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

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4

9

Syst

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Multi-Channel LC Example – 4 LC Channels

0 min

Syst

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2 min

3 min

1 min

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stem

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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

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1 Method 1

Method 2

0 min 3 min.

MS Data – Multiplexed

System diagram above is representative of a Transcend LX-2 system

Syst

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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

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RT: 120.78AA: 136RT: 23.46

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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

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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

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RT: 48.22AA: 1109 RT: 51.48

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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

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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)

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C:\Xcalibur\...\Sample001 11/14/13 11:30:06 Sample001N/CRT: 0.00 - 176.59 SM: 5G

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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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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

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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

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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

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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

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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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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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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

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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

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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?