Agilent Solutions for Ultra-High Performance LC/MS Ken Miller · 2016-08-30 · Ken Miller Global...

Agilent Solutions for Ultra-High

Performance LC/MS Ken MillerGlobal Director of Marketing, LC/MSS t b 29 2009September 29, 2009

Agilent Solutions - OverviewServing The Diverse Needs of the Mass Spec Community

• 1290 Infinity UHPLC• HPLC-Chip II• 7100 CESeparations

T h l • 7890A GC

• 7000A GC Triple Quad• 7700 Series ICP-MS

Technology

MS Detector• 6430 Triple Quad LC/MS• 6540 UHD Accurate Mass Q-TOF

• Mass Profiler Professional w/ Pathway Analysis

Technology

$0 3 BData Analysis / Mass Profiler Professional w/ Pathway Analysis• Personal Compound AMRT Databases• MassHunter Quant Analysis Enhancements

• Quick Start Solution Kits for LC GC LC/MS & GC/MS

$0.3 B34% of total revenue

ata a ys s /Application

Software

• Quick Start Solution Kits for LC, GC, LC/MS & GC/MS• Petrochemical• Food Safety• Environmental• Toxicology

• Metabolomics / Proteomics Standards & Workflows

Solutions

Agilent 1290 Infinity LCThe best liquid chromatography system for MS – EVER!

LowestDelay

Volume

Pump (w/o) mixer: 10 µLPump, Fixed Loop 20 µLPump, Fixed Loop, JetWeaver 55 µLVolume

Highest

p p µ

ALS precision for small volumes: <1.5% from 0.5-1µLm, <0.7% from 1-2µL,

0 2 % @ 2 20 l (40 l) **Precision <0.25% @ 2-20 µl (40 ul) **Pump Active Damping:

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

Best <0.002% carry-over with ChlorhexidineOptional needle seat backflushing with FlexCubeFixed Loop or Variable Loop Injections

BestAutosamplerPerformance

1200bar @ 2mL/min for highest resolution per timeReduced Ion & Matrix Suppression

HT-Solution for up to 2000 samples/day (ACR)Complete Integration and control from MassHunter

GreatestProductivity p g

Enables method conversion from/to any (U)HPLC

** preliminary Minimum Specs

1290 Infinity Binary Pump- A Peek Under The Hood!

Solvent selectionvalveDegasser

Jet Weaver

Multi-layerHeat exchanger

High ResolutionPump drives

Channel

ChannelB

PurgeValve

Silicon CarbidePistons

Channel A

1290 Infinity – Compatible with any HPLC and UHPLC

A new power range providing maximum performance, flexibility, compatibility and investment

protection1290 Infinity

1000Acquity

800 Dionex RSLC

Agilent RRLCThermoAccela

Shimadzu UFLCXR

Standard LC

0 1 2 3 4 5 ml/min

HPLC-Chip IIEnhanced Performance & Applications

Carbon Implantation Technology(patent pending)

• Optimized connections• Optimized connections• Improved precision• 2X extended Chip life – lower cost per analysis

New PhosphoChip

• Integrated TiO2 enrichment and analysis• Improved sensitivity

M h f t ti t lt• Much faster time-to-result

Powerful New Applications

• Combination with Agilent 6400-Series Triple Quads• Pharma:

• ADME/DMPK microdosing• Dried Blood Spotsp

• Targeted Protein/Peptide Quant

HPLC-Chip IIEnhanced Performance & Applications

Carbon Implantation Technology(patent pending)

• Optimized connections• Optimized connections• Improved precision• 2X extended Chip life – lower cost per analysis

New PhosphoChip

• Integrated TiO2 enrichment and analysis• Improved sensitivity

M h f t ti t lt• Much faster time-to-result

Powerful New Applications

• Combination with Agilent 6400-Series Triple Quads• Pharma:

• ADME/DMPK microdosing• Dried Blood Spotsp

• Targeted Protein/Peptide Quant

Relentless QQQ Innovation2006 2009

6430 Spring 2009 30 ms +/- Switching2006 - 2009

6460 Fall 2008 Agilent Jet Stream5-10X Sensitivity 100 ms +/- Switching2 MRM

g1 ms MRMs

6410B Winter 2008 500 ms Polarity SwitchingHotBox Option (2X)

2 ms MRMs

Dynamic MRMPeptide Optimizer

E l 2007 R ti 2 5 F t6410 Fall 2006500 f S iti it S

6410A Spring 20072,000 m/z Mass RangeChip Cube Compatible

Optimizer and DB

Excel 2007 Reporting 2-5x FasterMassHunter MS Optimizer

500 fg Sensitivity SpecHigh ReliablityVery Affordable

Compliance SW support for 21 CFR Part 11

MassHunter Quant DA – Batch at a GlanceParameter-less IntegratorE cel Reporting

for 21 CFR Part 11

Excel Reporting

6400 Triple Quadrupole Product LineExcellent Value and Performance

6410 Triple Quadrupole LC/MSRobust, Easy-to-Use.Overall lowest cost

• Axial Acceleration Collision Cell• Polarity switching

of ownership. • MRM analysis• Solid performance, good sensitivity

6430 Triple Quadrupole LC/MSFast, robust,sensitive quantitation.

• Fast optimization with Optimizer• Fast polarity switching

D i MRM h dIdeal for HPLC-Chip • Dynamic MRM methods• Complex multi-analyte analyses• Targeted Protein Quantitation

6460 Triple Quadrupole LC/MSThe world’s mostsensitive TripleQuadrupole LC/MS

• Agilent Jet Stream – extreme, sub-fg sensitivity!• Perfect for the most demanding applications• Fast polarity switchingQuadrupole LC/MS Fast polarity switching• Dynamic MRM methods

6540 UHD Accurate Mass Q-TOF LC/MS The Highest Performing Bench-top Q-TOF - By Far

Exceptional Ultra High Definition Performance…With No Trade-Offs

• Up to 40 000 resolution• Up to 40,000 resolution• Excellent isotopic fidelity• Mass measurement error down to 500 ppb• 5 orders of linear dynamic range • Femtogram level sensitivity with Agilent Jet Stream• Femtogram-level sensitivity with Agilent Jet Stream• FAST acquisition for UHPLC – up to 20 spectra/second

Made Possible by Continuing Technology Breakthroughs

• Ion Beam Compression (IBC) cools & focuses ion beam• Extended Flight Tube with Enhanced Mirror Technology (EMT)• New Photonis Fast Bipolar Detector

The Ultimate Qualitative Analysis SystemThe Ultimate Qualitative Analysis System

• Proteomics/Metabolomics• Non-targeted food/environmental screening• Impurity analysispu y a a ys s• Metabolite ID

MassHunter WorkstationOne software for all Agilent MS SystemsOne software for all Agilent MS Systems

Minimize learning (and maximize productivity)Minimize learning (and maximize productivity)with a common software for all of your

mass spec systems

Driving Applications Forward New MassHunter Software Tools Enable Huge Productivity Gains

High Throughput Quantitation

• MH Optimizer quickly and easily optimizes MS/MS signal• Dynamic MRM methods deliver robust assays fasterDynamic MRM methods deliver robust assays faster• Easy export to WATSON LIMS• Fast flexible Custom Reporting is 10X faster

High Throughput Targeted Screeningg g p g g

• Personal Compound Databases (PCD) use accurate mass and RT• Available for:

• Metabolomics 23,000 compoundsP ti id 1 600• Pesticides 1,600

• Toxicology/Forensics 7,000• New MS/MS Library Searching Capability

Proteomics / Metabolomics & Non-targeted Screeningg g

• New Mass Profiler Professional • Intuitive workflows• Powerful statistical tools rendered easy-to-apply

P th A hit t f di t bi h i l th i t ti• Pathway Architect for direct biochemical pathway interrogation

Ultra High Definition Optimizing all Analytical Dimensions

Signal Response

• Sensitivity• Dynamic Range• Linearity

Optimizing all Analytical Dimensions

Signal Response Linearity

• Peak Resolving Power• Peak Capacity• Peak Capacity• Separation Speed

Mass Spectrum

• Ionization Profile• Mass Accuracy• Isotopic Fidelity• Mass Resolving Power• Data Mining g• Acquisition Rate

g• Differential Profiling• Annotation

High Definition Mass Spec for Fast LCPractical Considerations

As peaks get narrower, the MS detector must scan faster.MS cycle times are reduced.

W1/2 = 0.63 sec

Excellent Data Quality:Avg W1/2 = 0.63 sec Avg W = 1 8 sec

3 Avg. W = 1.8 sec5 points across W1/2 13 points across WArea RSD [%] = 4.7SNR (3RMS) = 1672

Counts vs. Acquisition Time (min)

0.49 0.495 0.5 0.505 0.51 0.515 0.52 0.525 0.53 0.535 0.54 0.545 0.55 0.555 0.56

W = 1.8 sec

MS Cycle time = 122.5 ms

During each cycle, the MS system must:Triple Quad: analyze many MRM transitions and switch polarityTOF/Q-TOF: acquire as many accurate mass MS and MS/MS scans as possibleTOF/Q TOF: acquire as many accurate mass MS and MS/MS scans as possible

Agilent’s New 6430 and 6460 Triple QuadsThe ideal Triple Quads for Agilent’s new 1290 Infinity UHPLCp g y

High Pressure

1200bar @ 2mL/min fast chromatographyPressure

high resolution columns

Reduce run times by > 50%High Performance.

Reduce run times by > 50%Analyze > 1,000 samples per dayIncrease sensitivity with sharp peaks

1290/6460 Triple Quad 1290/6430 Triple Quad

6430 Triple Quad LC/MS6460 Triple Quad LC/MS 6430 Triple Quad LC/MS6460 Triple Quad LC/MS

Comparative sensitivity – 6430 vs 6460The 6460 is about 5 times more sensitive than 6430 for reserpineThe 6460 is about 5 times more sensitive than 6430 for reserpine

6460Typical S/N > 2,100:1

1 pg reserpinewhere noise = 1 X RMS

6430Typical S/N > 400:1Typical S/N > 400:1

1 pg reserpinewhere noise = 1 X RMS

Jet Stream Technology on 6460 Triple Quad: delivers more sensitivity with tighter spray plumedelivers more sensitivity with tighter spray plume

Super heated gas confines spray plume to increase analyte concentrations near the orifice with gas dynamicsconcentrations near the orifice with gas dynamics

Sub-femotgram sensitivity with 6460500 Att il l500 Attograms verapamil on-column

Power Range for 1290 Infinity LC–most powerful UHPLC

1200 This app falls in the “Power Range”

2.1mm ID 3 – 4.6mm ID

Power = Pressure x Flow Rate

Agilent 1290

This app falls in the Power Range

Vendor A

Agilent 1290 Infinity

800Vendor E

Agilent RRLCVendor C

Vendor D

Standard LC

0 1 2 3 4 5 ml/min

1290 Infinity - Ultra High Performance SeparationsChallenges for Quant sufficient sampling across peak

Challenges for Quant – sufficient sampling across peak

60 Typical Peak Width

~ 2 sec

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 18 200

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

Polarity Switching: – is it fast enough for UHPLC?A. Switch power supply from positive to negative B. Stabilize ion opticsC. Run negative MRM transitionsD Switch power supply from negative to positive

D. Switch power supply from negative to positiveE. Run positive MRM transitions

DetectorCollision Cell

Metabolism study with negative/positive MRMs:6460 Triple Quad with 30 millisecond polarity switching

+MRM (455.3 -> 165.1)Verapamil, 0.54 min

+MRM (455.3 -> 165.1) Verapamil

Flow rate 1.5 mL/min, 1,070 bar5x10

6Verapamil, 0.75 min

+MRM (455.3 -> 165.1)5x10

6Verapamil+MRM (455.3 -> 165.1)

Flow rate 1.0 mL/min, 850 bar

p Q p y g

5x10 +MRM (386.0 -> 122.1

Avg W1/2 = 0.44 sec 11 points across WRel. Area RSD [%] = 4.0

Buspirone,

5x10 +MRM (386.0 -> 122.1

Buspirone,

5x10 +MRM (386.0 -> 122.1

Buspirone,

5x10 +MRM (386.0 -> 122.1

Avg W1/2 = 0.44 sec 11 points across W

RSD [%] = 4.0

Buspirone

Avg W1/2 = 0.71 sec 15 points across WRel. Area RSD

Buspirone,+MRM (386.0 -> 122.1

Rel. Area RSD [%] = 2.9

RSD [%] = 2.9

Buspirone+MRM (386.0 -> 122.1

4x10 +MRM (272.2 -> 215.2)

p ,0.22 min

Dextromethorphan

4x10 +MRM (272.2 -> 215.2)

p ,0.22 min

Dextromethorphan

4x10 +MRM (272.2 -> 215.2)

p ,0.22 min

Dextromethorphan

4x10 +MRM (272.2 -> 215.2)

Buspirone

D t th h

RSD [%] = 4.2 0.5

Buspirone, 0.40 min

Dextromethorphan+MRM (272.2 -> 215.2)

Rel. Area RSD [%] =

Buspirone, 0.40 min

Avg W1/2 = 1.3 sec 35 points across WRel. Area RSD [%] =

Buspirone, 0.40 min

Avg W1/2 = 1.3 sec 35 points across WRSD [%] = 1.8

Buspirone

D t th h+MRM (272.2 -> 215.2)

3x10 -MRM (294 0-> 250 0

Dextromethorphan, 0.30 min

Avg W 1/2 = 0.98 sec 28 points across WRel. Area RSD [%] = 2.20.5

3x10 -MRM (294 0-> 250 0

DextromethorphanAvg W 1/2 = 0.98 sec 28 points across W

RSD [%] = 2.2

Analysis time < 0 8 min

-MRM (294.0 -> 250.0

-MRM (294.0 -> 250.0 Analysis time < 1 1 min

-MRM (294.0 -> 250.0

Dextromethorphan

-MRM (294.0 -> 250.0 Analysis time < 1 1 min3x10

-MRM (294.0 -> 250.0

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 1 1 1 2

Diclofenac, 0.73 minAvg W 1/2= 0.37 sec 9 points across WRel. Area RSD [%] = 7.8

-MRM (294.0 -> 250.0

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 1 1 1 2

-MRM (294.0 -> 250.0

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 1 1 1 2

-MRM (294.0 -> 250.0

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 1 1 1 2

DiclofenacAvg W 1/2= 0.37 sec 9 points across W

RSD [%] = 7.8

Analysis time < 0.8 min

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 1 1 1 20 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 1 1 1 20 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 1 1 1 20 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 1 1 1 2

Diclofenac, 1.04 min

( Analysis time < 1.1 minx10

( Analysis time < 1.1 min

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

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

Polarity switching with UHPLC & 6460 Triple Quad 30 msec polarity switching for sub-2 second peaks

3x103.8 - MRM (294.0 -> 250.0

30 msec polarity switching for sub 2 second peaks

DiclofenacAverage W 1/2 = 0.37 sec

9 points across W

9 points across 1.5 sec peak

Rel. Area RSD [%] = 7.8%

vs. [ ]

0.7 0.71 0.72 0.73 0.74 0.75 0.76 0.77

W = 1.5 sec

Polarity switching versus non-switched method Correlation of methods

Selected example:

Buspironer2 = 0.9999

Correlation of methods

r2 = 0.9914Selected example:

% Verapamil remaining Dextromethorphanr2 = 0.9848

t10Diclofenacr2 = 0.9946

ana t10

-sw t15

Pos/Neg switching analysis

00 10 20 30 40 50 60 70 80 90 100

No Time for “Time Segment Breaks” with UHPLC pesticide analysis with 1290/6460 LC/MS Systemp y y

C t A i iti TiCounts versus Acquisition Time

Dynamic MRM solves the crowded peak problem monitors ion transitions only when compounds elute

Const. Cycle Time:Concurrent MRMs

Variable dwell Time:

Dynamic MRM: fewer concurrent MRMs than with Time Segmentsimport retention timesadd a uniform retention time window – 0 5 min for exampleadd a uniform retention time window 0.5 min for example

300 Pesticides: 15 min Analysis with DMRM

Mean area RSD = 3.2% with Dynamic MRMCycle Time: 200 msecy

Eight Minute Analysis of 250 Pesticides 1290 Infinity UHPLC and 6460 Triple Quad& with DMRM

9.6 1 1500ppt, dynamic MRMPeak widths ~ 1 secondCycle time: 100 msec

8.2 Cycle time: 100 msecMin dwell: 2.5 msec

Zorbax Eclipse Plus C182.1 x 100mm (1.8μm)

3.193427

Counts vs. 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 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4

Target Screening of PPCPs in Water Matrices Using UHPLC and LC/MS/MS QQQ

Pharmaceuticals and Personal Care Products (PPCPs) refer, in general, to any product used by individuals for personal health or cosmetic reasons or used by

Using UHPLC and LC/MS/MS QQQ

agribusiness to enhance growth or health of livestock.

PPCPs comprise a diverse collection of thousands of chemical substances, including prescription and over-the-counter therapeutic drugs, veterinary

d f d idrugs, fragrances, and cosmetics.

EPA 1624 Method for Group 1 Pharmaceuticals46 pharmaceuticals – 22 minutes

2.1 x 100 mm column 3.5 micronEPA Standard Method—1200 RRLC

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 26

EPA 1624 Method: 1290 UHPLC/6460 Triple Quad 46 1146 pharmaceuticals – 11 minutes

2.1 x 100 mm column 1.8 micron1290 Infinity UHPLC0 6 mL/min at 750 Bar0.6 mL/min at 750 Bar

Pesticide application kit for 6400 Series Triples

G1733AA -- Pesticide DMRM Data Base Kit

Includes: 600 compound database aIncludes: 600-compound database, a positive and negative ion test mix with their analysis methods. The methods contain compound names, optimal settings, and retention times for the Dynamic MRM.

The Application Kit Quick Start GuideThe Application Kit Quick Start Guide shows how to run the test mixes and create a Dynamic MRM method.

Pesticide standard in the application kit

aminocarb ca r

thiabenzaz

dime m

rbofuran

metazach

pyraclodiazinon

imazapy

ethoate imazali

metoxuron

atrazinm

etosulam

molinat e

malathion

ostrobin

yr il e e n

1290 Infinity LC and the 6460 or 6430Triple Quad The Ideal, high-speed Quant System!, g p Q y

Sensitivity from femtogram to nanogramFast polarity switching – 30 millisecFast analysis times - < 2 min with good RSDs4,000 DMRMs for complex samples ,000 s o co p e sa p es> 1,000 Analyses per day

6430 Triple Quad LC/MS

6460 Triple Quad LC/MS

Agilent’s New 6540 Ultra High Definition QTOFResearch Performance in a Benchtop Formatp

40,000 Resolving Power<1 ppm MS <3 ppm MS/MS

Mass Accuracy20 Spectra/sp2 pg – 50:1 Reserpine S/N5 Decades in Spectrum Dynamic RangeExcellent Linearity and Isotopic FidelityExcellent Linearity and Isotopic FidelitySupports Standard ESI, Agilent Jet

Stream and HPLC-Chip

Unsurpassed Analytical Capacity in a Benchtop

6540 UHD

Enhanced Ion Flight Tube and Mirror TechnologyStable, Sensitive, High Resolution 6540 UHD

Q-TOF• 1ppm/°C Expansion Coefficient for Inner

Flight Tube virtually eliminates calibration d ift d t fli ht t b l ti

6530 Q-TOF

drift due to flight tube elongation.

• 2nd Order Temporal Focusing Ion Mirror uses high transmission Harp Grid for maximum sensitivity

Ion Mirror

DC Quad

Octopole 2

Ion Pulser

Detector

TurboMaking Research Grade Performance possible in a Benchtop Format

Ion Beam Compression (IBC)* TechnologyDrives Higher Resolutiong

Active Ion Beam Compression simultaneously maximizes ion transmission and reduces beam divergence

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 is achieved with Agilent’sAxial Ion Acceleration Technology applied to a taperedIon guide design.

* Patent pending

Making Research Grade Performance possible in a Benchtop Format

Next Generation – Ultra High Speed Detector

New Bipolar TOF Detector• New ultra fast and high Single Ion Response• New ultra fast and high

efficiency scintillator• New ultra fast response

PMT design continues

Single Ion Response~800 psec FWHM

PMT design continues the tradition of high dynamic range and detector lifetime

• Developed by Photonis with Agilent TOF TechnologyS ifi ll h

2 nsec/div

• Specifically enhances Resolution in 2Ghz Ext. Dynamic Range Mode

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

• 4 GHz Acquisition for Maximum Resolving Power and <1ppm

Dual Input Agilent pre-amplifiers

Resolving Power and <1ppm Mass Accuracy

• 5 Decades of in-Spectrum Dynamic Range from 2 Channel

4 GH (8 bit) A l Di it l C t

Picture of 4GHz boardGoes here

Dynamic Range from 2-Channel x 2 GHz Dual Gain Mode

• 4 GHz (8 bit) Analog-Digital-Converter Adapted from Agilent’s High Speed Oscilloscope Systems

• Ultra High Speed FPGAs process and• Ultra High Speed FPGAs process and store transients in real time

FPGAs 4 GHz Agilent ADC

6540 Ultra High Definition Q-TOFResolving Power – Across the Mass Range

R=42424622

R=387021221

R=43674

R=42750322

R=302182121

R=418251821

R=4040524212

R=40405R=39332 2721

R=37207

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

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

Resolution is Scan Rate Independentp

Enhancements in Resolving PowerHigh Resolution and Ext. Dynamic Range Modes

6540High Resolution

6540Ext. Dynamic Range

High Resolution

solving Po

6530High Resolution

15000Re 6530 Ext. Dynamic Range

0 500 1000 1500 2000 2500 3000m/z

6540 MS Resolution Is Invariant With Acquisition Rate - Pesticides ExampleRate Pesticides Example

azoxystrobin 404 m/z

cyproconazole 292 m/z

cyprodinil 226 m/z

diazinon 305 m/z

linuron 249 m/z

paclobutrazol 294 m/z

propazine 229 m/z

tetraconazole 372 m/z

tricyclazole 190 m/z

0 1 2 3 4 5 6 7 8 9 10 11 12

June 2009

Acquisition Rate (scans/second)

ASMS 2009 UHPLC 6540 QTOF lunch and learn

6540 Ultra High Definition QTOFMass Accuracy – Repetitive InjectionsMass Accuracy Repetitive Injections

+ESI EIC(609.28066) Scan Frag=240.0V Reserpine 40pgms3.d

40pg reserpine on-column, 10 injectionsScan Frag 240.0V Reserpine_40pgms3.d

1.2 609.28081Isotope 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

RunError (ppm)

1 0.962 ‐0.173 0.25

610.28425

4 1.41 1.48 612.28987 612.28941 0.75

5 0.14 0.21 613.29210 613.29203 0.11

3 0.254 0.025 0.396 0.137 0.018 0.52

611.28650

612.28987613.29210

9 0.0410 0.30

Mean 0.25

Std. Dev 0.32

0.0 0.25 0.5 0.75 1

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

609 610 611 612 613 614

Dev 0.32

250 ppb mass accuracy calibration and very accurate isotopic ratios

6540 Ultra High Definition QTOF Sensitivity – Full Scan MS Mode

609 280347

(M+H)+

1pg reserpine on-column

2609.280347

Ratio m/z Diff.Theor. Expt. Theor. Expt. (ppm)100.00 100.00 609.2807 609.2803 ‐0.5137.24 36.36 610.2839 610.2832 ‐1.258.59 8.39 611.2867 611.2878 1.77

Resolution ~ 33,000

S/N = 319 RMS

8.59 8.39 611.2867 611.2878 1.771.48 1.45 612.2894 612.2926 5.29

610.2831681

611.287790

612.292649

607 608 609 610 611 612 613 614 615 6160

0.0 0.5 1 1.5

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

Coeluting Metabolites With Parent Drug: Need Wide Dynamic RangeWide Dynamic Range

Five decades of response in a single scan

verapamil dihydroxy metabolite of verapamil

p y y p

2.8 455.290321(M+H)+

2.68 million counts

25 counts

400 counts

desmethyl

441.274704

471.284287487.279822

(M+H)+

desmethyl metabolite

monohydroxy metabolite

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

Resolving Isobaric Co-eluting Species

Isobaric Co-eluting Pair m/z ppm Difference

Differentiate by

RT No fragment m/z

294.13690

gTricyclazolePropazine

190.0433190.0667

123 X(1.3 min)

MethiocarbDiethofencarb fragment

226.0896226.1074

79 X X

Diethofencarb fragmentCyprodinil

226.1074226 1339

117 X(0 53 min)

Paclobutrazol0.44 ppmR=29,600

Cyproconazole-37Cl0 66 Cyprodinil 226.1339 (0.53 min)

Cyproconazole -37ClPaclobutrazol

294.1182294.1368

Clofentezine-37ClDiazinon

305.0618305.1083

Chlorfenvinphos 358.9768 177 X

294.118400.66 ppmR=30,500

Triflumuron 359.0405TetraconazoleAzoxystrobin fragment

372.0288372.0979

186 X X

Mass-to-Charge (m/z)

294.08294.10

294.12294.14

294.16294.18

•Resolved seven pairs of “isobaric coeluting species” (ICS) which have similar retention times and accurate mass values for their adduct ions or fragments

IMSC 2009 Infinity LC 6540 QTOF lunch and learn

August 2009Page 46

Identify These Pesticides By Isotopic Pattern And MassMass

cyproconazolecyproconazole

paclobutrazol

August 2009

Pesticides in Pepper Matrix, 10 ppb3 Scans/sec Acquisition Rate3 Scans/sec Acquisition Rate

256.05959Imidacloprid Triflumuron

163.05348(M+H)+

Methomyl

-0 90 ppm 258.05655

(M+H)+

-0.06 ppmR =28,900

(M+H)+268.15425

Diethofencarb

-0.33 ppmR =29 100

(M+H)+359.04026

-0.60 ppmR =31,700

Flufenoxuron

Acquisition Time (min)1 2 3 4 5 6 7 8 9

164 05652

-0.90 ppmR =24,200 (M+H)+

257.06237(M+H)+

259.05936(M+H)+

(M+H)+256 257 258 259 260

260.06128

269.15754(M+H)+

R =29,100 361.03783(M+H)+

360.04448(M+H)+

362.04095(M+H)+

(M+H)+

359 360 361 362 363

363.03121 489.04371(M+H)+

491.04126

-0.34 ppmR =34,500

164.05652(M+H)+ 165.04954

(M+H)+

163 164 165

(M+H)+

268 269 270

270.13975491.04126

(M+H)+490.04738

(M+H)+ 492.04627(M+H)+

489 490 491 4921.048

2.483 2.7802.549

2.872 3.1621.2131.232

1.318 1.522 1.8972.069

2.2332.233 2.470 2.589

2.8392.8651.700

Acquisition Time (min)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 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3

2.4642.4642.457

August 2009Page 48

Acquisition Time (min)

6530 vs. 6540: Comparison of Peptide Mass SpectraSpectra

+2 6530 Q-TOF

CCTKPESERMPCTEDYLSLILNR+2 6540 Q-TOF

+4+3 6530 Q-TOF 6530 Q-TOF4

+3 6530 Q-TOF 6530 Q TOF

6540 Q TOF+4+3 6540 Q-TOF 6540 Q-TOF

August 2009Page 49

Peptide Mapping: Serotransferrin

3x10 Peak width 1.1 sec27 d t i t

6 27 data points

Counts vs. Acquisition Time (sec)52 53 54 55

0.68 ppm

August 2009Page 50

Malaria MetabolomicsMalaria Metabolomics

RESULTS ANDBIOLOGICAL SIGNIFICANCE

Malaria Infected Red Blood Cell Study y

PCA analysis of all Red Blood Cell samples reveals separation based on pH of extraction solventseparation based on pH of extraction solvent

ID Browser Identified Arginine to be statistically differential in malaria infected cells

Pathway analysis in MPP showing differential abundances for three compounds in the urea cycle

L‐Arg (Arginine)Ornithine

abundances for three compounds in the urea cycle

OrnithineCitrulline

I f t d

InfectedBlood cells

Non infected

6540 Ultra High Definition Q-TOF

• Compatible with unmatched chromatographic peak capacity and resolving power of latest 1290 Infinity UHPLC technology

• Superb MS mass accuracy and resolution—invariant with acquisition rate

• Sophisticated identification software and algorithms—handle complex samples, overlapping isotopic patterns

Proteomics - From Discovery Mode to Validation

Peroxidase in Human Plasma

Discovery phase to Validation : MRM Selector

Biomarker validation workflow

• Run samples on Q-TOF for protein ID in d t d d t

• Search QTOF data using Spectrum Mill

Step-2Spectrum Mill • Import the MRM list

into QQQ Acquisition softwareR l QQQ

• Integrate the MRchromatograms

StepMass Prof

data-dependent MS/MS mode.

Step-1

using Spectrum Mill• Use Spectrum Mill

MRM Selector to create a list of MRM transitions with RT

• Run samples on QQQ in Dynamic MRM mode

Step-3

chromatograms• Import quantitatio

results into MPPperform statisticaanalysis

Q-TOF QQQ

Depleted Human Plasma Sample analysis

Replicate LC/MS runsHPLC – Chip / QTOFpSpiked with 0,0.5 and 5fmolper 0.5ug plasma

Data Dependent Protein IDs from Spectrum Mill

Spectrum Mill

MRM SelectorGenerates MRM method from discovery QTOF datay

Dynamic MRM

Dynamic MRMOverlaid 2000 MRM chromatograms acquired in a single run using Dynamic MRMOverlaid 2000 MRM chromatograms acquired in a single run using Dynamic MRM

Mass Profiler ProfessionalStatistical Processing of MRM Data

Four peptides from peroxidase were highlighted in green. The mean of 443 MRM abundances isabundances is displayed (black) to show the peptides from plasma did not vary f l t lfrom sample to sample.

All Samples B1 B2 B3 A1 A2 A3 M1 M2

Principle Component AnalysisMatrix and 2 different peroxidase levels

Samples at different peroxidase concentrations were correctly groupedgrouped together.

Hierarchical Clusteringcomparing different peroxidase concns.comparing different peroxidase concns.

A condition was generated with peroxidase concentration color-coded on the tree branches alongon the tree branches, along with the peptide features labeled on each row. The heat map is colored from blue to red where blue is

M1 M2 A1 A2 A3 B1 B2 B3

blue to red, where blue is low abundance and red is high abundance. The full view of all the features is on the left The zoom view is M1 M2 A1 A2 A3 B1 B2 B3the left. The zoom view is on the right.

Sensitivity : Peroxidase in plasma matrix (per 0.5ug)

matrix 5fmol 500amole

# RT Cycle Min. Max. # %RSD RSD# MRM window

ytime (ms)

dwell (ms)

concurrent MRM

%RSDArea RT

443 2 1000 16.5 50 2.5 0.038

443 1 1000 29.83 30 3.2 0.016

2000 2 1000 2.75 160 4.5 0.030

3293 1 10 0 2 18 18 4 0 02

Reproducibility of MS response

3293 1 1050 2.18 185 4.7 0.025p

and RT

The 1290 Infinity UHPLC + Agilent MS:The Best LC/MS Solution Ever!The Best LC/MS Solution Ever!

Triple Quad – 6460/6430/6410S iti it !Sensitivity!Productivity!1290 resolution/speedpDynamic MRM, 30ms pos/neg switching

TOF / Q-TOFSpeed! (narrow peaks, fast scanning MS, maximum data/unit time)C h i l i Ult Hi h R l tiComprehensive analysis – Ultra-High ResolutionProductivity!

THANK YOU!THANK YOU!

Agilent Solutions for Ultra-High Performance LC/MS Ken Miller · 2016-08-30 · Ken Miller Global...

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