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The Velos Pro: A Versatile, High Performance Ion Trap for Life Science Mass Spectrometry Users Meeting Somerset, October 12, 2011 Julie Horner, Ph. D.
2
Thermo Scientific Velos Pro
The LTQ-series linear ion traps have been established as the premier qualitative instruments on the market at their price point. The Velos Pro was custom designed for both Qualitative and Quantitative applications in the laboratory. CID, PQD, ETD, and now Trap-HCD fragmentation increases flexibility for structural elucidation
Fast, sensitive ion trap for the most demanding applications
Generation II ion optics improve robustness and reduce downtime.
Wide dynamic range, single digit RSD’s, great accuracy.
Upgrade to the power of Orbitrap
Thermo Scientific Velos Pro Dual-Pressure Linear Ion Trap
3
Velos Pro Technology Advancements
Feature Customer Benefit
1 UHPLC Cycle Time and High Data Rates
• More points across a chromatographic peak gives better quantitative performance • More MSN during a run • Now scanning at 66kDa/sec
2 Robust and Sensitive Generation II Ion Optics
• Improved robustness, reduce down time, all on the most sensitive ion trap. • High sensitivity S-Lens technology • Novel neutral beam blocking technology
3 Novel Detection System for superior Quantitation
• Single digit RSDs, up to 6 orders of linear dynamic range on the first ion trap designed for quantitation.
• New EM and electrometer detection system matched to handle the ultra-high peak currents from the VELOS trap.
4 Trap-HCD Fragmentation • Increases flexibility for structural elucidation • Trap-HCD fragmentation yields unique product ions for structural determinations.
4
Velos Pro: Rapid Scan Rate
Feature Customer Benefit
1 UHPLC Cycle Time and High Data Rates
• More points across a chromatographic peak gives better quantitative performance • More MSN during a run • Now scanning at 66kDa/sec
2 Robust and Sensitive Generation II Ion Optics
• Improved robustness, reduce down time, all on the most sensitive ion trap. • High sensitivity S-Lens technology • Novel beam blocking technology
3 Novel Detection System for superior Quantitation
• Single digit RSDs, up to 6 orders of linear dynamic range on the first ion trap designed for quantitation.
• New EM and electrometer detection system matched to handle the ultra-high peak currents from the VELOS trap.
4 Trap-HCD Fragmentation • Increases flexibility for structural elucidation • Trap-HCD fragmentation yields unique product ions for structural determinations.
5
Velos Pro: Dual Cell Technology
HPC Optimized for: •High efficiency ion trapping •Fast ion cooling •High efficiency fragmentation
•All ion trap mass spectrometers use helium to cool and fragment trapped ions.
•A high pressure of Helium is required for loading, cooling, and fragmenting ions.
•A low pressure of Helium is required for ejecting ions.
•Historically, all ion trap designs used a single, compromise, pressure for both processes.
•The Velos Pro uses two cells, each with the optimal pressure for the job at hand!
High Pressure Cell
Low Pressure Cell
LPC Optimized for: •High performance ion ejection
6.5 mTorr
0.3 mTorr
6
Velos Pro: Dual Cell Technology
Ion Injection
Mass Analysis
Ion Isolation
Ion Dissociation
n-1 1
0
50
100
150
200
250
300
350
LTQ XL LTQ VELOS LTQ VELOS w P/AGC
LTQ VELOS PRO
INTERSCAN TIME
OTHER OVERHEAD
MASS ANALYSIS (300-2000)
ACTIVATION
ISOLATION
ION INJECTION
PRESCAN
Since all parts of the scan function operate at the optimized pressure the system is:
4X faster for ion isolation 3X faster for ion fragmentation 1.3X greater fragmentation efficiency >90% trapping efficiency Fastest scanning trap in the world.
7
Ultra Zoom 27 Da/sec
Zoom 2.2 KDa/sec
Enhanced 10 KDa/sec
Normal 33 KDa/sec
Rapid 66 KDa/sec
New Scan Speed: Rapid Scan
0
50
100
150
200
250
300
350
LTQ XL LTQ VELOS LTQ VELOS w P/AGC
LTQ VELOS PRO
INTERSCAN TIME
OTHER OVERHEAD
MASS ANALYSIS (300-2000)
ACTIVATION
ISOLATION
ION INJECTION
PRESCAN
Ion Injection
Mass Analysis
Ion Isolation
Ion Dissociation
n-1 1
36,440 M/∆M
10,100 M/∆M
7,600 M/∆M
5,400 M/∆M
3,500 M/∆M
8
Advantage of Resolution: Ubiquitin 9+ Full MS/MS in Zoom Scan Avg 50 scans, 30 s
PeptideMix_003 4/1/2009 4:10:08 PM 0.5 ng/uL ea Ang I, Ubiquitin3 uL/min in 50/50 H20/ACN .1%FA after cal
PeptideMix_003 #990-1041 RT: 6.16-6.68 AV: 50 NL: 3.22E3F: ITMS + p ESI Z ms2 [email protected] [500.00-1500.00]
500 600 700 800 900 1000 1100 1200 1300 1400 1500m/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
1089.56
934.06
913.62
655.60 1142.00851.96
982.50
1039.22726.04 790.08 1184.801307.24639.58 832.92 1217.28
619.42520.34 1334.84 1414.96 1462.00
3+
5+
2+
6+
6+
9
Velos Pro Speed : Full Ion Tree in 1 second!
8 MSn Spectra in 1 second!
RT: 6.19 - 6.82
6.2 6.3 6.4 6.5 6.6 6.7 6.8 Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
6.44
10
Velos Pro: Generation II Optics
Feature Customer Benefit
1 UHPLC Cycle Time and High Data Rates
• More points across a chromatographic peak gives better quantitative performance • More MSN during a run • Now scanning at 66kDa/sec
2 Robust and Sensitive Generation II Ion Optics
• Improved robustness, reduce down time, all on the most sensitive ion trap. • High sensitivity S-Lens technology • Novel beam blocking technology
3 Novel Detection System for superior Quantitation
• Single digit RSDs, up to 6 orders of linear dynamic range on the first ion trap designed for quantitation.
• New EM and electrometer detection system matched to handle the ultra-high peak currents from the VELOS trap.
4 Trap-HCD Fragmentation • Increases flexibility for structural elucidation • Trap-HCD fragmentation yields unique product ions for structural determinations.
11
Velos Pro: High Sensitivity Generation I Ion Optics
S-Lens: Stacked Ring Ion Guide
Dual ID variable spacing Stacked Lenses • larger ID lenses capture the entire expansion
from the ion transfer tube • smaller ID lenses focus the ion beam through
the conductance limiting aperture • increasing spacing = increasing field
penetration to focus ion beam • small number of electrodes (18 total in one
assembly) Relatively Large Exit Lens Aperture versus
Skimmer 5-10x Transmission Improvement
12
Velos Pro: High Sensitivity Generation I Ion Optics
Parent
+O
-CH2
Base Peak Chromatogram
+2O
0 1 2 3 4 5 6 7 8 Time (min)
0
50
100 0
50
100 0
50
100
Rel
ativ
e Ab
unda
nce
0
50
100 0
50
100 469.35
485.32 485.30 312.47 399.23 469.35
485.32 485.30
485.31 485.27
455.31
501.29 501.22
501.19 501.09 501.39 500.21 501.29 500.25
NL: 2.21E7
NL: 2.21E7
NL: 3.52E6
NL: 1.63E6
NL: 2.00E5
Parent
+O
-CH2
Base Peak Chromatogram
+2O
0 1 2 3 4 5 6 7 Time (min)
0
50
100 0
50
100 0
50
100
Rel
ativ
e Ab
unda
nce
0
50
100 0
50
100 469.98
485.69 485.64 469.98
485.69 485.64
485.67 485.69
455.68
501.61
501.69 501.35 501.55 500.58
NL: 2.09E8
NL: 2.09E8
NL: 2.10E7
NL: 1.15E7
NL: 1.57E6
8
• 10 µm Maropitant • 60 min Incubation in Dog Liver Microsomes • 2.1x50mm 1.9 um Hypersil GOLD • 8 minute 10%-90% MeOH at 600 uL/min • Velos S-lens optics vs LTQ Tube Lens optics
10X in signal with
S-lens
LTQ XL with Tube Lens Velos Pro with S-Lens
13
“S-LENS” in Velos increased Sensitivity by 10 Bent Q0 Optics Designed to
Catch Neutrals Can Improve Robustness
Further
LTQ Velos - Generation I Ion Optics
14
To prevent material from building up on the rods, the Q0 quadrupole has been rotated 450. This opens up a new line of sight for the neutral beam.
A beam blocker is now able to capture the neutral beam out side the body of the ion optics. Neutrals can no longer reach the mass analyzer. This allows, for the first time, neutrals and ions to be separated in a linear optics design.
The “Rotated Quadrupole” with Neutral Beam Blocker
Neutral Blocker
15
Velos Pro Generation II Ion Optics
The “Rotated Quadrupole” with neutral beam blocker
The majority of material strikes the beam blocker outside of the ion path.
16
Velos Pro Generation II Optics Robust Signal Intensity
• Using the new rotated Q0, the Velos Pro is twice as robust as the LTQ Velos.
• The Velos Pro has increased robustness in complex matrices; e.g. crashed plasma.
0%
20%
40%
60%
80%
100%
0 10 20 Rela
tive
Inte
nsit
y al
l ion
s av
erag
ed
DHB Infused (mg)
Velos Pro Robustness
VELOS
PRO
2x increase in robustness
0.000
0.500
1.000
1.500
2.000
0 200 400 600 800 1000 1200 1400
Are
a Ra
tio
Crashed Plasma Injection Number
RSD: 2.07% Injections: >1300
17
Velos Pro: Ultra Linear Quantitation
Feature Customer Benefit
1 UHPLC Cycle Time and High Data Rates
• More points across a chromatographic peak gives better quantitative performance • More MSN during a run • Now scanning at 66kDa/sec
2 Robust and Sensitive Generation II Ion Optics
• Improved robustness, reduce down time, all on the most sensitive ion trap. • High sensitivity S-Lens technology • Novel beam blocking technology
3 Novel Detection System for superior Quantitation
• Single digit RSDs, up to 6 orders of linear dynamic range on the first ion trap designed for quantitation.
• New EM and electrometer detection system matched to handle the ultra-high peak currents from the VELOS trap.
4 Trap-HCD Fragmentation • Increases flexibility for structural elucidation • Trap-HCD fragmentation yields unique product ions for structural determinations.
18
Triple Quadrupole vs Velos Pro
Transmission Quadrupole Velos Pro
1. In the Velos Pro, the first trap acts like a filter to isolate the precursor ion while loading into the trap.
2. The precursor ions are then fragmented to produce product ions.
3. The product ions are then scanned in the second trap and detected.
1. In a triple quadrupole, the Q1 rod set acts like a filter to isolate the precursor ion.
2. The precursor ions are then fragmented to produce product ions.
3. The product ions are then filtered in Q3 and detected
19
Injection Waveforms
Velos symmetric rod stretch rf field: E=0 on axis
With new injection=isolation waveforms, the HPC acts just like a good single quadrupole!
Isolation profiles for Velos instruments during loading.
Even better isolation after loading. Is
olat
ion
Effic
ienc
y
0
20
40
60
80
100 m/z 524
-8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 m/z at Isolation q
20
Isolation before Analysis is Key
Transmission Quadrupole Velos Pro
21
Isolation while loading is key
200 400 600 800 1000 0.0
5.0x10 5
1.0x10 6
1.5x10 6
2.0x10 6
2.5x10 6
3.0x10 6
Inte
nsity
Mass
715 725 735 745 0.0
5.0x10 4
1.0x10 5
1.5x10 5 Select only
this ion while loading into the trap
22
Isolation while loading is key
720 725 730 735 740 745 750 0.0
5.0x10 3
1.0x10 4
1.5x10 4
2.0x10 4
2.5x10 4
3.0x10 4
3.5x10 4
Inte
nsity
Mass
Injection waveforms Off
Injection waveforms On
0 50 100 150 200 10 3
10 4
10 5
Effect of Injection Waveforms: MS/MS of m/z 735 Lactoperoxidase Digest
Inte
nsity
Scan
Off On
•Injection waveforms allow selective ion accumulation
•Injection waveforms prevent signal variation due to matrix based space charge
23
Isolation while loading is key
0 50 100 150 200 10 3
10 4
10 5
Effect of Injection Waveforms: MS/MS of m/z 735 Lactoperoxidase Digest
Inte
nsity
Scan
Off On
•Injection waveforms allow selective ion accumulation
•Injection waveforms prevent signal variation due to matrix based space charge
200 400 600 800 1000 1200 1400
Mass
Effect of Injection Waveforms: MS/MS of m/z 735 Lactoperoxidase Digest
Waveform OFF Low precursor intensity produces poor quality MS/MS
Waveform ON High precursor intensity produces high quality MS/MS
24
Faster Scan Rates – Higher Detected Ion Currents
Ultra Zoom 3700 usec/amu
Zoom 455 usec/amu
Enhanced 100 usec/amu
Normal 30 usec/amu
Rapid 15 usec/amu
36,440 M/∆M FWHM=.05
10,100 M/∆M FWHM=.18
7,000 M/∆M FWHM=.26
5,400 M/∆M FWHM=.34
3,500 M/∆M FWHM=.52
25
Continuous Dynode Electron Multiplier (CDEM) versus Discrete Dynode Electron Multiplier (DDEM)
Advantages: Larger Surface Area Larger linear Dynamic Range Longer life times
Advantages: Smaller Cost Effective
ions electrons Glass channel plate
Output electrons
26
Velos Pro – Novel Detection System
• Custom designed discrete dynode electron multipliers have larger surface area to produce an extended dynamic range with great RSDs
• New multiplier from ETP has slower aging and longer lifetimes than previous design
10 1004
6
8
10
12
Old Detection System New Detection System
Peak
Rati
oCurrent (µA)
Comparison of High End of Linear Range
• Novel 160 uA electrometer board designed to handle high peak currents found in fast scanning micropackets. (now 24bit)
~20 uA ~160 uA
27
Alprazolam
Y = 6852.68*X R^2 = 0.9918 W: 1/X
0 2000 4000 6000 8000 10000
pg on Column
0
10000000
20000000
30000000
40000000
50000000
60000000
70000000
Are
a
Velos Pro: Alprazolam 6 Order Quantitation
Pg On Column
RSD%
0.01 14.58
0.02 10.76
0.04 6.63
0.1 4.25
0.2 3.21 0.4 3.70
1 3.59
2 3.57
4 3.47
10 2.48
20 1.98
40 4.03
100 4.33
200 2.03
400 3.40
1000 3.85
2000 1.66
4000 1.27
10000 2.16
Y = 6852.68*X R^2 = 0.9918 W: 1/X
0 1 2 3 4 5 6 7 pg on Column
0
10000
20000
30000
40000
50000
Area
RSD 10 fg on Column: 14.58%
C:\j.horner\...\AlpFull-VelosPro194 4/1/2011 3:55:51 AM10000 pg on Column Velos Pro
RT: 0.00 - 0.80 SM: 7G
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
28
12 Compounds + 1 IS in plasma (5 min run)
Norcodeine 286.30 CID 4
Codeine-d3 303.37 CID 5
Hydrocodone 300.37 CID >5
Methampmetamine 150.24 CID 5
Methoxymethamphetamine 180.26 CID >5
Lidocaine 235.34 CID >5
Sulfamethazine 279.09 CID 4
Methylthioamphetamine IS 165.22 CID 4.2%
Bupropion 240.11 CID >5
Testosterone 289.43 CID 5
Terfenadine 472.32 CID 6
Propranolol 260.16 CID >5
Alprazolam 309.09 PQD >5
C:\j.horner\...\ASMS_VelosPro_Run2_088 4/9/2011 12:42:13 PM 200 pg/uL
RT: 0.00 - 4.40
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time (min)
0
10
20
30
40
50
60
70
80
90
100
3 Scan Events
4 Scan Events
1 Scan Event
1 Scan Event
2 Scan Events
2 Scan Events
IS
29
Codeine 20 fg – 2 ng 5 orders
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0 500 1000 1500 2000 pg on column
0
5
10
15
Area
Rat
io
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 pg on column
0.000
0.002
0.004
0.006
0.008
0.010
0.012
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 136 RT: 0.47 AV: 1 NL: 2.33E5 T: ITMS + c ESI Full ms2 [email protected] [80.00-305.00]
80 100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
214.98193
243.03616
285.11362
224.99274
182.98476
160.98511 254.06039 137.02051 111.12070
pg on Column %RSD
0.020 9.22
0.040 6.50
0.100 5.84
0.200 4.77
0.400 5.17
1.00 3.79
2.00 3.61
4.00 1.96
10.00 1.48
20.00 1.86
40.00 1.14
100.0 1.88
200.0 1.73
400.0 1.69
1000.0 2.65
2000.0 1.09
4000.0 2.27
10000.0 3.13
30
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0 500 1000 1500 2000 pg on column
0
5
10
15
Area
Rat
io
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 pg on column
0.000
0.002
0.004
0.006
0.008
0.010
0.012
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 136 RT: 0.47 AV: 1 NL: 2.33E5 T: ITMS + c ESI Full ms2 [email protected] [80.00-305.00]
80 100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
214.98193
243.03616
285.11362
224.99274
182.98476
160.98511 254.06039 137.02051 111.12070
pg on Column %RSD
0.020 9.22
0.040 6.50
0.100 5.84
0.200 4.77
0.400 5.17
1.00 3.79
2.00 3.61
4.00 1.96
10.00 1.48
20.00 1.86
40.00 1.14
100.0 1.88
200.0 1.73
400.0 1.69
1000.0 2.65
2000.0 1.09
4000.0 2.27
10000.0 3.13
Testosterone 100 fg – 10 ng 5 orders
pg on Column %RSD
0.100 36.18
0.200 12.50
0.400 5.28
1.00 1.41
2.00 1.16
4.00 2.40
10.00 2.85
20.00 1.99
40.00 2.02
100.0 3.16
200.0 1.96
400.0 2.80
1000.0 2.60
2000.0 2.63
4000.0 2.43
10000.0 1.03
Testosterone Y = 0.00122857*X R^2 = 0.9960 W: 1/X
0 5000 10000 0
2
4
6
8
10
12
Area
Rat
io
Testosterone Y = 0.00037205+0.00122824*X R^2 = 0.9960 W: 1/X
0.0 0.5 1.0 0.0000
0.0005
0.0010
0.0015
0.0020
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1248 RT: 4.00 AV: 1 NL: 5.26E4 F: ITMS + c ESI Full ms2 [email protected] [75.00-285.00]
80 100 120 140 160 180 200 220 240 260 280 m/z
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Inte
nsity
253.12668
271.13193
97.01697
109.03072 213.07745 189.07771
243.12128 157.01028 123.07123
31
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0 500 1000 1500 2000 pg on column
0
5
10
15
Area
Rat
io
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 pg on column
0.000
0.002
0.004
0.006
0.008
0.010
0.012
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 136 RT: 0.47 AV: 1 NL: 2.33E5 T: ITMS + c ESI Full ms2 [email protected] [80.00-305.00]
80 100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
214.98193
243.03616
285.11362
224.99274
182.98476
160.98511 254.06039 137.02051 111.12070
Testosterone Y = 0.00122857*X R^2 = 0.9960 W: 1/X
0 5000 10000 0
2
4
6
8
10
12
Area
Rat
io
Testosterone Y = 0.00037205+0.00122824*X R^2 = 0.9960 W: 1/X
0.0 0.5 1.0 0.0000
0.0005
0.0010
0.0015
0.0020
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1248 RT: 4.00 AV: 1 NL: 5.26E4 F: ITMS + c ESI Full ms2 [email protected] [75.00-285.00]
80 100 120 140 160 180 200 220 240 260 280 m/z
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Inte
nsity
253.12668
271.13193
97.01697
109.03072 213.07745 189.07771
243.12128 157.01028 123.07123
Alprazolam 40 fg – 10 ng >5 orders
Alprazolam Y = 0.0135765*X R^2 = 0.9973 W: 1/X
0 5000 10000 0
20
40
60
80
100
120
140
Area
Rat
io
Alprazolam Y = 0.0006305+0.0135759*X R^2 = 0.9973 W: 1/X
0.0 0.5 1.0 0.000
0.005
0.010
0.015
0.020
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1035 RT: 3.29 AV: 1 NL: 2.27E5 T: ITMS + c ESI Full ms2 [email protected] [85.00-305.00]
100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
281.01651
274.08139
204.97371 241.00223
164.92436 290.24725 197.05743 129.07735 104.12563
pg on Column %RSD
0.020 9.22
0.040 6.50
0.100 5.84
0.200 4.77
0.400 5.17
1.00 3.79
2.00 3.61
4.00 1.96
10.00 1.48
20.00 1.86
40.00 1.14
100.0 1.88
200.0 1.73
400.0 1.69
1000.0 2.65
2000.0 1.09
4000.0 2.27
10000.0 3.13
pg on Column %RSD
0.100 36.18
0.200 12.50
0.400 5.28
1.00 1.41
2.00 1.16
4.00 2.40
10.00 2.85
20.00 1.99
40.00 2.02
100.0 3.16
200.0 1.96
400.0 2.80
1000.0 2.60
2000.0 2.63
4000.0 2.43
10000.0 1.03
pg on Column %RSD
0.040 19.86
0.100 11.86
0.200 7.45
0.400 3.97
1.00 2.47
2.00 2.15
4.00 1.48
10.00 1.66
20.00 1.75
40.00 1.28
100.0 1.95
200.0 1.98
400.0 1.78
1000.0 3.67
2000.0 2.59
4000.0 1.79
10000.0 1.67
32
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0 500 1000 1500 2000 pg on column
0
5
10
15
Area
Rat
io
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 pg on column
0.000
0.002
0.004
0.006
0.008
0.010
0.012
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 136 RT: 0.47 AV: 1 NL: 2.33E5 T: ITMS + c ESI Full ms2 [email protected] [80.00-305.00]
80 100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
214.98193
243.03616
285.11362
224.99274
182.98476
160.98511 254.06039 137.02051 111.12070
Bupropion 10 fg – 2 ng >5 orders
Testosterone Y = 0.00122857*X R^2 = 0.9960 W: 1/X
0 5000 10000 0
2
4
6
8
10
12
Area
Rat
io
Testosterone Y = 0.00037205+0.00122824*X R^2 = 0.9960 W: 1/X
0.0 0.5 1.0 0.0000
0.0005
0.0010
0.0015
0.0020
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1248 RT: 4.00 AV: 1 NL: 5.26E4 F: ITMS + c ESI Full ms2 [email protected] [75.00-285.00]
80 100 120 140 160 180 200 220 240 260 280 m/z
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Inte
nsity
253.12668
271.13193
97.01697
109.03072 213.07745 189.07771
243.12128 157.01028 123.07123
Alprazolam Y = 0.0135765*X R^2 = 0.9973 W: 1/X
0 5000 10000 0
20
40
60
80
100
120
140
Area
Rat
io
Alprazolam Y = 0.0006305+0.0135759*X R^2 = 0.9973 W: 1/X
0.0 0.5 1.0 0.000
0.005
0.010
0.015
0.020
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1035 RT: 3.29 AV: 1 NL: 2.27E5 T: ITMS + c ESI Full ms2 [email protected] [85.00-305.00]
100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
281.01651
274.08139
204.97371 241.00223
164.92436 290.24725 197.05743 129.07735 104.12563
Bupropion Y = 0.0024576+0.0854631*X R^2 = 0.9948 W: 1/X
0 500 1000 1500 2000 0
20
40
60
80
100
120
140
160
Area
Rat
io
Bupropion Y = 0.0024576+0.0854631*X R^2 = 0.9948 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 0.00
0.02
0.04
0.06
0.08
0.10
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 611 RT: 1.99 AV: 1 NL: 1.99E6 F: ITMS + c ESI Full ms2 [email protected] [65.00-245.00]
80 100 120 140 160 180 200 220 240 m/z
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
Inte
nsity
183.94769
166.05928 223.09354 209.18298 147.07411 105.20074 79.29430
pg on Column %RSD
0.020 9.22
0.040 6.50
0.100 5.84
0.200 4.77
0.400 5.17
1.00 3.79
2.00 3.61
4.00 1.96
10.00 1.48
20.00 1.86
40.00 1.14
100.0 1.88
200.0 1.73
400.0 1.69
1000.0 2.65
2000.0 1.09
4000.0 2.27
10000.0 3.13
pg on Column %RSD
0.100 36.18
0.200 12.50
0.400 5.28
1.00 1.41
2.00 1.16
4.00 2.40
10.00 2.85
20.00 1.99
40.00 2.02
100.0 3.16
200.0 1.96
400.0 2.80
1000.0 2.60
2000.0 2.63
4000.0 2.43
10000.0 1.03
pg on Column %RSD
0.040 19.86
0.100 11.86
0.200 7.45
0.400 3.97
1.00 2.47
2.00 2.15
4.00 1.48
10.00 1.66
20.00 1.75
40.00 1.28
100.0 1.95
200.0 1.98
400.0 1.78
1000.0 3.67
2000.0 2.59
4000.0 1.79
10000.0 1.67
pg on Column %RSD
0.010 38.92
0.020 6.54
0.040 6.01
0.100 5.60
0.200 4.43
0.400 1.58
1.00 1.44
2.00 1.14
4.00 1.47
10.00 1.88
20.00 1.60
40.00 1.54
100.0 2.51
200.0 1.02
400.0 1.46
1000.0 1.35
2000.0 0.49
4000.0 1.50
10000.0 2.78
33
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0 500 1000 1500 2000 pg on column
0
5
10
15
Area
Rat
io
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 pg on column
0.000
0.002
0.004
0.006
0.008
0.010
0.012
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 136 RT: 0.47 AV: 1 NL: 2.33E5 T: ITMS + c ESI Full ms2 [email protected] [80.00-305.00]
80 100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
214.98193
243.03616
285.11362
224.99274
182.98476
160.98511 254.06039 137.02051 111.12070
Lidocaine 10 fg – 4 ng >5 orders
Testosterone Y = 0.00122857*X R^2 = 0.9960 W: 1/X
0 5000 10000 0
2
4
6
8
10
12
Area
Rat
io
Testosterone Y = 0.00037205+0.00122824*X R^2 = 0.9960 W: 1/X
0.0 0.5 1.0 0.0000
0.0005
0.0010
0.0015
0.0020
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1248 RT: 4.00 AV: 1 NL: 5.26E4 F: ITMS + c ESI Full ms2 [email protected] [75.00-285.00]
80 100 120 140 160 180 200 220 240 260 280 m/z
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Inte
nsity
253.12668
271.13193
97.01697
109.03072 213.07745 189.07771
243.12128 157.01028 123.07123
Alprazolam Y = 0.0135765*X R^2 = 0.9973 W: 1/X
0 5000 10000 0
20
40
60
80
100
120
140
Area
Rat
io
Alprazolam Y = 0.0006305+0.0135759*X R^2 = 0.9973 W: 1/X
0.0 0.5 1.0 0.000
0.005
0.010
0.015
0.020
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1035 RT: 3.29 AV: 1 NL: 2.27E5 T: ITMS + c ESI Full ms2 [email protected] [85.00-305.00]
100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
281.01651
274.08139
204.97371 241.00223
164.92436 290.24725 197.05743 129.07735 104.12563
Bupropion Y = 0.0024576+0.0854631*X R^2 = 0.9948 W: 1/X
0 500 1000 1500 2000 0
20
40
60
80
100
120
140
160
Area
Rat
io
Bupropion Y = 0.0024576+0.0854631*X R^2 = 0.9948 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 0.00
0.02
0.04
0.06
0.08
0.10
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 611 RT: 1.99 AV: 1 NL: 1.99E6 F: ITMS + c ESI Full ms2 [email protected] [65.00-245.00]
80 100 120 140 160 180 200 220 240 m/z
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
Inte
nsity
183.94769
166.05928 223.09354 209.18298 147.07411 105.20074 79.29430
Lidocaine Y = 0.0197037*X R^2 = 0.9907 W: 1/X
0 1000 2000 3000 4000 pg on Column
0
10
20
30
40
50
60
70
80
Area
Rat
io
Lidocaine Y = 0.000428793+0.0197027*X R^2 = 0.9907 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 pg on Column
0.000
0.005
0.010
0.015
0.020
0.025
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 331 RT: 1.06 AV: 1 NL: 5.30E5 F: ITMS + c ESI Full ms2 [email protected] [60.00-240.00]
60 80 100 120 140 160 180 200 220 240 m/z
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
Inte
nsity
86.02197
179.00397 217.00284 160.08882 109.14704 123.15810 68.07511
pg on Column %RSD
0.020 9.22
0.040 6.50
0.100 5.84
0.200 4.77
0.400 5.17
1.00 3.79
2.00 3.61
4.00 1.96
10.00 1.48
20.00 1.86
40.00 1.14
100.0 1.88
200.0 1.73
400.0 1.69
1000.0 2.65
2000.0 1.09
4000.0 2.27
10000.0 3.13
pg on Column %RSD
0.100 36.18
0.200 12.50
0.400 5.28
1.00 1.41
2.00 1.16
4.00 2.40
10.00 2.85
20.00 1.99
40.00 2.02
100.0 3.16
200.0 1.96
400.0 2.80
1000.0 2.60
2000.0 2.63
4000.0 2.43
10000.0 1.03
pg on Column %RSD
0.040 19.86
0.100 11.86
0.200 7.45
0.400 3.97
1.00 2.47
2.00 2.15
4.00 1.48
10.00 1.66
20.00 1.75
40.00 1.28
100.0 1.95
200.0 1.98
400.0 1.78
1000.0 3.67
2000.0 2.59
4000.0 1.79
10000.0 1.67
pg on Column %RSD
0.010 38.92
0.020 6.54
0.040 6.01
0.100 5.60
0.200 4.43
0.400 1.58
1.00 1.44
2.00 1.14
4.00 1.47
10.00 1.88
20.00 1.60
40.00 1.54
100.0 2.51
200.0 1.02
400.0 1.46
1000.0 1.35
2000.0 0.49
4000.0 1.50
10000.0 2.78
pg on Column %RSD
0.010 25.45
0.020 19.69
0.040 9.66
0.100 6.05
0.200 4.31
0.400 3.90
1.00 2.77
2.00 2.12
4.00 2.18
10.00 1.82
20.00 2.63
40.00 2.92
100.0 2.34
200.0 1.04
400.0 1.52
1000.0 1.89
2000.0 0.88
4000.0 2.80
10000.0 1.47
34
Methoxymethamphetamine 10 fg – 2 ng >5 orders
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0 500 1000 1500 2000 pg on column
0
5
10
15
Area
Rat
io
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 pg on column
0.000
0.002
0.004
0.006
0.008
0.010
0.012
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 136 RT: 0.47 AV: 1 NL: 2.33E5 T: ITMS + c ESI Full ms2 [email protected] [80.00-305.00]
80 100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
214.98193
243.03616
285.11362
224.99274
182.98476
160.98511 254.06039 137.02051 111.12070
Testosterone Y = 0.00122857*X R^2 = 0.9960 W: 1/X
0 5000 10000 0
2
4
6
8
10
12
Area
Rat
io
Testosterone Y = 0.00037205+0.00122824*X R^2 = 0.9960 W: 1/X
0.0 0.5 1.0 0.0000
0.0005
0.0010
0.0015
0.0020
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1248 RT: 4.00 AV: 1 NL: 5.26E4 F: ITMS + c ESI Full ms2 [email protected] [75.00-285.00]
80 100 120 140 160 180 200 220 240 260 280 m/z
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Inte
nsity
253.12668
271.13193
97.01697
109.03072 213.07745 189.07771
243.12128 157.01028 123.07123
Alprazolam Y = 0.0135765*X R^2 = 0.9973 W: 1/X
0 5000 10000 0
20
40
60
80
100
120
140
Area
Rat
io
Alprazolam Y = 0.0006305+0.0135759*X R^2 = 0.9973 W: 1/X
0.0 0.5 1.0 0.000
0.005
0.010
0.015
0.020
Are
a R
atio
Bupropion Y = 0.0024576+0.0854631*X R^2 = 0.9948 W: 1/X
0 500 1000 1500 2000 0
20
40
60
80
100
120
140
160
Area
Rat
io
Bupropion Y = 0.0024576+0.0854631*X R^2 = 0.9948 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 0.00
0.02
0.04
0.06
0.08
0.10
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1035 RT: 3.29 AV: 1 NL: 2.27E5 T: ITMS + c ESI Full ms2 [email protected] [85.00-305.00]
100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
281.01651
274.08139
204.97371 241.00223
164.92436 290.24725 197.05743 129.07735 104.12563
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 611 RT: 1.99 AV: 1 NL: 1.99E6 F: ITMS + c ESI Full ms2 [email protected] [65.00-245.00]
80 100 120 140 160 180 200 220 240 m/z
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
Inte
nsity
183.94769
166.05928 223.09354 209.18298 147.07411 105.20074 79.29430
Lidocaine Y = 0.0197037*X R^2 = 0.9907 W: 1/X
0 1000 2000 3000 4000 pg on Column
0
10
20
30
40
50
60
70
80
Area
Rat
io
Lidocaine Y = 0.000428793+0.0197027*X R^2 = 0.9907 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 pg on Column
0.000
0.005
0.010
0.015
0.020
0.025
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 331 RT: 1.06 AV: 1 NL: 5.30E5 F: ITMS + c ESI Full ms2 [email protected] [60.00-240.00]
60 80 100 120 140 160 180 200 220 240 m/z
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
Inte
nsity
86.02197
179.00397 217.00284 160.08882 109.14704 123.15810 68.07511
Methoxymethamphetamine Y = 0.0243242*X R^2 = 0.9964 W: 1/X
0 500 1000 1500 2000 0
10
20
30
40
50
Area
Rat
io
Methoxymethamphetamine Y = 0.0243242*X R^2 = 0.9964 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.000
0.005
0.010
0.015
0.020
0.025
0.030
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 302 RT: 0.98 AV: 1 NL: 6.60E5 F: ITMS + c ESI Full ms2 [email protected] [50.00-185.00]
60 80 100 120 140 160 180 m/z
0
100000
200000
300000
400000
500000
600000
Inte
nsity
149.01674
162.11177 121.06242 108.13741 138.03410 95.09702 60.31478 179.39780 71.17224
pg on Column %RSD
0.020 9.22
0.040 6.50
0.100 5.84
0.200 4.77
0.400 5.17
1.00 3.79
2.00 3.61
4.00 1.96
10.00 1.48
20.00 1.86
40.00 1.14
100.0 1.88
200.0 1.73
400.0 1.69
1000.0 2.65
2000.0 1.09
4000.0 2.27
10000.0 3.13
pg on Column %RSD
0.100 36.18
0.200 12.50
0.400 5.28
1.00 1.41
2.00 1.16
4.00 2.40
10.00 2.85
20.00 1.99
40.00 2.02
100.0 3.16
200.0 1.96
400.0 2.80
1000.0 2.60
2000.0 2.63
4000.0 2.43
10000.0 1.03
pg on Column %RSD
0.040 19.86
0.100 11.86
0.200 7.45
0.400 3.97
1.00 2.47
2.00 2.15
4.00 1.48
10.00 1.66
20.00 1.75
40.00 1.28
100.0 1.95
200.0 1.98
400.0 1.78
1000.0 3.67
2000.0 2.59
4000.0 1.79
10000.0 1.67
pg on Column %RSD
0.010 38.92
0.020 6.54
0.040 6.01
0.100 5.60
0.200 4.43
0.400 1.58
1.00 1.44
2.00 1.14
4.00 1.47
10.00 1.88
20.00 1.60
40.00 1.54
100.0 2.51
200.0 1.02
400.0 1.46
1000.0 1.35
2000.0 0.49
4000.0 1.50
10000.0 2.78
pg on Column %RSD
0.010 25.45
0.020 19.69
0.040 9.66
0.100 6.05
0.200 4.31
0.400 3.90
1.00 2.77
2.00 2.12
4.00 2.18
10.00 1.82
20.00 2.63
40.00 2.92
100.0 2.34
200.0 1.04
400.0 1.52
1000.0 1.89
2000.0 0.88
4000.0 2.80
10000.0 1.47
pg on Column %RSD
0.010 32.16
0.020 11.42
0.040 8.56
0.100 3.85
0.200 3.60
0.400 2.88
1.00 3.25
2.00 2.77
4.00 2.69
10.00 2.91
20.00 1.49
40.00 1.26
100.0 1.94
200.0 1.40
400.0 2.86
1000.0 1.37
2000.0 1.16
4000.0 1.41
10000.0 2.54
35
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0 500 1000 1500 2000 pg on column
0
5
10
15
Area
Rat
io
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 pg on column
0.000
0.002
0.004
0.006
0.008
0.010
0.012
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 136 RT: 0.47 AV: 1 NL: 2.33E5 T: ITMS + c ESI Full ms2 [email protected] [80.00-305.00]
80 100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
214.98193
243.03616
285.11362
224.99274
182.98476
160.98511 254.06039 137.02051 111.12070
Testosterone Y = 0.00122857*X R^2 = 0.9960 W: 1/X
0 5000 10000 0
2
4
6
8
10
12
Area
Rat
io
Testosterone Y = 0.00037205+0.00122824*X R^2 = 0.9960 W: 1/X
0.0 0.5 1.0 0.0000
0.0005
0.0010
0.0015
0.0020
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1248 RT: 4.00 AV: 1 NL: 5.26E4 F: ITMS + c ESI Full ms2 [email protected] [75.00-285.00]
80 100 120 140 160 180 200 220 240 260 280 m/z
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Inte
nsity
253.12668
271.13193
97.01697
109.03072 213.07745 189.07771
243.12128 157.01028 123.07123
Alprazolam Y = 0.0135765*X R^2 = 0.9973 W: 1/X
0 5000 10000 0
20
40
60
80
100
120
140
Area
Rat
io
Alprazolam Y = 0.0006305+0.0135759*X R^2 = 0.9973 W: 1/X
0.0 0.5 1.0 0.000
0.005
0.010
0.015
0.020
Are
a R
atio
Bupropion Y = 0.0024576+0.0854631*X R^2 = 0.9948 W: 1/X
0 500 1000 1500 2000 0
20
40
60
80
100
120
140
160
Area
Rat
io
Bupropion Y = 0.0024576+0.0854631*X R^2 = 0.9948 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 0.00
0.02
0.04
0.06
0.08
0.10
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1035 RT: 3.29 AV: 1 NL: 2.27E5 T: ITMS + c ESI Full ms2 [email protected] [85.00-305.00]
100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
281.01651
274.08139
204.97371 241.00223
164.92436 290.24725 197.05743 129.07735 104.12563
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 611 RT: 1.99 AV: 1 NL: 1.99E6 F: ITMS + c ESI Full ms2 [email protected] [65.00-245.00]
80 100 120 140 160 180 200 220 240 m/z
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
Inte
nsity
183.94769
166.05928 223.09354 209.18298 147.07411 105.20074 79.29430
Lidocaine Y = 0.0197037*X R^2 = 0.9907 W: 1/X
0 1000 2000 3000 4000 pg on Column
0
10
20
30
40
50
60
70
80
Area
Rat
io
Lidocaine Y = 0.000428793+0.0197027*X R^2 = 0.9907 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 pg on Column
0.000
0.005
0.010
0.015
0.020
0.025
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 331 RT: 1.06 AV: 1 NL: 5.30E5 F: ITMS + c ESI Full ms2 [email protected] [60.00-240.00]
60 80 100 120 140 160 180 200 220 240 m/z
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
Inte
nsity
86.02197
179.00397 217.00284 160.08882 109.14704 123.15810 68.07511
Methoxymethamphetamine Y = 0.0243242*X R^2 = 0.9964 W: 1/X
0 500 1000 1500 2000 0
10
20
30
40
50
Area
Rat
io
Methoxymethamphetamine Y = 0.0243242*X R^2 = 0.9964 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.000
0.005
0.010
0.015
0.020
0.025
0.030
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 302 RT: 0.98 AV: 1 NL: 6.60E5 F: ITMS + c ESI Full ms2 [email protected] [50.00-185.00]
60 80 100 120 140 160 180 m/z
0
100000
200000
300000
400000
500000
600000
Inte
nsity
149.01674
162.11177 121.06242 108.13741 138.03410 95.09702 60.31478 179.39780 71.17224
Propranolol 10 fg – 4 ng >5 orders pg on
Column %RSD
0.020 9.22
0.040 6.50
0.100 5.84
0.200 4.77
0.400 5.17
1.00 3.79
2.00 3.61
4.00 1.96
10.00 1.48
20.00 1.86
40.00 1.14
100.0 1.88
200.0 1.73
400.0 1.69
1000.0 2.65
2000.0 1.09
4000.0 2.27
10000.0 3.13
pg on Column %RSD
0.100 36.18
0.200 12.50
0.400 5.28
1.00 1.41
2.00 1.16
4.00 2.40
10.00 2.85
20.00 1.99
40.00 2.02
100.0 3.16
200.0 1.96
400.0 2.80
1000.0 2.60
2000.0 2.63
4000.0 2.43
10000.0 1.03
pg on Column %RSD
0.040 19.86
0.100 11.86
0.200 7.45
0.400 3.97
1.00 2.47
2.00 2.15
4.00 1.48
10.00 1.66
20.00 1.75
40.00 1.28
100.0 1.95
200.0 1.98
400.0 1.78
1000.0 3.67
2000.0 2.59
4000.0 1.79
10000.0 1.67
pg on Column %RSD
0.010 38.92
0.020 6.54
0.040 6.01
0.100 5.60
0.200 4.43
0.400 1.58
1.00 1.44
2.00 1.14
4.00 1.47
10.00 1.88
20.00 1.60
40.00 1.54
100.0 2.51
200.0 1.02
400.0 1.46
1000.0 1.35
2000.0 0.49
4000.0 1.50
10000.0 2.78
pg on Column %RSD
0.010 25.45
0.020 19.69
0.040 9.66
0.100 6.05
0.200 4.31
0.400 3.90
1.00 2.77
2.00 2.12
4.00 2.18
10.00 1.82
20.00 2.63
40.00 2.92
100.0 2.34
200.0 1.04
400.0 1.52
1000.0 1.89
2000.0 0.88
4000.0 2.80
10000.0 1.47
pg on Column %RSD
0.010 32.16
0.020 11.42
0.040 8.56
0.100 3.85
0.200 3.60
0.400 2.88
1.00 3.25
2.00 2.77
4.00 2.69
10.00 2.91
20.00 1.49
40.00 1.26
100.0 1.94
200.0 1.40
400.0 2.86
1000.0 1.37
2000.0 1.16
4000.0 1.41
10000.0 2.54
pg on Column %RSD
0.020 14.22
0.040 8.95
0.100 9.65
0.200 4.75
0.400 3.45
1.00 3.43
2.00 2.22
4.00 1.04
10.00 1.31
20.00 1.30
40.00 1.45
100.0 3.11
200.0 1.69
400.0 0.75
1000.0 3.19
2000.0 2.45
4000.0 1.20
10000.0 1.74
Propranolol Y = 0.025856*X R^2 = 0.9974 W: 1/X
0 1000 2000 3000 4000 0
20
40
60
80
100
Area
Rat
io
Propranolol Y = 0.00202443+0.0258514*X R^2 = 0.9974 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 785 RT: 2.52 AV: 1 NL: 3.04E5 F: ITMS + c ESI Full ms2 [email protected] [70.00-265.00]
80 100 120 140 160 180 200 220 240 260 m/z
0
50000
100000
150000
200000
250000
300000
Inte
nsity
182.98044
116.08667
156.95374 217.96509
98.04890 242.05054
132.03139 86.08720 200.04666 165.01280 225.06369 259.18817
36
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0 500 1000 1500 2000 pg on column
0
5
10
15
Area
Rat
io
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 pg on column
0.000
0.002
0.004
0.006
0.008
0.010
0.012
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 136 RT: 0.47 AV: 1 NL: 2.33E5 T: ITMS + c ESI Full ms2 [email protected] [80.00-305.00]
80 100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
214.98193
243.03616
285.11362
224.99274
182.98476
160.98511 254.06039 137.02051 111.12070
Testosterone Y = 0.00122857*X R^2 = 0.9960 W: 1/X
0 5000 10000 0
2
4
6
8
10
12
Area
Rat
io
Testosterone Y = 0.00037205+0.00122824*X R^2 = 0.9960 W: 1/X
0.0 0.5 1.0 0.0000
0.0005
0.0010
0.0015
0.0020
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1248 RT: 4.00 AV: 1 NL: 5.26E4 F: ITMS + c ESI Full ms2 [email protected] [75.00-285.00]
80 100 120 140 160 180 200 220 240 260 280 m/z
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Inte
nsity
253.12668
271.13193
97.01697
109.03072 213.07745 189.07771
243.12128 157.01028 123.07123
Alprazolam Y = 0.0135765*X R^2 = 0.9973 W: 1/X
0 5000 10000 0
20
40
60
80
100
120
140
Area
Rat
io
Alprazolam Y = 0.0006305+0.0135759*X R^2 = 0.9973 W: 1/X
0.0 0.5 1.0 0.000
0.005
0.010
0.015
0.020
Are
a R
atio
Bupropion Y = 0.0024576+0.0854631*X R^2 = 0.9948 W: 1/X
0 500 1000 1500 2000 0
20
40
60
80
100
120
140
160
Area
Rat
io
Bupropion Y = 0.0024576+0.0854631*X R^2 = 0.9948 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 0.00
0.02
0.04
0.06
0.08
0.10
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1035 RT: 3.29 AV: 1 NL: 2.27E5 T: ITMS + c ESI Full ms2 [email protected] [85.00-305.00]
100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
281.01651
274.08139
204.97371 241.00223
164.92436 290.24725 197.05743 129.07735 104.12563
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 611 RT: 1.99 AV: 1 NL: 1.99E6 F: ITMS + c ESI Full ms2 [email protected] [65.00-245.00]
80 100 120 140 160 180 200 220 240 m/z
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
Inte
nsity
183.94769
166.05928 223.09354 209.18298 147.07411 105.20074 79.29430
Lidocaine Y = 0.0197037*X R^2 = 0.9907 W: 1/X
0 1000 2000 3000 4000 pg on Column
0
10
20
30
40
50
60
70
80
Area
Rat
io
Lidocaine Y = 0.000428793+0.0197027*X R^2 = 0.9907 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 pg on Column
0.000
0.005
0.010
0.015
0.020
0.025
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 331 RT: 1.06 AV: 1 NL: 5.30E5 F: ITMS + c ESI Full ms2 [email protected] [60.00-240.00]
60 80 100 120 140 160 180 200 220 240 m/z
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
Inte
nsity
86.02197
179.00397 217.00284 160.08882 109.14704 123.15810 68.07511
Methoxymethamphetamine Y = 0.0243242*X R^2 = 0.9964 W: 1/X
0 500 1000 1500 2000 0
10
20
30
40
50
Area
Rat
io
Methoxymethamphetamine Y = 0.0243242*X R^2 = 0.9964 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.000
0.005
0.010
0.015
0.020
0.025
0.030
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 302 RT: 0.98 AV: 1 NL: 6.60E5 F: ITMS + c ESI Full ms2 [email protected] [50.00-185.00]
60 80 100 120 140 160 180 m/z
0
100000
200000
300000
400000
500000
600000
Inte
nsity
149.01674
162.11177 121.06242 108.13741 138.03410 95.09702 60.31478 179.39780 71.17224
Propranolol 10 fg – 4 ng >5 orders pg on
Column %RSD
0.020 9.22
0.040 6.50
0.100 5.84
0.200 4.77
0.400 5.17
1.00 3.79
2.00 3.61
4.00 1.96
10.00 1.48
20.00 1.86
40.00 1.14
100.0 1.88
200.0 1.73
400.0 1.69
1000.0 2.65
2000.0 1.09
4000.0 2.27
10000.0 3.13
pg on Column %RSD
0.100 36.18
0.200 12.50
0.400 5.28
1.00 1.41
2.00 1.16
4.00 2.40
10.00 2.85
20.00 1.99
40.00 2.02
100.0 3.16
200.0 1.96
400.0 2.80
1000.0 2.60
2000.0 2.63
4000.0 2.43
10000.0 1.03
pg on Column %RSD
0.040 19.86
0.100 11.86
0.200 7.45
0.400 3.97
1.00 2.47
2.00 2.15
4.00 1.48
10.00 1.66
20.00 1.75
40.00 1.28
100.0 1.95
200.0 1.98
400.0 1.78
1000.0 3.67
2000.0 2.59
4000.0 1.79
10000.0 1.67
pg on Column %RSD
0.010 38.92
0.020 6.54
0.040 6.01
0.100 5.60
0.200 4.43
0.400 1.58
1.00 1.44
2.00 1.14
4.00 1.47
10.00 1.88
20.00 1.60
40.00 1.54
100.0 2.51
200.0 1.02
400.0 1.46
1000.0 1.35
2000.0 0.49
4000.0 1.50
10000.0 2.78
pg on Column %RSD
0.010 25.45
0.020 19.69
0.040 9.66
0.100 6.05
0.200 4.31
0.400 3.90
1.00 2.77
2.00 2.12
4.00 2.18
10.00 1.82
20.00 2.63
40.00 2.92
100.0 2.34
200.0 1.04
400.0 1.52
1000.0 1.89
2000.0 0.88
4000.0 2.80
10000.0 1.47
pg on Column %RSD
0.010 32.16
0.020 11.42
0.040 8.56
0.100 3.85
0.200 3.60
0.400 2.88
1.00 3.25
2.00 2.77
4.00 2.69
10.00 2.91
20.00 1.49
40.00 1.26
100.0 1.94
200.0 1.40
400.0 2.86
1000.0 1.37
2000.0 1.16
4000.0 1.41
10000.0 2.54
pg on Column %RSD
0.020 14.22
0.040 8.95
0.100 9.65
0.200 4.75
0.400 3.45
1.00 3.43
2.00 2.22
4.00 1.04
10.00 1.31
20.00 1.30
40.00 1.45
100.0 3.11
200.0 1.69
400.0 0.75
1000.0 3.19
2000.0 2.45
4000.0 1.20
10000.0 1.74
Propranolol Y = 0.025856*X R^2 = 0.9974 W: 1/X
0 1000 2000 3000 4000 0
20
40
60
80
100
Area
Rat
io
Propranolol Y = 0.00202443+0.0258514*X R^2 = 0.9974 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 785 RT: 2.52 AV: 1 NL: 3.04E5 F: ITMS + c ESI Full ms2 [email protected] [70.00-265.00]
80 100 120 140 160 180 200 220 240 260 m/z
0
50000
100000
150000
200000
250000
300000
Inte
nsity
182.98044
116.08667
156.95374 217.96509
98.04890 242.05054
132.03139 86.08720 200.04666 165.01280 225.06369 259.18817
High Quality Full Scan MS/MS at EVERY
Data Point
37
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0 500 1000 1500 2000 pg on column
0
5
10
15
Area
Rat
io
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 pg on column
0.000
0.002
0.004
0.006
0.008
0.010
0.012
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 136 RT: 0.47 AV: 1 NL: 2.33E5 T: ITMS + c ESI Full ms2 [email protected] [80.00-305.00]
80 100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
214.98193
243.03616
285.11362
224.99274
182.98476
160.98511 254.06039 137.02051 111.12070
Testosterone Y = 0.00122857*X R^2 = 0.9960 W: 1/X
0 5000 10000 0
2
4
6
8
10
12
Area
Rat
io
Testosterone Y = 0.00037205+0.00122824*X R^2 = 0.9960 W: 1/X
0.0 0.5 1.0 0.0000
0.0005
0.0010
0.0015
0.0020
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1248 RT: 4.00 AV: 1 NL: 5.26E4 F: ITMS + c ESI Full ms2 [email protected] [75.00-285.00]
80 100 120 140 160 180 200 220 240 260 280 m/z
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Inte
nsity
253.12668
271.13193
97.01697
109.03072 213.07745 189.07771
243.12128 157.01028 123.07123
Alprazolam Y = 0.0135765*X R^2 = 0.9973 W: 1/X
0 5000 10000 0
20
40
60
80
100
120
140
Area
Rat
io
Alprazolam Y = 0.0006305+0.0135759*X R^2 = 0.9973 W: 1/X
0.0 0.5 1.0 0.000
0.005
0.010
0.015
0.020
Are
a R
atio
Bupropion Y = 0.0024576+0.0854631*X R^2 = 0.9948 W: 1/X
0 500 1000 1500 2000 0
20
40
60
80
100
120
140
160
Area
Rat
io
Bupropion Y = 0.0024576+0.0854631*X R^2 = 0.9948 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 0.00
0.02
0.04
0.06
0.08
0.10
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1035 RT: 3.29 AV: 1 NL: 2.27E5 T: ITMS + c ESI Full ms2 [email protected] [85.00-305.00]
100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
281.01651
274.08139
204.97371 241.00223
164.92436 290.24725 197.05743 129.07735 104.12563
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 611 RT: 1.99 AV: 1 NL: 1.99E6 F: ITMS + c ESI Full ms2 [email protected] [65.00-245.00]
80 100 120 140 160 180 200 220 240 m/z
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
Inte
nsity
183.94769
166.05928 223.09354 209.18298 147.07411 105.20074 79.29430
Lidocaine Y = 0.0197037*X R^2 = 0.9907 W: 1/X
0 1000 2000 3000 4000 pg on Column
0
10
20
30
40
50
60
70
80
Area
Rat
io
Lidocaine Y = 0.000428793+0.0197027*X R^2 = 0.9907 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 pg on Column
0.000
0.005
0.010
0.015
0.020
0.025
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 331 RT: 1.06 AV: 1 NL: 5.30E5 F: ITMS + c ESI Full ms2 [email protected] [60.00-240.00]
60 80 100 120 140 160 180 200 220 240 m/z
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
Inte
nsity
86.02197
179.00397 217.00284 160.08882 109.14704 123.15810 68.07511
Methoxymethamphetamine Y = 0.0243242*X R^2 = 0.9964 W: 1/X
0 500 1000 1500 2000 0
10
20
30
40
50
Area
Rat
io
Methoxymethamphetamine Y = 0.0243242*X R^2 = 0.9964 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.000
0.005
0.010
0.015
0.020
0.025
0.030
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 302 RT: 0.98 AV: 1 NL: 6.60E5 F: ITMS + c ESI Full ms2 [email protected] [50.00-185.00]
60 80 100 120 140 160 180 m/z
0
100000
200000
300000
400000
500000
600000
Inte
nsity
149.01674
162.11177 121.06242 108.13741 138.03410 95.09702 60.31478 179.39780 71.17224
Propranolol Y = 0.025856*X R^2 = 0.9974 W: 1/X
0 1000 2000 3000 4000 0
20
40
60
80
100
Area
Rat
io
Propranolol Y = 0.00202443+0.0258514*X R^2 = 0.9974 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 785 RT: 2.52 AV: 1 NL: 3.04E5 F: ITMS + c ESI Full ms2 [email protected] [70.00-265.00]
80 100 120 140 160 180 200 220 240 260 m/z
0
50000
100000
150000
200000
250000
300000
Inte
nsity
182.98044
116.08667
156.95374 217.96509
98.04890 242.05054
132.03139 86.08720 200.04666 165.01280 225.06369 259.18817
Terfenadine 10 fg - 10 ng 6 orders pg on
Column %RSD
0.020 9.22
0.040 6.50
0.100 5.84
0.200 4.77
0.400 5.17
1.00 3.79
2.00 3.61
4.00 1.96
10.00 1.48
20.00 1.86
40.00 1.14
100.0 1.88
200.0 1.73
400.0 1.69
1000.0 2.65
2000.0 1.09
4000.0 2.27
10000.0 3.13
pg on Column %RSD
0.100 36.18
0.200 12.50
0.400 5.28
1.00 1.41
2.00 1.16
4.00 2.40
10.00 2.85
20.00 1.99
40.00 2.02
100.0 3.16
200.0 1.96
400.0 2.80
1000.0 2.60
2000.0 2.63
4000.0 2.43
10000.0 1.03
pg on Column %RSD
0.040 19.86
0.100 11.86
0.200 7.45
0.400 3.97
1.00 2.47
2.00 2.15
4.00 1.48
10.00 1.66
20.00 1.75
40.00 1.28
100.0 1.95
200.0 1.98
400.0 1.78
1000.0 3.67
2000.0 2.59
4000.0 1.79
10000.0 1.67
pg on Column %RSD
0.010 38.92
0.020 6.54
0.040 6.01
0.100 5.60
0.200 4.43
0.400 1.58
1.00 1.44
2.00 1.14
4.00 1.47
10.00 1.88
20.00 1.60
40.00 1.54
100.0 2.51
200.0 1.02
400.0 1.46
1000.0 1.35
2000.0 0.49
4000.0 1.50
10000.0 2.78
pg on Column %RSD
0.010 25.45
0.020 19.69
0.040 9.66
0.100 6.05
0.200 4.31
0.400 3.90
1.00 2.77
2.00 2.12
4.00 2.18
10.00 1.82
20.00 2.63
40.00 2.92
100.0 2.34
200.0 1.04
400.0 1.52
1000.0 1.89
2000.0 0.88
4000.0 2.80
10000.0 1.47
pg on Column %RSD
0.010 32.16
0.020 11.42
0.040 8.56
0.100 3.85
0.200 3.60
0.400 2.88
1.00 3.25
2.00 2.77
4.00 2.69
10.00 2.91
20.00 1.49
40.00 1.26
100.0 1.94
200.0 1.40
400.0 2.86
1000.0 1.37
2000.0 1.16
4000.0 1.41
10000.0 2.54
pg on Column %RSD
0.020 14.22
0.040 8.95
0.100 9.65
0.200 4.75
0.400 3.45
1.00 3.43
2.00 2.22
4.00 1.04
10.00 1.31
20.00 1.30
40.00 1.45
100.0 3.11
200.0 1.69
400.0 0.75
1000.0 3.19
2000.0 2.45
4000.0 1.20
10000.0 1.74
pg on Column %RSD
0.010 20.75
0.020 8.69
0.040 7.21
0.100 4.36
0.200 2.87
0.400 2.34
1.00 1.22
2.00 2.32
4.00 2.14
10.00 2.15
20.00 1.54
40.00 2.13
100.0 2.28
200.0 3.69
400.0 1.76
1000.0 2.63
2000.0 3.94
4000.0 2.72
10000.0 1.93
Terfenadine Y = 0.102234*X R^2 = 0.9951 W: 1/X
0 5000 10000 0
200
400
600
800
1000
Area
Rat
io
Terfenadine Y = 0.00761644+0.102226*X R^2 = 0.9951 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.00
0.02
0.04
0.06
0.08
0.10
0.12
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1281 RT: 4.08 AV: 1 NL: 2.01E6 F: ITMS + c ESI Full ms2 [email protected] [125.00-470.00]
150 200 250 300 350 400 450 m/z
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
Inte
nsity
436.15707
454.16705
262.11255 230.19748 187.12711 380.28439 344.28711 289.16522
38
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0 500 1000 1500 2000 pg on column
0
5
10
15
Area
Rat
io
Codeine Y = 0.0100042*X R^2 = 0.9949 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 pg on column
0.000
0.002
0.004
0.006
0.008
0.010
0.012
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 136 RT: 0.47 AV: 1 NL: 2.33E5 T: ITMS + c ESI Full ms2 [email protected] [80.00-305.00]
80 100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
214.98193
243.03616
285.11362
224.99274
182.98476
160.98511 254.06039 137.02051 111.12070
Testosterone Y = 0.00122857*X R^2 = 0.9960 W: 1/X
0 5000 10000 0
2
4
6
8
10
12
Area
Rat
io
Testosterone Y = 0.00037205+0.00122824*X R^2 = 0.9960 W: 1/X
0.0 0.5 1.0 0.0000
0.0005
0.0010
0.0015
0.0020
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1248 RT: 4.00 AV: 1 NL: 5.26E4 F: ITMS + c ESI Full ms2 [email protected] [75.00-285.00]
80 100 120 140 160 180 200 220 240 260 280 m/z
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Inte
nsity
253.12668
271.13193
97.01697
109.03072 213.07745 189.07771
243.12128 157.01028 123.07123
Alprazolam Y = 0.0135765*X R^2 = 0.9973 W: 1/X
0 5000 10000 0
20
40
60
80
100
120
140
Area
Rat
io
Alprazolam Y = 0.0006305+0.0135759*X R^2 = 0.9973 W: 1/X
0.0 0.5 1.0 0.000
0.005
0.010
0.015
0.020
Are
a R
atio
Bupropion Y = 0.0024576+0.0854631*X R^2 = 0.9948 W: 1/X
0 500 1000 1500 2000 0
20
40
60
80
100
120
140
160
Area
Rat
io
Bupropion Y = 0.0024576+0.0854631*X R^2 = 0.9948 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 0.00
0.02
0.04
0.06
0.08
0.10
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1035 RT: 3.29 AV: 1 NL: 2.27E5 T: ITMS + c ESI Full ms2 [email protected] [85.00-305.00]
100 120 140 160 180 200 220 240 260 280 300 m/z
0
50000
100000
150000
200000
Inte
nsity
281.01651
274.08139
204.97371 241.00223
164.92436 290.24725 197.05743 129.07735 104.12563
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 611 RT: 1.99 AV: 1 NL: 1.99E6 F: ITMS + c ESI Full ms2 [email protected] [65.00-245.00]
80 100 120 140 160 180 200 220 240 m/z
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
Inte
nsity
183.94769
166.05928 223.09354 209.18298 147.07411 105.20074 79.29430
Lidocaine Y = 0.0197037*X R^2 = 0.9907 W: 1/X
0 1000 2000 3000 4000 pg on Column
0
10
20
30
40
50
60
70
80
Area
Rat
io
Lidocaine Y = 0.000428793+0.0197027*X R^2 = 0.9907 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 pg on Column
0.000
0.005
0.010
0.015
0.020
0.025
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 331 RT: 1.06 AV: 1 NL: 5.30E5 F: ITMS + c ESI Full ms2 [email protected] [60.00-240.00]
60 80 100 120 140 160 180 200 220 240 m/z
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
Inte
nsity
86.02197
179.00397 217.00284 160.08882 109.14704 123.15810 68.07511
Methoxymethamphetamine Y = 0.0243242*X R^2 = 0.9964 W: 1/X
0 500 1000 1500 2000 0
10
20
30
40
50
Area
Rat
io
Methoxymethamphetamine Y = 0.0243242*X R^2 = 0.9964 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.000
0.005
0.010
0.015
0.020
0.025
0.030
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 302 RT: 0.98 AV: 1 NL: 6.60E5 F: ITMS + c ESI Full ms2 [email protected] [50.00-185.00]
60 80 100 120 140 160 180 m/z
0
100000
200000
300000
400000
500000
600000
Inte
nsity
149.01674
162.11177 121.06242 108.13741 138.03410 95.09702 60.31478 179.39780 71.17224
Propranolol Y = 0.025856*X R^2 = 0.9974 W: 1/X
0 1000 2000 3000 4000 0
20
40
60
80
100
Area
Rat
io
Propranolol Y = 0.00202443+0.0258514*X R^2 = 0.9974 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 785 RT: 2.52 AV: 1 NL: 3.04E5 F: ITMS + c ESI Full ms2 [email protected] [70.00-265.00]
80 100 120 140 160 180 200 220 240 260 m/z
0
50000
100000
150000
200000
250000
300000
Inte
nsity
182.98044
116.08667
156.95374 217.96509
98.04890 242.05054
132.03139 86.08720 200.04666 165.01280 225.06369 259.18817
Terfenadine 10 fg - 10 ng 6 orders pg on
Column %RSD
0.020 9.22
0.040 6.50
0.100 5.84
0.200 4.77
0.400 5.17
1.00 3.79
2.00 3.61
4.00 1.96
10.00 1.48
20.00 1.86
40.00 1.14
100.0 1.88
200.0 1.73
400.0 1.69
1000.0 2.65
2000.0 1.09
4000.0 2.27
10000.0 3.13
pg on Column %RSD
0.100 36.18
0.200 12.50
0.400 5.28
1.00 1.41
2.00 1.16
4.00 2.40
10.00 2.85
20.00 1.99
40.00 2.02
100.0 3.16
200.0 1.96
400.0 2.80
1000.0 2.60
2000.0 2.63
4000.0 2.43
10000.0 1.03
pg on Column %RSD
0.040 19.86
0.100 11.86
0.200 7.45
0.400 3.97
1.00 2.47
2.00 2.15
4.00 1.48
10.00 1.66
20.00 1.75
40.00 1.28
100.0 1.95
200.0 1.98
400.0 1.78
1000.0 3.67
2000.0 2.59
4000.0 1.79
10000.0 1.67
pg on Column %RSD
0.010 38.92
0.020 6.54
0.040 6.01
0.100 5.60
0.200 4.43
0.400 1.58
1.00 1.44
2.00 1.14
4.00 1.47
10.00 1.88
20.00 1.60
40.00 1.54
100.0 2.51
200.0 1.02
400.0 1.46
1000.0 1.35
2000.0 0.49
4000.0 1.50
10000.0 2.78
pg on Column %RSD
0.010 25.45
0.020 19.69
0.040 9.66
0.100 6.05
0.200 4.31
0.400 3.90
1.00 2.77
2.00 2.12
4.00 2.18
10.00 1.82
20.00 2.63
40.00 2.92
100.0 2.34
200.0 1.04
400.0 1.52
1000.0 1.89
2000.0 0.88
4000.0 2.80
10000.0 1.47
pg on Column %RSD
0.010 32.16
0.020 11.42
0.040 8.56
0.100 3.85
0.200 3.60
0.400 2.88
1.00 3.25
2.00 2.77
4.00 2.69
10.00 2.91
20.00 1.49
40.00 1.26
100.0 1.94
200.0 1.40
400.0 2.86
1000.0 1.37
2000.0 1.16
4000.0 1.41
10000.0 2.54
pg on Column %RSD
0.020 14.22
0.040 8.95
0.100 9.65
0.200 4.75
0.400 3.45
1.00 3.43
2.00 2.22
4.00 1.04
10.00 1.31
20.00 1.30
40.00 1.45
100.0 3.11
200.0 1.69
400.0 0.75
1000.0 3.19
2000.0 2.45
4000.0 1.20
10000.0 1.74
pg on Column %RSD
0.010 20.75
0.020 8.69
0.040 7.21
0.100 4.36
0.200 2.87
0.400 2.34
1.00 1.22
2.00 2.32
4.00 2.14
10.00 2.15
20.00 1.54
40.00 2.13
100.0 2.28
200.0 3.69
400.0 1.76
1000.0 2.63
2000.0 3.94
4000.0 2.72
10000.0 1.93
Terfenadine Y = 0.102234*X R^2 = 0.9951 W: 1/X
0 5000 10000 0
200
400
600
800
1000
Area
Rat
io
Terfenadine Y = 0.00761644+0.102226*X R^2 = 0.9951 W: 1/X
0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.00
0.02
0.04
0.06
0.08
0.10
0.12
Are
a R
atio
C:\j.horner\...\ASMS_VelosPro_Run2_071 4/9/2011 9:37:03 AM 20 pg/uL
ASMS_VelosPro_Run2_071 # 1281 RT: 4.08 AV: 1 NL: 2.01E6 F: ITMS + c ESI Full ms2 [email protected] [125.00-470.00]
150 200 250 300 350 400 450 m/z
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
Inte
nsity
436.15707
454.16705
262.11255 230.19748 187.12711 380.28439 344.28711 289.16522
Velos Pro – Designed For Quantitation
39
Velos Pro: Trap-HCD Fragmentation
Feature Customer Benefit
1 UHPLC Cycle Time and High Data Rates
• More points across a chromatographic peak gives better quantitative performance • More MSN during a run • Now scanning at 66kDa/sec
2 Robust and Sensitive Generation II Ion Optics
• Improved robustness, reduce down time, all on the most sensitive ion trap. • High sensitivity S-Lens technology • Novel beam blocking technology
3 Novel Detection System for superior Quantitation
• Single digit RSDs, up to 6 orders of linear dynamic range on the first ion trap designed for quantitation.
• New EM and electrometer detection system matched to handle the ultra-high peak currents from the VELOS trap.
4 Trap-HCD Fragmentation • Increases flexibility for structural elucidation • Trap-HCD fragmentation yields unique product ions for structural determinations.
40
The Collision Cell You Never Knew You Had!
TRAP-HCD 1. Isolate parent ions in the HPC 2. Pass the ions back to the Q00 (100mTorr) at high energy. 3. Send fragments back to the Dual pressure trap for analysis.
41
connection to Exit Lens and Lens 0 Replace Square Quadrupole with Octopole for Q00
Advantages: • Higher Ion Capacity • Lower Mass Discrimination
42
Trap-HCD Technology
In General:
• CID is the most sensitive and has a wide range of efficiency.
• PQD is selective and has a very narrow optimum.
• Trap-HCD is typically ½ as efficient as CID. 200 400 600 800 1000
0.0
0.2
0.4
0.6
0.8
1.0
b) Transfer ITMS to MP00
Norm
alize
d In
tens
ity
Transfer Time (µs)200 300 400 500
0.0
0.2
0.4
0.6
0.8
1.0
Transfer MP00 to ITMS 350 C 300 C 250 C
Norm
alize
d In
tens
ity
Transfer Time (µs)
a)
The transfer of ions from the Ion Trap to the Octopole and back is fast and efficient.
CID (10 ms) NL: 9.41E4
PQD (0.1 ms) NL: 6.60E3
HCD (3 ms) NL: 6.65E3
43
Trap-HCD vs TSQ fragmentation
Velos Pro TSQ Vantage
40 60 80 100 120 140 160 180 200 220
m/z
HCD of Caffeine at Various Collision Energies on LTQ Velos Pro
Nor
mal
ized
Inte
nsity
% CE 75 65 45 35 25
40 60 80 100 120 140 160 180 200 220
m/z
Offset (V) 30 25 20 15 10
Nor
mal
ized
Inte
nsity
HCD of Caffeine at Various Collision Energies on TSQ Vantage
44
200 400 600 800 1000 1200 1400 1600 1800 2000 2200
0
100
200
300
400
5000
100
200
300
400
5000
100
200
300
400
500200 400 600 800 1000 1200 1400 1600 1800 2000 2200
Inte
nsity
Mass
CID
Inte
nsity
PQD
Inte
nsity
fHCD
MS/MS of m/z 1049 3+
Protein Quantitation – TMT Labeled Peptides
1. TMT intensities dramatically increased 2. Better ion statistics = better quantitation.
124 126 128 130 132 134
0
20
40
60
80
100
120
140
Inte
nsity
Mass
HCD PQD
45
Trap HCD – TMT Labeled Peptide Comparison
200 400 600 800 1000 1200
m/z
rCID PQD HCD QqQ
Nor
mal
ized
Inte
nsity
125 126 127 128 129 130 131 132 1330.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Norm
alize
d In
tens
ity
m/z
QqQ HCD
Compare Reporter Ion Regions for QqQ and Trap HCD Spectra
Compare MS/MS Activation Methods m/z 620 2+
Expected Ratio 10:4:1:1:4:10
46
Small Molecule Activation Comparison
100 150 200 250 m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
243.17
220.92 182.08
CID
50 100 150 200 250 m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
288.25
58.08
243.17
182.09
HCD
50 100 150 200 250 m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
288.25
243.09
182.00 226.00
PQD
CID
100 150 200 250 m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
186.08
124.08 156.00 204.00 92.08
HCD
50 100 150 200 250 m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
279.17
124.08
204.00
156.00 213.09 108.00 174.00
PQD
100 150 200 250 m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
279.17
186.00
124.08 156.00 213.08
Zolmitriptan
Sulfamethazine
Velos Pro for Metabolite Structure Identification and Quantitation
Trifluoropirazine Dextromethorphan
48
New Fragmentation Possibilities
Multiple Fragmentation Techniques
MSn and Fragment Heritage
Velos Pro:
Ability to use CID and HCD
combinations at multiple
different MSn levels.
LC-MS: Linear 10 minute gradient of Water:ACN w/ 0.1% Formic. Hypersil Gold aQ 50X2 1.9µ column 1.Full scan + CID only MSn 2.Full Scan + HCD MS2 + CID MS3 3.Full Scan + HCD MS2 + HCD MS3
Sample:
Trifluopirazine metabolites from
human liver S9 incubation
49
New Fragmentation Possibilities – HCD, CID MSn
Trifluopirazine_CID-CID #2411 RT: 6.50 AV: 1 NL: 2.07E4T: ITMS + p ESI d w Full ms2 [email protected] [100.00-420.00]
50 100 150 200 250 300 350 400m/z
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
Inten
sity
141.1
368.3
280.1 308.2348.3
262.0139.1 408.4340.2230.1210.2147.0
Trifluopirazine_CID-CID #2412 RT: 6.50 AV: 1 NL: 1.47E4T: ITMS + c ESI d w Full ms3 [email protected] [email protected] [50.00-155.00]
50 100 150 200 250 300 350 400m/z
0
2000
4000
6000
8000
10000
12000
14000
Inten
sity
113.1
70.298.2 113.9 146.2
Trifluopirazine CID MS2
408
Trifluopirazine CID MS3
408 141
Trifluopirazine_HCD-HCD #2817 RT: 6.43 AV: 1 NL: 1.95E5T: ITMS + p ESI d w Full ms2 [email protected] [50.00-420.00]
50 100 150 200 250 300 350 400m/z
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
Inten
sity
113.1
280.3
141.2
70.1
248.398.2 308.3
408.4239.384.1 368.4211.1 348.6184.9
Trifluopirazine HCD MS2
408 HCD fragmentation provides a
broader mass range of fragments. CID fragmentation provides similar
fragment detail with additional MSn steps.
Mass Frontier provides automated annotation of fragmentation spectra facilitating structure elucidation
Automatically assembled MSn Trees
Automatic annotation of fragmentatio
n
50
MSn Quantitation – Ultimate Selectivity in Real Matrix
MSn Quantitation
Ultimate Selectivity
Velos Pro:
Robust MSn fragmentation
and multiple fragmentation
techniques.
LC-MS: Linear 5 minute gradient of Water:MeOH w/ 0.1% Formic. Hypersil Gold aQ 50X2 1.9µ column CID (258) CID (210) MS3
Sample:
Human plasma standards with
Dextromethorphan and
Metabolites.
m/z 201 m/z 258
51
RT: 0.90 - 4.13 SM: 7G
1.0 1.5 2.0 2.5 3.0 3.5 4.0Time (min)
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
2.65
MS3 458 201 133
MSn Quantitation – Ultimate Selectivity in Real Matrix
MS2 458 201
Std 2 100 fg/µL 5 µL inj. Plasma • Without using MS3, detection and quantitation was not possible below 0.1 fg/µL.
• With MS3, quantitation was possible down to 0.01 fg/µL and detection possible below that.
• Triplicate Standard curves were prepared and analyzed, the limit of Quantitation and Detection as well as the relative standard deviation (RSD) are shown below.
Std pg/µL MS2 MS3
1000 8% 5%
100 5% 4%
10 4% 7%
1 11% 8%
0.1 LOQ/LOD, 12% 14%
0.01 X LOQ, 10%
0.005 X LOD
RT: 0.87 - 4.13 SM: 7G
1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time (min)
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
2.65
Velos Pro in Proteomics Discovery and Relative Quantitation
C. elegans Tandem Mass Tags
53
Proteomics Discovery Experiments
CID HCD
Velos Pro Benefits for Proteomics Discovery Experiments
• Rapid Scanning • Improves total numbers of proteins and
peptides identified • HCD
• Improves total numbers of proteins and peptides identified
• Low m/z fragment ions can confirm terminal residue identification
5816 6203
7477 8108
0
1500
3000
4500
6000
7500
9000
CID Normal Scan Rate
CID Rapid Scan Rate
HCD Normal Scan Rate
HCD Rapid Scan Rate
1 ug C. elegans loaded, 90 min gradient Multiconcensus Mascot 1%FDR
20%
Proteome Discoverer 1.3 CID/HCD Venn Diagram C. elegans
Peptides
CID
HCD
54
Proteomics Relative Quantitation with TMT
Velos Pro HCD gives: 1. Dramatic increase in TMT intensities 2. Better ion statistics = Better quantitation. 3. Low m/z sequence ions 4. Increased peptide identifications
TMT-HCD Experiments on Velos Pro • 12 protein mixture digest • Each peptide labeled with TMT 6-plex at 1:1:1:1:1:1 or
1:10:1:10:1:10 • 500 fmol each or 50:500 fmol loaded on column • 90 minute gradient 5% 30% ACN • Top 10 data dependent Trap-HCD, dynamic exclusion
repeat count 1
Results of Trap-HCD-TMT Experiments for 1:1:1:1:1:1 Labeled Peptides
Peptide Spectral Matches 4363
Number of Peptides Quantified 3269
Percent of Identified Peptides Quantifiable 75%
Average Protein Level CV 2.5%
HLVDEPQNLIK
C:\j.horner\...\fHCD-TMT-3-4-11_run21 fHCD 50% NCE TMT 500f 12-Prot equalM-top10-15sec-2 uscan
fHCD-TMT-3-4-11_run21 # 13182 RT: 44.24 AV: 1 NL: 2.46E4 T: ITMS + c NSI d Full ms2 [email protected] [50.00-1780.00]
124 126 128 130 132 134
m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
130.46 129.48 131.48 128.48 127.45 126.47
132.55 133.47
Proteome Discoverer 1.3
Velos Pro for Clinical Screening and Quantitative Analyses
Vitamin D2 and D3 Opiates
56
Vitamin D Standard Curves
Vitamin D2 Conc.
(ng/mL) %
Difference
3.3 1.54
7.5 -3.33
14.1 -0.24
29.7 -1.47
51.2 3.50
Y = 0.0978239+0.0818128*X R^2 = 0.9988 W: 1/X^2
0 5 10 15 20 25 30 35 40 45 50 55
ng/mL 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 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8
Are
a R
atio
Vitamin D2 & D3 Analysis Sample Preparation
LLE of plasma samples done at collaborator site
Chromatography Accela Open AS and Accela 1250 uHPLC pump Gradient and column duplicated from Collaborator Lab
Source APCI source conditions optimized for best signal to noise
Mass Spectrometry 3 Full Scan MS3 scan events
Data Processing All samples processed using LCQuan 2.6.1 Patient Sample results compared to Collaborator Lab
Y = 0.0421947+0.0442916*X R^2 = 0.9944 W: 1/X^2
0 5 10 15 20 25 30 35 40 45 50 55 ng/mL
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
Are
a R
atio
Vitamin D3 Conc.
(ng/mL) %
Difference
2.6 -3.10
5.3 6.49
12.0 -0.93
25.5 4.98
50.3 -7.43
Vitamin D3
Vitamin D2
57
Vitamin D3 23.6 ng/mL in plasma
0
10
20
30
40
50
60
70
80
90
100 2.59
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
257.20
201.17
147.10 239.17
NL: 1.62E4 P-45_082211#724 AV: 1 F: ITMS + c APCI corona Full ms3 [email protected] [email protected] [140.00-280.00]
Vitamin D Analysis in Patient Plasma Samples
0.000
10.000
20.000
30.000
40.000
50.000
60.000
0 10 20 30 40 50 60
Ther
mo
Fish
er V
elos
Pro
SF General Hospital
0.000
10.000
20.000
30.000
40.000
50.000
60.000
0 10 20 30 40 50 60
Ther
mo
Fish
er V
elos
Pro
SF General Hospital
MS3 Extracted Ion Chromatograms P45 Vitamin D2
Vitamin D3
Vitamin D2 1.0 ng/mL in plasma
0 1 2 3 4 5 Time (min)
0
10
20
30
40
50
60
70
80
90
100 2.63
150 200 250 m/z
0
10
20
30
40
50
60
70
80
90
100 251.19
269.26 199.20
NL: 2.41E3 P-45_082211#740 AV: 1 F: ITMS + c APCI corona Full ms3 [email protected] [email protected] [140.00-280.00]
58
Opiates Analysis • Sample Preparation
• 12 Opiates • 40X diluted urine
• Chromatography • Accela Open AS and Accela 1250 uHPLC
pump • 5 minute chromatographic method
• Source • Heated ESI
• Mass Spectrometry • 8 MS2 scan events, 2 MS3 scan events • Segmented method
• Data Processing • LC Quan 2.6.1
Morphine Full MS2 m/z 286.1
80 100 120 140 160 180 200 220 240 260 280 300 m/z
0 10 20 30 40 50 60 70 80 90
100
Rel
ativ
e A
bund
ance
229.00
268.09 211.00
183.08 286.09 173.00 239.08 147.00
255.09 123.00
201.08
Morphine C17H19NO3
Hydromorphone C17H19NO3
80 100 120 140 160 180 200 220 240 260 280 300 m/z
0 10 20 30 40 50 60 70 80 90
100 R
elat
ive
Abu
ndan
ce
185.00
229.00 211.00
243.08 286.09
183.08 199.08 268.09
Hydromorphone Full MS2 m/z 286.1
201.08
185.00
59
Alere Opiates Analysis Optimize Fragmentation for Best Sensitivity and Selectivity
Oxymorphone Fragmentation by CID, HCD
100 150 200 250 300 m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
284.09
MS2 CID @ 35%
302 [80-310]
100 150 200 250 300 m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
284.09
302.09 242.08 227.00
199.08 266.09 173.00 161.00
MS2 Wideband CID @ 28%
302 [80-310]
100 150 200 250 300 m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
284.00
227.00
242.00
302.00 256.00 199.00 161.00 188.00
MS2 HCD @ 40%
302 [80-310]
100 150 200 250 300 m/z
0
10
20
30
40
50
60
70
80
90
100 R
elat
ive
Abun
danc
e 242.08
227.00
266.09 199.08 173.00
224.08 256.09 284.09 161.00 209.00 188.08
MS3 CID @ 30%
302 284 [80-294]
• Sample Preparation • 12 Opiates • 40X diluted urine
• Chromatography • Accela Open AS and Accela 1250 uHPLC
pump • 5 minute chromatographic method
• Source • Heated ESI
• Mass Spectrometry • 8 MS2 scan events, 2 MS3 scan events • Segmented method
• Data Processing • LC Quan 2.6.1
Oxymorphone C17H19NO4
60
Alere Opiates Analysis
• Velos Pro sensitivity and scan speed are sufficient for Opiates screening and quantitation.
• Flexibility in selection of fragmentation affords highest sensitivity.
• Limits of quantification for all opiates < 10 ng/mL (estimated from S/N for low levels)
• Percent difference between known and calculated levels <=5% for all compounds.
Time (min)
0
20
40
60
80
100 0
20
40
60
80
100 0
20
40
60
80
100
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100 0
20
40
60
80
100
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
1.70
1.70
2.06
2.05
1.93
1.92
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Time (min)
0
20
40
60
80
100 0
20
40
60
80
100 0
20
40
60
80
100
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100 0
20
40
60
80
100 2.38
2.37
2.73
2.72
2.67
2.66
Morphine
Morphine-d6
Hydromorphone
Hydromorphone-d3
Oxymorphone
Oxymorphone-d3
Codeine
Codeine-d6
Hydrocodone
Hydrocodone-d6
Oxycodone
Oxycodone-d6
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0 50 100 150 200 250 300
Peak
Are
a Ra
tio
ng/mL in Urine
Morphine Hydromorphone Oxymorphone Codeine Hydrocodone Oxycodone
61
Alere Opiates Analysis
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0 50 100 150 200 250 300
Peak
Are
a Ra
tio
ng/mL in Urine
Morphine Hydromorphone Oxymorphone Codeine Hydrocodone Oxycodone
Compound Cal 1 Cal 2 Cal 3 Cal 4
50 ng/mL 75 ng/mL 150 ng/mL 300 ng/mL
Morphine
Average % Difference -3.98% -2.45% 0.91% 0.69%
%RSD 2.40% 4.9 3.1 3.2
Hydromorphone
Average % Difference -3.10% -5.43 -2.5 3.02
%RSD 4.40% 6.70% 4.30% 3.50%
Oxymorphone
Average % Difference -3.63 -4.87 -1.48 2.44
%RSD 5.20% 4.00% 1.40% 2.50%
Codeine
Average % Difference -0.09 -3.12 -5.05 3.32
%RSD 4.90% 5.50% 2.60% 2.30%
Hydrocodone
Average % Difference -1.83% -1.21% 0.18% 0.46%
%RSD 1.70% 4.80% 1.80% 3.80%
Oxycodone
Average % Difference 4.90% -1.86% -0.63% 0.12%
%RSD 3.40% 5.10% 2.40% 2.50%
• Velos Pro sensitivity and scan speed are sufficient for Opiates screening and quantitation.
• Flexibility in selection of fragmentation affords highest sensitivity.
• Limits of quantification for all opiates < 10 ng/mL (estimated from S/N for low levels)
• Percent difference between known and calculated levels <=5% for all compounds.
62
Velos Pro LC-MSn
Velos Pro LC-MSn
Accurate •All background ions are rejected – minimal space charge issues – maximum accuracy
Precise •Fast scanning and novel detection system yield single digit RSD’s at UHPLC speeds
Sensitive •HESI-II , S-LENS, and Dual pressure trap give superb low femtogram sensitivities with FULL SCAN product ion information
Robust •Generation II ion optics produce a robust and stable signal.
Versatile •CID, Trap-HCD, PQD, and ETD for maximum information from every sample.
