Making Sense of The LCMS Data Differences - David Weil

Making Sense of Differences in LCMS Data: Integrated Tools
David A. Weil
Agilent Technologies
MassHunter Overview Page 1 March 2008
How Clean is our Water?....

Mass Hunter Review Pitcon 2008 Page 2 March, 2008
Chemical Residue Analysis.. .. From the headlines..
Fungicides/Pesticides
PFOA/PFOS Persistent Chemical
PPCP Steroids and Antibiotics

Page 3
Residuals in Beverages
Mass Hunter Review Pitcon 2008 March, 2008
Challenges in Differential Analysis

Sample Preparation: Extraction Efficiency Separation Methods: Sharper the Better Ionization Efficiency Effects
ESI preferential Ionization of polar/acid/base compounds APCI for non-polar compounds Polarity Effects
Ionization Suppression: Matrix Effects Mass Spectrometry Sensitivity: Detection vs Relevant Data Analysis: Show me the Data
Agilents Solution for Differential Analysis

Objective: Detection - ID low-level Impurities in Drugs, Chemical Residues in Water, Food and Carbonated Beverages
Volatile Residues
GC/MS Analysis
Data Analysis Targeted Quant QQQ
Sample Preparation
Separations
Agilents New SPE Products
Non-Volatile Residues
LC/MS Analysis
Non-Targeted
Data Analysis
TOF/QTOF Impurity ID Profiling

The Match Game.

Choosing The Mass Spectrometer for the Task Sensitivity, Selectivity, Identification..
Unknown Identification Accurate Mass MS, MSMS MS/MS Sensitivity
Unknown Identification
Impurity Profiling by Accurate Mass
Target Analysis
Enhanced Sensitivity by MS/MS
Mass Hunter Review Pitcon 2008
Page 6
March, 2008
Agilent Suite of MassHunter Products

Qual Quant
Profiler
MetID
MassHunter QUAL MSD, QQQ, TOF, QTOF
Data Navigator
Method Editor Method Parameters
Qualitative Analysis: Compound Centric Data Mining and Navigation

Several automated algorithms to find compounds or sample components from LC/MS and LC/MS/MS datasets Linking of panes for easier browsing and navigation
Chromatogram
Navigation Tree
Spectrum
Qualitative Analysis: Molecular Feature Extraction
Compound Chromatogram
List of Compounds
Compound Spectrum
How To Determine Complexity of Sample/Matrix

Acquire MS or AutoMSMS Modes Base Peak Chromatogram Exclude Major Component Find Compounds by Molecular Feature Extraction
x10 6 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 x10 6 1.4 1.2 1 0.8 0.6 0.4 0.2 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 Counts vs. Acquisition Time (min) 6 6.5 7 7.5 8 8.5 9 + BPC(150.00000-800.00000 [-1]) Scan CoffeeSpiked12.d 1 1 + BPC(150.00000-800.00000) Scan CoffeeSpiked12.d 1 1
Base Peak Chromatogram with Exclude m/z 195.09145 +/- 100 ppm
Molecular Feature Extraction (MFE) Automated Data Reduction Software

Finds Features in TOF/QTOF Data
Original TIC Processed TIC 3D Plot No 3D Plot Before Coeluting Features Background
Data Reduced sum intensities of isotopes, adducts, clusters and multiply charges ions together.
Processing large amount of data with MFE

Display filtering:
- S/N > 2 - m/z 150 to 800 - [M+Na] and [M+NH4] Adducts
O rig in a lT IC TIC Original
1 5 Intensity (10^6)
- At least 2 ions - Relative intensity > 0.01%
5131 features
P rocessed TIC
15 Intensity (10^6)
Processed TIC
10
1 0
m in
0
10
12
14
m in
1 0
1 2
1 4
In MassHunter Qual
Select Find by Molecular Feature
Method Editor: Parameters for Molecular Feature
Display Filters
Mass Defect Filtering
Executed
Compound Table
Find Compounds by Molecular Feature

x10 6 Cpd 279:+ ECC Scan CoffeeSpiked12.d 2.8 2.6 2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2
0.5
1.5
2.5
3.5
4 4.5 5 5.5 Counts vs. Acquisition Time (min)
6.5
7.5
8.5
Over 280 Compounds Found in Coffee Spiked with Pesticide

Prednisolone: C21H28O5 360.193674
OH CH3
O OH CH 3
O O
CH3
2 year old Suspension
Sequential Neutral Water Losses Detected

x10
5
+ ECC Scan Prednisolone0002.d
1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 8.5 9 9.5 10 10.5 11 11.5 12 Counts vs. Acquisition Time (min) 12.5 13 13.5 14 13.421 23081.8
12 Potential Degradation Products in Prenisolone
CH2O Loss
-H2, -H2O, -CO2H2, +H2, +CH4+H2-O,

Molecular Formula Generation..
Technical Overview 5989-7409EN

Generate Formulas Parameters
Theoretical Isotope Pattern
Accurate Mass of Compounds
Identify Compounds by Database Searching
Search database by mass or by molecular formula
Select either exact mass or Personal Metlin database

Automated Compound Search Metlin Database
Mass Profiling Version 2.0

Processed TIC Processed TIC Processed Group TIC A (Impure) Aligns Data RT, Mass, Abundance Processed TIC Processed TIC Processed Group B TIC (Control)
Whats Changed? Mass/RT >2 Fold Change
Data RT/Mass Whats All Unique!! Impurity Control

How to Create a Project

Select 2 Groups for Differential Select 1 Group for Concentration Change
Need at least 3 replicates
Develop a Method
Select Retention Time and Mass Range For Small Molecules Exclude Multiply Charged Species Set Relative Intensity Limit Mass Defect Option Special Masses: Exclude ions present in blank Display only Targeted Ions
Results Filters
Application of Results Filters will reduce the number of features displayed in the graph To Display only Features with Greater than 2 fold change set Min Log2 value to 1.0 Relative Frequency filter by presence in samples. Exclude Saturated Features
Multiple Display Options

Mass vs RT (Average of Features)
Differential Display by Retention Time
Unique Features..
Single Group Example:

Compounds Spiked into Plant Extract
Feature Table Single Group Example
For Single Group: Large SD of abundance indicates compounds that have concentrations that vary over the samples
Graphical Display of Concentration Change of Feature

Change Plot Style to Colored by Feature
Abundance and Feature Details
Batch Chemical Composition and Database Search from Mass Profiler
Batch Molecular Formula Generation and Database Searching from Profiler
Web Searching of Compounds

NIST, ChemIDPlus, PubChem,
MetID Software For Impurity Profiling
MassHunter Workstation - Metabolite ID

Quickly Identify More Metabolites with Greater Confidence
; Powerful Sample-Control Comparison Algorithm based on Molecular
Feature
Extraction (MFE)
; Find Metabolites via Mass Defect Filter ; Empirical Formula Generation supports MS/MS data ;Confirm Metabolites via Noviatia Autoshift of MS/MS data ;Applications beyond Metabolites ;Impurity and Environment Profiling
Metabolite Table w/ Find + Confirm Results
Metabolite Chros + Spectra

Page 44
Molecular Formula Generation MS and MSMS Data

Mass Hunter Review Pitcon 2008 March, 2008
Overview MetID
1. Parent Compound: 2. Transformations: 3. Identification Criteria: MW, Formula and Structure List Proposed Degradation Products Vary Importance of Tests Molecular Feature Extraction For MSMS data
4. Find Compounds by MFE:
5. Find Compounds by AutoMSMS: 6. Sample Comparison: 7. Isotope Pattern Filtering: 8. Mass Defect Filtering: 9. EIC Generation:
Mass Profiler What is Different Best for Halogenated Species From Proposed Compounds Confirms Presence of Compounds
Method Input
Parent Compound Propose Degradation Products
Proposed Degradation Products Qualified by Mass Defect and Isotope Pattern
Lets Determine Composition of MW 411

Accurate Mass Generates Composition Acid Hydrolysis (H2O+CO2H-OH)
Accurate Mass of Fragments Ions
Overlay MSMS Spectrum Parent/Product
3D of MSMS Spectra Fragmentation Pattern
METLIN Personal Database Search Capabilities

Manual searches
Search on neutral or charged monoisotopic mass
User-specified search tolerance Optionally restrict by retention time Molecular formula
Compound name Retention time (user defined)
Batch and manual mass searches from Agilent products

GeneSpring MS MassHunter Qual Mass Profiler
Personal Metlin Database
METLIN Personal Database Initial View
User search mode
METLIN Manual Single Mass Search
Single mass search settings
Active metabolite structure
Hyperlinks to Metabolite websites
Metabolite search results
METLIN Batch Search
Load file for batch search
Active metabolite structure
Active mass
Active mass search results

METLIN Batch Summary
Active metabolite structure Batch summary best metabolite match
Exportable list of best metabolite search results

Creating a New METLIN Database
METLIN is read only and can not be changed
Create a new personal database from METLIN
Edit Metabolite Database Update Entry
Add retention time to existing metabolite
METLIN Mass Search With Retention Time Adds Specificity
Only 1 metabolite instead of 3 !!!!
Adding retention time eliminates isobaric metabolites
Edit Metabolite Database New Entry
Select add new then type new metabolite information
METLIN Search from Mass Hunter Qual

Identify compounds: search database
Specify the METLIN database Set search parameters
Name and CAS number Database and MFG results
Manual search options

Select ion in spectra Select MFE compound(s)
View detailed results
Batch search
Automate by Find and Identify Find compounds by MFE Search database Generate formula (optional)
Set Parameters
METLIN Searching From Mass Profiler

Mass Profiler METLIN searching is virtually the same as METLIN
Mass Profiler can optionally add MFG MFG is used to assist METLIN search METLIN results are exportable Database customization is done only in METLIN Retention time changes New compounds New database
METLIN search integrated into Mass Profiler
Summary
Accurate Mass and Reproducible Retention Time are Critical for Differential Analysis and Trace Level Impurity Detection Data Mining Software Tools Provide Unbiased Automated Data Analysis Feature Extraction: MassHunter Profiler: MetID: Find Compounds in Complex Matrix Differential Analysis Software
Metabolite ID and Impurity Profiling
Metlin Database: Personalize Destktop Database Searching
THANK YOU..

Making Sense of The LCMS Data Differences - David Weil

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Making Sense of The LCMS Data Differences - David Weil

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Авторское право:

Доступные форматы

Making Sense of Differences in LCMS Data: Integrated Tools

MassHunter Overview Page 1 March 2008

How Clean is our Water?....

Chemical Residue Analysis.. .. From the headlines..

PPCP Steroids and Antibiotics

Challenges in Differential Analysis

Mass Hunter Review Pitcon 2008 Page 4 March, 2008

Agilents Solution for Differential Analysis

Data Analysis Targeted Quant QQQ

Agilents New SPE Products

TOF/QTOF Impurity ID Profiling

The Match Game.

Agilent Suite of MassHunter Products

Mass Hunter Review Pitcon 2008 Page 7 March, 2008

MassHunter QUAL MSD, QQQ, TOF, QTOF

Method Editor Method Parameters

Mass Hunter Review Pitcon 2008 Page 8 March, 2008

Qualitative Analysis: Compound Centric Data Mining and Navigation

Mass Hunter Review Pitcon 2008 Page 9 March, 2008

Qualitative Analysis: Molecular Feature Extraction

Mass Hunter Review Pitcon 2008 Page 10 March, 2008

How To Determine Complexity of Sample/Matrix

Molecular Feature Extraction (MFE) Automated Data Reduction Software

Mass Hunter Review Pitcon 2008 Page 12 March, 2008

Processing large amount of data with MFE

- At least 2 ions - Relative intensity > 0.01%

Mass Hunter Review Pitcon 2008 Page 13 March, 2008

Mass Hunter Review Pitcon 2008 Page 14 March, 2008

Method Editor: Parameters for Molecular Feature

Mass Hunter Review Pitcon 2008 Page 15 March, 2008

Mass Defect Filtering

Mass Hunter Review Pitcon 2008 Page 16 March, 2008

Mass Hunter Review Pitcon 2008 Page 17 March, 2008

Find Compounds by Molecular Feature

4 4.5 5 5.5 Counts vs. Acquisition Time (min)

Over 280 Compounds Found in Coffee Spiked with Pesticide

Prednisolone: C21H28O5 360.193674

2 year old Suspension

Mass Hunter Review Pitcon 2008 Page 19 March, 2008

Sequential Neutral Water Losses Detected

+ ECC Scan Prednisolone0002.d

Mass Hunter Review Pitcon 2008 Page 20 March, 2008

12 Potential Degradation Products in Prenisolone

-H2, -H2O, -CO2H2, +H2, +CH4+H2-O,

Molecular Formula Generation..

Technical Overview 5989-7409EN

Generate Formulas Parameters

Mass Hunter Review Pitcon 2008 Page 23 March, 2008

Theoretical Isotope Pattern

Mass Hunter Review Pitcon 2008 Page 24 March, 2008

Accurate Mass of Compounds

Mass Hunter Review Pitcon 2008 Page 25 March, 2008

Identify Compounds by Database Searching

Search database by mass or by molecular formula

Select either exact mass or Personal Metlin database

Automated Compound Search Metlin Database

Mass Hunter Review Pitcon 2008 Page 27 March, 2008

Mass Profiling Version 2.0

Whats Changed? Mass/RT >2 Fold Change

Data RT/Mass Whats All Unique!! Impurity Control

How to Create a Project

Need at least 3 replicates