Agilent Technologies, Inc. · NYSE:A

Agilent Technologies — Technical Presentation (April 2015)

101 slides from Agilent Technologies's technical presentation, Apr 8, 2015

1. Slide 1: New Screening strategies with Exact Mass QTOF-MS LC & GC

   New Screening strategies with Exact Mass QTOF-MS LC & GC New tools and workflows for more efficiency and confidence Jaume C. Morales LCMS Product Specialist AccMass_Screening_Pesticides 08/04/2015 1

2. Slide 2: Accurate Mass Screening and Quantitation

   Accurate Mass Screening and Quantitation Different approaches and challenges • Target screening and quantitation - Limited list of target compounds - Reference compounds are available in the lab - Databases and libraries are available - Challenge: Efficient data review and quantitation • Qualitative

3. Slide 3: Accurate Mass Screening and Quantitation

   Accurate Mass Screening and Quantitation Different approaches and challenges • Target screening and quantitation - Limited list of target compounds - Reference compounds are available in the lab - Databases and libraries are available - Challenge: Efficient data review and quantitation • Qualitative

4. Slide 4: Dos enfoques de Screening

   Dos enfoques de Screening TARGET QUALITATIVE

5. Slide 5: Introduction: Current regulations and residue situation

   Introduction Current regulations and residue situation • EU regulation - Maximum residue levels regulated in commision regulation (EC) 396/2005 • appendix II – IV regulates >170 000 MRLs (matrix-pesticide combinations) - European and national monitoring programs, regulated by (EC) 396/2005 - 173 ale

6. Slide 6: SANCO guidelines for pesticides screening

   SANCO guidelines for pesticides screening Implementations for qualitative and quantitative methods • Qualitative multi-residue methods - To extend the analytical scope in a cost-effective way to analytes which have low probability to be present in samples - Screening Detection Level (SDL) for indivi

7. Slide 7: Introduction: Pesticide screening status today

   Introduction Pesticide screening – Status today • More than 1000 pesticides and pesticide metabolites need consideration • QuEChERS as „universal“ extraction procedure is widely accepted • Detection method of choice is chromatographic seperation and mainly QQQ detection • Current situation - 600 to

8. Slide 8: Introduction: Pesticide screening

   Introduction Pesticide screening – • More than 1000 pesticides and pesticide metabolites need consideration • QuEChERS as „universal“ extraction procedure is widely accepted • Detection method of choice is chromatographic seperation and mainly QQQ detection • Current situation - 600 to 800 compounds

9. Slide 9: HRMS QTOF Acquisition Modes

   HRMS QTOF ACQUISITION MODES QTOF Acquisition – MS and MS/MS Modes of Operation • MS Only – “TOF only” mode (RETROSPECTIVE but just MS [*]) • Data Directed (Targeted) MS/MS experiments (NON RETROSPECTIVE) • Import of Target mass lists from Target compounds List or Mass Profiler Professional software

10. Slide 10: Aportación de los sistemas de Tiempo de Vuelo

    Aportación de los sistemas de Tiempo de Vuelo El TOF es un cronómetro que mide el tiempo que tardan los diferentes iones en llegar al detector desde que se disparan en el PULSER. Los iones más ligeros llegan antes y los más pesados, más tarde. Ese tiempo se contrasta con una calibración del equipo t

11. Slide 11: HRMS TOF/QTOF

    HRMS TOF/QTOF TOF TOF Full Scan acquisition Tiapride 0.8 ppm QTOF Full Scan acquisition QTOF MS/MS spectrum Tiapride 0.8 ppm

12. Slide 12: HRMS TOF/QTOF – MS Scoring

    HRMS TOF/QTOF – MS SCORING Scoring based on Compounds Identification at MS level is based Monoisotopic mass (change in ppm) at least on three parameters : • monoisotopic mass accuracy • isotopes rel. abundance • isotopic spacing Isotopopic Distribution (Change in %) Matched with theoretical distribu

13. Slide 13: Is there a way to avoid the limitations of MS/MS?

    Is there a way to avoid the limitations of MS/MS ? Acquisition of all ions all the time using new All Ions MS/MS All Ions MS/MS  CID fragmentation without precursor selection Simultaneous acquisition of a low energy trace and one or more high energy traces (CID) AccMass_Screening_Pesticides 08/04/2

14. Slide 14: The Implementation of All Ions MS/MS in LC/MS

    The Implementation of All Ions MS/MS in LC/MS Acquisition with low & high collision energy channels Acquisition - Special method required - Chromatography is monitored in 2 channels (or more if desired) Low energy channel (CE = 0V) - Spectrum dominated by molecular ions and clusters Fragments High e

15. Slide 15: Example for all ions spectra

    Example for all ions spectra Carbofuran in multi-residue solvent standard Low energy spectrum AccMass_Screening_Pesticides 08/04/2015 16

16. Slide 16: Example for all ions spectra

    Example for all ions spectra Carbofuran in multi-residue solvent standard Low energy spectrum High energy spectrum AccMass_Screening_Pesticides 08/04/2015 17

17. Slide 17: Example for all ions spectra

    Example for all ions spectra Carbofuran in multi-residue solvent standard Low energy spectrum High energy spectrum AccMass_Screening_Pesticides 08/04/2015 18

18. Slide 18: Screening workflow using LC-(Q)TOF-MS

    Screening workflow using LC-(Q)TOF-MS Personal compound database and library AccMass_Screening_Pesticides 08/04/2015 19

19. Slide 19: Accurate Mass LC/MS Application Kits

    Accurate Mass LC/MS Application Kits Untargeted Screening with TOF or QTOF Pesticides Method -Test Mix: 253 compounds -DB: 1680+ compounds -Library: 750+ compounds Accurate Mass Veterinary Drugs Standards MS/MS -Test Mix: 146 compounds Library LC/MS -DB: 1150+ compounds Application -Library: 850+ co

20. Slide 20: All Ions MS/MS Features: Targeted Identification Using Agilent's PCDLs

    All Ions MS/MS Features: Targeted Identification Using Agilent’s PCDLs Personal Compound Database and Libraries (PCDLs) contain library searchable MS/MS spectra. All Ions MS/MS uses the product ions in the spectra to confirm the identification of hits in the screen—the result is fewer false positive

21. Slide 21: Qualitative screening using All Ions MS/MS

    Qualitative screening using All Ions MS/MS What is new about All Ions MS/MS ? • All Ions MS/MS offers a complete workflow for screening, verification and setup of a quantitation method • It features: - Support of TOF as well as QTOF - Easy setup of the acquisition method - Unique identification of p

22. Slide 22: Workflow of All Ions MS/MS acquisition

    Workflow of All Ions MS/MS acquisition Experimental setup of validation study LC-QTOF Data analysis All Ions MS/MS using acquisition „Find-by-Formula“ List of Suspects Yes Verified by Quant with verification by fragment coelution accurate mass metrics profile and qualifier fragments No AccMass_Scree

23. Slide 23: Experimental

    Experimental UHPLC and QTOF method parameters Agilent ZORBAX Eclipse Plus C18 RRHD UHPLC column 2.1 x 150 mm, 1.8 µm @ 30°C A: 5 mM NH4 formate + 0.1% formic acid Mobile phase B: 5 mM NH4 formate + 0.1% formic acid in methanol Min %B 0 5 0.2 5 2.2 30 Gradient program 10.5 100 13.0 100 13.5 5 Stop ti

24. Slide 24: All Ions MS/MS Targeted Screening Workflow

    All Ions MS/MS Targeted Screening Workflow Acquisition PCDL Qual Quant MassHunter Acquisition m/z, ion species Find by Formula on MS low channel Pesticide library (~450- Targets 500 compounds with spectra) High/Low Forensics Fragmentor /Tox library Voltage on TOF (2720 compounds and Q-TOF with spect

25. Slide 25: Qualitative screening using All Ions MS/MS

    Qualitative screening using All Ions MS/MS All Ions MS/MS workflow • Modified “Find by Formula Algorithm” searches compound based on database entry in the low energy domain AccMass_Screening_Pesticides 08/04/2015 26

26. Slide 26: All Ions MS/MS Targeted Screening Workflow

    All Ions MS/MS Targeted Screening Workflow Acquisition PCDL Qual Quant MassHunter Acquisition m/z, ion species Find by Formula on MS low channel Pesticide Find library (~450- Targets 500 EICs for compounds fragments on MS with spectra) high channel Extract High/Low Forensics Align EICs of Fragmentor

27. Slide 27: Qualitative screening using All Ions MS/MS

    Qualitative screening using All Ions MS/MS All Ions MS/MS workflow • Modified “Find by Formula Algorithm” searches compound based on database entry in the low energy domain • Uses PCDL spectra as source of fragments to look for in the high energy domain AccMass_Screening_Pesticides 08/04/2015 28

28. Slide 28: Qualitative screening using All Ions MS/MS

    Qualitative screening using All Ions MS/MS All Ions MS/MS workflow • Modified “Find by Formula Algorithm” searches compound based on database entry in the low energy domain • Uses PCDL spectra as source of fragments to look for in the high energy domain and compares elution profiles AccMass_Screenin

29. Slide 29: All Ions MS/MS Targeted Screening Workflow

    All Ions MS/MS Targeted Screening Workflow Acquisition PCDL Qual Quant MassHunter Acquisition m/z, ion species Find by Formula on MS low channel Pesticide Find library (~450- Targets 500 EICs for compounds fragments on MS with spectra) high channel Extract High/Low Forensics Align EICs of Fragmentor

30. Slide 30: Qualitative screening using All Ions MS/MS: Results overview

    Qualitative screening using All Ions MS/MS Results overview • Carbendazim was found with 5 valid qualifier fragments from the PCDL spectrum • Overlay of the EICs with the Coelution Plot shows excellent agreement between the products and precursor ion AccMass_Screening_Pesticides 08/04/2015 31

31. Slide 31: Qualitative screening using All Ions MS/MS: Results overview

    Qualitative screening using All Ions MS/MS Results overview AccMass_Screening_Pesticides 08/04/2015 32

32. Slide 32: Pesticides screening with accurate mass

    Pesticides screening with accurate mass Validation data for 3 different matrices Tomato Lemon Avocado Compound Blank 0.005 0.01 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Acetamiprid q u a life d q u a life d q u a life d q u a life d q u a life d q u a life d Aldicarb q u a life d q u a life d q

33. Slide 33: All Ions MS/MS Targeted Screening Workflow

    All Ions MS/MS Targeted Screening Workflow Acquisition PCDL Qual Quant MassHunter Acquisition m/z, ion species Find by Formula on MS low channel Pesticide Find library (~450- Targets 500 EICs for compounds fragments on MS with spectra) high channel Extract High/Low Forensics Align EICs of Fragmentor

34. Slide 34: Batch review for All Ions MS/MS results

    Batch review for All Ions MS/MS results Method setup from screening results AccMass_Screening_Pesticides 08/04/2015 35

35. Slide 35: Batch review for All Ions MS/MS results

    Batch review for All Ions MS/MS results Method setup from screening results Fragment ions with different CEs are used as qualifiers. AccMass_Screening_Pesticides 08/04/2015 36

36. Slide 36: All Ions MS/MS Targeted Screening Workflow

    All Ions MS/MS Targeted Screening Workflow Acquisition PCDL Qual Quant MassHunter Acquisition m/z, ion species Find by Formula on MS low channel Pesticide Find library (~450- Targets 500 EICs for compounds fragments on MS with spectra) high channel Quantify target with Extract additional fragment io

37. Slide 37: Quantitation of pesticides using accurate mass

    Quantitation of pesticides using accurate mass Batch review process including accurate mass metrics AccMass_Screening_Pesticides 08/04/2015 38

38. Slide 38: Quantitation of pesticides using accurate mass

    Quantitation of pesticides using accurate mass Batch review process using Compounds at a Glance Cal sample AccMass_Screening_Pesticides 08/04/2015 39

39. Slide 39: Pesticides screening and quantitation: Pirimicarb

    Pesticides screening and quantitation Apple sample from local market Pirimicarb Conc.: 120 µg/kg Mass error: -0.54 ppm TOF score: 99.2 Library score: 97.0 AccMass_Screening_Pesticides 08/04/2015 40

40. Slide 40: Pesticides screening and quantitation: Boscalid

    Pesticides screening and quantitation Apple sample from local market Boscalid Conc.: 20 µg/kg Mass error: -0.15 ppm TOF score: 97.5 Library score: 93.0 O NH Cl N Cl AccMass_Screening_Pesticides 08/04/2015 41

41. Slide 41: Pesticides screening and quantitation: Trifloxystrobin

    Pesticides screening and quantitation Apple sample from local market Trifloxystrobin Conc.: 10 µg/kg Mass error: -1.00 ppm TOF score: 99.3 Library score: 87.8 CH3 F F O N F O O H3C N CH3 O AccMass_Screening_Pesticides 08/04/2015 42

42. Slide 42: Fast and Fully Automated Multi-Residue Pesticide Screening using a GC-Q/TOF

    Fast and Fully Automated Multi-Residue Pesticide Screening in Fruit / Vegetable Extracts using a GC-Q/TOF CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

43. Slide 43: The Agilent 7200 Series GC-Q/TOF

    The Agilent 7200 Series GC-Q/TOF Triple Quadrupole MS + = Time of Flight MS Quadrupole Time of Flight MS 7890 + 7000 + 6500 = 7200 GC-Q/TOF CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

44. Slide 44: 7200 Q/TOF Components

    7200 Q/TOF Components NEW NEW Optics Removable 6500 Q/TOF Ion Source 7000B TQ Four stages of pumping Two 300 L/s Turbos CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

45. Slide 45: 7200 GC/Q-TOF Performance Specifications

    7200 GC/Q-TOF Performance Specifications • Resolving Power: > 12.5K at m/z 272 – 13K to 15K typical • Mass Accuracy: < 5 ppm at m/z 272 – < 2 ppm typical • MS Sensitivity >400:1 at m/z=272 • MS/MS Sensitivity is between SRM and Product Ion scan of a Tandem Quad MS • Dynamic Range: 3-5 orders of magn

46. Slide 46: The Implementation of All Ions MS/MS in LC/MS

    The Implementation of All Ions MS/MS in LC/MS Acquisition with low & high collision energy channels Acquisition – Special method required – Chromatography is monitored in 2 channels (or more if desired) Low energy channel (CE = 0V) – Spectrum dominated by molecular ions and clusters High energy chan

47. Slide 47: Screening Pesticides LC-Q/TOF vs GC-Q/TOF

    Screening Pesticides LC-Q/TOF vs GC-Q/TOF GC/MS EI+ LC/MS ESI+ CE = OV Low energy [M+H]+ CE = OV Malathion MWt = 330.0361 High energy Fragments CE > OV Spectrum dominated by [M]+ ion is the exception rather than the rule CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

48. Slide 48: Screening Pesticides with a GC-Q/TOF

    Screening Pesticides with a GC-Q/TOF • Accurate Mass provides added compound selectivity - Higher resolution provides added interference selectivity • TOF mode : Always full spectral data, Non-targeted acquisition • ‘Unlimited’ number of compounds can be screened (search accurate mass compound datab

49. Slide 49: Screening Pesticides with a GC-Q/TOF Objectives

    Screening Pesticides with a GC-Q/TOF Objectives • Similar functionality, look and feel as All Ions for LC/MS - Adapt MH Qual All Ions to process GC/MS EI mass spectra • Customizable UI, fast data processing, filtering result data, export found compounds for quantitation in MH Quant SW • Enable user

50. Slide 50: All Ions: Targeted Screening Workflow

    All Ions : Targeted Screening Workflow 1. Acquisition 2. PCDL 3. MH Qual SW 4. MH Quant SW GC-Q/TOF MH Acquisition m/z, ion species Find by Formula on MS Find Targets Compound library (compounds with EICs for fragments spectra and Quantify target with RTs) additional fragment ion Extract confirmatio

51. Slide 51: 7200 GC-Q/TOF RTL Pesticide Analyzer CF Method

    7200 GC-Q/TOF RTL Pesticide Analyzer CF Method 1. Acquisition Column (1) : 15m x 0.25mm ID x 0.25um HP-5MSUI (19091S-431UI) Column (2) : 15m x 0.25mm ID x 0.25um HP-5MSUI (19091S-431UI) ALS Same column configuration and flow rate for 40 minute and 20 minute methods, only difference is Oven Temperatu

52. Slide 52: PCDL: Personal Compound Database Library

    PCDL : Personal Compound Database Library 1. Acquisition 2. PCDL Accurate mass and Retention time Database + EI Accurate Mass Spectra • Name • Mol. Formula • Mono-isotopic exact mass • Locked RT • CAS # • Accurate mass spectrum CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2

53. Slide 53: User created GC/MS Accurate Mass PCDLs

    User created GC/MS Accurate Mass PCDLs 1. Run standard (RTL Method) 2. Extract compound spectra (Find by integration or by deconvolution) 3. Add compound information 4. Send spectra to PCDL PCDL Manager - view / edit entries CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

54. Slide 54: User Created PCDLs

    User Created PCDLs Find compounds by integration or by deconvolution Optional search of unit mass MS database CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

55. Slide 55: Steps to transfer the spectra to PCDL

    Steps to transfer the spectra to PCDL 1. Select Spectra 2. Right Mouse click 3. Specify PCDL 4. Press OK PCDL Manager - view / edit entries CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

56. Slide 56: Qualitative screening using All Ions MS

    Qualitative screening using All Ions MS 1. Acquisition 2. PCDL 3. MH Qual SW Gun Powder Tea CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

57. Slide 57: All Ions data processing - Method setup (1)

    All Ions data processing - Method setup (1) Formula Source • Specify PCDL • Use locked Retention time (If available) CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

58. Slide 58: All Ions data processing - Method setup (2)

    All Ions data processing - Method setup (2) Formula matching • Set acceptable mass tolerance for identifying molecular ion • If molecular ion found measure : Mass accuracy Isotope ratios Isotope spacing • Set mass window for fragment EICs CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-

59. Slide 59: All Ions data processing - Method setup (3)

    All Ions data processing - Method setup (3) Fragment Identification • Select number of fragment ions to be evaluated • Select minimum number of qualified fragments required to identify compound • Co-elution score is the major function to qualify compounds based on similarity of extracted ion peak sh

60. Slide 60: All Ions Results Overview GC-Q/TOF Tea extract

    All Ions Results Overview GC-Q/TOF Tea extract CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

61. Slide 61: All Ions Results Overview GC-Q/TOF Tea extract

    All Ions Results Overview GC-Q/TOF Tea extract 1 2 Target Compound List and Results Compound Identification Results 3 4 Pesticide screening GC-Q/TOF EICs and Co-Elution Plots Compound MS Spectrum Results 5 Compound Fragment Spectrum Results CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF G

62. Slide 62: Window 1 Target Compound list and Results

    Window 1 Target Compound list and Results CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

63. Slide 63: Window 2 Compound identification results

    Window 2 Compound identification results • RT difference • Mass accuracy of [M]+ (if present) • EIC Co-elution Scores based on : − Retention time − Peak shape (symmetry) − Peak width − Abundance CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

64. Slide 64: Window 3 Compound Chromatogram Results and EIC Co-Elution Plots

    Window 3 Compound Chromatogram Results and EIC Co-Elution Plots Overlayed EICs Co-elution plot CSEMP Pesticide Pesticide screening screening Contaminants in mussel GC-Q/TOF GC-Q/TOF GC-QQQ March 2011

65. Slide 65: Window 4 Compound MS Spectrum results

    Window 4 Compound MS Spectrum results • If molecular ion is detected, otherwise window is blank Observed isotope pattern for molecular ion cluster + Theoretical overlay from FBF spectrum Observed CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

66. Slide 66: Window 5 Compound Fragment Spectrum results

    Window 5 Compound Fragment Spectrum results Averaged mass spectrum across peak Qualifier fragments CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

67. Slide 67: Import and build MH Quant Method from MH Qual

    1. Acquisition 2. PCDL 3. MH Qual SW 4. MH Quant SW Import and build MH Quant Method from MH Qual Qualified targets MH Qual .cef file Quant method - Accurate mass EICs for quantifier Compound results and qualifier Counts x10 6 746.4838 (M+H)+ 5.5 5 4.5 - Relative responses 4 3.5 MH Quant 3 747.4871

68. Slide 68: All Ions Summary

    All Ions Summary • Continuing trend towards comprehensive screening methods using accurate mass instruments • Innovative data mining tools for identification of trace contaminants in complex matrices • All Ions integrated workflow for enhanced qualification of suspected compounds by additional fragm

69. Slide 69: Acknowledgements

    Acknowledgements Klaus Wilmers, Thorsten Bernsmann, Peter Fürst CVUA-MEL, Munster, Germany Noelia Belmonte, Milagros Mezcua, Amadeo R. Fernández-Alba EURL for Pesticides in Fruits and Vegetables Almeria, Spain CSEMP Pesticide screening Contaminants in mussel GC-Q/TOF GC-QQQ March 2011

70. Slide 70: Qualitative Screening using All Ions LC-MS/MS

    Qualitative Screening using All Ions LC-MS/MS Acquisition • Easy method setup • All Ions is faster than MS/MS • Chromatography is monitored in 2 channels (or more if desired) – A low energy channel ensures optimum molecular ion generation – A high enery channel produces fragments • support of TOF (f

71. Slide 71: Novel Ion Mobility Technology for LC/MS

    Novel Ion Mobility Technology for LC/MS Jaume C. Morales MS Product Specialist 08 de abril de 2015 73

72. Slide 72: Ion Mobility – A Brief History

    Ion Mobility – A Brief History… 2013 Agilent IMS QTOF 2006 Synapt Triwave 1997 G2 in 2009 Applications to G2S in 2011 Mass Spectrograph clusters & 1969 biomolecules Aston & Thomson 1919 Transport of Ions in Clemmer & Jarrold Gases 1905 McDaniel & Ion mobility Mason theory Paul Langevin 1872 - 1946 0

73. Slide 73: Mass Accuracy Does Not Equal Compound Identification

    Mass Accuracy Does Not Equal Compound Identification: Seven Golden Rules - Oliver Fiehn Number of Database Entries (Assuming Zero Mass Error) Empirical formula is not unique above mass m/z 100 (searching PubChem) Number of formula: ChemSpider mass search at m/z 400.3787 • 1 ppm mass error → 1742 ent

74. Slide 74: What Does Ion Mobility Bring to Mass Spectrometry?

    What Does Ion Mobility Bring to Mass Spectrometry? Separation • Ion Mobility resolves of many isomeric analytes otherwise impossible to determine by mass spectrometry alone. Improves Detection Limits • Ion Mobility dramatically reduces interference from other analytes and background. Confirmation •

75. Slide 75: Resolution Is Important!

    Resolution Is Important! Chromatographic Ion Mobility Mass ~seconds ~60 milli-seconds ~ 100 m seconds 08 de abril de 2015 77

76. Slide 76: It's All About Separation

    It’s All About Separation Chromatography Ion Mobility Mass ~seconds ~60 milli-seconds ~100 m seconds 08 de abril de 2015 78

77. Slide 77: Basic Operational Principle of Ion Mobility

    Basic Operational Principle of Ion Mobility 𝑒𝐸 For Conventional DC Uniform Field IMS 𝑣=𝐾𝐸 ∝ 𝑃 𝑇Ω VH Ion Mobility Cell VL Analyte Ions Detector Gating Optics Electric Field Stacked ring ion guide gives linear field t0 tdrift 08 de abril de 2015 79

78. Slide 78: Resolving Stereoisomers

    Resolving Stereoisomers α-glucose β-glucose Ion mobility enables separation of glucose stereoisomers

79. Slide 79: IM-QTOF Instrument Overview

    IM-QTOF Instrument Overview Novel Aspects Achieve New Performance Capabilities DC only drift tube to achieve lowest ion temperatures System sensitivity optimized using electrodynamic ion funnels Designed for both high Ion Mobility and Mass Resolution Ion Fragmentation can be selected using standard

80. Slide 80: IM-MS Operational Modes

    IM-MS Operational Modes • QTOF Only Mode • Mobility Separated Precursor Ion Mode • Mobility Separated All Ions Fragmentation Mode • Mobility Separated Targeted MS/MS Mode Mobility Fragmentation separation ON/OFF m/z selection ON/OFF 83 IM-QTOF workshop 01/28/14

81. Slide 81: New Agilent MassHunter IM-MS Browser

    New Agilent MassHunter IM-MS Browser •• Frame Navigation tool Frame viewer Visualizing Ion Mobility LC/MS Data • Heat map IMS/MS Frame Chromatogram View Selection Software IM Drift TIme Solutions for Improving your Productivity MS 1221.9906 1521.9711 1821.9521 2121.9332 922.0098 2421.9138 622.0294 2

82. Slide 82: New Agilent MassHunter IM-MS Browser

    New Agilent MassHunter IM-MS Browser Visualizing Ion Mobility LC/MS Data Ion mobility Frame Viewer provides linked views for easier navigation HEAT MAP 2721.8941 2722.8974 2723.9003 08 de abril de 2015 88

83. Slide 83: IMS QTOF - Overview

    IMS QTOF - Overview Applications - Software tools -Ω - Lipids - Carbohydrates ASMS 2013 Ion Mobility Abstracts 08 de abril de 2015 89

84. Slide 84: Published Collisional Cross Sections

    Published Collisional Cross Sections CCS CCS % CCS Mass Analyte Literature This Work Deviation New Analyte Ion IMS QTOF [Da] [Å2] [Å2] from Lit. [Å2] Colchicine1 399.4 196.2 196.2 ± 0.54 Å2 0% 5α-dihydrotestosterone (M+H)+ 181.6 ± 0. 0.6% 5α-dihydrotestosterone (M+Na)+ 201.5±1.0 Odansetron2 293.4 17

85. Slide 85: Collision Cross Section Correlates with Structure

    Collision Cross Section Correlates with Structure 91 IM-QTOF workshop 01/28/14

86. Slide 86: Structural Analysis of Ubiquitin Protein

    Structural Analysis of Ubiquitin Protein drift time (ms) [M+8H]8+ [M+7H]7+ [M+11H]11+ [M+6H]6+ [M+5H]5+ [M+9H]9+ [M+13H]13+ [M+10H]10+ [M+12H]12+ [M+14H]14+ 11+ 10+ 12+ 9+ 13+ 8+ 7+ 14+ 6+ 5+ m/z Structural studies of proteins possible with CCS calculation 92 IM-QTOF workshop 01/28/14

87. Slide 87: Cross Section Calculation of Ubiquitin Charge States

    Cross Section Calculation of Ubiquitin Charge States Ion Charge State CCS CCS experimental (Å2) literature (Å2) [M+5H]5+ 5 1196 [M+6H]6+ 6 1431, 1658 [M+7H]7+ 7 1755, 1886 1910 [M+8H]8+ 8 1966 1990 [M+9H]9+ 9 2008 2090 [M+10H]10+ 10 2114, 2197 2200 [M+11H]11+ 11 2239, 2348 2340 [M+12H]12+ 12 2412, 2

88. Slide 88: Reveal Greater Detail All Ions: Ondansetron, Colchicine, Reserpine

    Reveal Greater Detail All Ions: Ondansetron, Colchicine, Reserpine IM Reserpine Drift TIme Colchicine Ondansetron 400.1749 MS 609.2800 294.1597

89. Slide 89: Reveal Greater Detail All Ions: Ondansetron

    Reveal Greater Detail All Ions: Ondansetron 294.1597 170.0960 212.1064 184.1117 June 2013 95

90. Slide 90: Collision Cross Section Benchmark

    Collision Cross Section Benchmark Vanderbilt University • Tetraalkylammonium salts (TAA) • Proposed as an “ideal” ion mobility standard • Wide CCS range (TAA-4 to TAA-18; 100 to 400 Å2) TAA-5 • TAA salts do not form clusters N-(CH CH CH CH CH ) 2 2 2 2 • Literature CCS values exist N2 drift gas 3 4

91. Slide 91: Conformational Space Occupancy of Biomolecules: Class Association

    Conformational Space Occupancy of Biomolecules: Class Association by Trend Curves Size Shape Drift tube IMS allows Charge Class association Using a Synapt does NOT allow compound class association

92. Slide 92: Conformational Space Occupancy of Biomolecules

    Conformational Space Occupancy of Biomolecules lipids Hypothetical Ordering of Biomolecular Classes peptides Collision Cross Section (Å2) carbohydrates oligonucleotides Mass (Da) 08 de abril de 2015 98

93. Slide 93: Lipid nomenclature

    Lipid nomenclature Trivial nomenclature Palmitoleic acid Trivial names (or common names) are non-systematic historical names. Systematic names (or IUPAC names) derive from the standard IUPAC Rules for the Nomenclature of Organic Chemistry, published in 1979,[1] along with a Systematic (9Z)-octadecen

94. Slide 94: Lipid classes

    Lipid classes Main classes Examples of Glycerophospholipids Fatty acids Glycerolipids Glycerophospholipids Sphingolipids Sterol lipids Prenol lipids Saccharolipids Polyketides Source: Wikipedia 08 de abril de 2015 100

95. Slide 95: Cerebrosides

    Cerebrosides Cerebrosides are glycosphingolipids called monoglycosylceramides which are important components in animal muscle and nerve cell membranes. 08 de abril de 2015 101

96. Slide 96: Diseases Based on Sphingolipids

    Diseases Based on Sphingolipids Disease Deficient enzyme Accumulated products Niemann-Pick disease Sphingomyelinase Sphingomyelin in brain and RBCs Fabry disease α-galactosidase A Glycolipids in brain, heart, kidney Krabbe disease Galactocerebrosidase Glycolipids in oligodendrocytes Glucocerebroside

97. Slide 97: Lipid Analysis

    Lipid Analysis 70 60 PE oligomers (+1) Ion Mobility Drift Time (ms) 50 PE oligomers (+2) PE 41:N PE 64:N PE 39:N PE 62:N 40 PE 37:N PE 60:N PE 35:N PE 33:N 30 PE 23:N PE 21:N PE 19:N 20 10 L-α-phosphotidylethanolamines (PE) 0 0 500 1000 1500 2000 Mass (Da) 08 de abril de 2015 103

98. Slide 98: Lipid Analysis

    Lipid Analysis 70 60 PE oligomers (+1) Ion Mobility Drift Time (ms) 50 PE oligomers (+2) PE 41:N PE 64:N PE 39:N PE 62:N 40 PE 37:N PE 60:N PE 35:N PE 33:N 30 PE 33:(4-2) PE 35:(6-2) PE 37:(8-4) PE 39:(10-6) PE 23:N PE 21:N PE 19:N 20 +Na +K 10 740 750 760 770 780 790 800 810 820 Mass (Da) 0 0 500 1

99. Slide 99: Summary

    Summary Separation Compound Class CCS (Ω) Isomers Chromatography Natural Products Direct determination Small molecules Ion Mobility Finding outliers Excellent Precision Stereoisomers Size & Charge Shape & Biological Mass Resolution Accuracy vs. Literature Structural Isomers Activity Peak Capacity (~

100. Slide 100: ¿Preguntas?

     ¿Preguntas? New Screening strategies with Exact Mass QTOF-MS LC & GC AccMass_Screening_Pesticides 08/04/2015 106

101. Slide 101: jaume_morales@agilent.com

     jaume_morales@agilent.com AccMass_Screening_Pesticides 08/04/2015 107

Agilent Technologies — Technical Presentation (April 2015) on 6ix