Thermo Scientific MetWorks Metabolite Identification ….pdfof the most intense fragment ions, spectral tree (MS ninformation), and mass difference between parent and metabolite.

Part of Thermo Fisher Scientific

Enabling Confident Analysis of Metabolism Data

Thermo Scientific MetWorksMetabolite Identification

Software

m a s s s p e c t r o m e t r y

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, retentiontime, 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 theparent drug to identify common product ions and mass shifts related to neutral losses

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.

MetW

orks Metabolite Identification Softw

are

2

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 forconfident 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, MetWorkssoftware allows high-resolution, accurate-mass data from Orbitrap™, LTQ FT™,

Q Exactive™ and Exactive™ instruments to be fully leveraged.

3

Automatic Identificationof Expected Modifications

Choose from an extensive list of Phase I and II modifications

and create a custom list of single and combined expected

modifications for the parent drug of interest. During data

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.

Convenient reporting software displays full scan and product

ion mass spectra for each expected metabolite in order to

verify predicted biotransformations.

Expected Modifications ViewA 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 unmetabolizedLoperamide (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 figureabove. The full scan mass spectra (left) and product ion spectra (MS/MS, right) for demethylatedLoperamide are shown at the bottom of the figure.

Detecting Known Modifications

MetW

orks Metabolite Identification Softw

are

4

Component SubtractionUsing 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

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).

Simplify Identification ofUnknown 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 Detection ViewHighlighting the components at RT 4.4 minutes in Sample(metabolized Loperamide) and Control (unmetabolizedLoperamide) 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. Componentsunique to the sample are labeled in green and are shown in the panel labeled Potential Modifications. Data was obtained using the LTQ XLTM linear ion trap.

Detecting Unpredicted Modifications

5

Biotransformations cause predictable changes to the mass of

the parent drug. Mass defects arise since molecular masses

of metabolites are non-integer and deviate from nominal mass

values. This results in small changes of the mass defect between

the parent drug and its metabolites. The MetWorks Mass Defect

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 modifica-

tions of the parent drug. Consequently, this process automatically

removes a significant amount of background interference

originating from the matrix and unmasks low abundance

metabolite peaks. MetWorks software enables the use of

a combination of up to six Mass Defect Filters, such that

all the results are compiled into one chromatogram.

The effect of using single and multiple Mass DefectFilters on visualizing Oxime metabolites in rat hepatocytes. Unfiltered, single Phase I, single Phase IIand combined Phase I and Phase II filtered base peakchromatograms are shown. The parent drug Oxime ishighlighted. Data was obtained using the LTQOrbitrap™ hybrid mass spectrometer. Note: the y-axisintensity value is lower for Phase II MDF than the other chromatograms shown.

MetW

orks Metabolite Identification Softw

are

6

Multiple Mass Defect Filter

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 unexpecteddemethylated hydroxylated Loperamide metabolite at m/z 479.209597 identified by isotope pattern recognition, which includes a rearrangement anda modification. The bottom panel shows full scanMS of the metabolite indicating a distinct chlorineisotope pattern with a mass difference of two(highlighted in red) that was automatically recognizedby MetWorks software and determined to bedesmethyl-hydroxyl Loperamide. Data was obtainedusing the LTQ Orbitrap hybrid mass spectrometer.

7

Automatic Isotope Pattern Recognition

Spectral Correlations ViewChromatogram before (A) and after (B) spectral correlation indicates identification of an unpredicteddehydroxy 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) therearranged dehydroxy metabolite are shown with common product ions highlighted in blue. Data wasobtained using the LTQ XL linear ion trap.

MetW

orks Metabolite Identification Softw

are

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.

8

Automatic Trigger of Targeted MS/MSon Parent and Predicted Metabolites

The DDPT feature in MetWorks software automatically generates a

data-dependent parent time list based on parent and expected mod-

ification 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 avail-

able in the Acquire section of MetWorks software, for acquisition

on ion trap and Orbitrap hybrid instruments.

Data-Dependent Parent Time List enables on-the-flytargeted MS/MS.

Data-Dependent Parent Time List (DDPT)

9

MetW

orks Metabolite Identification Softw

are

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 metabo-lite of Loperamide is shown. Once metabolitestructure is confirmed using Mass Frontier’s frag-mentation schemes, product ion structures canbe assigned to the mass spectral peaks.

Confident Metabolite Identification

10

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 modifica-

tion, 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 ViewThe summary report is a table that containsthe Peak ID, retention time (RT), putativemetabolite, mass shift, formula change, theo-retical and measured m/z, ppm, peak area,relative abundance and parent normalizationinformation.

Comprehensive Data Visualization and Reporting

11

BR62428_E 04/12S

Tap our expertise throughout the life of your instrument. Thermo Scientific Services

extends its support throughout our worldwide network of highly trained and certified

engineers who are experts in laboratory technologies and applications. Put our team

of experts to work for you in a range of disciplines – from system installation, training

and technical support, to complete asset management and regulatory compliance

consulting. Improve your productivity and lower the cost of instrument ownership

through our product support services. Maximize uptime while eliminating the

uncontrollable cost of unplanned maintenance and repairs. When it’s time to

enhance your system, we also offer certified parts and a range of accessories

and consumables suited to your application.

To learn more about our products and comprehensive service offerings,

visit us at www.thermo.com.

Laboratory Solutions Backed by Worldwide Service and Support In addition to these offices, ThermoFisher Scientific maintains a network of representative organizationsthroughout the world.

Africa-Other+27 11 570 1840 • [email protected]+61 3 9757 4300 • [email protected]+43 1 333 50 34 0 • [email protected]+32 53 73 42 41 • [email protected]+1 800 530 8447 • [email protected]+86 10 8419 3588 • [email protected]+45 70 23 62 60 • [email protected] -Other+43 1 333 50 34 0 • [email protected] / Norway / Sweden+46 8 556 468 00 • [email protected]+33 1 60 92 48 00 • [email protected]+49 6103 408 1014 • [email protected]+91 22 6742 9434 • [email protected]+39 02 950 591 • [email protected]+81 45 453 9100 • [email protected] America+1 561 688 8700 • [email protected] East+43 1 333 50 34 0 • [email protected]+31 76 579 55 55 • [email protected] Zealand+64 9 980 6700 • [email protected]/CIS+43 1 333 50 34 0 • [email protected] Africa+27 11 570 1840 • [email protected]+34 914 845 965 • [email protected]+41 61 716 77 00 • [email protected]+44 1442 233555 • [email protected]+1 800 532 4752 • [email protected]

www.thermoscientific.com

Thermo Fisher Scientific, San Jose, CA USA is ISO Certified.

© 2007-12 Thermo Fisher Scientific Inc. All rights reserved.Mass Frontier and Fragmentation Library are trademarks ofHighChem, Ltd; Adobe is a registered trademark of AdobeSystems Incorporated; Microsoft is a registered trademarkof Microsoft Corporation. All other trademarks are the prop-erty of Thermo Fisher Scientific Inc. and its subsidiaries.

Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.