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The Q Exactive – A Benchtop Orbitrap Mass Spectrometer for DMPK
Presented to:5th EBF Open Meeting, Barcelona.
Simon Szwandt Ph.D.Market Development Manager– Pharma & CRO6th November, 2012.
2
Agenda
• Triple vs HR/AM• Instrumentation and HR/AM• Q Exactive Bioanalytical Data – Small Molecule and Biologics• Hepcidin Quantitation
3
The Exactive Family
• The Exactive Plus• HCD cell optional• Upgradeable to Q Exactive• Mass range to m/z 6000• Faster polarity switching• Rotated flatapole• New scan modes
• The Q Exactive• Resolving power >140,000• Parent ion selection capability• Spectral multiplexing• UHPLC and nanoflow capable
Superior selectivity & specificity due to HR/AM-MS and MS/MS
Why HR/AM in Bioanalysis?
5
Full Scan – The Most InformationC:\Xcalibur\...\Paroxetine-FS-002 8/3/2012 1:09:58 PMStd 1ng/mL - FS
RT: 0.00 - 3.00
0.0 0.5 1.0 1.5 2.0 2.5Time (min)
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2.14 2.170.25
2.45
2.40
2.60 2.62
1.97
0.34
1.60
1.401.32
1.150.48 1.040.18 0.62
NL:3.37E6TIC F: + c ESI Q1MS [150.000-400.000] MS Paroxetine-FS-002
C:\Xcalibur\...\Paroxetine-FS-002 8/3/2012 1:09:58 PMStd 1ng/mL - FS
RT: 0.00 - 3.00
0.0 0.5 1.0 1.5 2.0 2.5Time (min)
0
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2.15
2.602.552.400.25
2.352.102.69
1.96
2.751.891.85 2.79
1.791.73 2.830.30 1.49
1.390.33 1.290.18 0.49 1.161.05
NL: 2.08E4m/z= 329.58-330.58 F: + c ESI Q1MS [150.000-400.000] MS Paroxetine-FS-002
Triple Quadrupole Q ExactiveC:\Xcalibur\...\Paroxetine-QE-FS-03 8/13/2012 2:33:00 PM1
RT: 0.00 - 3.01
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0Time (min)
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0.24
2.632.622.50
2.642.49
2.652.342.33
2.30
2.162.15
0.340.34 2.061.981.87
1.841.792.751.74 2.811.701.56 2.871.491.351.271.140.850.37 0.51 0.59 0.730.11
NL:8.46E8TIC F: FTMS + p ESI Full ms [150.00-400.00] MS Paroxetine-QE-FS-03
C:\Xcalibur\...\Paroxetine-QE-FS-03 8/13/2012 2:33:00 PM1
RT: 0.00 - 3.01
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0Time (min)
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1.96
2.02 2.311.93
NL: 3.05E6m/z= 330.14835-330.15165 F: FTMS + p ESI Full ms [150.00-400.00] MS Paroxetine-QE-FS-03
6
SIM – The Highest Possible Signal
C:\Xcalibur\...\Paroxetine-SIM-002 8/3/2012 1:04:52 PMStd 1ng/mL - SIM
RT: 0.00 - 3.00
0.0 0.5 1.0 1.5 2.0 2.5Time (min)
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RT: 2.15AA: 36614 2.58
0.25
2.251.991.98
1.82
0.30 1.631.491.351.191.000.43
NL: 2.08E4m/z= 329.58-330.58 F: + c ESI Q1MS [330.079-330.081] MS ICIS Paroxetine-SIM-002
Paroxetine-QE-SIM-05_120813142428 8/13/2012 2:24:28 PM1
RT: 0.00 - 3.00
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0Time (min)
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RT: 1.96AA: 846873
NL: 5.56E5m/z= 330.14835-330.15165 F: FTMS + p ESI SIM ms [328.65-331.65] MS ICIS Paroxetine-QE-SIM-05_120813142428
Triple Quadrupole Q Exactive
7
Product Ion Scan
C:\Xcalibur\...\Paroxetine-PI-005 8/3/2012 1:35:31 PMStd 1ng/mL - PI
RT: 0.00 - 3.00
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8Time (min)
0
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60
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Rel
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unda
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2.15
0.25
2.582.56
2.392.23 2.612.04
2.68 2.731.94 2.77
1.800.31 1.751.57 2.901.541.14 1.320.990.07 0.46 0.72 0.950.61
NL:4.97E4TIC F: + c ESI Full ms2 330.081 [50.000-400.000] MS Paroxetine-PI-005
Paroxetine-PI-005 #270-281 RT: 2.11-2.19 AV: 12 NL: 3.91E3F: + c ESI Full ms2 330.081 [50.000-400.000]
50 100 150 200 250 300 350 400m/z
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Rel
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192.05
70.35 330.17
134.81123.07 151.17212.59108.98 178.02
311.73223.1097.15 163.15 237.33 365.9884.38 279.58 301.86 344.7657.12 383.96
Paroxetine-QE-MSMS-NoIS-001 8/13/2012 2:37:20 PM1
RT: 0.00 - 3.00
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0Time (min)
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2.51
2.27 2.292.262.24 2.300.24
0.23 2.572.322.232.63
2.21 2.342.642.342.20
2.652.18
1.88 1.961.88 2.16
1.86 2.151.84 2.121.82 2.11
1.801.77
2.701.731.681.550.28 2.761.381.03 1.280.940.340.06 0.69 0.850.60
NL: 3.68E6TIC F: FTMS + p ESI Full ms2 [email protected] [50.00-355.00] MS Paroxetine-QE-MSMS-NoIS-001
Paroxetine-QE-MSMS-NoIS-001 8/13/2012 2:37:20 PM1
Paroxetine-QE-MSMS-NoIS-001 #1317-1332 RT: 1.94-1.97 AV: 16 NL: 1.29E5F: FTMS + p ESI Full ms2 [email protected] [50.00-355.00]
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340m/z
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330.15046
192.11848
70.06580
57.07061 84.96029151.03907 297.0653098.98461
117.07027 279.05448178.10293163.09186
247.12213139.11194
222.99176
335.11606
271.08592 353.12676303.18369
208.11350 320.20065
Triple Quadrupole Q Exactive
8
MSMS (SRM Vs. MSMS)
C:\Xcalibur\...\Paroxetine-SRM-001 8/3/2012 12:09:43 PMStd 1ng/mL - SRM
RT: 0.00 - 3.00
0.0 0.5 1.0 1.5 2.0 2.5Time (min)
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RT: 2.18AA: 19656AH: 8804
NL: 8.81E3TIC F: + c ESI SRM ms2 330.081 [192.110-192.112] MS Genesis Paroxetine-SRM-001
Triple Quadrupole Q ExactiveParoxetine-QE-MSMS-NoIS-001 8/13/2012 2:37:20 PM1
RT: 0.00 - 3.00
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0Time (min)
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RT: 1.96AA: 237725
NL: 1.58E5m/z= 192.11752-192.11944 F: FTMS + p ESI Full ms2 [email protected] [50.00-355.00] MS ICIS Paroxetine-QE-MSMS-NoIS-001
9
Specificity = Resolution + Mass Accuracy
Resolution: 10k, 30k, 50k, 100k
279.12 279.14 279.16 279.18 279.20m/z
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Ethinyl-Estradiol, 279.17434
Butyl-Phthalate, 279.15909 (ubiquitous background ion)
54 ppmapart
10
HRAM or MS/MS
This permitted a direct comparison between the LC-HRMS and LC-MS/MS data. The data indicated that the selectivity of LC-HRMS exceeds LC-MS/MS, if high resolution mass spectrometry (HRMS) data is recorded with a resolution of 50,000 full width at half maximum (FWHM) and a corresponding mass window. This conclusion was further supported by experimental data (MS/MS based trace analysis).
This is to demonstrate that Exactive and HRAM with 50K resolution is equivalent or
better than QQQ.
11
Why HR/AM Q Exactive Large Molecule
12
Small vs Large Chemical Characteristics
Characteristic Small LargeEndogenous No Often YesSolubility Hydrophobic HydrophilicPurity Homogeneous HeterogeneousStability Chemical/enzymatic Immunologic/enzymaticStability Drug Drug + ReagentsBasis of Measurement Analyte (Direct) Antigen-Ab reaction (Indirect)Reagents Common and
availableUnique, not commercial or available
Calibration Curves Linear Non-LinearExpected/Common interferences
Yes/knowledge Unknown
Technology LC/MS LBA: ELISA, RIA, ECL, Multiplexing
Development Time Weeks Months
B. De Silva, Bristol-Myers Squibb
13
Real High Resolution to Increased Selectivity
Dilution curves
Multiplex SIM Mode:
> Separation of interfering signals: improvement of LOQ (200 x)> Increase in cycle time (~ 2 x)
Resolution : 70 000
Resolution : 140 000
18 amol
4.4 fmol
SDLAVPSELALLK Res : 70 000
0
20
40
60
80
100
682 682.5 683 683.5
682.371
682.397
682.705
682.899
683.039
0
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682 682.5 683 683.5
682.232
682.369
682.395
682.703
682.898
683.035
683.370
Zoom in
682.397 682.395682.39 682.39682.397 682.395
Res : 140 000
S. Gallien and B. Domon – Luxembourg Clinical Research, Strassen, Luxembourg
14
XIC for DIKCSNILLNNSGQIK at m/z 1007.0435(2+)
Not detected using Full MS
Detected and quantified using SIM MS
Sensitivity Gain by Selected Ion Monitoring (SIM)
• Spectrum noise is dependent on the ratio of compound within a certain ion population
• Spectrum noise is much less in SIM mode
• Sensitivity gain 5 – 10 x with SIM mode
• The gain will be higher in more complex matrices
15
Alprazolam, Full Scan ExperimentAlprazolam
Y = 6366.31+514.015*X R^2 = 0.9967 W: 1/X
0 2000 4000 6000 8000 10000fg/uL
0
500000
10000001500000
2000000
25000003000000
3500000
40000004500000
5000000
5500000
Are
a
0 50 100 150 200 250 300fg/uL
020000400006000080000
100000120000140000160000180000200000
Are
a
Zoom in 50 ppt- 100ppt
50 ppt – 10 ppb250 fg oc - 50 pg oc
16
Alprazolam SIM ExperimentAlprazolam
Y = -3135.8+552.216*X R^2 = 0.9982 W: 1/X
10 ppt – 10 ppb50 fg oc - 50 pg oc
0 2000 4000 6000 8000 10000fg/uL
0
1000000
2000000
3000000
4000000
5000000
6000000
Are
a
Zoom 10 ppt- 100ppt
0 20 40 60 80 100 120fg/uL
0100002000030000400005000060000700008000090000
100000110000120000
Are
a
50 ppt – 10 ppb250 fg oc - 50 pg oc
17
Q Exactive
18
High Throughput HR/AM Quantitation
QQ-Orbi
19
The Q Exactive: Hardware Innovations
HCD Cell
OrbitrapMassAnalyzer
S-lensIon Source
Enhanced FT
QuadrupoleMass Filter
C-Trap
20
Benchtop Orbitraps vs QToFs
• All high-resolution, accurate mass instruments trade in three “currencies”
• Scan speed (Hz) – also called “dwell time” which is familiar to triple quad users
• Resolution• Sensitivity
Speed
SensitivityResolutio
n
21
Benchtop Orbitraps vs QToFs
• ToF based instruments trade speed for sensitivity• Resolution is based on flight path so it is “fixed” – with a
mass dependence• You can scan at 100 Hz… but data compromised• Typical recommended full scan speed – 150-250 ms• This means ToF scan speeds are 4-10 Hz.
Speed
SensitivityResolutio
n
22
• Orbitrap based instruments trade speed and resolution• Resolution comes from more time spent measuring the
transient• Slow down to get more resolution• Sensitivity is “fixed” since we pre-fill the C-trap for up to
the Orbitrap scan time (it doesn’t “miss” any ions)
Speed
SensitivityResolutio
n
Benchtop Orbitraps vs QToFs
Speed
SensitivityResolutio
n
23
Plecanatide Peptide Analysis – Pyxant Laboratories
24
Plecanatide at 35K Resolution
25
Plecanatide at 70K Resolution
26
Plecanatide at 35K Resolution Chromatograms
Standard-1-01 - m/z= 841.32 + m/z= 841.82 RT: 3.04 - 3.79 NL: 7.97E4F: FTMS + p ESI SIM ms [840.50-842.50]
3.1 3.2 3.3 3.4 3.5 3.6 3.7Time (min)
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RT: 3.42AA: 131404
3.12
3.21
3.453.32 3.69 3.743.27 3.60 3.653.50
Standard-8-01 - m/z= 841.32 + m/z= 841.82 RT: 3.04 - 3.79 NL: 1.89E7F: FTMS + p ESI SIM ms [840.50-842.50]
3.1 3.2 3.3 3.4 3.5 3.6 3.7Time (min)
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RT: 3.41AA: 35229922
3.50 3.543.13 3.58 3.763.653.20 3.713.343.253.06 3.29
QC-0-01 - m/z= 841.32 + m/z= 841.82 RT: 2.76 - 3.51 NL: 7.33E4F: FTMS + p ESI SIM ms [840.50-842.50]
2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5Time (min)
0
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Rel
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RT: 3.13AA: 91151
3.43
3.453.16
3.33 3.393.183.09 3.283.48
2.99 3.363.313.212.822.78
QC-Low-01 - m/z= 841.32 + m/z= 841.82 RT: 3.05 - 3.80 NL: 1.36E5F: FTMS + p ESI SIM ms [840.50-842.50]
3.1 3.2 3.3 3.4 3.5 3.6 3.7Time (min)
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Rel
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RT: 3.42AA: 250387
3.113.14
3.17 3.23 3.303.20 3.633.523.273.08 3.69
Blank-02 - m/z= 841.32 + m/z= 841.82 RT: 3.10 - 3.85 NL: 1.33E4F: FTMS + p ESI SIM ms [840.50-842.50]
3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8Time (min)
0
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Rel
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RT: 3.47AA: 8199
3.74
3.57 3.77
3.513.32
3.423.35 3.63
3.213.403.12
3.703.17 3.26 3.30
3.60
QC-High-01 - m/z= 841.32 + m/z= 841.82 RT: 3.07 - 3.82 NL: 2.00E7F: FTMS + p ESI SIM ms [840.50-842.50]
3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8Time (min)
0
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Rel
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RT: 3.45AA: 33238161
3.533.15 3.57 3.62 3.65 3.743.19 3.703.27 3.31 3.803.373.233.12
QC 0 QC Low QC High
Blank 1 ng/mL 250 ng/mL
27
Plecanatide at 70K Resolution Chromatograms
Standard-1-01 - m/z= 841.32 SM: 3 RT: 2.95 - 3.70 NL: 8.10E4F: FTMS + p ESI SIM ms [840.50-842.50]
3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7Time (min)
0
5
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Rel
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RT: 3.33AA: 113704
3.142.99 3.203.02 3.463.08 3.43
3.383.18 3.583.04 3.633.533.483.11 3.26
Standard-8-01 - m/z= 841.32 SM: 3 RT: 2.99 - 3.74 NL: 1.05E7F: FTMS + p ESI SIM ms [840.50-842.50]
3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7Time (min)
0
5
10
15
20
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Rel
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ity
RT: 3.36AA: 19937890
3.50 3.56 3.60 3.713.64 3.683.15 3.303.05 3.193.02 3.09 3.26
Blank-02 - m/z= 841.32 SM: 3 RT: 2.94 - 3.67 NL: 3.29E4F: FTMS + p ESI SIM ms [840.50-842.50]
3.0 3.1 3.2 3.3 3.4 3.5 3.6Time (min)
0
5
10
15
20
25
30
35
40
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55
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Rel
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ity
3.33
3.113.193.143.08 3.603.03 3.553.22 3.412.97 3.37 3.613.01 3.29 3.50
3.513.473.26
QC-0-01 - m/z= 841.32 SM: 3 RT: 2.96 - 3.63 NL: 3.77E4F: FTMS + p ESI SIM ms [840.50-842.50]
3.0 3.1 3.2 3.3 3.4 3.5 3.6Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
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Rel
ativ
e In
tens
ity
3.37
3.413.17
3.273.553.23 3.33 3.60
3.46
3.10 3.193.15 3.583.43
3.02
QC-Low-01 - m/z= 841.32 SM: 3 RT: 3.00 - 3.75 NL: 1.82E5F: FTMS + p ESI SIM ms [840.50-842.50]
3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
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Rel
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ity
RT: 3.37AA: 294038
3.423.233.11 3.293.03 3.733.44 3.603.20 3.683.253.163.07 3.51 3.63
QC-High-01 - m/z= 841.32 SM: 3 RT: 3.02 - 3.77 NL: 9.93E6F: FTMS + p ESI SIM ms [840.50-842.50]
3.1 3.2 3.3 3.4 3.5 3.6 3.7Time (min)
0
5
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15
20
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Rel
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RT: 3.39AA: 19027842
3.49 3.54 3.693.62 3.753.203.10 3.313.04 3.243.15
QC High
Blank 1 ng/mL 250 ng/mL
QC 0 QC Low
28
841.0 841.5 842.0 842.5m/z
0
20
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60
80
1000
20
40
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Rel
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841.31789
841.81989
842.31868
841.68281842.47785
842.02495
841.12388
841.31988
841.82125
842.32102
High Resolution Ensures Accurate Quantification Using SIM
Theoretical Mass [M+2H]+= 841.31987
841.5 842.0 842.5m/z
0
20
40
60
80
1000
20
40
60
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100
Rel
ativ
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841.36617
841.81920
842.31919
841.44544
841.94947
841.82125
842.32102
ΔPPM = 1.2
Resolution = 35 K Resolution = 70 K
ΔPPM = 2.35
Actual
Theoretical
29
• Validated Quantitation of Hepcidin
A Validated Method for the Definitive Quantitation of Hepcidin-25 in Human
Serum by LC/MS using high resolution, accurate mass MS
• John E. Buckholz, Gary A. Schultz, Barry R. Jones, Kristen M. Bearup, Kathlyn M. Porter, Danielle J. Strong, Johnson Zhang
• Advion BioServices, Inc., Ithaca, NY
Hepcidin
AJP - GI February 2006, vol. 290 no. 2 G199-G203
•Hepcidin is a 25-amino acid peptide hormone and is the central regulator of iron metabolism making it an interesting biomarker for many applications.
•25 amino acids, folded over in a hairpin shape with 4 disulfide bridges at Cys7-Cys23, Cys10-Cys22, Cys11-Cys19, Cys13-Cys14
Internal Standard
•Mouse hepcidin molecular weight = 2752.02 g/mol
•Mouse Hepcidin Sequence = DTNFPICIFCCKCCNNSQCGICCKT
human hepcidin
MethodsPreparation and Extraction Procedure
• Protein precipitation extraction• Ostro block for phospholipid removal• 96 well format• Reversed phase chromatography• Q-Exactive • Human Hepcidin Sequence =
DTHFPICIFCCGCCHRSKCGMCCKT
Hepcidin Product Ion Mass Spectrum
33
200 400 600 800 1000 1200 1400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
698.2
644.0
1040.1
763.7 981.1501.2109.8 858.9 1145.0353.8
696 697 698 699 700 701m/z
Q1 Scan[M+4H]4+
Isotope pattern confirmationhuman hepcidin
EIIHEP-BM_PSAE_067 10/13/2010 10:57:26 PM 20 67 EIIHEP PSAE STD8 2 1
559 560m/z
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
558.8130z=?
559.0131z=?
559.2131z=?
559.4131z=?
559.6130z=?
560.0130z=?
558.8123z=5
559.0122z=5
559.2120z=5
559.4119z=5
559.6117z=5
560.0107z=5
NL:2.62E2
C113 H170 N34 O31S 9 +H: C113 H173 N34 O31S 9
p (gss, s /p:40) Chrg4,1R: 40000 Res.Pwr . @FWHM
NL:2.68E5EIIHEP-BM_PSAE_067#240-249 RT: 3.32-3.41 AV: 10 T: FTMS {1,1} + p ESI Full lock ms [400.00-1800.00]
698 700m/z
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
698.2644z=?
698.5145z=?
698.7645z=?
699.0145z=?
699.2645z=?
699.7644z=?
698.2641z=4
698.5137z=4
698.7634z=4
699.0133z=4
699.2635z=4
699.7630z=4
NL:5.23E3
C113 H170 N34 O31S 9 +H: C113 H173 N34 O31S 9
p (gss, s /p:40) Chrg4,1R: 40000 Res.Pwr . @FWHM
NL:4.77E5EIIHEP-BM_PSAE_067#240-249 RT: 3.32-3.41 AV: 10 T: FTMS {1,1} + p ESI Full lock ms [400.00-1800.00]
930 931 932 933m/z
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
930.6834z=?
931.0169z=?
931.3502z=?
931.6836z=?
932.0169z=?
930.6846z=3
931.0180z=3
931.3512z=3
931.6843z=3
932.0175z=3
NL:2.62E2
C113 H170 N34 O 31S 9 +H: C113 H173 N34 O 31S 9
p (gss, s /p:40) Chrg4,1R: 40000 Res.Pwr . @FWHM
NL:1.07E5EIIHEP-BM_PSAE_067#240-249 RT: 3.32-3.41 AV: 10 T: FTMS {1,1} + p ESI Full lock ms [400.00-1800.00]
Theoretical mass spectrum @ 40,000 FWHM resolution parameter
Experimental mass spectrum at 256 ng/mL
[M+5H]5+
[M+5H]5+
[M+4H]4+
[M+4H]4+
[M+3H]3+
[M+3H]3+
34
HRAM hepcidin @ 30,000 FWHMTop 6 isotopes summed using +/- 5 ppm mass tolerance
35
0.0 1.0 2.0 3.0 4.0 5.0
Time (min)
4.09E5
8.05E5
1.67E5
[M+5H]5+
[M+4H]4+
[M+3H]3+
Human Hepcidin extracts from serum
QQQ m/z 698.2 > m/z 1040.0
Q-E SIM – [M+4H]4+ Top 6 Sum
3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0Time (min)
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
7.28E1
4.21E2
1.20E3
3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0Time (min)
0
20
40
60
80
100R
elat
ive
Abu
ndan
ce0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
1.89E5
1.80E5
1.77E5
Blank
Std 12ng/mL
Std 24ng/mL
Blank
Std 12ng/mL
Std 24ng/mL
0123456789
1011
0 200 400 600 800 1000 1200 1400 1600 1800 2000
Inst
rum
ent R
espo
nse
Nominal Conc.
Linear Dynamic Range HRAM-MS
human hepcidin using 140,000 FWHM resolution
0.000.020.040.060.080.100.120.140.160.180.20
0 5 10 15 20 25 30 35
Inst
rum
ent R
espo
nse
Nominal Conc.
Concentration, ng/mL
Concentration, ng/mL
LINEAR - Weighting Factor = 1/X**2 Slope = 0.005439Intercept = -0.003694r-Squared = 0.9927 LLOQ = 2.0 ULOQ = 2048.0 Mean bias = 5.3Standard deviation = 4.9
37
Accuracy and Precision HRAM-MS human hepcidin using 140,000 FWHM acquisition parameter
LLOQQC LQC MQC HQC ULOQQC 50xDilQCTheor. Conc., ng/mL 2 6 1024 1564 2048 50000
Mean 2.2 6.7 1067.5 1589.3 2160.2 52576.7
S.D. 0.2 0.4 38.7 32.9 55.8 1828.9
%CV 9.1 6 3.6 2.1 2.6 3.5
%RE 110 111.7 104.2 101.6 105.5 105.2
Replicates, n 6 6 6 6 6 6
38
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fragmentation.• Increased mass range good for large molecule quantitation. • Q Exactive can be used for quantitative bioanalysis• Sensitivity (S/N) can be improved• Easy to operate and proven to be potentially beneficial for assays
including: – Difficult to fragment– High background– Multiple charges– High mass
40
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High Throughput HR/AM Quantitation
Exactive Plus Orbitrap Q Exactive Orbitrap
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Thermo Posters at ASMS 2012
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ASMS – Q Exactive Pharma/Biopharma
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using High Resolution Accurate Mass Spectrometry, Kevin Cook1; Rose Herbold1; Keeley Murphy 1; Panos Hatsis 2, 1Thermo Fisher Scientific, San Jose, CA; 2Novartis, Cambridge, MA
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• Oral: MOC pm - Integrated Qualitative and Quantitative LC-MS for Small Molecule Analysis• Development of a Higher Throughput Metabolite Screening Assay in Early ADME Profiling
Using Generic HRMS Acquisition and Automated Data Processing, Anthony Paiva1; Cheryl Klakouski1; Tatyana Zvyaga1; Dieter Drexler1; Jonathan Josephs 1; Harold Weller1; Wilson Shou 1; Ismael Zamora2, 1Bristol-Myers Squibb Company, Wallingford, CT; 2Lead Molecular Design, San Cugat del Valles, Spain
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• Utility of High-Resolution-Accurate-Mass to Minimize Interference in Simultaneous Determination of Ribavirin and its Phosphate Metabolites in an In Vitro Assay, Cong Wei; James E. Grace Jr.; Tatyana Zvyaga; Dieter M. Drexler, Bristol-Myers Squibb Company, Research&Development, Wallingford, CT
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ASMS – Q Exactive Pharma/Biopharma
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• Quantitation of Oligonucleotides In Human Plasma Using Q-Exactive Orbitrap High Resolution MS, Weiwei Yuan1; Laixin Wang1; Min Meng1; Jessica Wang2; Kevin Cook 2; Patrick Bennett2, 1Tandem Labs, Salt Lake City, UT; 2Thermo Fisher Scientific, San Jose, CA
• Robust Ultra High Sensitivity Quantitative Analysis using Low Flow LC/MS/MS and Reduced Phospholipid Matrix Effects. Myth or Reality?, David Humphries1; Roger N. Hayes1; Kevin Cook2; Mark Dreyer2; Xiang He2; Subodh Nimkar2; Patrick Bennett2, 1MPI Research, Mattawan, MI; 2Thermo Fisher Scientific, San Jose, CA
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ASMS – Q Exactive Pharma/Biopharma• Evaluation of Resolution/Scan Speed Limitations for Quantitation of Small
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ASMS – Q Exactive Pharma/Biopharma
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• LC/SRM Has Come of Age to Quantitate Proteins: Amyloid β isoforms measured in Human Cerebrospinal Fluid and Compared to ELISA, Kwasi Mawuenyega; Tom Kasten; Vitaliy Ovod; Kelly Moor; Ling Munsell; Rose Connors; Wendy Sigurdson; Randall Bateman, Washington University School of Medicine, Saint Louis, MO
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47
Acknowledgements
• Thermo Fisher Scientific• Patrick Bennett• Kevin Cook• Zhiqi Hao• Keeley Murphy• Jessica Wang• TFS Demo Labs
• Advion Bioservices Inc.• John Buckholz• Gary Schultz• Barry Jones• Kristen Bearup• Kathlyn Porter• Danielle Strong• Johnson Zhang
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