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
g
PurgeValve
Silicon CarbidePistons
Page 4
Channel A
1290 Infinity – Compatible with any HPLC and UHPLC
1200
bar
A new power range providing maximum performance, flexibility, compatibility and investment
protection1290 Infinity
1000Acquity
600
800 Dionex RSLC
400
Agilent RRLCThermoAccela
Shimadzu UFLCXR
0
200
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.
4x10
3
3.5
4
4.5
W1/2 = 0.63 sec
Excellent Data Quality:Avg W1/2 = 0.63 sec Avg W = 1 8 sec
1.5
2
2.5
3 Avg. W = 1.8 sec5 points across W1/2 13 points across WArea RSD [%] = 4.7SNR (3RMS) = 1672
0
0.5
1
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
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”
bar
2.1mm ID 3 – 4.6mm ID
Power = Pressure x Flow Rate
Agilent 1290
1000
This app falls in the Power Range
Vendor A
Agilent 1290 Infinity
600
800Vendor E
400
Agilent RRLCVendor C
Vendor D
0
200
Standard LC
0 1 2 3 4 5 ml/min
1290 Infinity - Ultra High Performance SeparationsChallenges for Quant sufficient sampling across peak
mAU60
Challenges for Quant – sufficient sampling across peak
50
60 Typical Peak Width
~ 2 sec
30
40
10
20
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
Q3Q1
D. Switch power supply from negative to positiveE. Run positive MRM transitions
a
DetectorCollision Cell
Q3Q1
a
DetectorCollision Cell
Metabolism study with negative/positive MRMs:6460 Triple Quad with 30 millisecond polarity switching
5x108
+MRM (455.3 -> 165.1)Verapamil, 0.54 min
5x108
+MRM (455.3 -> 165.1)Verapamil, 0.54 min
5x108
+MRM (455.3 -> 165.1)Verapamil, 0.54 min
5x108
+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, 0.75 min
+MRM (455.3 -> 165.1)5x10
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
2
4
6
5x10 +MRM (386.0 -> 122.1
Avg W1/2 = 0.44 sec 11 points across WRel. Area RSD [%] = 4.0
Buspirone,
2
4
6
5x10 +MRM (386.0 -> 122.1
Avg W1/2 = 0.44 sec 11 points across WRel. Area RSD [%] = 4.0
Buspirone,
2
4
6
5x10 +MRM (386.0 -> 122.1
Avg W1/2 = 0.44 sec 11 points across WRel. Area RSD [%] = 4.0
Buspirone,
2
4
6
5x10 +MRM (386.0 -> 122.1
Avg W1/2 = 0.44 sec 11 points across W
RSD [%] = 4.0
Buspirone
2
4
6
5x10
Avg W1/2 = 0.71 sec 15 points across WRel. Area RSD
[ 2.9
Buspirone,+MRM (386.0 -> 122.1
2
4
6
5x10
Avg W1/2 = 0.71 sec 15 points across W
Rel. Area RSD [%] = 2.9
Buspirone,+MRM (386.0 -> 122.1
2
4
6
5x10
Avg W1/2 = 0.71 sec 15 points across W
Rel. Area RSD [%] = 2.9
Buspirone,+MRM (386.0 -> 122.1
2
4
6
5x10
Avg W1/2 = 0.71 sec 15 points across W
RSD [%] = 2.9
Buspirone+MRM (386.0 -> 122.1
1
2
3
4x10 +MRM (272.2 -> 215.2)
p ,0.22 min
Dextromethorphan
Avg W1/2 = 0.70 sec 20 points across WRel. Area RSD [%] = 4.21
2
3
4x10 +MRM (272.2 -> 215.2)
p ,0.22 min
Dextromethorphan
Avg W1/2 = 0.70 sec 20 points across WRel. Area RSD [%] = 4.21
2
3
4x10 +MRM (272.2 -> 215.2)
p ,0.22 min
Dextromethorphan
Avg W1/2 = 0.70 sec 20 points across WRel. Area RSD [%] = 4.21
2
3
4x10 +MRM (272.2 -> 215.2)
Buspirone
D t th h
Avg W1/2 = 0.70 sec 20 points across W
RSD [%] = 4.2 0.5
1
1.5
4x10
Avg W1/2 = 1.3 sec 35 points across WRel. Area RSD [%] = 1.8
Buspirone, 0.40 min
Dextromethorphan+MRM (272.2 -> 215.2)
0.5
1
1.5
4x10
Avg W1/2 = 1.3 sec 35 points across W
Rel. Area RSD [%] =
Buspirone, 0.40 min
Dextromethorphan+MRM (272.2 -> 215.2)
0.5
1
1.5
4x10
Avg W1/2 = 1.3 sec 35 points across WRel. Area RSD [%] =
Buspirone, 0.40 min
Dextromethorphan+MRM (272.2 -> 215.2)
0.5
1
1.5
4x10
Avg W1/2 = 1.3 sec 35 points across WRSD [%] = 1.8
Buspirone
D t th h+MRM (272.2 -> 215.2)
0.5
1
1.5
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
1
1.5
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
1
1.5
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
1
1.5
2
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
1
2
3
3x10
Avg W1/2 = 1.3 sec 35 points across WRel. Area RSD [%] = 2.7
Dextromethorphan, 0.53 min
-MRM (294.0 -> 250.0
1
2
3
3x10
Avg W1/2 = 1.3 sec 35 points across WRel. Area RSD [%] = 2.7
Dextromethorphan, 0.53 min
-MRM (294.0 -> 250.0 Analysis time < 1 1 min
1
2
3
3x10
Avg W1/2 = 1.3 sec 35 points across WRel. Area RSD [%] = 2.7
Dextromethorphan, 0.53 min
-MRM (294.0 -> 250.0
1
2
3
3x10
Avg W1/2 = 1.3 sec 35 points across WRSD [%] = 2.7
Dextromethorphan
-MRM (294.0 -> 250.0 Analysis time < 1 1 min3x10
1
2
3
4
-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
3x10
1
2
3
4
-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
3x10
1
2
3
4
-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
3x10
1
2
3
4
-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
x10
1
2
3
4
Avg W1/2 = 0.52 sec 11 points across WRel. Area RSD [%] = 6.5
Diclofenac, 1.04 min
(x10
1
2
3
4
Avg W1/2 = 0.52 sec 11 points across WRel. Area RSD [%] = 6.5
Diclofenac, 1.04 min
( Analysis time < 1.1 minx10
1
2
3
4
Avg W1/2 = 0.52 sec 11 points across WRel. Area RSD [%] = 6.5
Diclofenac, 1.04 min
(x10
1
2
3
4
Avg W1/2 = 0.52 sec 11 points across WRSD [%] = 6.5
Diclofenac, 1.04 min
( Analysis time < 1.1 min
Counts 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
Counts 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
Counts 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
Counts 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 1.2Counts 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
Counts 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
Counts 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
Counts 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
2.6
3
3.4
Tim
e (m
in)
DiclofenacAverage W 1/2 = 0.37 sec
9 points across W
1.4
1.8
2.2
. Acq
uisi
tion
9 points across 1.5 sec peak
Rel. Area RSD [%] = 7.8%
0 2
0.6
1
1.4
Cou
nts
vs. [ ]
0.2
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
80
90
100
alys
is t5
t10Diclofenacr2 = 0.9946
40
50
60
70
witc
hed
ana t10
t15
10
20
30
Non
-sw t15
t25
t35
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
y p
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
Page 27
Eight Minute Analysis of 250 Pesticides 1290 Infinity UHPLC and 6460 Triple Quad& with DMRM
2x10
8.2
8.4
8.6
8.8
9
9.2
9.4
9.6 1 1500ppt, dynamic MRMPeak widths ~ 1 secondCycle time: 100 msec
y p
6 2
6.4
6.6
6.8
7
7.2
7.4
7.6
7.8
8
8.2 Cycle time: 100 msecMin dwell: 2.5 msec
4 2
4.4
4.6
4.8
5
5.2
5.4
5.6
5.8
6
6.2
Zorbax Eclipse Plus C182.1 x 100mm (1.8μm)
2 2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
4.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
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
X103
4.55
5.56
6.5
1.52
2.53
3.54
00.5
1
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
zole
ethoate imazali
metoxuron
atrazinm
etosulam
hlor
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
Page 34
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
, , g
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
Making Research Grade Performance possible in a Benchtop Format
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
Making Research Grade Performance possible in a Benchtop Format
6540 Ultra High Definition Q-TOFResolving Power – Across the Mass Range
9225
x10
9
R=42424622
R=387021221
R=43674
4
4.5
5
5.5
1521
R=42750322
R=302182121
R=418251821
R=4040524212
2.5
3
3.5
4
R=40405R=39332 2721
R=37207
0
0.5
1
1.5
2
Counts vs. Mass-to-Charge (m/z)
200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800
0
Resolution is Scan Rate Independentp
Enhancements in Resolving PowerHigh Resolution and Ext. Dynamic Range Modes
40000
45000
g y g
6540High Resolution
30000
35000
40000
ower
6540Ext. Dynamic Range
High Resolution
20000
25000
solving Po
6530High Resolution
6530
10000
15000Re 6530 Ext. Dynamic Range
0
5000
0 500 1000 1500 2000 2500 3000m/z
6540 MS Resolution Is Invariant With Acquisition Rate - Pesticides ExampleRate Pesticides Example
30000
35000
25000
30000
azoxystrobin 404 m/z
15000
20000
Res
olut
ion
cyproconazole 292 m/z
cyprodinil 226 m/z
diazinon 305 m/z
linuron 249 m/z
paclobutrazol 294 m/z
5000
10000
paclobutrazol 294 m/z
propazine 229 m/z
tetraconazole 372 m/z
tricyclazole 190 m/z
0
5000
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
Page 42
6540 Ultra High Definition QTOFMass Accuracy – Repetitive InjectionsMass Accuracy Repetitive Injections
+ESI EIC(609.28066) Scan Frag=240.0V Reserpine 40pgms3.d
5x10
40pg reserpine on-column, 10 injectionsScan Frag 240.0V Reserpine_40pgms3.d
0.511
1
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
0 4
0.6
0.8
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
0
0.2
0.4
611.28650
612.28987613.29210
9 0.0410 0.30
Mean 0.25
Std. Dev 0.32
Counts vs. Acquisition Time (min)
0.0 0.25 0.5 0.75 1
0
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
3x10
609 280347
(M+H)+
4x10
1pg reserpine on-column
2609.280347
2
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
1
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
0
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
6
p y y p
6x10
2
2.2
2.4
2.6
2.8 455.290321(M+H)+
2.68 million counts
25 counts
400 counts
1
1.2
1.4
1.6
1.8
2
desmethyl
0
0.2
0.4
0.6
0.8
1
441.274704
471.284287487.279822
(M+H)+
desmethyl metabolite
monohydroxy metabolite
0
Counts vs. Mass-to-Charge (m/z)435 440 445 450 455 460 465 470 475 480 485 490 495 500 505
Page 45
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)
X
MethiocarbDiethofencarb fragment
226.0896226.1074
79 X X
Diethofencarb fragmentCyprodinil
226.1074226 1339
117 X(0 53 min)
X X
Paclobutrazol0.44 ppmR=29,600
Cyproconazole-37Cl0 66 Cyprodinil 226.1339 (0.53 min)
Cyproconazole -37ClPaclobutrazol
294.1182294.1368
63 X
Clofentezine-37ClDiazinon
305.0618305.1083
300 X
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
3
cyproconazolecyproconazole
44
paclobutrazol
Page 47 August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Pesticides in Pepper Matrix, 10 ppb3 Scans/sec Acquisition Rate3 Scans/sec Acquisition Rate
256.05959Imidacloprid Triflumuron
TIC
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
1.331
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
2.661
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
2.865
2.4642.4642.457
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
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
+4
+3 6530 Q-TOF 6530 Q TOF
6540 Q TOF+4+3 6540 Q-TOF 6540 Q-TOF
August 2009Page 49
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Peptide Mapping: Serotransferrin
3x10 Peak width 1.1 sec27 d t i t
2
4
6 27 data points
Counts vs. Acquisition Time (sec)52 53 54 55
0.68 ppm
August 2009Page 50
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Malaria MetabolomicsMalaria Metabolomics
RESULTS ANDBIOLOGICAL SIGNIFICANCE
Page 51
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
pHpH
2
7
9
ID Browser Identified Arginine to be statistically differential in malaria infected cells
Page 54
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
2500
InfectedBlood cells
0
500
1000
1500
2000
2500
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
Page 57
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
Page 58
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
Page 59
Spectrum Mill
MRM SelectorGenerates MRM method from discovery QTOF datay
Page 60
Dynamic MRM
Page 61
Dynamic MRMOverlaid 2000 MRM chromatograms acquired in a single run using Dynamic MRMOverlaid 2000 MRM chromatograms acquired in a single run using Dynamic MRM
Page 62
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
Page 63
Principle Component AnalysisMatrix and 2 different peroxidase levels
Samples at different peroxidase concentrations were correctly groupedgrouped together.
Page 64
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.
Page 65
Sensitivity : Peroxidase in plasma matrix (per 0.5ug)
M A B
matrix 5fmol 500amole
# RT Cycle Min. Max. # %RSD RSD# MRM window
(min)
ytime (ms)
dwell (ms)
concurrent MRM
%RSDArea RT
(min)
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
Page 66
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!