Ultra High Definition LC/MS

Transcription

1 Ultra High Definition LC/MS Achieving the utmost information from today s complex analyses Dr. John Fjeldsted General Manager LC/MS Division Agilent Technology Santa Clara, CA

2 What is Ultra High Definition? Ultra High Definition is the combination of uncompromising performance in All Dimensions of the analytical measurement. Ultra High Definition LC/MS achieves the Maximum Qualitative and Quantitative Information possible. MassHunter Informatics TM Chromatography Mass Spectrometry Comprehensive Sample Information

3 Ultra High Definition Optimizing all Analytical Dimensions Sensitivity Dynamic Range Signal Response Linearity Peak Resolving Power Peak Capacity Separation Speed Mass Spectrum Ionization Profile Software Mass Accuracy Data Mining Isotopic Fidelity Differential Profiling Mass Resolving Power Annotation Acquisition Rate

4 Agilent 1290 Infinity LC Attributes for MS - Infinitely Better for LC/MS Lowest Delay Volume Pump (w/o) mixer: 10 µl Pump, Fixed Loop 20 µl Pump, Fixed Loop, JetWeaver 55 µl Highest Precision Best Autosampler Performance ALS precision for small volumes: <1.5% from 0.5-1µLm, <0.7% from 1-2µL, 2-20 µl (40 ul) ** Pump Active Damping: RT stability < 0.2 % (1.5 min runs)** <0.002% carry-over with Chlorhexidine Optional needle seat backflushing with FlexCube Fixed Loop or Variable Loop Injections Greatest Productivity 2mL/min for highest resolution per time Reduced Ion & Matrix Suppression HT-Solution for up to 2000 samples/day (ACR) Complete Integration and control from MassHunter Enables method conversion from/to any (U)HPLC

5 1290 Infinity Compatible with any HPLC and UHPLC bar A new power range providing maximum performance, flexibility, compatibility and investment protection Infinity 1000 Acquity 800 Dionex RSLC Thermo Accela Agilent RRLC Shimadzu UFLCXR Standard LC ml/min

6 1290 Infinity Binary Pump How it looks like inside Degasser Solvent selection valve Jet Weaver High Resolution Pump drives Multi-layer Heat exchanger Silicon Carbide Pistons Channel A Purge Valve Channel B December 15, 2009

7 1290 Infinity - Ultra High Performance Separations Maximizing Throughput Fast screening of 220 pesticides in 90 seconds Peak Width ~ 0.7 sec FWHM sec

8 1290 Infinity - Ultra High Performance Separations Maximizing High Peak Capacity mau Peak Capacity = 540 Peak Width ~ 2 sec FWHM min Peptide Map of Tryptic Digest of BSA run on Agilent RRHT Zorbax SB-C18, 2.1x150mm, 1.8µ

9 HPLC-Chip: Polymer Microfluidics developed in Agilent Labs µ-fluidic holes Laser ablation of polyimide film ZORBAX 300SB-C18, 5µm 75µm, 43 mm length Registration holes µ-filter Laser Ablated Cylindrical nanospray emitter Enrichment Column 40 nl ZORBAX 300SB C18 Electrical Interface to Internal Grounding Electrode Robust spray performance under all solvent conditions Polyimide nanospray emitter lifetime measured in weeks MS inlet orifice Counter electrode

10 Agilent s New 6540 Ultra High Definition QTOF Research Performance in a Benchtop Format 40,000 Resolving Power <1 ppm MS <2 ppm MS/MS Mass Accuracy 20 Spectra/s 2 pg 50:1 Reserpine S/N 5 Decades in Spectrum Dynamic Range Excellent Linearity and Isotopic Fidelity Supports Standard ESI, Agilent Jet Stream and HPLC-Chip Unsurpassed Analytical Capacity in a Benchtop The result of RELENTLESS INNOVATION

11 Theory of High Resolving Power Highest resolving power in a Benchtop R m FWHM peak 2 TOF t t 2 2 TA 2 PW 2 R TA Turn-around time term PW Detector pulse width term R Residual term Agilent TOF, Model s t TA, ns t PW, ns t R, ns t, ns Resolving Power , ,000 *Values are for m/z 2722

12 Enhanced Ion Flight Tube and Mirror Technology Stable, Sensitive, High Resolution 1ppm/C Expansion Coefficient for Inner Flight Tube virtually eliminates calibration drift due to flight tube elongation. 2 nd Order Temporal Focusing Ion Mirror uses high transmission Harp Grid for maximum sensitivity Ion Mirror 6530 Q-TOF 6540 UHD Q-TOF Octopole 2 Detector DC Quad Ion Pulser Turb o

13 Precise Ion Beam Acceleration Focusing Maximizing Transmission and Resolving Power Autotune algorithms optimize resolving power and transmission by adjusting among other voltages: Ion Mirror Mid-Mirror Puller Offset Accelerator Focus Octopole 2 Detector DC Quad Accelerator Focus Puller, Puller Offset Pusher

14 Turn-around Time A closer look Ion Mirror Puller Ground Pusher Velocity reducing flight time Velocity increasing flight time Octopole 2 Turn-around time is the delay for an ion to overcome incoming velocity away from the time of flight measurement. DC Quad Detector

15 Challenge: Reduce Turn-around Time and Preserve Ion Transmission Standard ion optic elements cannot achieve a reduction in phase space (i.e. product of beam divergence and size). Smaller size, but divergent beam increases spread in initial ion velocity in TOF ion pulser Creating a simple RF ion guide funnel looks like a good idea. Large Aperture Entrance Small Aperture Exit

16 Challenge: Reduce Turn-around Time and Preserve Ion Transmission Cut-away view Retarding Vector Because the rods are angled-in there is a vector component of the pseudo potential well force that pushes the ions back out the funnel. Retarding Vector + = Net positive ion motion Applying a voltage gradient along the length of each rod creates an Axial Acceleration Potential which overcomes the retarding force vector of the pseudo-potential well.

17 Challenge: Reduce Turn-around Time and Preserve Ion Transmission Collision Cooling removes energy from the ions and allows the beam to collapse in size and not increase in divergence. Once again the Axial Acceleration Potential is critical to replace the energy losses and prevent beam stall-out.

18 Ion Beam Compression (IBC)* Technology Drives Higher Resolution without Sensitivity Loss! Collision Cell Axial Ion Acceleration Technology applied to a tapered ion guide Compressed beam reduces turn-around time and achieves 40,000 resolving power. * Patent pending

19 Next Generation Ultra High Speed Detector New Bipolar TOF Detector New ultra fast and high efficiency scintillator New ultra fast response PMT design continues the tradition of high dynamic range and detector lifetime Developed by Photonis with Agilent TOF Technology Specifically enhances Resolution in 2Ghz Ext. Dynamic Range Mode 2 nsec/div Single Ion Response ~800 psec FWHM

20 Ultra High Speed Acquisition From Agilent s Leadership in GHz Speed Electronics 4 GHz Acquisition for Maximum Resolving Power and <1ppm Mass Accuracy 5 Decades of in-spectrum Dynamic Range from 2-Channel x 2 GHz Dual Gain Mode Dual Input Agilent pre-amplifiers Picture of 4GHz board Goes here 4 GHz (8 bit) Analog-Digital-Converter Adapted from Agilent s High Speed Oscilloscope Systems Ultra High Speed FPGAs process and store transients in real time FPGAs 4 GHz Agilent ADC

21 6540 Ultra High Definition QTOF Resolving Power Across the Mass Range m/z 622 and isotopes 5 x R= R= R= Counts vs. Mass-to-Charge (m/z) R=30218 R= R= R= R= R= Counts vs. Mass-to-Charge (m/z) Scan Rate Independent

22 6540 Ultra High Definition QTOF Resolving Power Small Proteins Horse Heart Myoglobin (MW Da) Average resolution of these peaks R = Heme

23 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) A A A A A Counts vs. Mass-to-Charge (m/z)

24 Verapamil: Major And Minor Metabolites (Phase I) 6 x10 2 verapamil 1 TIC 1 x x x x desmethyl monohydroxy dihydroxy Isopropyl dealkylation Counts vs. Acquisition Time (min)

25 Coeluting Metabolites With Parent Drug: Need Wide Dynamic Range Five decades of response in a single scan verapamil dihydroxy metabolite of verapamil 6 x counts (M+H) million desmethyl metabolite monohydroxy metabolite counts (M+H) Counts vs. Mass-to-Charge (m/z) 25 counts

26 Metabolite ID Results

27 6430 and 6460 Triple Quad LC/MS Maximizing Performance and Throughput Optimized: Design: Software: Rapid MRM Transitions Fast +/- Switching 6430 Std ESI /HPLC Chip 6460 Agilent Jet Stream Dual turbo pumping 6430 mass range is 2250m/z 6460 mass range is 3000m/z Sub millisecond collision cell clearance Ultra Sensitive ADC for wide dynamic range and single ion detection Dynamic MRM (DMRM) Optimizer (small molecule and peptide) MRM Optimizer Database MRM Builder (from Spectrum Mill) Quantification SW

28 Agilent Jet Stream Ion Generation Gas Dynamics View Enhanced efficiency nebulizer Nebulizing gas Super-heated sheath gas 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

29 Agilent Jet Stream In Action Observing Thermal Focusing Scattered light shows good collimation, but without heat there is high droplet density Less light scattering shows increased droplet desolvation and high ion production. Start temperature = 25 ºC Stop temperature = 400 ºC

30 Arb. Units. Axial Acceleration Collision Cell Sub-millisecond ion transport times Axial acceleration overcomes memory or cross-talk effects Beam Turn-off Characteristics mz922 mz Sub millisecond ion transport sec 350 sec microseconds

31 Ultra High Definition LC/MS Moving from Discovery to Validation 6400 Series QQQ New Collision Cell Incorporates Axial Acceleration for High Speed MS/MS Analysis Octopole 1 Quad Mass Filter (Q1) Quad Mass Filter (Q3) Lens 1 and 2 Collision Cell 10KV Detector Rough Pump Turbo 1 Over 12 Years in LC/MS Atmospheric Sampling and Patented Orthogonal Geometry - Result in Industry Leading Sensitivity and Robustness Turbo 1 Turbo 1 Rough Pump Octopole 1 Turbo 1 Quad Mass Filter (Q1) Lens 1 and 2 Turbo Series Q-TOF Collision Cell Turbo 1 Octopole 2 DC Quad Ion Pulser Turbo 2

32 QTOF - Discovery and QQQ - Validation Produce Common Fragment Ions x10 x Intens. x ESI Product Ion ( min, 2 scans) Frag=175.0V CID@21.1 (637.6[z=3] -> **) syn-100fmol.d ESI Product Ion ( min, 18 scans) Frag=120.0V CID@21.0 ( > **) peptide5-ce21.d Peptide: RPCFSALEVDETYVPK (m/z 637.6, +3) b6 y6 b6 y MS2(637.8), min #344-# B13 ++ b7 b Counts vs. Mass-to-Charge (m/z) y7 y7 b b y9 y9 Q-TOF QQQ Ion Trap / Orbitrap m/z

33 Moving From Discovery to Validation Having the right tools Spectrum Mill Protein ID via Database Search, Mass Gap Search MRM Builder Targets the most intense transitions Peptide Optimizer Maximizing Sensitivity for each Peptide MRM Database MS Acquisition Direct Import from MRM Database Dynamic MRM Acquisition maximizes Duty Cycle and Sensitivity

34 Agilent 6400 Series Triple Quads with HPLC- Chip/QQQ LCMS Technology Nanospray chip configuration brings new era in high-precision, highsensitivity quantification NanoLC system for analytical chromatography HPLC Chip Cube system CapLC pump for sample loading on enrichment column QQQ LCMS Sensitivity: down to low amol Dynamic range: up to 10 5

35 HPLC-QQQ - Low amol limit of Quantitation range Peroxidase spiked into human serum 10 amol peroxidase in 1ug serum 100 amol peroxidase in 1ug serum 1 fmol peroxidase in 1ug serum 10 fmol peroxidase in 1ug serum

36 External Quantitation Curve of Peroxidase Peptide DTIVNELR From 10 amol to 10 fmol Spiked into Human Serum 10amol 1fmol 100amol

37 Thank You