QTOF Small Molecule Applications Update April 10 th, 2008 David A. Weil David_weil@agilent.com Application Scientist, Schaumburg, IL December 6 th, 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 (QTOF@googlegroups.com ) 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
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
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
Mass Profiling A Powerful Workflow 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 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 GC/MS LC/MS Find molecula r features MFE (LC/MS) AMDIS (GC/MS) Compare sample sets Mass Profiler GeneSpring MS Identification Spectrum Mill Molecular Formulas DB Search (METLIN) Library Search Validation QQQ MRM GeneSpring MS
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) Compound list or Molecular feature list RT m/z Abund 2.11 195.1745 21000 2.11 257.2566 33550 2.34 224.2134 11784
MassProfiler Dependent on MFE Feature Extraction Don t extract all the compounds only Important ones How to Mine for Gold Generated a Base Peak Chromatogram Input Mass Range of Compounds Exclude Reference Ions Input m/z to 4 decimal places Set window to 20 ppm Run Standards to Determine RT Information
Good Quality *.MHD Gives Good Quality Profiler 1. Centroid versus Profile data 2. S/N versus Total Counts Typically use 100 counts Too high may not detect C13 peak Profile S/N 10 First Pass 3. Focused MHD generation Limit Retention Time Limit m/z Range 4. Ion Species (NH 4 If buffer) 5. Check Box if High Salt 6. Check if Free Radicals/Quats December 6 th, 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
Find Compounds by Molecular Feature
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
Mass Profiling Software Processed Processed TIC Processed TIC Group TIC A (Impure) Aligns Data RT, Mass, Abundance Processed Processed TIC Processed TIC Group B TIC (Control) What s Changed? Mass/RT >2 Fold Change What s All Unique!! Data RT/Mass Impurity Control
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
Graphical Displays
Mass Profiler Reproducibility of Results Reproducible Retention Time Stability Spread in ChromatographyRT Spread of Peak in Time
Mass Profiler Reproducibility of Results Outlier Sample or Feature
Blue Unique Group 2 and Red Unique Group 1
Mass Profiler Examples
Pharmaceuticals Personal Care Products in Water Problem: Solution: ID and Profile Changes in Chemical Residues Found in Streams Throughout North America RRLC/QTOF with Exact Mass Chemical Residue Mass 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 6 th, 2007
Pharmaceutical Personal Care Products in Water QTOF MFE/Profiling Experiment (44 Compounds) Neutral Masses of Potential Residues in Water 151.06333 Acetaminophen 176.09496 Cotinine 180.04226 Aspirin 180.06473 1,7-dimethylxanthine 194.08038 Caffeine 201.03607 Thiabendazole 214.03967 Clofibic acid 230.09429 Naproxen 236.09496 Carbamaepine 239.10769 Busproprion 239.15214 Albuterol 250.15698 Gemifrozil 252.11572 Cimetidine 253.05211 Sulfamethoxazole 254.09429 Ketoporfeb 255.16231 Diphenhydramine 267.12593 Venlafaxine 270.02452 Sulfamethizole 274.14298 Trimethoprim 278.08375 Sulfamethazine 284.01347 Sulfachloropyridazine 287.95116 Triclosan 293.05000 Norsertraline 295.01668 Diclofenav 295.11840 Norfluoxetine 296.96447 HCTZ 297.11873 Duloxetine 299.15214 Codeine 305.07380 Sertraline 308.10486 Wararin 309.13405 Fluxetine 310.07358 Sulfadimethoxine 313.97805 Triclocarban 314.14126 Ranitidine 318.15551 Fluvoxamine 324.16379 Citalopram 329.14272 Paroxetine 330.00772 Furosemide 344.10084 Dehydroifedipine 348.16852 Enalaprilat 413.98602 Miconazole 414.16133 Diltiazem 418.27192 simvastatil 573.51210 Erythromycin
Mass Profiling > 20,000 features S/N >2 RI 0.001% M ass vs. Retention Tim e 1000 900 800 700 Mass 600 500 400 300 200 100 0 0 2 4 6 8 10 12 14 16 R eten tion T ime (min ) Group 4 vs 10 December 6th, 2007
Inclusion List Utilized to Reduce Display +/- 0.005 daltons 450 Mass vs. Retention Time 400 350 Mass 300 250 200 150 0 2 4 6 8 10 12 14 16 Retention Time (min) Selecting any point on graph gives detailed MW, Empirical Formula info
RT 9.63 C 15 H 12 N 2 O Carbamaepine > 5X Higher in Sample 10 > 4
Impurity Analysis: Drug Delivery Product Extractable 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
Comparison Sample/Control BPC at 500 µl/min x10 6 1.5 1.4 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 + BPC Scan SampleAutoMSMS_New0002.d 1 1 Sample x10 5 5.5 5 4.5 + BPC Scan ControlAutoMSMS_New2.d 1 1 Control 4 3.5 3 2.5 2 1.5 1 0.5 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 Counts vs. Acquisition Time (min)
Mass Profiler to ID Differences: Sample/Control 515 Features, RI > 0.01%, in all replicates
174 Unique Features in Sample 88 Unique Features in Control
C 16 H 19 N 3 SO 5 Degradation Products of Amoxicillin and AmoxClavin MW 365.10454 NH 2 Source Tablet 875mg New Tablet 500mg 09/07 Sample Preparation HO O NH O N S OH CH 3 CH 3 O 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 December 6 th, 2007
Degradation of Amoxicillin S/N 5:1, RI > 0.01%, Mass 200-600, RT 1-11 Minutes 835 Features
Amoxicillin Degradation Products Isotope Pattern, Background Subtraction, # Ions
Features only in Degraded Drug
Composition and Database Searching
Plant Extract with 16 Spiked Components
Plant Extract 16 Compounds Scaling Factor Total Conc Changes Rat input/output
Plant Extract 16 Compounds
Mass Profiler- How to Use
Mass Profiler The Basics Double Click Mass Profiler Icon 1 for Single Group Comparison 2 for Differential Analysis
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
Set Up Mass Profiler Method *.mpm is Extension of Mass Profiler Project
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
Mass Profiler How to Import Exclusion List Sort Table By Height/Volume Right Click on Table Select EXPORT From EXCEL Copy Masses From Table
Mass Profiler Exclusion List of Masses Paste List of Masses From Blank Into Exclude Masses
Mass Profiler Alignment
Mass Profiler Results Filters
Mass Profiler Special Case No Replicates For Example without Replicates Min Differential Score MUST be set to ZERO before loading *.MHD Files
Feature Identification Set Parameters for MFG
Identify Composition of Features Right Click any feature in Graph Right Click any feature in Table
Individual Feature Results # Compositions and Database Hits
Identification Summary Report
Feature Details
Exact Mass/Formula Database Searching
Export Feature Information
Questions and Answers
METLIN Personal Metabolite Database Software
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
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
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 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
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 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)
Questions and Answers Thank You
Upcoming Proteomics eseminars April 24, 2008, 11:00 a.m. EDT Biomarker Discovery by Targeted and Profiling Proteomics Professor Rainer Bischoff, Analytical Biochemistry, University of Groningen May 8, 2008, 11:00 a.m. EDT Protein Analysis Using CAD/ETD Ion Trap Tandem Mass Spectrometry Professor Ole Norregard Jensen, Protein Research Group at University of Southern Denmark June 26, 2008, 11:00 a.m. EDT Peptide Quantitation With An Agilent 6410 QQQ System Ning Tang, Application Scientist, Agilent Technologies, Inc.