30 eV collision energy (laboratory frame). The MS/MS behavior of the protonated hydroxylamine 1b greatly differs from fragmentation patterns described for several glutathione conjugates, characterized in positive ion mode tandem mass spectrometry by the loss of the 129 Da pyroglutamic acid neutral [13][14][15][16][17]. This suggests that a nitroso function on the thiol moiety would completely modify dissociation of the ion in the gas-phase.…”

“…In particular, in the positive ion mode, a main dissociation reaction consists of the elimination of a 129 Da neutral corresponding to a pyroglutamic acid molecule. As a result, detection of glutathione conjugates is often performed using a 129 Da neutral loss MS/MS experiments [13][14][15][16][17]. Nevertheless, different classes of glutathione conjugates, such as aliphatic thioether adducts ionized in the positive ion mode, were shown to behave differently upon collisioninduced dissociation (CID) [18,19].…”

Orbitrap mass spectrometry: Instrumentation, ion motion and applications

“…The LTQOrbitrap has also been used to analyze intact small (3-10 kDa) (Bredehöft, Schänzer, & Thevis, 2008;Thevis et al, 2006a;Thevis, Thomas, & Schänzer, 2008b), medium (10-25 kDa) (Macek et al, 2006;Waanders, Hanke, & Mann, 2007) and large ($150 kDa) (Zhang & Shah, 2007) proteins where the MS n capability of the LIT coupled with the high performance features of the orbitrap can facilitate unambiguous determination of the charge states of fragment ions, as well as identification of PTMs by database searching. The high performance attributes of this hybrid mass spectrometer have also found applications in environmental chemistry (Barceló & Petrovic, 2007;Krauss & Hollender, 2008), drug and metabolite analysis (Breitling, Pitt, & Barrett, 2006;Cuyckens et al, 2008;Ding et al, 2007;Karu et al, 2007;Li et al, 2007a;Lim et al, 2007Lim et al, , 2008Madalinski et al, 2008;Nielen et al, 2007;Peterman et al, 2006;Ruan et al, 2008;Thevis et al, 2005;Thevis, Kamber, & Schänzer, 2006b;Thevis, Krug, & Schänzer, 2006c;Thevis et al, 2006dThevis et al, ,e, 2007aThevis et al, , 2008aZhu et al, 2007), lipidomics (Davis et al, 2008;Ejsing et al, 2006;Schwudke et al, 2007;Silipo et al, 2008), small-molecule (Chen et al, 2007b) and reaction product (Jamin et al, 2007) identification, as well as molecular structure characterization using hydrogen/deuterium exchange measurements (Chen et al, 2007a;...…”