Untargeted Screening on the X500R QTOF R. E. Haufler, Paul Winkler, Andrei Schreiber, SCIEX, Concord, ON Canada NEMC August 2016 RUO-MKT-11-4397
Outline X500R technology and performance X500R QTOF Scan modes Application Examples 2 2016 AB Sciex
X500R Technology and Performance
Super simplified TOF At t=0, all ions are accelerated to the detector Acceleration is constant energy, all ions achieve the same K.E. Since KE=0.5*mass*velocity 2, velocity is mass dependent, therefore the time to impinge on the detector is mass dependent Ions enter the accelerator high m/z low m/z E E=0 Accelerate Detect 4 2016 AB Sciex
Resolution is a key requirement Necessary for mass accuracy, but does not guarantee mass accuracy For differentiation of nearby peaks Helps with sensitivity in TOF intensity 160 140 ±1ppm 120 30K 100 17K 80 60 40 20 0-50 -25 0 25 50 Shift from centroid (ppm) Causes of resolution loss Jitter timing and voltage Path-length variation detector, mechanical variation Trajectory variation ion position, ion velocity variation 5 2016 AB Sciex
Mass Resolution The time-of-flight equation Geometry mirror ground NTOF detector TOF y y 0 2 TOF 2 y y 0 d 2 f ( d1, d3, d6, d8, V1, V 3, V 5) Conditions for 2 nd order focusing dff f ( d1, d3, d6, d8, V1, V 3, V 5) liner ground pulser Y Z 3 high voltages ground Shift from mean TOF (ns) grid 6 Pusher plate 4 v y 0 2 0-2 -4-6 -8 0 5 10 15 location in accelerator (mm) X 6 2016 AB Sciex
ESI QTOF Geometry Parameter Liner length Mirror Length Mirror Stage 1 length Mirror Stage 2 length Accelerator AC section length Accelerator DC section length Liner potential Mirror Plate potential Value 495mm 102mm 36mm 66mm 12mm 60mm +/-6kV -/+2.1kV Mirror Grid Potential 0 Pusher plate potential Beam energy -/+1.6kV 22eV Effective flight path length 2.35m Actual path length 1.95m 7 2016 AB Sciex
Space velocity correlation focusing 80 85 90 65 75 70 Correlated position and velocity eliminates turnaround time problem Direction of acceleration Resolution and spectral line shapes in the reflecting time-of-flight mass spectrometer with orthogonally injected ions, V. V. Laiko and A. F. Dodonov, RCMS 8 720-726 (1994). Improved resolution and substantially higher sensitivity on a QTOF mass spectrometer, A. Loboda, I. V. Chernushevich, and Nic Bloomfield, 57 th ASMS conference 2009. 8 2016 AB Sciex Z=distance from collision cell aperture to point of acceleration into TOF analyzer
NTOF Analyzer Field-free zone 2 grids This field is always on Double grid prevents field penetration OFF state This field switches on and off ON state Single pulse only, pusher only, no puller 9 2016 AB Sciex
Mass Resolution Detector tilter 32,400 31,600 30,800 30,000 29,200 28,400 27,600 32,400 31,600 30,800 30,000 29,200 28,400 27,600 Resolutio 40000-50000 30000-40000 10 2016 AB Sciex
Resolution Performance, MS mode detector m/δm(fwhm) 46000 44000 42000 40000 38000 36000 +1 +2 +3 34000 32000 400 500 600 700 800 900 1000 m/z(amu) +1 +2 +3 11 2016 AB Sciex m/z z Δm(amu) Δt(ps) m/δm 922.005 +1 0.027 785 34668 785.838 +2 0.018 332 43042 524.228 +3 0.012 269 43371
Resolution Stability 45,000 40,000 35,000 m/δm(fwhm) 30,000 25,000 20,000 15,000 10,000 5,000 0 12 2016 AB Sciex 0 4 8 12 Hours 16 20 24
Mass Accuracy Resolution is nothing without mass accuracy 1 Mass accuracy means confidence in the indicated peak position is accurate and stable. A narrow high resolution peak can be an unreliable indicator of peak position. 1 Bob Haufler, NEMC 2016 13 2016 AB Sciex
Mass Accuracy: Thermal effects 5 C change at t=0 The high voltage power supplies are independently thermally stabilized 14 2016 AB Sciex
Mass Accuracy - Statistical Each sample was acquired every 20 minutes. 15 2016 AB Sciex
Mass Accuracy - Systematic Linear calibration equation, 2 coefficients: and 0 mass a t 2 t 0 2 1.5 1 mass error (ppm) 0.5 0-0.5-1 -1.5-2 0 200 400 600 800 1000 1200 1400 m/z (amu) GluFib MSMS ALILTLVS MSMS TOFCAL MS GluFib=[Glu1]-fibrinopeptide B 16 2016 AB Sciex
Sensitivity no beam aperturing 17 2016 AB Sciex
X500R Scan Modes and Data Acquisition
QTOF scan modes Mode Q1 Collision cell TOF Workflow TOFMS Open, all ions pass Off All ions detected (precursors) Targeted, suspect and unknown screening TOFMSMS Set to target precursor On All fragments detected Targeted, suspect and unknown screening SWATH Scanning 25 amu window On All fragments detected Suspect and unknown screening MRM HR Set to precursor On All fragments detected Targeted quantitation and confirmation IDA Scan mode Time Step 1 TOFMS Survey Scan-Obtain precursor ion list Step 2 TOFMSMS Obtain fragment MS of 1 st precursor Step 3 TOFMSMS Obtain fragment MS of 2 nd precursor Step 4 TOFMSMS Obtain fragment MS of 3 rd precursor 0.1-0.5 sec 0.05-0.1 sec 0.05-0.1 sec 0.05-0.1 sec Total ~1sec Cycles repeatedly during chromatography 19 2016 AB Sciex
Untargeted Analysis Known unknowns (suspect screening) spectra exists in library (MSMS and possibly retention time) Library match Formula finder Isotope pattern MSMS pattern Compare retention time if LC method is standard Obtain standard for confirmation and quantitation Unknowns (unknown screening) no spectra exists in library Formula finder Isotope pattern ChemSpider MSMS pattern Obtain standard based on ChemSpider results for confirmation 20 2016 AB Sciex
IDA details - Data Acquisition Setup 21 2016 AB Sciex
IDA Data 22 2016 AB Sciex
IDA Data 23 2016 AB Sciex
Sample - Control Comparison in SCIEX OS Software Quick example: unknown screening of food dye compounds Select samples Select control 24 2016 AB Sciex
Unknown Screening in SCIEX OS Software Setup: Peak finding Library searching Traffic lights Formula finding Peak detection sensitivity (fast to exhaustive) 25 2016 AB Sciex
Results Review and Filtering in SCIEX OS Software Table is Filtered by Formula Finding Results and Sorted by Area Control XIC TOF-MS MS/MS Sample XIC TOF-MS MS/MS 26 2016 AB Sciex Sample was diluted with water/sugar solution (10.000x) to simulate dissolved cake icing
Review of Formula Finding Results in SCIEX OS Software Evaluation of TOF-MS and MS/MS Error and ChemSpider Hit Count Formula with 5 Hits Zoom Link to ChemSpider 27 2016 AB Sciex
Review of ChemSpider Results in SCIEX OS Software Automatic In-silico Fragmentation of Structure Found in ChemSpider MS/MS 28 2016 AB Sciex Tentative identification of Erythrosine in RED food color
Examples
Three Isomers Analysis by MRM HR 3 compounds with same formula but separated by LC conditions 30 2015 AB Sciex
Sebuthylazine MSMS Spectra 31 2015 AB Sciex
Suspect screening: water samples Cocaine human metabolite: Benzoylecgonine Caffeine Sildenafil (viagra) Water samples from lakes, rivers, drinking water and a few other sources around the world. 32 2015 AB Sciex
Benzoylecgonine in Different Water Samples 0.300 0.250? Berlin 0.200 0.150 0.100 0.050 100 ng/l Zermatt Canoe Zürich Philadelphia Rio de Janeiro Roma Santa Fe Scarborough Toronto Wien St. Pete Beach Creeks Drinking water Lakes 0.000 33 2015 AB Sciex Rivers BE and other drug metabolites are detected in rivers in major cities, concentration can be used to estimate consumption. Sea
Caffeine in Different Water Samples 40.000 35.000 30.000? Philadelphia 25.000 20.000 15.000 10.000 5.000 100 ng/l Mountain hut in Switzerland Vienna Drinking water Lakes Zürich Berlin Roma Rio de Janeiro Dresden Wien St. Pete Beach 0.000 Creeks Rivers Sea Caffeine or other compounds consumed in large amounts are markers for human activity, i.e. artificial sweetener 34 2015 AB Sciex
Sildenafil in Different Water Samples 0.100 0.090 100 ng/l 0.080 0.070 0.060 0.050 Wien 0.040 0.030 0.020 0.010 Creeks Holy water from a church in Roma Drinking water? Lakes Rio de Janeiro Rivers LOQ 0.000 Sea Concentration of sildenafil detected were very low (<10 ppt), one exception! 35 2015 AB Sciex
Data of Holy Water (Direct Injection 100 µl) Compound µg/l LOQ (µg/l) Acetaminophen 9.1 0.010 Benzoylecgonine (metabolite of cocaine) 0.47 0.001 Caffeine 38 0.010 Carbamazepine 0.21 < 0.001 Codeine 0.050 0.001 Dextromethorphan 0.021 0.001 Diazepam 0.003 0.001 EDDP (metabolite of methadone) 0.001 0.001 Erythromycin 1.7 0.050 Morphine 0.15 0.005 Sildenafil 0.015 0.005 Thiabendazole 0.016 < 0.001 36 2015 AB Sciex
Summary The X500R technology and performance Technology to achieve high mass resolution (>30,000 resolution) N-geometry. Detector tilter. Space-velocity correlation focusing. Technology to achieve high mass accuracy(<1ppm accuracy for m/z>50amu) Thermal stabilization. Simplified accelerator with no puller. X500R scan modes IDA for suspect and untargeted screening. SWATH for suspect and untargeted screening. MRM HR for targeted quantification and confirmation. Application examples Unknown food dye compound MRM HR identification of identical m/z isomers Detection of human activity compounds in various water samples 37 2015 AB Sciex
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