WW Seminar Tour

Spring 2006

Introducing the Agilent G6410A Triple QuadrupoleMass Spectrometer

Michael Zumwalt

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

Collision Cell

Ion source

WW Seminar Tour

Spring 2006

Overview of QQQ HardwareQQQ Mechanical Layout

a

Leveraged w/ Q & QTOF

Common w/ QTOF

Quad Mass Filter (Q3)

HED Detector

Quad Mass Filter (Q1)

Collision CellLens 1 and 2

Rough Pump

Turbo Pump

Turbo Pump

Turbo Pump

Quad Mass Filter (Q1)

Collision CellLens 1 and 2

WW Seminar Tour

Spring 2006

MRM (Multiple Reaction Monitoring)

170 210 250 290

210

222

268 280165

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

Collision Cell

Spectrum with backgroundions (from ESI)

Q1 lets onlytarget ion 210 pass through

190 210

210

Collision cell breaks ion 210 apart

150 170 190 210

210158

191

Q3 monitors onlycharacteristic fragments 158 and 191 from ion 210 for quant and qual.

160

158

190

191

no chemical background

WW Seminar Tour

Spring 2006

Autotune and Calibration SystemBuilt-in calibrant delivery system is unique to Agilent

Built-in calibrantdelivery systemused for fastauto-calibration

calibrant solutions

WW Seminar Tour

Spring 2006

Calibrant delivered to MS via the API Interface

Calibrant Delivery System

to waste

from calibrant bottles

nitrogen

to MS spray chamber

from LC pump

Valve can also be utilized to divert

flow from the HPLC via software

WW Seminar Tour

Spring 2006

The analyzer story - minimizing noise

1. Agilent's outstanding Patented orthogonal spray sources maximize ionization while minimizing solvent and matrix noise

2. Counter-Current Drying gas Cold Dielectric capillary...enhances desolvation while minimizing chemical noise.

3. RF Octopole guide……High efficiency ion capture while optimizing wide mass bandwidth ion transmission

4. L2 RF…….greatly enhances high mass ion transmission. Patented!

5. Quad 1….hyperbolic quads optimize ion transmission and spectral resolution

6. RF quadrupole segment…..enhances ion transmission into and out of the collision cell

7. Collision cell….high pressure collision cell with linear acceleration optimizes MS.MS fragmentation while eliminating cross-talk even at very low dwell times. Small diameter high frequency hexapole assembly assists with capturing and focusing fragmented ions.

8. Quad 2 ….hyperbolic quads optimize ion transmission and spectral resolution

9. Off axis matching dual High Energy Dynode detector with Log amp signal compression……..permits high gain with rapid polarity switching, long life and low noise. Off axis design allows neutrals to pass without hitting detector.

10. Multiplier has extremely long life since ions never touch the surface, only electrons.

a

Rough Pump Edwards E2M28 Single 3 stage Edwards

Turbo Pump

2

3 4 5 6 7 810

6

9

1

WW Seminar Tour

Spring 2006

Agilent G1978A Multimode Source

Liquid enters the grounded nebulizer

A charged aerosol is made in the ESI Zone

The aerosol is dried by IR lamps

Neutral analytes and ESI charged analytes pass through the APCI Zone

ESI and APCI ions enter the capillary

Capillary

HPLC inlet

Nebulizer

Drying gas

Corona needle

ESI Zone

APCI ZoneThermalcontainer

WW Seminar Tour

Spring 2006

Agilent Multimode Source - Compound Detection CompoundIon Polarity (+) (-) (+) (-) (+) (-)

Acetazolamide 0 + 0 + 0 +Butyl 4-aminobenzoate + 0 + 0 + 0Cortisone + + + 0 + +Gemfibrozil 0 + 0 + 0 +Hexahydro-...-dione + 0 + 0 + 0Hydroflumethiazide 0 + 0 + 0 +Indole + 0 0 0 + 0Iodipamide 0 0 0 + + +Labetalol + + + + + +Lidocaine + 0 + 0 + 0Morin + + + + + +Paclitaxel 0 0 + 0 + 0Phenylbutazone + + + + + +Procainamide + 0 + 0 + 0Progesterone + 0 + 0 + 0Sulfamethoxazole + + + + + +Tolazamide + + + + + +Uracil 0 + 0 0 0 +Detected by Polarity

67% 56% 67% 50% 78% 61%

Detected by Source

89% 89% 100%

Dedicated APCI Dedicated ESI Simultaneous

WW Seminar Tour

Spring 2006

Environmental Compounds, Alternating Positive/Negative Mixed Mode Ionization

5 ng each analyte; 2.1 x 150 mm Zorbax XDB-C18, 3.5 µ, 60 °C, water/methanol gradient (3-90% methanol) with 1mM ammonium acetate, scan mode m/z 130-330; sample dissolved in 80:20 water/methanol containing 1% acetic acid. * denotes impurity

Positive mixed mode TIC,primarily [M+H]+ and [M+Na]+

min5 10 15 20 25 30 35

DiquatParaquat(respond in ESI only)

Oxamyl Methomyl

Fenuron

Monuron

*

Simazine

Propoxur

AtratonPropham

Fluometuron

Carbaryl

AminocarbCarbofuran

TerbutylazineSiduron

Methiocarb PropazineAmetrynSiduron

Secbumeton

LinuronPrometon

SimetrynAtrazine

Diuron

Prometryn

TerbutrynNeburon

ChlorprophamBarban

Mexacarbate

Negative mixed mode TIC,primarily [M-H]- and [M+acetate]-

min5 10 15 20 25 30 35

4-Nitrophenol(responds best in APCI)

Dicamba2,4-D

Monuron2,4,5-T** * *

*Fluometuron

Diuron

Linuron

NeburonSiduron

WW Seminar Tour

Spring 2006

Variables include:

• Number of poles (i.e. quad, hex, octo)

• Inscribed diameter (R0)

• Drive Frequency

Evaluation included:

• Theoretical modeling

– Calculation, Simulations

• Experimental results

Broad Mass Range Transmission and Transmission Efficiency – Why a Hexapole?

Mass Range Transmission (octo > hex > quad)

0

0.2

0.40.6

0.8

1

1.2

0 500 1000 1500Mass (m/z)

Tran

smis

sion

Quadrupole Hexapole Octopole

Ion focusing: quad > hex > octo

WW Seminar Tour

Spring 2006

Collision Cell Design and Electrical Drive Specifics

Acceleration Potential

to 10 V

RF Voltage = 10 - 550 v

• Hexapole construction

• 2R0 = 4.5 mm

• Length = 150 mm

• Experimental press. range = 0.1 – 20mTorr

• In coming ion energy range = 0 – 250eV

• Accelerating potential range = 0 – 10V

• RF Drive voltage range 10 – 550V

WW Seminar Tour

Spring 2006

Beam Turn-off Characteristics

10

100

1000

10000

100000

-500 0 500 1000 1500 2000

microseconds

Arb

. Uni

ts

mz922 mz118

Collision Cell Clearing Profile

Collision gas = 7mTorr 0 V collision energy 5 V Applied Axial Potential

600µsec

350µsec

WW Seminar Tour

Spring 2006

Fast MRM Selection

Transition 1 Transition 2Q1 = 622m/z Q1 = 600m/z (no signal)CE = 30V CE = 30VAAP = 7V AAP = 7VQ3 = 540m/z Q3 = 540m/z5msec settling 5msec settling5msec sampling 5msec sampling

3x10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Abundance vs. Acquisition Time (Min)0.029 0.03 0.031 0.032 0.033 0.034 0.035 0.036 0.037 0.038 0.039 0.04 0.041 0.042 0.043 0.044 0.045

+EIC (540.0000 m/z) MRM (600.0000 m/z); from crosstalk-03.d

Sample:Tuning Soln,Infused

Transition 1

Transition 2

WW Seminar Tour

Spring 2006

Dwell Time Evaluation 200 pg Alprazolam 100, 20, 5 ms dwell times

2.250.740.61% RSD

132021360514860Area

5 ms20 ms100 msDwell

5 ms dwell

WW Seminar Tour

Spring 2006

The Analyzer Story - Detector aspects:

9. Off axis matching dual High Energy Dynode detector with Log amp signal compression……..permits high gain with rapid polarity switching, long life and low noise. Off axis design allows neutrals to pass without hitting detector.

10. Multiplier has extremely long life since ions never touch the surface, only electrons.

a

Rough Pump Edwards E2M28 Single 3 stage Edwards

Turbo Pump

2

3 4 5 6 7 810

6

9

1

WW Seminar Tour

Spring 2006

The Analyzer Story - Pump aspects:

Newly designed manifold with a new single 3-stage turbomolecular pump, backed by a single mechanical (rough) pump.

3-staged partitioning removes the need for two turbomolecular pumps.

a

Rough Pump Edwards E2M28 Single 3 stage Edwards

Turbo Pump

2

3 4 5 6 7 810

6

9

1

WW Seminar Tour

Spring 2006

Reserpine SensitivitySpecification: 500 fg with S/N 20:1, %RSD < 5

100pg

500pg

50pg

10pg5pg1pg0.5pg0.1pgBlank

1pg

0.5pg

0.1pgBlank

WW Seminar Tour

Spring 2006

Reserpine Dilution Series (in mobile phase)Demonstration of Sensitivity via a Standard

min10 20 30 40 50 60

0

200

400

600

800

1000

1200

4.18

2 6.32

0

8.36

6

10.4

65

12.4

69

16.7

18

18.7

43

20.8

31

22.8

79

24.9

78

29.1

42

31.1

96

33.2

98 35.3

59 37.4

28

41.6

28 43.6

85

45.7

64

47.8

34

49.9

07

54.0

91 56.1

60

58.2

29

60.2

91

62.3

59

100fg200fg

500fg

1000fg

2000fg

0

2000

4000

6000

8000

10000

12000

0 500 1000 1500 2000 2500

Sample Amount (fg)

Peak

Are

a (a

rb. u

nits

)

r2 = 0.987

Reserpine (609>195) EIC, acquiring 3 transitions

WW Seminar Tour

Spring 2006

Alprazolam Calibration Results1pg – 10 ng, Positive ESI Mode

1 pg

5 pg

10 pg

5 ng

10 ng

2 ng

1 ng

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000

Abun

danc

e

Amount (pg)

Alprazolam Linearity

Y=77.2x +1593.4R2=0.9974

WW Seminar Tour

Spring 2006

New 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

WW Seminar Tour

Spring 2006

Full Integration: The Lab, Business Applications, OpenLAB, the Company Network

IntranetIntranet

InternetInternet

GC

CE

LC

Lab-on-a Chip

GC/MS

MS WorkstationICP-MS

UV-vis

ELNPDF

Images

DrawingsLIMSResearch

MS Office LegacyData

The Lab Business Applications

Agilent OpenLAB (Laboratory Informatics)

WW Seminar Tour

Spring 2006

Data Acquisition

Instrumentstatus

Data display

Acquisitionsettings

Sequence list

WW Seminar Tour

Spring 2006

Auto or Manual Tune

WW Seminar Tour

Spring 2006

Triple (QQQ) Reporting (Excel) - Tune

WW Seminar Tour

Spring 2006

• Macros in Excel Adding• Add tables and graphics

via tool buttons• Drag and drop

additional columnsfrom XML results

• Formatting of Fontand numbers in Excel

• Print Preview in Excel

Reporting (Excel)

WW Seminar Tour

Spring 2006

Agilent Quantitative Analysis

Batch processing

WW Seminar Tour

Spring 2006

Quantitative View - Method Editor

WW Seminar Tour

Spring 2006

• All peak list level and peak level info available• Visualization of baseline regression• Handling of merged peaks• Peak validation – Quality Factor and ‘Traffic Light Coloring’

Parameter-Free Integrator – Optimal for QQQ data

SD

Baseline Offset

Discarded Peak

Accepted PeaksPeak List (XML)

Info messages

WW Seminar Tour

Spring 2006

New “Parameterless” Integrator

• Handles low level spiky data (statistically sets peak start and end)• Auto adjustment of threshold - Eliminates need for manual re-integration of peaks for low-level MRM signals

WW Seminar Tour

Spring 2006

THC Qualifier ion normalized THC Qualifier ion un-normalized

THC in QuantAnalysisTHC retention time < 4.5 min.

D0 quant ion

D3 quant ion

Overlap of quantand qual ion areas

WW Seminar Tour

Spring 2006

Triple (QQQ) CurveFit Assistant

WW Seminar Tour

Spring 2006

Calibration curve data point information

WW Seminar Tour

Spring 2006

Calibration curve data point zooming

QC (blue triangle) at 2 ng/mL

QC (blue triangle) at 5 ng/mL

WW Seminar Tour

Spring 2006

MRM transitions

Assay # 2, Cocaine, etcCoc, quant = 304.1 > 182.0Coc, qual = 304.1 > 82.0D3 - Coc (ISTD), quant = 307.1 > 185.1D3 - Coc (ISTD), qual = 307.1 > 85.1MDMA, quant = 194.1 > 163.0MDMA, qual = 194.1 > 135.0D5 - MDMA (ISTD), quant = 199.1 > 165.0D5 - MDMA (ISTD), qual = 199.1 > 135.0Meth, quant = 150.1 > 91.0Meth, qual = 150.1 > 119.0D5 - Meth (ISTD), quant = 155.1 > 92.0D5 - Meth (ISTD), qual = 155.1 > 121.0Amp, quant = 136.1 > 93.0Amp, qual = 136.1 > 119.0D5 - Amp (ISTD), quant = 141.1 > 93.0D5 - Amp (ISTD), qual = 141.1 > 124.0

Analytical Conditions – MS

WW Seminar Tour

Spring 2006

Using Agilent Qualitative Analysis for Coc, Meth, Amp, & MDMA

EICs for all 16 MRMs definedand specified for peak integration

Qualitative AnalysisMethod Editor

WW Seminar Tour

Spring 2006

Using Agilent Qualitative Analysis for Coc, Meth, Amp, & MDMA

All 16 MRM transition EICs generated and integrated automatically

All peaks elute< 1.5 min!

Coc

MDMA

Meth

Amp

d3-Coc

d5-MDMA

d5-Meth

d5-Amp

WW Seminar Tour

Spring 2006

G6410A Agilent QQQ

• Outstanding sensitivity for its price class– 0.5 pg RESERPINE on column at 20:1 S/N and 5% RSD with 3 simultaneous MRMs running

concurrently – utilizing Autotune parameters for mass analyzer calibration.

• 1635 amu mass range (m/z 15 – 1650)• Powerful and reliable AutoTune analyzer calibration software• New, Powerful and flexible Quantitation Software• New “Parameterless” integrator• Intelligent Qualitative Data Analysis Software• Flexible reporting based on Excel• Single point LC and MS instrument control• ESI, APCI, and MultiMode sources available