Drug bioactivation leading to the formation of reactive species capable of 
covalent binding to proteins represents an important cause of drug-induced 
toxicity. Reactive metabolite detection using in vitro microsomal incubations is 
a crucial step in assessing potential toxicity of pharmaceutical compounds. The 
most common method for screening the formation of these unstable, electrophilic 
species is by trapping them with glutathione (GSH) followed by liquid 
chromatography/mass spectrometry (LC/MS) analysis. The present work describes 
the use of a brominated analog of glutathione, N-(2-bromocarbobenzyloxy)-GSH 
(GSH-Br), for the in vitro screening of reactive metabolites by LC/MS. This 
novel trapping agent was tested with four drug compounds known to form reactive 
metabolites, acetaminophen, fipexide, trimethoprim and clozapine. In vitro rat 
microsomal incubations were performed with GSH and GSH-Br for each drug with 
subsequent analysis by liquid chromatography/high-resolution mass spectrometry 
on an electrospray time-of-flight (ESI-TOF) instrument. A generic LC/MS method 
was used for data acquisition, followed by drug-specific processing of accurate 
mass data based on mass defect filtering and isotope pattern matching. GSH and 
GSH-Br incubations were compared to control samples using differential analysis 
(Mass Profiler) software to identify adducts formed via the formation of 
reactive metabolites. In all four cases, GSH-Br yielded improved results, with a 
decreased false positive rate, increased sensitivity and new adducts being 
identified in contrast to GSH alone. The combination of using this novel 
trapping agent with powerful processing routines for filtering accurate mass 
data and differential analysis represents a very reliable method for the 
identification of reactive metabolites formed in microsomal incubations.