m a s s
s p e c t r o m e t r y
Thermo Scientific MetWorks
Metabolite Identification
Software
Enabling Confident Analysis of Metabolism Data
Part of Thermo Fisher Scientific
MetWorks Metabolite Identification Software
Software for the Confident Analysis of Metabolism Data
Thermo Scientific MetWorks with Mass Frontier™ software provides
a simple and complete solution for identifying drug metabolites.
MetWorks™ software automates and simplifies
identification of Phase I and Phase II metabolites
Automatically identify and generate ion chromatograms for
all expected metabolites. Detect and identify unpredicted
modifications with advanced data analysis tools:
• Analyze sample and controls and utilize MSn spectra, retention
time, and m/z to target putative metabolites
• Refine metabolite identification using Mass Defect Filtering
of high mass accuracy data
• Incorporate isotope pattern recognition of both natural
and artificial isotopes
• Correlate MS/MS and MS3 spectra with those from the
parent drug to identify common product ions and mass shifts
related to neutral losses
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MetWorks Simplifies Data Visualization and Reporting
MetWorks data analysis complements the Data Dependent™ and Dynamic Exclusion™
capabilities of all Thermo Scientific ion trap mass spectrometers for simultaneous
determination of metabolic stability and structural identification.
MetWorks provides capabilities for
confident analysis of metabolism data
• Detecting Known Modifications
• Detecting Unpredicted Modifications
• Multiple Mass Defect Filter
• Automatic Isotope Pattern Recognition
• Spectral Correlation
• Data-Dependent Parent Time List (DDPT)
• Confident Metabolite Identification
• Comprehensive Data Visualization and Reporting
MetWorks software is compatible with all Thermo Scientific mass spectrometers
capable of performing MS/MS and MSn fragmentation. In addition, MetWorks
software allows high-resolution, accurate-mass data from Orbitrap™, LTQ FT™,
Q Exactive™ and Exactive™ instruments to be fully leveraged.
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MetWorks Metabolite Identification Software
Detecting Known Modifications
Automatic Identification
of Expected Modifications
Choose from an extensive list of Phase I and II modifications
Convenient reporting software displays full scan and product
and create a custom list of single and combined expected
ion mass spectra for each expected metabolite in order to
modifications for the parent drug of interest. During data
verify predicted biotransformations.
acquisition, the user has the option to specify a custom
MS/MS list to target metabolites. During data processing,
MetWorks software will automatically generate Extracted
Ion Chromatograms (EIC) for each expected metabolite.
Expected Modifications View
A custom list of expected modifications was
used for the analysis of Loperamide data from
the Thermo Scientific LTQ XL linear ion trap.
Extracted ion chromatograms for unmetabolized
Loperamide (m/z 477.289) and its demethylated
(m/z 463.379) and hydroxylated (m/z 493.297)
metabolites are shown at the top of the figure
above. The full scan mass spectra (left) and
product ion spectra (MS/MS, right) for demethylated
Loperamide are shown at the bottom of the figure.
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Detecting Unpredicted Modifications
Simplify Identification of
Unknown Drug Metabolites
MetWorks software provides the flexibility to filter data in order to
identify low- and high-abundance peaks related to the parent drug.
These masses may arise from metabolites which were not initially
predicted or included in a targeted mass list. MetWorks software
provides a number of different software filters to help detect these
components so that automated MSn fragmentation can be
triggered and structural identification confirmed.
Component Subtraction
Using Controls
Component subtraction can remove the effect of matrix or non-drug
related interferences from the sample data and uncover unpredicted
metabolites. A MetWorks algorithm compares components found
in Sample and Control using composite MSn data in addition to
Component Detection View
Highlighting the components at RT 4.4 minutes in Sample
(metabolized Loperamide) and Control (unmetabolized
Loperamide) total ion chromatograms (top two panels)
links the components to their composite MSn spectra
(bottom panel). Since the RT (4.4 minutes), m/z (437.30)
and product ion spectra (MS/MS) of these two components
are identical in both Sample and Control, they were
automatically labeled in red in the chromatograms
and can be excluded from further analysis. Components
unique to the sample are labeled in green and are
shown in the panel labeled Potential Modifications.
Data was obtained using the LTQ XL linear ion trap.
TM
full scan retention time (RT) and m/z values.
Using these criteria, Components in the
Sample and Control that are the same can
automatically be excluded from further
analysis, while components that are unique
to the sample are automatically highlighted
in green for easy visualization. These
components are potential drug metabolites.
If a Control data file is not available, another
option is to perform the background
subtraction using the solvent (not shown).
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MetWorks Metabolite Identification Software
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Multiple Mass Defect Filter
Biotransformations cause predictable changes to the mass of
removes a significant amount of background interference
the parent drug. Mass defects arise since molecular masses
originating from the matrix and unmasks low abundance
of metabolites are non-integer and deviate from nominal mass
metabolite peaks. MetWorks software enables the use of
values. This results in small changes of the mass defect between
a combination of up to six Mass Defect Filters, such that
the parent drug and its metabolites. The MetWorks Mass Defect
all the results are compiled into one chromatogram.
Filter takes advantage of the high mass accuracy data from the
Orbitrap-based and LTQ FT Ultra™ instruments and the known mass
defects for different biotransformations. After data acquisition, a
software filter removes ions with mass defects that are outside a
user-defined mass defect window based on all possible modifications of the parent drug. Consequently, this process automatically
The effect of using single and multiple Mass Defect
Filters on visualizing Oxime metabolites in rat
hepatocytes. Unfiltered, single Phase I, single Phase II
and combined Phase I and Phase II filtered base peak
chromatograms are shown. The parent drug Oxime is
highlighted. Data was obtained using the LTQ
Orbitrap™ hybrid mass spectrometer. Note: the y-axis
intensity value is lower for Phase II MDF than the other
chromatograms shown.
Automatic Isotope Pattern Recognition
Drug candidates which contain elements such as bromine and
chlorine have easily distinguishable isotope patterns which are not
common in endogenous metabolites found in body fluids. Drugs can
also be artificially labeled using stable isotopes including 13C, 2H,
and 15N. For metabolism studies, these unique isotope patterns can
act as intrinsic or extrinsic signatures that can be used to efficiently
detect drug metabolites. MetWorks software can filter full scan
mass spectra based on the changes in mass and relative abundance
of these isotopes and automatically display extracted ion
chromatograms representing these isotope patterns that
indicate potential metabolites.
The top panel shows the EIC for an unexpected
demethylated hydroxylated Loperamide metabolite
at m/z 479.209597 identified by isotope pattern
recognition, which includes a rearrangement and
a modification. The bottom panel shows full scan
MS of the metabolite indicating a distinct chlorine
isotope pattern with a mass difference of two
(highlighted in red) that was automatically recognized
by MetWorks software and determined to be
desmethyl-hydroxyl Loperamide. Data was obtained
using the LTQ Orbitrap hybrid mass spectrometer.
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MetWorks Metabolite Identification Software
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Spectral Correlation
Spectral correlation analysis compares MS/MS or MS3 spectra
between the sample (metabolized drug) and a reference (typically
parent drug). The MetWorks correlation algorithm searches for
product ion spectral components that are the same in metabolized
and unmetabolized drugs (common product ions) or different through
a consistent mass shift that indicates a chemical modification such
as an adduct. The related parent ions are automatically highlighted
as metabolites of interest.
Spectral Correlations View
Chromatogram before (A) and after (B) spectral
correlation indicates identification of an unpredicted
dehydroxy rearrangement of Loperamide at RT of 4.04
minutes with m/z of 455.188. The MS3 spectra of (C)
unmetabolized Loperamide (reference) and (D) the
rearranged dehydroxy metabolite are shown with
common product ions highlighted in blue. Data was
obtained using the LTQ XL linear ion trap.
Data-Dependent Parent Time List (DDPT)
Automatic Trigger of Targeted MS/MS
on Parent and Predicted Metabolites
The DDPT feature in MetWorks software automatically generates a
data-dependent parent time list based on parent and expected modification list in the MetWorks method file (.mbx file). This list is used
by the instrument method on the fly for acquiring targeted MS/MS–
all from within MetWorks software. This unique feature is available in the Acquire section of MetWorks software, for acquisition
on ion trap and Orbitrap hybrid instruments.
Data-Dependent Parent Time List enables on-the-fly
targeted MS/MS.
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MetWorks Metabolite Identification Software
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Confident Metabolite Identification
Once putative metabolites are identified, the location of the functional
modification and the assignment of overall structure can be facilitated
using Mass Frontier software. Chemical Structures representing the
possible sites of modification can be created and fragmented in silico
using general mechanistic fragmentation rules and the extensive
Fragmentation Library™ that contains literature-based fragmentation
schemes. The fragment ions generated using this approach can be
directly compared to those acquired during LC-MSn analysis.
Mass spectral peaks can be automatically annotated with their
corresponding structures.
Structural annotation of N-demethylated metabolite of Loperamide is shown. Once metabolite
structure is confirmed using Mass Frontier’s fragmentation schemes, product ion structures can
be assigned to the mass spectral peaks.
Comprehensive Data Visualization and Reporting
The MetWorks Report View can be customized to display the
Summary Report or the Standard MetReport. The Summary Report
reports all the expected metabolites found in one summary table.
The Standard MetReport compiles all potential metabolites you
selected from all results views into a single view. The Standard
MetReport view may include elemental formula, type of modification, structure, retention time, m/z, associated chromatograms, a list
of the most intense fragment ions, spectral tree (MSn information),
and mass difference between parent and metabolite.
The Report View can be used to create reports in Adobe PDF,
Microsoft Word, and Microsoft Excel formats.
MetWorks Summary
Report View
The summary report is a table that contains
the Peak ID, retention time (RT), putative
metabolite, mass shift, formula change, theoretical and measured m/z, ppm, peak area,
relative abundance and parent normalization
information.
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