GSH-trapping

Zheng, J., Ma, L., Xin, B., Olah, T., Humphreys, W., and Zhu, M. (2007). Screening and Identification of GSH-trapped reactive metabolites using hybrid triple quadruple linear ion trap mass spectrometry. Chem. Res. Toxicol. 20 757-766. 

Mass Defect Shifts of GSH-Trapped Reactive Metabolites That Are Formed via Common P450-Mediated Bioactivation Reactions... 

FIGURE 10.10 Analysis of [ H]GSH trapped reactive metabolites by HPLC with TopCount (Zhu et al., 2005b). A nonlabeled drug (50 mM) was incubated with a mixture of GSH (1 mM) and trace [ H]GSH (1-2 p,Ci/mL) in human liver microsomes. After precipitating proteins, the samples were analyzed by HPLC (1 mL/min) with TopCount (4wlls/min, 10-min counting time) (a) Radioactivity profile of the incubation. Ml and M2 were GSH-trapped reactive metabolites and (b) Radioactivity profile of a control incubation sample (without NADPH). 

Another surprise was that interstrand cross-Unks were also formed independently of O2. However, this was initially rationalized by demonstrating that O2 reacted reversibly with 102 (Scheme 43). Nonlinear regression analysis of the ratio of thymidine to oxygenated products (eg 107) as a function of GSH concentration provided an estimated rate constant for GSH trapping of 102 (fecsH = 6.9 X 10 M s ) consistent with expectations for reaction of an alkyl radical with the thiol.The accuracy of the thiol trapping rate constant validated the extracted rate constant for O2 loss from 106 to reform 102 (feo2 = d.4 s ), which is consistent with rate constants reported for O2 loss from other peroxyl radicals. 

As an alternative approach, pMRM—EPI was developed for detection and characterization of GSH-trapped reactive metabolites using a 4000 QTRAP (Zheng et al., 2007). In the analysis, pMRM transitions are constructed from the protonated molecules of potential GSH adducts to their product ions derived from neutral losses of 129 and 307 Da in the positive ion mode. Once a predicted GSH adduct is detected, acquisition of its MS/MS spectrum in the positive ion mode is triggered (Fig. 15.8Z)). The MRM survey scan can be set to monitor up to 100 transitions with the 4000 QTRAP without a signifieant loss of sensitivity. The effectiveness and reliability of this approach were evaluated using several model compounds known to undergo bioactivation in HLM. Results showed that the MRM-based approaeh... 

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