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Rev. 1.3
MassHunter TOF/QTOF Users Meeting
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
1
Rev. 1.3
Qualitative Analysis Workflows
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
5
•Workflows in Qualitative Analysis allow the user to only see and work with the
areas and dialog boxes they need for their specific tasks
•A Workflow consists of three parts:
– A default method for the Workflow with the parameters and report templates
already set for that type of task.
– A specified layout of the user interface that only shows the windows and
table columns that the user needs to see.
– A new section at the top of the Method Explorer (called a workflow section)
which groups together the tabs a user may need while performing a task:
Rev. 1.3
Applying a Workflow
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
7
•“Configuration Configure for Workflow” in the menu bar
•You will see a warning that applying the Workflow will make changes
Rev. 1.3
Using a Workflow
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
8
•After applying the Workflow, go through each tab in the workflow section
sequentially to adjust the parameters for your task:
Rev. 1.3
Isotope Distribution Calculator
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
12
•There is a standalone Isotope Distribution Calculator included with Qualitative
Analysis at:
C:\Program Files\Agilent\MassHunter\Workstation\Qual\B.0x.00\Bin\IsotopeDistributionCalculator.exe.
•Or under Start menu under:
•In B.03.01 or later: All Programs Agilent MassHunter Workstation Qualitative Analysis B.0x.00 Tools
•It allows you to model the theoretical abundances and spacing of any particular
compound:
Rev. 1.3
How To Determine Complexity of Sample/Matrix
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
14
Base Peak Chromatogram with Exclude m/z 195.09145 +/- 100 ppm
• Acquire MS or AutoMSMS Modes
• Base Peak Chromatogram Excludes Major Components – reference masses
• Find Compounds by Molecular Feature Extraction (MFE)
Rev. 1.3
Find Compounds by Molecular Feature Results
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
15
Over 280 Compounds Found in Coffee Spiked with Pesticide
Rev. 1.3
Molecular Feature Extractor (MFE) Transforming Data into Chemical Information
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
31
Raw data Background noise
removed
Individual m/z peaks
grouped into isotope
clusters
Isotope
clusters
grouped into
molecular
features
Identification, Quantification, Differential Analysis are performed on chemically qualified
compound data
Rev. 1.3
Find by Molecular Feature
13 May 2015 19
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
Method Editor – MFE
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
20
Rev. 1.3
Method Editor – MFE (cont’d)
13 May 2015 21
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
Method Editor – MFE (cont’d)
13 May 2015 22
Similar compounds (e.g.
metabolites) will have similar mass
defects, so we can use the Mass
Defect tab to filter on that.
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
MFE Results
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
23
Rev. 1.3
Find Compounds by Molecular Feature Results
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
24
Over 280 Compounds Found in Coffee Spiked with Pesticide
Rev. 1.3
Compound List
Use found compounds for further analysis:
•Use Molecular Formula Generator (MFG) on list.
•Search a Database and match findings
•Upload the MFE results to Mass Profiler or GeneSpring MS for analysis.
13 May 2015 25
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
Find Compounds by Formula
13 May 2015 26
Find Compounds in analysis by inputting:
•Formulas separated by “,”
•CEF file with Formulas
•A database with Formulas (.csv, .mtl, .cdb)
•Worklist Formulas
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
Find Compounds by Formula - Options
Input Formula Source
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
13 May 2015 28
Rev. 1.3
Find Compounds by Formula – Sample Purity
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
29
• Enables the user to do Sample Purity (not Peak Purity) calculations based on
the same algorithms as LC/MS ChemStation
• Sample Purity compares a peak against all other peaks in the data file to
determine the estimated purity of the compound
After doing a Find
Compounds by
Formula, pure
compounds are
marked green and
impure are marked
yellow
Rev. 1.3
Find Compounds by Formula – System Suitability
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
30
• Integrate (UV) and Find by Formula – Sample Purity
• It allows the user to calculate system suitability values on a UV chromatogram
based on the chosen Pharmacopoeia
• The results are displayed in the “Integration Peak List.”
Rev. 1.3
System Suitability Calculations Can Be Done for
MS, MS/MS, UV and GC Chromatograms
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
31
Rev. 1.3
Find Compounds by Formula Results
13 May 2015 33
Compounds Found Formulas Found
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
Find Compounds by Auto MS/MS
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
34
Auto MS/MS
• Data Acquisition function used when the m/z value of the precursor ion is unknown prior
to analysis. The instrument acquires a mass spectrum then determines ions meeting
preset criteria for precursor ions. MS/MS spectra are taken for each identified precursor.
The instrument then switches back to MS operation.
Find by Auto MS/MS
• Data Analysis function used to automatically identify compounds in MS/MS data and
create averaged MS and MS/MS spectra for each compound. The functionality is an
easy way to “mine” information from complex data.
Reference Mass
MS Scan - Precursor
MS/MS Scan – Product Ions
Precursor Ion
Precursor Ion Reference Mass
Product Ions
Rev. 1.3
Start with MS/MS Data
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Page 35
Very complex data.
Difficult to obtain
information.
Rev. 1.3
Find by Auto MS/MS Algorithm
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
37
User-specified excluded ions and persistent background are removed from the
Auto MS/MS data file. The remaining spectra are then are divided into groups
that represent scans from the same compound.
This is done (first for positive ion scans, then for negative ion scans) by finding
scans that:
•Have the same precursor m/z within a user-specified tolerance, and
•Have retention times within a user-specified window of each other, and
•Have a TIC value that exceeds a user-specified threshold
•Best Value is to Find Compounds Based on Product Ion Filters
Rev. 1.3
Auto MS/MS Parameters
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
38
Appropriate Symmetry
for Q-TOF:
10-100 ppm
Data to be found
As of B.03.01 - Can filter
results by fragments
Exclude Reference Masses
Rev. 1.3
Find by Auto MS/MS Results
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
39
MS
MS/MS
Rev. 1.3
Compound List
13 May 2015 40
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
Find Compound by Integration
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
42
• New function that is similar to Integrate and Extract Peak Spectra.
• Results are in the form of Compounds rather than Peaks and User Spectra.
Rev. 1.3
Find Compound by Integration
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
43
Best used when compounds are chromatographically separated.
Creates Compounds which may be exported as a CEF file.
Better labeling after library search than User Spectra:
• Easer to understand
• Finds more low level ions
Rev. 1.3
Find Compound by Integration
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
44
Integrator and Peak Filter parameter settings are unique to Find Compounds by
Integration.
Rev. 1.3
Find Compound by Integration
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
45
Exclude Mass(es), Peak Spectrum Extraction, Charge State & Results are shared
with other method sections.
Rev. 1.3
Find Compound by Integration
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
46
Specify the type of chromatogram to extract & integrate:
• Will extract the chromatogram even if it is already present.
• Duplicate TIC when run.
• Not required to extract the signal in File Open Action.
• Cannot be used on “user defined” chromatograms (example EIC).
Rev. 1.3
Find Compound by Integration
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
47
Optional - Extract additional chromatogram (not integrated)
Optional - Restrict m/z range on BPC
Rev. 1.3
Find Compound by Integration
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
48
Adjust Delay Time
• Will adjust the RT of other signals compared to MS.
• Applied to GC, TWC, etc. signals when signal is extracted, not after.
Rev. 1.3
Find Compound by Integration
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
49
When “User Interface Configuration” has “LC” checked the Adjust Delay Time feature
is active when extracting signals!
Rev. 1.3
Find by Targeted MS/MS Algorithm
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
50
This algorithm extracts a chromatogram for each targeted mass in the data file. This
chromatogram is integrated and if the peaks are not eliminated by one of the filters on
this tab, a compound is generated.
The algorithm uses the targeted masses input in the acquisition method.
Rev. 1.3
Find by Targeted MS/MS Results
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
51
Rev. 1.3
Find by Targeted MS/MS
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
52
Can extract
MS/MS spectra based on
collision energy
Rev. 1.3
Identify Compounds
13 May 2015 53
Generate Formulas after Find Compounds
Search a Database or Library after
Find Compounds
*Sequence editing
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
Database Searching
Search Mode
13 May 2015 54
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
Automated Compound Search METLIN Database
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
55
Rev. 1.3
Accurate Mass Database vs. Library
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
57
Database: contains an accurate mass,
possibly a retention time, and other
compound attributes such as CAS ID and
the chemical structure for each
compound.
Accurate Mass Library: contains the
same information as a Database plus
MS/MS spectra.
By convention, Databases are kept in
D:\MassHunter\databases
and Libraries are kept in
D:\MassHunter\Library
Libraries from Agilent have the suffix “Lib”
in the name, e.g. SulfasLib.cdb
Rev. 1.3
Accurate Mass Database vs. Library (cont.)
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
58
When users search an Accurate Mass
Database, they are searching for a specific
accurate mass (e.g. 156.0761) against the
accurate masses listed in the Database.
When users search a Library, they are
searching for a spectrum against the
spectra in the Library.
Unit Mass Library Search: accessed by
turning on the “GC” Separation Type in the
User Interface Configuration dialog box.
The Library Editor for Unit Mass Libraries
can be accessed by installing Quantitative
Analysis.
Rev. 1.3
Accurate Mass Database Searching
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
59
If an Accurate Mass Database Search
finds a compound, it will place the
chemical structure in the Product Ion
Spectrum and display the structure in the
Structure Viewer pane (must be toggled
On).
Any links to outside information sources
about the compound (e.g. Chemical
Abstract Service #, CAS) are active —
click on them to go to a web site.
Rev. 1.3
Accurate Mass Library Searching
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
60
If an Accurate Mass Library Search finds a
compound, it will place the chemical
structure in the Product Ion Spectrum and
display the structure in the Structure
Viewer pane (if it is toggled On).
It is also possible to display a difference
spectrum for the Library hit by toggling the
Difference Results button to On in the
main toolbar.
Acquired Spectrum
Library Spectrum Difference Spectrum
Rev. 1.3
All Ions MS/MS
13 May 2015 61
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
Why All Ions MS/MS?
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
62
-The EU’s SANCO 12495 compliant screening requires:
– At least 2 product ions
– Final confirmation with a reference compound
-Fully developed workflow in All Ions MS/MS for compound confirmation and quant method setup.
– Easy Acquisition method setup
– Targeted identification of product ions using Agilent’s MS/MS libraries
– Chromatographic confirmation of product ions (coelution score)
– Rapid Quant method development—automatically selects two high quality product ions for each compound, eliminating tedious manual steps
-Be prepared for unexpected compounds in the sample
-QQQ-like easy acquisition method setup
-Productivity tool for faster analysis
- Identification and fragment finding done in MH Qualitative Analysis
- Routine quantitation and confirmation done in MH Quantitative Analysis
Rev. 1.3
Quantify target with
additional fragment
ion confirmation
All Ions MS/MS Targeted Screening Workflow
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
63
Acquisition PCDL Qual Quant
MassHunter Acquisition
High/Low
Fragmentor
Voltage on TOF
and Q-TOF
Pesticide
library 700+
compounds
with spectra)
Forensics
/Tox library
(2720
compounds
with spectra )
High/Low
Collision Energy
on Q-TOF
Find by Formula
on MS low
channel
EICs for
fragments on MS
high channel
Correlate qualified
fragments with
target
Find
Extract
Qualify and confirm
fragments
Export
Align EICs of
fragments with
parent EIC
Targets
m/z, ion species
Quant
Report
Qual
Report
Rev. 1.3
All Ions MS/MS
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
64
• Use and combine PCDL libraries with All Ions MS/MS.
• Export information and automatically create quant methods in seconds.
• Reduce processing time from one day to a matter of hours.
0
200
400
600
800
1000
1200
1400
Without All Ions With Allonsm
inu
tes
Processing time
Quant
Qual
Rev. 1.3
All Ions MS/MS Setup
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
65
Minimum Co-elution
score
Turn on All Ions MS/MS
using the current
Formula Source PCDL
Rev. 1.3
All Ions MS/MS can distinguish between isomers
13 May 2015 66
Codeine Hydrocodone
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
All Ions MS/MS can distinguish between isomers
13 May 2015 67
Codeine
Hydrocodone
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
All Ions MS/MS can distinguish between isomers
13 May 2015 68
Codeine
Hydrocodone
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
All Ions MS/MS: Chromatographic Confirmation of
Product Ions
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
69
Overlaid low and high
energy chromatograms
Co-elution curves—the
precursor and product
ions agree exactly on
their apex!
Rev. 1.3
New Compound Details View in Qual rev. B.06.00
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
70
New
chromatogram
and spectrum
windows that
show data from
one compound
only.
Data belonging
to one compound
can be overlaid
easily and
quickly.
Rev. 1.3
Quality Score for MFE
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
71
Rev. 1.3
Molecular Formula Generator - Calculate
Compound Formulas
NH
O
HNH2 N
O
S
H
CH3
CH3
H COOH
H
OH
NH
O
HNH2 NH
S
H
CH3
CH3
H COOH
H
O OH
OHOH
NH
NH
O
O
NH
S
CH3
CH3
H COOH
1 2 3
1 Amoxicillin, 2 Amoxicillin penicillinoic acid, 3 Amoxicillin penilloic acid
You can generate the molecular formula for each selected compound.
First, find the Compounds, then use MFG.
Example: Structure Elucidation of Degradation Products of the
Antibiotic Drug Amoxicillin
13 May 2015 72
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
Molecular Formula Generator
13 May 2015 73
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
Rev. 1.3
Molecular Formula Generator Algorithm
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
74
• Determine a mass in the compound to use for MFG:
– The program looks at clusters of ions and applies rules for isotopic
abundance and mass spacing (mass accuracy and resolution are important
to achieve this).
• Apply user knowledge of samples to limit the possible formula:
– Users fill out the Allowed Species dialog box with their knowledge of the
chemistry of their samples.
• Rule out formulas that are chemically impossible (e.g. CH7)
• Calculate probability scores for each candidate formula based on:
– Calculated vs. measured mass
– Calculated vs. measured abundance ratios
– Calculated vs. measured mass intervals of isotopes (isotope spacing)
Rev. 1.3
Molecular Formula Generator Algorithm (cont.)
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
75
•Rank and display each candidate formula based on its probability score
•For calculations done in conjunction with MFE, calculate an abundance-weighted
average cross score for all species (adducts, dimers, charge states) associated
with a compound. These are all listed in the Compound List table
Rev. 1.3
Molecular Formula Generator Algorithm – MS/MS
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
76
If MS/MS data are available, the algorithm uses it for greater confidence
in formula generation:
• The algorithm generates tentative formulas for each MS/MS fragment (product
ion).
• Precursor formulas with no related product ions are eliminated
– For a precursor formula to be valid, it must generate product ions that make sense
• Proposed precursor formulas are scored and ranked based on their fit with the
product ions
– Algorithm uses coverage (explanation of how the precursor fragmented) and mass
error of product ions
• A single score is provided that combines both MS and MS/MS data
Rev. 1.3
Molecular Formula Generator Algorithm – MS/MS
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
77
Note the scores in the Compound List table below:
– Score (various)
– MS Score (MS-only)
– MS/MS (goodness-of-fit of product ions formulas to precursor formula)
As of Qual rev. B.02.00, scores are absolute (Best is not fixed at 100)
for comparison across formulae and samples.
Rev. 1.3
Major Features of Qual B.06.00
13 May 2015
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
111
All Ions MS/MS
Compound Details View
New Quality score for MFE
Support for new tMRM files
Enhanced Molecular Formula Generation
New Find Compound Algorithm : Find by Integration
Annotate fragment ions with fragment formula generation
Save results faster
Export format changes