New breath related topics: sample collection for exhaled breath condensate and aerosol, development of real-time medical alerts, measurement of artificial atmospheres, and analysis of legalized cannabis product

Abstract

Exhaled breath technology is expanding beyond conventional gas-phase analysis, and conversely, methodology from other disciplines is finding applications in breath research. Recently, the authors attended conferences that incorporated new technologies into ‘breath related’ applications. The first was the International Submarine Air Monitoring and Air Purification (SAMAP) held in Uncasville Connecticut, November 2017, and the second was the Pittcon Conference and Exposition (Pittcon) held in Orlando, Florida, February 2018. Herein, we report some of the new topics and ideas encountered, ranging from very specific submarine related res

Background

The use of exhaled breath as a bioanalytical fluid is expanding beyond conventional gas-phase analysis, and conversely, methodology from other disciplines is finding applications in breath research. As such, branching out beyond the parochial bounds of focused breath meetings has value in developing the field. Recently, the authors attended conferences that incorporated new technologies into ‘breath related’ applications. The first was the International submarine air monitoring and air purification (SAMAP) held in Uncasville Connecticut, November 2017, and the second was the Pittcon Conference and Exposition (Pittcon) held in Orlando Florida, February 2018. They essentially span the range from small/focused to giant/eclectic. Neither of these conferences focused on biomonitoring, yet both had content relevant to exhaled breath technologies. In the following accounts, we report on those topics that might stimulate thought on breath research based on highlights of exposition booths and presentations encountered by the authors, as well as myriad personal discussions with attendees.

SAMAP

SAMAP is the international forum for military submarine technology; the meeting is held every year or two, depending on availability of a host organization; in 2017, the meeting took place near the Submarine Naval base in Groton Connecticut with the support of US Navy.

The subtitle was ‘Resurgence in Submarine Shipbuilding’ which is a reference to the current US Navy program in building Virginia class fast-attack submarines (2 per year) to a planned total of 49 submarines by 2032, replacing the existing Los Angeles class and gearing up for building the newest Columbia class ballistic missile submarines starting in 2021. Also of significance, the British Royal Navy is currently constructing their Astute class submarines and the French Navy is building Barracuda class nuclear attack submarines. The Royal Australian Navy is embarking on a replacement for the Collins class submarines with large conventional submarines based on the shortfin French Barracuda class, which are expected to enter service in the early 2030s. Since 2005 the Japanese Navy has been building a new Sōryū class of diesel-electric submarines of their own design with Kockums Stirling engine air-independent propulsion for extended dived endurance. As such, new engine and life support technologies are becoming a major topic in the international submarine faring community, and provide an intriguing look into human sustainability in artificial environments.

Of breath related subjects, the exhalation and subsequent removal of carbon dioxide (CO₂) and the production of oxygen (O2) are the most prevalent issues in SAMAP conferences. However, over the years, exposure monitoring, breath analysis, and pulmonary measurements have become important topics as well, demonstrating the reciprocity in the submarine and breath research communities (Pleil and Hanzel 2012). SAMAP also encompasses artificial atmospheres control in other applications, in particular for space flight missions. In fact, breath analysis may take on an increasingly important role in long-term space flight (e.g., International Space Station (ISS) and Mars mission) for monitoring human health (NASA 2017). This year’s meeting had a series of presentations introducing novel air monitoring to the ISS as well retrospective considerations as to the continuing progress of analytical equipment designed for environments with power, size, and resupply constraints. As such, the focus was on the adaptation of commercial off the shelf technologies, especially chemical and optical sensors. We observed that the development of rugged/miniaturized air monitoring devices required onboard military submarines and spacecraft may also serve as a valuable research resource for exhaled breath monitors, especially those for outpatient and at home use.

Biomonitoring played a small role at this meeting. Some of the presentations included dermal exposure and bio-measurements of diesel and torpedo fuel (Otto fuel), as well as lubricating and hydraulic fluids, and the various CO₂ scrubbers including monoethanolamine (MEA), soda lime and lithium hydroxide. Additionally, the concept of monitoring exposure and health state using exhaled breath aerosol (EBA) was presented, as were the relationships among exposures and pulmonary function variables.

Pittcon meeting

Pittcon is the largest analytical chemistry meeting in the United States, attracting about 18 000–20 000 attendees each year from all over the world. Like SAMAP, the focus at Pittcon is not on breath or biomarkers. Despite that, the sheer size guarantees that all manner of instrumentation, methods, studies, etc are presented that are related to breath biomonitoring. In addition, Pittcon also has an extensive exposition of 2000–3000 vendors wherein attendees can interact with hardware and technical experts; the graphic below shows just a small part of the exposition floor.

The Pittcon meeting is comprised of technical talks, poster sessions, instrument exposition, and conferee networking sessions. In addition, there are hands-on demonstrations, plenary lectures, and awards ceremonies. Herein, we touch upon some of the more relevant events related to breath analysis, chosen subjectively by the authors. This article is part of a long series of Pittcon reports going back to 2010. The two most recent, from Pittcon 2016 and 2017, have focused on breath topics regarding cellular respiration and National Security applications, respectively (Pleil et al 2016, 2017). This report has more focus on new technologies dealing with exhaled breath condensate and aerosols (EBC and EBA), as well as a selection of instrumentation-based advances, medical topics, and concepts regarding point of use analyses that will likely influence the future of breath research. In the following, we describe topics from the conference of particular interest to the authors.

Exhaled breath condensate and aerosols

The implementation of condensed phase breath was discussed in assorted platforms and poster sessions. One presentation showed that aerosols could be extracted from various mask materials and analyzed for semi- and non-volatile compounds that represent health state and potential exposures. This technique is advantageous because it is non-invasive, and sampling can be conducted with little inconvenience to the subject/patient. An assortment of compounds was observed in the mask materials, including fatty acids, cytokines, drugs, and exogeneous compounds from commercial products, such as plasticizers. Other relevant presentations related inflammatory cytokines in breath aerosols with changes in cardio-pulmonary function and demonstrated a new extractive technique for EBC and other complex liquids using a refluxing approach that efficiently extracts highly polar organic compounds from aqueous matrices.

Medical applications

Technologies were presented that have direct breath application for assessing medical state and tracking health state. One presentation discussed instrumentation to monitor the breath of drivers to warn of impending adverse events such as drowsiness, epileptic seizure or diabetic event. Breath biomarkers of drowsiness and fatigue were reported, identifying potential VOC biomarkers that can be monitored using a sensor placed on the steering wheels of cars as a safety function. Another presentation discussed occupational exposure monitoring of firefighters using wristbands worn at the station and during active fires. Air concentrations measuring ppm levels of VOCs and polycyclic aromatic hydrocarbons (PAHs) above regulation were detected, indicating improvements were needed for firefighter breathing apparatus and protective gear. Another presentation described a breath analysis system for real-time tracking of anesthetics metabolites (in this case prilocaine) to control dose administration. The main metabolite of prilocaine, o-toluidine, was monitored in pig models using PTR-TOF-MS. A method for detection of aliphatic amines by PTR-TOF-MS was also reported. Amines in breath can be indicative of disease state, but they are difficult to analyze due to their high reactivity. The newly developed method resulted in successful analysis of trimethylamine as well as methyl- and di-methylamine. A presentation discussing how breath biomarkers can be used to detect pathogens for biosecurity applications was also of interest. The headspace of bacteria was monitored to detect bacteria-specific volatiles, and volatile nitrogen- and sulfur-containing compounds were detected as well as methyl ketones and alcohols.

Mass spectrometry (MS)

MS advances have often occurred to foster breath applications that rely on reactive metabolites. The newest methods are now coupling real-time analysis with high resolution MS (HR-MS) to provide rapid and accurate identifications without the need for chromatography. HR-MS is an emerging technology for breath analysis as it provides an additional dimension to standard identification strategies (Pleil and Isaacs 2016). A hands-on demonstration by Ionicon was provided on the exposition floor wherein a real-time system based on proton transfer reaction (PTR)-MS allowed attendees to test their own breath. LECO introduced innovative time-of flight technology that could be interfaced with GC (gas chromatography) and GC × GC instrumentation; Shimadzu Scientific Instruments Inc. demonstrated a variety of MS based instruments for organics analysis.

Cannabis analysis research

In a previous Pittcon (2016), there was a concerted effort to develop impairment diagnostics for automobile drivers under the influence of cannabis, analogous to the breath tests for alcohol intoxication. In 2018, the focus shifted to testing cannabis as a consumer product. In the US, 9 states have legalized marijuana for recreational use and a total of 29 states now have some form of decriminalized or legalized medical marijuana. Similarly, many countries now have decriminalized cannabis, or have stopped rigorously enforcing existing statutes. As such, US States and international jurisdictions are now requiring cannabis safety and potency analyses for which the analytical chemistry community is developing laboratory tests based on various MS instrumentation. There were whole sessions devoted to cannabis testing methodology, including genetic strain, identification of terpenes, plant metabolites, adjuvant pesticides and herbicides, and the content of cannabinoids, especially the psychoactive ingredient, tetrahydrocannabinol (THC). Current research includes potency testing and semi-/non-targeted analysis of confounding compounds such as pesticides using LC- and GC-MS/MS and high resolution time-of-flight (TOF)-MS. Significant inconsistencies have been observed between cannabinoid potency values reported by commercial labs, revealing the need for accurate and consistent testing methods. A diode array detection method was proposed to reduce the interference of terpenes during cannabinoid potency testing. Throughout these presentations, there was an undercurrent of thought regarding the ultimate approaches for detecting impairment; however, the major focus right now is on identifying the product and its chemical composition. This work will ultimately have implications for breath analysis, as biomarker compounds besides THC will be required to assess intoxication because THC is too long lived in the human system to serve as a probative marker of current status.

Conferee networking

Conferee networking sessions are an interesting concept at Pittcon conferences. The general idea is to have a thought leader in a field propose a session, and then the conference lists it as part of the program. Some of the featured sessions this year were:

• Quality in the cannabis industry

• Non-invasive Biomedical Analysis—How Can Innovative Instrumentation Promote Biomedical Research And Medical Applications?

• Detection Techniques For Chemical Contaminants And Pesticides In Food And Pharmaceutical Raw Material

• Expediting Method Development in Pharma

• Challenges in Managing Analytical Instruments in An Academic Environment

• Challenges of the Fentanyl Epidemic

These sessions represent the microcosm of trending topics for the current year. As mentioned above, the quality assurance of the newest consumer product, (or pharmaceutical), cannabis, is of high interest. The second topic was of particular interest to the breath research community: non-invasive Biomedical Analysis—How Can Innovative Instrumentation Promote Biomedical Research and Medical Applications? was organized and hosted by Jochen Schubert (Rostock Medical University, Germany), a founding member of JBR and the International Association of Breath Research. The participants discussed new instruments that could have profound implications to the broadening practice of breath analysis for outpatient testing, in particular optical spectroscopy, immunochemistry, electrochemical sensor arrays, e-noses, and other technologies that could be miniaturized to bring to the subject/patient. They did not lose sight of the standard technologies such as real-time MS, gas chromatography, etc, however the consensus was that the future is awaiting methods with utility that are fairly easy to use and relatively inexpensive.

A second central issue was the regular use of breath related technologies by the medical community. Certainly the research community has embraced breath as an important biological medium. However, mainstream (Western) medicine tends to be very conservative, and it is difficult to get instruments close to patients especially in the operating room. Furthermore, there are concerns about privacy, collection, and use of metadata, as well as the need for Institutional Review Board approvals for developing mainstream methods for medicine. The consensus, however, was that these obstacles are worth overcoming; real-time and bedside analyses are of great value in efficient monitoring of health state, and breath is a preferred biological medium for its non-invasive and easily accessible properties.

Ensuing discussions considered the broad view of the purpose of breath testing instruments. The participants created the following list:

• Diagnostics: case-control and pattern recognition-based methods for disease prediction before standard medical tests can.

• Environmental assessments: determining internal dose, environmental contamination, and protecting public health.

• Infections: preclinical diagnosis and subsequent monitoring progression of antibiotics treatment.

• Tracking medication: non-invasive monitoring of pharmaceuticals treatment.

• Rapid onset events: predicting convulsion, seizure, fainting (syncope), heart attack, psychotic break, and stroke.

• Chronic disease: monitoring health state; assessing treatment response for cancer, asthma, cardiovascular disease, kidney disease, inflammation, and chronic obstructive pulmonary disease.

These subjects are a broad wish list for future research. The consensus was that one of the biggest challenges is gaining acceptance of new technologies at the medical practitioner’s level, and that training and standardization with new instrumentation will be required at the institutional level.

Sampling technologies

The automation of sample recollection onto sorbent tubes was described by various researchers. Of note was one application wherein PAHs could be recovered from sorbent tubes with minimal carryover. Such recoveries are an import step forward for the analysis of semi-volatile compounds from the environment and from breath aerosols. Several sessions and talks featured improvements and current applications of solid-phase microextraction (SPME). New coatings for SPME fibers to improve retention of high molecular weight compounds were investigated, as were new technologies for rapid analysis of small sample volumes using SPME-MS.

Summary

Both SAMAP and Pittcon conferences offer opportunities for attendees to encounter different viewpoints that could lead to valuable contributions to their areas of research. For example, the concepts from the submarine community revolve around the technology of dealing with the balance of inhaled O₂ and exhaled CO₂; this has direct implications for breath research in inhalation toxicology and environmental exposures, especially for the pharmacokinetics of metabolism. The concerns stemming from an artificial and confined environment extend beyond oxygen content; there are no opportunities to ‘open the window’ to get fresh air, or to go to the manufacturer for resupply of expendables or spare parts. From SAMAP, we learn how to ruggedize instruments, build simpler and smaller machines, and reduce resupply requirements.

In contrast to the specific focus of SAMAP, Pittcon is much more eclectic. Here, one never knows where the next inspiration will strike. At Pittcon 2018, the concepts of non-invasive monitoring, cannabis analysis, and medical alerts were the most intriguing for the authors in relation to breath research. The opportunity to self-test ones own breath using a real-time PTR-MS instrument was also enlightening. Overall, Pittcon allows the random walk through analytical science that inspires new ideas and applications.