1. PTR-TOF-MS: A New Instrument For Real-Time Analysis Of Multi-Component Systems: Applications To Food Analysis Rob Linforth, Annie Blisset, Andy Taylor Dept. of Food Science University of Nottingham Caroline Lamont-Smith, Steve Mullock & Fraser Reich Kore Technology Ltd. Ely, Cambs

2. Talk: Outline Overview of the Proton Transfer Reactor method Why a PTR-TOF-MS? The Instrument Some Example Data Summary First a confession: This talk is heavy on the instrument description, its properties and possibilities of the instrument, because the speaker is one of the team that designed the instrument.

3. PTR: Proton Transfer Reactions Essentially, PTR is a subset of Chemical Ionisation. It was defined and refined by Werner Lindinger in the 1990’s, and resulted in the first commercially available instrument (Ionicon) Aim is to achieve ‘Soft’ ionisation so as not to fragment the molecule(s) of interest: less fragments = less mass spectral ‘noise’. In positive ion mode, most frequently used reaction is: H 3 O + + R H 2 O + RH + For this protonation reaction to occur, the proton affinity of R must be greater than that of water The ionisation probability is almost the same as the collision cross-section, so the aim is to induce sufficient collisions to ensure efficient analyte ionisation

4. PTR: Proton Transfer Reactions With H 3 O + CompoundPA (kJ/mol) Oxygen421 Nitrogen493 Carbon Dioxide541 Sulphur Dioxide672 Water691 Hydrogen Sulphide709 Benzene750 Methanol754 Toluene784 Naphthalene803 Ethyl Acetate836 Tri ethyl phosphate909 Compounds above water in the proton affinity table will not be ionised, whereas compounds below will The good news is that most VOCs have higher proton affinities than water and will receive a proton from H 3 O + Generally, larger molecules have larger proton affinities

5. Linearity of Response Provided that [RH+]<< [H 3 O+], the H 3 O+ signal does not change with analyte concentration, and the detected analyte intensity is linear with concentration: [RH+] = [H 3 O+] 0 (1-e -k(R)t )  [H 3 O+] 0 [R] kt 0-50ppm H 2 S calibration: Raw data plot (k is rate constant for the reaction, and t is time to travel through the drift tube) H 2 S proton affinity= 709kJ/mol

6. Soft Ionisation vs. Electron Impact Ionisation 70eV EI spectrum of Ethyl Butyrate Molecular Ion appears at mass 116 m/z Significant fragmentation to lower masses

7. Soft Ionisation vs. Electron Impact Ionisation PTR-TOF-MS Spectrum of 5ppm Ethyl Butyrate Molecular Ion at 117 m/z (molecular ion + proton) Very little fragmentation

8. Charge Transfer In The PTR Reactor Need to induce sufficient collisions: H 3 O + and analyte mixed in a reactor tube (drift cell) Voltage gradient across reactor Pressure ~ 1 mbar, approximately 2000 collisions down 10cm length reactor Aim is to have dilute analyte, so that analyte molecules do not collide with anything except H 3 O +. This keeps the ionisation scheme and calibration simple, unlike in Ion Mobility Spectrometry (complex inter-analyte charge transfer reactions)

9. The PTR Reactor and the E/n Ratio E is the voltage gradient down the reactor in V/cm n is the gas density in molecules / cc (1mbar in reactor, typically) E/n in Townsend; 1 Townsend = 10 -17 cm 2 / Vs Lindinger et al. (1998) showed that 120-140 T is ideal for non- fragmentation of organic molecules If E/n too high, fragmentation of molecule occurs If E/n too low, clustering of water molecules occurs and can present a problem for certain species

10. The PTR Reactor and the E/n Ratio Tri-ethyl phosphate, protonated mass 182 130 Townsend 195 Townsend – loss of the ethyl groups (28 mass units) 260 Townsend PO 4 + proton

11. Why Use A TOF-MS? Parallel detection means no analytical price to pay for monitoring many species, unlike a sequential analyser such as a quadrupole or magnetic sector TOF Cycle frequency typically 20-50 kHz = high data rate Full mass spectra to several hundred masses with 4-5 orders dynamic range in one second Possibility of ‘real time’ data analysis with < 100ms resolution, multiple species and sensible counting statistics Ability to interrogate a data set ‘retrospectively’: intensity of any species as a function of time, mass spectra for a variety of ‘time slices’ Parallel detection and full mass spectra more suitable to software data reduction techniques, e.g. principle component analysis

12. Why Use A TOF-MS? This mass spectral plot, acquired with a PTR-Quad-MS (a sequential scanning device) took 40 seconds A full mass spectrum to several hundred m/z can be acquired in less than a second with a PTR-TOF-MS Mass spectrum from breath (U. Nijmegan)

13. TOF-MS: Accurate Mass Capability 30.00 m/z NO + 31.99 m/z O 2 + 33.04 m/z CH 3 OH 2 + 36.04 m/z (H 2 O) 2 +

14. Downside Of A TOF-MS? Main drawback is analysis of a continuous analyte stream: When the TOF cycle has started, no further ions can be injected without resulting in multiple overlapping spectra ‘Duty cycle’ describes the % of the analyte stream that is sampled In an orthogonal TOF (analyte stream enters TOF source at 90°), the duty cycle can reach 10%: when the Kore TOF source pulses, it ‘empties out’ ~5-10 microseconds worth of ions. Usage is therefore 5µs in 50µs, I.e. 10% Currently we are observing up to 500kc/s of Hydronium at the detector, approximately half that reported by the PTR-Quad (single ion mode). Prospect of improving duty cycle using ‘Hadamard’ methods – high frequency random pulsing of source with mathematical deconvolution of spectra - still not really proven as a workable solution

15. TOF MS: Orthogonal Pulsing TOF Source Continuous Sample Detector Ions Extracted Light Ions Heavy Ions Ions of different masses within a single ‘cycle’ arrive at the detector at different times according to the relation: K.E. = mv 2 /2 Ions with m/z = 1000 has flight time ~20-50  s, therefore ‘cycle time’ = 20-50  s, so typical pulsing frequency = 20-50 kHz VoltsSource Off t Source On 0 -2kV Extractor pulses to –400v TOF Source Variant on ‘Wiley – Maclaren’ source. Compensation for ion position within source

16. TOF MS: Orthogonal Pulsing TOF Source Overview schematic of the instrument: TOF source to detector

17. TOF MS: Orthogonal Pulsing TOF Source Overview schematic of the instrument GD Source 2mbar PTR Reactor 1mbar Transfer Optics 10 -4 mbar Mass spectrometer and detector 10 -6 - 10 -7 mbar

18. Mass Spectral Analysis: Applications to Food Analysis University of Nottingham Department of Food Science Professor Andy Taylor Dr. Rob Linforth Annie Blisset “Breath-by-breath” Research Main technique in lab: APCI Wanted to add PTR-TOF-MS Instrument recently delivered; has both PTR and APCI functionality Data acquired at Nottingham by one of us (FR) with Rob Linforth and Annie Blisset

19. Breath-by-Breath Analysis Of Juicy Fruit Gum The principle compound released during chewing of Juicy Fruit gum is ethyl butyrate, molecular mass 116 Soft tube inserted lightly into the nostril of subject Breathing normally, the mass spectrometer begins acquiring data. Most of the exhaled breath passes out into open space, but a side-mounted capillary pipe samples the breath into the proton transfer reactor.

20. Breath-by-Breath Analysis: Release of Ethyl Butyrate Idealised appearance of breath markers, such as acetone, as exhalation occurs Release of flavours during mastication Signal intensity Time First data set: take ‘raw data set’ (all ion flight times recorded as a function of elapsed experiment time) Integrate the ethyl butyrate protonated ion (mass 117) every second

21. Breath-by-Breath Analysis At Different Data ‘Granularity’ Reconstruction of ethyl butyrate signal with 1 second integration time Reconstruction of Acetone signal with 1 second integration time Breathing rate ~5/min

22. Breath-by-Breath Analysis At Different Data ‘Granularity’ Reconstruction of ethyl butyrate signal with 0.25 second integration time- further structure emerging still Reconstruction of ethyl butyrate signal with 0.5 second integration time- further structure emerging

23. Breath-by-Breath Analysis At Different Data ‘Granularity’ Reconstruction of ethyl butyrate signal with 0.125 second integration time Reconstruction of ethyl butyrate signal with 0.0625 second integration time: no further structure emerging

24. Breath-by-Breath Analysis: Different Person Reconstruction of Acetone signal with 0.125 second integration time Note different breathing rate ~10/min for different person Overlay of ethyl butyrate signal with acetone

25. Full Mass Spectra From 125ms Time Slices 34.00 –34.125 seconds 34.50 –34.625 seconds Logscale data Acetone and Ethyl butyrate

26. Mass Spectrum From Headspace of Freshly Macerated Tomatoes 3 hexenol 3 hexenal

27. 3-Hexenol vs. Hexanal, Both C 6 H 12 O Hexanal: protonated masses at 101, 83, 55 3-Hexenol: protonated masses at 101, 83, 59 and 55 In previous slide, masses 101 and 83 were identified as 3- Hexenol, but in truth there is the possibility that 101 and 83 can be due to Hexanal.

28. 3-Hexenol vs. Hexanal, Both C 6 H 12 O Is it possible to find an E/n value that will give a mass 101 for one compound but not the other? Hexanal3 Hexenol 83 101 No ‘threshold phenomenon’ observed Also, no E/n found for 3-Hexenol at which intensity of 101>83 Possibility of modulating PTR voltage and using software tools for compound differentiation? More work required, clearly!

29. Summary PTR-MS instruments based on quadrupole mass spectrometers are now widely used in studies of environmental and atmospheric chemistry as well as food and medical applications A TOF-MS increases the possibilities of real-time analysis down to <100ms “data granularity”, with full mass spectral acquisition in each time slice A TOF-MS permits any ion chromatogram to be re-constructed from the data set A TOF-MS can be operated at higher mass resolutions than a quadrupole mass spectrometer. Even at relatively low mass resolution the mass accuracy is better than 30 millimass units Greater mass range capability, with no discrimination against high masses up to several hundred Daltons