The risk of idiosyncratic toxicity is of major concern to both pharmaceutical companies and regulatory bodies, especially because the low frequency of occurrence means that it can be missed by preclinical assessment and clinical trials. One possible cause of idiosyncratic toxicity is the formation of reactive metabolites, which are capable of covalent modification of proteins and nucleic acids via reactions such as nucleophilic substitution. It has been suggested that these types of reactions can contribute to idiosyncratic toxicity due to the interruption of certain cellular processes. While the process and causes of drug-induced idiosyncratic toxicity are not understood fully, the formation of reactive metabolites appears to be associated with various toxicological events. It is therefore desirable for pharmaceutical companies to screen for the formation of reactive metabolites.
Reactive–electrophilic metabolites are known to conjugate with the endogenous tripeptide, glutathione (γ-glutamyl-cysteinylglycine [GSH]), either spontaneously or through catalysis by GSH-S-transferases present in the cytosol and endoplasmic reticulum. Therefore, the presence of GSH-conjugated metabolites is an indication of the formation of reactive metabolites. As a result, it is widely accepted that the identification and characterization of GSH conjugates represents a valuable indirect approach for the identification of chemically reactive intermediates formed during the metabolism of both xenobiotic and endogenous compounds.
The complexity of different biological matrices combined with the low abundance of possible toxic GSH conjugates creates the need for highly selective and sensitive analytical tools for detection and identification of these types of metabolites.MS-Based Approaches to Metabolite ID
With its sensitivity, selectivity, speed, and robustness, mass spectrometry (MS) coupled with high performance liquid chromatography (HPLC) has evolved into the preferred method for metabolite identification. Advances in software and hardware have resulted in a variety of techniques for tandem MS (MS-MS) that enable identification, quantitation, and characterization of many metabolites; however, many of the approaches are tedious and time-consuming.
Workflows commonly have consisted of sequential ion scans for metabolite identification followed by additional stages of MS for characterization, and extensive offline data analysis for confirmation. Different platforms offer varying attributes that are desirable to these tasks, but they can also possess drawbacks including limited sensitivity, throughput, structural information, and reliability.
Such strategies have included employing two MS systems — a triple-quadrupole system to find metabolites, via the benefit of fast selective scan functions such as precursor ion and neutral loss scans, and a separate ion-trap system to identify and characterize the metabolites, via the fast, high-sensitivity full scans that are possible on an ion-trap system. Samples would be split and run on the separate instruments sequentially, with the information generated on the triple-quadrupole system used to drive characterization on the ion-trap system. Other systems have relied on splitting the HPLC flow into two separate systems to generate information simultaneously. While the information in this type of configuration is acquired simultaneously, the data is generated into two separate files that must then be interrogated manually.
Hybrid Triple Quadrupole–Linear Ion-Trap MS
The triple-quadrupole scan modes on the hybrid system provide a selective method for identification of structurally similar metabolites even in the presence of complex biological backgrounds. Information based on the parent drug structure fragmentation can be utilized in the application of parent structure precursor ion scans and neutral-loss scans. A precursor ion scan essentially will find all of the masses in a sample that have the ability to lose a selected charged fragment related to the parent molecule. Neutral-loss scans select for those species that lose a characteristic uncharged fragment.
Ion-trap scan modes on a hybrid system provide the full-scan MS and MS-MS data often needed for metabolite identification. IDA mode is used most commonly, which allows data acquired in the survey scan (be it precursor, neutral loss, MRM, or full-scan MS) to select the ions for MS-MS acquisition "on the fly." In this manner, MS-MS data can be acquired automatically without the need to know parent drug information in advance.
Wen and colleagues (1) recently described a high-throughput approach to screening and characterization of reactive metabolites that takes advantage of both the unique hybrid system scan functions and the polarity switching capability of the instrument. Using the negative precursor ion scan to trigger the acquisition of positive enhanced product ion spectra, they developed a highly sensitive and efficient method for the detection and characterization of GSH-trapped reactive metabolites.
They found that by setting the negative precursor ion scan as the survey to monitor for the anion at m/z 272, they were able to detect a range of GHS conjugates. In their experiments, four of the seven clozapine GSH conjugates they detected with this method had not been reported previously in the literature. This unique screening strategy is extremely beneficial because the experiment design utilizes a selective negative precursor ion scan that is universal in finding GSH conjugates. It is followed by a positive ion full-scan MS-MS, enabling them to obtain structural information regarding GSH adducts, within a single run. An additional benefit they found with this new method was that they did not experience any false positives in human liver microsome (HLM) incubation.