Qualitative Analysis Workflows
Howard Sanford
Stephen Harnos
With Tom Barrett & Matt Leyden & Kevin Costalunga
Qualitative Analysis Workflows
1
MassHunter Qualitative Analysis Webinar Series
Navigator
• User centric
• Interactive Browsing
–
Walk the Chromatogram
–
Spectrum Preview
• Spectrum ID
–
Library/Database Search
–
Molecular Formula Generator
• No concept of compounds
• No Feature Finding – ‘Find by…’
• What does the chromatography look
like? System suitability, S/N, plates…
• What does the spectrum look of each
peak look like?
• Can the spectrum be identified by
searching a library?
Qualitative Analysis Workflows
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Navigator and Workflows
MassHunter Qualitative Analysis Software B.08.00 SP1
Workflows
• Compound centric
• Automated Routine Workflows
Sample Purity
Compound Discovery
Compound Identification
• Feature Finding – ‘Find by…’
• No Spectrum Preview
• No ‘User Spectra’
• All Spectra are compound based.
• What compounds can be found and
identified?
Navigator Workflows
Qualitative Analysis Workflows
3
MassHunter Qualitative Analysis Software B.08.00 SP1
Navigator and Workflows
File opens in Workflows.
The sample chromatogram(s) extracted.
Spectra not extracted.
Method is NOT passed to other context.
File opens in Navigator.
The sample chromatogram(s) are extracted.
No compound results.
Method is NOT passed to other context.
Qualitative Analysis Workflows
Workflow Definitions
• Target/Suspect Screening
• Compound Discovery
• Sample Purity
• Custom
Compound Mining Algorithms
• Find by Integration
• Find by Chromatogram Deconvolution
• Molecular Feature Extraction
• Find by Formula
Compound Identification
• Library and Databases
Generating Reports
Basic Qualitative Analysis features were covered in the previous webinar available at
www.agilent.com/en-us/training-events/eseminars/236e
Qualitative Analysis Workflows
4
Topics
MassHunter Qualitative Analysis Software B.08.00 SP1
Qualitative Analysis Workflows
5
Workflows Default Layout
MassHunter Qualitative Analysis Software B.08.00 SP1
Method Editor is a tab.
Sample Table
Compound List
Compound
Identification
Results
Structure
Viewer
Sample
Chromatogram
Results
Compound
Chromatogram
and Spectrum
Results
Open Data File Dialog Box
• Options
– Load Worklist Method
– Load Results Method
– Use Current Method
• Sample Information
• Load result data
• Run method workflow
Qualitative Analysis Workflows
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Open Data File
Workflows
Qualitative Analysis Workflows
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Adaptive User Interface– simplifies Method Editor
Method Editor
MS Levels MS or MS/MS
Qualitative Analysis Workflows
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User Interface Configuration
• Check Show Advanced Settings
from the Configuration menu to
access the User Interface
Configuration.
• Also shows advanced Method
Editor features.
Separation types
(Check GC or LC)
Unit Mass (SQ, TQ)
Accurate Mass (TOF, QTOF)
Open Method
• Load the method relevant to the data set.
• Method contains recommended/default
parameters.
• Method Editor values can be modified.
• Saves time developing specific values.
• Good starting point.
• Default-GCMS-SQ.M → GCMS and GC QQQ
• Default-GCMS.M →GC QTOF
• Default-LCMS.M → LCMS
Qualitative Analysis Workflows
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Open Method
Qualitative Analysis Methods
• Intended for routine and automated
analysis.
– Select a workflow.
– Select the mining algorithm.
– Develop method parameters.
• Limited tools for manual spectral
extraction.
– No spectral preview.
– Manual extraction always creates a
compound.
• Double-click on sample in Sample Table
to process data files with loaded
method.
• Compound report only (no analysis
reports).
Qualitative Analysis Workflows
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Definition
Workflows
A workflow is an automated sequence of steps
to complete a task and generate results.
• Workflow is the table of contents.
• All the steps are defined via the Method Editor.
• Default workflows depend upon data set.
• Workflows
– Target/Suspect Screening
– Sample Purity
– Compound Discovery
– Custom
• The workflow parameters can be saved as part
of the Qualitative Analysis Workflows method.
Qualitative Analysis Workflows
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Definition
Workflows
Can save
processing time.
The Method Editor groups method sections to match workflows.
• Method Automation via workflows.
• Target/Suspect Screening
• Compound Discovery
• Sample Purity
• Custom
• Compound Identification
• Spectra
• Chromatograms
Qualitative Analysis Workflows
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Method Groupings
Workflows
Compound Mining defines the algorithm to be run to find compounds.
• All the steps are defined via the Method Editor.
• Mining Algorithms depend upon data set selected.
• Mining Algorithms depend upon workflow selected.
Qualitative Analysis Workflows
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Definition
Compound Mining
All features may not be visible
with the Adaptive User
Interface.
Auto-Select Compound Mining defines the algorithm to be run to find compounds.
• Based on data set loaded.
• Not always present, for example MRM data.
• Available with only Target/Suspect Screening and Compound Discovery.
• Good starting point.
Qualitative Analysis Workflows
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Auto-Select Compound Mining
Compound Mining
“I know what I want, is it there?
• Useful with accurate mass data (TOF,
QTOF).
• Requires a library or a database
– List of compound formulas (formulae)
– In the form of cdb, csv, cef files
– Can enter the neutral mass
• Mining Algorithms (data dependent)
– Find by Formula
– Find by Fragment (accurate mass data)
• Are the fragments logical losses?
– Find by MRM
•
Used with MRM data (QQQ)
Require RT match
Only report qualified compounds
Qualitative Analysis Workflows
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Target/Suspect Screening
Workflows
“I know what I want, how pure is it?”
• Uses high resolution data.
• Requires a library or a database
– List of compound formulas (formulae)
– In the form of cdb, csv, cef files
• Mining Algorithms (data dependent)
– Find by Formula
•
Adds sample purity calculations
•
Adds the area percent calculations and
report.
Require RT match
Only report qualified compounds
Qualitative Analysis Workflows
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Sample Purity
Workflows
“I don’t know what I have, find the
compounds and identify them”
• Library or database is optional.
– Usually one database or library is specified.
– List of compound formulas (formulae)
– In the form of cdb, csv, cef files
• Compound Mining
– Auto Select Compound Mining
– Find by Molecular Feature
– Find by Auto MS/MS
– Find by Targeted MS/MS
– Find by Integration
– Find by Chromatogram Deconvolution
Qualitative Analysis Workflows
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Compound Discovery
Workflows
“I know what I need to do, generate the
compounds!”
• Selecting Custom provides Available Actions.
• Define and refine the actions in Method
Editor.
• Available for all datasets.
Qualitative Analysis Workflows
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Custom
Workflows
Steps must be logical
• Method Editor reveals 3 algorithms
– Find by Formula
– Find by Fragments
– Find by MRM
• Selecting the ‘Find by’ in the Method Editor
opens additional parameters.
Qualitative Analysis Workflows
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Target/Suspect Screening Method Editor
Compound Mining
• Only 2 Method Editor choices
• Sample Purity parameters
• Find by Formula
– Useful for LC/TOF or QTOF data.
– Requires a formula database.
Qualitative Analysis Workflows
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Sample Purity Method Editor
Compound Mining
• Multiple Compound Mining algorithms
available.
• Find by Auto MS/MS and Find by Targeted
MS/MS specific to QTOF data.
Qualitative Analysis Workflows
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Compound Discovery Method Editor (1)
Compound Mining
• Find by Chromatogram Deconvolution
– Similar to Quantitative Analysis and
Unknowns Analysis.
Qualitative Analysis Workflows
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Compound Discovery Method Editor (2)
Compound Mining
• Component of the Method Editor
–
Establishes database or library to use.
Qualitative Analysis Workflows
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Definition
Compound Identification
• Load the data file Evaldemo2.D
– Simple data file.
– GCMS SQ data.
• Load the method Default-GCMS-SQ.M.
• Set the Workflow to Compound Discovery.
• Select Find by Integration for the Compound Mining.
• Add the NIST17.L.
• Review the Find by Integration parameters.
• Run Method Workflow or run icon.
Qualitative Analysis Workflows
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Example
Find by Integration
Qualitative Analysis Workflows
25
Results
Find by Integration
Let’s take a moment for questions on Workflows and
Compound Mining Algorithms
Up Next:
Data Review
MassHunter Webinar Series
26
• Sample Table shows information about the
sample.
– Workflow column shows most recent workflow
applied to data file.
• Results
– Four compounds identified (4 found in library).
– Saved Results Method.
• Must be saved and shows Qualitative Analysis
parameters.
• Workflow
– Workflow used and library searched.
Qualitative Analysis Workflows
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Data Review
Sample Table
• Acquisition
Information about acquisition parameters.
• Toolbar
Add/Remove Columns
Run method workflow
Can run on multiple samples
Reprocess sample
Can reprocess single samples or
reprocess with a different workflow.
Sample Information
• Compound List shows 4 compounds
• Four compounds identified
• Context menu yields numerous options for formatting
– Add/Remove Columns
– Auto Size all Columns
– Sorting
– Copying, printing and exporting
• Toolbar
– Hide any currently empty columns
– Auto Size All Columns
– Select previous and next compounds
Qualitative Analysis Workflows
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Compound List
Data Review
• Additional options are available in the context menu.
– Identify Compound(s)
• Initiates Identification workflow for selected compounds.
– Search Using NIST MS Program
– Add/Edit Manual Identification
• Overwrites current Name column.
– Send Spectra to PCDL database.
– Initiate Quantitation
• Useful with MRM data
• Launches Quantitative analysis and transfers transitions
and collision energies to the quantitation database.
Qualitative Analysis Workflows
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Compound List
Data Review
Super Headers can be moved as group.
Qualitative Analysis Workflows
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Super Header vs. Header
Compound List
Sub Headers can be moved with the super header.
• Compound Identification Results more
detailed information about compounds.
– Detailed information about identified
compound.
• May be tabbed window in default layout.
• Shows 5 library hits.
• Sub level shows additional information
about identified compound.
• Context menu yields numerous options for
formatting.
– Add/Remove Columns
– Auto Size all Columns
– Sorting
– Copying, printing and exporting
• Toolbar
– Hide any currently empty columns
– Auto Size All Columns
Qualitative Analysis Workflows
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Compound Identification Results
Data Review
Qualitative Analysis Workflows
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Sample Chromatogram Results
Data Review
• Sample Chromatogram Results.
– Can be overlaid or list modes
• Toolbar
– Autoscale X-axis and Y-axis
– Autoscale X-axis
– Autoscale Y-axis
– Unzoom (multiple levels)
– Autoscale Y-axis during zoom
– List mode or overlaid mode
– Maximum number of panes
– Compound overlay mode
– Extract Chromatograms (allows TIC,
BPC, EIC, etc. to be extracted.
– Scale Chromatogram Off
– Scale to Largest in Each
Chromatogram
– Integration Peak List
– Chromatogram Display Options
– Print
Qualitative Analysis Workflows
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Compound Chromatogram Results
Data Review
• Compound Chromatogram Results
– Displays the TCC
– Can be overlaid or list modes
• Toolbar
– Autoscale X-axis and Y-axis
– Autoscale X-axis
– Autoscale Y-axis
– Unzoom (multiple times)
– Autoscale Y-axis during zoom
– List mode or overlaid mode
– Maximum number of panes
– Show Legend in Overlaid Mode
– Range Select tool
– Annotation Mouse Tool
– Scale to Largest in Each Chromatogram
– Integration Peak List
– Coelution Plot
– Chromatogram Display Options
– Print
Qualitative Analysis Workflows
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Compound MS Spectrum Results
Data Review
• Compound MS Spectrum Results.
– Can be overlaid or list modes
• Toolbar
– Autoscale X-axis and Y-axis
– Autoscale X-axis
– Autoscale Y-axis
– Unzoom (multiple times)
– Autoscale Y-axis during zoom
– List mode or overlaid mode
– Maximum number of panes
– Show Predicted Isotope Distribution
– Range Select tool
– Annotation Mouse Tool
– Delta Mass Caliper tool
– Spectrum Peak List
– Spectrum Display Options
– Print
Qualitative Analysis Workflows
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Spectral Difference Results
Data Review
• Spectral Difference Results
• View > Difference
Results activates this
window
• Toolbar
– Autoscale X-axis and Y-
axis
– Autoscale X-axis
– Linked Y-axis mode
Extracted Spectrum
Mirror Plot
Library Spectrum
Qualitative Analysis Workflows
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Structure Viewer
Data Review
• Composed of two tabs
• Structure tab
• MOL Text
– Must be available in library
– Easily copied to clipboard
Let’s take a moment for questions on Data Review
Up Next:
Find by Chromatogram Deconvolution
MassHunter Webinar Series
37
• One of the ‘Find by…..’ algorithms.
• Useful to determine what is in a sample.
• Optional identification through the Compound
Identification > Identification Workflow in the Method
Editor.
Qualitative Analysis Workflows
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Case Study
Find by Chromatogram Deconvolution
TIC
Compound 1
Compound 2
Compound 3
One gaussian peak but actually
composed of three compounds.
An average spectrum is created over the entire or selected RT range.
An EIC is extracted for each mass in the average spectrum.
Each EIC is integrated using the Agile integrator and “EIC peaks” are created.
The “EIC peaks” are grouped together based on common RT and each set of “EIC peaks” become
“Compounds”.
The “EIC peak” shape is used to create a Compound peak shape.
Filters can remove small ions and “compounds” with only a few small ions.
The relative intensity (area) of the “EIC peaks” and the mass of the EIC are used to create a clean
spectrum of the Compound.
Qualitative Analysis Workflows
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Case Study
Find by Chromatogram Deconvolution
The algorithm creates Extracted Ion Chromatograms (EICs) across a small
time range around the apex spectrum RT.
Qualitative Analysis Workflows
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Case Study
Find by Chromatogram Deconvolution
50
170
280
310
75
185
160
50
170
280
75
185
310
TIC
160
Uncleaned apex mass spectrum
Qualitative Analysis Workflows
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Case Study
Find by Chromatogram Deconvolution
280
185
Cleaned mass spectra
170
75
50
310
160
75
170
185
280
50
160
310
•RT window size factor
– Key parameter that controls grouping
– Smaller number → more compounds
– Possible ‘fake’ compounds
• Excluded m/z
• Spectrum peak threshold
• SNR threshold
• Extraction window
– Mass defect → -0.3 to +0.7 Low resolution
– High resolution in ppm.
• Component shape
– Upper 75% of peak is considered.
Qualitative Analysis Workflows
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Deconvolution Parameters
Find by Chromatogram Deconvolution
•Mass filters are applied after the EICs are organized into
compounds.
– Remove small ions from the compounds based on absolute
height or relative height of base peak or both.
• Compounds Filters are applied to the compounds based
on absolute area or relative area of the largest compound
or both.
• Results
– Clear previous results is unchecked allows multiple algorithms
to be applied.
Qualitative Analysis Workflows
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Deconvolution Parameters
Find by Chromatogram Deconvolution
Let’s take a moment for questions on Find by Chromatogram
Deconvolution
Up Next:
Molecular Feature Extraction
MassHunter Webinar Series
44
MFE (Molecular Feature Extraction)
• Untargeted analysis for a unique molecular entity based on three dimensional array of retention time,
mass and abundance in GC or LC MS data. Operates on raw scan data.
– Evaluates the data as a three dimensional array based on retention time, mass and abundance.
– Removes persistent or slowly changing background.
– Looks for masses with a common elution profile at approximately the same time.
– Masses are grouped into compounds.
– Co-eluting interferences are resolved.
– Isotopic cluster are determined and grouped (accurate mass).
– Charge state assignments and molecular adducts are recognized (multi-charged species and ESI).
– 2D/3D visualization of data.
– Chemical characterization and identification.
Qualitative Analysis Workflows
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Case Study
Find by Molecular Feature
MFE (Molecular Feature Extraction)
Qualitative Analysis Workflows
46
Case Study
Find by Molecular Feature
398.6
398.8
399.0
399.2
399.4
399.6
Drift Time (ms) vs. m/z
38
39
40
41
398
399
400
401
402
403
404
Drift Time (ms) vs. m/z
36
37
38
39
40
41
42
Identify the highest point in the three
dimensional array.
MFE (Molecular Feature Extraction)
Qualitative Analysis Workflows
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Case Study
Find by Molecular Feature
398.6
398.8
399.0
399.2
399.4
399.6
Drift Time (ms) vs. m/z
38
39
40
41
398
399
400
401
402
403
404
Drift Time (ms) vs. m/z
36
37
38
39
40
41
42
Identify the highest point in the three dimensional
array.
Expand the array to include neighboring data
points.
MFE (Molecular Feature Extraction)
Qualitative Analysis Workflows
48
Case Study
Find by Molecular Feature
398.6
398.8
399.0
399.2
399.4
399.6
Drift Time (ms) vs. m/z
38
39
40
41
398
399
400
401
402
403
404
Drift Time (ms) vs. m/z
36
37
38
39
40
41
42
Identify the highest point in the three
dimensional array.
Expand the array to include neighboring data
points.
Once the bounds of the array have been
established, save the 3D peak array.
Resolve co-eluting interferences.
Chemical characterization and subsequent
identification.
Reiteratively repeat the process.
MFE (Molecular Feature Extraction)
Qualitative Analysis Workflows
49
Case Study
Find by Molecular Feature
398.6
398.8
399.0
399.2
399.4
399.6
Drift Time (ms) vs. m/z
38
39
40
41
398
399
400
401
402
403
404
Drift Time (ms) vs. m/z
36
37
38
39
40
41
42
Identify the next highest point in the three
dimensional array.
After completed, reiteratively process the
next highest point in the array.
MFE (Molecular Feature Extraction)
• The algorithm effectively processes the three dimensional array based on retention time, mass and
abundance.
• The algorithm does not evaluate the information based on spectra or chromatograms.
• The process is reiteratively applied to the entire data array until no peak remains above background.
• Noise is effectively removed.
• Highly computationally intensive but very effective in identifying compounds.
Qualitative Analysis Workflows
50
Case Study
Find by Molecular Feature
Qualitative Analysis Workflows
51
Parameters (GC/MS)
Find by Molecular Feature
• Method Editor >
Compound Discovery >
Find by Molecular
Feature.
• Composed of 3 tabs.
• Limited to small molecules
(chromatographic) for EI
GC/MS.
• Quality score is the
algorithm’s estimation of
quality of the feature.
• MS/MS Spectrum for GC
QTOF data.
Qualitative Analysis Workflows
52
Parameters (LC/MS) (1)
Find by Molecular Feature
• Different parameters to
accommodate the different
data set.
• Composed of multiple tabs.
• Various target data types.
– Choose applicable to data.
• Ion species
– Choose expected adducts
based on conditions.
– Can add and delete
additional adducts.
Qualitative Analysis Workflows
53
Parameters (LC/MS) (2)
Find by Molecular Feature
• Charge State
– Choice of 5 Isotope models.
– Choose model appropriate to
dataset.
– Can set a range of charge
states.
• Compound Filters same.
• Mass Filters
– Targeted list.
– When checked, other items
available to refine results.
– Can also create an exclusion
list.
Qualitative Analysis Workflows
54
Parameters (LC/MS) (3)
Find by Molecular Feature
• Mass Defect tab
– Not applicable to large
molecules (proteins, oligos).
– Useful for halogen containing
and hetero atoms.
– Compounds outside the mass
defect tolerance are excluded
from the results.
• Results tab is the same as
before.
Qualitative Analysis Workflows
55
Results
Find by Molecular Feature
Let’s take a moment for questions on Molecular Feature
Extraction (MFE)
Up Next:
Find by Formula and Compound Identification
MassHunter Webinar Series
56
Qualitative Analysis Workflows
57
Parameters (Accurate Mass)
Find by Formula
• Find by Formula
– Targeted analysis.
– Numerous tabs are available to
refine parameters.
– Requires a target source and
optional RT matches.
– Can confirm fragment ions ->
Fragment Confirmation tab.
– Uses MS scan data but can be
used on MS/MS data but ignores
MS/MS spectra.
Qualitative Analysis Workflows
58
Results
Find by Formula
Adduct
confirmation
Identification Workflow
• Identify by Library/Database search
– Optional search
– Select numerous libraries.
• Search all libraries / databases
– Search all libraries or stop when hit is
found.
• Identify by Formula Generation
– Optional generation of formulas
Qualitative Analysis Workflows
59
Identification Workflow
Compound Identification
Qualitative Analysis Workflows
60
Database Search Settings
Compound Identification
• Numerous tabs
• Define database
settings.
Library Search Settings
• Search Criteria
– Adjust Score
• RT Scoring
– Retention time or
retention index.
• Peak Filters
• Tolerances
– Accurate mass values
Qualitative Analysis Workflows
61
Library Search Settings
Compound Identification
• Available from a Desktop icon.
• Installed with Quantitative
Analysis.
• Most useful for low resolution
data.
– Most common NIST library.
• Can review and search
commercial libraries in .L format.
– From Edit > Search
– 3 conditional statements
• Can create user generated
libraries.
– Can copy entries from Qualitative
Analysis, Quantitative Analysis
and Unknowns Analysis.
Qualitative Analysis Workflows
62
Library Editor
Database and Library Tools
Installed from a stand-alone
application
• Useful for high resolution MS/MS
data
• Handles both CDB and CDBL
• Can have multiple spectra with
various collision energies (CE).
• Works with ion mobility data.
Qualitative Analysis Workflows
63
PCDL Manager
Database and Library Tools
Generate Formulas
• Allowed Species
– Elements and limits.
– Use for narrowing possible hits.
– Can add elements.
Qualitative Analysis Workflows
64
Generate Formulas
Compound Identification
Method Automation > Reports
• Printed or electronic format
available.
• Report can be saved to data file or
directory.
• Auto generate file name.
Qualitative Analysis Workflows
65
Generating Compound Reports
Reports
Templates
• Excel Reporting or PDF based
reporting.
• Various templates are available.
• Organized based on workflows.
Qualitative Analysis Workflows
66
Generating Compound Reports
Reports
Layout & Contents
• Allow further customization of the report.
Qualitative Analysis Workflows
67
Generating Compound Reports
Reports
Let’s take a moment for questions on Compound
Identification and Reporting
Up Next:
Training Resources
MassHunter Webinar Series
68
Training Resources
LCMS Data Analysis Resources DVD
69
Training Resources
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