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

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.

MassHunterTMInformatics

Chromatography

Comprehensive Sample

InformationMass Spectrometry

Signal Response

Mass Spectrum

Ultra High Definition

Optimizing all Analytical Dimensions

Software

• Data Mining

• Differential Profiling

• Annotation

• Ionization Profile

• Mass Accuracy

• Isotopic Fidelity

• Mass Resolving Power

• Acquisition Rate

• Sensitivity

• Dynamic Range

• Linearity

• Peak Resolving Power

• Peak Capacity

• Separation Speed

Agilent 1290 Infinity LC – Attributes for MS - Infinitely Better for LC/MS

Lowest

Delay

Volume

Highest

Precision

Pump (w/o) mixer: 10 µL

Pump, Fixed Loop 20 µL

Pump, Fixed Loop, JetWeaver 55 µL

<0.002% carry-over with Chlorhexidine

Optional needle seat backflushing with FlexCube

Fixed Loop or Variable Loop Injections

1200bar @ 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

ALS precision for small volumes:

<1.5% from 0.5-1µLm, <0.7% from 1-2µL,

<0.25% @ 2-20 µl (40 ul) **

Pump Active Damping:

RT stability < 0.2 % (1.5 min runs)**

Best

Autosampler

Performance

Greatest

Productivity

1290 Infinity

0

200

400

600

800

1000

1200

0 1 2 3 4 5

1290 Infinity – Compatible with any HPLC and UHPLC

bar

ml/min

Standard LC

Acquity

Agilent RRLCThermo

Accela

Shimadzu UFLCXR

Dionex RSLC

A new power range providing maximum performance,

flexibility, compatibility and investment protection

1290 Infinity Binary PumpHow it looks like inside

December 15, 2009

Channel

A

Channel

B

Jet Weaver

Multi-layer

Heat exchanger

Solvent selection

valveDegasser

Purge

Valve

Silicon Carbide

Pistons

High Resolution

Pump drives

0 10 20 30 40 50 60 70 80 90 sec

1.00

0.75

0.50

0.25

0.00

1290 Infinity - Ultra High Performance Separations Maximizing Throughput

Fast screening of 220 pesticides

in 90 seconds

Peak Width

~ 0.7 sec FWHM

1290 Infinity - Ultra High Performance SeparationsMaximizing High Peak Capacity

Peptide Map of Tryptic Digest of BSA run on Agilent RRHT Zorbax SB-C18, 2.1x150mm, 1.8µ

min2 4 6 8 10 12 14 16 18 20

mAU

0

10

20

30

40

50

60

Peak Capacity = 540

Peak Width ~ 2 sec FWHM

HPLC-Chip: Polymer Microfluidics

developed in Agilent Labs

µ-fluidic holes

µ-filter

Registration holes

Enrichment Column 40 nl ZORBAX 300SB C18

Electrical Interface to Internal Grounding Electrode

ZORBAX 300SB-C18, 5µm 75µm, 43 mm length

Laser Ablated Cylindrical

nanospray emitter

MS inlet

orifice

Counter

electrodeRobust spray performance under all solvent conditions

Polyimide nanospray emitter lifetime measured in weeks

Laser ablation of polyimide film

Agilent’s New 6540 Ultra High Definition QTOFResearch 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

Theory of High Resolving PowerHighest resolving power in a Benchtop

Turn-around time term

t

TOF

FWHM

mR

peak 2

2222

RPWTAt

TA

PW

R

Detector pulse width term

Residual term

Agilent Model

TOF, s tTA, ns tPW, ns tR, ns ∆t, ns

Resolving Power

6520 92 1.1 2.1 1.6 2.8 16,5006540 152 0.5 0.9 1.6 1.8 42,000

*Values are for m/z 2722

6540 UHD

Q-TOF

6530

Q-TOF

• 1ppm/C Expansion Coefficient for Inner

Flight Tube virtually eliminates calibration

drift due to flight tube elongation.

• 2nd Order Temporal Focusing Ion Mirror

uses high transmission Harp Grid for

maximum sensitivity

Enhanced Ion Flight Tube and Mirror TechnologyStable, Sensitive, High Resolution

DC Quad

Octopole 2

Ion Pulser

Ion Mirror

Detector

Turbo

Ion Mirror

Detector

Precise Ion Beam Acceleration FocusingMaximizing Transmission and Resolving Power

• Autotune algorithms optimize

resolving power and transmission

by adjusting among other

voltages:

• Mid-Mirror

• Puller Offset

• Accelerator Focus

DC Quad

Octopole 2

Accelerator Focus

Puller , Puller Offset

Pusher

DC Quad

Octopole 2

Ion Mirror

Detector

Turn-around TimeA closer look

DC Quad

Octopole 2

DC Quad

Octopole 2

DC Quad

Octopole 2

DC Quad

Octopole 2

Puller

Pusher

Ground

Turn-around time is the delay

for an ion to overcome

incoming velocity away from

the time of flight measurement.

Velocity reducing flight time

Velocity increasing flight time

• Standard ion optic elements cannot achieve a reduction in

phase space (i.e. product of beam divergence and size).

Challenge: Reduce Turn-around Time and

Preserve Ion Transmission

Large

Aperture

Entrance

Small

Aperture

Exit

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.

• 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.

Challenge: Reduce Turn-around Time and

Preserve Ion Transmission

Retarding Vector

Cut-away view

• 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.

+ =

Net positive ion motion

Retarding Vector

• Collision Cooling removes energy from the ions and allows the beam to

collapse in size and not increase in divergence.

Challenge: Reduce Turn-around Time and

Preserve Ion Transmission

• Once again the Axial Acceleration Potential is critical to replace the

energy losses and prevent beam stall-out.

Ion Beam Compression (IBC)* TechnologyDrives Higher Resolution without Sensitivity Loss!

Axial Ion Acceleration Technology

applied to a tapered ion guideCollision Cell

Compressed beam reduces

turn-around time and achieves

40,000 resolving power.

* Patent pending

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

Single Ion Response

~800 psec FWHM

2 nsec/div

Ultra High Speed AcquisitionFrom Agilent’s Leadership in GHz Speed Electronics

• 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

Picture of 4GHz board

Goes here

FPGAs

Dual Input Agilent

pre-amplifiers

4 GHz Agilent ADC

• 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

6540 Ultra High Definition QTOF

Resolving Power – Across the Mass Range

5x10

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

200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800

922

R=42424622

R=38702

1221

R=43674

1521

R=42750322

R=302182121

R=418251821

R=404052421

R=39332 2721

R=37207

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

Scan Rate Independent

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

622.00822

623.01059

624.01219 625.01471

m/z 622 and isotopes

6540 Ultra High Definition QTOFResolving Power – Small Proteins

Average resolution of these peaks

R = 38045

10+

16+

15+

14+13+

12+11+

19+

18+

17+

20+

22+

21+

23+

24+

25+

26+

Heme

Horse Heart Myoglobin (MW 16951 Da)

6540 Ultra High Definition QTOFMass Accuracy – Repetitive Injections

+ESI EIC(609.28066)

Scan Frag=240.0V Reserpine_40pgms3.d

0.511

Counts vs. Acquisition Time (min)

0.0 0.25 0.5 0.75 1

5x10

0

0.2

0.4

0.6

0.8

1

1.2609.28081

610.28425

611.28650

612.28987

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

609 610 611 612 613 614

613.29210

Isotope Obs % Calc % Obs m/z Calc m/z Diff (ppm)

A 100 100 609.28081 609.28066 0.25

A+1 35.87 37.24 610.28425 610.28393 0.52

A+2 8.58 8.59 611.28650 611.28671 -0.34

A+3 1.41 1.48 612.28987 612.28941 0.75

A+4 0.14 0.21 613.29210 613.29203 0.11

Run

Error (ppm)

1 0.96

2 -0.17

3 0.25

4 0.02

5 0.39

6 0.13

7 0.01

8 0.52

9 0.04

10 0.30

Mean 0.25

Std. Dev 0.32

40pg reserpine on-column, 10 injections

6x10

1

2 1

5x10

0

5

1

4x10

0

2.5

51

2x10

0

5

1

3x10

0

1

1

Counts vs. Acquisition Time (min)

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4

Verapamil: Major And Minor Metabolites (Phase I)

desmethyl

Isopropyl

dealkylation

monohydroxy

dihydroxy

TICverapamil

6x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8 455.290321

(M+H)+

441.274704

471.284287487.279822

(M+H)+

Counts vs. Mass-to-Charge (m/z)435 440 445 450 455 460 465 470 475 480 485 490 495 500 505

Coeluting Metabolites With Parent Drug: Need

Wide Dynamic RangeFive decades of response in a

single scan

2.68 million

counts

25 counts

verapamil dihydroxy metabolite of verapamil

desmethyl

metabolite

monohydroxy

metabolite

400

counts

Metabolite ID Results

6430 and 6460 Triple Quad LC/MSMaximizing Performance and Throughput

Optimized: Rapid MRM TransitionsFast +/- Switching6430 Std ESI /HPLC Chip6460 Agilent Jet Stream

Design: Dual turbo pumping6430 mass range is 2250m/z6460 mass range is 3000m/zSub millisecond collision cell clearanceUltra Sensitive ADC for wide dynamic range and single ion detection

Software: Dynamic MRM (DMRM)Optimizer (small molecule and peptide)MRM Optimizer DatabaseMRM Builder (from Spectrum Mill)Quantification SW

Agilent Jet Stream Ion Generation Gas Dynamics View

The super-heated sheath gas

collimates the nebulizer spray and

creates a dramatically “brighter source”

Nozzle voltage

Resistive sampling

capillary

Nebulizing gas

Super-heated sheath gas

Heated drying gas

Enhanced efficiency nebulizer

Patent Pending

Scattered light shows

good collimation, but

without heat there is high

droplet density

Start temperature = 25 ºC Stop temperature = 400 ºC

Less light scattering

shows increased droplet

desolvation and high ion

production.

Agilent Jet Stream In ActionObserving Thermal Focusing

Beam Turn-off Characteristics

10

100

1000

10000

100000

-500 0 500 1000 1500 2000

microseconds

Arb

. U

nit

s .

mz922 mz118

Beam Turn-off Characteristics

10

100

1000

10000

100000

-500 0 500 1000 1500 2000

microseconds

Arb

. U

nit

s .

mz922 mz118

600 sec

350 sec

Sub millisecond

ion transport

Axial Acceleration Collision CellSub-millisecond ion transport times

•Axial acceleration overcomes memory or cross-talk effects

Ultra High Definition LC/MSMoving from Discovery to Validation

Over 12 Years in LC/MS

Atmospheric Sampling

and Patented

Orthogonal Geometry -

Result in Industry

Leading Sensitivity and

Robustness

Rough Pump

Octopole 1

Turbo 1

Turbo 1

Turbo 1

Quad Mass Filter (Q1)

Collision Cell

Lens 1 and 2

Quad Mass Filter (Q3)

10KV Detector

Ion Pulser

Turbo 2

Octopole 1

DC Quad

Rough Pump

Turbo 1

Turbo 1

Turbo 1

Quad Mass Filter (Q1)

Collision CellLens 1 and 2

Octopole 2

New Collision Cell Incorporates

Axial Acceleration for High Speed

MS/MS Analysis

6500 Series Q-TOF

6400 Series QQQ

0

3x10

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

2.5

+ESI Product Ion (4.023-4.037 min, 2 scans) Frag=175.0V CID@21.1 (637.6[z=3] -> **) syn-100fmol.d

719.3 851.4

961.5

832.4

481.2343.2

607.3506.3 1079.5691.3648.3416.7 804.4

4x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

+ESI Product Ion (4.765-4.832 min, 18 scans) Frag=120.0V CID@21.0 (637.6 -> **) peptide5-CE21.d

719.1

961.2480.9

832.2

350.8 416.5

530.6 1079.2607.1

301.8

QTOF - Discovery and QQQ - Validation Produce Common Fragment Ions

343.0 481.1530.8

588.2 653.4703.5

736.4

785.1

834.6

961.5

5. +MS2(637.8), 3.9-4.1min #344-#365

0.0

0.5

1.0

1.5

2.0

2.5

7x10

Intens.

300 400 500 600 700 800 900 1000 1100 1200 1300 m/z

Ion Trap / OrbitrapB13++

Counts vs. Mass-to-Charge (m/z)300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350

QQQ

b8

y6

b6

y7

b7

y9

Peptide: RPCFSALEVDETYVPK (m/z 637.6, +3)

Q-TOF

b8

y6

b6

y7

b7

y9

Moving From Discovery to ValidationHaving 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

Agilent 6400 Series Triple Quads with HPLC-

Chip/QQQ LCMS TechnologyNanospray chip configuration brings new era in high-precision, high-

sensitivity quantificationNanoLC system for

analytical chromatography

HPLC Chip Cube system

QQQ LCMS

CapLC pump for sample

loading on enrichment column

Sensitivity: down to low amol

Dynamic range: up to 105

HPLC-QQQ - Low amol limit of Quantitation rangePeroxidase spiked into human serum

10 fmol peroxidase in 1ug serum

1 fmol peroxidase in 1ug serum

100 amol peroxidase in 1ug serum

10 amol peroxidase in 1ug serum

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

100amol

1fmol10amol

Thank You