New Objective, Inc., Woburn, MA USA · • Emitter: uncoated, 360 µm OD x 20 µm ID x 10 µm tip...

1
©2013 New Objective, Inc. All rights reserved. Trademarks are the properties of their respective companies. Introduction Nanospray 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 MS 1 . 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/LIT Instrumentation 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, [Glu 1 ]-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 Rinsing Amanda 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 Average TIC Injecon 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 Injecon 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 Average Intensity 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time, 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 Intensity, cps 4.07 4.23 3.79 3.65 0.65 1.07 10.12 7.95 12.02 14.64 14.97 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/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 Intensity, cps 433.5 786.5 445.5 880.8 875.3 524.3 705.3 884.8 429.6 519.6 467.6 483.5 525.2 430.6 532.7 649.4 409.6 458.7 708.1 797.4 683.9 676.6 469.5 537.4 886.6 442.7 503.5 412.5 712.8 655.8 557.6 611.8 721.9 805.9 805.4 784.7 593.6 679.9 475.5 529.8 894.8 863.1 968.9 489.4 900.3 853.8 405.8 944.9 752.7 800.6 835.7 990.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time, 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 Intensity, cps 4.60 4.14 4.05 3.67 3.34 5.42 5.91 2.29 7.27 8.58 1.67 16.53 13.69 10.08 9.58 11.32 19.40 18.27 0.46 15.73 17.49 12.13 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/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 Intensity, cps 433.4 786.3 705.4 880.9 875.0 524.3 445.4 711.6 884.5 427.6 708.1 649.8 676.6 413.3 446.4 483.7 721.9 655.9 437.6 671.7 797.9 467.4 513.8 647.6 463.7 532.6 595.8 788.5 425.7 693.7 885.8 714.3 662.1 805.1 557.6 687.0 457.5 739.0 876.6 894.6 642.8 857.4 507.8 527.6 782.1 989.9 616.3 960.0 495.5 930.4 545.8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time, 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 9.0e7 9.2e7 Intensity, cps 4.54 4.32 3.89 3.77 3.57 8.73 0.70 10.50 11.61 13.81 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/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 Intensity, cps 875.3 786.5 433.5 445.4 884.8 881.0 705.3 524.2 519.6 429.5 483.3 711.2 513.6 888.8 464.3 532.7 430.4 409.6 708.2 676.9 557.5 503.6 886.3 649.4 401.6 467.4 453.4 534.6 894.4 593.6 805.4 857.5 718.8 784.8 777.4 611.7 684.7 560.5 728.1 944.8 477.6 968.9 816.2 870.7 976.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time, 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 3.4e7 3.5e7 Intensity, cps 4.04 4.28 3.77 4.81 3.44 5.42 3.27 18.12 2.80 2.31 12.66 19.77 17.55 15.74 14.66 6.37 0.42 0.84 13.18 12.55 18.79 8.98 10.76 10.08 16.55 7.34 7.97 1.36 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/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 Intensity, cps 433.4 786.4 445.5 875.6 524.4 705.4 880.8 513.8 676.7 885.1 483.6 649.3 429.3 798.2 519.5 710.5 534.6 464.3 409.5 458.4 557.6 453.4 707.9 647.9 415.5 554.1 778.2 718.7 593.6 784.9 421.9 806.7 632.7 882.6 489.1 588.0 503.6 889.0 669.4 766.5 968.7 944.9 627.5 680.1 470.2 858.5 923.5 769.3 826.7 989.9 Results With Emitter Rinsing Effects of Positioning on Signal Results Without Emitter Rinsing Ion Trap Methods & Results A - START A - START B - FINISH B - FINISH C - EMITTER C - EMITTER Host PC XYZ Stage Rinsing station with catch basin Mass spec LC pump A 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 Mass spec 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 #30 Total ion current and spectrum INJECTION #20 Total ion current and spectrum INJECTION #418 Total ion current and spectrum INJECTION #420 Total 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 1000 m/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 Intensity, cps 445.4 447.4 786.5 483.5 453.5 409.5 429.4 519.6 484.5 787.3 437.6 537.5 413.5 532.5 557.7 711.8 739.9 593.4 797.2 639.8 677.7 463.7 633.6 805.3 487.0 765.9 854.1 898.2 875.7 944.6 986.1 924.2 966.2 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/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 Intensity, cps 786.5 409.4 483.7 712.1 453.6 524.6 532.6 467.5 415.7 739.9 445.4 697.1 875.5 766.0 470.7 429.6 722.0 731.5 557.3 423.4 475.8 507.4 823.9 515.7 655.9 788.0 421.6 638.0 668.4 585.9 455.3 761.2 617.8 896.1 708.0 846.9 479.6 981.8 549.8 884.9 954.5 491.2 529.8 749.3 651.8 932.2 800.0 835.8 440.8 408.3 663.7 628.6 574.0 471.7 779.1 701.9 899.1 592.6 849.0 769.6 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/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 Intensity, cps 445.4 786.5 483.4 409.4 453.5 429.5 467.6 519.5 454.6 469.5 437.6 524.8 532.1 740.0 557.7 711.9 797.7 593.6 683.8 771.8 805.5 640.1 677.8 633.8 507.7 481.7 432.4 853.9 898.2 874.9 926.0 944.7 970.2 Position 1 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Time, 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 Intensity, cps 4.09 3.09 2.12 1.14 3.72 1.86 Position #1 Position #3 Position #4 Position #5 Position #7 Position #8 Position #9 Position #6 Position #2 Position 1 Position 4 Position 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 7.0 Time (min) 6.32 5.49 3.59 2.30 3.55 3.13 2.11 1.93 6.08 5.27 4.90 1.78 5.12 6.47 3.79 4.66 3.85 6.75 6.90 4.60 2.47 1.70 4.00 6.93 0.91 6.03 0.75 0.65 0.50 0.97 0.04 5.69 5.95 4.16 2.72 2.95 1.41 4.39 7.19 7.34 1.19 NL: 3.52E9 NL: 1.67E8 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z 558.59 433.13 195.19 837.19 649.00 757.41 559.40 649.72 758.44 837.92 725.15 408.12 433.94 571.19 319.65 196.13 660.98 1296.55 513.25 784.40 856.90 257.60 1036.46 1334.53 1000.48 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 7.0 Time (min) 1.53 4.50 3.04 6.00 4.72 4.87 4.90 0.06 0 2 . 6 9 3 . 5 3.31 3.85 5.05 5.48 2.25 0.92 6.93 2.21 6.61 6.44 0.17 3.48 0.37 1.86 3.10 3.98 2.49 1.82 0.82 0.65 6.83 3.79 2.17 0.95 6.98 5.89 4.43 2.92 5.70 4.28 7.19 2.66 7.34 1.05 1.23 NL: 1.26E9 NL: 3.32E7 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z 558.63 433.14 195.20 648.99 837.21 757.42 559.46 838.05 649.77 433.90 758.43 725.16 2 2 . 1 7 5 6 2 . 9 7 3 849.03 196.17 555.26 1296.58 876.97 319.71 238.13 1036.35 795.16 1320.42 999.99 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 7.0 Time (min) 6.19 3.19 4.68 0.15 4.72 6.27 1.67 3.34 1.77 0.18 4.85 6.87 6.70 5.47 6.31 3.71 3.50 0.37 5.24 1.92 2.39 6.96 2.24 3.77 0.71 3.90 0.89 0.99 2.50 7.09 1 6 . 5 5 1 . 4 1.15 5.87 6 6 . 2 5 4 . 1 7.20 4.38 2.77 NL: 1.88E9 NL: 1.10E8 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z 558.60 433.12 195.19 837.19 648.98 757.40 559.35 837.97 649.64 758.43 725.20 643.48 433.88 379.27 848.93 196.17 1296.54 319.64 876.98 555.22 1036.40 254.99 785.30 927.60 1334.33 1096.57 1249.31 1189.04 1454.00 m/z 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 7.0 Time (min) 4.68 6.20 3.22 6.98 2.21 0.11 0.62 4.90 1.68 3.35 5.15 6.55 0.69 3.82 3.50 0.15 6.40 5.30 6.87 2.47 5.49 0.32 1.76 3.97 2.06 0.97 8 3 . 4 5 9 . 2 1 6 . 2 2 4 . 1 7.34 5.69 4.15 7.19 1.31 5.95 NL: 1.72E9 NL: 1.10E8 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z 558.61 433.13 195.18 837.19 648.99 757.42 559.42 837.97 649.67 758.39 643.50 433.87 725.13 379.20 856.33 1296.56 196.16 513.21 343.11 238.08 897.65 1028.32 785.48 1074.82 1320.48 1187.70 979.35 1394.80 1447.86 WITH RINSING Injection #1 Injection #650 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 1mm 1mm 1mm 1mm 1mm 1mm 1mm 1mm

Transcript of New Objective, Inc., Woburn, MA USA · • Emitter: uncoated, 360 µm OD x 20 µm ID x 10 µm tip...

Page 1: New Objective, Inc., Woburn, MA USA · • Emitter: uncoated, 360 µm OD x 20 µm ID x 10 µm tip (New Objective, Inc.) • HPLC: Eksigent nanoLC·2D (AB SCIEX) • Autosampler: HTC

©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

1mm

1mm1mm

1mm

1mm

1mm

1mm

1mm