Ultra-High Sensitivity in Triple Quadrupole LC MS MS Performance.ppt · · 2016-09-11Ultra-High Sensitivity in Triple Quadrupole LC/MS/MSTriple Quadrupole LC/MS/MS ... 6460 triple

Ultra-High Sensitivity in Triple Quadrupole LC/MS/MSTriple Quadrupole LC/MS/MS

Performance

John HughesSr. Applications ConsultantAgilent Technologies

with contributions fromLinda Côté, John Fjelsted, Frank Kuhlman Ning Tang

Page 1 Our measure is your success. QQQ talk LCMS US TourWinter 2009

Frank Kuhlman, Ning Tang

The Agilent 6460: Cutting Edge QQQ Performance

With…

Agilent Jet Stream Technology

Unmatched sensitivity

Workflow improvementsThe industry’s most

100fg reserpine sensitivity with less than 10% RSD !

Faster method development The industry’s mostsensitive QQQ

g p y

Fast Pos/Neg switching

Faster MRMs and more MRMs per time segment

New Optimizer software enables faster MS/MS method development


Breaking the “fg barrier” with the new 6460A Triple Quad LC/MS/MSnew 6460A Triple Quad LC/MS/MS

Performance of Agilent QQQ models

Femtogram barrier

Performance of Agilent QQQ models

6410-High

6460

6410

6410 High Sensitivity

10-1 100 101 102 103

Femtograms


Femtograms

Agilent 6460 QQQ PerformanceShattering the Femtogram Barrier – 500 Attogramsg g g

Verapamil 500 attogramsVerapamil, 500 attograms

1.8 μm C18, 2.1 x 50mm0.4 mL/min

Breakthrough Sensitivity6460 triple quadrupole with Agilent Jet Stream technology breaks the femtogram barrier shown here with


6460 triple quadrupole with Agilent Jet Stream technology breaks the femtogram barrier, shown here with 500 attograms of verapamil injected on-column, using unit resolution for both Q1 and Q3.

Agilent 6460 QQQ Performance –500 attograms verapamil and extended dynamic linear g p yrange

VerapamilVerapamil

Five Decades of Linearity6460 triple quad with Agilent Jet Stream technology exhibits outstanding performance with 5 decades of


6460 triple quad with Agilent Jet Stream technology exhibits outstanding performance with 5 decades of linearity from sub-femtogram to 100 picograms of verapamil injected on-column.

Why focus on improving the efficiency of ESI?

Ionization and Transmission Efficiency in an Electrospray Ionization–Mass Spectrometry Interface Jason S. Pagea, Ryan T. Kellya, Keqi Tanga and Richard D. Smith, Biological Sciences Division Pacific Northwest National Laboratory RichlandBiological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA

Journal of the American Society for Mass SpectrometryVolume 18, Issue 9, September 2007, pages 1582-1590

“Ion transmission efficiency, also defined as the fraction of ES current that t th l h t diti ll b li it d b l t thenters the mass analyzer, has traditionally been limited by losses at the

mass spectrometer inlet and at the skimmer [[7] and [21]]. It has been estimated that only about one out of every 103–105 analyte ions generated by ESI at atmospheric pressure is actually detected usinggenerated by ESI at atmospheric pressure is actually detected using present instrument designs [[7], [10] and [22]].”


Agilent Jet Stream* Ion Generation Gas Dynamics Viewy

N b li iNebulizing gas

“S h d” h h

Enhanced efficiency nebulizer

Nozzle voltage

“Super-heated” sheath gas

The super-heated sheath gas

g

Heated drying gas

e supe eated s eat gascollimates the nebulizer spray and creates a dramatically “brighter” source Resistive sampling

capillary*


*Patent Pending

Agilent Jet Stream animation

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


Agilent Jet Stream In ActionObserving Thermal Gradient FocusingObserving Thermal Gradient Focusing

Fast-forward video of sheath gas heating f bi t t 400°C 8 i t


from ambient to 400°C over 8 minutes

Agilent Jet Stream available on 6460 QQQ 6x30 TOF/QTOFWhat else is new for these systems?

Octopole 1

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

6460 QQQ6530 QTOF

Collision Cell

Lens 1 and 210KV

Detector

2nd T bRough Pump

Turbo 1 Turbo 1 Turbo 1 Turbo 22nd Turbo

Resistive Capillary*

Increased turbo stage 1 pumping conductance

Agilent Jet Stream*

Octopole 1

Quad Mass Filter (Q1) Octopole 2

Capillary pumping conductanceJet Stream

Ion PulserDC QuadCollision CellLens 1 and 2

*Patent Pending


Turbo 2Rough Pump

Turbo 1 Turbo 1 Turbo 1

Reserpine sensitivity comparisons6460 QQQ: > >> Signal/Noise vs 6410 QQQ6460 QQQ: > >> Signal/Noise vs. 6410 QQQ for 500 femtogram injections on columnNoise = 3x RMS noise

SNR = 36:1

6410 QQQ10x improvement

New 6460 QQQSNR = 354:1

6 x 102 6 x 104


Limits of Detection (Estrone 3- sulfate) in Negative Ion ModeNegative Ion Mode100 fg on-column LOD 25 femtogram

100 fg on-column LOD 0.9 femtogram!

SNR (0.370min) > 3303.0

2.5

x103

2.0

1.55

1.0

0 5

6410 6460 S b F t LOD!

0.5

0


6410 6460 Sub-Femtogram LOD!

6460 Triple Quad compared to 6410 Triple Quad6410 Triple Quad:

Tramadol (LOD 23 fg) Phencyclidine (LOD 54 fg)Propoxyphene (LOD 33 fg)

Tramadol (LOD 3 fg) Propoxyphene (LOD 2 fg) Phencyclidine (LOD 3 5 fg)

6460 Triple Quad:

Tramadol (LOD 3 fg) Propoxyphene (LOD 2 fg) Phencyclidine (LOD 3.5 fg)

6460 8 S/N 6460 16 S/N 6460 15 S/N


6460 8 x more S/N 6460 16 x more S/N 6460 15 x more S/N

Five Orders of Linearity with new 6460 Triple Quad Alprazolam 22 femtograms to 22 nanograms!Quad Alprazolam 22 femtograms to 22 nanograms!

12 calibration levels

Y =0.13371x – .000076 R2 = 0 99912R = 0.99912


Agilent Jet Stream PerformanceRuggedness & Reproducibility – 6460 QQQ

5 pg injections of Alprazolam in human plasma extract

gg p y QQQ500 Injections of Alprazolam in Spiked Human Plasma Extract, ~ 10hrs.

250000

300000

1.2

1.4Internal Standard = 1.45 % RSD for amount

150000

200000

a R

espo

nse

0 6

0.8

1.0

ve R

espo

nse

External Standard = 3.76% RSD for areas

50000

100000

Are

a

0.2

0.4

0.6

Rel

ativ

00 50 100 150 200 250 300 350 400 450 500

Injection Number

0.0


6460 QQQ performance: some additionalsome additional application examples


Cyprodinil: LOD 21 femtogramswith 100 msec +/- switching and 5 msec dwell times

1x10

8

8.5

9

+ MRM (226.0 -> 118.0) 11_50MRM_Blank-r001-r002.d

Blank

100 fg, S/N=14

Positive ion

5.5

6

6.5

7

7.5

8 Blank

4

4.5

5

1x10+ MRM (226.0 -> 118.0) 14_50MRM_STD_50ppt-r001.d Noise (PeakToPeak) = 2.92; SNR (2.35min) = 14.0

7

8

9* 2.35

50

4

5

6

1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9


Counts vs. Acquisition Time (min)1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

Chloramphenicol: LOD 50 femtogramswith 100 msec +/- switching and 5 msec dwell times

1x10

5 6

5.8

6- MRM (321.0 -> 152.0) 01_10MRM_Blank-r010.d

Blank

250 fg, S/N=15.7

Negative ion

4 6

4.8

5

5.2

5.4

5.6 Blank

4.2

4.4

4.6

1x10- MRM (321.0 -> 152.0) 04_10MRM_STD_50ppt-r001.d Noise (PeakToPeak) = 1.02; SNR (1.50min) = 15.7

5

5.2

5.4

5.6

5.8

6

* 1.5015

4.2

4.4

4.6

4.8

1 3 1 4 1 5 1 6 1 7 1 8 1 9


Counts vs. Acquisition Time (min)1.3 1.4 1.5 1.6 1.7 1.8 1.9

Agilent 6460 QQQTrace analysis of pesticidesTrace analysis of pesticides

JetstreJetstre

3x10

0.7

0.8

0.9

1+ TIC MRM (** -> **) 50ppt-r001.d

1 1 2 2 3 3 4 4 5 5

Prometon

Ametryn

Prometryn Promazine

amamResultResultss0 1

0.2

0.3

0.4

0.5

0.6

SimazineAtrazine

y

Terbutryn

ss0.1

Counts vs. Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15

Solvent Std, 1uL injection, 50ppt (50 fg each onSolvent Std, 1uL injection, 50ppt (50 fg each on--column)column)


Agilent 6460 QQQTrace analysis of pesticides

1x10

7

8 BlankBlank+ TIC MRM (** -> **) pinch_blank1-r001.d

1 1 2 2 3 3 4 4 5 5

Trace analysis of pesticides

2

3

4

5

6

0

1

3x10

33.25

3.53.75

4

+ TIC MRM (** -> **) pinch_100ppt1-r001.d

1 1 2 2 3 3 4 4 55

Sample Prometon Prometryn Promazine

0 50.75

11.25

1.51.75

22.25

2.52.75

Sa p e

SimazineAtrazine

Ametryn

Terbutryn

0.250.5

Counts vs. Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15

Herbicides in Spinach Matrix Herbicides in Spinach Matrix --1uL injection, 100ppt level1uL injection, 100ppt level


(100 fg each on(100 fg each on--column)column)

Agilent MassHunter Workstation

More Innovations in M S t tWorkstation Mass Spectrometry Software


MassHunter OptimizerAutomated MRM Method Development SoftwareAutomated MRM Method Development Software

T diti l MS/MS th d d l tTraditional MS/MS method development:• Manual optimization of even three parameters, for dozens of compounds

=> MANY Days to WEEKS of tedious, interactive work

WITH Optimizer:• Optimization can be fully automated for multiple compounds

> O f DAYS f tt d d k!=> One or a few DAYS of unattended work!

Compound-specific optimization for MRM experiments• Selection and optimization of precursor and product ions• Selection and optimization of precursor and product ions• Supports optimization with multiple methods (e.g. pos then neg)• Creation of a compound database with optimized parameters for re-use


MassHunter OptimizerAdvantages over previous solutionsAdvantages over previous solutions

Utmost flexibility via support of all common optimization modes:M l i f i ( i )• Manual infusion (syringe pump)

• Automatic infusion (via loop injection at lower flows)• Flow injection analysis without column (FIA)• Analysis with column (multiple compounds per run)

Optimization WITH column for highest success:• Infusion or FIA can result in 20% of cmpds not optimizing

• Optimizer includes analysis WITH column using fast LC.

V i 2 l 2009 A t ti ti f LC/MS th d fVersion 2, early 2009: Automatic creation of LC/MS method for large numbers of compounds from Compound Database, using “Scheduled MRM”


MassHunter OptimizerCompound Entry & Method Setupp y p


MassHunter OptimizerStep 2 – Setup Precursor Ion SelectionStep 2 – Setup Precursor Ion Selection

• Define up to 4 adducts and/or charge states for each polarity• Define exclusion of ions based on m/z or intensity


MassHunter OptimizerStep 3 – Setup Product Ion SelectionStep 3 – Setup Product Ion Selection

• Optimizer will find the 4 most abundant product ions• Define minimum product ion mass• Define exclusion of ions based on m/z intensity or neutral losses


Define exclusion of ions based on m/z, intensity or neutral losses

MassHunter OptimizerStep 4 – Setup Optimization ParametersStep 4 – Setup Optimization Parameters

• Setup Sample introduction mode• Setup Sample introduction mode– Manual or automatic infusion, Injection with or without column

• Select data file directory and one or multiple LC/MS method(s)S f C


• Select range and step size for Fragmentor and Collision Energy

MassHunter OptimizerStep 5 – Execute Automatic OptimizationStep 5 – Execute Automatic Optimization

FragVColl E

Run 1: The precursor ion is selected based on user criteria (adduct p (ions, charge states) and Fragmentor voltage is optimized

(optional): fine adjustment of Fragmentor voltage Run 2: Coarse product ion scan finds the largest 4 product ions with p g p

corresponding Collision Energy (default CE 0, 10, 20, 30, 40).(optional) fine adjustment of Collision Energy

Post Run: Populates Compound Database with the 4 best transitionsw/ optimum Fragmentor Voltages and Collision Energies

Prints Optimization Report


MassHunter OptimizerStep 6 –View Results for ProjectStep 6 –View Results for Project

• Results for the project can be immediately viewed after optimization


MassHunter OptimizerStep 7 – Build LC/MS method from Cmpd DBStep 7 – Build LC/MS method from Cmpd DB

• Select compounds in database after filtering• Select transitions for each compound (all or individual)


• Easily drop into template LC/MS method

MassHunter “Scheduled MRM”Increased Utility and PerformanceIncreased Utility and PerformanceNew applications require quantitation of 100 – 1000 compounds in one MRM method !co pou ds o e et od• Food and environmental analysis (e.g. pesticides)• Targeted quantitation of proteins via peptides (proteomics)

WITHOUT Scheduled MRM:• Need to manually set up multiple time segments to maximize dwell times• Tedious to set up; problematic if changes in retention times

WITH Scheduled MRM:A t ti t f l i ti t ith t i t ti• Automatic setup of overlapping time segments without user intervention

• Fewer MRMs per unit time results in longer dwell time => incr sensitivity• Unaffected by chromatographic time shifts


y g p

Scheduled MRMIncreased Utility and PerformanceIncreased Utility and Performance

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

Time segments Time segments MRM

Compounds (x/block)

Cycle Time (sec)50 800.5 0.8 1 0.7

100 70

Scheduled MRM

2x shorter cycle times better for narrow chromatographic peaks, more

Max Coincident

Cycle Time (sec) 0.2 0.4 0.4 0.320 40 40 30


2x shorter cycle times better for narrow chromatographic peaks, more analytes, longer dwell time per analyte.

Application example for new software features –Optimizer and Scheduled MRMOptimizer and Scheduled MRMPeptide quantitation from protein digests for protein biomarker studies

•SpectrumMill Peptide Selector to choose useful peptides

•QQQ Optimizer to create MRM methods for quantitation•QQQ Optimizer to create MRM methods for quantitation

•HPLC-Chip MS/MS for rapid and reliable quant with small sample quantitiessample quantities

•MassHunter Quantitative Analysis for efficient data processing


Agilent HPLC-Chip/QQQ LCMS TechnologyNanospray chip configuration brings new era in high sensitivity quantitation

NanoLC system forl ti l h t h

HPLC Chip Cube system

analytical chromatography

CapLC pump for sampleloading on enrichment column

6410 QQQ LCMS

Sensitivity: down to low amolDynamic range: up to 105


y g p

Chromatographic PerformanceProtein Digest Mixture g

3

6

x107

Intensity

EIC m/z 985 615.7 secs

Conventional nanospray LC/MS

Conventional nanospray LC/MS

1

2

2

4

EIC m/z 985.6

0

3

0

2.0x108

6 secsHPLC-Chip/MS HPLC-Chip/MS

1

2

0.5

1.0

1.5

32% increase

EIC m/z 985.6

Red ced MS comple it + red ced ioni ation competition = impro ed ID

0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5Time [min] 10 12 14 16 18 20 22 Time [min]

0.0


Reduced MS complexity + reduced ionization competition = improved ID

MassHunter Optimizer – Compound(s) setup


MassHunter Optimizer – optimization results for peptides


Limit of Quantitation in the Low Amol RangePeroxidase 10 amol to 10 fmol spiked into 1 µg 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


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

100amol

1fmol10amol

R2 = 0 9988R = 0.9988


Excellent Reproducibility of MS ResponseHSA Peptide LVNEVTEFAK from 10 amol to 1 pmol (n=6)p p ( )

0.12

0.14

0.16

0.04

0.06

0.08

0.1

RSD

0

0.02

0.01

0.02

0.05 0.1 0.2 0.5 1 2 5 10 20 50 100

200

500

1000

fmolfmol

R2 0 9975

All RSDs are within 15%

R2 = 0.9975


All RSDs are within 15%

New 6460 Triple Quad LC/MS

Breaks the femtogram detection barrier for many compounds

New Ionization Technology – Agilent Jet StreamNew Ionization Technology – Agilent Jet Stream

Higher Signal Strength, great RSDs even in low fg region

New Acquisition Software Scheduled MRMsNew Acquisition Software - Scheduled MRMs

New Method Development Tool – MH Optimizer

A il t i itt d t ti i ti iAgilent is committed to continuous innovation in:Mass analyzer technologyBrighter sourcesI ti ftInnovative software



Documents

Ultra-High Sensitivity in Triple Quadrupole LC MS MS Performance.ppt · · 2016-09-11Ultra-High Sensitivity in Triple Quadrupole LC/MS/MSTriple Quadrupole LC/MS/MS ... 6460 triple