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?... Page 2
Chemical Residue Analysis.... From the headlines.. PFOA/PFOS Persistent Chemical Fungicides/Pesticides PPCP Steroids and Antibiotics Residuals in Beverages Page 3
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 Page 4
Agilent s 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 Sample Preparation Separations Targeted Quant QQQ Agilent s New SPE Products Non-Volatile Residues LC/MS Analysis Non-Targeted TOF/QTOF Impurity ID Profiling Data Analysis Page 5
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 Page 6
Agilent Suite of MassHunter Products Qual Quant Profiler MetID Page 7
MassHunter QUAL MSD, QQQ, TOF, QTOF Data Navigator Method Editor Method Parameters Page 8
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 Chromatogram Linking of panes for easier browsing and navigation Navigation Tree Spectrum Page 9
Qualitative Analysis: Molecular Feature Extraction Compound Chromatogram List of Compounds Compound Spectrum Page 10
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 + BPC(150.00000-800.00000) Scan CoffeeSpiked12.d 1 1 x10 6 1.4 1.2 1 0.8 0.6 0.4 0.2 + BPC(150.00000-800.00000 [-1]) Scan CoffeeSpiked12.d 1 1 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 Counts vs. Acquisition Time (min) Base Peak Chromatogram with Exclude m/z 195.09145 +/- 100 ppm Page 11
Molecular Feature Extraction (MFE) Automated Data Reduction Software Finds Features in TOF/QTOF Data 3D 3D Plot Plot Before No Coeluting Features Background Original TIC Processed TIC Data Reduced sum intensities of isotopes, adducts, clusters and multiply charges ions together. Page 12
Processing large amount of data with MFE Display filtering: - S/N > 2 -m/z150 to 800 - [M+Na] and [M+NH 4 ] Adducts - At least 2 ions - Relative intensity > 0.01% 5131 features Processed TIC Intensity (10^6) 15 10 5 Original TIC TIC Intensity (10^6) 15 Processed TIC 10 5 0 min 2 4 6 8 10 12 14 min 0 2 4 6 8 10 12 14 Page 13
In MassHunter Qual Select Find by Molecular Feature Page 14
Method Editor: Parameters for Molecular Feature Display Filters Page 15
Mass Defect Filtering Page 16
Executed Compound Table Page 17
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 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 Counts vs. Acquisition Time (min) Over 280 Compounds Found in Coffee Spiked with Pesticide Page 18
Prednisolone: C 21 H 28 O 5 360.193674 OH CH 3 O CHOH 3 O O CH 3 O 2 year old Suspension Page 19
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 13.421 23081.8 0.1 0 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 Counts vs. Acquisition Time (min) Page 20
12 Potential Degradation Products in Prenisolone CH 2 O Loss -H 2, -H 2 O, -CO 2 H 2, +H 2, +CH 4 +H 2 -O, Page 21
Molecular Formula Generation.. Technical Overview 5989-7409EN Page 22
Generate Formulas Parameters Page 23
Theoretical Isotope Pattern Page 24
Accurate Mass of Compounds Page 25
Identify Compounds by Database Searching Search database by mass or by molecular formula Select either exact mass or Personal Metlin database Page 26
Automated Compound Search Metlin Database Page 27
Mass Profiling Version 2.0 Processed Processed TIC Processed TIC Group TIC A (Impure) Processed Processed TIC Processed TIC Group B TIC (Control) Aligns Data RT, Mass, Abundance What s Changed? Mass/RT >2 Fold Change What s All Unique!! Data RT/Mass Impurity Control Page 28
How to Create a Project Select 2 Groups for Differential Select 1 Group for Concentration Change Need at least 3 replicates Page 29
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 Page 30
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 Page 31
Multiple Display Options Mass vs RT (Average of Features) Page 32
Differential Display by Retention Time Page 33
Unique Features.. Page 34
Single Group Example: Compounds Spiked into Plant Extract Page 35
Feature Table Single Group Example For Single Group: Large SD of abundance indicates compounds that have concentrations that vary over the samples Page 36
Graphical Display of Concentration Change of Feature Change Plot Style to Colored by Feature Page 37
Abundance and Feature Details Page 38
Batch Chemical Composition and Database Search from Mass Profiler Page 39
Batch Molecular Formula Generation and Database Searching from Profiler Page 40
Web Searching of Compounds NIST, ChemIDPlus, PubChem, Page 41
MetID Software For Impurity Profiling Page 42
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 Page 43
Metabolite Table w/ Find + Confirm Results Metabolite Chros + Spectra Molecular Formula Generation MS and MSMS Data Page 44
Overview MetID 1. 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 Page 45
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Method Input Parent Compound Propose Degradation Products Page 47
Proposed Degradation Products Qualified by Mass Defect and Isotope Pattern Let s Determine Composition of MW 411 Page 48
Accurate Mass Generates Composition Acid Hydrolysis (H 2 O+CO 2 H-OH) Accurate Mass of Fragments Ions Page 49
Overlay MSMS Spectrum Parent/Product Page 50
3D of MSMS Spectra Fragmentation Pattern Page 51
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 Page 52
Personal Metlin Database Page 53
METLIN Personal Database Initial View User search mode Page 54
METLIN Manual Single Mass Search Single mass search settings Active metabolite structure Hyperlinks to Metabolite websites Metabolite search results Page 55
METLIN Batch Search Load file for batch search Active metabolite structure Active mass Active mass search results Page 56
METLIN Batch Summary Active metabolite structure Batch summary best metabolite match Exportable list of best metabolite search results Page 57
Creating a New METLIN Database METLIN is read only and can not be changed Create a new personal database from METLIN Page 58
Edit Metabolite Database Update Entry Add retention time to existing metabolite Page 59
METLIN Mass Search With Retention Time Adds Specificity Only 1 metabolite instead of 3!!!! Adding retention time eliminates isobaric metabolites Page 60
Edit Metabolite Database New Entry Select add new then type new metabolite information Page 61
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 Set Parameters Database and MFG results View detailed results Page 62
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 Page 63
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 Page 64
THANK YOU.. Page 65