Identifying organic compounds in exhaled breath aerosol: Non-invasive sampling from respirator surfaces and disposable hospital masks

Highlights

• A method for exhaled breath aerosol collection was developed using masks materials.

• Surgical-style paper and hard-surface plastic masks were investigated.

• Elevated cytokines showed that human compounds were extracted from masks.

• Compounds were from industrial, medical, food, pesticide, and care product sources.

Abstract

Exhaled breath aerosol (EBA) is an important non-invasive biological medium for detecting exogenous environmental contaminants and endogenous metabolites present in the pulmonary tract. Currently, EBA is typically captured as a constituent of the mainstream clinical tool referred to as exhaled breath condensate (EBC). This article describes a simpler, completely non-invasive method for collecting EBA directly from different forms of hard-surface plastic respirator masks and disposable hospital paper breathing masks without first collecting EBC. The new EBA methodology bypasses the complex EBC procedures that require specialized collection gear, dry ice or other coolant, in-field sample processing, and refrigerated transport to the laboratory. Herein, mask samples collected from different types of plastic respirators and paper hospital masks worn by volunteers in the laboratory were analyzed using high resolution-liquid chromatography-mass spectrometry (HR-LC-MS) and immunochemistry. The results of immunochemistry analysis revealed that cytokines were collected above background on both plastic respirator surfaces and paper hospital masks, confirming the presence of human biological constituents. Non-targeted HR-LC-MS analyses demonstrated that larger exogenous molecules such as plasticizers, pesticides, and consumer product chemicals as well as endogenous biochemicals, including cytokines and fatty acids were also detected on mask surfaces. These results suggest that mask sampling is a viable technique for EBA collection to assess potential inhalation exposures and endogenous indicators of health state.

Introduction

Breath-based testing has a long history focused on analyzing gas-phase compounds for environmental exposures or for indications of health-state via preclinical biomarkers (Pleil, 2016; Wallace & Pleil, 2018). More recently, the concept of condensed-phase breath collection, referred to as exhaled breath condensate (EBC), has extended the analyte range beyond organic gases to include water-soluble inorganic constituents, as well as a wide range of compounds that are otherwise difficult to collect due to polarity and/or low volatility (Ahmadzai et al., 2013; Kubáň & Foret, 2013; Zamuruyev et al., 2018). EBC has been collected to assess airway pH for tracking asthma status (Accordino et al., 2008; Morton, Henry, & Thomas, 2006; Ojoo, Mulrennan, Kastelik, Morice, & Redington, 2005). A wide variety of compounds are also part of the EBC fluid that could complement, or even replace some blood or urine assays (Stiegel, Pleil, Sobus, Morgan, & Madden, 2015; Wallace, Kormos, & Pleil, 2016). EBC became a robust biological medium used for a wide variety of environmental and clinical biomonitoring applications (Barbara et al., 2018; Marie-Desvergne et al., 2016; Peralbo-Molina, Calderón-Santiago, Priego-Capote, Jurado-Gámez, & Castro, 2016a; Sauvain, Hohl, Wild, Pralong, & Riediker, 2014).

Certainly, EBC has some logistical advantages over blood sampling in that there is little, if any, infectious waste generated, and that no specialized medical personnel are required. However, there are some challenges for collecting EBC in the field that revolve around infrastructure and subject/patient time. EBC sample collection requires specialized equipment, dry ice or other coolant, and a clinical/laboratory space to process frozen condensate into liquid samples (Wallace & Pleil, 2018; Zamuruyev et al., 2018). Subjects are required to perform breathing maneuvers generally for 10 min, each with previously sterilized sampling gear. Although the resulting EBC samples are of low-volume (1–2 mL of liquid), like blood, they need to be shipped to the laboratory at sub-ambient temperature to maintain integrity (Ahmadzai et al., 2013; Wallace & Pleil, 2018).

The proposed solution to streamlining EBC sampling is based on the observation that many of the analytes of interest are likely entrained in liquid aerosols formed during exhalation, and thus could be expected to form films on surfaces or become entrained within filters. Termed exhaled breath aerosol (EBA), it represents a small fraction (<0.1%) of the EBC containing larger organic constituents (Hayes et al., 2016). Aerosols have been classified as particles ≤5 μm, while respiratory droplets are those >5 μm (Duguid, 1946; Siegel, 2007; Zhang, Leung, Cowling, & Yang, 2018). So, even when dried, it was presumed that EBA films and deposits would retain the semi- and non-volatile fraction of EBC. Certainly, polar volatile organic compounds (PVOCS) and robust measures of pH would be lost in this method, but the strategic advantages could outweigh the loss. The philosophical aspects of EBC and EBA sampling have been described in the literature (Pleil & Wallace, 2018; Pleil, Wallace, & Madden, 2018; Wallace & Pleil, 2018). Traditional methods for EBC sampling typically involve breathing through a long, chilled tube to condense the aqueous fraction of exhaled breath (Ahmadzai et al., 2013; Davis, Fowler, & Montpetit, 2019; Kubáň & Foret, 2013; Mutlu, Garey, Robbins, Danziger, & Rubinstein, 2001; Wallace & Pleil, 2018; Winters et al., 2017). Methods for EBA sampling include collection on filters, use of commercial sampling devices, and particle impaction (Beck, Olin, & Mirgorodskaya, 2016; Kintz, Mathiaux, Villéger, & Gaulier, 2016; Mikheev & Morozov, 2018; Seferaj et al., 2018; Wallace & Pleil, 2018). Fig. 1 is a diagram of how all breath constituents relate.

Based on previous research using EBC, it is expected that certain groups of analytes would be preserved in EBA samples. These non-volatile particles are contained within the fraction of EBC, as shown in Fig. 1. The most likely compounds are the heavier (non-volatile) organic molecules representing exogenous exposures to pesticides, consumer products (fragrances, cosmetics), cooking, drugs/medications, combustion sources (fuel, cigarettes), and the various endogenous metabolic compounds representative of human biology, including fatty acids, proteins, surfactants, and cytokines in part derived from lung epithelial lining fluid (Beck, Stephanson, Sandqvist, & Franck, 2013; Davis, Fowler, & Montpetit, 2019; Ljungkvist et al., 2015, 2017; Oldham et al., 2017; Phares, Collier, Zheng, & Jung, 2017; Soares et al., 2018; Tinglev et al., 2016; Trefz et al., 2017; Ullah, Sandqvist, & Beck, 2018). In addition, EBA contains protein and DNA remnants of organisms (e.g., bacteria, viruses, fungal spores) that could be indicative of infection (Heo, Lim, Kim, & Lee, 2017; Morozov et al., 2018; Patrucco et al., 2019; Yao, 2018; Zhang et al., 2018; Zheng, Chen, Yao, & Li, 2018). There is also the possibility that semi-volatile compounds may be collected on mask surfaces, such as aldehydes, alcohols, and other organic compounds that have been previously observed in EBC (Peralbo-Molina, Calderón-Santiago, Priego-Capote, Jurado-Gámez, & De Castro, 2016b; Peralbo-Molina et al., 2016a; Pleil, Hubbard, Sobus, Sawyer, & Madden, 2008).

In our laboratory, the original concept for exploiting EBA evolved from solving practical and logistical problems in biomonitoring. The initial work along these lines came from the observation that disposable filters from pulmonary function instruments (spirometers) might be a valuable resource for assessing infections. As these filters are routinely discarded in a clinical setting, they appeared to be a pragmatic source of interesting biological specimens. A second application was derived from the evaluation of exposures and health states from occupational settings wherein standard biomonitoring could not be done easily. For example, it is not practical to intrude on firefighters or military pilots during their activities, but one could easily sample the internal surfaces of their masks afterwards. Finally, we found that standard disposable paper masks worn in hospitals could be used as a non-invasive sampling method for the general public. The masks only need to be worn for 10 min to collect EBA and do not affect normal activities. An overview of these applications has been published recently (Pleil et al., 2018).