Breath and Blood Metabolomics: A Comparative Study Using SESI-HRMS/MS and UHPLC-ESI-HRMS/MS

Abstract

Breath metabolomics enables noninvasive and rapid acquisition of metabolic information by detecting volatile organic compounds (VOCs) in exhaled breath. Secondary electrospray ionization high-resolution tandem mass spectrometry (SESI-HRMS/MS) offers the highest coverage for detecting breath metabolites among current real-time breath analysis techniques. Although it has been generally recognized that metabolites in breath originate from the blood, a molecular-level understanding of the characteristics of metabolites in both breath and blood remains insufficient. In this study, nontargeted analyses of breath and blood samples from 11 healthy volunteers were performed using SESI-HRMS/MS and ultrahigh performance liquid chromatography electrospray ionization high-resolution tandem mass spectrometry (UHPLC-ESI-HRMS/MS), respectively. Tandem mass spectrometry was employed for metabolite annotation. Twenty-six breath-unique metabolites and 73 blood-unique metabolites were identified. Besides, seven metabolites were found in both breath and blood, including 7-oxabicyclo [2.2.1] heptane, levulinic acid, indole, pyroglutamic acid, malic acid, glutamic acid, and histidine. Intriguingly, the correlation of these metabolites between breath and blood was low (r < 0.4 or p > 0.05). Among all the confirmed metabolites, breath metabolites exhibit higher volatility according to their water-gas partition coefficient (log P _w/g) compared to blood metabolites. In addition, gender-derived differences in breath were significantly smaller than blood. In summary, this study indicates that breath metabolites are likely to offer complementary information on blood metabolites. When combined with blood metabolomics, this would be advantageous for the appropriate application of breath metabolomics in life sciences, such as in biomarker discovery.

Keywords: SESI-HRMS/MS; UHPLC-ESI-HRMS/MS; blood metabolomics; breath metabolomics; metabolite identification; metabolomics comparison.

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Breath (top) and blood (bottom) metabolomics study flow. Breath and blood samples were collected from 11 subjects (6 males and 5 females). Breath samples were analyzed in real-time using SESI-HRMS/MS, which was completed within 10 min for each sample. The m/z-int matrix was obtained via BreathXplorer. Blood samples underwent pretreatment and were detected by UHPLC-ESI-HRMS/MS. The entire process took approximately 16 h. The m/z-int matrix was extracted using MS-DIAL. Metabolites in both breath and blood were identified based on MS/MS matched to the NIST20 and MoNA databases. The identified metabolites were then utilized to investigate the similarities and differences between breath and blood metabolomics. Finally, an analysis of differences in metabolome between males and females was conducted.

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(A) Exact mass distributions of breath and blood features; (B) intensity distributions of breath and blood features; (C) Venn analysis of exact mass of breath and blood features.

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(A) Comparison of seven features with identical MS/MS in breath and blood and (B) MS/MS match results of glutamic acid and histidine in breath and blood with NIST20 and MoNA database.

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Spearman correlation analysis of nine features (seven identified and two nonidentified) detected in both breath and blood.

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Distribution of exact mass and log P _w/g of breath, blood, and common metabolites.

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PLS-DA score plot between males and females in (A) breath and (B) blood.