Contemporary human breath related topics: aerosols, saliva, and HR-MS bioinformatics from Pittcon 2019

Abstract

Researchers from the International Association of Breath Research and the scientific board of the Journal of Breath Research (JBR) attended the annual Pittsburgh Conference and Exposition (Pittcon) in March 2019 in Philadelphia, PA, USA. Herein we report on a series of breath related topics from the meeting featuring sessions on exhaled breath aerosols and development of saliva analysis as a complement to non-invasive breath monitoring. Other contemporary breath related subjects, including bioinformatics, metabolomics, and forensics are also discussed.

Introduction

The annual Pittsburgh Conference and Exposition (Pittcon) is one the largest worldwide meetings of analytical chemistry instrumentation and applications. Pittcon attracts researchers from all over the world and is unique for us in the breath community in that it is the only non-specialized meeting we generally attend as a group. This year Pittcon hosted International Association of Breath Research (IABR) and Journal of Breath Research (JBR) researchers from Pisa, Italy, Rostock, Germany, Innsbruck, Austria, as well as US researchers from California, Virginia, Arizona, and North Carolina.

The 2019 Pittcon was held at the Philadelphia Convention Center with approximately 12 500 attendees from 80 countries, 155 technical sessions, an aggregate of 80 additional poster, networking and workshop sessions, and 750 corporate exhibitors on the exposition floor. Pittcon 2019 featured two main sessions directly related to breath diagnostics research. These were a symposium entitled ‘Human Exhaled Breath Aerosol (EBA) Sampling and Analysis: Applications for Clinical Diagnostics and Environmental Exposure Studies,’ and a workshop session entitled ‘Saliva Analysis: Non-Invasive Technologies for Drug Testing, Diagnosing Infection, and Assessing Inflammatory and Health Status.’ In addition, there were numerous standalone presentations and posters affecting breath research that will be reported herein. We also had numerous opportunities to interact with our colleagues beyond what was formally presented, and often such casual conversation resulted in new ideas and potential collaborations. Additionally, we could serve the breath research community by developing awareness of breath-based applications in attendees that may not have previously considered this biological medium as a diagnostic tool.

Certainly, this report is not fully comprehensive, but is part of an ongoing series describing breath topics at Pittcon as they evolve (e.g. Pleil et al 2017, Pleil and Wallace 2018). From these, we see that Pittcon is very broad and complex, and we, the authors, could not be everywhere. However, we have put together this report to describe what our colleagues presented, and to give the readership an idea of new topics we encountered that may broaden our own perspectives. In the following, we describe briefly the aerosols and saliva sessions, related presentations of colleagues, and our own observations from interacting with attendees.

EBA symposium

Recent innovations for exhaled breath analysis have expanded beyond the gas-phase and are now including the liquid exhaled breath condensate (EBC), and further focusing on the smaller subset of EBA that are continually exhaled. Dr Joachim Pleil of US EPA, Research Triangle Park, NC USA and Dr Jens Herbig of Ionicon Analytik, Innsbruck, Austria hosted this symposium that brought together experts regarding a series of new EBA applications. The first presentation by Dr Herbig discussed new technology for direct aerosols analysis using real-time proton transfer reaction-time-of-flight mass spectrometry (PTR-TOF MS). Professor Michael Davis from Virginia Commonwealth University Hospital Richmond VA, USA, presented safety considerations and diagnostic applications for collecting breath EBC and EBA directly from hospital ventilator circuits. Dr M Ariel Geer Wallace, of US EPA, Research Triangle Park, NC, USA presented a study regarding pre-clinical cancer diagnostics using EBA collected on standard hospital masks and compared case-control results using canine olfaction, immunochemistry, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-high resolution-mass spectrometry (LC-HR-MS). One of the newest promising technologies for non-targeted analysis of EBA and EBC using two-dimensional GC (GC × GC) and HR-MS was presented by Professor Heather Bean of Arizona State University, Phoenix, AZ, USA. The symposium finished with a talk by Professor Cristina Davis of University of California, Davis, USA describing new technology Micro-electro-mechanical systems for collecting and concentrating EBA directly for analysis. Overall, these talks spanned the range of the newest EBA technologies that were proposed in a recent perspective published in JBR (Pleil et al 2018).

Saliva workshop and conferee networking

The use of direct saliva collection and analysis is gaining traction as a non-invasive biological medium for environmental and diagnostic surveillance according to the organizer of these sessions, Professor Fabio DiFrancesco of University of Pisa, Italy. This new approach has a direct link to breath research, especially with respect to polar organic compounds that are partitioned in the gas–liquid interface of the mouth. Additionally, condensed phase analytes found in exhaled breath are also present in saliva. One unique feature of saliva is the direct linkage to circulating blood constituents through the salivary glands wherein biological molecules are transported from blood through passive diffusion across cell membranes, ultrafiltration through intercellular spaces or active transport through protein channels. The relative efficiency of these mechanisms depends on the sampling approach, and Dr Tommaso Lomonaco of University of Pisa, Italy showed how saliva composition may change upon use of different sampling procedures similarly to effects on breath sampling. Some of the current applications of saliva analysis are the assay of illicit and pharmaceutical drug levels, as well as for hormone levels. Therapeutic drug monitoring exploits the fact that only small molecules (<1900 Dalton) can reach saliva. Since most drugs in blood are bound to large proteins (e.g. albumin), salivary concentrations only mirror the unbound fraction in blood, which is the one effecting the pharmacological action.

Non-invasive and fast sampling makes saliva analysis appealing for roadside test of illicit drugs. Professor Erin Baker of North Carolina State University, NC, USA showed a high-throughput analytical method coupling solid phase micro-extraction (SPME) with ion mobility spectrometry-mass spectrometry for analysis of complex mixtures perfectly suitable for this application, whereas Professor Shannon Krauss of the University of Virginia, USA developed a centrifugal microfluidic system for the colorimetric detection of explosives and illicit drugs.

Because saliva is easily accessible like breath, it does not require specialized personnel or produce hazardous waste. These characteristics foster the use of sensors for self-monitoring purposes. Professor Fabio Di Francesco mentioned results of HEARTEN, an EU project developing sensor technologies measuring salivary lactate and breath acetone to prevent the flare up of heart failure crisis. The use of diagnostic saliva sensors was also discussed by Devon Pawley of University of Miami, Florida, USA who is developing lateral flow strips for the detection of salivary biomarkers related to Zika virus.

General breath-related presentations and posters

In a broadly technical conference like Pittcon, we expect to find a variety of breath-related topics represented among the thousand or more technical presentations and posters. We highlight a selection of these here:

Selected presentations

Dr Lara Phelps of US EPA, Research Triangle Park, NC, USA discussed some important research objectives of the agency, including the development of sensors for personal use, indoor/outdoor air quality, and industry/energy applications to monitor emissions. The development of new emission measurement tools, such as crowdsourcing odor applications, are being investigated at this time, providing opportunity for future breath related research.

The breath research from University Medical Center of Rostock, Germany, was well represented in a series of talks and poster presentations. Ann-Christin Bischoff investigated volatile organic compounds (VOC) emissions over stem cells in vitro using needle-trap micro-extraction, GC-MS and GCxGC-ToF for up to 200 substances. These results were amended by the flash talk and poster of Julia Bartels presenting data of VOC monitoring over mouse embryonic stem cells. Selina Traxler reported on the recognition of viral infections by means of VOC pattern in well-controlled animal models (pigs). This talk was complemented by a flash talk/poster presented by Radost Saß who applied NTD-GC-MS of VOCs over human cell cultures and demonstrated that co-infection with virus and bacteria had distinct effects onto VOC patterns over infected human cells.

Professor Boris Mizaikoff (Ulm University, Germany) presented current and future concepts based on mid-infrared (MIR) diagnostic platforms for sensing of breath patterns in condensed and vapor phase media at ppm-ppb levels. Recently emerging strategies use innovative waveguide technologies such as substrate-integrated hollow waveguides, and planar semiconductor waveguides in combination with highly efficient light sources to facilitate compact yet robust MIR platforms for label-free chemical and biological sensing.

Selected posters

Dr Laura McGregor (Sep-solve Analytical, Peterborough, UK) presented improved pre-concentration techniques in combination with secondary cold trap and GCxGC- ToF analysis with combined soft/ conventional ionization to improve sensitivity and enhance identification of biometric (VOC) patterns that could be used for case-control diagnostics.

Giovanni Pugliese (University Medical Center of Rostock, Germany) developed and optimized a PTR-TOF-MS based method for real-time determination of N-containing compounds in the breath. In a nutrition study, he demonstrated that breath VOC concentration changes may provide a non-invasive insight into protein metabolism.

Chantrell Frazier (Florida International University (FIU), Miami, FL, USA) used headspace (HS)-SPME-GC-MS VOC analyses in conjunction with LH-PCR to screen microbial communities at the same time point of VOC extraction, to determine the correlation between microbiota and the human scent profile. Optimization of these combined techniques could improve one touch DNA analysis in various areas of the body for both forensic and medical interest. In a related poster, Alice Breia Boone (FIU) showed that results of VOC analyses of body odors may be just as useful as fingerprints and DNA in the attempt to identify individuals.

Bio-informatics and metabolomics

In the broadest sense, measurements in any biological media need to be interpreted to address specific problems. The challenge is that even the most general non-targeted measurement methods will only detect subsets of all chemicals in a complex sample, depending on the particular sampling/analysis methods. In addition to such qualitative results, instruments and methods need to be tuned and calibrated to perform quantitative analyses requiring external standards. For example, Professor David Wishart of University of Alberta, Edmonton, Canada explained that LC-HR-MS analyses of human biofluids typically identify only about 10% of chemical features in complex samples such as human blood or plasma. Furthermore, only 160 million out of the 2.4 billion chemically feasible formulae (<2000 Dalton) are included in the PubChem/CAS Registry, and just 1.5 million LC/GC-MS spectra collected on ~15 different platforms are available. Dr Wishart developed the freely available Human Metabolome Database (http://hmdb.ca/). Dr Antony Williams, US EPA, NC, USA, presented an update of the US EPA CompTox Chemicals Dashboard which focuses on chemicals of interest to the EPA including substances in commerce, human metabolites, and many other types, many of which are related to human toxicity. To date, the CompTox Chemicals Dashboard has aggregated data for ~875 000 chemical substances including physicochemical properties, human hazard data, in vitro bioactivity data and links to other public website resources (https://comptox.epa.gov/dashboard). Additional databases for use in HR-MS non-targeted analyses were also presented, including DrugBank, FoodB, ContaminantDB, and BioTransformer as well as databases for prediction of drug metabolism, such as Meteor Nexus, ADMET Predictor, Metabolizer, and MetabolExpert.

e-cigarettes, PFAS, GSR and forensics

Other new topics from Pittcon 2019 that may ultimately impact breath research were related to external exposures. Conventional tobacco use, especially cigarette smoking, has long served as a baseline for inhalation toxicology and exhaled breath analysis, especially with respect to pre-clinical disease diagnostics. Additional consumer products considered at Pittcon 2019 were e-cigarettes. These are not as yet well-defined chemically and so provide a great opportunity for novel research. E-cigarette liquid flavorings have been found to contain varying levels of nicotine, the measured concentrations of which were found to differ from those posted on the label. Also of concern are the levels of ethanol that were detected in some of the e-cigarette liquids. The inhalation of ethanol through e-cigarettes could have significant consequences for individuals who are under the legal drinking age or trying to avoid consumption for other reasons. This could lead to unintentional alcohol consumption that could lead to impairment, vehicle accidents, and legal consequences.

The per- and polyfluoro-alkyl substances (PFAS) are a class of chemicals used to modify consumer products and clothing for anti-stain, anti-stick, and anti-wrinkle properties. They are also considered ubiquitous environmental pollutants that are generally measured in blood and environmental media. Posters at Pittcon presented novel measurement technologies for assessing PFAS exposures in EBC.

A third intriguing subject concerned assays of gunshot residue (GSR), body fluids, and sexual lubricants for criminal forensics. Discussions with researchers revealed that there may be blood and breath biomarkers of metals in GSR could be used to defeat countermeasures (washing) of skin wipes during analysis. Additionally, methods are being developed to distinguish between the microbiome signatures of seven common body fluids found at crime scenes, and male and female samples were successfully separated. Other researchers presented the creation of a database for sexual assault lubricants that was developed using direct analysis in real time-time of flight mass spectrometry (DART-TOFMS), GC-MS, and Fourier-transform infrared spectroscopy methods to classify lubricants based on unique components and chemical signatures. Rapid and accurate identification of body fluids and sexual lubricants will speed up forensic analyses during criminal investigations.

Opioids diagnostics

Some of the intriguing aspects of Pittcon are the ‘accidental’ encounters in the hallways between sessions, the exposition floor, or simply over coffee wherein we talk about speculative topics that have not as yet been presented. One of these is the use of exhaled breath to monitor opioids status, both for prescribed and for illicit usage. There is now new technology proposed for non-invasive monitoring of opioids chemicals and their metabolites in real-time, with potential applications for forensics and criminal justice. Based on these discussions, we have recruited the researchers to submit their work to JBR and hope to publish this research soon.

Summary

In this report, we have described a number of fascinating subjects, both directly and indirectly related to the JBR readership. We were particularly intrigued by the rapid advancement of publicly available databases for biological media, environmental and toxicological chemicals, and their metabolites, and their user-friendly interfaces. For example; in a recent article in JBR (Pleil et al 2018) we had used the DSSTox database to illustrate the methodology for unknowns in HR-MS analysis; at the time, this database contained ~33 000 curated compounds. At this writing, the number of curated entries has increased to 875 000! These databases are becoming a valuable tool for non-targeted analysis, especially for the breath community in research of condensed phase EBC and EBA samples using HR-MS analytical tools. Another interesting topic is the inclusion of saliva analysis along with blood, breath and urine; we now are developing an understanding of saliva as an additional window into circulating blood via the salivary glands.

Pittcon 2019 served as a platform for presenting new breath-related research to a wide audience from the analytical chemistry community. In return, we had the opportunity to gain insights from this eclectic collection of researchers to inspire ideas for future investigations and develop contacts with new colleagues across a wide range of research expertise.