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

Mass Hunter Review Pitcon 2008 Page 4 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 5 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 7 March, 2008

MassHunter QUAL MSD, QQQ, TOF, QTOF

Data Navigator

Method Editor Method Parameters

Mass Hunter Review Pitcon 2008 Page 8 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 9 March, 2008

Qualitative Analysis: Molecular Feature Extraction

Compound Chromatogram

List of Compounds

Compound Spectrum

Mass Hunter Review Pitcon 2008 Page 10 March, 2008

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
Mass Hunter Review Pitcon 2008 Page 11 March, 2008

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.

Mass Hunter Review Pitcon 2008 Page 12 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 13 March, 2008

In MassHunter Qual
Select Find by Molecular Feature

Mass Hunter Review Pitcon 2008 Page 14 March, 2008

Method Editor: Parameters for Molecular Feature

Display Filters

Mass Hunter Review Pitcon 2008 Page 15 March, 2008

Mass Defect Filtering

Mass Hunter Review Pitcon 2008 Page 16 March, 2008

Executed

Compound Table

Mass Hunter Review Pitcon 2008 Page 17 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 18 March, 2008

Prednisolone: C21H28O5 360.193674

OH CH3

O OH CH 3

O O

CH3

2 year old Suspension

Mass Hunter Review Pitcon 2008 Page 19 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 20 March, 2008

12 Potential Degradation Products in Prenisolone

CH2O Loss

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

Mass Hunter Review Pitcon 2008 Page 21 March, 2008

Molecular Formula Generation..

Technical Overview 5989-7409EN

Mass Hunter Review Pitcon 2008 Page 22 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 26 March, 2008

Automated Compound Search Metlin Database

Mass Hunter Review Pitcon 2008 Page 27 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 28 March, 2008

How to Create a Project

Select 2 Groups for Differential Select 1 Group for Concentration Change

Need at least 3 replicates

Mass Hunter Review Pitcon 2008 Page 29 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 30 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 31 March, 2008

Multiple Display Options

Mass vs RT (Average of Features)

Mass Hunter Review Pitcon 2008 Page 32 March, 2008

Differential Display by Retention Time

Mass Hunter Review Pitcon 2008 Page 33 March, 2008

Unique Features..

Mass Hunter Review Pitcon 2008 Page 34 March, 2008

Single Group Example:

Compounds Spiked into Plant Extract

Mass Hunter Review Pitcon 2008 Page 35 March, 2008

Feature Table Single Group Example

For Single Group: Large SD of abundance indicates compounds that have concentrations that vary over the samples
Mass Hunter Review Pitcon 2008 Page 36 March, 2008

Graphical Display of Concentration Change of Feature

Change Plot Style to Colored by Feature

Mass Hunter Review Pitcon 2008 Page 37 March, 2008

Abundance and Feature Details

Mass Hunter Review Pitcon 2008 Page 38 March, 2008

Batch Chemical Composition and Database Search from Mass Profiler

Mass Hunter Review Pitcon 2008 Page 39 March, 2008

Batch Molecular Formula Generation and Database Searching from Profiler

Mass Hunter Review Pitcon 2008 Page 40 March, 2008

Web Searching of Compounds

NIST, ChemIDPlus, PubChem,

Mass Hunter Review Pitcon 2008 Page 41 March, 2008

MetID Software For Impurity Profiling

Mass Hunter Review Pitcon 2008 Page 42 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 43 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 45 March, 2008

Mass Hunter Review Pitcon 2008 Page 46 March, 2008

Method Input
Parent Compound Propose Degradation Products

Mass Hunter Review Pitcon 2008 Page 47 March, 2008

Proposed Degradation Products Qualified by Mass Defect and Isotope Pattern

Lets Determine Composition of MW 411

Mass Hunter Review Pitcon 2008 Page 48 March, 2008

Accurate Mass Generates Composition Acid Hydrolysis (H2O+CO2H-OH)

Accurate Mass of Fragments Ions

Mass Hunter Review Pitcon 2008 Page 49 March, 2008

Overlay MSMS Spectrum Parent/Product

Mass Hunter Review Pitcon 2008 Page 50 March, 2008

3D of MSMS Spectra Fragmentation Pattern

Mass Hunter Review Pitcon 2008 Page 51 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 52 March, 2008

Personal Metlin Database

Mass Hunter Review Pitcon 2008 Page 53 March, 2008

METLIN Personal Database Initial View

User search mode

Mass Hunter Review Pitcon 2008 Page 54 March, 2008

METLIN Manual Single Mass Search

Single mass search settings

Active metabolite structure

Hyperlinks to Metabolite websites

Metabolite search results

Mass Hunter Review Pitcon 2008 Page 55 March, 2008

METLIN Batch Search

Load file for batch search

Active metabolite structure

Active mass

Active mass search results

Mass Hunter Review Pitcon 2008 Page 56 March, 2008

METLIN Batch Summary

Active metabolite structure Batch summary best metabolite match

Exportable list of best metabolite search results

Mass Hunter Review Pitcon 2008 Page 57 March, 2008

Creating a New METLIN Database

METLIN is read only and can not be changed

Create a new personal database from METLIN

Mass Hunter Review Pitcon 2008 Page 58 March, 2008

Edit Metabolite Database Update Entry

Add retention time to existing metabolite

Mass Hunter Review Pitcon 2008 Page 59 March, 2008

METLIN Mass Search With Retention Time Adds Specificity

Only 1 metabolite instead of 3 !!!!

Adding retention time eliminates isobaric metabolites

Mass Hunter Review Pitcon 2008 Page 60 March, 2008

Edit Metabolite Database New Entry

Select add new then type new metabolite information

Mass Hunter Review Pitcon 2008 Page 61 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 62 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 63 March, 2008

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

Mass Hunter Review Pitcon 2008 Page 64 March, 2008

THANK YOU..

Mass Hunter Review Pitcon 2008 Page 65 March, 2008