Application of a fluorous derivatization method for characterization of glutathione-trapped reactive metabolites with liquid chromatography-tandem mass spectrometry analysis

The glutathione (GSH) trapping assay is commonly utilized for the screening and characterization of reactive metabolites produced by drug metabolism. This study describes a fluorous derivatization method for a more sensitive and selective analysis of reactive metabolites trapped by GSH using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, the GSH-trapped reactive metabolites, which were obtained after incubation of the test compounds with human liver microsome (HLM) in the presence of GSH and NADPH, were derivatized using the perfluoroalkylamine reagent through oxazolone chemistry. Since this reaction enabled the selective modification of the cz-carboxyl group in GSH, the structural compositions of the metabolites were not affected by the derivatization. Furthermore, the selective analysis of the resulting derivatives could be performed using perfluoroalkyl-modified stationary phase LC separation via the interaction between the perfluoroalkyl-containing compounds, such as fluorous affinity, followed by detection with the precursor ion and/or enhanced product ion scan modes in MS/MS. Finally, we demonstrated the applicability of this method by analyzing perfluoroalkyl derivatives of some drug metabolites trapped by GSH in HLM incubation. (C) 2020 Elsevier B.V. All rights reserved.