masshunter tof/qtof users meeting - agilent · 2016. 9. 4. · rev. 1.3 qualitative analysis...

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

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•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

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•“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

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•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

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•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

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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

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Over 280 Compounds Found in Coffee Spiked with Pesticide

Rev. 1.3

Molecular Feature Extractor (MFE) Transforming Data into Chemical Information

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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

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Rev. 1.3

Method Editor – MFE

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Rev. 1.3

Method Editor – MFE (cont’d)

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Rev. 1.3

Method Editor – MFE (cont’d)

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Similar compounds (e.g.

metabolites) will have similar mass

defects, so we can use the Mass

Defect tab to filter on that.


Rev. 1.3

MFE Results

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Rev. 1.3

Find Compounds by Molecular Feature Results

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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.

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Rev. 1.3

Find Compounds by Formula

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Find Compounds in analysis by inputting:

•Formulas separated by “,”

•CEF file with Formulas

•A database with Formulas (.csv, .mtl, .cdb)

•Worklist Formulas


Rev. 1.3

Find Compounds by Formula - Options

Input Formula Source


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Rev. 1.3

Find Compounds by Formula – Sample Purity

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• 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

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• 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

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Rev. 1.3

Find Compounds by Formula Results

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Compounds Found Formulas Found


Rev. 1.3

Find Compounds by Auto MS/MS

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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

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Very complex data.

Difficult to obtain

information.

Rev. 1.3

Find by Auto MS/MS Algorithm

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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

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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

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MS

MS/MS

Rev. 1.3

Compound List

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Rev. 1.3

Find Compound by Integration

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• 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


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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


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Integrator and Peak Filter parameter settings are unique to Find Compounds by

Integration.

Rev. 1.3


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Exclude Mass(es), Peak Spectrum Extraction, Charge State & Results are shared

with other method sections.

Rev. 1.3


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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


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Optional - Extract additional chromatogram (not integrated)

Optional - Restrict m/z range on BPC

Rev. 1.3


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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


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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

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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

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Rev. 1.3

Find by Targeted MS/MS

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Can extract

MS/MS spectra based on

collision energy

Rev. 1.3

Identify Compounds

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Generate Formulas after Find Compounds

Search a Database or Library after

Find Compounds

*Sequence editing


Rev. 1.3

Database Searching

Search Mode

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Rev. 1.3

Automated Compound Search METLIN Database

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Rev. 1.3

Accurate Mass Database vs. Library

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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.)

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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

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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

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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

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Rev. 1.3

Why All Ions MS/MS?

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-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

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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

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• 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

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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

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Codeine Hydrocodone


Rev. 1.3


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Codeine

Hydrocodone


Rev. 1.3


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Codeine

Hydrocodone


Rev. 1.3

All Ions MS/MS: Chromatographic Confirmation of

Product Ions

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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

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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

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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

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Rev. 1.3

Molecular Formula Generator

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Rev. 1.3

Molecular Formula Generator Algorithm

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• 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.)

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•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

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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

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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

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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

masshunter tof/qtof users meeting - agilent · 2016. 9. 4. · rev. 1.3 qualitative analysis...

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