title: A fragmentation-based method for the differentiation of glutathione conjugates by high-resolution mass spectrometry with electrospray ionization.
abstract: Idiosyncratic reactions are one of the major causes of drug treatment limitations or market withdrawals, and likely involve the formation of reactive metabolites. Because of their unstable nature, reactive species are usually discovered as stable conjugates by glutathione (GSH) trapping rather than by direct detection. The GSH conjugates are then detected by neutral loss scanning of 129 Da or precursor ion scanning at m/z 272, but the conjugation sites can only be identified by comparison with reference standards. In the present study, the fragmentation behaviors of 52 GSH conjugates belonging to five structural classes (aliphatic, aromatic, benzylic, disulfide, and thioester) were investigated in both positive and negative electrospray ionization modes by high-resolution mass spectrometry with suitable collision energies such that the relative abundance of the parent ion was approximately 50% of the most abundant product ion. Several structural-diagnostic fragmentations were identified: aliphatic conjugates gave i/j-type ions upon cleavage of the C-S bond between the drug and GSH, and d/k-type ions formed by the cleavage of the cysteinyl C-S bond, with approximately equal intensity, in both positive and negative modes, whereas aromatic conjugates only possessed d/k-type ions, and benzylic conjugates primarily yielded i/j-type ions. Disulfide conjugates typically produced dehydrogenated GS(-) fragment ions ([i-2H]-type) in negative mode, and thioester conjugates displayed sequential losses of pyroglutamic acid and water ([e-H2O]-type) in positive mode. A fragmentation-based method was thus established to facilitate the discrimination of these five classes of GSH conjugates, thereby providing insight into the bioactivation mechanisms and supporting lead optimization.
authors.affiliation: Center for Drug Metabolism and Pharmacokinetics Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Cen
authors.initials: C
authors.lastname: Xie
authors.forename: Dafang
authors.initials: D
authors.lastname: Zhong
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Chen
mesh_terms: D005591:Chemical Fractionation / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D004220:Disulfides / Q000737:chemistry; D005978:Glutathione / Q000032:analysis* / Q000737:chemistry*; D007477:Ions; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D004220:Disulfides; D007477:Ions; D005978:Glutathione
keywords: C/Q-TOF MS, ultra-high performance liquid chromatography-quadrupole-time of flight-mass spectrometry; CE; CID; Detection; ESI; Electrospray ionization-mass spectrometry; FAB; Fragmentation pattern; GSH; Glutathione conjugates; HLM s; LC-MS/MS; N-acetylcysteine; NAC; NADPH; Structural differentiation; UHPL; collision energy; collision-induced dissociation; electrospray ionization; fast-atom bombardment; glutathione; human liver microsomes; liquid chromatography-tandem mass spectrometry; β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt