Application of stable isotope labeled glutathione and rapid scanning mass spectrometers in detecting and characterizing reactive metabolites

Abstract

The formation of reactive metabolites from a number of compounds was studied in vitro using a mixture of non-labeled and stable isotope labeled glutathione (GSH) as a trapping agent. GSH was labeled by incorporating [1,2-(13)C(2),(15)N]glycine into the tripeptide to give an overall increase of 3 Da over the naturally occurring substance. Detection and characterization of reactive metabolites was greatly facilitated by using the data-dependent scanning features of the linear ion trap mass spectrometers to give complimentary and confirmatory data in a single analytical run. A comparison was made by analyzing the samples simultaneously on a triple-stage quadrupole mass spectrometer operated in the constant neutral loss mode. The compounds studied included 2-acetamidophenol, 3-acetamidophenol, 4-acetamidophenol (acetaminophen), and flufenamic acid. GSH adducts for each of these compounds produced a characteristic pattern of 'twin ions' separated by 3 Da in the mass spectral data. This greatly facilitated the detection and characterization of any GSH-related adducts present in the microsomal extracts. Furthermore, characterization of these adducts was greatly facilitated by the rapid scanning capability of linear ion trap instruments that provided full-scan, MS/MS and MS(3) data in one single analysis. This method of detecting and characterizing reactive metabolites generated in vitro was found to be far superior to any of the existing methods previously employed in this laboratory. The combination of two techniques, stable isotope labeled glutathione and linear ion traps, provided a very sensitive and specific method of identifying compounds capable of producing reactive metabolites in a discovery setting. The complimentary set of mass spectral data (including full-scan, MS/MS and MS(3) mass spectra), obtained rapidly in a single analysis with the linear ion trap instruments, greatly accelerated identification of metabolically bioactivated soft spots on the molecules. This in turn enabled chemists to rapidly design out the potential metabolic liability from the back-up compounds by making appropriate structural modifications.

An algorithm for thorough background subtraction from high‐resolution LC/MS data: application for detection of glutathione‐trapped reactive metabolites

“…7 In addition, stable-isotope labeled GSH has been used to improve the analytical efficiency of the assay. 8,9 Recently, highresolution mass spectrometry-based mass defect filtering 10 has been used to facilitate the screening and identification of GSH-trapped reactive metabolites. 11 All these techniques target a specific behavior or property of GSH adducts to facilitate their detection.…”

Regulatory Control or Oxidative Damage? Proteomic Approaches to Interrogate the Role of Cysteine Oxidation Status in Biological Processes

“…These include neutral loss scanning mass spectrometry for a loss of the 129 Da pyroglutamic acid from endogenous glutathione adducts (93), or treatment of cells with glutathione ethyl ester (GEE) combined with precursor ion scanning of 300 m/z to detect the GEE linked analyte (94). In addition, treating cells with stable-isotope labeled GSH improves selective detection of glutathionylated molecules because they appear as a pair of light and heavy ions (95,96).…”

Neuroimaging - Clinical Applications

“…Terminal acetylenes are well known to be mechanism-based CYP-inactivators (Testa & Jenner, 1981) and there is an increasing body of information suggesting that internal acetylenes can be activated by CYPs (Fontana et al, 2005;Foroozesh et al, 1997;Shimada et al, 2007) or even undergo uncatalyzed addition of glutathione Mutlib et al, 1999). Mutlib et al reported that incubation of MPEP with triple-labeled glutathione gave compounds with molecular weights and fragmentations consistent with both activated and unactivated addition of GSH to the alkyne (Mutlib et al, 2005). These events are potential sources for hepatic or idiosyncratic toxicity.…”