agilent’s lc/ms portfolio and applications examples · agilent’s lc/ms portfolio and...

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.

Agilent 6000 Series LCMS

Pa

Ultra High Definition LC/MS The Most Comprehensive Answer To Your Urgent Analytical Question

Signal Response

Mass Spectrum

• Sensitivity • Dynamic Range • Linearity

• Ionization Profile • Mass Accuracy • Isotopic Fidelity • Mass Resolving Power • Acquisition Rate

• Peak Resolving Power • Peak Capacity • Separation Speed

…a few words about chromatography

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LC - MS

Preparative LC Capillary & Nano LC

Standard LC Ultra High Pressure LC

High Sensitivity HPLC-Chip/MS

Agilent High Performance Infinity Series LC: Best in class “Front End” systems for MS

The industry’s most comprehensive LC portfolio to configure an integrated LC/MS system

Agilent LC-MS column configurations:

Column Type Column I.D. Column Lengths Particle Sizes Flow Rate

Range

Nano 0.075, 0.10 mm 50, 150 mm 3.5 um 100 – 600

nL/min

Capillary 0.3, 0.5 mm 35, 150, 250 mm 3.5, 5 um 1 – 10 uL/min

MicroBore 1.0 mm 30, 50, 150 mm 3.5, 5 um 30 – 60 ul/min

NarrowBore 2.1 mm 15 – 250 mm 1.8, 3.5, 5 um 0.1 – 0.3 mL/min

Solvent Saver 3.0 mm 50 – 250 mm 3.5, 5 um 0.3 – 1 mL/min

Analytical 4.6 mm 15 – 250 mm 1.8, 3.5, 5um 1 – 1.5 mL/min

… plus Prep columns – 9.4, 21.2, 30, and 50 mm id

Nano LC Capillary LC

Analytical LC Prep LC

Chip LC

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.

1971, 5930A 2005, 5975A

GC/MS 1971 - 2011

LC/MSD, LC/MSD Ion Trap

LC/MSD TOF

1994, 2000

LC/MS 1980 - 2011 2003

Agilent’s > 40 year heritage in mass spectrometry 2008, 7000A

LC/QQQ

LC/QTOF

2006

1976, 5992A

1994, 4500 Series

ICP-MS 1994 - 2011

2012 8800 QQQ

2000, 7500 Series

Direct Liquid Injection 1980, 5985 SQ Thermospray, 5987 Particle Beam, 5988

ESI on 5989 MS Engine

2011 7200A

A big leap forward in 2006

Over 15 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


10KV Detector

Ion Pulser

Turbo 2

Octopole 1

DC Quad

Rough Pump

Turbo 1

Turbo 1

Turbo 1


Collision Cell Lens 1 and 2

Octopole 2

Rough Pump

Octopole 1

Turbo 1

Turbo 1

Turbo 1


Lens 1 and 2 10KV

Detector

Collision cell incorporates axial acceleration for high speed MS/MS analysis

6500 Series Q-TOF 6400 Series QQQ

6100 Series SQ

Agilent offers a range of LC/MS systems

Single quad

Triple quad

Ion trap

TOF QTOF

3/26/2013 Page 11

Introduction to the Agilent LC/MS Portfolio Agilent Restricted

Agilent 6000 Series LC/MS portfolio in 2013

1290 series UHPLC

Comprehensive Range of Software

6200 series TOF

6100 series Quad

6500 series Q-TOF

6400 series QQQ

1 Model 3 Models

4 Models 3 Models

Agilent 6000 Series LC/MS portfolio

6200 series TOF

6100 series Quad

6500 series Q-TOF

6400 series QQQ

High Resolution

Low Resolution

Sing

le S

tage

Tandem

Stage

Qualitative

Quantitative

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

Mass Hunter Extensive application support

• Confirm identities and identify variants of proteins BioConfirm

• Identify metabolites Metabolite ID

• Used for expression profiling Mass Profiler

• Most comprehensive database made available on PC METLIN Personal Metabolite Database

• Chemometrics software to visualize large sample sets Mass Profiler Professional

• Fast analysis of large amounts of LC/MS data Analytical Studio Reviewer

• Walk-up operation for instruments Easy Access

• Identify proteins and peptides by fast database searches Spectrum Mill

Full integration: the lab, business applications, OpenLAB, the company network

Intranet

Internet

GC

CE

LC

Lab-on-a Chip

GC/MS

ICP-MS

UV-vis

ELN PDF Images

Drawings LIMS Research

MS Office Legacy Data

The Lab Business Applications

Agilent OpenLAB (Laboratory Informatics)

Masshunter Workstation

LC/MS – Ion sources Wide range of sampling conditions Sources are interchangeable between mass spectrometer platforms

Multimode source • Flow rates up to 2 mL/min • Simultaneous operation

HPLC-Chip/MS • Nanoelectrospray without the fuss • Reproducible response and retention

times • On-chip enrichment for speed and

sensitivity

Multimode HPLC-Chip

APCI ESI NanoESI

APPI AP-MALDI

6100 Series Single Quad

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

6100 Series Single Quadrupole LCMS

6120 6130 6150

Easiest Flexible Performance

6100 Series Single Quadrupole LCMS

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Agilent Jet Stream Technology

June 2008, Denver Page 23

Agilent Restricted 23

Agilent Jet Stream

Enter slide show mode to see the animation on this page.

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

Agilent Jet Stream Parameters • Sheath Gas Flow • Sheath Gas Temperature • Nozzle Voltage • Nebulizing Pressure • Chamber Voltage

Agilent Jet Stream Ion Generation Gas Dynamics View

Page 24

Heating the Sheath Gas in Jet Stream, time lapse

Start Temperature 25oC at t = 0 min

Page 25

Narrower Spray Beam

Stop Temperature 400oC at t = 8 min

2R

Page 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)

Phthalates in Children’s Toys

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6400 Series Triple quadrupole

31

Ancient proverb: On a dark night you can see more stars

Why a Triple Quad ?

Why a Triple Quad?

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Multiple reaction monitoring (MRM)

Quad Mass Filter (Q3) Quad Mass Filter (Q1)

Collision Cell

170 210 250 290

210

222

268 280 165

Spectrum with background ions (from ESI)

Q1 lets only target ion 210 pass through

190 210

210

Collision cell breaks ion 210 apart

150 170 190 210

210 158

191

Q3 monitors only characteristic fragments from ion 210 for quant and qual.

160

158

190

191

no chemical background

Eight min Dynamic MRM Analysis 6460 QQQ - 250 Pesticide Screen.

500 ppt, dynamic MRM Retention Time Window = 12 sec Peak widths ~ 1 second

Zorbax Eclipse Plus C18 2.1 x 100 mm (1.8 μm)

ASMS 2010 Agilent Introduces iFunnel Technology

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Agilent Jet Stream

• Thermal confinement of ESI plume

• Efficient desolvation to create gas phase ions

• Creates an ion rich zone

N E W I F U N N E L T E C H N O L O G Y

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

iFunnel Technology captures 6x more ions

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

6 Bore Capillary

Hexabore atmospheric sampling….

Ion Funnel Operation

RF Drive RF Voltage focuses the ions to the center.

DC Voltage accelerates the ions to the exit. DC Drive

Unfocused Ions and Gas Enter

Focused Ions Out

Ma38

Two-stage Ion Funnel can handle the gas load

Stage 1 8 - 12 Torr

Stage 2 1 - 3 Torr

Stage 1 offset deflects the high quantity of gas exiting the hexabore capillary

Line of Sight

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

2006 2007 2008 2009 2010

6410 6460

6490 SN

R 1

pg

rese

rpin

e

New 6490 advances in sensitivity 50 fold in 4 years

Acquisition Time (min) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

X101

4.2

4.3

4.4

4.5

Res

pons

e

Blank

6490/iFunnel: Zeptomolar detection limits 100 attograms Verapamil on-column (200 zeptomoles)

+ MRM (455.3 -> 164.9)

Unprecedented six logs of linearity with 6490

Y = 65283 x – 0.390412 R2 = 0.9976

Femtograms

Area

R

espo

nse

1

10

100

1000

10000

100000

1000000

10000000

0.1 1 10 100 1000 10000 100000

100 attogram to 100 picogram Verapmil on-column

Estradiol in Human Plasma

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6400 Series Triple Quad

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6420

6430

6460

6490

6200 Series TOF

Formulas for TOF performance

Resolving power = (m/z)/w1/2

1000/0.025 = 40,000

922/0.022 = 41,909

Mass accuracy ( rel. mass error)

(1000.000-1000.001)/1000)x106=1 ppm

Therefore: 1 ppm @ 1000 m/z is 0.001Da (1 mDa)

921.5 922.0 922.5 923.0 923.5 924.0 924.5 m/z, amu

923.0121

W1/2 = 0.022

610actual

measured - actual accuracy Mass 1aa

+TOF MS: 5.474 to 5.547 min from steroid2TOFap42.wiff Agilent, subtracted (4.696 to ... Max. 1.2e5 counts.

120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, amu

0.00

1.00e4

2.00e4

3.00e4

4.00e4

5.00e4

6.00e4

7.00e4

8.00e4

9.00e4

1.00e5

1.10e5

1.16e5

Inte

ns

ity

, c

ou

nts

289.2159

271.2051

269.1893287.1997

Epitestosterone

Resolving power: 6890

actual 289.2162 meas 289.2159

-1.06 ppm

922.0092

XIC of +TOF MS: 334.4 to 335.4 amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff Max. 2.0e5 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

6.8e4

Inte

nsi

ty,

cps

123.06

123.05

NPAMOZ at unit mass resolution 335.1352 , -0.3 + 0.7

NPAMOZ 335.1352 +/- 0.1


0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

Inte

nsi

ty,

cps

123.06

S/N = 70:1


0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

9000

Inte

nsi

ty,

cps

NPAMOZ 335.1352 +/- 100 ppm (335.09939 - 335.16641)

S/N = 103:1


0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

Inte

nsi

ty,

cps

123.05

NPAMOZ 335.1352 +/- 50 ppm (335.11614 - 335.14966)

S/N = 156:1

NPAMOZ 335.1352 +/- 20 ppm (335.1262-335.1396)


0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Time, min

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500

1600

1700

1800

1900

2000

2100

2200

2300

2400

2500

2600

2700

2800

2900

3000

3100

3200

Inte

nsi

ty,

cps

123.05

S/N = 214:1

LC/ESI-TOF MS, using accurate mass

250.0 250.5 251.0 251.5 252.0 252.5 253.0 253.5 m/z, amu

0.0

4.0e4

8.0e4

1.2e5

1.6e5

2.0e5

2.4e5

250.0434

252.0406

251.0473

Table of possible chemical formulas for m/z 250.0434 (with < 2 ppm error)

Formula Calculated m/z mDa error error (ppm) DBE C10 H13 N2 F Cl2 250.0434 -0.0336 -0.1346 4

C9 H13 N2 O F P Cl 250.0432 0.1410 0.5640 4 C5 H12 N5 F2 Cl2 250.0432 0.1661 0.6642 0.5

C5 H16 N3 O F P2 Cl 250.0435 -0.1715 -0.6858 -0.5 C3 H7 N10 O2 Cl 250.0436 -0.2487 -0.9949 5 C4 H13 N3 O7 Cl 250.0436 -0.2541 -1.0165 -0.5

C4 H12 N5 O F2 P Cl 250.043 0.3407 1.3629 0.5 C6 H16 N3 F P Cl2 250.0437 -0.3461 -1.3845 -0.5 C10 H17 O P2 Cl 250.0437 -0.3712 -1.4848 3

C13 H10 N O F Cl 250.0429 0.4535 1.8138 8.5



C9 H13 N2 O F P Cl 250.0432 0.1410 0.5640 4 C5 H12 N5 F2 Cl2 250.0432 0.1661 0.6642 0.5



C13 H10 N O F Cl 250.0429 0.4535 1.8138 8.5

C10 H13 N2 F Cl2

C5 H12 N5 F2 Cl2

C6 H16 N3 F P Cl2

Eliminate compounds with two Cl


C9 H13 N2 O F P Cl 250.0432 0.1410 0.5640 4 C5 H12 N5 F2 Cl2 250.0432 0.1661 0.6642 0.5



C13 H10 N O F Cl 250.0429 0.4535 1.8138 8.5

C5 H16 N3 O F P2 ClC3 H7 N10 O2 ClC4 H13 N3 O7 Cl

C4 H12 N5 O F2 P Cl

Eliminate compounds with low C count



C9 H13 N2 O F P Cl 250.0432 0.1410 0.5640 4 C5 H12 N5 F2 Cl2 250.0432 0.1661 0.6642 0.5



C13 H10 N O F Cl 250.0429 0.4535 1.8138 8.5

C9 H13 N2 O F P Cl

C10 H17 O P2 Cl

Apply the ‘Nitrogen Rule’

10 -> 1 compound


Molecular Formula Generator

57

Molecular Formula Generator

58

Formula Generation…three criteria

Page 59

monoisotopic mass

Isotope spacing

Isotope distribution

Scoring based on…

1. Monoisotopic Mass

2. Isotope Spacing C10H9ClN4O2S

3. Isotope Distribution

17 min.

3 min.

1.5 min.

Agilent 6230 TOF Pesticide Screening 224 pesticides, 50 pg on column

Thiabendazole

Peak Width=0.8 sec 23 data points

-1.8 ppm

Spectral Resolution &

Isotopic Fidelity

6500 Series QTOF design

QTOF 6500 Series

6510 6520

MMA > 2ppm RP > 20K MS2 Speed 10 Hz

Ion Beam Compression (IBC) Technology Drives Higher Resolution

Active Ion Beam Compression is achieved with Agilent’s Axial Ion Acceleration Technology applied to a tapered Ion guide design.

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 simultaneously maximizes ion transmission and reduces beam divergence

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6540 UHD

Ultra High Definition QTOF

6540 Ultra High Definition QTOF Mass 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

5 x10

0

0.2

0.4

0.6

0.8

1

1.2 609.28081

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)

1 100 100 609.28081 609.28066 0.25

2 35.87 37.24 610.28425 610.28393 0.52

3 8.58 8.59 611.28650 611.28671 -0.34

4 1.41 1.48 612.28987 612.28941 0.75

5 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

250 ppb mass accuracy calibration and very accurate isotopic ratios

6540 Ultra High Definition QTOF Sensitivity – Full Scan MS Mode- 1 picogram

3 x10

0

1

2 609.280347

(M+H)+

610.283168

611.287790

612.292649

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

607 608 609 610 611 612 613 614 615 616

4 x10

0

1

2

Counts vs. Acquisition Time (min)

0.0 0.5 1 1.5

S/N = 319 RMS

Ratio m/z Diff. Theor. Expt. Theor. Expt. (ppm) 100.00 100.00 609.2807 609.2803 -0.51 37.24 36.36 610.2839 610.2832 -1.25 8.59 8.39 611.2867 611.2878 1.77 1.48 1.45 612.2894 612.2926 5.29

Resolution ~ 33,000

Excellent sensitivity, accuracy and resolution at 1 pg on column

6 x10

0

0.5

1

1.5

2

2.5

3

3.5

4

Counts vs. Mass - to - Charge ( m/z ) 322 322.5 323 323.5 324 324.5 325 325.5 326

322.05432 20535

323.05854 20415 324.05976

21517 325.06230

21835 326.20440

17254

6 x10

0

0.5

1

1.5

2

2.5

3

3.5

4

Counts vs. Mass - to - Charge ( m/z ) 322 322.5 323 323.5 324 324.5 325 325.5 326

322.05432 20535

323.05854 20415 324.05976

21517 325.06230

21835 326.20440

17254

6540 Ultra High Definition QTOF Wide Dynamic Range

Peak height

5,000,000

Dynamic Range:

G2421A Tune Mix – m/z 322 1 sec spectrum

5x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

2.5

2.75

Counts vs. Mass-to-Charge (m/z)323 323.5 324 324.5 325 325.5 326 326.5

323.0585420415

324.0597621517

325.0623021835

323.3331316656

324.3249911265

326.2044017254

5x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

2.5

2.75

Counts vs. Mass-to-Charge (m/z)323 323.5 324 324.5 325 325.5 326 326.5

323.0585420415

324.0597621517

325.0623021835

323.3331316656

324.3249911265

326.2044017254

4x10

0

0.5

1

1.5

2

2.5

3

3.5

4

Counts vs. Mass-to-Charge (m/z)324 324.5 325 325.5 326 326.5

324.0597621517

325.0623021835324.32499

11265326.20440

17254

4x10

0

0.5

1

1.5

2

2.5

3

3.5

4

Counts vs. Mass-to-Charge (m/z)324 324.5 325 325.5 326 326.5

324.0597621517

325.0623021835324.32499

11265326.20440

17254

3x10

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

Co325 325.5 326 326.5

325.0623021835

326.2044017254

3x10

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

Co325 325.5 326 326.5

325.0623021835

326.2044017254

+ESI Scan (#27) Frag =180.0V 2GHz_accF.d +ESI Scan (#27) Frag =180.0V 2GHz_accF.d

5,000,000 100

= 50,000

Peak Height

100

Dynamic Range

Page 68

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

6550 iFunnel Q-TOF LC/MS System

ASMS 2010

Sensitivity

• Dramatically improved quantitative capabilities • New Qual/Quan Workflows • Superior metabolite and protein detection • Non-targeted compound screening

Comprehensive Performance Enhancements

• Mass Resolution >40,000 • 50 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

New 6550 iFunnel QTOF 10X Sensitivity Gain Enables Applications

TOF and QTOF Fundamentals

March 2007 Page 71

Agilent QTOF Design Incorporates Cumulative Hardware Innovations

Ion optics Common with Q & QQQ

Flight tube Common with TOF

Octopole 1

DC Quad

Collision cell Common with QQQ

Rough Pump

Turbo Turbo Turbo


Collision Cell Lens 1 and 2

Octopole 2

Ion Pulser

Ion Mirror

Detector

Turbo

Targeted MS/MS mode for Caffeine

N

NH

+ N

NO

CH3O

CH3

CH3

NC+

N

NO

CH3

CH3

C8H11N4O2+

+ 2.84 ppm

C6H8N3O+ - 0.09 ppm

Calculate chemical formula given accurate mass measurement and using elements C, H, N, and O

C3H5N2+

- 23.15 ppm

Proposed structures

CH +

N

N

N

CH3

CH3

C5H8N3+

- 3.91 ppm

N

CH+

N

CH369.0463

110.0717

138.0662

195.0871

MS-MS Data Resolved

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ID Browser: Identification of Statistically Differential Compounds

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

Thank you !

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