Stable-Isotope Trapping and High-Throughput Screenings of Reactive Metabolites Using the Isotope MS Signature

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Abstract
A highly efficient method has been developed to detect and identify reactive metabolites, using stable-isotope trapping combined with ESI-MS/MS neutral loss scanning. A mixture of glutathione (GSH, γ-glutamylcysteinylglycine) and the stable-isotope labeled compound (GSX, γ-glutamylcysteinylglycine-13C2-15N) was used at an equal molar ratio to trap reactive metabolites generated in microsomal incubations. Samples resulting from incubations were cleaned and concentrated by SPE, followed by LC−MS/MS analyses using constant neutral loss scanning for 129 Da (the γ-glutamyl moiety) to detect formed GSH conjugates. Unambiguous identification of glutathione adducts was greatly facilitated by the presence of a unique MS signature of a prominent isotopic doublet that differs in mass by 3 Da. Further structural characterization of conjugates was achieved with high confidence by subsequently acquiring MS/MS spectra that were featured by neutral losses of 75 and 129 Da for GSH adducts and 78 and 129 Da for isotopic GSX adducts. The reliability of this method was vigorously validated using a number of compounds known to form reactive metabolites. Superior sensitivity was demonstrated by the capability of the current approach to identify reactive metabolites at low abundance. Because of the unique isotopic MS signature, ultrafast analyses of reactive metabolites were accomplished by direct injection of cleaned samples into mass spectrometers for neutral loss scanning. More importantly, this study has demonstrated the feasibility of the current method for completely automated detection of reactive metabolites via computer-assisted pattern recognition.