Rapid screening and characterization of glutathione-trapped reactive metabolites using a polarity switch-based approach on a high-resolution quadrupole orbitrap mass spectrometer

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Abstract
Formation of reactive metabolites that are capable to react with macromolecules could contribute to drug-induced toxicity. As part of early drug screening strategy to support small molecule structure-activity relationship analysis, glutathione (GSH) trapping is commonly used for the detection of reactive metabolites. When trapped, the GSH conjugates can be characterized using mass spectrometry (MS)-based methods. In the present study, an efficient method was developed for rapid identification and characterization of GSH-trapped metabolites with a single run using a quadrupole orbitrap high-resolution mass spectrometer. The selective ion monitoring of m/z 272.0888, a characteristic product ion corresponding to deprotonated γ-glutamic-dehydroalanyl-glycine in the negative ionization mode, was applied as a survey scan leveraging all ion fragmentation mode using in-source collision-induced dissociation. Detection of the extracted product ions within 5.0 Δppm mass accuracy indicated the presence of putative GSH conjugates. Incorporation of fast polarity switching option and multiple data-dependent acquisition scans in a single cycle allowed the determination of accurate mass and multiple MS/MS spectra of GSH conjugates in both negative and positive ionization modes, which featured rich fragments for structural characterization. The effectiveness of this method was evaluated with four model compounds including acetaminophen, clozapine, diclofenac, and nefazodone in both liver microsome and cryopreserved hepatocyte incubations. Successful characterization of multiple GSH conjugates in each case validated this method. Overall, this approach provided a sensitive tool for rapid detection and characterization of GSH conjugates in vitro. Notably, this method could be suitable for high-throughput screening of reactive metabolites in the early drug discovery process.

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