1. TOF and QTOF Fundamentals March 2007 Page 1 Fundamentals of TOF and QTOF Dr. Patrick (Pat) Perkins R&D scientist Agilent Technologies Life Sciences and Chemical Analysis Santa Clara, CA

2. TOF and QTOF Fundamentals March 2007 Page 2 Agenda Time-of-Flight (TOF) Mass Spectrometry basics Mass measurement Sources of variability in the measurement Minimizing the variability in the measurement Quadrupole Time-of-Flight (QTOF) MS Ion optics, collision cell design and performance

3. TOF and QTOF Fundamentals March 2007 Page 3 Definitions Used With TOF m/z—”mass-to-charge ratio”. The mass of an ion divided by its charge, the actual result produced by all mass spectrometers. For ions with a single charge (most small molecules), this is its isotopic molecular weight plus any adduct (proton, sodium,…). Proteins and peptide ions are in general multiply-charged, and therefore additional math (deconvolution) is used to determine their molecular weights. oa—”orthogonal acceleration”. Ions are accelerated (“pulsed”) down the flight tube in a direction perpendicular to their entry into the TOF analyzer. This minimizes the effects of ion generation and transmission on the mass measurement. ppm—”parts-per-million”, a measure of the error in the experimental mass assignment, compared to the known (or theoretical) value. Resolution—a dimensionless value indicating the ability of the mass analyzer to separate (resolve) ions. Higher values mean better resolving power. Several definitions exist, but the most common is M/  M, the m/z value of the ion divided by its peakwidth expressed in m/z units. Transient—one packet of ions pulsed down the flight tube and detected. Data from many pulses, typically ~10,000, are summed to create a spectrum.

4. TOF and QTOF Fundamentals March 2007 Page 4 Basic Ideal TOF Mass Spectrometer KE = ½ m v 2 v = d/t t = m ½ * d/(2KE) ½ constant Ideally, to measure the mass, Measure the time and use m = A * t 2 Practically, t = m ½ * d/(2KE) ½ + t 0 2 constants m = A * (t – t 0 ) 2 Detector V d Vacuum

5. TOF and QTOF Fundamentals March 2007 Page 5 Sources of Variability in Measurement (Deviations from Ideality) Detector V d Vacuum ???

6. TOF and QTOF Fundamentals March 2007 Page 6 Sources of Variability in Measurement (Deviations from Ideality) Ions don’t all start at the same plane in space relative to flight direction Ions aren’t sitting still and have different velocities relative to flight direction Temperature fluctuations change flight distance Voltage pulse is not instantaneous or of invariant shape Ions farther from voltage pulse element(s) obtain a higher velocity Change in temperature/humidity alter pulse electronics Poor vacuum scatters ions Detector and/or method of detection alters arrival times Inaccurate method(s) of locating the maximum of the mass peaks Detector V d Vacuum ???

7. TOF and QTOF Fundamentals March 2007 Page 7 Minimizing the Variability in Measurement (Approximating Ideality) Orthogonal acceleration Reflectron Beam shaping/conditioning prior to pulsing Higher order focusing Temperature control Humidity control Analog-to-Digital Conversion (ADC) vs. Time-to-Digital Conversion (TDC) Calibration Reference mass addition Sophisticated algorithms to locate mass peak centroids

8. TOF and QTOF Fundamentals March 2007 Page 8 Agilent TOF Schematic PulserDetector Vacuum ~1*10 -7 Torr Internal support structure with low coefficient of thermal expansion 1 meter flight tube, 2 meter flight path Reflectron (ion mirror) with self- compensating thermal design Optics for beam shaping and conditioning Flight acceleration orthogonal to ion introduction Turbopumps

9. TOF and QTOF Fundamentals March 2007 Page 9 A Time-of-Flight Scan PulserDetector 1.Pulse ions every 100 microseconds 2.Measure at detector each nanosecond (1 GHz) 3.100,000 data points in each transient 4.Sum ~10000 transients into one scan (1 scan/second operation) 5.Produces spectra with excellent ion statistics 20 µsec – m/z 118 46 µsec – m/z 622 90 µsec – m/z 2421

10. TOF and QTOF Fundamentals March 2007 Page 10 TOF Instruments: Excellent Mass Accuracy and High Resolution Simultaneously Advantages of High Resolution and Mass Accuracy Excellent mass accuracy provides a high degree of confidence in compound identification High resolution allows distinguishing peaks separated by only small m/z values High resolution allows selective detection of desired (“targeted”) compounds in the presence of interferences

11. TOF and QTOF Fundamentals March 2007 Page 11 Greater Resolution Does Not Necessarily Yield Better Mass Accuracy A TOF MS at 10,000 resolution cannot resolve between an ion with one C 13 isotope and one with one N 15 isotope. FTMS at 100,000+ resolution can. A TOF MS delivers < 3 ppm mass accuracy, while FTMS is often not as good under the same sample conditions

12. TOF and QTOF Fundamentals March 2007 Page 12 Why is Accurate Mass Useful? Accurate masses give possible elemental compositions 1 10 100 1000 0.10.050.010.0050.0010.00050.0001 Error in Mass Accuracy (amu) 176 386 882 1347 1672 5687 Possible formulas MW

13. TOF and QTOF Fundamentals March 2007 Page 13 Accurate Mass in Small Molecules Reserpine (C 33 H 40 N 2 O 9 ) has a protonated ion at 609.28066 Single quad reports mass to +/- 0.1 = 165 ppm Number of possible formulas using only C, H, O & N: 165 ppm (quad)209 10 ppm13 5 ppm7 3 ppm4 2 ppm2 Accurate mass reduces risk of investing effort on the wrong molecule (drug discovery and development)

14. TOF and QTOF Fundamentals March 2007 Page 14 Data Analysis – Mass Assignment Note: ADC sampling rate is 1 GHz

15. TOF and QTOF Fundamentals March 2007 Page 15 Data Analysis – Mass Assignment Note: ADC sampling rate is 1 GHz

16. TOF and QTOF Fundamentals March 2007 Page 16 Data Analysis – Mass Assignment Note: ADC sampling rate is 1 GHz

17. TOF and QTOF Fundamentals March 2007 Page 17 4 GHz ADC: What Is It? 4 GHz ADC for TOF and QTOF based on proprietary Agilent oscilloscope technology –Resolution improved to 9,000 @ 118 m/z 14,000 @ 322 m/z 16,000 @ 2722 m/z –In-spectrum dynamic range of 4.5 decades (ten-fold increase) –Extend mass range to 20,000 m/z –Retrofit to existing systems FPGA Master Agilent TALON ADC 4GSPS 8-BIT 4GSPS BUS PULSE Frontend_A Frontend_B CONTROL BUS CLOCK FPGA Slave 2GSPS BUS Dual Gain Front End SIGNAL Real Time Signal Processing and Summing Memory (32 staggered ADCs) FAST DATA OUT

18. TOF and QTOF Fundamentals March 2007 Page 18 4 GHz ADC Resolution Example: Two Compounds Nominally 195 m/z Separated By 0.036 Da FIA, 2 scans/sec (6713 transients/scan) 2 IRM averaged over five scans Butyl paraben [M+H] + 195.101571 m/z Methyl 5-acetylsalicylate [M+H] + 195.065185 m/z 4 GHz board, 1 GHz rate 6,100 FWHM resolution4 GHz8,900 FWHM resolution Error = + 0.8 ppm Error = - 0.9 ppm 4 GHz, transient peak picking 14,000 FWHM resolution 0.036 Da 4 GHz board, 1 GHz rate 6,100 FWHM resolution Error = + 0.8 ppm Error = - 0.9 ppm 4 GHz, high resolution mode 0.036 Da 14,000 FWHM resolution 4 GHz8,900 FWHM resolution

19. TOF and QTOF Fundamentals March 2007 Page 19 Automatic Tuning and Calibration Automated tuning done infrequently Automated calibration done daily

20. TOF and QTOF Fundamentals March 2007 Page 20 View of Calibration Results

21. TOF and QTOF Fundamentals March 2007 Page 21 Inlets – Electrospray Ion Source Dual sprayer design for sample and mass reference compound Reference Sprayer Analytical Sprayer

22. TOF and QTOF Fundamentals March 2007 Page 22 Automated Internal Reference Mass Correction

23. TOF and QTOF Fundamentals March 2007 Page 23 Empirical Formula Confirmation Report Formula input by submitter and system calculates monoisotopic mass Extracted ion chromatogram covering specified adducts Mass spectrum for major peak Zoomed spectrum covering adduct range Calculated mass error results

24. TOF and QTOF Fundamentals March 2007 Page 24 Expanded View of Results

25. TOF and QTOF Fundamentals March 2007 Page 25 TDC versus ADC Time to Digital Conversion only measures time of arrival of first ion at a given m/z value More sample means more ions means earlier arrival Requires higher acquisition rate than ADC and peak intensity matching to accurately assign mass Reduced dynamic range Analog to Digital Conversion (ADC) records time and number of ions arriving Sample concentration does not impact (maximum) arrival time Provides wider dynamic range

26. TOF and QTOF Fundamentals March 2007 Page 26 Dynamic Range Example for TOF Technology: 400x Difference in Abundance

27. TOF and QTOF Fundamentals March 2007 Page 27 Limits of Mass Accuracy Basic Calibration Error (1 – 2 ppm base) Chemical Interferences (0 – 10 ppm additive) Ion Statistics (0 – 25 ppm additive) Observed Mass Measurement Accuracy

28. TOF and QTOF Fundamentals March 2007 Page 28 Precision of the Mass Measurement in TOF Depends on Number of Ions Sampled

29. TOF and QTOF Fundamentals March 2007 Page 29 High Throughput Chemical Library Analysis Analysis of 140 real screening compounds under ultra-fast RRLC/TOF conditions (90s cycle time  1000 samples/day): Results from automated formula confirmation report – no manual interaction! LC: Water/ACN(0.1%TFA), 5-100%B in 0.7min, 60°C, 1.5ml/min UV-Detection: 210 – 500 nm, 80Hz MS-Detection: 120-1200Da, 8Hz, Split 1:7.5 Injection: 1µl, online sample dilution by injector program (determined the cycle time!)

30. TOF and QTOF Fundamentals March 2007 Page 30 High Throughput Chemical Library Analysis Mass error histogram of the analysis of 140 real screening compounds under ultra-fast LC conditions (90s cycle time  1000 samples/day): 2 outliers not shown, 16 compounds could not be ionized by ESI+ !

31. TOF and QTOF Fundamentals March 2007 Page 31 Mass Accuracy – Environmental Extremes Injecting every 15 minutes, from 11º - 36º C and 10 - 95 %Relative Humidity

32. TOF and QTOF Fundamentals March 2007 Page 32 NPAMOZ at Unit Mass Resolution 335.1352 -0.7+0.3 (similar to quadrupole) ???

33. TOF and QTOF Fundamentals March 2007 Page 33 NPAMOZ 335.1352 +/- 0.1 (300 ppm) S/N = 70:1

34. TOF and QTOF Fundamentals March 2007 Page 34 NPAMOZ 335.1352 +/- 100ppm (335.09939 - 335.16641) S/N = 103:1

35. TOF and QTOF Fundamentals March 2007 Page 35 NPAMOZ 335.1352 +/- 20ppm (335.1262 - 335.1396) S/N = 214:1

36. TOF and QTOF Fundamentals March 2007 Page 36 Why QTOF? All the benefits of TOF plus accurate mass information on the substructure fragments produced by MS/MS and selectivity of accurate mass product ions yields even greater confidence in the confirmation of the structure and even better selectivity for quantitation

37. TOF and QTOF Fundamentals March 2007 Page 37 The analysis of impurities in Amoxicillin by Q-TOF Degradation of Amoxicillin 160.0432 366.1325 Confirmation of the identity of the degradation product by accurate mass measurement in MS and MS/MS mode with subsequent empirical formula calculation Optimization of collision energy for targeted Q-TOF MRM and calibration for quantitative analysis of degradation products

38. TOF and QTOF Fundamentals March 2007 Page 38 Agilent QTOF Design Incorporates Cumulative Hardware Innovations

39. TOF and QTOF Fundamentals March 2007 Page 39 Innovations in Front-End Ion Optics Deliver Better Sensitivity Across a Broad Mass Range 10X sensitivity advantage Key components contributing to sensitivity Dielectric capillary Small diameter octopole ion guide High frequency RF octopole Lens 2 RF (transmission of higher masses) Hyperbolic post-filter and quadrupole

40. TOF and QTOF Fundamentals March 2007 Page 40 Innovations in Front-End Ion Optics Deliver Better Sensitivity Across a Broad Mass Range, cont. 10X sensitivity advantage Lens 2 RF (transmission of higher masses) Hyperbolic post-filter and quadrupole

41. TOF and QTOF Fundamentals March 2007 Page 41 1 st Quadrupole Beam shaper HEXAPOLE RODS Entrance lens Exit lens Beam shaper 3 rd Quadrupole Agilent 6410 Collision Cell Position

42. TOF and QTOF Fundamentals March 2007 Page 42 Collision Cell Design and Electrical Drive Acceleration Potential to 10 V RF Voltage = 10-550 v Hexapole construction 2R0 = 4.5 mm Length = 150 mm Experimental press. range = 0.1 – 20mTorr In coming ion energy range = 0 – 250eV Accelerating potential range = 0 – 10V RF Drive voltage range 10 – 550v

43. TOF and QTOF Fundamentals March 2007 Page 43 Transmission Velocity (Latency) Results Simulation Studies 3-D ion optics modeling (100-600  sec) Experimental Results Fast precursor ion selection (SRM) Linked scan latency Gated ion beam measurements

44. TOF and QTOF Fundamentals March 2007 Page 44 Dwell time evaluation 200 pg Alprazolam 100, 20, 5 ms dwell times Dwell100 ms20 ms5 ms Area148601360513202 % RSD0.610.742.25 5 ms dwell

45. TOF and QTOF Fundamentals March 2007 Page 45 Collision Cell Clearing Profile 0 V collision energy 5 V Applied Axial Potential 600  sec 350  sec

46. TOF and QTOF Fundamentals March 2007 Page 46 Broad Mass Range Transmission and Transmission Efficiency – Why Hexapole? Variables include: Number of poles (i.e. quad, hex, octo) Inscribed diameter (R 0 ) Drive Frequency Evaluation included: Theoretical modeling –Calculation, Simulations Experimental results

47. TOF and QTOF Fundamentals March 2007 Page 47 Full Mass Range With Single Parameter Set on QTOF (m/z 100 – 3000), No Switching

48. TOF and QTOF Fundamentals March 2007 Page 48 Mass Accuracy in MS/MS Mode MS Calibration is Maintained over Broad Collision Energy 4.8 ppm 1.3 ppm 0.9 ppm 1.6 ppm 1.2 ppm 3.2 ppm 0.7 ppm CE=150.5 eV @ 10 mTorr 3.4 ppm 5.1 ppm 3.8 ppm 1.6 ppm 1.2 ppm 2.1 ppm 1.4 ppm 0.2 ppm 0.4 ppm 0.7 ppm2.5 ppm CE=66.5 eV @ 10 mTorr 2.6 ppm 2.3 ppm 2.6 ppm 2.0 ppm

49. TOF and QTOF Fundamentals March 2007 Page 49 Internal Reference Mass Correction Fundamentals: The header of each acquired spectrum has reference mass corrected “a” and “t0” coefficients. They are calculated based on the presence of user selectable ions in each MS spectrum. Running averaging is supported. Extensions to MS/MS operation: MS/MS (DDE): Survey scan coefficients are copied into the headers of the subsequent MS/MS spectra. MS/MS (Manual/Targeted): MS scans are auto inserted into the specified MS/MS spectra acquisition cycle. The updated coefficients of these “background” generate MS spectra are copied into the headers of the subsequent MS/MS spectra.

50. TOF and QTOF Fundamentals March 2007 Page 50 STD ESI: 0.25 mL/min flow Q1=256 (2.5 wide), CE=10v, 167 (0.1 wide) EIC MS/MS spectrum at 1 pg. Background subtracted 200 fg 500 fg 1000 fg 2000 fg Dilution Series of Diphenhydramine on Q-TOF

51. TOF and QTOF Fundamentals March 2007 Page 51 Benchmarking - Small Molecule Sensitivity 200 pg MDMA (3,4-methylenedioxymethamphetamine)

52. TOF and QTOF Fundamentals March 2007 Page 52 1001502002503003504004505005506006507007508008509009501000 m/z, amu 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 187.06339 333.19855 268.11402 175.12511480.25381 684.35544246.160 95 785.83143 231.12109 Other Q-TOF At Detection Limit 10020030040050060070080090010001100120013001400150016001700 m/z, amu 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000 6200 6400 72.0791 684.3291 187.0662 813.3683 333.1794 480.2426 175.1143 240.1288 120.0774 1056.4458 942.4062 230.0715 1285.5122 1171.4684 87.0529 627.3093 497.1879 102.0522 337.1420 268.1223 924.3967 740.2681 1039.4248 355.1508 612.2114 515.1935 316.1450 159.0733 1268.4917 1570.6306 1154.4547 785.8255 1296.4777 853.2747 1022.3982 409.128555.0531 303.2225482.2451 667.2974 Agilent Q-TOF 10-20X above Detection Limit Benchmarking – Peptide MS/MS Sensitivity ESI infusion, Glu-Fib, MS/MS of 876 m/z