Agilent s LC/MS Portfolio and Applications Examples

Transcription

1 Agilent s LC/MS Portfolio and Applications Examples Michael Scott LC/MS Product Specialist Agilent Technologies March 2013 For Research Use Only. Not for use in diagnostic procedures.

2 Agilent 6000 Series LCMS

3 Ultra High Definition LC/MS The Most Comprehensive Answer To Your Urgent Analytical Question Signal Response Sensitivity Dynamic Range Linearity Peak Resolving Power Peak Capacity Separation Speed Mass Spectrum Ionization Profile Mass Accuracy Isotopic Fidelity Mass Resolving Power Acquisition Rate

4 a few words about chromatography LC - MS

5 Agilent High Performance Infinity Series LC: Best in class Front End systems for MS Preparative LC Standard LC Ultra High Pressure LC Capillary & Nano LC High Sensitivity HPLC-Chip/MS The industry s most comprehensive LC portfolio to configure an integrated LC/MS system

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7 Agilent LC-MS column configurations: Column Type Column I.D. Nano Capillary MicroBore 0.075, 0.10 mm 0.3, 0.5 mm 1.0 mm Column Lengths Particle Sizes 50, 150 mm 3.5 um 35, 150, 250 mm 30, 50, 150 mm NarrowBore 2.1 mm mm 1.8, 3.5, 5 um Chip LC Nano LC Capillary LC Solvent Saver 3.0 mm mm 3.5, 5 um Analytical 4.6 mm mm 1.8, 3.5, 5um plus Prep columns 9.4, 21.2, 30, and 50 mm id Flow Rate Range nl/min 3.5, 5 um 1 10 ul/min 3.5, 5 um ul/min Analytical LC ml/min ml/min ml/min Prep LC

8 Agilent s > 40 year heritage in mass spectrometry 1971, 5930A GC/MS , 5975A 2008, 7000A ICP-MS , 4500 Series 1976, 5992A A 2000, 7500 Series QQQ LC/MS Direct Liquid Injection 1980, 5985 SQ ESI on 5989 MS Engine 1994, LC/QQQ Thermospray, 5987 Particle Beam, 5988 LC/MSD TOF 2006 LC/MSD, LC/MSD Ion Trap LC/QTOF Over 37,000 GC/MS, 4,500 ICP-MS and 6,500 LC/MS systems through June 2012 Agilent (and previously, as HP) has sold more quadrupole MS systems than any other mfr.

9 A big leap forward in 2006 Rough Pump Quad Mass Filter (Q1) Octopole 1 Turbo 1 Lens 1 and 2 Turbo Series SQ 10KV Detector Turbo 1 Collision cell incorporates axial acceleration for high speed MS/MS analysis 6500 Series Q-TOF 6400 Series QQQ Quad Mass Filter (Q1) Octopole 1 Quad Mass Filter (Q3) Lens 1 and 2 Collision Cell 10KV Detector Rough Over 15 Years in LC/MS Pump atmospheric sampling and patented orthogonal geometry - Result in industry leading sensitivity and robustness Turbo 1 Turbo 1 Rough Pump Turbo 1 Quad Mass Filter (Q1) Octopole 1 Turbo 1 Lens 1 and 2 Turbo 1 Collision Cell Turbo 1 Octopole 2 DC Quad Ion Pulser Turbo 2

10 Agilent offers a range of LC/MS systems Single quad Triple quad Ion trap TOF QTOF Page 11 Introduction to the Agilent LC/MS Portfolio Agilent Restricted 3/26/2013

11 Agilent 6000 Series LC/MS portfolio in series UHPLC 6500 series Q-TOF 1 Model 3 Models 6200 series TOF Comprehensive Range of Software 3 Models 6400 series QQQ 4 Models 6100 series Quad

12 Agilent 6000 Series LC/MS portfolio High Resolution Qualitative Single Stage 6200 series TOF 6500 series Q-TOF Tandem Stage Quantitative 6100 series Quad Low Resolution 6400 series QQQ

13 Agilent MassHunter Workstation Software Instrument Control Real-time monitoring Method set-up Autotune Qualitative Analysis Chromatographic results Spectral results Find compounds Quantitative Analysis User filters Compound results Calibration curve

14 Mass Hunter Extensive application support BioConfirm Confirm identities and identify variants of proteins Metabolite ID Identify metabolites Mass Profiler METLIN Personal Metabolite Database Mass Profiler Professional Used for expression profiling Most comprehensive database made available on PC Chemometrics software to visualize large sample sets Analytical Studio Reviewer Fast analysis of large amounts of LC/MS data Easy Access Walk-up operation for instruments Spectrum Mill Identify proteins and peptides by fast database searches

15 Full integration: the lab, business applications, OpenLAB, the company network The Lab Business Applications GC/MS GC MS Office Legacy Data UV-vis LC PDF ELN Images ICP-MS Lab-on-a Chip CE Masshunter Workstation LIMS Research Drawings Agilent OpenLAB (Laboratory Informatics) Intranet Internet

16 LC/MS Ion sources Wide range of sampling conditions Sources are interchangeable between mass spectrometer platforms Multimode HPLC-Chip Multimode source Flow rates up to 2 ml/min Simultaneous operation APCI ESI NanoESI HPLC-Chip/MS Nanoelectrospray without the fuss Reproducible response and retention times On-chip enrichment for speed and sensitivity APPI AP-MALDI

17 6100 Series Single Quad

18 Quadrupole Operation Principles Each m/z value has a unique DC and RF potential that will allow that ion to be passed through the quadrupole analyzer

19 6100 Series Single Quadrupole LCMS Easiest Flexible Performance

20 6100 Series Single Quadrupole LCMS

21 Agilent Jet Stream Technology Page 22

22 Agilent Jet Stream Enter slide show mode to see the animation on this page. Page 23 Agilent Restricted 23 June 2008, Denver

23 Agilent Jet Stream Ion Generation Gas Dynamics View Nebulizing gas Enhanced efficiency nebulizer Super-heated sheath gas Agilent Jet Stream Parameters Sheath Gas Flow Sheath Gas Temperature Nozzle Voltage Nebulizing Pressure Chamber Voltage Nozzle voltage Heated drying gas The super-heated sheath gas collimates the nebulizer spray and creates a dramatically brighter source Patent Pending Resistive sampling capillary Page 24

24 Heating the Sheath Gas in Jet Stream, time lapse Start Temperature 25 o C at t = 0 min Page 25

25 Narrower Spray Beam 2R Stop Temperature 400 o C at t = 8 min Page 26

26 Herbicides in Water Anglian Water Laboratories, Huntingdon, UK ppb For clean matrix, LC/MSD (single quad) is a very useful and inexpensive tool for screening and quantitation)

27 Phthalates in Children s Toys

28 6400 Series Triple quadrupole

29 Why a Triple Quad? Ancient proverb: On a dark night you can see more stars 31

30 Why a Triple Quad?

31 Multiple reaction monitoring (MRM) Quad Mass Filter (Q1) Quad Mass Filter (Q3) Collision Cell Spectrum with background ions (from ESI) Q1 lets only target ion 210 pass through 210 Collision cell breaks ion 210 apart Q3 monitors only characteristic fragments from ion 210 for quant and qual no chemical background

32 Eight min Dynamic MRM Analysis 6460 QQQ Pesticide Screen. 500 ppt, dynamic MRM Retention Time Window = 12 sec Peak widths ~ 1 second Zorbax Eclipse Plus C x 100 mm (1.8 μm)

33 ASMS 2010 Agilent Introduces ifunnel Technology

34 N EW I F UNNEL TECHNOLOGY ifunnel Technology captures 6x more ions Agilent Jet Stream Thermal confinement of ESI plume Efficient desolvation to create gas phase ions Creates an ion rich zone Hexabore Capillary 6 capillary inlets Samples 6X more ion rich gas from the source Captures the majority of the gas from the source region Dual Ion Funnel Removes the gas but captures the ions Removes neutral noise Extends turbo pump life

35 Hexabore atmospheric sampling. 6 Bore Capillary Six bores, Half the restriction means 6 times the amount of atmospheric gas sampled AND 5 10X the number of ions sampled over wide mass range. But how do we handle all the extra gas molecules? Monolith With 6 Bores

36 Ion Funnel Operation RF Voltage focuses the ions to the center. RF Drive Unfocused Ions and Gas Enter Focused Ions Out DC Voltage accelerates the ions to the exit. DC Drive 38

37 Two-stage Ion Funnel can handle the gas load Stage 1 offset deflects the high quantity of gas exiting the hexabore capillary Line of Sight Stage Torr Stage Torr

38 New 6490 advances in sensitivity 50 fold in 4 years SNR 1 pg reserpine

39 6490/iFunnel: Zeptomolar detection limits 100 attograms Verapamil on-column (200 zeptomoles) + MRM ( > 164.9) X10 1 Response Blank Acquisition Time (min)

40 Unprecedented six logs of linearity with attogram to 100 picogram Verapmil on-column Y = x R 2 = Area Response Femtograms

41 Estradiol in Human Plasma

42 6400 Series Triple Quad

43 6200 Series TOF

44 Formulas for TOF performance Resolving power = (m/z)/w 1/2 1000/0.025 = 40, /0.022 = 41,909 Mass accuracy ( rel. mass error) actual - measured Mass accuracy 10 actual ( )/1000)x10 6 =1 ppm Therefore: m/z is 0.001Da (1 mda) W 1/2 = m/z, amu 6 In te n sity, co u n ts +TOF MS: to min from steroid2tofap42.wiff Agilent, subtracted (4.696 to e5 1.10e5 1.00e5 9.00e4 8.00e4 7.00e4 6.00e4 5.00e4 4.00e4 3.00e4 2.00e4 1.00e Epitestosterone Resolving power: 6890 actual meas ppm Max. 1.2e5 counts m/z, amu

45 NPAMOZ at unit mass resolution , XIC of +TOF MS: to amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff 6.8e4 Max. 2.0e5 cps e4 6.0e4 5.5e4 5.0e4 4.5e4 In te n sity, cp s 4.0e4 3.5e4 3.0e e4 2.0e4 1.5e4 1.0e Time, min

46 NPAMOZ /- 0.1 XIC of +TOF MS: to amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff 3.4e4 Max. 1.7e5 cps e4 3.0e4 2.8e4 2.6e4 S/N = 70:1 2.4e4 2.2e4 In te n sity, cp s 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e Time, min

47 NPAMOZ /- 100 ppm ( ) XIC of +TOF MS: to amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff Max. 9.3e4 cps S/N = 103: In te n sity, cp s Time, min

48 NPAMOZ /- 50 ppm ( ) XIC of +TOF MS: to amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff Max. 4.2e4 cps S/N = 156: In te n sity, cp s Time, min

49 NPAMOZ /- 20 ppm ( ) XIC of +TOF MS: to amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff Max. 1.0e4 cps. In te n sity, cp s S/N = 214: Time, min

50 LC/ESI-TOF MS, using accurate mass 2.4e e5 1.6e5 1.2e5 8.0e e m/z, amu

51 Table of possible chemical formulas for m/z (with < 2 ppm error) Formula Calculated m/z mda error error (ppm) DBE C10 H13 N2 F Cl C9 H13 N2 O F P Cl C5 H12 N5 F2 Cl C5 H16 N3 O F P2 Cl C3 H7 N10 O2 Cl C4 H13 N3 O7 Cl C4 H12 N5 O F2 P Cl C6 H16 N3 F P Cl C10 H17 O P2 Cl C13 H10 N O F Cl

52 Table of possible chemical formulas for m/z (with < 2 ppm error) Eliminate compounds with two Cl Formula Calculated m/z mda error error (ppm) DBE C10 H13 N2 F Cl C9 H13 N2 O F P Cl C5 H12 N5 F2 Cl C5 H16 N3 O F P2 Cl C3 H7 N10 O2 Cl C4 H13 N3 O7 Cl C4 H12 N5 O F2 P Cl C6 H16 N3 F P Cl C10 H17 O P2 Cl C13 H10 N O F Cl

53 Table of possible chemical formulas for m/z (with < 2 ppm error) Eliminate compounds with low C count Formula Calculated m/z mda error error (ppm) DBE C10 H13 N2 F Cl C9 H13 N2 O F P Cl C5 H12 N5 F2 Cl C5 H16 N3 O F P2 Cl C3 H7 N10 O2 Cl C4 H13 N3 O7 Cl C4 H12 N5 O F2 P Cl C6 H16 N3 F P Cl C10 H17 O P2 Cl C13 H10 N O F Cl

54 Table of possible chemical formulas for m/z (with < 2 ppm error) Apply the Nitrogen Rule Formula Calculated m/z mda error error (ppm) DBE C10 H13 N2 F Cl C9 H13 N2 O F P Cl C5 H12 N5 F2 Cl C5 H16 N3 O F P2 Cl C3 H7 N10 O2 Cl C4 H13 N3 O7 Cl C4 H12 N5 O F2 P Cl C6 H16 N3 F P Cl C10 H17 O P2 Cl C13 H10 N O F Cl > 1 compound

55 Molecular Formula Generator

56 Molecular Formula Generator

57 Formula Generation three criteria Scoring based on 1. Monoisotopic Mass 2. Isotope Spacing monoisotopic mass Isotope distribution C 10 H 9 ClN 4 O 2 S 3. Isotope Distribution Isotope spacing Page 59

58 Agilent 6230 TOF Pesticide Screening 224 pesticides, 50 pg on column 17 min. 3 min. Thiabendazole Peak Width=0.8 sec 23 data points 1.5 min ppm Spectral Resolution & Isotopic Fidelity

59 6500 Series QTOF design

60 QTOF 6500 Series MMA > 2ppm RP > 20K MS2 Speed 10 Hz

61 Ion Beam Compression (IBC) Technology Drives Higher Resolution Active Ion Beam Compression simultaneously maximizes ion transmission and reduces beam divergence Exit from collision cell Into slicer and time-of-flight pulser region Narrowed beam slits enables a mass resolving power of 40K Active Ion Beam Compression is achieved with Agilent s Axial Ion Acceleration Technology applied to a tapered Ion guide design.

62 Ultra High Definition QTOF 6540 UHD

63 6540 Ultra High Definition QTOF Mass Accuracy Repetitive Injections 40pg reserpine on-column, 10 injections +ESI EIC( ) Scan Frag=240.0V Reserpine_40pgms3.d Counts vs. Acquisition Time (min) Error Run (ppm) Mean 0.25 Std. Dev 0.32 x Isotope Obs % Calc % Obs m/z Calc m/z Diff (ppm) Counts vs. Mass-to-Charge (m/z) 250 ppb mass accuracy calibration and very accurate isotopic ratios

64 6540 Ultra High Definition QTOF Sensitivity Full Scan MS Mode- 1 picogram 4 x10 S/N = 319 RMS Excellent sensitivity, accuracy and resolution at 1 pg on column 3 x10 (M+H)+ 2 2 Resolution ~ 33, Ratio m/z Diff. Theor. Expt. Theor. Expt. (ppm) Counts vs. Acquisition Time (min) Counts vs. Mass-to-Charge (m/z)

65 6540 Ultra High Definition QTOF Wide Dynamic Range G2421A Tune Mix m/z sec spectrum Dynamic Range: 5,000, Peak height 5,000,000 6 x = 50,000 5 x x Counts vs. Mass-to-Charge (m/z) x Co Peak Height Counts vs. Mass-to-Charge (m/z) Counts vs. Mass - to - Charge ( m/z ) +ESI Scan (#27) Frag =180.0V 2GHz_accF.d

66 Dynamic Range Page 68

67 ifunnel Technology Revolutionizes Ion Sampling Proven10X Sensitivity Breakthrough For The 6490 QQQ Agilent Jet Stream Hexabore Capillary Dual Stage Ion Funnel 10X Sensitivity Breakthrough for 6550 QTOF

68 New 6550 ifunnel QTOF 10X Sensitivity Gain Enables Applications Sensitivity Dramatically improved quantitative capabilities New Qual/Quan Workflows Superior metabolite and protein detection Non-targeted compound screening Comprehensive Performance Enhancements Mass Resolution >40, spectra /sec MS and 33 spectra/sec MS/MS 5 orders of linear dynamic range <1 ppm MS mass accuracy; <2 ppm MS/MS Unrivalled sensitivity 6550 ifunnel Q-TOF LC/MS System ASMS 2010

69 Agilent QTOF Design Incorporates Cumulative Hardware Innovations Flight tube Common with TOF Collision cell Common with QQQ Ion optics Common with Q & QQQ Ion Mirror Octopole 1 Quad Mass Filter (Q1) Octopole 2 Detector Lens 1 and 2 Collision Cell DC Quad Ion Pulser Rough Pump Turbo Turbo Turbo Turbo TOF and QTOF Fundamentals Page 71 March 2007

70 Targeted MS/MS mode for Caffeine Calculate chemical formula given accurate mass measurement and using elements C, H, N, and O C 6 H 8 N 3 O ppm CH 3 N C 5 H 8 N ppm CH O C + N CH 3 N H C + N N N C 3 H 5 N ppm CH + N N CH 3 CH C 8 H 11 N 4 O ppm H 3 C O + N N H CH 3 O N N CH 3 Proposed structures

71 MS-MS Data Resolved

72 ID Browser: Identification of Statistically Differential Compounds

73 6500 Series QTOF Portfolio 6520 Femtomole sens ppm MMA >20K RP 10Hz MSMS 6540 UHD Femtomole sens ppb MMA >40K RP 20Hz MSMS 6550 ifunnel Attomole sens ppb MMA >40K RP 33Hz MSMS

74 Thank you!