by Dr. Ching Wu, Founder and CEO, Excellims
Breath analysis has considerable potential as a truly non-invasive tool for clinical diagnosis and research.1 Volatile compounds in exhaled breath can reveal insights into a patient’s metabolic and nutritional state to support the elucidation of disease progression or to determine the effect of medication. Breath analysis can also pick up microbial infection and robustly detect intoxication or certain types of poisoning. Gas chromatography-mass spectrometry (GC-MS) is the traditional, routine method for such analyses but there is considerable appetite for change. GC-MS is slow, relies on having an appropriate specialty gas supply and is also associated with high false positives for certain applications.
High performance ion mobility spectrometry (HPIMS) and HPIMS-MS platforms are prized for their ability to analyze rapidly and at the point of need. Equipped with a flexible ionization source, these platforms can be used to measure liquids, in configurations comparable to HPLC-MS, or gases, using corona discharged ionization or secondary electrospray ionization (SESI), in configurations comparable to GC-MS. This article examines the potential of HPIMS as a technique for breath analysis within the lab and as a potential clinical diagnostic, highlighting its capabilities for robust, real-time measurement at low cost. Experimental data illustrate the application of HPIMS paired with SESI to detect peppermint via breath analysis.
Introducing HPIMS/HPIMS-MS for Breath Analysis
An ion mobility spectrometer separates ions in the gas phase on the basis of size and shape, or, more precisely, collision cross area, as shown in Figure 1. In contrast, a mass spectrometer detects analytes on the basis of mass (mass to charge ratio). Integrating IMS and MS together therefore brings orthogonal techniques to bear for more powerful analysis, enabling differentiation on the basis of both mass and molecular structure. This complementarity has been recognized since the 1970s and IMS is routinely integrated in commercial MS instrumentation.
Figure 1: Key components of an HPIMS system (left) with a schematic showing principle of operation (right).
However, there has been considerable progress over recent years with respect to refining IMS technology for greater sensitivity and resolution, portability, and standalone operation. IMS is an inherently rapid technique with separation complete within ~50 milliseconds.2 Advances in sample introduction, ionization and separation performance have capitalized on this core benefit to deliver optimized, independent platforms for routine use.
Modern, commercial HPIMS technology delivers high resolution, due to features such as an innovative atmospheric drift-tube design, in a compact design. Figure 2 illustrates the performance that is now accessible. The data shown are for the two trisaccharide isomers melezitose and raffinose - compounds that MS would fail to differentiate because of their identical molecular weight. In contrast, HPIMS provides clear differentiation with a resolving power of 85. These results demonstrate the potential of HPIMS as an independent technique but also provide a good example of the complementary nature of HPIMS, relative to MS. State-of-the-art HPIMS-MS systems capitalize on this complementarity with portable technology offering in-field performance that cannot be matched by combining other separation techniques (e.g. GC, LC) with MS.
Figure 2: Ion mobility separation of two trisaccharide isomers, melezitose and raffinose compounds, demonstrating high resolving power greater than 80.
Pairing electrospray ionization (ESI) with HPIMS extends application to an almost limitless range of analytes. With this “soft ionization” technique, liquid samples are volatilized from the tip of a needle by applying a strong voltage. For gaseous samples, the resulting ion stream enables the application of SESI, the ionization of gas molecules via contact with the ions produced by ESI. The benefits of SESI are well-recognized with published data highlighting its application for the detection of explosives - down to very low levels,3,4 - and bacteria, for example, to support disease detection.5 Combining SESI with HPIMS results in application of the most sensitive gas ionization technique and rapid analysis, creating a compelling solution for the detection of analytes at very low concentrations in breath. To summarize, for breath analysis, SESI-HPIMS enables:
- Rapid, effectively real-time measurement
- Portable instrumentation suitable for use at the point of need
- High sensitivity and resolution
- An accessible total cost of ownership, with minimal consumable costs and notable solvents relative to GC-MS.
The following study provides proof of performance using peppermint as an example analyte. Peppermint is a readily accessible, easy to use analyte for breath analysis already being used to develop a benchmarking test for breath analysis,6 making it a sound choice for testing.
Demonstrating Performance: Detecting Peppermint in Exhaled Breath
Breath analysis was carried out using a commercial HPIMS platform (HPIMS GA2200, Excellims, Acton, MA) designed for use in the field or production environment. Samples taken from a test participant following the consumption of peppermint Altoids were compared with samples of lab air to determine the ability of the instrument to detect components in the breath. The HPIMS was used with a gas line positioned behind the second electrode; SESI was used to prepare a suitable gas sample for analysis, using methanol acidifed with formic acid for the initial ESI. The experiment was a proof-of-concept study with no attempt made to optimize SESI conditions.
Figure 3 shows HPIMS traces for the ESI blank (purple), to provide a baseline measurement of acidified methanol, the SESI blank (blue), to provide a baseline measurement in the absence of peppermint, analyzed using the same SESI conditions as the breath sample, and for the breath sample itself (black). Four major components are detected via this analysis along with six additional minor components. The blue trace shown in Figure 4 elucidates the peaks with improved resolving power for better separation of the major components.
Figure 3: HPIMS traces for the ESI blank (purple), lab air (SESI blank - blue) and a breath sample after peppermint Altoid consumption (black) demonstrate the potential of HPIMS for breath analysis.
This simple study highlights the potential of HPIMS in combination with SESI for breath analysis. At least 10 unique peaks were observed in the breath samples, indicating the successful ionization of chemical species via the SESI mechanism. A key point to highlight is that with SESI, compounds with a charge affinity lower than the selected ESI solvent - in this case methanol - are masked since there will be no charge transfer. Optimization of the spray and solvent matrix, and of the gas inlet position, would therefore be expected to improve the sensitivity observed and would be clear areas of development of the method.
Figure 4: Baseline subtraction improves resolution for the four major components (upper image) while a zoomed in view more clearly identified the six minor additional components (lower image).
Conclusion
Breath analysis is a somewhat underutilized tool, relative to the analysis of, for example, blood and urine. Better platforms for breath analysis could help to change this and enable the more effective use of this truly non-invasive technique, which enjoys high patient acceptability. HPIMS, as used in this study, and HPIMS-MS technology have considerable potential within this context. Fast, sensitive, cost-efficient and compact, such technology can be used at the point-of-need to provide results in less than a minute. Pairing this technology with SESI for highly sensitive gas ionization provides a compelling potential solution for routine breath analysis.
References
1 Beale DJ et al ‘A Review of Analytical Techniques and Their Application in Disease Diagnosis in Breathomics and Salivomics Research’ Int. J. Mol. Sci. 2017, 18, 24; doi:10.3390/ijms18010024
2 Wu C et al ‘Delivering the Power of Ion Mobility Spectrometry – Mass Spectrometry to the Point of Analysis’ Chromatography Today Feb/March 2021
3 Zamora D et al ‘Reaching a Vapor Sensitivity of 0.01 Parts per Quadrillion in the Screening of Large Volume Freight’ Anal. Chem. 2018, 90, 2468−2474
4 Martinez-Lozano P et al ‘Secondary Electrospray Ionization (SESI) of Ambient Vapors for Explosive Detection at Concentrations Below Parts Per Trillion’ J Am Soc Mass Spectrom 2009, 20, 287–294
5 Bregy L et al ‘Differentiation of oral bacteria in in vitro cultures and human saliva by secondary electrospray ionization – mass spectrometry. Nature Scientific Reports, Oct 2015 5:15163 | DOI: 10.1038/srep15163
6 Henderson B et al. ‘A benchmarking protocol for breath analysis: the peppermint experiment’ J. Breath Res. 14 (2020) 046008
About the Author: Dr. Ching Wu is the founder and chief executive officer of Excellims. He has been working on all aspects of ion mobility spectrometry and mass spectrometry technology for over 25 years, from initial instrument design/product development to exploring new applications and business development. Before founding Excellims, he served as the research leader for GE Security's chem/bio and explosives detection business, where he was responsible for many government funded and GE internal programs to support the business growth. He also previously served as mass spectrometry software manager at Bruker Daltonics, managing software development projects for Bruker’s time of flight mass spectrometer product lines. He has both Ph.D. and MS degrees in chemistry from Washington State University, an MS in computer science also from Washington State University, and an MS in chemical engineering from Yokohama National University. Dr. Wu has authored more than 90 peer reviewed papers and patents/patent applications in ion mobility spectrometry and mass spectrometry field.