New Objective, Inc., Woburn, MA USA · • Emitter: uncoated, 360 µm OD x 20 µm ID x 10 µm tip...
Transcript of New Objective, Inc., Woburn, MA USA · • Emitter: uncoated, 360 µm OD x 20 µm ID x 10 µm tip...
©2013 New Objective, Inc. All rights reserved. Trademarks are the properties of their respective companies.
IntroductionNanospray has become an essential tool in high-sensitivity MS, but limited robustness and reproducibility have historically challenged the adoption of nanospray in quantitative applications with triple quadrupole mass spectrometry. MS-based biomarker quantitation places strict requirements on the analytical performance of nanobore LC-MS not only in the need for reproducibility and robustness but also due to the intrinsically complex nature of the samples used in these workflows. Automated emitter positioning with rinsing has been previously demonstrated to improve spray stability and analyte response on a 3D ion trap MS1. Here we investigate the utility of automated tip rinsing to improve emitter spray stability and data quality on a hybrid triple quadrupole/LIT MS equipped with a heated interface blanketed by a laminar flow of nitrogen gas.
Methods - QQQ/LITInstrumentation
• Mass Spectrometer: 4000 Q TRAP (AB SCIEX)
- Scan Settings: Q1 MS Scan; 400 – 1000 Da; profile mode; unit resolution
- Compound parameters: Declustering potential – 70; Entrance potential - 10
- Source/Gas parameters: Curtain gas - 10.0; Ion spray voltage - 2200 kV, Ion source gas - 3.0; Interface heater temperature -150°C
• Source: Digital PicoView DPV-450 nanospray source (New Objective, Inc.)
• Emitter: uncoated, 360 µm OD x 20 µm ID x 10 µm tip (New Objective, Inc.)
• HPLC: Eksigent nanoLC·2D (AB SCIEX)
• Autosampler: HTC PAL (Leap Technologies)
Reagents
• Tip Rinse: 50% water/50% methanol, gravity flow (~50 µL/min.)
• Mobile Phase A: 0.1% formic acid in water (JT Baker)
• Mobile Phase B: 0.1 formic acid in acetonitrile (JT Baker)
• Peptides: Angiotensin I, Angiotensin II, [Glu1]-Fibrinopeptide B, Insulin Chain B (Sigma-Aldrich)
Flow Injection
• Flow Rate: 300 nl/min.; 90% mobile phase A/10% mobile phase B
• Analyte: 500 fmol/µL 4 peptide mixture in 70% mobile phase A; 30% mobile phase B
• Injection: 1 µL loop
Instrumentation• Ion-trap mass spectrometer equipped with Harvard
Biosciences syringe pump (LCQ Deca, Thermo Scientific)• Nanospray source (Digital PicoView 150, New Objective)• 1100 Capillary pump operated in Normal Mode (Agilent)• PicoFrit emitter (360 μm OD, 75 μm ID, 15 μm tip ID,
New Objective) Reagents• 0.1% Formic acid in water (J.T. Baker)• 0.1% Formic acid in acetonitrile (J.T. Baker)• Methanol (J.T. Baker)• 250 ng/µL Caffeine, MW 194.19 Da (Sigma-Aldrich)• 5 pmol/µL Bradykinin fragment 1-7, MW 756.39 Da
(Sigma-Aldrich)• 5 pmol/µL Angiotensin I, MW 1296.48 Da (Sigma-Aldrich)• 5 pmol/µL Neurotensin, MW 1672.92 Da (Sigma-Aldrich)• tert-Butyl methyl ether (MTBE, Sigma-Aldrich)• Liquid-liquid extracted canine plasma with heparin (Harlan Bioproducts)
Conclusions• Demonstrated benefits of automated tip rinsing on two different instrument platforms
• Validated regular monitoring of TIC RSD as a good indicator of system performance
• Observed finite and variable analyte recovery and emitter performance when regular automated tip rinsing is not enabled
• Enabled robustly stable nanospray and reproducible data using remote controlled automated tip rinsing at regular intervals
Evaluating Nanospray Ruggedness on a Curtain Gas-Triple Quadrupole MS Equipped with Emitter RinsingAmanda Berg, Helena Svobodova, Ben Ngo, Gary Valaskovic
New Objective, Inc., Woburn, MA USA
Digital PicoView DPV-450 nano-spray source installed on an AB SCIEX 4000 Q TRAP instrument.
PV Acquire v2.0.0 software package which accompanies the Digital PicoView system. The automated Digital Divert mode is active, as shown in the image.
No Rinse
Rinse
4.00E+06
2.40E+07
4.40E+07
6.40E+07
8.40E+07
1.04E+08
0 50 100 150 200 250 300 350 400 450
Aver
age
TIC
Injection Number
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
35.00%
40.00%
45.00%
0 50 100 150 200 250 300 350 400
TIC
RSD
Injection Number
No Rinse
Rinse
0.00E+00
2.00E+07
4.00E+07
6.00E+07
8.00E+07
1.00E+08
1.20E+08
1.40E+08
1.60E+08
Neurotensin: No rinsing
Caffeine: No rinsing
With rinsing
With rinsing
1 50 100 150 200 250 300 350 400 450 500 550
Number of Injections
Ave
rage
Inte
nsity
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Time, min
0.0
5.0e6
1.0e7
1.5e7
2.0e7
2.5e7
3.0e7
3.5e7
4.0e7
4.5e7
5.0e7
5.5e7
6.0e7
6.5e7
7.0e7
7.5e7
8.0e7
8.5e7
8.9e7
Inte
nsity
, cps
4.07 4.23
3.79
3.65
0.65 1.0710.127.95 12.02 14.64 14.97
400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, amu
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.1e5
Inte
nsity
, cps
433.5
786.5
445.5
880.8
875.3
524.3
705.3884.8
429.6 519.6467.6 483.5 525.2430.6 532.7 649.4409.6 458.7 708.1797.4683.9676.6469.5 537.4 886.6442.7 503.5412.5 712.8655.8557.6 611.8 721.9 805.9805.4784.7593.6 679.9475.5 529.8 894.8863.1 968.9489.4 900.3853.8405.8 944.9752.7 800.6 835.7 990.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Time, min
0.00
5.00e6
1.00e7
1.50e7
2.00e7
2.50e7
3.00e7
3.50e7
4.00e7
4.50e7
5.00e7
5.50e7
6.00e7
6.50e7
7.00e7
7.50e7
8.00e7
8.50e7
9.00e7
9.50e7
1.00e8
1.05e8
1.10e8
1.15e8
1.20e8
Inte
nsity
, cps
4.604.14
4.05
3.67
3.34
5.42
5.912.29 7.27
8.581.67 16.5313.6910.089.58 11.32 19.4018.270.46 15.73 17.4912.13
400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, amu
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
4.0e5
4.2e5
4.4e5
4.6e5
4.8e5
Inte
nsity
, cps
433.4
786.3
705.4
880.9
875.0524.3445.4 711.6 884.5427.6
708.1649.8
676.6413.3 446.4483.7 721.9655.9437.6 671.7 797.9467.4 513.8 647.6463.7 532.6 595.8 788.5425.7 693.7 885.8714.3662.1 805.1557.6 687.0457.5 739.0 876.6 894.6642.8 857.4507.8 527.6 782.1 989.9616.3 960.0495.5 930.4545.8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Time, min
0.0
5.0e6
1.0e7
1.5e7
2.0e7
2.5e7
3.0e7
3.5e7
4.0e7
4.5e7
5.0e7
5.5e7
6.0e7
6.5e7
7.0e7
7.5e7
8.0e7
8.5e7
9.0e79.2e7
Inte
nsity
, cps
4.544.32
3.89
3.77
3.57
8.730.70 10.50 11.61 13.81
400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, amu
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e5
8.1e5
Inte
nsity
, cps
875.3
786.5433.5
445.4
884.8
881.0
705.3524.2
519.6
429.5 483.3711.2513.6
888.8464.3 532.7430.4409.6708.2676.9557.5503.6 886.3649.4401.6 467.4453.4 534.6 894.4593.6 805.4 857.5718.8 784.8777.4611.7 684.7560.5 728.1 944.8477.6 968.9816.2 870.7 976.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Time, min
0.0
2.0e6
4.0e6
6.0e6
8.0e6
1.0e7
1.2e7
1.4e7
1.6e7
1.8e7
2.0e7
2.2e7
2.4e7
2.6e7
2.8e7
3.0e7
3.2e7
3.4e73.5e7
Inte
nsity
, cps
4.044.28
3.774.81
3.44
5.42
3.27
18.122.802.31 12.66 19.7717.5515.7414.666.370.42 0.84 13.1812.55 18.798.98 10.7610.08 16.557.34 7.97
1.36
400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, amu
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
2.0e5
2.1e5
2.2e5
2.3e5
2.4e5
2.5e5
2.6e5
2.7e5
Inte
nsity
, cps
433.4
786.4
445.5
875.6
524.4
705.4880.8
513.8676.7 885.1483.6 649.3429.3 798.2519.5 710.5534.6464.3409.5 458.4 557.6
453.4 707.9647.9415.5 554.1 778.2718.7593.6 784.9421.9 806.7632.7 882.6489.1 588.0503.6 889.0669.4 766.5 968.7944.9627.5 680.1470.2 858.5 923.5769.3 826.7 989.9
Results With Emitter RinsingEffects of Positioning on Signal Results Without Emitter Rinsing Ion Trap Methods & Results
A - START A - STARTB - FINISH B - FINISHC - EMITTER C - EMITTER
Host PC
XYZ Stage
Rinsing station with catch basin
Massspec
LC pump
Host PC
XYZ Stage
Rinsing station with catch basin
Massspec
LC pumpA
B
A) System schematic during run (spraying) B) Picture of emitter position relative to curtain plate inlet on instrument during data collec-tion.
Host PC
XYZ Stage
Rinsing station with catch basin
Massspec
LC pump
Host PC
XYZ Stage
Rinsing station with catch basin
Massspec
LC pump
A
B
A) System schematic between injections (not spraying). A contact closure from the LC trig-gers the movement of the XYZ stage to the rinse station position. B) Picture of emitter di-verted from the inlet and parked under rinse station in between injections.
A) Spray image collected at injection 20; B) Spray image collected at injection 420; C) Photo of emitter at 32X magnification taken after 420 injections (140 hours run time)
The automated XYZ stage and software control of Digital PicoView enables facile tuning for optimal signal and spray stability on the 4000 QTrap interface.
A) Spray image collected at injection 1; B) Spray image collected at injection 420; C) Photo of emitter at 32X magnification taken after 418 injections (139 hours run time)
Plot of average total ion current (TIC) over 418 replicate injections for two data sets: with tip rinsing and without tip rinsing. The average total ion current was calculated for a 0.5-minute time segment (4.0 – 4.5 minutes).
Plot of total ion current relative standard deviation (RSD) over 418 replicate injections for two data sets: with tip rinsing and without tip rinsing.
SPRAY INSTABILITY The TIC RSD showed a change beginning at injection 252 reflecting an increase in spray instability. The autosampler was injecting air. Spray stability was restored, as indicated by the TIC RSD, once the air was flushed from the system.
INJECTION #30Total ion current and spectrum
INJECTION #20Total ion current and spectrum
INJECTION #418Total ion current and spectrum
INJECTION #420Total ion current and spectrum
Photo of PicoFrit emitter taken after 650 injections without tip rinsing using op-tical microscopy
Photo of PicoFrit emitter taken after 650 injections with rinsing using optical microscopy.
Left: Comparative data plot of average intensity per injection for two data sets, one collected with rinsing and one without. The average intensity for two differ-ent ions, neurotensin MH2+, 837.2 Da and caffeine MH+, 195.2 Da, is plotted for each data set.
400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, amu
0.00
5.00e4
1.00e5
1.50e5
2.00e5
2.50e5
3.00e5
3.50e5
4.00e5
4.50e5
5.00e5
5.50e5
6.00e5
6.50e5
7.00e5
7.50e5
8.00e5
8.50e5
9.00e5
9.50e5
1.00e6
1.05e6
1.10e6
1.15e6
1.20e6
1.25e6
1.30e6
Inte
nsity
, cps
445.4
447.4
786.5483.5
453.5409.5 429.4
519.6484.5 787.3
437.6 537.5413.5 532.5 557.7 711.8 739.9593.4 797.2639.8 677.7463.7 633.6 805.3487.0 765.9 854.1 898.2875.7 944.6 986.1924.2 966.2
400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, amu
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
4.0e5
4.2e5
4.4e5
4.6e5
4.8e5
5.0e5
Inte
nsity
, cps
786.5
409.4
483.7 712.1453.6 524.6
532.6
467.5415.7 739.9445.4697.1 875.5766.0
470.7429.6722.0 731.5557.3423.4 475.8 507.4 823.9515.7 655.9 788.0421.6 638.0 668.4585.9455.3 761.2617.8 896.1708.0 846.9479.6 981.8549.8 884.9 954.5491.2 529.8 749.3651.8 932.2800.0 835.8440.8408.3 663.7628.6574.0471.7 779.1701.9 899.1592.6 849.0769.6
400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, amu
0.0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
1.0e6
1.1e6
1.2e6
1.3e6
1.4e6
1.5e6
1.6e6
Inte
nsity
, cps
445.4
786.5483.4
409.4453.5
429.5 467.6
519.5454.6 469.5437.6 524.8 532.1 740.0557.7 711.9 797.7593.6 683.8 771.8 805.5640.1 677.8633.8507.7481.7432.4 853.9 898.2874.9 926.0 944.7 970.2
Position 1
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min
0.0
5.0e6
1.0e7
1.5e7
2.0e7
2.5e7
3.0e7
3.5e7
4.0e7
4.5e7
5.0e7
5.5e7
6.0e7
6.5e7
7.0e7
7.5e7
8.0e7
8.4e7
Inte
nsity
, cps
4.09
3.092.12
1.14
3.72
1.86
Position #1
Position #3
Position #4
Position #5Position #7
Position #8
Position #9
Position #6
Position #2Po
sitio
n 1
Posi
tion
4
Posi
tion
9
Position 4
Position 9
Stable spray at beginning of run
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)
6.325.493.592.30 3.553.132.111.93 6.085.274.901.78 5.12 6.473.79 4.663.85 6.75 6.904.602.471.70 4.00 6.930.91 6.030.750.650.50 0.970.04
5.69 5.954.162.72 2.951.41 4.39 7.19 7.341.19
NL: 3.52E9
NL: 1.67E8
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
558.59
433.13195.19
837.19649.00 757.41
559.40 649.72 758.44 837.92725.15408.12 433.94 571.19319.65196.13 660.98 1296.55513.25 784.40 856.90257.60 1036.46 1334.531000.48 1139.07 1253.11 1382.18 1496.34
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)
1.534.503.04
6.00
4.72 4.874.900.06 02.693.53.31 3.85 5.05 5.482.250.92 6.932.21 6.616.440.17 3.480.37 1.86 3.10 3.982.491.820.820.65 6.833.792.170.95
6.98
5.894.432.92 5.704.28 7.192.66 7.341.05 1.23
NL: 1.26E9
NL: 3.32E7
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
558.63
433.14
195.20648.99 837.21
757.42559.46 838.05649.77433.90 758.43725.1622.17562.973 849.03196.17 555.26 1296.58876.97319.71238.13 1036.35795.16 1320.42999.99 1065.63 1139.58 1201.38 1384.35 1451.03
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)
6.325.493.592.30 3.553.132.111.93 6.085.274.901.78 5.12 6.473.79 4.663.85 6.75 6.904.602.471.70 4.00 6.930.91 6.030.750.650.50 0.970.04
5.69 5.954.162.72 2.951.41 4.39 7.19 7.341.19
NL: 3.52E9
NL: 1.67E8
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
558.59
433.13195.19
837.19649.00 757.41
559.40 649.72 758.44 837.92725.15408.12 433.94 571.19319.65196.13 660.98 1296.55513.25 784.40 856.90257.60 1036.46 1334.531000.48 1139.07 1253.11 1382.18 1496.34
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)
1.534.503.04
6.00
4.72 4.874.900.06 02.693.53.31 3.85 5.05 5.482.250.92 6.932.21 6.616.440.17 3.480.37 1.86 3.10 3.982.491.820.820.65 6.833.792.170.95
6.98
5.894.432.92 5.704.28 7.192.66 7.341.05 1.23
NL: 1.26E9
NL: 3.32E7
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
558.63
433.14
195.20648.99 837.21
757.42559.46 838.05649.77433.90 758.43725.1622.17562.973 849.03196.17 555.26 1296.58876.97319.71238.13 1036.35795.16 1320.42999.99 1065.63 1139.58 1201.38 1384.35 1451.03
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)
6.193.19 4.680.15
4.72 6.271.67 3.341.770.18 4.85 6.876.705.47 6.313.713.500.37 5.241.92 2.39 6.962.24 3.770.71 3.900.89 0.992.50
7.0916.551.41.15 5.8766.254.1 7.204.382.77
NL: 1.88E9
NL: 1.10E8
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
558.60
433.12
195.19
837.19648.98757.40
559.35 837.97649.64 758.43725.20643.48433.88379.27 848.93196.17 1296.54319.64 876.98555.22 1036.40254.99 785.30 927.60 1334.331096.57 1249.311189.04 1454.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)
4.686.203.22 6.982.210.11 0.62 4.901.68 3.35 5.15 6.550.69 3.823.500.15 6.405.30 6.872.47 5.490.32 1.76 3.972.060.97
83.459.216.224.1 7.345.694.15 7.191.31 5.95
NL: 1.72E9
NL: 1.10E8
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
558.61
433.13
195.18837.19648.99 757.42
559.42 837.97649.67 758.39643.50433.87 725.13379.20 856.33 1296.56196.16 513.21343.11238.08 897.65 1028.32785.48 1074.82 1320.481187.70979.35 1394.80 1447.86
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)
6.193.19 4.680.15
4.72 6.271.67 3.341.770.18 4.85 6.876.705.47 6.313.713.500.37 5.241.92 2.39 6.962.24 3.770.71 3.900.89 0.992.50
7.0916.551.41.15 5.8766.254.1 7.204.382.77
NL: 1.88E9
NL: 1.10E8
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
558.60
433.12
195.19
837.19648.98757.40
559.35 837.97649.64 758.43725.20643.48433.88379.27 848.93196.17 1296.54319.64 876.98555.22 1036.40254.99 785.30 927.60 1334.331096.57 1249.311189.04 1454.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)
4.686.203.22 6.982.210.11 0.62 4.901.68 3.35 5.15 6.550.69 3.823.500.15 6.405.30 6.872.47 5.490.32 1.76 3.972.060.97
83.459.216.224.1 7.345.694.15 7.191.31 5.95
NL: 1.72E9
NL: 1.10E8
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
558.61
433.13
195.18837.19648.99 757.42
559.42 837.97649.67 758.39643.50433.87 725.13379.20 856.33 1296.56196.16 513.21343.11238.08 897.65 1028.32785.48 1074.82 1320.481187.70979.35 1394.80 1447.86
WITH RINSING
Inje
ctio
n #1
Inje
ctio
n #6
50
WITHOUT RINSING
1. Berg, AL; Marshall-Waggett, CJ; Valaskovic, GA; Proceedings of the 57th Conference on Mass Spectrometry and Allied Topics, Philadelphia, PA, May 29 - June 5, 2009
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