Development of a commercial add-on for ultrasensitive real-time mass

spectrometric analysis of vapors

Novartis Day 11th September, Zurich (Switzerland)

C. Barrios-Collado1,2,3, D. García-Gómez1, M. Gaugg1, R. Zenobi1, G. Vidal-de-Miguel1,2*, P. Martínez-Lozano Sinues1*

1 ETH Zürich, Switzerland; 2 SEADM S.L., Boecillo, Spain; 3 University of Valladolid, Valladolid, Spain*email: guillermo.vidal@seadm.com / guillermo.vidal@org.chem.ethz.ch / pablo.martinez@org.chem.ethz.ch

1. Overview

7. Conclusions

8. Acknowledgements

9. References

4. Drugs detection

3. Pilot tests: smokers

6. Analysis of aldehydes

5. Light-induced plant metabolism

• With this add-on any commercial API-MS without any modification can be deployed for the sensitive and real-time analysis of trace gases.

• We provide some examples of application for the universal LFSESI on the detection of drugs, suggesting that such system can be suitable for real-time pharmacokinetic studies, real time breath analysis or deciphering the language of plants by analyzing their released VOCs.

• First real breath analysis with the new platform was a pilot test with smokers and non-smokers [11].

• Around 1000 features were detected.• Compounds over 900 Da were detected, which expands the available

state-of-the-art on-line breath analyzer range.• We found compounds correlated with smoking frequency.• Breathprints allowed 100% accuracy at smoking/non-smoking status

prediction.

• Exhaled breath contains relevant metabolites that may reflect the biochemical activity within a subject.

• However, in contrast to other biofluids (e.g. plasma), the analysis of breath remains far less explored.

• Secondary Electrospray Ionization (SESI) in tandem with Atmospheric Pressure ionization Mass Spectrometry (API MS) has achieved sensitivities in the sub-ppt range for polar vapors such as drugs, explosives and breath [1-7].

• Low-Flow SESI (LF-SESI), was developed in tandem with a Differential Mobility Analyzer (DMA), and in explosive detection applications reached sensitivities at the sub-ppq range [8-9]. Figure 1 shows the LFSESI configuration.

• We developed a real-time breath analysis platform via MS by implementing an add-on LFSESI [10] on a commercial mass spectrometer atmospheric pressure inlet. Here we present some applications.

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Figure 2 Orbitrap Mass Spectrometer with LFSESI [10]:1. Mechanical alignment; 2. Thermal insulation surrounding the core; 3. High voltage electronics; 4. Electrospray vial holder; 5. Transfer line; 6. Electrospray positioning; 7. Temperature control connection for core and transfer line

• Aldehydes and furans in breath in real time were studied [15,16].• High volatile aldehydes (less than six carbon atoms), not detected in

exhaled breath condensate studies, were identified.• Figure 7 shows detection of aldehydes in breath in real time.

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Figure 7 Real time analysis of aldehyde in Exhaled Breath Condensate [15]

We gratefully acknowledge Dr. Juan Zhang (Novartis AG) for the donation of the LTQ Orbitrapinstrument used in this study and the European Community’s Seventh Framework Programme (FP7-2013-IAPP) for funding the project “Analytical Chemistry Instrumentation Development” (609691). We are indebted to Christoph Bärtschi (ETH workshop) for his assistance machining the ion source and Myriam Macía for assisting during the development phase.

1. C. Wu, W. F. Siems, H. H. Hill Jr. Secondary Electrospray Ionization Ion Mobility Spectrometry / Mass Spectrometry of Illicit Drugs. Anal. Chem 72, no. 2 (2000).

2. J. F de la Mora. Ionization of Vapor Molecules by an Electrospray Cloud. Int. J. Mass Spectrometry 300, no. 2-3 (2011)

3. P. Martínez-Lozano, J. Rus, G. F de la Mora, M. Hernández, J. F de la Mora; Secondary Electrospray Ionization (SESI) of ambient vapors for explosive detection at concentrations bellow parts per trillion. American J. Mass Spectrometry (2009).

4. L. Meier, C. Berchtold, S. Schmid and R. Zenobi; Sensitive detection of drug vapors using an ion funnel interface for secondary electrospray ionization mass spectrometry; J. of Mass Spectrometry, 2012 May;47(5):555-9

5. M. Tam, H. H. Hill Jr. Secondary Electrospray Ionization – Ion Mobility Spectrometry for Explosive Vapor Detection. Anal. Chem. 76, 2741-2742 (2004).

6. P. Martínez-Lozano, J. Fernández de la Mora. Electrospray ionization of volatiles in breath. Int. J. Mass Spectrometry, 265 (1):68-72 (2007).

7. C. Berchtold, L. Meier, R. Zenobi. Evaluation of Extractive Electrospray Ionization and atmospheric pressure chemical ionization for the detection of narcotics in breath. Int. J. Mass Spectrometry, vol. 299, issues 2-3, p. 145-150 (2011).

8. G. Vidal-de-Miguel, M. Macía, P. Pinacho, et. al. Low Sample Flow Secondary Electro-Spray Ionization, improving vapor ionization efficiency. Anal. Chem. (2012).

9. G. Vidal-de-Miguel, D. Zamora, M. Amo, A. Casado, G. F de la Mora, J. F de la Mora; Method for Detecting Atmospheric Vapors at Parts per Quadrillion (PPQ) Concentrations. USPTO

10. C. Barrios-Collado, G. Vidal-de-Miguel, P. Martínez-Lozano Sinues; Numerical Modelling and Experimental Validation of a Universal Secondary Electrospray Ionization Source for Mass Spectrometric Gas Analysis in Real-Time. Submitted to Sensors and Actuators B: Chemical.

11. M. Gaugg, D. García-Gómez, C. Barrios-Collado, G. Vidal-de-Miguel, M. Kohler, R. Zenobi, P. Martinez-Lozano Sinues; Expanding metabolite coverage of real-time breath analysis by coupling a universal secondary electrospray ionization source and high resolution mass spectrometry – a pilot study on tobacco smokers. Submitted to J. Breath Research

12. X. Li, P. Martínez-Lozano Sinues, R. Dallmann, L. Bregy, M. Hollmén, S. Proulx, et al., Drug pharmacokinetics Determined by Real-Time Analysis of Mouse Breath Angew Crem Int Ed (2015)

13. D. Tholl, W. Boland, A. Hanse, F. Loreto, U. S. R. Röse, J.-P. Schnitzler; Practical approaches to plant volatile analysis The Plant Journal (2006) 45, 540–560

14. J. K. Holopainen, J. Gershenzon; Multiple stress factors and the emission of plant VOCs Trends in plant science (2010)

15. D. García-Gómez, P. Martínez-Lozano, C. Barrios-Collado, G. Vidal-de-Miguel, M. Gaugg, R. Zenobi; Identification of 2-Alkenals, 4-Hydroxy-2-alkenals, and 4-Hydroxy-2,6-alkadienals in exhaled breath condensate by UHPLC-HRMS and in breath by real-time HRMS. Anal. Chem. (2015)

16. D. García-Gómez, L. Bregy, C. Barrios-Collado, G. Vidal-de-Miguel, R. Zenobi Real-time high-resolution tandem mass spectrometry identifies furan derivatives in exhaled breath. Anal. Chem. (2015)

• The breath analysis platform consists basically on a heated asymmetric LFSESI chamber which is coupled to the MS atmospheric pressure interface. A 2-axis micrometric positioning system provides fine mechanical alignment between the LFSESI and the MS inlet capillary. Also, the axial position of the electrospray tip can be optimized manually. Figure 2 shows the LFSESI coupled to an MS.

2. Architecture

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inlet

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Figure 1 LFSESI configuration: Electrospray ions (blue) transfer charge to sample vapors (yellow) which become ionized (green) and get into the MS.

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Integrated signal vs gas phase concentration

MelatoninPropofolAcetaminophenPentobarbitalMidazolam

y = 1313.4 x0.8566

R2 = 0.9701

y = 193.34 x1.2135

R2 = 0.9566

y = 368.34 x0.8078

R2 = 0.9757

y = 0.2012 x1.9375

R2 = 0.9963

y = 1084.7 x0.6707

R2 = 0.988

• Sensitivity was tested towards vapors of common drugs [10].• Drugs were injected into a nitrogen flow of 0.2 L/min.• Figure 4 shows that the system was able to detect these drugs from

concentrations of tenths of ppt in the gas phase, with a linear response across three orders of magnitude.

• Such low concentrations are deemed to be necessary to be detected in exhaled breath of small animals such as mice [12].

Figure 4 Detection of several drugs of interest

Figure 3 Differences between smokers and non-smokers; a) overlaid breath mass spectra of all subjects in the region around the feature at m/z 114.0733; b) box-plot of the average intensities per subject, split into smokers and non-smokers. This compound was significantly increased in smokers; c) linear regression between the peak intensities of the feature at m/z 114.0733 and smoking frequency.

233.00 233.05 233.10 233.15 233.20 233.25 233.30 233.35 233.40 233.45

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233.13

[M+H] = 233.13

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179.14

[M+H] = 179.14

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[M+H] = 152.07

227.00 227.05 227.10 227.15 227.20 227.25 227.30 227.35 227.40 227.45

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Figure 5 Spectra of several compounds of interest:

a) Melatonin (m/z = 233);b) Propofol (m/z = 179);

c) Acetaminophen (m/z = 152);d) Pentobarbital (m/z = 227); e)Midazolam (m/z = 326) [10]

Figure 6 Evolution of some VOCs of interest emitted by plants

• Plants release VOCs as part of their own metabolism or to send warning messages to other plants [13].

• SESI-MS can be used to decipher the language of plants [14].

• Experiments with begonia show more than 1000 different compounds detected.

• Figure 6 shows the set-up to analyze plant VOCs. Figure 6 shows the temporal evolution along two days of some basic plant VOCs related with photosynthesis. Figure 6 Experimental set-up for

analysis of VOCs emitted by plants

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Isoprene C5H8

Monoterpene C10H16

Monoterpene alcohol C10H18O

Sesquiterpene C15H24

Sesquiterpene alcohol C15H26O