Just Breathe: Detecting Diseases with Exhaled Breath

Title: Analysis of a Broad Range of Carbonyl Metabolites in Exhaled Breath by UHPLC-MS

Authors: Zhenzhen Xie, James D. Morris, Stephanie J. Mattingly, Saurin R. Sutaria, Jiapeng Huang, Michael H. Nantz, and Xiao-An Fu

Journal: Analytical Chemistry (ACS)

Year: 2023

Featured image by Brett Jordan from Flickr.

Our lungs do much more than inhale and exhale — your own breath can tell you if you’re susceptible to diseases. Detecting elevated levels of volatile organic compounds (VOCs) in exhaled breath can assist in identifying biomarkers of certain conditions such as adult asthma, chronic obstructive pulmonary disease, and lung cancer. This can lead to early detection and treatment of these diseases! Moreover, measuring exhaled breath for disease biomarkers is beneficial as the process is non-invasive and efficient.

Zhenzhen Xie and his research team from the University of Louisville (Louisville, KY) developed a novel ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS) method combined with a silicon microreactor for detecting carbonyl VOCs in exhaled breath.1 The method detected six subgroups of carbonyl compounds (two subgroups were identified for the first time), with acetone being the most abundant compound and aldehydes and saturated ketones being detected at higher levels than previous studies. This method has the potential for the quantitative detection of disease biomarkers.

Typically, gas chromatography-mass spectrometry (GC-MS) is used for analyzing relevant biomarkers, such as acetone, benzene, and ethanol, for detecting lung cancer in exhaled breath. However, using GC-MS requires sample pretreatment, which can cause loss of metabolites and contamination issues. Additionally, GC-MS analysis is not effective for real-time measurement, meaning the technique can’t tell what compounds are in the exhaled breath at that very moment. While other methods like proton transfer reaction-MS have been implemented to avoid these problems, these options don’t involve non-volatile chromatography or the use of spectral libraries to compare spectra results to database spectra. Advanced mass spectrometry methods such as Fourier transform-MS have been successful in precise analysis of exhaled breath. However, this method does not allow the separation of isomers, which distinguishes the differences between carbonyl compounds with the same atoms but different molecular arrangements.

Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS) provides chromatographic separations for more precise detection, access to MS spectral libraries for comparing experimental spectra to literature spectra, and potential for isomeric separations. Xie et al. used UHPLC-MS with a silicon microreactor to detect a wide range of carbonyl VOCs in exhaled breath and analyzed six subgroups of carbonyl compounds, compounds that are noted to have high levels in the breath of lung cancer patients.

To directly capture the carbonyl compounds, UHPLC-MS was combined with a silicon microreactor to “see” the compounds. For instance, you can’t actively see bubbles inside a glass of tap water. But when you blow into a straw in the glass, a quick reaction happens: bubbles rise and pop at the surface of the water. Similarly, the UHPLC-MS can’t “see” the carbonyl VOCs in exhaled breath without a reaction. Enter the “straw” in this research study: a silicon microreactor. When exhaled breath is blown onto the silicon microreactor, an oximation reaction occurs between the carbonyl compounds in the exhaled breath and the coating on the microreactor. The UHPLC-MS can then “see”, or detect, the carbonyl VOCs.

To make sure the silicon microreactor worked at an optimal level for measuring compounds in exhaled breath, the scientists performed a test run using ambient air. After spiking the air with deuterated carbonyl VOCs, researchers determined the microreactor’s capture efficiency decreased with an increase in flow rate and an increase in the molecular weight of the compounds. They decided to fix the flow rate at 7mL/min for >90% capture efficiency in the exhaled breath experiments.

Once the flow rate was set, the researchers put the UHPLC-MS method to the test. Twenty healthy human subjects exhaled into 1L Tedlar bags through Teflon tubes. After the collected breath traveled through the silicon microreactor to create measurable carbonyl VOCs, the compounds were washed out with methanol. UHPLC separated the VOCs, and tandem MS detected and confirmed the identities of the VOCs. The experimental method is graphically shown in Figure 1.


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Figure 1. Xie et al.’s graphical abstract of the UHPLC-MS experimental method: (1) patient exhales breath with carbonyl VOCs; (2) 2-(aminooxy)ethyl-N,N,N-trimethylammonium (ATM)-coated microreactor reacts with the compounds; (3) the compounds are oximated into adducts measurable by UHPLC-MS; (4) results are displayed in a chromatogram; (5) results are subsequently displayed mass spectrum (adapted from ref. 1).

The UHPLC chromatogram showed retention times of the carbonyl standards matched well with the carbonyl VOCs from the exhaled breath, meaning carbonyl compounds were indeed in the breath. The m/z measurements from the MS spectra for carbonyl standards (e.g. 58.0658 – 161.1284 m/z for hydroxy-acetaldehyde) also matched with the carbonyl VOCs from exhaled breath, such as shown in Figure 2. Additionally, researchers utilized fragmentation trees, such as shown in Figure 3, to further confirm the identity of the compounds.

Figure 2. Extracted chromatograms and MS spectra from a carbonyl standard (top) and exhaled breath sample (bottom) identified as (fragmented) hydroxy-acetaldehyde (reprinted from ref. 1). 

The most exciting discovery was that the method detected carbonyl isomers in exhaled breath – this had not been achieved by any other analytical method in the literature! Furthermore, hydroxy-aldehydes and hydroxy-ketones were detected for the first time, which are compounds both used as biomarkers for detecting lung cancer.

Acetone was the most detected VOC overall and formaldehyde was the most aldehyde VOC detected. Because acetone levels in the human body can be affected by factors such as diet, exercise, and pulmonary functions, acetone is a relevant biomarker for diagnosing lung cancer and diabetes. Additionally, aldehyde detection was higher in exhaled breath in this research study compared to previous studies. Aldehydes are relevant biomarkers for pulmonary diagnoses including lung cancer and chronic obstructive pulmonary disease (COPD). Finally, more saturated ketones were detected in exhaled breath compared to previous studies – ketones are also relevant as lung cancer biomarkers.


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Figure 3. Fragmentation pathway tree based on the MS/MS spectrum of heptanal, a biomarker for detecting lung cancer. Each tree “branch” points to a fragment of the heptanal compound (reprinted from ref. 1).

Xie et al. utilized UHPLC-MS coupled with a silicon microreactor to detect carbonyl volatile organic compounds relevant for identifying respiratory diseases in exhaled breath. Six subgroups of carbonyl compounds were identified, including two subgroups previously unidentified in the literature. The method achieved higher detection of relevant biomarker compounds such as acetone and saturated ketones. This novel method has a high potential for quantitative analysis of carbonyl VOCs in exhaled breath. Providing accurate quantification of these biomarkers may improve breath analysis for determining symptoms of conditions in lung cancer, COPD, and other respiratory diseases.


Figures reprinted with permission from: Xie, Z., Morris, J., Mattingly, S., Sutaria, S., Huang, J., Nantz, H., and Fu, X. A Novel Strategy Based on Permanent Protein Modifications Induced by Formaldehyde for Food Safety Analysis. 2023. Analytical Chemistry. Copyright 2023. American Chemical Society.

Reference 1: Xie et al. Analysis of a Broad Range of Carbonyl Metabolites in Exhaled Breath by UHPLC-MS. Anal. Chem. 2023, 95(9), 4344-4352.

DOI: https://doi.org/10.1021/acs.analchem.2c04604

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