MassHunter TOF/QTOF Users Meeting

Transcription

1 MassHunter TOF/QTOF Users Meeting 1

2 Qualitative Analysis Workflows Workflows in Qualitative Analysis allow the user to only see and work with the areas and dialog boxes they need for their specific tasks A Workflow consists of three parts: A default method for the Workflow with the parameters and report templates already set for that type of task. A specified layout of the user interface that only shows the windows and table columns that the user needs to see. A new section at the top of the Method Explorer (called a workflow section) which groups together the tabs a user may need while performing a task: 5

3 Applying a Workflow Configuration Configure for Workflow in the menu bar You will see a warning that applying the Workflow will make changes 7

4 Using a Workflow After applying the Workflow, go through each tab in the workflow section sequentially to adjust the parameters for your task: 8

5 Isotope Distribution Calculator There is a standalone Isotope Distribution Calculator included with Qualitative Analysis at: C:\Program Files\Agilent\MassHunter\Workstation\Qual\B.0x.00\Bin\IsotopeDistributionCalculator.exe. Or under Start menu under: In B or later: All Programs Agilent MassHunter Workstation Qualitative Analysis B.0x.00 Tools It allows you to model the theoretical abundances and spacing of any particular compound: 12

6 How To Determine Complexity of Sample/Matrix Acquire MS or AutoMSMS Modes Base Peak Chromatogram Excludes Major Components reference masses Find Compounds by Molecular Feature Extraction (MFE) Base Peak Chromatogram with Exclude m/z /- 100 ppm 14

7 Find Compounds by Molecular Feature Results Over 280 Compounds Found in Coffee Spiked with Pesticide 15

8 Molecular Feature Extractor (MFE) Transforming Data into Chemical Information Raw data Background noise removed Individual m/z peaks grouped into isotope clusters Isotope clusters grouped into molecular features Identification, Quantification, Differential Analysis are performed on chemically qualified compound data 31

9 Find by Molecular Feature 19

10 Method Editor MFE 20

11 Method Editor MFE (cont d) 21

12 Method Editor MFE (cont d) Similar compounds (e.g. metabolites) will have similar mass defects, so we can use the Mass Defect tab to filter on that. 22

13 MFE Results 23

14 Find Compounds by Molecular Feature Results Over 280 Compounds Found in Coffee Spiked with Pesticide 24

15 Compound List Use found compounds for further analysis: Use Molecular Formula Generator (MFG) on list. Search a Database and match findings Upload the MFE results to Mass Profiler or GeneSpring MS for analysis. 25

16 Find Compounds by Formula Find Compounds in analysis by inputting: Formulas separated by, CEF file with Formulas A database with Formulas (.csv,.mtl,.cdb) Worklist Formulas 26

17 Find Compounds by Formula - Options Input Formula Source 28

18 Find Compounds by Formula Sample Purity Enables the user to do Sample Purity (not Peak Purity) calculations based on the same algorithms as LC/MS ChemStation Sample Purity compares a peak against all other peaks in the data file to determine the estimated purity of the compound After doing a Find Compounds by Formula, pure compounds are marked green and impure are marked yellow 29

19 Find Compounds by Formula System Suitability Integrate (UV) and Find by Formula Sample Purity It allows the user to calculate system suitability values on a UV chromatogram based on the chosen Pharmacopoeia The results are displayed in the Integration Peak List. 30

20 System Suitability Calculations Can Be Done for MS, MS/MS, UV and GC Chromatograms 31

21 Find Compounds by Formula Results Compounds Found Formulas Found 33

22 Find Compounds by Auto MS/MS Auto MS/MS Data Acquisition function used when the m/z value of the precursor ion is unknown prior to analysis. The instrument acquires a mass spectrum then determines ions meeting preset criteria for precursor ions. MS/MS spectra are taken for each identified precursor. The instrument then switches back to MS operation. Find by Auto MS/MS Data Analysis function used to automatically identify compounds in MS/MS data and create averaged MS and MS/MS spectra for each compound. The functionality is an easy way to mine information from complex data. MS Scan - Precursor Reference Mass Precursor Ion Reference Mass Product Ions MS/MS Scan Product Ions Precursor Ion 34

23 Start with MS/MS Data Very complex data. Difficult to obtain information. Page 35

24 Find by Auto MS/MS Algorithm User-specified excluded ions and persistent background are removed from the Auto MS/MS data file. The remaining spectra are then are divided into groups that represent scans from the same compound. This is done (first for positive ion scans, then for negative ion scans) by finding scans that: Have the same precursor m/z within a user-specified tolerance, and Have retention times within a user-specified window of each other, and Have a TIC value that exceeds a user-specified threshold Best Value is to Find Compounds Based on Product Ion Filters 37

25 Auto MS/MS Parameters Data to be found As of B Can filter results by fragments Appropriate Symmetry for Q-TOF: ppm Exclude Reference Masses 38

26 Find by Auto MS/MS Results MS MS/MS 39

27 Compound List 40

28 Find Compound by Integration New function that is similar to Integrate and Extract Peak Spectra. Results are in the form of Compounds rather than Peaks and User Spectra. 42

29 Find Compound by Integration Best used when compounds are chromatographically separated. Creates Compounds which may be exported as a CEF file. Better labeling after library search than User Spectra: Easer to understand Finds more low level ions 43

30 Find Compound by Integration Integrator and Peak Filter parameter settings are unique to Find Compounds by Integration. 44

31 Find Compound by Integration Exclude Mass(es), Peak Spectrum Extraction, Charge State & Results are shared with other method sections. 45

32 Find Compound by Integration Specify the type of chromatogram to extract & integrate: Will extract the chromatogram even if it is already present. Duplicate TIC when run. Not required to extract the signal in File Open Action. Cannot be used on user defined chromatograms (example EIC). 46

33 Find Compound by Integration Optional - Extract additional chromatogram (not integrated) Optional - Restrict m/z range on BPC 47

34 Find Compound by Integration Adjust Delay Time Will adjust the RT of other signals compared to MS. Applied to GC, TWC, etc. signals when signal is extracted, not after. 48

35 Find Compound by Integration When User Interface Configuration has LC checked the Adjust Delay Time feature is active when extracting signals! 49

36 Find by Targeted MS/MS Algorithm This algorithm extracts a chromatogram for each targeted mass in the data file. This chromatogram is integrated and if the peaks are not eliminated by one of the filters on this tab, a compound is generated. The algorithm uses the targeted masses input in the acquisition method. 50

37 Find by Targeted MS/MS Results 51

38 Find by Targeted MS/MS Can extract MS/MS spectra based on collision energy 52

39 Identify Compounds Search a Database or Library after Find Compounds Generate Formulas after Find Compounds *Sequence editing 53

40 Database Searching Search Mode 54

41 Automated Compound Search METLIN Database 55

42 Accurate Mass Database vs. Library Database: contains an accurate mass, possibly a retention time, and other compound attributes such as CAS ID and the chemical structure for each compound. Accurate Mass Library: contains the same information as a Database plus MS/MS spectra. By convention, Databases are kept in D:\MassHunter\databases and Libraries are kept in D:\MassHunter\Library Libraries from Agilent have the suffix Lib in the name, e.g. SulfasLib.cdb 57

43 Accurate Mass Database vs. Library (cont.) When users search an Accurate Mass Database, they are searching for a specific accurate mass (e.g ) against the accurate masses listed in the Database. When users search a Library, they are searching for a spectrum against the spectra in the Library. Unit Mass Library Search: accessed by turning on the GC Separation Type in the User Interface Configuration dialog box. The Library Editor for Unit Mass Libraries can be accessed by installing Quantitative Analysis. 58

44 Accurate Mass Database Searching If an Accurate Mass Database Search finds a compound, it will place the chemical structure in the Product Ion Spectrum and display the structure in the Structure Viewer pane (must be toggled On). Any links to outside information sources about the compound (e.g. Chemical Abstract Service #, CAS) are active click on them to go to a web site. 59

45 Accurate Mass Library Searching If an Accurate Mass Library Search finds a compound, it will place the chemical structure in the Product Ion Spectrum and display the structure in the Structure Viewer pane (if it is toggled On). Acquired Spectrum It is also possible to display a difference spectrum for the Library hit by toggling the Difference Results button to On in the main toolbar. Library Spectrum Difference Spectrum 60

46 All Ions MS/MS 61

47 Why All Ions MS/MS? -The EU s SANCO compliant screening requires: At least 2 product ions Final confirmation with a reference compound -Fully developed workflow in All Ions MS/MS for compound confirmation and quant method setup. Easy Acquisition method setup Targeted identification of product ions using Agilent s MS/MS libraries Chromatographic confirmation of product ions (coelution score) Rapid Quant method development automatically selects two high quality product ions for each compound, eliminating tedious manual steps -Be prepared for unexpected compounds in the sample -QQQ-like easy acquisition method setup -Productivity tool for faster analysis - Identification and fragment finding done in MH Qualitative Analysis - Routine quantitation and confirmation done in MH Quantitative Analysis 62

48 All Ions MS/MS Targeted Screening Workflow Acquisition PCDL Qual Quant MassHunter Acquisition m/z, ion species Find by Formula on MS low channel High/Low Fragmentor Voltage on TOF and Q-TOF High/Low Collision Energy on Q-TOF Pesticide library 700+ compounds with spectra) Forensics /Tox library (2720 compounds with spectra ) Qual Report Targets Find EICs for fragments on MS high channel Align EICs of fragments with parent EIC Extract Correlate qualified fragments with target Qualify and confirm fragments Export Quantify target with additional fragment ion confirmation Quant Report 63

49 minutes All Ions MS/MS Use and combine PCDL libraries with All Ions MS/MS. Export information and automatically create quant methods in seconds. Reduce processing time from one day to a matter of hours Processing time Quant Qual 0 Without All Ions With Allons 64

50 All Ions MS/MS Setup Turn on All Ions MS/MS using the current Formula Source PCDL Minimum Co-elution score 65

51 All Ions MS/MS can distinguish between isomers Hydrocodone Codeine 66

52 All Ions MS/MS can distinguish between isomers Hydrocodone Codeine 67

53 All Ions MS/MS can distinguish between isomers Hydrocodone Codeine 68

54 All Ions MS/MS: Chromatographic Confirmation of Product Ions Overlaid low and high energy chromatograms Co-elution curves the precursor and product ions agree exactly on their apex! 69

55 New Compound Details View in Qual rev. B New chromatogram and spectrum windows that show data from one compound only. Data belonging to one compound can be overlaid easily and quickly. 70

56 Quality Score for MFE 71

57 Molecular Formula Generator - Calculate Compound Formulas You can generate the molecular formula for each selected compound. First, find the Compounds, then use MFG. Example: Structure Elucidation of Degradation Products of the Antibiotic Drug Amoxicillin HO H H H O COOH O OH COOH HN H NH NH 2 N CH H 2 NH CH 3 O N 3 S NH NH S CH 3 S CH 3 H H H N O H O H O HO HO H COOH CH 3 CH 3 1 Amoxicillin, 2 Amoxicillin penicillinoic acid, 3 Amoxicillin penilloic acid 72

58 Molecular Formula Generator 73

59 Molecular Formula Generator Algorithm Determine a mass in the compound to use for MFG: The program looks at clusters of ions and applies rules for isotopic abundance and mass spacing (mass accuracy and resolution are important to achieve this). Apply user knowledge of samples to limit the possible formula: Users fill out the Allowed Species dialog box with their knowledge of the chemistry of their samples. Rule out formulas that are chemically impossible (e.g. CH 7 ) Calculate probability scores for each candidate formula based on: Calculated vs. measured mass Calculated vs. measured abundance ratios Calculated vs. measured mass intervals of isotopes (isotope spacing) 74

60 Molecular Formula Generator Algorithm (cont.) Rank and display each candidate formula based on its probability score For calculations done in conjunction with MFE, calculate an abundance-weighted average cross score for all species (adducts, dimers, charge states) associated with a compound. These are all listed in the Compound List table 75

61 Molecular Formula Generator Algorithm MS/MS If MS/MS data are available, the algorithm uses it for greater confidence in formula generation: The algorithm generates tentative formulas for each MS/MS fragment (product ion). Precursor formulas with no related product ions are eliminated For a precursor formula to be valid, it must generate product ions that make sense Proposed precursor formulas are scored and ranked based on their fit with the product ions Algorithm uses coverage (explanation of how the precursor fragmented) and mass error of product ions A single score is provided that combines both MS and MS/MS data 76

62 Molecular Formula Generator Algorithm MS/MS Note the scores in the Compound List table below: Score (various) MS Score (MS-only) MS/MS (goodness-of-fit of product ions formulas to precursor formula) As of Qual rev. B.02.00, scores are absolute (Best is not fixed at 100) for comparison across formulae and samples. 77

63 Major Features of Qual B All Ions MS/MS Compound Details View New Quality score for MFE Support for new tmrm files Enhanced Molecular Formula Generation New Find Compound Algorithm : Find by Integration Annotate fragment ions with fragment formula generation Save results faster Export format changes 111