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1 The world leader in serving science
Pittcon John Fink
So, How Fast is Your LC/MS Analysis?
2
So, How Fast is Your LC/MS Analysis?
• We will shows examples of…
• Ultra-fast LC/MS solutions
• Ultra-fast LC/MS • Pharma and Clinical Research applications • Chromatography only and ultra-fast online clean-up
3
Benefits of Ultra-Fast LC/MS
• Get results sooner
• Get more data in same or shorter time
• Save money
• Potential to save solvents • Free up capacity on expensive equipment
4
Obstacles of Ultra-Fast LC/MS
• Is your equipment even robust enough?
• Was it designed to run 24/7? • Are you overstressing it, require constant maintenance?
• Will data quality be sacrificed by going faster? • Expensive to buy additional systems for more capacity
5
Solution – Multi-Channel LCUltra-Fast Throughput
• Multiple, independent, but parallel LC channels
• All separate, but flow into one mass spec detector
• Time staggered injections, so no data is ever missed
6
Multi-Channel LC Technology
• All channels are independent • Separate pumps, injectors, tubing, columns
• Time staggering, valve switching all effortlessly controlled by user-friendly software
7
Method and Gradient Example – LC-MS
0 min 4 min
Acquisition: compounds elute to MS during this 45s window
gradient
8
Multi-Channel LC Increases Throughput
All coordinated and controlled by Thermo Scientific™ Aria™ software
System diagram above is representative of a Thermo Scientific™ Transcend™ LX-4 system
1 min 4 min.
Syst
em
1
3 min .
4 min .
2 min
Syst
em
2 Sy
stem
3
Each LC system only requires the MS during
the elution step
Idle Time
Acquisition Window
Idle Time
Syst
em
4
9
Syst
em
4
Multi-Channel LC Example – 4 LC Channels
0 min
Syst
em
1
2 min
3 min
1 min
Syst
em
2 Sy
stem
3
0 min
During this 4 min window – 4 samples were completed
Equals 60 samples/hour versus 15 samples/hour (without Multiplexing)
Final Collected MS Data
Individual data files
Staggered Injections
10
Data on LX-4 System – Compare Method Data Window
1 LC
LX-4
3.75 samples in the time it takes to run 1 sample
11
Multiple Method Ultra-Throughput
Two methods running simultaneously
0 min 3 min.
0 min 3 min.
Syst
em
1 Method 1
Method 2
0 min 3 min.
MS Data – Multiplexed
System diagram above is representative of a Transcend LX-2 system
Syst
em
2
12
Multi-Channel LC with Aria Software
1. Import or Create a batch/sequence of samples
2. Select which LC channel(s) to run the samples or select “Any Available”
3. Submit batch!
More batches can be submitted at any time in any order and the system will automatically multiplex where possible.
13
Benefits of Multi-Channel LC
• Accelerate results • Getting answers/data faster
Throughput
Less Cost
Flexibility
Ease of Use
• Handles multiple methods • Many options available
• Intuitive, quick learning curve
• Reduce cost of capital equipment • Reduce cost per sample
14 The world leader in serving science
Case Study – High Throughput QQQ Screening
15
High Throughput Screening Application
• Equipment • Transcend LX-4 System 4-Channel LC • Aria OS software • Thermo Scientific™ TSQ Vantage™ mass spectrometer
• Application • Four-channel LC separation for high-throughput, simultaneous
analysis of a compound of interest for drug discovery • Compound: 1-minute method with a 15-second data collection window
• Same method runs on separate channels, simultaneously
• Quadruple throughput
Chromatography is performed in this short time !
16
HTS, 15s Injections – Throughput RT: 0.00 - 139.82
0 20 40 60 80 100 120Time (min)
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RT: 120.78AA: 136RT: 23.46
AA: 120
RT: 113.27AA: 95.09
RT: 14.83AA: 91.95
RT: 31.00AA: 91.17 RT: 126.91
AA: 81.14RT: 48.24AA: 68.21
RT: 100.64AA: 68.13
RT: 67.22AA: 55.48
RT: 36.73AA: 56.75
RT: 87.66AA: 52.46
120.77
RT: 120.78AA: 6352RT: 23.46
AA: 5456
RT: 14.83AA: 4177
RT: 113.27AA: 4494
RT: 31.00AA: 4038 RT: 126.91
AA: 3452RT: 48.24AA: 2949
RT: 68.30AA: 2997
RT: 13.74AA: 3002
RT: 100.64AA: 2868
RT: 36.73AA: 2483
RT: 87.66AA: 2369
NL: 3.31E3TIC MS ICIS SPL1_Well_001
NL: 9.86E2TIC F: - c ESI SRM ms2 403.500 [151.500-152.500] MS SPL1_Well_001
NL: 3.31E3TIC F: - c ESI SRM ms2 417.500 [151.500-152.500] MS ICIS SPL1_Well_001
RT: 43.98 - 55.62
44 45 46 47 48 49 50 51 52 53 54 55Time (min)
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RT: 48.24AA: 68.21RT: 47.16
AA: 62.70RT: 48.25AA: 49.29
RT: 46.79AA: 50.86 RT: 51.09
AA: 41.83
RT: 48.22AA: 1109 RT: 51.48
AA: 1026RT: 47.15AA: 930 RT: 50.00
AA: 864RT: 48.92AA: 771 RT: 52.54AA: 725RT: 53.63AA: 744
RT: 44.62AA: 721
RT: 45.70AA: 711
RT: 54.72AA: 652
RT: 48.24AA: 2949RT: 47.16
AA: 2753 RT: 50.01AA: 2408RT: 46.79
AA: 2221RT: 44.63AA: 2066RT: 48.94AA: 2072
RT: 52.89AA: 1993
RT: 50.42AA: 1878
RT: 53.64AA: 1834
RT: 51.09AA: 1787 RT: 54.73
AA: 1488RT: 45.71AA: 1546
NL: 1.62E3TIC MS ICIS SPL1_Well_001
NL: 6.18E2TIC F: - c ESI SRM ms2 403.500 [151.500-152.500] MS ICIS SPL1_Well_001
NL: 1.62E3TIC F: - c ESI SRM ms2 417.500 [151.500-152.500] MS ICIS SPL1_Well_001
17
Ultra-Fast LC/MS
• Able to screen 384 well plate (384 samples) in 96 minutes
• 24 plate capacity
• Redundant systems
• Intelligent software
18 The world leader in serving science
Example – High Resolution Accurate Mass Drug Discovery Workflows
19
Transcend II LX-4 UHPLC System with Q Exactive MS
20
Transcend II, LX-4 System Hardware
• Four Thermo Scientific™ Dionex™ UltiMate™ 3000 HPG-3400RS pumps
• Dual-arm CTC autosampler with DLW • Transcend II VIM with selector and detector
bypass valves
21
LC Method and Sample - 4 Lipids
• Four lipids in a 100ng/mL neat standard (Lipid 862 used as IS) • BEH C8, 2.1x50 mm, 1.7 μm columns • MP A: 5mM Ammonium Formate + 0.2% Formic Acid (aq) • MP B: 5mM Ammonium Formate + 0.2% Formic Acid in 1:1 ACN:MeOH
Before LC Method Optimization: Constant 0.750 mL/min Flow Rate Loading at 10:90 A:B Step Gradient to 5:95 A:B Re-equilibration back to 10:90 A:B Elution within a ~36s data window 5-μL 100% organic sample injection
After LC Method Optimization: Variable 1 – 0.5 mL/min Flow Rate Loading at 10:90 A:B Step Gradient to 5:95 A:B Re-equilibration back to 10:90 A:B Elution within a 21s data window 10-μL 90% organic sample injection
22
3 Method Trials
Test #
Data Window
(secs)
# of
Injections
% RSD
(peak area ratio)
1
21
(multiple data files)
1000
3.94 (L_806) 2.93 (L_890) 2.55 (L_888)
2
21
(single data file)
384 x 3
5.83 (L_806) 4.52 (L_890) 4.34 (L_888)
3
9
(single data file)
384
2.84 (L_888)
23
C:\Xcalibur\...\Sample001 11/14/13 11:30:06 Sample001N/CRT: 0.00 - 176.59 SM: 5G
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RT: 137.72AA: 492410SN: 1567
NL: 3.54E4m/z= 806.54173-806.54979 MS ICIS Sample001
NL: 9.64E4m/z= 862.60405-862.61267 MS ICIS Sample001
NL: 2.39E5m/z= 890.63521-890.64411 MS ICIS Sample001
NL: 6.05E5m/z= 888.61957-888.62845 MS ICIS Sample001
RT: 103.06 - 104.81 SM: 5G
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RT: 103.70AA: 86764SN: 304
RT: 104.16AA: 77207SN: 293 RT: 104.54
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RT: 103.19AA: 7985SN: 34
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RT: 103.34AA: 219431SN: 293
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NL: 2.67E4m/z= 806.54173-806.54979 MS ICIS Sample001
NL: 7.35E4m/z= 862.60405-862.61267 MS ICIS Sample001
NL: 1.82E5m/z= 890.63521-890.64411 MS ICIS Sample001
NL: 4.62E5m/z= 888.61957-888.62845 MS ICIS Sample001
Single Data File (21s DW) 384 Sample Injections
24
384 Sample Injections – Individual Data Files (Four-Channel View) – 21 Second Data Window
C:\Xcalibur\...\4_data2088 11/09/13 15:32:38
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NL:2.49E4m/z= 806.53769-806.55383 MS ICIS 4_data2086
NL:6.26E4m/z= 862.59973-862.61699 MS ICIS 4_data2086
NL:1.51E5m/z= 890.63075-890.64857 MS ICIS 4_data2086
NL:4.04E5m/z= 888.61512-888.63290 MS ICIS 4_data2086
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NL:2.71E4m/z= 806.53769-806.55383 MS ICIS 4_data2087
NL:7.70E4m/z= 862.59973-862.61699 MS ICIS 4_data2087
NL:1.94E5m/z= 890.63075-890.64857 MS ICIS 4_data2087
NL:4.78E5m/z= 888.61512-888.63290 MS ICIS 4_data2087
RT: 0.00 - 0.35 SM: 5G
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NL:2.63E4m/z= 806.53769-806.55383 MS ICIS 4_data2088
NL:6.33E4m/z= 862.59973-862.61699 MS ICIS 4_data2088
NL:1.50E5m/z= 890.63075-890.64857 MS ICIS 4_data2088
NL:4.03E5m/z= 888.61512-888.63290 MS ICIS 4_data2088
RT: 0.00 - 0.35 SM: 5G
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NL:2.63E4m/z= 806.53769-806.55383 MS ICIS 4_data2088
NL:6.33E4m/z= 862.59973-862.61699 MS ICIS 4_data2088
NL:1.50E5m/z= 890.63075-890.64857 MS ICIS 4_data2088
NL:4.03E5m/z= 888.61512-888.63290 MS ICIS 4_data2088
25
File (9 sec Data Window) 384 Sample Injections – Single Data
E:\Q-Exactive Data\...\Sample1537 11/21/2013 3:17:55 PM Sample1537N/C
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RT: 44.26AA: 597256SN: 1650
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NL:9.95E4m/z= 862.59973-862.61699 M S ICIS Sample1537
NL:6.13E5m/z= 888.61512-888.63290 M S ICIS Sample1537
RT: 53.13 - 54.13 SM: 5G
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NL:8.52E4m/z= 862.59973-862.61699 M S ICIS Sample1537
NL:5.11E5m/z= 888.61512-888.63290 M S ICIS Sample1537
Scaling to one-minute RT: 53.13 – 54.13 mins.
26
Four LC Channels Running….
27
Aria OS Batch File Viewer: Pressure Trace Overlay
28
Aria OS Batch Status View
29
Overview of Acquisition Styles – 384 Samples/Batch
Acquisition Style
Data Window Length (secs)
LC Method Length (mins)
Chromatography Batch Run Time (mins) Injections per minute
Standard LC/MS w/ 384
Data Files
N/A
1.6
Baseline separation of 3
Analytes
~614.4
0.63
LX-4 Multiplexing & 384 Data Files
21
1.6
Baseline separation of 3
Analytes
222.74
1.7
LX-4 Multiplexing & One Data File
21
1.6
Baseline separation of 3
Analytes
176.59
2.2
LX-4 Multiplexing & One Data File
9
1.0
Co-Elution of 2
Analytes
107.25
4.0
30 The world leader in serving science
Ultrafast Screening of Antiretrovirals: TurboFlow Technology and Multi-Channel LC
31
LC System
• Transcend LX-2 system • Two channels
32
LC Method View
33
AS Method View
34
LCMS Method Details
• Transcend LX-2 system with CTC single-arm DLW • Thermo Scientific™ TurboFlow™ column (0.5x50 mm) used in 1D setup • Analytical column: single guard column located at MS source • Mobile Phases:
1. 10 mM Ammonium Formate w/ 0.05% Formic Acid (Loading) 2. Acetonitrile w/ 0.1% Formic Acid (Eluting) 3. 45:45:10 Acetonitrile:Isopropanol:Acetone (TurboFlow column wash)
• Injection volume: 30 μL • LC Method Length: 100 seconds • Thermo Scientific™ Q Exactive™ MS with HESI source • Warfarin used as both Positive and Negative ESI mode IS • 30 sec MS data window necessary for simultaneous Positive/Negative switching • Use of Detector ByPass to allow for high flow rates of TurboFlow technology • Multiplexing sample clean-up yet singular analytical separation
35
TurboFlow Technology for Fast Online Clean Up
• TurboFlow technology can eliminate off-line sample prep • Directly inject biological or dirty matrices • Typically used in 2D-LC • Improve data quality by reducing ion suppression and interferences
• Uses • Size exclusion
• Large molecules are not retained and go directly to waste • Do not have time to diffuse with high flow rates
• Chemistry • Capture analytes with affinity for column’s stationary phase
− Retain targeted analytes
• Result • Rapid, automated, sample cleanup of unwanted large molecules and
retention of specific analytes of interest
36
Standard Curve Comparisons In Three Matrices
• ACN Std curve – diluted 1:3 with aqueous IS solution
• Plasma Std curve – diluted 1:3 with aqueous IS solution
• Plasma Std curve – crashed 1:3 with ACN IS solution
• Whole Blood Std curve – crashed 1:3 with ACN IS solution
37
Nevirapine: ACN Standard Curve
38
Nevirapine: Plasma Dilution Standard Curve
39
Nevirapine: Plasma ACN-Crashed Standard Curve
40
Nevirapine: Whole Blood ACN-Crashed Curve
41
250 ng/mL Standard Comparison Across Four Matrix Preps (Full MS)
Diluted ACN ACN-crashed Plasma Diluted Plasma
ACN-crashed Whole Blood
Nevirapine [M+H]+
Saquinavir [M+H]+
Zidovudine [M-H]-
Efavirenz [M-H]-
C:\Xcalibur\...\StdA_250_003 6/18/2013 6:02:47 PMJHopkins Plasma Curve w/ Warf and Morph-d3 IS added (Water dilution)RT: 0.00 - 0.53 SM: 7G
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NL: 1.54E7m/z= 267.12326-267.12594 F: FTMS + p ESI Full ms [250.00-700.00] MS ICIS StdA_250_004
NL: 4.90E7m/z= 671.38819-671.39491 F: FTMS + p ESI Full ms [250.00-700.00] MS ICIS StdA_250_004
NL: 8.93E3m/z= 266.08815-266.09081 F: FTMS - p ESI Full ms [250.00-350.00] MS ICIS StdA_250_004
NL: 1.37E6m/z= 314.01854-314.02168 F: FTMS - p ESI Full ms [250.00-350.00] MS ICIS StdA_250_004
NL: 2.87E7m/z= 309.11059-309.11369 F: FTMS + p ESI Full ms [250.00-700.00] MS ICIS StdA_250_004
NL: 2.24E7m/z= 307.09604-307.09912 F: FTMS - p ESI Full ms [250.00-350.00] MS ICIS StdA_250_004
RT: 0.00 - 0.52 SM: 7G
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NL: 5.59E6m/z= 267.12326-267.12594 F: FTMS + p ESI Full ms [250.00-700.00] MS ICIS StdA_250_003
NL: 2.82E7m/z= 671.38819-671.39491 F: FTMS + p ESI Full ms [250.00-700.00] MS ICIS StdA_250_003
NL: 1.97E4m/z= 266.08815-266.09081 F: FTMS - p ESI Full ms [250.00-350.00] MS StdA_250_003
NL: 7.60E5m/z= 314.01854-314.02168 F: FTMS - p ESI Full ms [250.00-350.00] MS ICIS StdA_250_003
NL: 3.49E6m/z= 309.11059-309.11369 F: FTMS + p ESI Full ms [250.00-700.00] MS ICIS StdA_250_003
NL: 3.51E7m/z= 307.09604-307.09912 F: FTMS - p ESI Full ms [250.00-350.00] MS ICIS StdA_250_003
RT: 0.00 - 0.52 SM: 7G
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NL: 4.78E6m/z= 267.12326-267.12594 F: FTMS + p ESI Full ms [250.00-700.00] MS ICIS StdA_250_004
NL: 1.74E7m/z= 671.38819-671.39491 F: FTMS + p ESI Full ms [250.00-700.00] MS ICIS StdA_250_004
NL: 3.82E3m/z= 266.08815-266.09081 F: FTMS - p ESI Full ms [250.00-350.00] MS ICIS StdA_250_004
NL: 6.82E5m/z= 314.01854-314.02168 F: FTMS - p ESI Full ms [250.00-350.00] MS ICIS StdA_250_004
NL: 1.19E7m/z= 309.11059-309.11369 F: FTMS + p ESI Full ms [250.00-700.00] MS ICIS StdA_250_004
NL: 3.16E7m/z= 307.09604-307.09912 F: FTMS - p ESI Full ms [250.00-350.00] MS ICIS StdA_250_004
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NL: 4.15E6m/z= 267.12326-267.12594 F: FTMS + p ESI Full ms [250.00-700.00] MS ICIS StdA_250_004
NL: 9.82E6m/z= 671.38819-671.39491 F: FTMS + p ESI Full ms [250.00-700.00] MS ICIS StdA_250_004
NL: 3.29E3m/z= 266.08815-266.09081 F: FTMS - p ESI Full ms [250.00-350.00] MS ICIS StdA_250_004
NL: 6.49E5m/z= 314.01854-314.02168 F: FTMS - p ESI Full ms [250.00-350.00] MS ICIS StdA_250_004
NL: 1.46E7m/z= 309.11059-309.11369 F: FTMS + p ESI Full ms [250.00-700.00] MS ICIS StdA_250_004
NL: 3.86E7m/z= 307.09604-307.09912 F: FTMS - p ESI Full ms [250.00-350.00] MS ICIS StdA_250_004
42
Antiretroviral Throughput with Online Cleanup
• Ultra-fast throughput was achieved by three things… • Using TurboFlow technology for Quick-Elute fast online clean-up
• Multi-channel LC enabled us to run four samples at a time • Allowing four samples in the time of one typical UHPLC sample
• The Q Exactive MS enabled full-scan, accurate mass
43
Thermo Scientific Transcend II LX-4 Platform
• Capable of standard method development and HPLC/UHPLC
or a high-throughput workflow
• Four LC systems utilize one mass spec
• Single data file or multiple data files
• Software tools for troubleshooting
• Rugged, easy-to-maintain LC solution
Fast Workflow-Driven Versatile User-Friendly Cost-Effective
44
Q Exactive MS: The Ultimate Routine Quan/Qual LC-MS/MS Instrument
• Ideal QUANT + QUAL routine solution with highest quantification capabilities and identification confidence
• Sensitivity: ~ high-end triple quadrupole mass spec
• Mass Accuracy: 3 ppm external calibration, 1ppm internal calibration for at least one week
• Resolution: 140K resolution
• Speed: 12Hz @ 17.5K, 7Hz @ 35K, 3Hz @ 70K. No loss in sensitivity.
• Scan-to-scan Polarity Switching: 1Hz @ 35K
• Spectrum Multiplexing and All Ion Fragmentation
• MS/MS or MS3: HCD with precursor or in-source CID
45
Summary – So, How Fast is Your LC/MS Analysis?
• Demonstrated ultra-fast LC/MS • Pharma and Clinical Research applications • Chromatography alone and TurboFlow technology for ultra-fast online
clean-up
• Transcend systems drastically increase throughput, while keeping costs low
• Robustness is maintained since systems are not overstressed
• Take your best LC method and run it on a Transcend system to increase throughput
So, How Fast is Your LC/MS Analysis?So, How Fast is Your LC/MS Analysis?Benefits of Ultra-Fast LC/MSObstacles of Ultra-Fast LC/MSSolution – Multi-Channel LCUltra-Fast ThroughputMulti-Channel LC TechnologyMethod and Gradient Example – LC-MSMulti-Channel LC Increases ThroughputMulti-Channel LC Example – 4 LC ChannelsData on LX-4 System – Compare Method Data WindowMultiple Method Ultra-ThroughputMulti-Channel LC with Aria Software Benefits of Multi-Channel LCCase Study – High Throughput QQQ ScreeningHigh Throughput Screening ApplicationHTS, 15s Injections – ThroughputUltra-Fast LC/MSExample – High Resolution Accurate Mass Drug Discovery WorkflowsTranscend II LX-4 UHPLC System with Q Exactive MSTranscend II, LX-4 System HardwareLC Method and Sample - 4 Lipids 3 Method TrialsSingle Data File (21s DW) 384 Sample Injections384 Sample Injections – Individual Data Files �(Four-Channel View) – 21 Second Data WindowFile (9 sec Data Window) �384 Sample Injections – Single DataFour LC Channels Running….Aria OS Batch File Viewer: Pressure Trace OverlayAria OS Batch Status ViewOverview of Acquisition Styles – 384 Samples/BatchUltrafast Screening of Antiretrovirals: �TurboFlow Technology and �Multi-Channel LCLC SystemLC Method ViewAS Method ViewLCMS Method DetailsTurboFlow Technology for Fast Online Clean UpStandard Curve Comparisons In Three MatricesNevirapine: ACN Standard CurveNevirapine: Plasma Dilution Standard CurveNevirapine: Plasma ACN-Crashed Standard Curve Nevirapine: Whole Blood ACN-Crashed Curve 250 ng/mL Standard Comparison Across Four Matrix Preps (Full MS) Antiretroviral Throughput with Online Cleanup Thermo Scientific Transcend II LX-4 PlatformQ Exactive MS: The Ultimate Routine Quan/Qual LC-MS/MS InstrumentSummary – So, How Fast is Your LC/MS Analysis?