Reactive Metabolite Trapping and Metabolite Identification using Multiple Reagents andSpecific Survey Scans on a Rapid Scanning Linear Ion Trap

MP529

James A. Ferguson1, Jenny Moshin1, LaHoma Easterwood2, Stanley Rosenberg1, and Sai Y. Chang3 – 1Applied Biosystems, 500 Old Connecticut Path, Framingham, MA 01701, 2CellzDirect, 1624 Headway Circle, Austin, TX 78754, 3MSMS Science, 20 Ranch Road, Sedona, AZ 86336

ABSTRACTRESULTS

Figure 1. Structure of Troglitazone (TGZ)
CH3

Troglitazone (TGZ)—was incubated with three different reactive metabolite trapping reagents—glutathione (GSH), glutathione
ethyl ester (GSH-EE), and N-acetyl cysteine (NAC). Five different scan functions were used to look for reactive metabolites inthe incubated samples. Data was acquired using information-dependent acquisition (IDA) methods on a rapid scanning hybrid
linear ion trap instrument, taking advantage of the ability to rapidly switch from specific triple quadrupole (TQ) mode MS/MS scans
to sensitive linear ion trap (LIT) mode MS and MS/MS scans for confirmation.

H3COO

TGZ Metabolites found in samples incubated with GSH-EE bypMRM-triggered IDA experiments

Table 4.

TGZ Metabolites found in samples incubated with NAC by pMRMtriggered IDA experiments

Table 5.

Summary of TGZ Metabolites found by the various trappingreagents

OHOCH3

Table 3.

CH3

S

NHO

INTRODUCTION

Table 1.

TGZ Metabolites found in samples incubated with GSH by pMRMtriggered IDA experiments

The formation of reactive metabolites through hepatic metabolism is considered a major liability for drug candidates. Accordingly,emphasis has been placed on sensitive, reliable, and high-throughput detection and identification of these metabolites early in theADME process.
*Metabolites 1 through 5 are well-documented in the literature

The most common trapping reagent for reactive metabolites is glutathione. However, other reagents such as GSH ethyl ester andN-acetylcysteine1-3, among others, can provide additional coverage and different modes of trapping. Selective triple quadrupole
scans such as neutral loss, precursor, and MRM can be used as survey scans to detect the trapped species. In this study, weevaluate the use of multiple survey scans on a fast scanning hybrid quadrupole linear ion trap system to detect reactive
metabolites trapped by three different reagents.
The precursor and neutral loss scans included semi-targeted (based on the fragmentation of the parent drug) and more highlytargeted (based on the fragmentation pattern of the trapping reagent). In order to cover all of the possibilities, five different survey
methods were created. The most specific was pMRM (predictive MRM), based on the fragmentation pattern of the parent drugand expected metabolites. In addition to the predictive MRMs automatically generated by LightSight® software, extra MRMs wereadded to account for the expected neutral loss (NL) of 129 from GSH and GSH-EE and loss of 42 from NAC. Many of themetabolites showed fragments from both the trapping reagent-specific NL and fragment(s) expected from the fragmentation of theparent compound. Slightly less specific were precursor and neutral loss survey scans specific to each trapping reagent. ForGSH, the specific precursor was m/z 272 in the negative ion mode and the neutral loss was 129 in positive ion mode. For GSHEE, the negative pre-cursor was m/z 300 (the ethyl addition to the molecule is conserved in the fragment) while the neutral loss
remained 129. For NAC, the negative precursor was m/z 128 and the positive neutral loss was 42, both of which can haveinterferences. The final two methods were precursor and neutral loss scans based on fragmentation of the parent drug, andtherefore, not specific for the trapping reagents.

MATERIALS AND METHODS
Incubations—Human liver microsomes (1 mg/mL) were incubated on ice with alamethicin (50 µg/mg of protein) for 15 minutes prior toadding to sample tubes containing 100 mM potassium phosphate buffer, pH 7.4, 5 mM saccharic acid 1,4-lactone, 1 mM magnesium
chloride, and 1 mM trapping reagent (glutathione, N acetylcysteine, or glutathione ethyl ester) and the analytes (concentrations of 10 and 1µM in the final reaction volume). Samples were pre-incubated for 10 minutes at 37 °C prior to initiation of the reaction by the addition of 5mM UDPGA and 1 mM NADPH. Aliquots (250 µL) of each sample were removed at 0, 30, 60, and 120 minutes and quenched with 2
volumes of ice cold acetonitrile. The samples were centrifuged at 10,000×g for 30 minutes and the resulting supernatant was transferred toclean deep-well tubes and snap frozen. The samples were stored at -80 °C prior to analysis.
Mass Spectrometry—Data was acquired using the AB Sciex QTRAP® 5500 LC/MS/MS system and Analyst® 1.5 software (AppliedBiosystems/MDS Analytical Technologies, Foster City, CA). The QTRAP 5500 system was operated in information-dependent acquisition(IDA) mode with TQ mode survey scans and LIT mode enhanced resolution and enhanced product ion scans for confirmation. Data wasprocessed with LightSight and Analyst software packages.

CONCLUSIONSThe table above correlates the metabolites found by each of the various trapping
reagents. Metabolites M1-M5 are well-documented in the literature and all were
found, although not all were found by each trapping reagent. M6 (unexplained asof yet) was found in samples incubated with GSH and in those incubated withNAC. M7 appears to be di-oxidation followed by NAC adduction and was foundonly in samples incubated with NAC.

Note: Areas in the 2-hour peak area column of all tables are shown in boldface black if theyincreased and in boldface red if they decreased significantly from their corresponding 1-hourareas.
Table 1 lists metabolites found in the 1- and 2-hour incubations of TGZ at 1 µM with GSH using pMRM
as the survey experiment with added MRMs for the NL of 129 Da. The list was limited to expected
metabolites related to GSH adducts previously reported in the literature for TGZ4. The biotransformations listed in boldface (lines 13, 14, 16, & 17) were expected. In addition to the expected GSH-2Hadducts, the list included their GSH (addition of 307 rather than 305 Da) counterparts.
In addition to the expected GSH-2H (305) adducts, MRMs for adducts resulting for their intact GSH(307) counterparts were included in the method—75 MRMs total—and found the mass shift of 297 Da(line 15). This metabolite at m/z 739 could not be the [M+2] isotope of the expected 737 Da addition as
their retention times differ. It might, however, be due to in-source loss of 42 Da from the m/z 781
metabolite, as they have the same retention time, although this loss would be hard to rationalize. Theproduct ion spectrum of this potential metabolite includes the expected m/z 165, 191, and 219 ionsfrom the parent drug as well as the m/z 610 due to NL of 129 Da.

Table 2.

Table 3 lists the metabolites found in the 1- and 2-hour incubations of TGZ at 1 µM with GSH-EE using
pMRM as the survey experiment with added MRMs for the NL of 129 Da. The biotransformations listed
in boldface (lines 15-21) were expected as analogous to the GSH adducts found in the literature (table1). More investigation needs to be done to see if there really are three separate m/z 749 metabolites.
Note that GSH-EE trapped the oxidative metabolite (line 20) whereas it was not detected in the samples incubated with GSH (table 1). In the other experiments, the GSH-EE specific precursor scanfound the m/z 775 metabolite corresponding to the m/z 745 metabolite on line 4 in table 2.

Figure 2. Product ion spectrum of the potential
TGZ metabolite at m/z 739 found byGSH Trapping

Table 4 lists the metabolites found in the 1- and 2-hour incubations of TGZ at 1 µM with NAC using
pMRM as the survey experiment with added MRMs for the NL of 42 Da. The biotransformations listed
in boldface (lines 14, 15, 17, & 19) were expected. In addition to the expected NAC-2H adducts, thelist included their NAC (addition of 163 rather than 161 Da) counterparts. Once more, this found a new
mass shift of 153 Da (line 16), which couldn’t be the [M+2] isotope of the expected 151 Da addition as
the retention times differ. This Δ of 153 Da is equivalent to the m/z 739 peak in the GSH incubation.
However, this metabolite does not have the same RT as m/z 637 and so can not be loss of 42 Da from637 Another unexpected mass shift is the 193 Da adduct on line 18 corresponding to di-oxidation andthen NAC conjugation. The expected addition from the literature would be 195 Da (line 19), but thearea for the addition of 195 and it’s retention time suggest it may be an isotope of the 193 Da adduct.

Figure 3. Product ion spectrum of the TGZ
metabolite at m/z 595 found by NAC
Trapping

Figure 4. Product ion spectrum of the TGZ
metabolite at m/z 635 found by NAC
Trapping

Using the QTRAP 5500 System allows for rapid discovery at low incubation levels(1 µM) using multiple survey scans including MRMs with very low dwell (3-5 ms)
and pause times (1-3 ms) and confirmation by enhanced product ion scans in thelinear ion trap for comparing full scan product ion spectra of the metabolite to theparent drug.

FUTURE WORK
● spiking the in vitro incubations into plasma and bile.
● further structural analysis on the unexplained metabolites● extending the study to other drugs● running this type of analysis on samples obtained in vivo.

TGZ Metabolites found in samples incubated with GSH by IDA
experiments using other survey scans (Precursor and NeutralLoss)

REFERENCES

Liquid Chromatography—The HPLC system consisted of an CBM-20A system controller, a SIL-20AD HT autosampler, two LC-20AD
pumps, and a CTO-20A column oven, all from Shimadzu (Columbia, MD).
Chromatographic Conditions
Column: Imtakt Cadenza CD-C18 2.1 × 50 mm, 3 µm
Mobile Phase A: 95% Water, 5% acetonitrile, 0.1% formic acid
2 mM ammonium acetate
Mobile Phase B: 95% Acetonitrile, 5% water, 0.1% formic acid,2 mM ammonium acetate
Flow rate: 0.3 to 0.4 mL/min
Injection Volumes: 5 & 10 µL
Gradient: See graph

The best mode for finding the trapped reactive metabolites was, as expected, thepMRM IDA. Addition of the extra MRM for the loss of 129 from GSH and GSHEE
or 42 from NAC added to the ability to detect and confirm the metabolites.

1. Soglia, J. R., Harriman, S. P., Zhao, S., Barberia, J., Cole, M. J., Boyd, J. G., Contillo, L. G.,
J. Pharm. Biomed. Anal. 2004, 36, 105-16.
2. Staack, R. F., Hopfgartner, G., Anal. Bioanal. Chem. 2007, 388, 1365–1380.

Table 2 lists the parent and glutathione metabolites found in the 1- and 2-hour incubations of TGZ
using the other four survey scans. Using a precursor scan based on the parent drug’s fragmentation,
the parent, one of the metabolites which is apparently an adduct lost in the source (see line 7, table 1)
and the GSH adduct at m/z 747 were found. Using a precursor scan based on the negative m/z 272
precursor scan, only the GSH adduct was found (note the 2Da mass difference due addition of a protonin positive ion mode and subtraction in negative ion mode). The other adducts were formed at a lower
level and may not have exceeded the intensity needed to be found by the precursor scan. Neither theneutral loss survey based on the parent drug’s fragmentation nor the NL of 129 Da picked up any GSH
metabolites.

3. Wen, B., Fitch, W. L. J. Mass Spectrom. 2009, 44, 90-100.
4. Kassahun, K., Pearson, P. G., Tang, W., McIntosh, I., Leung, K., Elmore, C., Dean, D., Wang, R.,Doss, G., Baillie, T. A., Chem. Res. Toxicol., 2001, 14, 62-70.

TRADEMARKS/LICENSING
This is figure shows the product ion spectra of the metabolite atm/z 739 (top panel) and of TGZ (bottom panel). The inset shows
the fragments common to both spectra. The m/z 610 ion in themetabolite’s spectrum corresponds to the loss of 129 expectedfrom a glutathione adduct.

This is figure shows the product ion spectra of the metabolite at m/z595 (top panel) and of TGZ (bottom panel). The inset shows thefragments common to both spectra.

This is figure shows the product ion spectra of the metabolite atm/z 635 (top panel) and of TGZ (bottom panel). The inset shows
the fragments common to both spectra.

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