title: High-throughput screening and characterization of reactive metabolites using polarity switching of hybrid triple quadrupole linear ion trap mass spectrometry.
abstract: A highly sensitive and efficient method has been developed for detection and characterization of glutathione (gamma-glutamyl-cysteinylglycine, GSH)-trapped reactive metabolites using a negative precursor ion (PI) as the survey scan to trigger the acquisition of positive enhanced product ion (EPI) spectra on a triple quadrupole linear ion trap mass spectrometer. The negative precursor ion scan step was carried out monitoring the anion at m/z 272, corresponding to deprotonated gamma-glutamyl-dehydroalanyl-glycine originating from the glutathionyl moiety. Because of the uniqueness and abundance of the anion at m/z 272, this single survey scan exhibited broad utility in the detection of unknown GSH conjugates. Further structural characterization was achieved by analyzing positive MS2 spectra that featured rich fragments without mass cutoff and were acquired in the same liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. The effectiveness and reliability of this approach was evaluated using a number of model compounds in human liver microsomal incubations, including acetaminophen, clozapine, diclofenac, imipramine, meclofenamic acid, and ticlopidine. As a result, the PI-EPI approach revealed the presence of known adducts and, in many instances, identified additional conjugates that had not been reported previously. In comparison to the widely used neutral loss (NL) scanning analysis, this approach provided superior sensitivity and selectivity for different types of GSH conjugates. More importantly, the PI-EPI approach is suitable for high-throughput screening of reactive metabolites in the drug discovery process.
authors.affiliation: Department of Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Princeton, New Jersey 08543, USA. bo.wen@roche.com
authors.forename: Bo
authors.initials: B
authors.lastname: Wen
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.forename: Sidney D
authors.initials: SD
authors.lastname: Nelson
authors.forename: Mingshe
authors.initials: M
authors.lastname: Zhu
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry; D000929:Antidepressive Agents, Tricyclic / Q000737:chemistry; D014150:Antipsychotic Agents / Q000737:chemistry; D003024:Clozapine / Q000737:chemistry; D004563:Electrochemistry; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism*; D007099:Imipramine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013988:Ticlopidine / Q000737:chemistry
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D000929:Antidepressive Agents, Tricyclic; D014150:Antipsychotic Agents; D010975:Platelet Aggregation Inhibitors; D005978:Glutathione; D003024:Clozapine; D007099:Imipramine; D013988:Ticlopidine