title: Dual negative precursor ion scan approach for rapid detection of glutathione conjugates using liquid chromatography/tandem mass spectrometry.
abstract: Screening for conjugates formed by the tripeptide glutathione with new chemical entities is an essential step during the drug discovery process, as the formation of these conjugates serves as an indicator for the presence of reactive electrophilic intermediates. To increase the selectivity and throughput of this analysis, various mass spectral scan types have evolved over time. Historically, samples were analyzed under positive ionization conditions for the neutral loss of m/z 129 (loss of the pyroglutamic acid moiety from glutathione); however, more recently, negative precursor ion scanning for the loss of m/z 272 (deprotonated gamma-glutamyl-dehydroalanyl-glycine from glutathione) has emerged as a more selective tool. Further increasing the selectivity, we report on an extension of this methodology by incorporating a simultaneous dual negative precursor ion scan for two commonly observed ion fragments from glutathione conjugates, m/z 272 and 254 (the dehydrated form of m/z 272). This negative dual precursor ion scan methodology was first validated using substrates known to undergo metabolic bioactivation (diclofenac, carbamazepine, and 3-methyl indole) and has then been applied to the routine analysis of proprietary compounds undergoing active lead optimization. In comparison to alternative scan methodologies, the increased selectivity offered by this simultaneous dual precursor method results in a reduction in the generation of false positive results as well as reduced data interpretation time.
authors.affiliation: Preclinical Drug Discovery, Cardiovascular and Urogenital Center of Excellence for Drug Discovery, GlaxoSmithKline, King of Prussia, PA 19406, USA. mukesh.k.mahajan@gsk.com
authors.forename: Mukesh K
authors.initials: MK
authors.lastname: Mahajan
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Evans
mesh_terms: D000818:Animals; D000838:Anions; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005978:Glutathione / Q000032:analysis* / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D000838:Anions; D005978:Glutathione