qtof small molecule applications update david a. weil

QTOF Small Molecule Update

December 6th, 2007

QTOF Small Molecule Applications Update

April 10th, 2008

David A. Weil

[email protected] Scientist, Schaumburg, IL


December 6th, 2007

Agenda• Goal – Share Best Practices, Updates Software/Hardware• Communication

• Past Recorded Webex’s available on website • Recorded Video’s on use of MassHunter Qual and MassHunter Quant• QTOF User Sponsored Email Group ([email protected] )

Today’s Topics:• Care and Feeding of QTOF/TOF

• Differential and Impurity Profiling

• Overview of MassHunter MassProfiler Software

• Overview of Personal Metlin Database Software

• Questions and Answer Period


December 6th, 2007

Care and Feeding of QTOF/TOF

• Defragment Hard Drives (Weekly)– Use Diskeeper to Defragment Drives– If Errors – Run Check Disk for Errors Program

• Check LC Components Firmware– If older version update LC Firmware


December 6th, 2007

Care and Feeding of QTOF/TOF (Cont.)

• Software Updates– MassHunter Qual and Protein Decovolution Patch 1

• MassHunter Quant (B.01.04)

• MassHunter Qual (B.01.03) Patch 1

• Metabolite ID B.01.01

• New April 2008– Mass Profiler B.01.00

– METLIN Personal Metabolite Database B.01.01


December 6th, 2007

Mass Profiling – A Powerful Workflow

Molecular Feature: • a discrete molecular entity defined by the combination of

RT, mass and response in an LC/MS or GC/MS analysis• typically represents a known or unknown compound

LC/MS or GC/MS Analysis

IdentificationFind molecular features

Compare sample sets

Validation

Mass Profiling: • is used to find differences between sample sets• originally developed for proteomics/metabolomics • can be applied to many analytical problems where finding

differences between samples is important• is done using statistical analysis

Mass ProfilerGeneSpring MS

MFE (LC/MS)AMDIS (GC/MS)

Spectrum MillMolecular FormulasDB Search (METLIN)Library Search

GC/MSLC/MS

QQQ MRMGeneSpring MS


December 6th, 2007

Molecular Feature Extractor (MFE) – How it works

• Map signals in the 3-dim. space in time and mass at the MS level. IMPORTANT: High resolution in mass and time!

• Remove areas which only contain noise, and NO signals.• Identify all mass signals with a common RT (narrow time window)• Combine mass signals with common RT and chemical relation (isotope,

adduct, dimer, higher charge state) => Molecular feature or Compound.• Create Extracted Compound Chromatograms (ECC) and Compound Mass

Spectra from ions associated to a molecular feature (compound)

RT m/z Abund… … …

… … …

… … …2.11 195.1745 210002.11 257.2566 33550

2.34 224.2134 11784

… … …

Compound list orMolecular feature list


December 6th, 2007

MassProfiler Dependent on MFE Feature Extraction

Don’t extract all the compounds only Important ones

How to Mine for Gold…Generated a Base Peak ChromatogramInput Mass Range of Compounds

Exclude Reference IonsInput m/z to 4 decimal placesSet window to 20 ppm

Run Standards to Determine RT Information


December 6th, 2007

Good Quality *.MHD Gives Good Quality Profiler

1. Centroid versus Profile data2. S/N versus Total Counts

Typically use 100 countsToo high may not detect C13 peakProfile S/N 10 First Pass

3. Focused MHD generationLimit Retention TimeLimit m/z Range

4. Ion Species (NH4 If buffer)5. Check Box if High Salt 6. Check if Free Radicals/Quats


December 6th, 2007

Good Quality *.MHD Gives Good Quality Profiler

7. Charge State = 1 to detect only singly charged species

8. Last Tabs only for Display in MassHunter QUAL

9. *.ProPos from Profile

10. *.CentPos from Centroid


December 6th, 2007

Find Compounds by Molecular Feature


December 6th, 2007

NEW Software – Mass Profiler B.01.00 G3297AA

• Identify similarities and differences in features across multiple analyses/samples from *.MHD data files generated from MassHunter Qualitative Analysis Software

• Aligns and normalizes features (RT, m/z and intensity)• Identify composition of features from Molecular Formula

Generation (MFG) and AMRT database searching• Compatible with Agilent TOF and QTOF Products• Includes Example Data Files, Quick Start Guide• Minimal Statistic in Mass Profiler (Student t test) therefore best

results with at least 3 replicates of “Good/Bad” sample and a blank run

• Cost $3,150


December 6th, 2007

Mass Profiling Software

Processed TICProcessed TICProcessed TICGroup A (Impure)

Processed TICProcessed TICProcessed TICGroup B (Control)

Aligns Data RT, Mass,

Abundance

All Data RT/Mass

What’s Changed? Mass/RT >2 Fold Change

What’s Unique!! Impurity Control


December 6th, 2007

Mass Profiler B.01.00 – Highlights

• Impurity Profiling/Differential Analysis Software• Graphical Displays

– Mass vs Retention Time– Log2 Ratio (Group1/Group2) vs Retention Time– Log2 Abundance Group 2 vs Log2 Abundance Group 1– Unique to Group 1 vs RT and Unique to Group 2 vs RT (New)

• Feature Identification (NEW)– Molecular Formula Generation and AMRT database– Single Feature or Batch Processing (summary report)– Web Internet Database Searching


December 6th, 2007

Graphical Displays


December 6th, 2007

Mass Profiler – Reproducibility of Results

Spread in ChromatographyRT

Spread of Peak in Time

Reproducible Retention Time Stability


December 6th, 2007

Mass Profiler – Reproducibility of Results

Outlier Sample or Feature


December 6th, 2007

Blue Unique Group 2 and Red Unique Group 1


December 6th, 2007

Mass Profiler Examples


December 6th, 2007

Pharmaceuticals Personal Care Products in Water

Problem: ID and Profile Changes in Chemical Residues Found in Streams Throughout North America

Solution: RRLC/QTOF with Exact Mass Chemical ResidueMass Profiler ID Differences Between Streams

Critical Component:Automated Data Analysis.. 20,000 33 Features

Outcome: Ability to ID ppb level Chemical Residues in Water


December 6th, 2007

Pharmaceutical Personal Care Products in WaterQTOF MFE/Profiling Experiment (44 Compounds)

308.10486 Wararin309.13405 Fluxetine310.07358 Sulfadimethoxine313.97805 Triclocarban314.14126 Ranitidine318.15551 Fluvoxamine324.16379 Citalopram329.14272 Paroxetine330.00772 Furosemide344.10084 Dehydroifedipine348.16852 Enalaprilat413.98602 Miconazole414.16133 Diltiazem418.27192 simvastatil573.51210 Erythromycin

151.06333 Acetaminophen176.09496 Cotinine180.04226 Aspirin180.06473 1,7-dimethylxanthine194.08038 Caffeine201.03607 Thiabendazole214.03967 Clofibic acid230.09429 Naproxen236.09496 Carbamaepine239.10769 Busproprion239.15214 Albuterol250.15698 Gemifrozil252.11572 Cimetidine253.05211 Sulfamethoxazole

254.09429 Ketoporfeb255.16231 Diphenhydramine267.12593 Venlafaxine270.02452 Sulfamethizole274.14298 Trimethoprim278.08375 Sulfamethazine284.01347 Sulfachloropyridazine287.95116 Triclosan293.05000 Norsertraline295.01668 Diclofenav295.11840 Norfluoxetine296.96447 HCTZ297.11873 Duloxetine299.15214 Codeine305.07380 Sertraline

Neutral Masses of Potential Residues in Water


December 6th, 2007

Mass Profiling > 20,000 features S/N >2 RI 0.001%

M ass vs. Retention Tim e

0 2 4 6 8 10 12 14 16Retention Time (min)

0

100

200

300

400

500

600

700

800

900

1000

Mas

s

Group 4 vs 10


December 6th, 2007

Inclusion List Utilized to Reduce Display+/- 0.005 daltons

Mass vs. Retention Time

0 2 4 6 8 10 12 14 16Retention Time (min)

150

200

250

300

350

400

450

Mas

s

Selecting any point on graph gives detailed MW, Empirical Formula info


December 6th, 2007

RT 9.63 C15H12N2O Carbamaepine > 5X Higher in Sample 10 > 4


December 6th, 2007

Impurity Analysis: Drug Delivery ProductExtractable Materials in Saline Solutions

• Objective: Identify Molecular Weight, Formula Structural Information of Impurities

• Method: RRLC, MFE/Profiler, Top 50 for Targeted MS/MS

• Results: > 30 Compounds ID, mass error


December 6th, 2007

Comparison Sample/Control BPC at 500 µL/min

6x10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

+ BPC Scan SampleAutoMSMS_New0002.d

1 1

5x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5+ BPC Scan ControlAutoMSMS_New2.d

1 1

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 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5

Sample

Control


December 6th, 2007

Mass Profiler to ID Differences: Sample/Control515 Features, RI > 0.01%, in all replicates


December 6th, 2007

174 Unique Features in Sample88 Unique Features in Control


December 6th, 2007

Degradation Products of Amoxicillin and AmoxClavin

C16H19N3SO5

MW 365.10454Source• Tablet 875mg New• Tablet 500mg 09/07

Sample Preparation• Tablets Dissolved in 40mL Methanol

Water with 0.1% Formic Acid• Syndicated 20 minutes• Spin 5 minutes 14000 RPM• Dilute 100 and 1000 fold with water

S

O

OHO

OOH

N

NH

NH2

CH3

CH3


December 6th, 2007

Degradation of Amoxicillin

S/N 5:1, RI > 0.01%, Mass 200-600, RT 1-11 Minutes 835 Features


December 6th, 2007

Amoxicillin Degradation Products

Isotope Pattern, Background Subtraction, # Ions


December 6th, 2007

Features only in Degraded Drug


December 6th, 2007

Composition and Database Searching


December 6th, 2007

Plant Extract with 16 Spiked Components


December 6th, 2007

Plant Extract – 16 Compounds

Scaling Factor

Total Conc Changes

Rat input/output


December 6th, 2007

Plant Extract – 16 Compounds


December 6th, 2007

Mass Profiler- How to Use


December 6th, 2007

Mass Profiler – The Basics…

Double Click Mass Profiler Icon

“1” for Single Group Comparison “2” for Differential Analysis


December 6th, 2007

Mass Profiler – Load MHD Files

*.MHD Data File Location

Select “Add” then add in the *.MHD files in Groups

Holding Down ctrl key select all *.MHD to add

Then Select “OK”


December 6th, 2007

Set Up Mass Profiler “Method”

*.mpm is Extension of Mass Profiler Project


December 6th, 2007

Mass Profiler – Edit Method

1. Limit Mass Range and RT

2. For Small Molecule Apps Multiply Charge Forbidden

3. For Biomolecules Apps Multiply Charged Required

4. Abundance to 0.1 to 0.01%

5. Number of Ions >=2

6. Isotope Pattern Matching


December 6th, 2007

Mass Profiler – How to Import Exclusion List

Sort Table By Height/Volume

Right Click on TableSelect “EXPORT”

From EXCEL Copy Masses From Table


December 6th, 2007

Mass Profiler – Exclusion List of Masses

Paste List of Masses From Blank Into Exclude Masses


December 6th, 2007

Mass Profiler – Alignment


December 6th, 2007

Mass Profiler – Results Filters


December 6th, 2007

Mass Profiler – Special Case – No Replicates

For Example without ReplicatesMin Differential Score MUSTbe set to ZERO before loading*.MHD Files


December 6th, 2007

Feature Identification – Set Parameters for MFG


December 6th, 2007

Identify Composition of Features

Right Click any feature in Graph

Right Click any feature in Table


December 6th, 2007

Individual Feature Results# Compositions and Database Hits


December 6th, 2007

Identification Summary Report


December 6th, 2007

Feature Details


December 6th, 2007

Exact Mass/Formula Database Searching


December 6th, 2007

Export Feature Information


December 6th, 2007

Questions and Answers


December 6th, 2007

METLIN Personal Metabolite Database Software


December 6th, 2007

METLIN Personal Database G6825AA

Metabolite-specific database for metabolomics research

• Database installed on a PC • Contains ~15000 compounds• Manual and batch searches

– Query based on monoisotopic mass

• Customizable– Add compounds– Assign chromatographic retention

times to metabolites– Create subset databases

• Works with other Agilent software• Cost: $3759


December 6th, 2007

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


December 6th, 2007

METLIN Personal Database Initial View

User search mode


December 6th, 2007

METLIN Manual Single Mass Search

Metabolite search results

Active metabolite structureSingle mass search settings

Hyperlinks to Metabolite websites


December 6th, 2007

METLIN Batch Search

Load file for batch search

Active mass

Active metabolite structure

Active mass search results


December 6th, 2007

METLIN Batch Summary

Active metabolite structure

Batch summary – best metabolite match

Exportable list of best metabolite search results


December 6th, 2007

Creating a New METLIN Database

Create a new personal database from METLIN

METLIN is read only and can not be changed


December 6th, 2007

Edit Metabolite Database – Update Entry

Add retention time to existing metabolite


December 6th, 2007

METLIN Mass Search With Retention Time Adds Specificity

Adding retention time eliminates isobaric metabolites

Only 1 metabolite instead of 3 !!!!


December 6th, 2007

Edit Metabolite Database – New Entry

Select add new then type new metabolite information


December 6th, 2007

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 numberDatabase and MFG results

Set Parameters

View detailed results


December 6th, 2007

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


December 6th, 2007

Summary

• Care and Feeding of QTOF/TOF– Remember to Defrag Hard Drives Weekly – Software updates available

• Mass Profiler B.01.00 $3,150– Differential Analysis Software– Batch Molecular Formula Generation and Database Searching

• METLIN Personal Metabolite Database B.01.00 $3,759– Accurate Mass Retention Time Database (AMRT)– Convert *.CSV to MTL Formats– User Generated Databases (Plant Extract, Pesticides, AA’s)


December 6th, 2007

Questions and AnswersThank You


December 6th, 2007

Upcoming Proteomics eSeminars

April 24, 2008, 11:00 a.m. EDTBiomarker Discovery by Targeted and Profiling ProteomicsProfessor Rainer Bischoff, Analytical Biochemistry, University of Groningen

May 8, 2008, 11:00 a.m. EDTProtein Analysis Using CAD/ETD Ion Trap Tandem Mass SpectrometryProfessor Ole Norregard Jensen, Protein Research Group at University of Southern Denmark

June 26, 2008, 11:00 a.m. EDTPeptide Quantitation With An Agilent 6410 QQQ SystemNing Tang, Application Scientist, Agilent Technologies, Inc.

qtof small molecule applications update david a. weil

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