Making Sense of Differences in LCMS Data: Integrated Tools

Transcription

1 Making Sense of Differences in LCMS Data: Integrated Tools David A. Weil Agilent Technologies MassHunter Overview Page 1 March 2008

2 How Clean is our Water?... Page 2

3 Chemical Residue Analysis.... From the headlines.. PFOA/PFOS Persistent Chemical Fungicides/Pesticides PPCP Steroids and Antibiotics Residuals in Beverages Page 3

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 Page 4

5 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

6 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

7 Agilent Suite of MassHunter Products Qual Quant Profiler MetID Page 7

8 MassHunter QUAL MSD, QQQ, TOF, QTOF Data Navigator Method Editor Method Parameters Page 8

9 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

10 Qualitative Analysis: Molecular Feature Extraction Compound Chromatogram List of Compounds Compound Spectrum Page 10

11 How To Determine Complexity of Sample/Matrix Acquire MS or AutoMSMS Modes Base Peak Chromatogram Exclude Major Component Find Compounds by Molecular Feature Extraction x BPC( ) Scan CoffeeSpiked12.d 1 1 x BPC( [-1]) Scan CoffeeSpiked12.d Counts vs. Acquisition Time (min) Base Peak Chromatogram with Exclude m/z /- 100 ppm Page 11

12 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

13 Processing large amount of data with MFE Display filtering: - S/N > 2 -m/z150 to [M+Na] and [M+NH 4 ] Adducts - At least 2 ions - Relative intensity > 0.01% 5131 features Processed TIC Intensity (10^6) Original TIC TIC Intensity (10^6) 15 Processed TIC min min Page 13

14 In MassHunter Qual Select Find by Molecular Feature Page 14

15 Method Editor: Parameters for Molecular Feature Display Filters Page 15

16 Mass Defect Filtering Page 16

17 Executed Compound Table Page 17

18 Find Compounds by Molecular Feature x10 6 Cpd 279:+ ECC Scan CoffeeSpiked12.d Counts vs. Acquisition Time (min) Over 280 Compounds Found in Coffee Spiked with Pesticide Page 18

19 Prednisolone: C 21 H 28 O OH CH 3 O CHOH 3 O O CH 3 O 2 year old Suspension Page 19

20 Sequential Neutral Water Losses Detected x ECC Scan Prednisolone0002.d Counts vs. Acquisition Time (min) Page 20

21 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

22 Molecular Formula Generation.. Technical Overview EN Page 22

23 Generate Formulas Parameters Page 23

24 Theoretical Isotope Pattern Page 24

25 Accurate Mass of Compounds Page 25

26 Identify Compounds by Database Searching Search database by mass or by molecular formula Select either exact mass or Personal Metlin database Page 26

27 Automated Compound Search Metlin Database Page 27

28 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

29 How to Create a Project Select 2 Groups for Differential Select 1 Group for Concentration Change Need at least 3 replicates Page 29

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 Page 30

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 Page 31

32 Multiple Display Options Mass vs RT (Average of Features) Page 32

33 Differential Display by Retention Time Page 33

34 Unique Features.. Page 34

35 Single Group Example: Compounds Spiked into Plant Extract Page 35

36 Feature Table Single Group Example For Single Group: Large SD of abundance indicates compounds that have concentrations that vary over the samples Page 36

37 Graphical Display of Concentration Change of Feature Change Plot Style to Colored by Feature Page 37

38 Abundance and Feature Details Page 38

39 Batch Chemical Composition and Database Search from Mass Profiler Page 39

40 Batch Molecular Formula Generation and Database Searching from Profiler Page 40

41 Web Searching of Compounds NIST, ChemIDPlus, PubChem, Page 41

42 MetID Software For Impurity Profiling Page 42

43 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

44 Metabolite Table w/ Find + Confirm Results Metabolite Chros + Spectra Molecular Formula Generation MS and MSMS Data Page 44

45 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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47 Method Input Parent Compound Propose Degradation Products Page 47

48 Proposed Degradation Products Qualified by Mass Defect and Isotope Pattern Let s Determine Composition of MW 411 Page 48

49 Accurate Mass Generates Composition Acid Hydrolysis (H 2 O+CO 2 H-OH) Accurate Mass of Fragments Ions Page 49

50 Overlay MSMS Spectrum Parent/Product Page 50

51 3D of MSMS Spectra Fragmentation Pattern Page 51

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 Page 52

53 Personal Metlin Database Page 53

54 METLIN Personal Database Initial View User search mode Page 54

55 METLIN Manual Single Mass Search Single mass search settings Active metabolite structure Hyperlinks to Metabolite websites Metabolite search results Page 55

56 METLIN Batch Search Load file for batch search Active metabolite structure Active mass Active mass search results Page 56

57 METLIN Batch Summary Active metabolite structure Batch summary best metabolite match Exportable list of best metabolite search results Page 57

58 Creating a New METLIN Database METLIN is read only and can not be changed Create a new personal database from METLIN Page 58

59 Edit Metabolite Database Update Entry Add retention time to existing metabolite Page 59

60 METLIN Mass Search With Retention Time Adds Specificity Only 1 metabolite instead of 3!!!! Adding retention time eliminates isobaric metabolites Page 60

61 Edit Metabolite Database New Entry Select add new then type new metabolite information Page 61

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 Set Parameters Database and MFG results View detailed results Page 62

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 Page 63

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: MassHunter Profiler: MetID: Find Compounds in Complex Matrix Differential Analysis Software Metabolite ID and Impurity Profiling Metlin Database: Personalize Destktop Database Searching Page 64

65 THANK YOU.. Page 65