A high‐throughput liquid chromatography/tandem mass spectrometry method for screening glutathione conjugates using exact mass neutral loss acquisition

Abstract

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 common 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 advantage 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.

Nucleophile addition of reduced glutathione on 2-methyl-2-nitroso compound: A combined electron paramagnetic resonance spectroscopy and electrospray tandem mass spectrometry study

“…In particular, dehydration of m/z 322.1 was observed to occur from a 30 eV collision energy (laboratory frame). The MS/MS behavior of the protonated hydroxylamine 1b greatly differs from fragmentation patterns described for several glutathione conjugates, characterized in positive ion mode tandem mass spectrometry by the loss of the 129 Da pyroglutamic acid neutral [13][14][15][16][17]. This suggests that a nitroso function on the thiol moiety would completely modify dissociation of the ion in the gas-phase.…”

“…In particular, in the positive ion mode, a main dissociation reaction consists of the elimination of a 129 Da neutral corresponding to a pyroglutamic acid molecule. As a result, detection of glutathione conjugates is often performed using a 129 Da neutral loss MS/MS experiments [13][14][15][16][17]. Nevertheless, different classes of glutathione conjugates, such as aliphatic thioether adducts ionized in the positive ion mode, were shown to behave differently upon collisioninduced dissociation (CID) [18,19].…”

High‐resolution chromatography/time‐of‐flight MSE with in silico data mining is an information‐rich approach to reactive metabolite screening

“…[14] The method can suffer from poor selectivity resulting in detection of false positives, [15] but this can be alleviated by employing accurate mass analyzers to monitor the more specific neutral loss of 129.0426 u. [16] Several classes of GSH conjugates do not fragment by loss of 129 u, [3,15] however, and therefore cannot be detected by this method, so additional scan types monitoring neutral loss of 307 u, intact GSH, or 147 u, loss of 129 u followed by rapid loss of water, may be employed. [15] Precursor (PC) ion scans of m/z 272, deprotonated g-glutamyl-dehydroalanyl-glycine, [17] and m/z 254, the corresponding water loss, can be employed in negative mode.…”

Drug Metabolite Profiling and Identification by High-resolution Mass Spectrometry

“…This example illustrates the use of HR-MS and HR-MS/MS data in complete metabolite identification. There has been some progress in the development of software that allows automated assignment of product ion structures based on HR-MS/MS data to aid in metabolite structure determination (65,66).…”