Development ofthe World’s Most

SensitiveTriple Quad MS

LC/MS DivisionAgilent TechnologySanta Clara, CA

Triple Quad? 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

Q U A N T I TAT I O N - S E N S I T I V I T Y

“…only about one out of every 103 to 105 ions generated by ESI at atmospheric pressure

is actually detected using present instrument designs.”

(Page, Smith, et al; JASMS, 2007, 18, 1582-1590)

3

6490 Triple Quadrupole LC/MS System with iFunnel Technology

N E W 6 4 9 0 T R I P L E Q U A D R U P O L E L C / M S

Zeptomole Sensitivity is here!Much higher performance in a

Much smaller box!iFunnel Technology• A new paradigm for atmospheric ion sampling• 10X sensitivity breakthrough for ESI• Improves assay robustness; simplifies sample prep

Curved, Tapered Collision Cell• Efficient ion transmission; Reduced noise• Compact design (25% smaller)

New scanning capabilities• 0.4 m/z precursor isolation reduces interference• Dynamic MRM• Data dependent scanning

4

Agilent Jet Stream

• Thermal confinement of ESI plume

• Efficient desolvation to create gas phase ions

• Creates an ion rich zone

5

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 10X 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 10X more ions

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 samplingcapillary

Nebulizing gas

Super-heated sheath gas

Heated drying gas

Enhanced efficiency nebulizer

Patent Pending

6

7

SIX bores

HALF as long

Six bores, Half the restriction means…

• Nearly 12x the amount of atmospheric gas sampledAND

• 5 - 10x the number of ions sampled over wide mass range.

But how do we handle all the extra gas molecules?

Compare:Standard

Capillary is180mm

6 Bore Capillary

Agilent’s Hexabore Atmospheric Sampling…. The Most Sensitive…. By Far.

Hexabore Sampling Capillary

• Hexabore sampling capillary increases ion sampling efficiency up to 10X compared to single bore

• Capillary length is reduced by 50% • minimize high mobility ion loss• pulls in more ion rich gas

Ion sampling gains are most dramatic with the combination of both Agilent Jet Stream technology with the hexabore sampling capillary

8

High Pressure Stage 1

Low Pressure Stage 2

ExistingRF IonGuide

Agilent’s Two Stage Ion Funnel Can Handle the Gas Load

Stage 18-12 Torr

Stage 21-3 Torr

Stage 1 offset breaks up the high pressure gas exiting the Hexabore Capillary

9

Novel Curved and Tapered Collision Cell DesignEfficient Ion Transmission over wide mass rangeReduces transmission of ionizer generated noise

Larger (5 mm)

EntranceAperture

Accepts more Ions

Smaller (3.5mm)

ExitApertureFocusesIons into

MS2

RF & DCVoltages Set

Confinement and Axial Acceleration

10

Improved Ion Sampling Efficiencies with iFunnel

Agilent Jet Stream positive ion

200 400 600 800 1000 1200 1400m/z

10x

14x11x

10x7x

113

302 1034

200 400 600 800 1000 1200 1400

1222

922622

322

118

m/z

5x5x

7x

7x7x 6490

6460

Agilent Jet Stream negative ion

11

New 6490 Increases Signal but also Reduces Noise

6X gain in signal shown in previous slide translates into 10X gain in signal-to-noise for alprazolam relative to the 6460 as shown below

6460S/N = 1,157:1 where noise = 1xRMS

6490S/N = 11,640:1 where noise = 1xRMS

12

1 pg Chloramphenicol (8X) 1 pg Acid red 4 (9X)

6460 BB1: CAT CConly

Triton

Relative Increase insignal of Triton vs. 6460

6 x

3x103

0

1

2

0.5 1 1.5 2Acquisition Time (min)

6460

64903

00.10.20.30.40.50.60.70.80.9

11.1

0.2 0.4 0.6 0.8 1Acquisition Time (min)

6490 with iFunnel vs 6460 in negative ion mode

x103

6490

6460

13

AT T O G R A M D E T E C T I O N L I M I T S W I T H T H E 6 4 9 0

100 attograms of verapamilinjected on column

Six orders of linear dynamic range(100 attograms to 100 picograms)

14

Unprecedented Six logs of Linearity with 6490

Y = 65283 x – 0.390412R2 = 0.9976

Femtograms

Are

a R

espo

nse

1

10

100

1000

10000

100000

1000000

10000000

0,1 1 10 100 1000 10000 100000

100 Attogram to 100 Picogram varapmil on-column

15

New 6490 Triple installation Spec: S/N > 10,000:1 so the 6490 should be able to detect 1 fg ?

1 pg reserpine on-column S/N > 22,605:1

16

6490 Triple - Attogram Sensitivity for Reserpine1 fg of reserpine injected on-column

Acquisition Time [min]

S/N scales as expected

NO gaming the noise spec

17

Agilent Triple Quads: 2006 – 2010Relentless increase in signal-to-noise specs

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

2006 2007 2008 2009 2010

64106430

6460

6490SN

R 1

pg

rese

rpin

e

18

Res

erpi

ne R

espo

nse

(Sig

nal/N

oise

)

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

2004 2005 2006 2007 2008 2009 2010

AB/SciexAgilent

New Agilent 6490

Agile

nt 6

460

Agile

nt 6

430

Agile

nt 6

410

T R I P L E Q U A D R U P O L E I N S T R U M E N T S P E C I F I C AT I O N S

19

2.5 Femtogram on-columnArea RSD = 23.7% (n=6)Mean accuracy = 104

50 pg/mL Fluticasone in plasma with 6460

Acquisition Time (min)0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4

*1.534 min.1x10

4.2

4.3

4.4

4.5

4.6

4.7

4.8

4.9

5

5.1

5.2

5.3

MRM unit / unit resolution (0.7 amu / 0.7 amu)

O

CH3

F

F

OH

H

CH3

OS

F

O

CH3O

CH3

Fluticasone propionate

20

5 pg/mL Fluticasone in plasma with new 64902.5 Femtogram on-column

Area RSD = 8.7% (n=5)Mean accuracy = 104

Acquisition Time [min]

0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

1x10

4.2

4.3

4.4

4.5

4.6

4.7

4.8

4.9

5

5.1

5.2

5.3

1.765 min.

O

CH3

F

F

OH

H

CH3

OS

F

O

CH3O

CH3 MRM enhanced / unit resolution (0.4 amu / 0.7 amu)

Fluticasone propionate 1x10

4.2

4.3

4.4

4.5

4.6

4.7

4.8

4.9

5

5.1

5.2

5.3

21

1x10

5

6

7

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4

Q1 Unit (0.7 amu)Q2 Unit (0.7 amu)

The Value of Enhanced Resolution on 6490Fluticasone propionate (inhalant) in human plasma 2.5 fg on-column)

Q1 Enhanced (0.4 amu)Q2 Unit (0.7 amu)

1x10

5

6

7

Acquisition Time (min)0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4

Enhanced Resolution gives increased selectivity for analytes in complex matrices

22

Agilent Dynamic MRM application kitsOur approach to make you more productive

G1733AA - Pesticide DMRMData Base Kit

• Database with conditions, transitions, and retention times for 600 compounds

• Pre-configured methods for 300 pesticides for different LC configurations

• ZORBAX Eclipse Plus UHPLC column

• Small suite positive and negative ion test mix for method validation and adjustment

• Extensive documentation and free trial SampliQ QuEChERS kits

Multi residue pesticide method Compounds included in method

313 compounds including 9 isomers

Acephat Butocarboxim-sulfoxid Desmedipham Fenamiphos-sulfon Flusilazol Metamitron Oxadiazon Proquinazid Terbutylazin

Acetamiprid Buturon DMSA Fenamiphos-sulfoxid Flutolanil Metazachlor Oxadixyl Prosulfocarb Tetraconazol

Acrinathrin Cadusaphos Dichlofluanid Fenarimol Flutriafol Metconazol Oxamyl Pymetrozin Thiabendazol

Aldicarb Carbaryl Dichlorvos Fenazaquin Foramsulfuron Methabenzthiazuron Oxasulfuron Pyraclostrobin Thiacloprid

Aldicarb-sulfon Carbendazim Diclofop-methyl Fenbuconazol Formetanat-Hydrochlorid Methacrifos Oxydemeton-sulfon Pyrazophos Thiamethoxam

Aldicarb-sulfoxid Carbofuran 3 hydroxy Dicrotophos Fenbutatinoxid Formothion Methamidophos Paclobutrazol Pyridaben Thifensulfuron-methyl

Alloxydim Carbofuran Diethofencarb Fenhexamid Fosthiazat Methidathion Paraoxon-methyl Pyridaphenthion Thiodicarb

Amidosulfuron Carbosulfan Difenoconazol Fenobucarb Fuberidazol Methiocarb Penconazol Pyridat Thiofanox sulfon

Aminopyralid Carfentrazone-ethyl Difenoxuron Fenoxaprop free acid Furathiocarb Mthiocarb-sulfon Pencycuron Pyrifenox Thiofanox sulfoxid

Amitraz Chlorantraniliprol Diflubenzuron Fenoxycarb Halosulfuron-methyl Methiocarb-sulfoxid Pendimethalin Pyrimethanil Thiofanox

Asulam Chlorfenvinphos Diflufenican Fenpiclonil Haloxyfop free acid Methomyl Pethoxamid Pyriproxifen Thiophanat

Atrazin Chlorfluazuron Dimefuron Fenpropimorph Hexaconazol Methoxyfenozid Phenmedipham Pyroxsulam Thiophanat-methyl

Avermectin B1a Metabolite Chloridazon (Pyrazon) Dimethenamid Fenpyroximat Hexaflumuron Metobromuron Phenthoat Quinalphos Tolclophos-methyl

Avermectin B1a Chlorimuron-ethyl Dimethoat Fenthion Hexythiazox Metolachlor Phorat Quinmerac Tolylfluanid

Avermectin B1b Chloroxuron Dimethomorph Fenthion-oxon Imazalil Metosulam Phosalon Quinoxyfen Topramezone

Azimsulfuron Chlorsulfuron Dimoxystrobin Fenthion-oxon-sulfon Imidacloprid Metoxuron Phosmet Quizalfop free acid Tralkoxydim

Azinphos-ethyl Chlozolinat Diniconazol Fenthion-oxon-sulfoxid Indoxacarb Metrafenon Phosmet-oxon Quizalofop-ethyl Triadimefon

Azinphos-methyl Chromafenozid Dioxathion Fenthion-sulfon Ipconazol Metribuzin Phosphamidon Rimsulfuron Triadimenol

Azoxystrobin Clethodim Diuron Fenthion-sulfoxid Iprodion Metsulfuron-methyl Phoxim Rotenone Triasulfuron

Beflubutamid Clofentezin EPN Flazasulfuron Iprovalicarb Mevinphos Picoxystrobin Sethoxydim Triazofos

Benalaxyl Clomazon Epoxyconazol Flonicamid Isoproturon Molinat Pinoxaden Siduron Tribenuron-methyl

Benfuracarb Clopyralid Ethaboxam Florasulam Isoxaflutol Monocrotophos Piperonyl butoxid Silthiopham Trichlorfon

Bensulfuron-methyl Clothianidin Ethiofencarb Fluazifop free acid Kresoxim methyl Monolinuron Pirimicarb Simeconazol Tricyclazol

Benthiavalicarb Cyazofamid Ethiofencarb-sulfon Fluazifop-P-butyl Lenacil Monuron Pirimiphos-methyl Spinosad Trifloxystrobin

Bifenazat Cycloxydim Ethiofencarb-sulfoxid Flufenacet Linuron Myclobutanil Pirmicarb-desmethyl Spirotetramat Triflumizol

Bifenox Cyhexatin Ethion Flufenoxuron Lufenuron Napropamid Prochloraz Spiroxamin Triflumuron

Bispyribac Cymoxanil Ethiprol Flumetsulam Malaoxon Neburon Profenophos Sulfosulfuron Triflusulfuron-methyl

Bitertanol Cyproconazol Ethirimol Fluometuron Mandipropamid Nicosulfuron Promecarb Tebuconazol Triforin

Bromacil Cyprodinil Ethofumesat Fluopicolid Mecarbam Nitenpyram Propamocarb Tebufenozid Trinexapac-ethyl

Bromuconazol Cyromazin Ethoprophos Fluoroglycofen-ethyl Mepanipyrim Novaluron Propaquizafop Tebufenpyrad Trit iconazol

Bupirimat Daminozid Etofenprox Fluoxastrobin (E) Mesosulfuron-methyl Nuarimol Propargite Teflubenzuron Tritosulfuron

Buprofezin DEET Famoxadon Fluquinconazol Mesotrione Ofurace Propiconazol Tembotrion Zoxamid

Butocarboxim Demeton-S-methyl Fenamidon Fluroxypyr Metaflumizone Omethoat Propoxur Tepraloxydim

Butoxycarboxim Demeton-S-methyl-sulfon Fenamiphos Flurtamon Metalaxyl Orthosulfamuron Propyzamid Terbutryn

Pesticide MRM Data Base G1733AA

Page 25

Multi residue pesticide method Comparison of MRM and Dynamic MRM

Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Compounds (10msec / Compound

Cycle Time (msec)

Max Coincident

Cycle Time (msec)

MRM

50 80

400 400 400 40020 40 40

Dynamic MRM

500 800 1000

30

Time Segment 1 Time Segment 2 Time Segment 3 Time Segment 4

700100 70

• 2 x shorter cycle times supports narrow chromatographic peaks, more analytes or longer dwell per analyte.

Multi residue pesticide method Dynamic MRM simulation

Time >>>

Abu

ndan

ce>>

>

ConcurrentCompounds

Multi residue pesticide method MRM traces for tomato extract spiked at 10 µg/kg

5x10

0

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

1.5

1.6

1.7

1.8

+ESI MRM Frag=380.0V CID@4.0 (116.0000 -> 70.1000) tomato 5 pg_uL.d

Counts vs. Acquisition Time (min)7.6 7.7 7.8 7.9 8 8.1 8.2 8.3 8.4 8.5

more than 160 MRMtransitions in 1 min

Dynamic MRM viewer – display of concurrent MRMsQuantitative analysis of pesticides (dMRM)

Page 29

Compounds at a glance – peak review in MH Quant

Results for black teaMRM traces for black tea extract spiked at 0.5 pg/µl

nearly all compounds were found

equates to 2.5 µg/kg in the sample 25% of the maximum residue limit

Results for black teaCoverage of method for black tea

0

50

100

150

200

250

300

350

0,5 2,5 5 25 50 250 500

262298 302 312 312 312 312

concentration in µg/kg in black tea

number of compounds dectected in spiked black tea

All compounds found in black tea

MRL

Results for black teaDimethoate/Omethoate (No. 1 in “Check Your Scope” ranking)

0.5 µg/kg 2.5 µg/kg 5 µg/kg 25 µg/kg 50 µg/kg

Omethoate

Dimethoate

Results for black teaOxamyl and Carbendazim (High in “Check Your Scope” ranking)

0.5 µg/kg 2.5 µg/kg 5 µg/kg 25 µg/kg 50 µg/kg

Oxamyl

Carbendazim

Results for tomato extractCoverage of method for tomato matrix

0

50

100

150

200

250

300

350

0,1 0,5 1 5 10 50 100

281308 309 313 313 313 313

concentration in µg/kg in tomato

number of compounds dectected in spiked tomato

All compounds found

MRL

Ultra Trace Levels of Pesticides in Potable Water

Simetryn S/N = 9

Atraton S/N =: 819

Simazine S/N = 18

Secbumeton S/N = 51

Prometon S/N = 40

Atrazine S/N = 28

Triazine herbicides analyzed at 50 parts per quadrillion

6490 Triple Quad with on-line enrichment

36

Triggered MRM – What Problem Does tMRM Solve?

Gap in MRM trace, numerically filled in.Typically > 100 msec

Data dependent product ion scans leaves gaps in MRM• mix of raw and synthetic points• higher RSDs and higher LOQs

DDA –scan switch to PIS

Targeted Quant + Compound Identification

37

Triggered MRM data acquisitionNew acquisition mode for enhanced confirmation• Triggered MRM is new data dependant acquisition mode for the

confirmation of target compounds• When the signal heights of the primary MRMs exceed a given threshold up

to 9 additional MRM transitions are triggered• Triggered MRM makes use of the well established Dynamic MRM

functionality• Triggered MRM has advantages over data dependant product ion scans

because:• It is more sensitive due to longer dwell times per transition and due to the ideal

collision energies for each transition• Peak shapes of the primary transitions are not compromised due to constant cycle

times• In spectra fragment ratios are very constant due to relatively long dwell times per

transition• Triggered MRM spectra can be saved in an user defined library and can

be searched from the qualitative and quantitative software.

tMRM for up to 8 Confirmatory Ion Transitions- when primary compound MRM triggers confirmatory MRMs

Plot counts versus m/z for product ions “Composite MS/MS spectra for compound b”

coun

ts

100 200 300

Product %MRMIon Scan Sensitivity

TimeQTRAP

5500 ~ 100 msec ~ 10%

Vantage ~ 150 msec ~ 2%Xevo ~ 150 msec ~ 2%

6490 ~ 30 msec 100%

Faster compound ID at higher sensitivity with tMRM

39

Triggered MRM data acquisitionMethod parameters

1. Primary and secondary MRM transitions are imported from Optimizer database (in total up to 10 MRMs per compound)

2. Thresholds for triggering are specified based on response of primary transitions at LOQ3. Number of repeats for triggering is specified – typically 5 repeats are sufficient for

reproducible MRM spectra4. Cycle time stays constant to allow for reproducible quantitation whereas dwell times are

maximized based on number of concurrent MRMs

1.2.

3.4.

Quantitative analysis of pesticidesUser editable Triggered MRM spectra library for pesticides

Quantitative analysis of pesticidesAutomatic creation of triggered MRM library from MH Quant

Quantitative analysis of pesticidesTriggered MRM traces of tebufenpyrad in QC sample (50 ppb)

2x10

0

0.2

0.4

0.6

0.8

1

Cpd 1: Tebufenpyrad: +ESI MRM:361 (9.611-9.752 min, 15 Scans) Frag=165.0V CID@28.0 (334.2…

147.1000

117.0000

132.100091.1000 105.1000

2x10

-1

-0.5

0

0.5

1

Cpd 1: Tebufenpyrad: +ESI MRM:361 (9.611-9.752 min, 15 Scans) Frag=165.0V CID@28.0 (334.2…

147.1000

117.0000132.100091.1000 105.1000

2x10

0

0.2

0.4

0.6

0.8

1

Tebufenpyrad C18H24ClN3O + MRM CAS_Library.mslibrary.xml

117.0000 145.0000

132.100091.1000 105.1000

Counts vs. Mass-to-Charge (m/z)75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170

Library match score: 96.75

Quantitative analysis of pesticidesLibrary match for tebufenpyrad in QC sample

Acquired TMRM spectrum

TMRM library spectrum

Comparison Acquired vs.TMRM library spectrum

Quantitative analysis of pesticidesLibrary match for matrix compound in ginger extract

Acquired TMRM spectrum

TMRM library spectrum

Comparison Acquired vs.TMRM library spectrum

Quantitative analysis of pesticidesBatch-at-a-glance – quantitation of tebuthiuron in chamomile extract

Signal for tebuthiuron for quantifier and qualifier- RT slightly higher than reference RT but within accepted RT range- Qualifier ratio higher than expected (183% of expected value)

Quantitative analysis of pesticidesTriggered MRM traces of tebuthiuron in QC sample (50 ppb)

2x10

0

0.25

0.5

0.75

1

Cpd 14: Tebuthiuron: +ESI MRM:370 (6.070-6.250 min, 19 Scans) Frag=105.0V CID@12.0 (229.10…

172.1000

62.000089.1000

74.0000 116.0000157.1000

2x10

-1

-0.5

0

0.5

1

Cpd 14: Tebuthiuron: +ESI MRM:370 (6.070-6.250 min, 19 Scans) Frag=105.0V CID@12.0 (229.10…

172.1000

62.0000 89.100074.0000 116.0000 157.1000

2x10

0

0.25

0.5

0.75

1

Tebuthiuron C9H16N4OS + MRM CAS_Library.mslibrary.xml

172.1000

62.0000 116.000089.100074.0000 157.1000

Counts vs. Mass-to-Charge (m/z)60 70 80 90 100 110 120 130 140 150 160 170

Library match score: 97.93

Quantitative analysis of pesticidesLibrary match for tebuthiuron in spiked chamomile extract

Quantitative analysis of pesticidesLibrary match for matrix compound in spiked chamomile extract

200 zeptomoles

verapamilInjected on-column

Acquisition Time (min)

+ MRM (455.3 -> 164.9)

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1x10

4.2

4.3

4.4

4.5

6490 Achieves Zeptomolar Detection Limits

50

The 6490 has Zeptomole Capabilitywhat is a zeptomole?

Photo of new 6490 taken from 1 meter distanceHow far away is 1021 meters?

51

The Milky Way Galaxy as seen from Hubble1021 meters is at the edge of the known universe

52