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

Abstract

Drugs can be metabolically activated to soft and hard electrophiles, which are readily trapped by glutathione (GSH) and cyanide (CN), respectively. These adducts are often detected and structurally characterized using separate tandem mass spectrometry methods. We describe a new method for simultaneous screening of GSH and CN adducts using precursor ion (PI) and neutral loss (NL) scansdependent product ion spectral acquisition and data mining tools on an triple quadrupole linear ion trap mass spectrometry. GSH, potassium cyanide, and their stable isotope labeled analogues were incubated with liver microsomes and a test compound. Negative PI scan of m/z 272 for detection of GSH adducts and positive NL scans of 27 and 29 Da for detection of CN adducts were conducted as survey scans to trigger acquisition of enhanced resolution (ER) spectrum and subsequent enhanced product ion (EPI) spectrum. Post-acquisition data mining of EPI data set using NL filters of 129 and 27 Da was then performed to reveal the GSH adducts and CN adducts, respectively. Isotope patterns and EPI spectra of the detected adducts were utilized for identification of their molecular weights and structures. The effectiveness of this method was evaluated by analyzing reactive metabolites of nefazodone formed from rat liver microsomes. In addition to known GSH-and CN-trapped reactive metabolites, several new CN adducts of nefazodone were identified. The results suggested that current approach is highly effective in the analysis of both soft and hard reactive metabolites and can be used as a high-throughput method in drug discovery.

Post‐acquisition analysis of untargeted accurate mass quadrupole time‐of‐flight MSE data for multiple collision‐induced neutral losses and fragment ions of glutathione conjugates

“…For ezetimibe, several monohydroxylated GSH adducts could be observed most likely involving the aniline substructure motif . Nefazodone formed three GSH conjugates in agreement with previous reports . The small conjugate with MW 449.07 Da likely corresponds to GSH conjugation to the chloro‐aniline part of nefazodone in addition to N‐dealkylation and oxidative deamination.…”

“…[1] Nefazodone formed three GSH conjugates in agreement with previous reports. [23,35] The small conjugate with MW 449.07 Da likely corresponds to GSH conjugation to the chloro-aniline part of nefazodone in addition to N-dealkylation and oxidative deamination. The compound tolcapone showed three GSH adducts which have been reported to be formed in microsomal incubates.…”

“…[15][16][17][18] Methods using mixtures with stable-isotope-labeled trapping reagents in concert with isotope pattern analysis as well as methods using mixtures of different kinds of trapping reagents have been published and proved to be useful tools to assess reactive metabolites in vitro. [19][20][21][22][23][24] The advantage of high-resolution accurate mass fragmentation data is that the accurate mass change of the neutral loss of pyroglutamic acid (129.0426 Da) could be used for post-acquisition data analysis to identify GSH adducts taking advantage of positive ionization and in some cases more informative MS 2 fragment ions for structural elucidation work. Alternatively, data-dependent scan methods can be set up using a narrow (e.g.…”

Metabolism and Bioactivation of The Tricyclic Antidepressant Amitriptyline in Human Liver Microsomes and Human Urine

“…GSH and NAC are often used to trap reactive metabolites [39][40][41]. The MIM-EPI scan function was applied for the analysis of the incubation sample of amitriptyline with excessive amount of NAC/GSH in HLM.…”

The busulfan metabolite EdAG irreversibly glutathionylates glutaredoxins

“…Fragmentation of the peptide corresponding to VVVFIKPT C PYCR clearly reveals EdAG adduction at cys-23 (bold underlined) for Grx-1. In addition to the ions resulting from fragmentation at the peptide backbone, a neutral loss of the pyroglutamic acid (loss of 129 m/z) is observed for many fragments, consistent with the γ-glutamyl linkage on EdAG, which is a well-established fragmentation for GSH adducts (34, 35). The ions corresponding to loss of pyroglutamic acid from the EdAG-adducted peptide are labeled with ‘y x - pGlu’ in Figure 4.…”