RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2005; 19: 798–804 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1855 A high-throughput liquid chromatography/tandem mass spectrometry method for screening glutathione conjugates using exact mass neutral loss acquisition Jose Castro-Perez 1 *, Robert Plumb 2 , Li Liang 3 and Eric Yang 3 1 Waters Corporation (MS Technology Center), Floats Road, Manchester M23 9LZ, UK 2 Waters Corporation, 34 Maple Street, Milford, MA 01757-3696, USA 3 Worldwide Bioanalysis, Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA 19406, USA Received 24 November 2004; Revised 20 January 2005; Accepted 20 January 2005 Chemically reactive metabolites may cause hepatotoxicity and as a result liver failure or other adverse side reactions. Therefore, this is a vital topic of interest because early reactive metabolite screening may prevent compound failure at a later stage. In order to address this issue, a screening assay has been developed to detect the formation of reactive metabolites by using glutathione as a trapping reagent, which will allow us to search for phase I metabolites and also glutathiones during in vitro metabolite screening using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with exact mass. Glutathione conjugations when fragmented by the mass spectrometer give a com- mon loss corresponding to the pyroglutamic acid moiety, which can be monitored. Until recently, this work has been carried out with triple quadrupole technology using nominal mass. The advan- tage of the hybrid quadrupole time-of-flight mass spectrometer is the selectivity and sensitivity that can be achieved. Exact neutral loss detection is achieved via sequential low- and high-energy MS acquisitions. After detection of the loss of the pyroglutamic acid moiety, using a window of 20 mDa on the high-energy scan, MS/MS is carried out on the parent mass of interest to confirm the common neutral loss. Copyright # 2005 John Wiley & Sons, Ltd. Over the years, many marketed compounds have been withdrawn due to adverse drug reactions. 1 It has been esti- mated that adverse drug reactions rank among one of the leading causes of death in the United States. 2–4 Idiosyn- cratic drug reactions, also classified as type B (unpredict- able) reactions, account for about 20% of all adverse drug reactions. 5 Although a direct link between toxicity and for- mation of reactive metabolites has not been established, there is much circumstantial evidence indicating that idio- syncratic drug reactions are due to reactive metabolites. 6–12 Due to the inability of predicting which reactive metabo- lites will be toxic and which will not, one general approach to reduce the idiosyncratic reactions has been to minimize reactive metabolite formation by appropriate structural modification during the lead optimization stage. 7 Research over the past three decades in the field of biological reac- tive intermediates has yielded a wealth of information on the functional groups that may be converted by either phase I or phase II enzymes into electrophilic metabolites. 8 Despite the rather extensive literature on the mechanisms by which drugs and other foreign compounds undergo metabolic activation, it is likely that numerous functional groups which have not hitherto been recognized as precur- sors to reactive intermediates also can undergo bioactiva- tion. Therefore, the detection and characterization of chemically reactive metabolites has become imperative in the discovery and development of drug candidates in pharmaceutical research. Most reactive metabolites are electrophilic species and, as such, are susceptible to covalent binding with nucleophilic portions of proteins, DNA, and cellular glutathione (GSH). Such reactions in vivo may lead to enzyme deactivation, genotoxicity, hapten formation triggering immune responses, compromised cellular function due to GSH depletion and increased oxidative stress, apoptosis, cumu- lative damage to long-lived proteins, and ultimately cause cell death. 9–11 A variety of techniques are available to indicate reactive metabolite formation. Most prevalently used are GSH depletion assays, time-dependent P450 inhibition assays, covalent binding of radiolabeled mate- rial, and GSH trapping of reactive species with analysis using liquid chromatography/tandem mass spectrometry (LC/MS/MS). 13,14 Glutathione (GSH) is a tripeptide present in mammalian systems. It plays an important role in the detoxification of electrophilic foreign compounds and chemically reactive intermediates, which may arise during the biotransformation of xenobiotics. Various efforts have been made to identify reactive metabolites by the use of chemical trapping agents, Copyright # 2005 John Wiley & Sons, Ltd. *Correspondence to: J. Castro-Perez, Waters Corporation (MS Technology Center), Simons Way, Atlas Park, Manchester M22 5PP, UK. E-mail: jose_m_castro-perez@waters.com