Screening and characterization of reactive metabolites using glutathione ethyl ester in combination with Q-trap mass spectrometry.
The present study describes a new analytical approach for the detection and characterization of chemically reactive metabolites using glutathione ethyl ester (GSH-EE) as the trapping agent in combination with hybrid triple quadrupole linear ion trap mass spectrometry. Polarity switching was applied between a negative precursor ion (PI) survey scan and the positive enhanced product ion (EPI) scan. The negative PI scan step was carried out monitoring the anion at m/z 300, corresponding to deprotonated gamma-glutamyl-dehydroalanyl-glycine ethyl ester originating from the GSH-EE moiety. Samples resulting from incubations in the presence of GSH-EE were cleaned and concentrated by solid-phase extraction, followed by the PI-EPI analysis. Unambiguous identification of GSH-EE-trapped reactive metabolites was greatly facilitated by the unique survey scan of the anion at m/z 300, which achieved less background interference, in particular, from endogenous glutathione adducts present in human liver microsomes. Further structural characterization was achieved by analyzing positive MS(2) 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, amodiaquine, carbamazepine, 4-ethylphenol, imipramine and ticlopidine. In addition, iminoquinone reactive metabolites of mianserin were trapped and characterized for the first time using this method. Compared to neutral loss (NL) scanning assays using GSH as the trapping agent, the results have demonstrated superior selectivity, sensitivity, and reliability of this current approach.
Wen
Bo
B
Department of Drug Metabolism and Pharmacokinetics, Roche Palo Alto, Palo Alto, CA 94304, USA. bo.wen@roche.com
Fitch
William L
WL
D000082:Acetaminophen / Q000032:analysis; D000655:Amodiaquine / Q000032:analysis; D002220:Carbamazepine / Q000032:analysis; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D007099:Imipramine / Q000032:analysis; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D055432:Metabolomics / Q000379:methods*; D008862:Microsomes, Liver / Q000737:chemistry / Q000378:metabolism; D010636:Phenols / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013988:Ticlopidine / Q000032:analysis
D010636:Phenols; D000655:Amodiaquine; D002220:Carbamazepine; D000082:Acetaminophen; C042431:S-ethyl glutathione; C042291:4-ethylphenol; D005978:Glutathione; D007099:Imipramine; D013988:Ticlopidine