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

Abstract

Electrophilic reactive metabolite screening by liquid chromatography/mass spectrometry (LC/MS) is commonly performed during drug discovery and early-stage drug development. Accurate mass spectrometry has excellent utility in this application, but sophisticated data processing strategies are essential to extract useful information. Herein, a unified approach to glutathione (GSH) trapped reactive metabolite screening with high-resolution LC/TOF MS(E) analysis and drug-conjugate-specific in silico data processing was applied to rapid analysis of test compounds without the need for stable- or radio-isotope-labeled trapping agents. Accurate mass defect filtering (MDF) with a C-heteroatom dealkylation algorithm dynamic with mass range was compared to linear MDF and shown to minimize false positive results. MS(E) data-filtering, time-alignment and data mining post-acquisition enabled detection of 53 GSH conjugates overall formed from 5 drugs. Automated comparison of sample and control data in conjunction with the mass defect filter enabled detection of several conjugates that were not evident with mass defect filtering alone. High- and low-energy MS(E) data were time-aligned to generate in silico product ion spectra which were successfully applied to structural elucidation of detected GSH conjugates. Pseudo neutral loss and precursor ion chromatograms derived post-acquisition demonstrated 50.9% potential coverage, at best, of the detected conjugates by any individual precursor or neutral loss scan type. In contrast with commonly applied neutral loss and precursor-based techniques, the unified method has the advantage of applicability across different classes of GSH conjugates. The unified method was also successfully applied to cyanide trapping analysis and has potential for application to alternate trapping agents.

Characterization of Human Cytochrome P450s Involved in the Bioactivation of Clozapine

“…Occasional cases of liver injury in patients treated with CLZ might be the result of local bioactivation to a reactive nitrenium ion by hepatic P450s (Maggs et al, 1995). In addition, bioactivation of CLZ to a reactive iminium ion, which can be trapped by cyanide, has been demonstrated in microsomal incubations (Argoti et al, 2005;Rousu et al, 2009;Li et al, 2011;Barbara and Castro-Perez JM, 2011) and might contribute to hepatotoxicity. Although these reactive CLZ metabolites have been demonstrated in many in vitro studies using HLM, only very limited information is available on the role of individual P450s in bioactivation of CLZ .…”

“…In addition, formation of iminium ions resulting from dehydrogenation of the piperazine-ring of CLZ, DMCLZ, and CLZ-NO were demonstrated using cyanide, which is able to trap hard electrophiles (Rousu et al, 2009;Argoti et al, 2005;Li et al, 2011, Barbara andCastro-Perez, 2011). Theoretically, two different iminium ions can be formed by dehydrogenation of the two different pairs of nitrogen-methylene bonds of the piperazine-ring (Fig.…”

“…Many studies have shown that CLZ is bioactivated by HLM to a reactive nitrenium ion (Maggs et al, 1995;Pirmohamed et al, 1995;Damsten et al, 2008;Wen et al, 2008;Dragovic et al, 2010;Barbara and Castro-Perez, 2011). In addition, formation of iminium ions resulting from dehydrogenation of the piperazine-ring of CLZ, DMCLZ, and CLZ-NO were demonstrated using cyanide, which is able to trap hard electrophiles (Rousu et al, 2009;Argoti et al, 2005;Li et al, 2011, Barbara andCastro-Perez, 2011).…”

Simultaneous Screening of Glutathione and Cyanide Adducts Using Precursor Ion and Neutral Loss Scans-Dependent Product Ion Spectral Acquisition and Data Mining Tools

“…Recently, Barbara et al reported a LC-time-offlight (TOF) MS E post-acquisition data mining method for reactive metabolite screening. Post-acquisition mining using mass defect filter (MDF) that detect drug metabolites based on predicable ranges of their mass defect values, automated comparison of sample and control, and time-alignment of high-and low-energy MS E data enabled detection of reactive metabolites without the need for stable-or radio-isotopelabeled trapping agents [29].…”

High-Resolution Mass Spectrometry Elucidates Metabonate (False Metabolite) Formation from Alkylamine Drugs during In Vitro Metabolite Profiling

“…Because the complete m/z range data set is acquired for each sample, these workflows support the analysis of unknowns, making them useful tools for qualitative a priori characterization of metabolites. The generic full-scan ToF MS E workflow with postacquisition data mining has been described in detail for screening for reactive metabolites (Barbara and Castro-Perez, 2011).…”