Using Electron Activated Dissociation (EAD) To Characterize Challenging Metabolites
App Note / Case Study
Last Updated: April 13, 2023
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Published: March 15, 2023
Credit: iStock
Glucuronidation is an essential metabolic pathway for drug clearance and its characterization is crucial for optimizing the efficiency and safety of drug candidates.
However, the identification and characterization of glucuronide-drugs conjugates can be challenging using by MS/MS alone, as the glucuronic acid bond is often labile.
This application note presents a quick and robust soft-spot identification procedure using diagnostic fragments from electron activated dissociation (EAD).
Download this app note to discover how EAD:
- Enhances the sensitivity of MS/MS workflows
- Enables a comprehensive characterization of critical glucuronide metabolites
- Helps to implement a streamlined data processing
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For research use only. Not for use in diagnostics procedures.
Confident characterization and identification of glucuronide
metabolites using diagnostic fragments from electron
activated dissociation (EAD)
Comprehensive characterization of challenging metabolites using EAD on the ZenoTOF 7600 system
Rahul Baghla, Eshani Nandita
SCIEX, USA
This technical note demonstrates the comprehensive
characterization and confident identification of glucuronide
metabolites from hepatocyte incubations of midazolam. An
orthogonal fragmentation mechanism was applied to generate
diagnostic fragment ions for confident identification of
glucuronide metabolites using electron activated dissociation
(EAD). Several key glucuronide conjugations were identified,
including aromatic/aliphatic hydroxylation, o-glucuronide
conjugation and N-dealkylated midazolam N-glucuronide. A
streamlined workflow was developed to efficiently characterize
and identify conjugated structures during drug metabolism
studies.
Glucuronide conjugation can be challenging to characterize
thoroughly by MS/MS alone, as the glucuronic acid bond is often
labile, both in the ionization source and the collision cell of mass
spectrometers.3 One of the significant challenges when
implementing a soft-spot analysis approach is the ability to
produce data promptly in alignment with the pace of the drug
discovery process. This technical note demonstrates a quick and
robust soft-spot identification procedure using a novel orthogonal
fragmentation mechanism, EAD, on the ZenoTOF 7600 system
(Figure 1). Sites of midazolam glucuronide conjugation were
predicted using Mass-MetaSite software. Structural
determination was performed using unique EAD fragments.
Key features for metabolite identification
using the ZenoTOF 7600 system
• Comprehensive characterization and confident
identification: Achieve comprehensive characterization and
identification of glucuronide metabolites from hepatocyte
incubations of midazolam using the ZenoTOF 7600 system.
• Site-specific identification: Acquire diagnostic fragments to
easily identify the site of metabolism for glucuronide
metabolites with EAD. EAD is the only non-CID/HCD
fragmentation mechanism that works well on singly charged
molecules.
• Detection of low-level metabolites: Identify all critical
metabolites present in drug metabolism studies with
enhanced MS/MS sensitivity provided by the Zeno trap.
• Streamlined data processing: Develop confident structuremetabolic stability relationships for drug products utilizing a
quick, easy-to-use methodology from acquisition to analysis.
Drug metabolism characterization is essential for optimizing
pharmacokinetics (PK), pharmacodynamics (PD) and safety
profiles of drug candidates in the drug discovery and
development process. Drugs must reach the site of action to
Figure 1. EAD enables the confident identification of glucuronide
conjugation using diagnostic fragments. CID generated two possible
sites of glucuronide conjugation. However, the power of EAD provides sitespecific characterization that narrows it down to a single metabolite
candidate. Here, the glucuronide conjugation was localized on the
imidazole ring.
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For research use only. Not for use in diagnostics procedures.
elicit their pharmacological effects after administration. If results
indicate inferior PK properties—such as high clearance, short
half-life (t1/2) and/or low bioavailability—PD effects will likely be
sub-optimal. In vitro metabolism studies in human and animal
tissue preparations are valuable for identifying major metabolism
pathways.1
Glucuronidation is the most critical phase II metabolic pathway
responsible for clearing many endogenous and exogenous
compounds. In addition to being an essential detoxification
mechanism for structurally diverse drugs, glucuronidation also
leads to a short duration of action and loss of pharmacological
activity. The prediction of glucuronidation is crucial for the earlystage characterization of drug clearance properties in humans to
improve PK results.2
Methods
Sample preparation: Midazolam was incubated at 37°C in
human hepatocytes at a starting concentration of 5 µM. Samples
were removed from incubation and quenched with acetonitrile at
0-, 30-, 60-, 90-, 120- and 240-minute intervals.
Chromatography: LC separation was performed on a
Phenomenex Kinetex Polar C18 column (2.1 x 100 mm, 2.6 µm,
100 Å) at a column temperature of 40°C. Mobile phase A was
0.1% (v/v) formic acid in water and mobile phase B was 0.1%
(v/v) formic acid in acetonitrile. An injection of 5 µL was
subjected for analysis.
The chromatographic gradient conditions are summarized in
Table 1.
Mass spectrometry: The samples were analyzed using the data
dependent acquisition (DDA) method with Zeno CID DDA and
Zeno EAD DDA on the ZenoTOF 7600 system. The method
conditions are summarized in Table 2.
The source and gas conditions are summarized in Table 3.
Data processing: SCIEX OS software 3.0 was used for data
acquisition. Mass-MetaSite software was used for the prediction
of biotransformation sites using Zeno CID DDA and Zeno EAD
DDA data.4-9
EAD provides positional information on
glucuronide conjugation sites
Zeno DDA data provided excellent MS/MS coverage for TOF MS
peaks of interest in both CID and EAD acquisitions. MassMetaSite software automatically predicted the metabolites based
on MS1 data and performed structural elucidation by comparing
the precursor and metabolite-specific fragment ions. The 240-
minute incubation sample showed a significant peak for
midazolam N-glucuronide at a retention time of 4.21 minutes.
Zeno CID DDA did not indicate any specific fragments for
midazolam N-glucuronide (Figure 2). Instead, fragments from
CID originated from the primary midazolam structure. The
Mass-MetaSite software predicted 2 possible sites of metabolism
Table 1. Chromatographic gradient.
Time
(min)
Mobile phase A
(%)
Mobile phase B
(%)
0.0 95 5
1.0 95 5
7.0 5 95
9.0 5 95
9.1 95 5
10 95 5
Table 2. Zeno DDA parameters.
Parameter Setting
Method duration 10 min
TOF MS start-stop mass 100–1000 Da
Maximum candidate ions 5
Accumulation time (TOF MS) 0.1 s
TOF MS/MS start-stop mass 50–1000 Da
Accumulation time (TOF MS/MS) 0.1 s
Collision energy (CID) 40 V
Collision energy spread (CID) 15 V
Electron kinetic energy (EAD) 12 eV
Electron beam current (EAD) 6000 nA
Table 3. Source and gas conditions.
Parameter Setting
Curtain gas 40 psi
Ion source gas 1 55 psi
Ion source gas 2 65 psi
CAD gas 7
Ion spray voltage 5500 V
Source temperature 500°C
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For research use only. Not for use in diagnostics procedures.
with CID MS/MS spectra. EAD showed unique fragments at
m/z 309.0582 and m/z 354.0796 and confirmed N-glucuronide
conjugation on the imidazole ring. Another metabolite with
aromatic/aliphatic hydroxylation and o-glucuronide conjugation
Figure 2. Comparing results from CID MS/MS spectra (top) with EAD MS/MS spectra (bottom) for precursor ion m/z 502.1176. Fragment ions in
red originate from the primary structure of midazolam, while fragment ions in yellow are from glucuronide conjugation. EAD results show a more
significant presence of fragment ion information from the glucuronide conjugation compared with CID data. Fragment ions (m/z 309.0582 and m/z
354.0796) generated using EAD localized the glucuronide conjugation on the imidazole ring.
CID MS/MS Spectra
Observed mass – 244.0318
Mass error -2.46 ppm
Observed mass – 291.1170
Mass error -1.37 ppm
Observed Mass – 326.0854
Mass error 0.31 ppm
EAD MS/MS spectra
Observed Mass – 309.0582
Mass error 2.24 ppm
Observed Mass – 310.0553
Mass error -3.55 ppm
Observed Mass – 354.0796
Mass error 2.26 ppm
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For research use only. Not for use in diagnostics procedures.
was detected at a retention time of 4.40 minutes. Due to the
absence of any glucuronide-specific fragments with CID, four
possible sites of metabolism on the benzene ring of midazolam
were predicted (Figure 3). EAD indicated metabolite-specific
fragments at m/z 311.0610, m/z 324.0706, m/z 342.0796,
m/z 370.0737 and m/z 442.0980.
Therefore, information from EAD enabled the identification of
the peak as 1-hydroxymidazolam o-glucuronide (Figure 4).
EAD provided rich MS/MS spectra, enabling the identification
of an N-dealkylated midazolam N-glucuronide metabolite. EAD
spectra included all fragments generated using CID along with
a glucuronide-specific fragment at m/z 386.0716, confirming
the site of conjugation (Figure 5).
The ZenoTOF 7600 system demonstrated excellent mass
accuracy for the workflow. All metabolites and fragments were
identified with <10 ppm error. This enabled the confident
identification of critical metabolites present in an in vitro
metabolism study of midazolam. Furthermore, identification of
all critical metabolites was easily performed with the
improvement in MS/MS sensitivity provided by the Zeno trap
on the ZenoTOF 7600 system.
Gathering information on structure metabolism relationships is
critical for developing chemical strategies for stabilization.10
This method demonstrates a robust soft-spot identification
procedure for characterization and identification of glucuronide
conjugates using EAD on the ZenoTOF 7600 system coupled
with Mass-MetaSite software.
Figure 3. EAD provides site-specific identification of aromatic/aliphatic hydroxylation and o-glucuronide conjugation. With CID, most fragment
ions (m/z 203.0373, m/z 304.0625, m/z 324.0709 and m/z 342.0814) originated from the primary structure of midazolam. Therefore, it lacks positional
information for hydroxylation and glucuronide conjugation. As a result, CID proposed 4 possible sites of metabolism on the benzene ring, indicative of
aromatic hydroxylation. With EAD, confident identification of aliphatic hydroxylation and o-glucuronidation was possible as fragment ions provided sitespecific information from the conjugation structure. Fragment ions m/z 311.0610, m/z 324.0706, m/z 342.0796, m/z 370.0737 and m/z 442.0980
indicated that the modification was present on the methyl group on the imidazole ring.
Aromatic/Aliphatic Hydroxylation - Glucuronidation
CID
EAD
1-hydroxymidazolam o-glucuronide
Observed Mass – 203.0373
Mass error -0.98 ppm
Observed Mass – 304.0625
Mass error 3.62 ppm
Observed Mass – 324.0709
Mass error -3.39 ppm
Observed Mass – 342.0814
Mass error -2.92ppm
Observed Mass – 311.0610
Mass error 3.21 ppm
Observed Mass – 324.0706
Mass error -2.46 ppm
Observed Mass – 342.0796
Mass error 2.33 ppm
Observed Mass – 370.0737
Mass error 4.32 ppm
Observed Mass – 442.0980
Mass error -3.62 ppm
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For research use only. Not for use in diagnostics procedures.
Figure 4. EAD MS/MS spectra provides site-specific identification of aliphatic hydroxylation and o-glucuronide conjugation, confirming the
peak as 1-hydroxymidazolam o-glucuronide.
Figure 5. Identification of an N-dealkylated midazolam N-glucuronide metabolite using EAD. With CID, the fragment ions indicate the presence of
a dealkylated midazolam N-glucuronide structure. However, EAD generated fragment ion information for the dealkylated midazolam N-glucuronide
metabolite structure, including a glucuronide-specific fragment at m/z 386.0702, verifying the site of conjugation.
EAD MS/MS spectra
Observed mass – 370.0737
Mass error 4.32 ppm
Observed Mass – 442.0980
Mass error -3.62 ppm
EAD
CID
Glucuronidation (tertiary amine) – N Dealkylation
Observed Mass – 234.0498
Mass error -7.69 ppm
Observed Mass – 310.0553
Mass error -3.55 ppm
Observed Mass – 311.0590
Mass error 8.36 ppm
Observed Mass – 358.0750
Mass error 0.83 ppm
Observed Mass – 516.0981
Mass error -2.52 ppm
Observed Mass – 386.0716
Mass error -3.63 ppm
Observed Mass – 340.0636
Mass error 3.23 ppm
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For research use only. Not for use in diagnostics procedures.
Conclusions
• Comprehensive characterization and identification of critical
glucuronide metabolites from hepatocyte incubations of
midazolam were demonstrated on the ZenoTOF 7600
system.
• Diagnostic fragment ions were used to identify the site of
metabolism for several glucuronide metabolites using EAD.
• A highly sensitive workflow enables the detection of lowlevel metabolites and can be easily adapted for in vivo
metabolism studies with the enhanced sensitivity provided
by the Zeno trap.
• A streamlined data processing method was utilized for ease
of data reduction and development of confident structuremetabolic stability relationships for drugs.
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