Enhanced Detection and Characterization of Glutathione-Trapped Reactive Metabolites by Pseudo-MS 3 Transition Using a Linear Ion Trap Mass Spectrometer Xiaochun Zhu,* Mike Hayashi, and Raju Subramanian Pharmacokinetics and Drug Metabolism, Amgen Inc., Thousand Oaks, California 91320, United States * S Supporting Information ABSTRACT: We present a simple and label-free approach to characterize glutathione (GSH)-trapped reactive metabolites from a single LC-MS analysis employing a linear ion trap mass spectrometer. The GSH specific fragment anion m/z 272 was first generated from the nonselective in-source fragmentation of intact conjugates. GSH conjugates were then detected by selected reaction monitoring (SRM) of the anion pair m/z 272 → 179 or 210. The resultant SRM peaks represented putative GSH conjugates which were then further characterized from their MS and MS 2 data acquired in both positive and negative ion modes. The method is demonstrated with test compounds that are all known to form GSH conjugates. M etabolic bioactivation of a drug can generate reactive intermediates that are capable of covalently modifying cellular biomolecules and are implicated in drug induced toxicity. 1 Reactive metabolites are usually detected by trapping them with L-glutathione (GSH) followed by characterization of the resultant conjugate(s) with liquid chromatography (LC)- mass spectrometry (MS). Many approaches have been developed based on attributes of different MS platforms. Methods implemented on a triple quadrupole or Q-Trap mass spectrometer include constant neutral loss scan, 2 precursor ion scan, 3-5 and MRM, 6 all based on monitoring characteristic GSH conjugate fragmentation. Methods utilizing high reso- lution mass spectrometry (HRMS) implemented on TOF and Orbitrap platforms typically include data processing and filtering techniques. These techniques include comparison with control samples, 7-10 background subtraction, 7,11 mass defect filtering, 7-10,12 and mass extraction 13 to detect and characterize GSH conjugates. Ion trap mass spectrometers have been employed for the detection of GSH conjugates, and published methods 14-16 have the disadvantage of requiring the use of a mixture of natural and stable isotope labeled GSH as the trapping agent. We recently reported a sensitive and selective method called XoPI 13 to screen for and characterize GSH conjugates using a hybrid Orbitrap spectrometer. The method relied on fragments produced from nonselective in-source collision-induced dis- sociation (SCID; also called in-source fragmentation) and detection of accurate mass of GSH conjugate product ion at m/z 272.0888, representing the deprotonated gamma-glutamyl- dehydroalanyl-glycine (γ-EdAG). The presence of γ-EdAG was identified from an extracted product ion chromatogram of m/z 272.0888 (referred to as XPIC 272.0888 ). 13 Its precursor GSH conjugate was then confirmed and characterized from the corresponding full scan MS and MS 2 spectra collected in alternate scans of the same chromatographic peak. The XoPI method requires a HRMS instrument such as a hybrid Orbitrap or TOF MS. In this rapid report, we demonstrate an effective and isotope-label-free approach for the detection of GSH conjugates using a unit resolution linear ion trap mass spectrometer. γ-EdAG can be identified from its characteristic product ion. Figure 1 shows the negative ion mode HRMS tandem mass spectrum obtained by wideband activation of the γ-EdAG ion generated from an amodiaquine GSH conjugate A5 (Figure 2) under SCID. It is essentially the MS 3 spectrum of the GSH conjugate; however, since this spectrum was not an MS 3 Received: July 31, 2012 Published: August 24, 2012 Figure 1. γ-EdAG HRMS tandem mass spectrum (MS 2 ; pseudo-MS 3 spectrum of the GSH conjugate A5) obtained by wideband activation of m/z 272 ion in the negative ion mode. The proposed assignment of the observed fragments is shown on the inset γ-EdAG structure. Rapid Report pubs.acs.org/crt © 2012 American Chemical Society 1839 dx.doi.org/10.1021/tx300339u | Chem. Res. Toxicol. 2012, 25, 1839-1841