Breath Biopsy: combining Thermal Desorption-Gas Chromatography with High Resolution Mass Spectrometry for improved sensitivity and selectivity in untargeted breath analysis

Importance of the Topic

Breath analysis of volatile organic compounds (VOCs) offers a non-invasive window into human metabolic processes. Untargeted metabolomic profiling of exhaled breath can reveal biomarkers for disease diagnosis, monitoring and environmental exposure assessment. However, the complexity of breath matrices and the wide concentration range of VOCs demand highly sensitive and selective analytical methods.

Objectives and Study Overview

This study introduces an analytical workflow combining thermal desorption–gas chromatography with a high-resolution Orbitrap mass spectrometer for untargeted breath analysis. The main goals were to demonstrate:

• Reliable pre-concentration and capture of breath VOCs
• Extensive dynamic range and ppt-level sensitivity in a single run
• Improved compound identification and quantitation for metabolomic applications

Methodology

Breath sampling was performed using a ReCIVA Breath Sampler to collect 1.5 L of exhaled air onto proprietary cartridges. Samples underwent thermal desorption in a TD100-xr autosampler, transferring analytes onto a 30 m × 0.32 mm column via splitless injection. Chromatographic separation used a TRACE 1310 GC oven with a temperature program optimized for VOC resolution. Mass spectral acquisition employed an Exactive GC Orbitrap system, operating at 60 000 resolving power across five orders of magnitude dynamic range. Both variable-energy electron ionization (VeV EI) and positive/negative chemical ionization (CI) modes were applied. Data processing integrated Thermo Scientific Xcalibur 4.0 and TraceFinder 4.1 software with High Resolution Filtering (HRF) and retention index scoring to reduce false positives and enhance identification confidence.

Instrumentation Used

• ReCIVA Breath Sampler with pressure and CO₂ monitoring
• Breath Biopsy Cartridges for VOC pre-concentration
• TD100-xr thermal desorption autosampler
• TRACE 1310 GC oven and Quadrex column (30 m × 0.32 mm × 3.00 µm)
• Exactive GC Orbitrap mass spectrometer with VeV EI and CI capabilities
• Thermo Scientific Xcalibur 4.0 and TraceFinder 4.1 software

Main Results and Discussion

Mass accuracy better than 1 ppm was consistently achieved across chromatographic peaks, enabling precise deconvolution and differentiation of co-eluting analytes. Ion ratios remained stable over a wide concentration range, supporting reliable quantitation. The application to 12 breath samples stratified by smoking status demonstrated high fold changes for 2,5-dimethylfuran and toluene in current smokers, confirming these as robust smoking markers. High-resolution filtering and retention-index matching substantially reduced false positive features, yielding dense yet curated feature lists suitable for untargeted metabolomics.

Benefits and Practical Applications

• Simultaneous detection of high-abundance and trace VOCs in one analysis
• Enhanced specificity through sub-ppm mass accuracy and alternative ionization
• Robust feature curation with HRF and retention index scoring
• Effective biomarker discovery demonstrated for smoking status differentiation
• Scalable approach for clinical metabolomics, QA/QC and environmental monitoring

Future Trends and Opportunities

Advances may include integration of additional ionization techniques to further elucidate molecular structures, real-time breath analysis for dynamic monitoring, and application of machine-learning algorithms to high-density data sets. Clinical validation studies could expand breath biopsy to broader disease biomarker discovery and personalized health monitoring.

Conclusion

The combined thermal desorption–GC Orbitrap platform delivers unprecedented sensitivity, selectivity and dynamic range for untargeted breath metabolomics. High-resolution filtering and complementary ionization modes enhance compound identification and data quality, positioning the workflow as a powerful tool for biomarker discovery and non-invasive diagnostics.

Reference

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2. S. Capone et al. Chromatographic Analysis of VOC Patterns in Exhaled Breath from Smokers and Nonsmokers. Biomed. Chromatogr. 2018, 32(4), 1-11.
3. S. M. Gordon et al. Volatile Organic Compounds as Breath Biomarkers for Active and Passive Smoking. Environ. Health Perspect. 2002, 110(7), 689-698.
4. M. Castellanos et al. 2,5-Dimethylfuran as Validated Biomarker of Smoking Status. Nicotine Tob. Res. 2018.