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.

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;...…”

Simultaneous Screening of Glutathione and Cyanide Adducts Using Precursor Ion and Neutral Loss Scans-Dependent Product Ion Spectral Acquisition and Data Mining Tools

“…A number of derivatives of GSH such as GSH ethyl ester, dansyl-GSH, brominated GSH, or quaternary ammonium GSH have also been utilized to facilitate MS detection or quantitation [13][14][15][16]. Introduction of stable isotope-labeled GSH analogues such as 13 C 2 15 N-GSH into an incubation medium provides GSHtrapped reactive metabolites with unique isotope patterns that can be used either in isotope pattern-dependent scan or post-acquisition data mining [17][18][19][20]. Hard reactive intermediates cannot be efficiently trapped by GSH or its derivatives.…”

“…In contrast, detection of GSH and CN adducts of reactive metabolites by HR-MS relays on characteristics of protonated molecules of the adducts, including their predictable ranges of mass defects or unique isotope patterns. In addition, background subtraction processing that extracts ion species present in accurate full MS spectra of a test samples but not in a control samples is applied for detection of GSH adducts in vitro and in animals [15,20,28]. Recently, Barbara et al reported a LC-time-offlight (TOF) MS E post-acquisition data mining method for reactive metabolite screening.…”

Metabolism and Excretion of an Oral Taxane Analog, [14C]3′-tert-Butyl-3′-N-tert-butyloxycarbonyl-4-deacetyl-3′-dephenyl-3′-N-debenzoyl-4-O-methoxy-paclitaxel (BMS-275183), in Rats and Dogs

“…Stable isotope-labeled drugs are widely used in many pharmacokinetic and drug metabolism studies including as standards in bioanalytical analysis, in determination of bioavailability, in drug metabolism profiling, in elucidation of drug biotransformation mechanisms, and in metabolite identification (Baillie and Rettenmeier 1986;Abramson et al, 1996;Baillie et al, 2001;Tong et al, 2006;Lim et al, 2008). The commonly used stable isotopes are 2 H, 13 C, 15 N, 18 O, and 34 S. The isotopic clusters generated from the mixture of natural and synthetically introduced isotopes can help the detection and identification of the drug-related compounds in complex biological matrices.…”