Enhanced Screening of Glutathione-Trapped Reactive Metabolites by In-Source Collision-Induced Dissociation and Extraction of Product Ion Using UHPLC-High Resolution Mass Spectrometry

A selective and sensitive approach, called extraction of product ion (XoPI) method, was developed for the detection of l-glutathione (GSH)-trapped reactive metabolites employing an Orbitrap high resolution mass spectrometer. Fragmentation of GSH conjugates in the negative ion mode leads to a product ion, deprotonated γ-glutamyl-dehydroalanyl-glycine (m/z 272.0888). As a means of utilizing this property, negative ion high resolution MS data were collected from in vitro incubations by monitoring ions from m/z 269.5 to 274.5 under in-source collision-induced dissociation. Extraction of product ions at m/z 272.0888 ± 5 ppm from this data resulted in a chromatogram exhibiting deprotonated γ-glutamyl-dehydroalanyl-glycine as the major peaks with no or very few interferences. Therefore, peaks in this extracted product ion chromatogram potentially came from GSH-trapped reactive metabolites. The GSH conjugate parent ions were then confirmed in the corresponding full scan MS data, and their structures were identified from their MS2 fragmentation patterns. The effectiveness of the approach was assessed with four model compounds, amodiaquine, clozapine, diclofenac, and fipexide, all well-known to form GSH-trapped reactive metabolites, following incubation in human liver microsomes supplemented with β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt (NADPH) and GSH. The results from XoPI method were compared to two other commonly employed liquid chromatography–mass spectrometry (LC-MS) methods: precursor ion scan method and mass defect filter method. Overall, the XoPI method was more selective and sensitive in detecting the GSH conjugates. Many GSH conjugates previously not reported were detected and characterized in this study.