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
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
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,
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
A hybrid triple quadrupole–linear ion-trap LC–MS-MS system (QTRAP system, Applied Biosystems, Foster City, California) (Figure
1) has been shown to be a powerful analytical tool for in vitro and in vivo metabolite detection and identification, including
determining the presence of and characterization of GSH conjugates. The unique ability of this hybrid system to combine highly
selective triple-quadrupole scan modes with fast, sensitive ion-trap MS-MS scans allows for optimal detection of drug metabolites
as well as characterization and confirmation, in the same LC–MS run. The variety of survey modes (full-scan MS, precursor
ion scans, neutral loss scans, and MRM), coupled with full-scan MS-MS confirmation via information-dependent acquisition (IDA),
allows for a broad, sensitive, and effective system for metabolite detection.
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.
There are also several well-documented types of precursor and neutral-loss scan modes that can be applied specifically to
certain types of phase II metabolites. Though there are several well-documented types of scan modes used, particularly for
finding GSH metabolites, the most common are the neutral loss scan of 129 in positive ion mode and the precursor of m/z 272 scan in negative ion mode (Figure 2). Another well-known triple-quadrupole scan mode is multiple reaction monitoring
(MRM). MRM is an extremely sensitive mode in which the desired precursor ion is selectively permitted into the collision cell
(q2), and a specific product ion is filtered in the third quadrupole for confirmation and quantitation, if desired. A LINAC
collision cell will permit the monitoring of up to 300 MRM transitions in a single experiment. MRM also has been shown to
be excellent for GSH-conjugate detection, particularly when the Q3 transition is determined from the neutral loss of 129 from
the expected conjugate Q3 mass.
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.