Agilent Users Meeting
September 17th, 2008
Agenda
• Goal – Share Practices, Assistance to Network of Agilent Users
Today’s Topics:
• Care and Feeding Agilent LCMS Systems
• Data Acquisition: Optimize TOF/QTOF, Methods Development
• MassHunter Qual: Flexible Data Mining
• Overview MassProfiler Software
• Quant/Qual (HRAMS Quant): Setup and Use
• MassHunter Bioconfirm Software
• Questions and Answer Period
Agilent Users Meeting
September 17th, 2008
Care and Feeding of QTOF/TOF
Current Versions of Software
• MassHunter Acquisition (B.05)
• MassHunter Quant (B.06)
• MassHunter Qual (B.06)
• MH Mass Profiler (B.03)
• MassHunter BioConfirm (B.06)
Agilent Users Meeting
September 17th, 2008
Data Acquisition Overview
How to Improve Performance
• Optimize Source Parameters:
– Drying Gas Temperature, Gas Flow, and Neb - LC Condition and
Compound Dependent
– Fragmenter Voltage - Compound Dependent
• Optimize Ion Transmission in over the Mass Range
– QTOF For Low Mass Analytes < 200 Change Quad Amu Gain to 100
– TOF/QTOF Fragmentor
• Review How to Calibrate/Tune Q(TOF)
• Optimize Acquisition – Scan the Proper Mass Range
– Saturation and Acquisition Mode
– Set the Proper Acquisition Rate for Chromatographic Peaks
Agilent Users Meeting
September 17th, 2008
Optimization of ESI Source Parameters non-Ion
Funnel TOF/QTOF
VCap – Entrance of the Capillary, 2000 to 4500V / 5000VMAb
(3500V) Pos, (3000V) Neg
Dependent on compound and MW Concentrated samples (µg/µL) lower VCap
Drying Gas - Dependent on LC flow rate 10-13 L/min
Gas Temp - Start at high temperature work down-usually 275-325C
Skimmer - Voltage on skimmer and lens after capillary 65V
Fragmentor- Not Same as QQQ/Quad, Compound Dependent
Vary fragmentor/scan (30V steps) 100V to 240V
FIA with Injection Program
Sheath Gas- Dependent on LC flow rate 10-12 L/min
Shields Up!
Dual ESI Source Schematic
Vcap
Fragmentor
Nebulizer Pressure
Drying Gas Temperature and Flow
Drying Gas Flow high water needs higher flow if too low, spikes in spectra from droplets, dirty cap Drying Gas Temperature higher for low vapor pressure solvents Usually 275 - 350C Vcap optimize with FIA (2000-6000) start with 3000 V in negative mode, look for high chamber current or
blue glow (indicates corona): reduce Vcap if this happens
Electrospray Spray Chamber Settings
HPLC
Flow Rate
(µ/min)
Nebulizer
Pressure
(psi)
Drying Gas
Flow
(l/min)
Drying Gas
Temp
(C)
1 – 10 10 – 15 7 150 - 250
10 – 50 15 – 20 8 - 10 150 - 250
50 – 200 20 – 40 10 - 12 200 - 325
200 – 500 25 – 45 12 325
500 – 1000 50 – 60 12 - 13 350
Vcap
Corona
current
Nebulizer
Pressure
Fragmentor
Drying gas
Temperature
and Flow
Heater
APCI Spray Chamber Settings
HPLC
Flow Rate
(µl/min)
Nebulizer
Pressure
(psi)
Drying Gas
Flow
(l/min)
Drying Gas
Temp
(oC)
200 – 500 30-40 5 - 8 300-350
500 - 1000 40-60 8*-13* 350
APCI
Vap Temp
(oC)
Corona Current
(nA)
Capillary
Voltage
(V)
300 – 500 4000 (pos) & 20,000 (neg) 3500
* Use enough dry gas, but don’t remove reagent
Overview of the Multimode Source
Capillary
HPLC inlet
Nebulizer
Drying gas
Corona needle
ESI Zone
APCI Zone
Thermal container
To convert a method developed with any other source, all that is needed is to go to method and run control and change the Method spray chamber to MM-ES+APCI. Spray chamber values will be the same as for APCI
IR
Multimode Spray Chamber Settings
Mode Nebulizer
Pressure
(psi)
Drying Gas
Flow
(l/min)
Drying Gas
Temp
(oC)
APCI 30-60 5 - 8 300-350
ESI 30-60 8-13 325
APCI/ESI
Vap Temp
(oC)
Corona Current
APCI
(nA)
Capillary
Voltage
(V)
150(ESI)
250(APCI)
4000 (pos) & 20,000 (neg) 3500
Capillary
HPLC inlet
Nebulizer
Drying gas
Corona needle
ESI Zone
APCI Zone
Thermal container
Agilent Jetstream Technology (AJT) source
Non-ion funnel small molecule
The super-heated sheath gas collimates the nebulizer spray and presents more ions to the MS inlet.
*Nozzle voltage
Resistive sampling capillary exit: FragV
Nebulizing gas: pressure
*Sheath gas: flow and temperature
Drying gas:
flow and temperature
Resistive sampling capillary entrance: Capillary V
12
*New parameters unique to AJT source
Parameter
Starting Conditions
Agilent Jetstream
Pos/Neg
Nebulizer pressure 35-45 psi
Drying gas flow 10-12 LPM
Drying gas temp 275°C/325°C
Sheath gas flow 10-12 LPM
Capillary voltage 3500V/3000V
Nozzle voltage (AJT) 0V/1500V
Pos - low/Neg - higher
Fragmentor voltage
(cpd-dependent)
100-175V QTOF/TOF
Drying Gas Experiment on Agilent Jet Stream
Source 5l/min
Drying Gas Experiment on Agilent Jet Stream
Source 8l/min
Drying Gas Experiment on Agilent Jet Stream
Source 10l/min
Drying Gas Experiment on Agilent Jet Stream
Source at 12l/min
Drying Gas Experiment on Agilent Jet Stream Source
12l/min Note: a Higher Vcap increases High m/z response
Agilent Jetstream Technology (AJT) source
Non-ion funnel intact protein
The super-heated sheath gas collimates the nebulizer spray and presents more ions to the MS inlet.
*Nozzle voltage
Resistive sampling capillary exit: FragV
Nebulizing gas: pressure
*Sheath gas: flow and temperature
Drying gas:
flow and temperature
Resistive sampling capillary entrance: Capillary V
18
*New parameters unique to AJT source
Parameter
Starting Conditions
Agilent Jetstream
Pos/Neg
Nebulizer pressure 55 psi
Drying gas flow 10 LPM
Drying gas temp 350°C
Sheath gas flow 10-12 LPM
Capillary voltage 4500-5500V
Nozzle voltage (AJT) 2000V
Fragmentor voltage
(cpd-dependent)
225-400V QTOF, TOF
Note that “quad amu” will need to be increased 100-250amu with old quad drive in quad tuning parameters for QTOF
Agilent Jetstream Technology (AJT) source
Ion funnel intact protein
The super-heated sheath gas collimates the nebulizer spray and presents more ions to the MS inlet.
*Nozzle voltage
Resistive sampling capillary exit:
Nebulizing gas: pressure
*Sheath gas: flow and temperature
Drying gas:
flow and temperature
Resistive sampling capillary entrance: Capillary V
19
*New parameters unique to AJT source
Parameter
Starting Conditions
Agilent Jetstream
Pos/Neg
Nebulizer pressure 55 psi
Drying gas flow 15LPM
Drying gas temp 250°C
Sheath gas flow 12 LPM
Sheath gas temp 250-300
Capillary voltage 4500-5500V
Nozzle voltage (AJT) 2000V
Fragmentor voltage
(cpd-dependent)
NA
Note that “quad amu” will need to be increased 100-250amu with old quad drive
in quad tuning parameters for QTOF
Agilent Jetstream Technology (AJT) source
Ion funnel small molecule
The super-heated sheath gas collimates the nebulizer spray and presents more ions to the MS inlet.
*Nozzle voltage
Resistive sampling capillary exit:
Nebulizing gas: pressure
*Sheath gas: flow and temperature
Drying gas:
flow and temperature
Resistive sampling capillary entrance: Capillary V
20
*New parameters unique to AJT source
Parameter
Starting Conditions
Agilent Jetstream
Pos/Neg
Nebulizer pressure 35-45 psi
Drying gas flow 15-17LPM
Drying gas temp 200-250°C
Sheath gas flow 10-12 LPM
Sheath gas temp 200-400°C
Capillary voltage 3500V/2500V
Nozzle voltage (AJT) 0-500V/1500-2000V
Fragmentor voltage
(cpd-dependent)
NA
APCI source
Ion funnel small molecule
21
Parameter
Starting Conditions
Agilent Jetstream
Pos/Neg
Nebulizer pressure 20-30 psi
Drying gas flow 14-15LPM
Drying gas temp 150-200°C
Vap Temp 350-400°C
Sheath gas temp NA
Capillary voltage 3500V/2000V
Chamber current 35 µa
Fragmentor voltage
(cpd-dependent)
NA
Vcap
Corona
current
Nebulizer
Pressure
Fragmentor
Drying gas
Temperature
and Flow
Heater
Ion Funnel Settings for Different Applications
6550 Tune (default)
6550 Tune
Peptide/Protein
6550Tune very
small molecule
(m/z <120) and
fragile adduct
6550 Generic
small molecule
6490 Tune
(default)
6490 Tune
generic small
molecule
6490 Tune Fragile
Compound and
Adduct
Funnel DC 50 50 30 50 15 15 15
HP Funnel Voltage Drop 150 150 100 150 180 180 130
HP Rf Voltage 200 200 90 150 200 200 150
LP Funnel Voltage Drop 100 100 50 100 100 100 90
LP Rf Voltage 100 100 40 60 110 110 40
Fragmenter to 175 Fragmentor to 325
Quad amu 100 Quad amu 120
These are suggested starting funnel parameters and will need optimization. Settings can be very analyte specific. They can be used “as is” should a user need to find out very quickly if it is worth the time to optimize the parameters further. These parameters may not be the same as listed from other sources that have been optimized for a particular analyte or class of analytes.
Agilent Users Meeting
September 17th, 2008
Vary Fragmentor with FIA Injection Program
Multiple Methods or Scan Functions
Auto Sampler Injection Program
• REMOTE Startpulse
• REPEAT 5 times
• VALVE bypass
• DRAW Def Amount from Sample, def. speed, def offset
• VALVE mainpass
• WAIT 0.75 min
• END REPEAT
Vary Fragmentor Voltage
• Right Click in Expt Area and select “Add
Experiment”
• Vary Fragmentor Voltage by 20-40V per
Experiment
• Acquire Data using Injector Program
• Determine Optimum Voltage from EIC as a
Function of Fragmentor Voltage
Agilent Users Meeting
September 17th, 2008
Extracted Ion Chromatograms:
Advanced Tab “Fragmentor Setting”
Review How to Tune and Calibrate the Q(TOF)
Agilent Users Meeting
September 17th, 2008
Entering Calibration and Tuning
In Mass Hunter Workstation Data Acquisition, select the
Tune Context.
September 17th, 2008
Agilent Users Meeting
Set Instrument Mode for Acquisition and
Calibration • Mass Range: Low (1700 m/z), Standard (3200 m/z) or
High (20000)
• Instrument Modes:
• High Resolution (4GHz)
• 4GHz (High Resolution disabled)
• Extended Dynamic Range (2GHz)
• High Mass Range (2 GHz)
Advanced showing
Agilent 6520
September 17th, 2008
Agilent Users Meeting
Ultra High Speed Acquisition From Agilent’s Leadership in GHz Speed Electronics (Integration and active sampling
to TOF detection)
Picture of 4GHz board
Goes here
FPGAs
Dual Input Agilent
pre-amplifiers
4 GHz Agilent ADC
• 4 GHz Acquisition for Maximum
Resolving Power and <1ppm
Mass Accuracy
• 5 Decades of in-Spectrum
Dynamic Range from 2-Channel x
2 GHz Dual Gain Mode
• Allows changing from Highest
Resolution to Highest Dynamic
Range without changing
Sensitivity
Brings Research Grade HRAMS Performance in a routine format
Best Performance Obtained by not Overscanning
the Chromatographic Peaks: 4-peaks elute 0.6 to
0.9min and 10 scans/sec gives 24scans/peak
Calibration
• Calibrate daily to weekly.
• Generates fifth order polynomial to
accurately assign masses over entire
mass range.
•Takes 10 –15 seconds.
•6550 B.05 should use Extended
Calibration
1. Turn on MS.
2. Select Instrument State.
3. Select Bottle B.
4. Press Calibrate.
5. Apply
September 17th, 2008
Agilent Users Meeting
Calibration Result - Mass Calibration Curve
The Mass Calibration Curve
is generated by selecting the
Calibrate button.
The calibration curve shows
the time-to-mass fit of the
polynomial correction across
the full mass range.
Apply accepts new TOF
Mass Coefficients to
polynomial mass correction
function.
Residual Error display often
exhibits polynomial “wave”
pattern.
September 17th, 2008
Agilent Users Meeting
Calibration Result - Detailed Residual Plot
Detailed Residual Plot shows:
• uncorrected mass error in ppm (blue diamonds).
• corrected mass error in ppm (red squares) after applying fifth-order
polynomial mass correction.
• actual calibration curve – green line.
September 17th, 2008
Agilent Users Meeting
Optimization for Low Mass Operation
1. Calibrate using the entire mass range (High 3200 m/z).
2. Perform an AutoTune.
3. Instrument State – change mass range from 3200 to
1700.
4. Deselect higher masses not in new range from mass list.
5. Re-calibrate.
September 17th, 2008
Agilent Users Meeting
Mass Calibration Error Reduced
•The errors have been reduced.
•Must have 6 points.
•No gain with fewer points.
September 17th, 2008
Agilent Users Meeting
Automatic and Manual Tuning
Automated tuning for most users
Manual tuning for diagnostics and special applications
September 17th, 2008
Agilent Users Meeting
Order of Tuning Choices
(Check TOF Tune)
• If necessary, do…
(Quick TOF Tune)
• If necessary, do…
(TOF Tune)
• If necessary, do…every few months
Initial TOF Tune
(Check Quad Tune)
• If necessary, do…
(Quad Tune)
• If necessary, do…at PM time (Yearly)
Initial Quad Tune
September 17th, 2008
Agilent Users Meeting
Autotunes – TOF and Quad Autotune
TOF Autotune steps are displayed while the system tunes.
Autotune Report
September 17th, 2008
Agilent Users Meeting
Save Tune File When Changing Context
Make certain to specify correct tune file in acquisition method.
Or, save and load tune files in the Instrument State tab.
September 17th, 2008
Agilent Users Meeting
Agilent Users Meeting
September 17th, 2008
Agilent Users Meeting
September 17th, 2008
**First, try reducing Nebulizer pressure
**
Agilent Users Meeting
September 17th, 2008
New Makeup Procedure for Reference Solution
Changing from Acetonitrile to Methanol Solution
• 2 mL of HP121 into 98 mL of 95/5 MeOH/Water
• 0.8 mL of HP921 into 99.2 mL of 95/5 MeOH/Water
• Using a 500 mL volumetric flask, mix 25 mL of HP121 and 921 into 450
mL of 95/5 MeOH/Water with 0.2% Acetic Acid.
• Internal Reference Mass Correction – Two Ions (1 < m/z 350, second 500 amu higher), One ion OK
– (m/z 121.050873, 149.02332, 322.048121, 922.009798, 2421.91339)
– Do not use any ion with an interference
– Neb Pressure at 5 psi, B.05 not revealed
Better for Negative ion performance and for use with APCI and Multimode sources without changing reference solution.
New Ref Mass Solutions AJT
Improved recipes for ref mass abundances above sample background (>100k) Adjust quantities as necessary to ensure both ref masses in both pos and neg modes are greater than 75,000 counts throughout the gradient, with both UNX and plasma extracts.
uL per 100mL of 95/5 MeOH/water, reagents from Agilent Ref Mass kit:
AJST sources
Ammonium TFA 40 Purine 720 HP-0921 300
dual-nebulizer AJST source; roughly half the concentration needed vs UIRM introduction.
Ammonium TFA 20 Purine 300 HP-0921 75 (reduce to 50uL if 922 still >150k at both ends of gradient)
Behavior of the these compounds in the ref mass systems is not linear with concentration; some empirical adjustment might be required for each instrument, hence dedicated ref mass bottles with good labeling are required for each instrument.
Agilent Users Meeting
September 17th, 2008
Addition of Negative Ion Reference Ion
• From Acquisition
• MS TOF Tab
• General Tab
– Ion Polarity – Negative
• Ref Mass Tab
– Right Click “Add”
– Input Mass 966.000725
– Input Mass 980.016375
Reference Solution can also be introduced using a Binary or
Isocratic with a 100:1 splitter designed by Agilent. Flow rate of
LC set at between 0.5-1.0 mL/min and gives very stable flow.
Agilent Users Meeting
September 17th, 2008
My Mass Error is Above 3 ppm What should I do?
• Check for Interference from Chemical Background
– Change Chromatography
– Create Narrow (+/- 10ppm) Extracted Ion Chromatogram with subtraction
• Check Resolution of Peak in Spectrum Peak List
• Check for Saturated Peak – Peak List or “*” in Mass Spectrum
Agilent Users Meeting
September 17th, 2008
Poor Mass Accuracy Example – Unresolved Peaks
“Spectrum Peak List 1 Table – Resolution Column”
EIC m/z 337.09847
EIC m/z 338.17521
BPC
1.59 ppm error
Agilent Users Meeting
September 17th, 2008
10000 transients/sec = 10000@1scan/sec*255 =2.6 x 10^6, 5000@2scan/sec, 3333@3scan/sec 10000 transients/sec - 10000*255=2.5x10^6 @ 1scan/sec (no worries 400k)
- 5000*255=1.2x10^6 @2scan/sec (no worries 200k)
- 3333*255=8.4x10^5 @3scan/sec (no worries 166k)
- 2500*255=6.3x10^5 @4scan/sec (no worries 125k)
- 1000*255=2.5x10^5 @10scan/sec (no worries 40k)
Agilent Users Meeting
September 17th, 2008
Agilent Users Meeting
September 17th, 2008
Compare Manual versus Automated Analysis “Find Compounds by Feature Extraction”
Manual Spectral Subtraction
Automated Spectral Extraction
AutoMS/MS Features In Acquisition B.04 and
B.05 Acquisition
July 2011 52
Topics
The Decision Engine (“DE”)
Variable number of transients
– TIC target (MS/MS TIC abundance)
– Use MS/MS abundance limit
– Reject precursors that cannot reach target TIC
Purity
Isotope model
Multiple collision energies
Directed MS/MS
July 2011 53
Decision Engine
586.33420
464.79648
561.27494418.91250 1221.99064650.79033
726.36857363.25277
Counts vs. Mass-to-Charge (m/z)300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300
575 580 585 590
3+ charge state
580.60775
719.3670786.09825
645.83725302.09533
783.89034462.21703525.27282
935.42894187.072131178.58929
Counts vs. Mass-to-Charge (m/z)250 500 750 1000 1250 1500
681.34779586.33438
86.09667 863.43679
314.17494499.24340940.89651
728.30139157.13583
1048.493531184.64735
Counts vs. Mass-to-Charge (m/z)250 500 750 1000 1250
675 680 685 690
2+ charge state
MS/MS
MS/MS
July 2011 54
Quad Isolation Behavior Medium isolation (4 m/z) and wide isolation (9 m/z) modes
– 400.0000 m/z, 1+, medium isolation: window centered at 401.7000 m/z, isolates
approximately 399.7 to 403.7 m/z
– 400.0000 m/z, 2+, medium isolation: window centered at 401.4000 m/z, isolates
approximately 399.4 to 403.4 m/z
– Charge states >3 treated like 3; charge state ‘unknown’ (0) treated like 2
Narrrow (1.3 m/z) mode
– Isolation centered at requested m/z value
Values stored in .xml file (can be changed, use care!)
Center of window = monoisotopic m/z + (charge state * -0.3) + (window width / 2)
399 399.5 400 400.5 401 401.5 402 402.5 403 403.5 404
1+
399 399.5 400 400.5 401 401.5 402 402.5 403 403.5 404
2+
Decision Engine Flowchart
Centroid
spectrum
Eliminate peaks
below storage
threshold
Find isotope
groups by
applying isotope
model, “de-ring”,
determine
charge state
Rank by charge
state and/or
abundance
Profile
spectrum
Eliminate
candidates with
undesired charge
states and below
precursor
threshold
Calculate purity,
apply stringency,
rerank
Apply variable
number of
transients cutoff
Schedule
top N
Apply active
exclusion criteria
Match with
preferred list,
rerank
Limit to
directed list
typical
directed
preferred
LCMS Default Conditions for “Average Analysis”
0.4-0.6ml/min and 5-8min Gradient Time
LCMS Default Conditions for “Walkup Self-Serve
MS” 0.4-0.6ml/min and 5-8min Gradient Time
LCMS All Ions MSMS Experiment Low energy
parent ion scan
LCMS All Ions MSMS Experiment High energy
fragmentation scan
LCMS Setting up AutoMSMS experiment
Precursor Threshold Selection for MSMS
TIC Target: Summary
63 July 2011
Lower TIC target
Higher acquisition rate
Higher TIC target
Higher MS/MS spectral quality
•Trying to strike the compromise between too many poor quality MS/MS spectra, and
too few high quality spectra
•‘Just enough quality’
•Optimum value differs by experimental goals
Variable Number Of Transients
Power Ramp Correlation Plot—6530 Q-TOF M
S/M
S to
tal io
n c
urr
en
t/tr
an
sie
nt (lo
g s
ca
le)
MS precursor abundance/transient (log scale)
July 2011 64
Use MS/MS Accumulation Time Limit
This is the maximum number of transients
Unchecked: the maximum number of transients is the DE limit (16383, ~1.5 seconds on a 6530)
July 2011 65
**
Reject Precursors That Cannot Reach Target TIC
•From precursor abundance, calculate number of transients needed to
achieve TIC Target
•If number of MS/MS transients in Spectral Parameters tab is exceeded, it is
likely to yield a poorer quality product ion spectrum, so don’t use this
precursor
July 2011 66
Purity Feature and Isotope Model
Avoid isolating multiple precursors / minor precursors which produce
mixed (“chimeric”) product ion spectra
Purity: minimum acceptable percentage of target precursor cluster
Stringency: weighting factor for the ‘contaminants’
Purity re-orders abundance sorting (higher purity precursors move ahead
of higher abundance precursors with lower purity)
Disable purity: 0 stringency, 0 cutoff
600 601 602 603 604
Isolation window
July 2011 67
Collision Energy Tab Examples
3 CE fixed (e.g., small molecule libraries)
Suggested default
value for peptides
More aggressive fragmentation
for 2+ charge state peptides
July 2011 69
Watch for Acquisition Centroid Threshold Set Too
Low: periods of no automsms
Cause:
Too many mass peaks in the MS1 spectrum, exceeding the maximum number of isotope
groups permitted for the DE to handle. All isotope groups are then assigned as “unknown
charge state”, and the unknown charge state is excluded from sampling for this analysis.
Therefore, no autoMS/MS occurs until the number of isotope groups decreases below the
limit.
Solution: increase the centroid storage threshold so fewer isotope groups are being detected
and processed
July 2011 70
Directed MS/MS, A Special Mode of AutoMS/MS
•Box is checked: Directed MS/MS “Do autoMS/MS, but only if the precursor
candidate is in this table”.
•Box is unchecked: Preferred MS/MS “Do autoMS/MS, and if the precursor
candidate is in this table, move it to the top of the precursor candidate list.”
•Alternative concept—targeted MS/MS but using autoMS/MS selection criteria
NOTE: The preferred/directed table is executed in the order listed. No sorting is
applied to items in this table.
July 2011 71
MFE Results
12 February 2014
AN-CE-LCMS-2-086-B: Advanced MassHunter Qualitative and Quantitative Analysis
72
Compounds in a Compound List Exported for
Acquisition
Compounds showing H+ and Na+ limited to just H+
(For H+ and NH4+ may have 2 precursors/cmpnd)
Setup Precursor Charge States (Calculated from
Neutral Mass)
Choose Ion Species (Added to Charge States)
Imported into Acquisition
Agilent Users Meeting
September 17th, 2008
Summary
• Care and Feeding of QTOF/TOF
– Remember to Defrag Hard Drives Weekly
– Software updates available
• Mass Profiler B.01.00 $3,150
– Differential Analysis Software
– Batch Molecular Formula Generation and Database Searching
• METLIN Personal Metabolite Database B.01.00 $3,759
– Accurate Mass Retention Time Database (AMRT)
– Convert *.CSV to MTL Formats
– User Generated Databases (Plant Extract, Pesticides, AA’s)
Agilent Users Meeting
September 17th, 2008
Questions and Answers
Thank You
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