Stable-isotope trapping combined with mass spectrometry (MS) neutral loss scanning has recently been developed as a high-throughput method for the in vitro screening of major reactive metabolites. In fact, detection and identification of minor reactive metabolites are equally important since the minor metabolites, even though at low levels, may be highly reactive and also play an important role in drug-induced adverse reactions. In this study, 2-acetylthiophene, clozapine, troglitazone and 7-methylindole were selected as model compounds to further validate the advantages of this method for rapid detection and structural characterization of minor glutathione (GSH) adducts derived from reactive metabolites. The utility of the current method was clearly demonstrated by successful identification of novel reactive metabolites at low levels and also minor ones either masked by non-specific responses or co-eluted with other conjugates. In comparison with existing methods, this method is sensitive, efficient, and suitable for rapid screening and more complete profiling of reactive metabolites.

Metabolism and bioactivation of 3-methylindole (3MI) were investigated in human liver microsomes. The metabolism of two deuterium-labeled analogues of 3MI permitted a relatively unambiguous identification of multiple metabolites and glutathione (GSH) adducts of reactive intermediates. A total of eight oxidized metabolites were detected, five of which were assigned as previously identified 3-methyloxindole, 3-hydroxy-3-methylindolenine, 3-hydroxy-3-methyloxindole, 5-hydroxy-3-methylindole, and 6-hydroxy-3-methylindole. Among the three new metabolites, one was either 4- or 7-OH-3-methylindole, and the other two were derived from additional oxidation on the phenyl ring of 3-methyloxindole. When GSH was added to the microsomal incubations, seven conjugates that had molecular ions corresponding to the incorporation of GSH and an atom of oxygen at m/z 453 (group I) were produced, and two additional conjugates had molecular ions at m/z 437 that corresponded to the incorporation of GSH with no additional oxygen (group II). Two conjugates in group I (m/z 453) were apparently derived by GSH addition to the 5,6-epoxide metabolite of 3-methyloxindole. These two GSH adducts were tentatively identified as 5-(glutathione-S-yl)-3-methyloxindole and 6-(glutathione-S-yl)-3-methyloxindole. The most abundant conjugate in group I was identified as 3-(glutathione-S-yl)-3-methyloxindole, which substantiated the presence of the putative 2,3-epoxy-3-methylindole intermediate. The remaining four adducts in group I were likely formed by conjugation of GSH at different positions of the phenyl ring, possibly via oxidation of 5-hydroxy-3-methylindole and 6-hydroxy-3-methylindole to two very interesting new electrophilic benzoquinone imine intermediates. For the group II conjugates (m/z 437), two isomers were identified as 2-(glutathione-S-yl)-3-methylindole and 3-(glutathione-S-yl-methyl)-indole. The former adduct was primarily derived from the 2,3-epoxide intermediate by thiol conjugation followed by dehydration. The latter adduct was consistent with our previously published work on the dehydrogenation of 3MI. In those studies, we showed that the reactive intermediate, 3-methylenenindolenine, was formed by hydrogen abstraction at the methyl group and was trapped with GSH. The putative dehydrogenation bioactivation mechanism is also substantiated by the finding that CYP2E1 selectively generated 2-(glutathione-S-yl)-3-methylindole but did not produce 3-(glutathione-S-yl-methyl)-indole. In summary, the results not only confirmed the formation of 2,3-epoxide-3-methylindole in human liver microsomes but also suggested that the phenolic metabolites of 3-methylindole were dehydrogenated to previously uncharacterized reactive intermediates.

Glutathione (GSH) has been widely used for in vitro trapping and subsequently detecting reactive metabolites using liquid chromatography-mass spectrometry. A major drawback of GSH is its low trapping efficiency for "hard" reactive metabolites such as reactive aldehydes. In the present study, a bifunctional trapping agent (gamma GSK, gamma-glutamylcysteinlysine) is investigated as an alternative of GSH for simultaneous trapping both "hard" and "soft" reactive metabolites. In microsomal incubations, soft and hard reactive metabolites are captured by conjugation to the free thiol and the amine group of gamma GSK, respectively, resulting in formation of stable peptide adducts. Similar to GSH conjugates, all gamma GSK adducts derived from both soft and hard reactive metabolites contain a gamma-glutamyl moiety and, thus, undergo a neutral loss of 129 Da under collision-induced dissociation. As a result, an NL MS/MS scan can be utilized as a generic method for rapid detecting of both hard or soft reactive metabolites. As demonstrated by a number of model compounds, this approach, in combination with the isotope trapping technique, is reliable, sensitive, and efficient and can be potentially utilized as a high-throughput method for screening and rapid identification of both soft and hard reactive metabolites. In comparison with other methods, this approach is highly efficient and suitable in drug discovery for screening a wide variety of compounds for different reactive metabolites.

Constant neutral loss (CNL) and precursor ion (PI) scan have been widely used for the in vitro screening of glutathione conjugates derived from reactive metabolites, but these two methods are only applicable to triple quadrupole or hybrid triple quadrupole mass spectrometers. Additionally, the success of CNL and PI scanning largely depends on structure and CID fragmentation pathways of GSH conjugates. In the present study, a highly efficient methodology has been developed as an alternative approach for high-throughput screening and structural characterization of reactive metabolites using the linear ion trap mass spectrometer. In microsomal incubations, a mixture of glutathione [GSH, gamma-glutamyl-cystein-glycin] and the stable-isotope labeled compound [GSX, gamma-glutamyl-cystein-glycin-(13)C2-(15)N] was used to trap reactive metabolites, resulting in formation of both labeled and unlabeled conjugates at a given isotopic ratio. A mass difference of 3.0 Da between the natural and labeled GSH conjugate (mass tag) at a fixed isotopic ratio constitutes a unique mass pattern that can selectively trigger the data-dependent MS(2) scan of both isotopic partner ions, respectively. In order to eliminate the response bias of GSH adducts in the positive and negative mode, a polarity switch is executed between the mass tag-triggered data dependent MS(2) scan, and thus ESI- and ESI+ MS(2) spectra of both labeled and nonlabeled GSH conjugates are obtained in a single LC-MS run. Unambiguous identification of glutathione adducts was readily achieved with great confidence by MS(2) spectra of both labeled and unlabeled conjugates. Reliability of this method was vigorously validated using several model compounds that are known to form reactive metabolites. This approach is not based on the appearance of a particular product ion such as MH(+) - 129 and anion at m/z 272, whose formation can be structure-dependent and sensitive to the collision energy level; therefore, the present method can be suitable for unbiased screening of any reactive metabolites, regardless of their CID fragmentation pathways. Additionally, this methodology can potentially be applied to triple quadrupole or hybrid triple quadrupole mass spectrometers.