Xevo TQ-S for Bioanalysis · ©2010 Waters Corporation . Compound name: OH Prog Correlation...
Transcript of Xevo TQ-S for Bioanalysis · ©2010 Waters Corporation . Compound name: OH Prog Correlation...
©2010 Waters Corporation
Xevo TQ-S for Bioanalysis
©2010 Waters Corporation
Key innovations
• High Performance
• Stepwave • RADAR
• Scan Wave
• Versatility
• Ion Sources
• Simplicity of Operation
• Engineered Simplicity
©2010 Waters Corporation
Xevo TQD
Xevo TQD Fast data acquisition
Routine UPLC-MS/MS
RADAR acquisition mode
Universal ion source architecture
Xevo TQ MS
Xevo TQ-S
Frost and Sullivan Product Quality Leadership Award 2011
Xevo TQ-S Highest sensitivity
Fastest data acquisition
ScanWave Enhanced product ion scanning
RADAR acquisition mode
Universal ion source architecture
The Xevo TQ family Built on the same foundations
Best new product, ASMS 2008
Xevo TQ MS More sensitive than TQD
ScanWave Enhanced product ion scanning
Fast data acquisition
RADAR acquisition mode
Universal ion source architecture
Ultra high performance targeted quantification with information rich qualitative supporting data
Routine targeted quantification with information rich qualitative supporting data
High performance targeted quantification with information rich qualitative supporting data
©2010 Waters Corporation
Xevo TQ-S
Ultra high sensitivity for targeted quantification
©2010 Waters Corporation
Time0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
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100
Time0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
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Xevo TQ
Xevo TQ-S Enhanced sensitivity 129X increase in peak area 25X increase in signal:noise
An increase in sensitivity? Rel
ativ
e io
n a
bundan
ce
Desompressin (Peptide)
UPLC/MRM, ESI +
©2010 Waters Corporation
An increase in sensitivity?
Fenuron ESI+ 30 7Metamitron ESI+ 32 15Acephate ESI+ 27 7Chlortoluron ESI+ 27 8Aldicarb ESI+ 27 6Demeton S Methyl ESI+ 26 9Phoxim ESI+ 64 19Kresoxim Methyl ESI+ 64 4Azinphos Methyl ESI+ 42 6Azoxystrobin ESI+ 45 4Dimethoate ESI+ 23 10Acetamiprid ESI+ 30 28Fluticasone ESI+ 30 3Formoterol ESI+ 39 4Nefadazone ESI+ 28 3Desmopressin ESI+ 129 25Salmeterol ESI+ 41 8Alprazolam ESI+ 21 13Reserpine ESI+ 25 5Ibuprofen ESI- 13 16Prostaglandin E2 ESI- 30 37
Mean Difference 38 11
Relative Peak Area
Relative S:N
Compound NameIonisation
Mode
©2010 Waters Corporation
Xevo TQ Xevo TQ-S
Larger sampling orifice
How did we increase sensitivity?
Problems associated with a larger sampling orifice:
1)A disperse ion cloud
2) Higher levels of matrix contamination
©2010 Waters Corporation
Ion Block Design
Sampling cone aperture — Increased to 0.8mm diameter
o Approx 5x increase in gas/ion flow
New ion block design
— No supplemental pumping
— All ions (and gas) enter StepWave guide
o Approx 200x increase in gas flow
o Up to 200x increase in ion flux
©2010 Waters Corporation
Ele
ctri
c Fi
eld
Diffuse Ion
Cloud
Designed to deal with problems associated with a larger sampling orifice
Problems associated with a larger sampling orifice:
1)A disperse ion cloud
2) Higher levels of matrix contamination
©2010 Waters Corporation
Ele
ctri
c Fi
eld
Diffuse Ion
Cloud
Maximising signal
Maximising robustness
Designed to deal with problems associated with a larger sampling orifice
©2010 Waters Corporation
SIMION Model
No Electric Field
N2 Flow
1 mbar N2
©2010 Waters Corporation
With Electric Field (25 V between guides)
N2 Flow
E
SIMION Model
1 mbar N2 T-Wave
T-Wave
©2010 Waters Corporation
Ultra High Sensitivity?
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
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0
1000.83
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
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100
2fg of
Verapamil Time
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
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100
0.83
Solvent
Blank
0.1fg
6 replicates
RSD<20%
UPLC/MRM of Verapamil (solvent standard) using an
ACQUITY prepared for trace analysis
220 Zeptomoles
©2010 Waters Corporation
Compound name: OH ProgCorrelation coefficient: r = 0.999149, r^2 = 0.998298Calibration curve: 3386.22 * x + 23.6767Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Conc0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000
Re
sp
on
se
0
500000
1000000
1500000
2000000
2500000
3000000
APCI+, Hydroxprogesterone
Triplicate injections of Standards
50fg to 5,000pg on column
5 orders of magnitude
Correlation coefficient>0.995
Deviation<15%
Linearity of Response?
©2010 Waters Corporation
Assay Robustness
Verapamil, 10pg/µL spiked into supernatant from
2:1 ACN:Plasma protein precipitation.
ACQUITY BEH 2.1x 50mm
1000 on column injections
RSD of peak areas < 5%
©2010 Waters Corporation
10ul loop injection 0.5pg/ul Verapamil in human plasma/ACN_2
Time0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25
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100TC8_100721_1135 MRM of 1 Channel ES+
455 > 165 (Verapamil)2.66e7
0.83
Assay Robustness
Verapamil, 0.5pg/µL spiked
into supernatant from 2:1 ACN:Plasma protein
precipitation.
10µL injections
ACQUITY BDH 2.1x 50mm
>2000 on column injections
RSD of peak areas < 5%
Plasma injection 1
Plasma injection 1000
Plasma injection 2000
©2010 Waters Corporation
Xevo TQ-S
Information rich qualitative supporting
data
©2010 Waters Corporation
Rapid MS to MRM Switching
©2010 Waters Corporation
m/z100 200 300 400 500 600 700 800 900 1000
%
0
100
m/z100 200 300 400 500 600 700 800 900 1000
%
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100 253.3
123.0
293.3
397.3441.4
619.6485.5
329.3
214.1
100.1149.3 279.3
251.1
415.3
363.4
546.4468.6
600.7
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
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%
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Targeted compound MRM positive ESI Fluticasone
in plasma
Matrix Profile MS full scan negative ESI
Matrix Profile MS full scan positive ESI
Qu
alita
tive
Qu
an
tita
tive
Rapid MS to MRM Switching
©2010 Waters Corporation
Method Development: Comparison of PPT to SPE Extract
Protein Precipitation
MRM
Full Scan (Extracted
Interference)
Protein precipitation Monitor Alprazolam MRM 309->281 7.03e6
SPE Monitor Alprazolam MRM 309->281 1.56e6
SPE Background ms scan 2.61e7
Protein precipitation Background ms scan 2.44e8
©2010 Waters Corporation
Method Development: Choosing between two gradients
Time0.50 1.00 1.50 2.00 2.50 3.00
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1.95
2.792.32
2.19
1.65 2.36
2.29 2.60 2.962.78
Phospholipids184>184
Radar Plasma Blank
Radar Solvent Blank
Fluticasone501>293
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25
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0.97
1.08
1.61
0.831.17 1.29
1.271.17
1.00
1.791.56
1.97 2.23
Phospholipids184>184
Radar Plasma Blank
Radar Solvent Blank
Fluticasone501>293
Gradient method 2 5-95% MeOH in 2.0 minutes
Gradient method 1 5-95% MeOH in 0.70 minutes
©2010 Waters Corporation
Simultaneous MRM and Full Scan Detection of Metabolites
MRM of Drug
Full Scan of Metabolites
Metabolites
©2010 Waters Corporation
RADAR & StepWave
RADAR — Monitor matrix complexity — RADAR enables understanding of matrix complexity — Intelligent decision making when assessing matrix effects — Intelligent decision making when developing methods
StepWave — Extra sensitivity in MRM mode — Reduce matrix effects by simple dilution of complex extracts — Maintain ability to achieve LOQs
©2010 Waters Corporation
Xevo TQ-S
Information rich qualitative supporting
data
ScanWave
©2010 Waters Corporation
ScanWave Technology
Travelling Wave
DC Barrier
RF Barrier
Potential Energy
Low m/z Ion
High m/z Ion
Intermediate m/z Ion
Storage Region
ScanWave Region
Travelling Wave
Travelling Wave
Accumulation
Transfer
High m/z Ejection
Medium m/z Ejection
©2010 Waters Corporation
Standard Product Ion Scanning
Continuous output of ions by cell
Most ions filtered out by MS2
Very inefficient
©2010 Waters Corporation
ScanWave Enhanced Product Ion Scanning
Ion accumulation
Mass selective ion ejection
Synchronised with scanning of MS2
©2010 Waters Corporation
StepWave Combined with ScanWave
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00
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100S/N:RMS=60.14
m/z50 100 150 200 250 300 350 400 450 500 550 600 650
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100195
174
105148
236 397365 448
ScanWave enhanced
Product ion spectrum
UPLC/MS/MS
50fg Reserpine
on column
ScanWave enhanced
Product ion scanning
chromatogram
(m/z 195)
A combination providing leading-edge
spectral MS/MS sensitivity
©2010 Waters Corporation
PIC Scan Acquisition
MRM Acquisition
PIC SCAN:
MRM data is used as a specific trigger for the acquisition of a ScanWave enhanced product ion spectrum.
Eliminates the need for 2 analytical runs for confirmation
Rapid Mode Switching Precursor Ion Confirmation Scan
©2010 Waters Corporation
ScanWave Technology
MRM
MRM ScanWave DS
A product ion confirmation (PIC) scan is a useful tool for method development, or when there may be two peaks within a single MRM transition
©2010 Waters Corporation
An Alternate Approach to Full Scan Profiling of Metabolites
Screen for selected panel of metabolites — Commonly reported metabolites
— User definable
Predicts metabolite MRMs based upon Parent Compound MSMS
Generates MANY MRMs — 1 msec dwell
With RADAR, full scan MS may be added to the experiment
©2010 Waters Corporation
( ) +
P
F1
F2 F3 ( )
P M
F1
F2+M F3
P+M > F1 P+M > F1+M P+M > F2 P+M > F2+M P+M > F3 P+M > F3+M
iMRMs :
©2010 Waters Corporation
List of Potential Metabolites from Text
File
MS Source and Analyzer
Conditions
iMRM Method
Spectra of Parent Automatically Obtained from
MassLynx
Select Metabolites from list of probable
transformations
Identify Product Ions From Parent Compound
Calculate MRMs and Write MS Method
File
MSMS of Parent Compound
Automatic Product Ion Selection
Develop Theoretical MRMs to Investigate
©2010 Waters Corporation
iMRM method
©2010 Waters Corporation
[Benefits] — Metabolites detected with
maximum sensitivity
— Spectra acquired in the time scale of UPLC peak
— Data acquired in one analytical run, no need for confirmatory experiment
Simple Metabolite Detection and Confirmation
Time0.50 1.00 1.50 2.00 2.50
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18_4_022 5: MRM of 1 Channel ES+ 458.195 > 339.845 (AZ1_Hydroxylation)
3.77e5
m/ z1 00 12 0 1 40 1 60 18 0 2 00 2 20 24 0 2 60 28 0 30 0 3 20 34 0 36 0 3 80 40 0 42 0 4 40 46 0
%
0
1 00
18 _4 _0 23 3 ( 2. 07 8) 5: Pro du c t I on s o f 4 58 ES+45 8. 19 5 > 3 39 .84 5 (AZ1_ Hy dro x y la tion )
1.9 2e 5326
315
340
354
m/ z1 00 12 0 14 0 16 0 18 0 2 00 2 20 2 40 26 0 28 0 30 0 3 20 3 40 3 60 38 0 40 0 42 0 44 0 4 60
%
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1 00
18 _4 _0 25 5 ( 2. 14 0) 3: Pro du c t I on s o f 4 42 ES+44 2.2 > 32 5. 84 7 ( AZ1_ Par en t)
8.9 8e 6326
313
340
425
Time0.50 1.00 1.50 2.00 2.50
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18_4_025 3: MRM of 1 Channel ES+ 442.2 > 325.847 (AZ1_Parent)
1.08e8
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18_4_022 1: MRM of 1 Channel ES+ 416.148 > 325.847 (AZ1_Dealkylation)
5.66e6
m/ z1 00 1 20 14 0 16 0 1 80 20 0 22 0 2 40 2 60 28 0 30 0 3 20 340 36 0 3 80 4 00 42 0
%
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1 00
18 _4 _0 23 4 ( 1. 97 6) 1: Pro du c t I on s o f 4 16 ES+4 16 .1 48 > 32 5. 84 7 ( AZ1 _D ea lk y la tion )
5.5 6e 5326
313298
340
Time0.50 1.00 1.50 2.00 2.50
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18_4_022 2: MRM of 1 Channel ES+ 428.184 > 325.829 (AZ1_Demethylation)
3.58e6
m /z10 0 12 0 140 1 60 1 80 20 0 22 0 24 0 260 2 80 3 00 3 20 34 0 36 0 380 4 00 4 20
%
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18_ 4_ 023 3 (1 .87 1) 2 : Pro du ct I ons of 428 ES+42 8.1 84 > 3 25 .82 9 ( AZ1 _De me thy la tio n)
3. 92 e4326
147235
176
340
Dealkylation Demethylation
Dosed Compound
MRM 442.2>326
HydroxylatedMetabolite
MRM 458.2>340
PIC Spectrum
PIC Spectrum
PIC Spectrum
PIC Spectrum
©2010 Waters Corporation
ScanWave & StepWave
ScanWave — Enhanced product ion spectra — Confirm target compound quantitative data with PICS — Indicate presence of related metabolites
StepWave — Enhances signals in all ionisation modes — Highest quality of spectra MS/MS data for structural
confirmation
©2010 Waters Corporation
Xevo TQ-S
Versatility
&
Simplicity of operation
©2010 Waters Corporation
IntelliStart™ Automated MS Resolution &
Calibration Checks
IntelliStart™ Automated MRM
Method Developer
IntelliStart™ Automated LC/MS
System Check
IntelliStart™ Automated System
Monitoring.
Automatically ensure the system is ready
to run
Quanpedia Automated MRM Time Window &
Data Rate Scheduling
Simplicity of Operation
©2010 Waters Corporation
Solvent + Analyte and Internal Standard
Extracted Matrix + Analyte and Internal Standard
A= 100
A= 90
A= 95
A= 85
XICAnalyte XICInternal Standard
MF = Amatrix/Asolvent
MF = 90/100 = .90
MF = Amatrix/Asolvent
MF = 85/95 = .89
IS Normalized MF = MFAN/MFIS
IS Normalized MF = .90/.89 = 1.01
Spiked Method Calculating a Simple Matrix Factor
©2010 Waters Corporation
Alternate Approach for Calculating Matrix Factors
AN and ISinjected through the column
2) Inject Solvent Blank
3) Inject Extracted Matrix Blank
LC Column
1) Determine Analyte Profile
Solvent Blank injected with post column infusion of AN and IS
Extracted Blank injected with post column infusion of AN and IS
MRM Signal
AN and IS infused post-column
Analyte = AN Internal Standard = IS
©2010 Waters Corporation
Automating Matrix Factor Calculations: Integrated Intellistart Fluidics
©2010 Waters Corporation
Time0.40 0.60 0.80 1.00 1.20 1.40
%
1 Time1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60
%
1
Matrix Factor Calculation Results (MassLynx 4.1 SCN 737
©2010 Waters Corporation
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Source versatility
©2010 Waters Corporation
A common ion source platform
Designed to be compatible with HPLC, UPLC and GC applications
Designed for performance, usability & serviceability.
XEVO Family Ion Sources
©2010 Waters Corporation
Source versatility
DART DESI
ASAP
APGC TRIZAIC™ Source with nanoTile
©2010 Waters Corporation
Simple and tool-free Allowing you to change between ion source options in minutes.
Universal source architecture Enables the widest range of ionization techniques to be utilized
ESI, APCI, ESCi
Nano-flow, TRIZAIC
APPI, ASAP, APGC
Compatible with third party sources (DESI, DART, LDTD, etc)
Simplicity of Operation & Versatility
©2010 Waters Corporation
Xevo TQ-S
Summary
©2010 Waters Corporation
Xevo TQ-S Key MS Technologies
StepWave — Large increase in sensitivity (all acquisition modes)
— Designed to be robust to sample matrix contamination
RADAR — Targeted MRM at the same time as full scan MS
ScanWave — Enhanced product ion spectra
©2010 Waters Corporation
Xevo TQ-S Designed to Meet Customer Needs
Ultra high sensitivity to make:
— Detection of banned substances easier
— Monitoring of low exposure drugs easier
— Blood spot analysis easier
— It possible to reduce matrix effects by simple sample dilution
— It possible to work with small sample volumes
Information rich supporting data
©2010 Waters Corporation
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Thank You
Xevo TQ-S
Consider Quantitative Assays Previously Beyond Your Reach