Making Sense of the LCMS Data Differences -David Weil

MassHunter Overview

March 2008Page 1

Making Sense of Differences in LCMS Data: Integrated Tools David A. Weil

Agilent Technologies

Mass Hunter Review Pitcon 2008

March, 2008Page 2

How Clean is our Water?....


March, 2008Page 3

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

PFOA/PFOS Persistent Chemical

PPCP Steroids and Antibiotics

Fungicides/Pesticides

Residuals in Beverages


March, 2008Page 4

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


March, 2008Page 5

Agilents Solution for Differential Analysis

Data AnalysisLC/MS

Analysis

Sample Preparation

GC/MS Analysis

Separations

Quant QQQ

Volatile Residues

Non-Volatile Residues

TOF/QTOFImpurity IDProfiling

Targeted

Non-TargetedAgilents New SPE Products

Data Analysis

Objective: Detection - ID low-level Impurities in Drugs, Chemical Residues in Water, Food and Carbonated Beverages


March, 2008Page 6

The Match Game.Choosing The Mass Spectrometer for the Task

Sensitivity, Selectivity, Identification..

Unknown IdentificationImpurity Profiling by Accurate Mass

Target AnalysisEnhanced Sensitivity by MS/MS

Unknown IdentificationAccurate Mass MS, MSMS

MS/MS Sensitivity


March, 2008Page 7

Agilent Suite of MassHunter Products

Qual Quant

Profiler MetID


March, 2008Page 8

MassHunter QUAL MSD, QQQ, TOF, QTOF

Method Parameters

Method Editor

Data Navigator


March, 2008Page 9

Qualitative Analysis: Compound Centric Data Mining and Navigation

Navigation Tree

Chromatogram

Spectrum

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


March, 2008Page 10

Qualitative Analysis: Molecular Feature Extraction

List of Compounds

Compound Spectrum

Compound Chromatogram


March, 2008Page 11

How To Determine Complexity of Sample/Matrix

6x10

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+ BPC(150.00000-800.00000) Scan CoffeeSpiked12.d

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+ BPC(150.00000-800.00000 [-1]) Scan CoffeeSpiked12.d

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Counts vs. Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9

Base Peak Chromatogram with Exclude m/z 195.09145 +/- 100 ppm

Acquire MS or AutoMSMS Modes Base Peak Chromatogram Exclude Major Component Find Compounds by Molecular Feature Extraction


March, 2008Page 12

3D Plot Before3D Plot Before

Molecular Feature Extraction (MFE)Automated Data Reduction Software

Original TICProcessed TIC

3D Plot No Background

Data Reduced sum intensities of isotopes, adducts, clusters and multiply charges ions together.

Finds Features in TOF/QTOF Data Coeluting Features


March, 2008Page 13

Processing large amount of data with MFEDisplay filtering:

- S/N > 2

- m/z 150 to 800

- [M+Na] and [M+NH4] Adducts

2 4 6 8 10 12 14

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Original TIC

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- At least 2 ions

- Relative intensity > 0.01%

5131 features

Original TIC Processed TIC


March, 2008Page 14

In MassHunter QualSelect Find by Molecular Feature


March, 2008Page 15

Method Editor: Parameters for Molecular Feature

Display Filters


March, 2008Page 16

Mass Defect Filtering


March, 2008Page 17

Executed

Compound Table


March, 2008Page 18

Find Compounds by Molecular Feature6x10

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Cpd 279:+ ECC Scan CoffeeSpiked12.d

Counts vs. Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9

Over 280 Compounds Found in Coffee Spiked with Pesticide


March, 2008Page 19

Prednisolone: C21H28O5 360.193674

OHOH

OO

O

O

CH3

CH3

CH3

2 year old Suspension


March, 2008Page 20

Sequential Neutral Water Losses Detected5x10

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Counts vs. Acquisition Time (min)8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14


March, 2008Page 21

12 Potential Degradation Products in Prenisolone

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

CH2O Loss


March, 2008Page 22

Molecular Formula Generation..

Technical Overview 5989-7409EN


March, 2008Page 23

Generate Formulas Parameters


March, 2008Page 24

Theoretical Isotope Pattern


March, 2008Page 25

Accurate Mass of Compounds


March, 2008Page 26

Identify Compounds by Database Searching

Search database by mass or by molecular formula

Select either exact mass or Personal Metlin database


March, 2008Page 27

Automated Compound Search Metlin Database


March, 2008Page 28

Mass Profiling Version 2.0

Processed TICProcessed TICProcessed TICGroup A (Impure)

Processed TICProcessed TICProcessed TICGroup B (Control)

Aligns Data RT, Mass,

Abundance

All Data RT/Mass

Whats Changed? Mass/RT >2 Fold Change

Whats Unique!! Impurity Control


March, 2008Page 29

How to Create a ProjectSelect 2 Groups for DifferentialSelect 1 Group for Concentration Change

Need at least 3 replicates


March, 2008Page 30

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


March, 2008Page 31

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


March, 2008Page 32

Multiple Display OptionsMass vs RT (Average of Features)


March, 2008Page 33

Differential Display by Retention Time


March, 2008Page 34

Unique Features..


March, 2008Page 35

Single Group Example:Compounds Spiked into Plant Extract


March, 2008Page 36

Feature Table Single Group Example

For Single Group: Large SD of abundance indicates compounds that have concentrations that vary over the samples


March, 2008Page 37

Graphical Display of Concentration Change of Feature

Change Plot Style to Colored by Feature


March, 2008Page 38

Abundance and Feature Details


March, 2008Page 39

Batch Chemical Composition and Database Searchfrom Mass Profiler


March, 2008Page 40

Batch Molecular Formula Generation and Database Searching from Profiler


March, 2008Page 41

Web Searching of CompoundsNIST, ChemIDPlus, PubChem,


March, 2008Page 42

MetID Software For Impurity Profiling


March, 2008Page 43

MassHunter Workstation - Metabolite IDQuickly 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


March, 2008Page 44

Molecular Formula Generation MS and MSMS Data

Metabolite Table w/ Find + Confirm Results

MetaboliteChros + Spectra


March, 2008Page 45

Overview MetID1. Parent Compound: MW, Formula and Structure

2. Transformations: List Proposed Degradation Products

3. Identification Criteria: Vary Importance of Tests

4. Find Compounds by MFE: Molecular Feature Extraction

5. Find Compounds by AutoMSMS: For MSMS data

6. Sample Comparison: Mass Profiler What is Different

7. Isotope Pattern Filtering: Best for Halogenated Species

8. Mass Defect Filtering: From Proposed Compounds

9. EIC Generation: Confirms Presence of Compounds


March, 2008Page 46


March, 2008Page 47

Method Input

Parent Compound Propose Degradation Products


March, 2008Page 48

Proposed Degradation ProductsQualified by Mass Defect and Isotope Pattern

Lets Determine Composition of MW 411


March, 2008Page 49

Accurate Mass Generates CompositionAcid Hydrolysis (H2O+CO2H-OH)

Accurate Mass of Fragments Ions


March, 2008Page 50

Overlay MSMS Spectrum Parent/Product


March, 2008Page 51

3D of MSMS Spectra Fragmentation Pattern


March, 2008Page 52

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


March, 2008Page 53

Personal Metlin Database


March, 2008Page 54

METLIN Personal Database Initial View

User search mode


March, 2008Page 55

METLIN Manual Single Mass Search

Metabolite search results

Active metabolite structureSingle mass search settings

Hyperlinks to Metabolite websites


March, 2008Page 56

METLIN Batch Search

Load file for batch search

Active mass

Active metabolite structure

Active mass search results


March, 2008Page 57

METLIN Batch Summary

Active metabolite structure

Batch summary best metabolite match

Exportable list of best metabolite search results


March, 2008Page 58

Creating a New METLIN Database

Create a new personal database from METLIN

METLIN is read only and can not be changed


March, 2008Page 59

Edit Metabolite Database Update Entry

Add retention time to existing metabolite


March, 2008Page 60

METLIN Mass Search With Retention Time Adds Specificity

Adding retention time eliminates isobaric metabolites

Only 1 metabolite instead of 3 !!!!


March, 2008Page 61

Edit Metabolite Database New Entry

Select add new then type new metabolite information


March, 2008Page 62

METLIN Search from Mass Hunter Qual

Identify compounds: search database

Specify the METLIN database Set search parameters

Manual search options

Select ion in spectra Select MFE compound(s)

Batch search

Automate by Find and Identify Find compounds by MFE Search database Generate formula (optional)

Name and CAS number Database and MFG results

Set Parameters

View detailed results


March, 2008Page 63

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


March, 2008Page 64

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: Find Compounds in Complex Matrix MassHunter Profiler: Differential Analysis Software MetID: Metabolite ID and Impurity Profiling Metlin Database: Personalize Destktop Database Searching


March, 2008Page 65

THANK YOU..

Documents

Making Sense of the LCMS Data Differences -David Weil