ORBITRAP Mass Spectrometer An Ultimate Qual and Quan Machine - PowerPoint PPT Presentation

ORBITRAP Mass Spectrometer An Ultimate Qual and Quan Machine Pongsagon Pothavorn Scispec Co., Ltd.

Information Rich Data

Accurate Mass in Life Science Resolution

FT Ultra - ICR

Brukers FT-ICR

Waters Ion Mobility

LC-MS solutions for all analytical challenges • Best LC-MS Portfolio

Induced by ion packets moving inside the trap Ions trapped in an electrostatic field • • Central electrode kept on high voltage • Outer electrode is split and able to pick up an image current induced by ion packets moving inside the trap r { } z k = ⋅ − + ⋅ 2 2 2 U ( r , z ) z r / 2 R ln( r / R ) m m 2 φ

Ion Injection and Formation of Ion Rings • An ion packet of a selected m/z enters the field • Increasing voltage squeezes ions • Voltage stabilises and ion trajectories are also stabilized • Angular spreading forms a ROTATING RING (r, φ ) (r,z)

Fourier Transform-based The moving ion rings induce an image current on outer electrodes • The frequency of harmonic oscillations is proportional to ions’ m/z •

Orbitrap and Nuclear Magnetic Resonance (NMR) • Free Induction Decay (FID) Time Domain ->Fourier Transform -> Spectrum (Frequency Domain)

Strategies for Analysis Organic Contaminants Known Known unknowns Unknown Target Screening Non-Target Screening Rapid and sensitive screening methods able to assign positive hits undoubtedly to particular organic compounds

Typical Mass Accuracy Type of MS Mass accuracy Utility for Quadrupole 0.1 µ Identify Traps 0.1 µ Identify TOF 0.0001 µ Empirical formula/ composition Sector 0.0001 µ Empirical formula/ composition FT-MS 0.0001 µ Empirical formula/ composition

Mass Accuracy Good ( ) • The precision of which the mass is measured error = 786.6003 – 786.60124 x 10 6 by the mass spectrometer. 786.60124 786.6003 (-1.19 ppm) • Typical way of reporting mass error in ppm 786.70 (+124 ppm) (relative measure) or mDa (absolute measure) Not so Good ( ) 786.60124 ( ) 100 error = 786.7000 – 786.60124 x 10 6 90 Mass error = Measured – Exact Mass x 10 6 80 786.60124 Exact Mass 70 60 787.60463 50 40 C = 12.0000 30 788.60773 20 789.61068 10 O = 15.9949 H = 1.0078 786 788 790 792 794 S = 31.9721 N = 14.0031

Mass Resolution • Ability of a mass spectrometer to distinguish between ions of nearly equal m/z ratios (isobars). Low Resolution 100 R = 786.6 = 1,000 90 80 70 0.786 60 Δm 50 40 = 0.786 30 20 10 786 788 790 792 794 High Resolution 100 90 80 R = 786.6 = 100,000 m - measured mass 70 60 0.007 Δm 50 Δ m - peak width measured at 50% peak 40 = 0.007 30 intensity (Full Width Half Maximum) 20 10 786 788 790 792 794

Commercial High Resolution MS Technology Race New Tribrid Orbitrap 500000 Tribrid Orbitrap Orbitrap Tof / QTof 450000 400000 FWHM) LIT-Orbitrap ETD 350000 ORBITRAP’s spectacular climb on (FW in performance in a decade! 300000 Q-Orbitrap* ution 250000 New Q-Orbitrap olut esol res 200000 ass r 150000 Mas Quad Orbitrap 100000 Entry Q-Orbitrap Ion Trap-Orbitrap 50000 First Q-Tof Bendix Tof 0 1955 1965 1975 1985 1995 2005 2015 Time Ti e prog ogres ession ( on (year ear)

Isobaric Pesticides Thiamethoxam: [M+H] + = C 8 H 11 ClN 5 O 3 S (292.02656) Parathion: [M+H] + = C 10 H 15 NO 5 PS (292.04031)

Isobaric Pesticides 3:1 Mix

Resolution – Why Is It Important? • Enables accurate mass • Increases confidence of identification • Improves quantitative accuracy • Gives access to qualitatively different information

Average Mass

How’s About Mass Accuracy • Average Mass = summing the average atomic masses of the constituent elements, H 2 O; 1.00794 + 1.00794 + 15.9994 = 18.01528. • Exact Mass = summing the masses of the individual isotopes of the molecule, H2O; 1.0078 + 1.0078 + 15.9994 = 18.0106. The Others Stories; • Isotopomer (Isotopic Isomer) = same type of isotope but difference in position, CH 3 CHDCH 3 vs CH 3 CH 2 CH 2 D • Isotopologues = difference in isotope in the molecules, H 2 O HOD • Monoisotopic = sum of masses in molecule. Using of most abundance or stable isotope.

Mass Accuracy – What for? H = 1.0078 O = 15.9949 C = 12.0000 N = 14.0031 S = 31.9721 Mass measured Tolerance [Da] Suggestions Calc Mass 32.0 +/- 0.2 O 2 31.9898 CH 3 OH 32.0261 N 2 H 4 32.0374 S 31.9721 32.02 +/- 0.02 CH 3 OH 32.0261 N 2 H 4 32.0374 32.0257 +/- 0.002 CH 3 OH 32.0261

Determine Fine Isotopic Pattern

Mass Accuracy across the Elution Profile • 21 scans per elution peak • External calibration Ma Mass A ss Accura racy y [p [ppm] m] RT: 1.72 - 1.96 477.23016 3 100 90 2 80 Relative Abundance 1 70 [ppm] 60 0 err [ 50 40 -1 30 -2 20 10 -3 775 780 785 790 795 800 Scan # can # 0 1.75 1.80 1.85 1.90 1.95 Time (min)

Average Isotope Ratio Variation

Mass Accuracy

Resolving Power and Mass Accuracy

Long-term mass accuracy with external calibration

Advantage • Easy method development for multi-residue analysis especially in complex matrices • Easy troubleshooting with detection of all adducts, degradation and contaminants • Higher detection specification • Simultaneous Qual and Quan analysis

Comparison

Non-Targeted Screening or Newborn Ideal • High isolation power for higher discrimination • High precision for accurate mass identification • High resolution for more identification • High mass stability for a long lasting mass calibration • MS n • Library availability for easy interpretations

Orbitrap Analyzer - the ‘Heart’ of a Mass Spectrometer 1.5 x 1.2 x Standard Orbitrap High-field Orbitrap

Resolution VS m/z

Resolving Power

Orbitrap VS QToF

Labelling Techniques

Intact Protein Analysis • Complete charge state envelope of IgG ‘Humira’ • Major glycosylation forms are baseline separated • Relative intensity reproducibility within a few percent

Intact Protein Analysis • Mass measurement accuracy • Average error for 34 measurements 6.9 ppm • Standard deviation 6.4 ppm Confirmation of protein primary structure

Sequence Confirmation of mAB • ETD fragmentation of an intact IgG ‘Humira’ • Resolution settings 240,000 for fragment detection • Increased sequence coverage • Localization of modifications (deamidation)

What do we gain by selected ion monitoring? Signal visibility is dependent, whether a signal is visible • above the spectrum noise Spectrum noise is dependent on the ratio of compound • within a certain ion population 195.0876 N=248402.81 NL: 1.94E8 Gain in sensitivity (7x) 100 [150.00-2000.00] Lowest detected 80 6000 Full MS signal/scan 60 S/N = 745 5000 250330 40 20 ectrum) um) 4000 0 195.0877 (spect N=20741.58 NL: 1.12E8 3000 100 Caffeine [190.10-200.10] S/N ( 80 Relative Abundance 2000 Lowest detected 60 SIM (10amu) signal/scan 40 1000 S/N = 5400 28240 20 0 0 195.082 195.084 195.086 195.088 195.09 195.092 195.094 Sensitivity gain 5 – 10 x with SIM mode S/N (FMS) S/N (SIM10)

Full Scan Spectrum of Atenolol AZ_1000ng_ml_100k_1e6_HypersilGoldPFP #246 RT: 3.46 AV: 1 SB: 1 3.25 NL: 1.36E6 T: FTMS + p ESI Full ms [140.00-1800.00] 267.16994 C 14 H 23 O 3 N 2 -1.42629 ppm 100 90 80 Atenolol 70 C14 H22 N2 O3 Relative Abundance M+H = 267.17030 60 50 40 195.08837 214.09034 251.12632 30 C 10 H 13 O 3 N C 10 H 23 O 2 N K Na -3.18542 ppm 2.05409 ppm 289.15189 20 229.14414 C 14 H 22 O 3 N 2 Na 158.02816 C 10 H 24 O 2 N K -1.30423 ppm 185.11564 1.22476 ppm 10 C 8 H 18 O 3 Na 149.02451 4.44492 ppm 292.93153 0 140 160 180 200 220 240 260 280 300 m/z

Full Scan Spectrum of Pyridoxine AZ_1000ng_ml_100k_1e6_HypersilGoldPFP #92 RT: 1.27 AV: 1 SB: 1 1.04 NL: 1.86E6 T: FTMS + p ESI Full ms [140.00-1800.00] 170.08135 C 8 H 12 O 3 N 1.03533 ppm 100 90 80 144.98848 Pyridoxine C8 H11 N O3 70 M+H = 170.08117 Relative Abundance 60 50 157.03556 40 168.02009 30 C 11 H 4 O 2 152.07113 C 8 H 10 O 2 N -2.90528 ppm 3.45525 ppm 20 162.97397 145.98632 159.03166 171.08468 10 146.98632 C 9 H 5 O 2 N 153.01050 143.99090 173.07839 149.94382 1.12952 ppm 166.99569 154.00835 0 140 145 150 155 160 165 170 175 m/z

Alprazolam, Full Scan Experiment Alprazolam Y = 6366.31+514.015*X R^2 = 0.9967 W: 1/X 5500000 50 ppt – 10 ppb 5000000 250 fg oc - 50 pg oc 4500000 4000000 3500000 Zoom in 50 ppt- 100ppt 200000 Area 3000000 180000 2500000 160000 140000 2000000 Area 120000 100000 1500000 80000 60000 40000 1000000 20000 0 500000 0 50 100 150 200 250 300 fg/uL 0 0 2000 4000 6000 8000 10000 fg/uL

Alprazolam SIM Experiment Alprazolam Y = -3135.8+552.216*X R^2 = 0.9982 W: 1/X 6000000 10 ppt – 10 ppb 5000000 50 fg oc - 50 pg oc 4000000 Zoom 10 ppt- 100ppt 120000 Area 110000 3000000 100000 90000 80000 Area 70000 2000000 60000 50000 40000 30000 1000000 20000 10000 0 0 20 40 60 80 100 120 fg/uL 0 0 2000 4000 6000 8000 10000 fg/uL

A Switch is on from QqQ to Orbitrap