Isobaric metabolite interferences and the requirement for close examination of raw data in addition to stringent chromatographic separations in liquid chromatography/tandem mass spectrometric analysis of drugs in biological matrix.

In addition to matrix effects, common interferences observed in liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses can be caused by the response of drug-related metabolites to the multiple reaction monitoring (MRM) channel of a given drug, as a result of in-source reactions or decomposition of either phase I or II metabolites. However, it has been largely ignored that, for some drugs, metabolism can lead to the formation of isobaric or isomeric metabolites that exhibit the same MRM transitions as parent drugs. The present study describes two examples demonstrating that interference caused by isobaric or isomeric metabolites is a practical issue in analyzing biological samples by LC/MS/MS. In the first case, two sequential metabolic reactions, demethylation followed by oxidation of a primary alcohol moiety to a carboxylic acid, produced an isobaric metabolite that exhibits a MRM transition identical to the parent drug. Because the drug compound was rapidly metabolized in rats and completely disappeared in plasma samples, the isobaric metabolite appeared as a single peak in the total ion current (TIC) trace and could easily be quantified as the drug since it was eluted at a retention time very close to that of the drug in a 12-min LC run. In the second example, metabolism via the ring-opening of a substituted isoxazole moiety led to the formation of an isomeric product that showed an almost identical collision-induced dissociation (CID) MS spectrum as the original drug. Because two components were co-eluted, the isomeric product could be mistakenly quantified and reported by data processing software as the parent drug if the TIC trace was not carefully inspected. Nowadays, all LC/MS data are processed by computer software in a highly automated fashion, and some analysts may spend much less time to visually examine raw TIC traces than they used to do. Two examples described in this article remind us that quality data require both adequate chromatographic separations and close examination of raw data in LC/MS/MS analyses of drugs in biological matrix.

Use of a trapping agent for simultaneous capturing and high-throughput screening of both "soft" and "hard" reactive metabolites.

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.

Metabolism and bioactivation of 3-methylindole by human liver microsomes.

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.

Rapid detection and characterization of minor reactive metabolites using stable-isotope trapping in combination with tandem mass spectrometry.

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.

Bioactivation of 4-methylphenol (p-cresol) via cytochrome P450-mediated aromatic oxidation in human liver microsomes.

It has previously been proposed that 4-methylphenol (p-cresol) is metabolically activated by oxidation of the methyl group to form a reactive quinone methide. In the present study a new metabolism pathway is elucidated in human liver microsomes. Oxidation of the aromatic ring leads to formation of 4-methyl-ortho-hydroquinone, which is further oxidized to a reactive intermediate, 4-methyl-ortho-benzoquinone. This bioactivation pathway is fully supported by the following observations: 1) one major and two minor glutathione (GSH) adducts were detected in microsomal incubations of p-cresol in the presence of glutathione; 2) a major metabolite of p-cresol was identified as 4-methyl-ortho-hydroquinone in microsomal incubations; 3) the same GSH adducts were detected in microsomal incubations of 4-methyl-ortho-hydroquinone; and 4) the same GSH adducts were chemically synthesized by oxidizing 4-methyl-ortho-hydroquinone followed by the addition of GSH, and the major conjugate was identified by liquid chromatography-tandem mass spectrometry and NMR as 3-(glutathione-S-yl)-5-methyl-ortho-hydroquinone. In addition, it was found that 4-hydroxybenzylalcohol, a major metabolite derived from oxidation of the methyl group in liver microsomes, was further converted to 4-hydroxybenzaldehyde. In vitro studies also revealed that bioactivation of p-cresol was mediated by multiple cytochromes P450, but CYP2D6, 2E1, and 1A2 are the most active enzymes for formation of quinone methide, 4-methyl-ortho-benzoquinone, and 4-hydroxybenzaldehyde, respectively. Implications of the newly identified reactive metabolite in p-cresol-induced toxicity remain to be investigated in the future.