Breath Biopsy with TD-GC-Orbitrap: a non-invasive approach for disease detection

Importance of the Topic

Breath Biopsy using thermal desorption–gas chromatography–Orbitrap mass spectrometry offers a non-invasive window into human metabolism by measuring volatile organic compounds (VOCs) in exhaled breath. As metabolic changes precede overt symptoms, breath analysis enables earlier disease detection, treatment monitoring and exposure assessment without the discomfort or risk of blood or tissue sampling. The high sensitivity and specificity of Orbitrap-based detection extend the reach of breathomics to ultra-trace biomarkers, supporting precision diagnostics and personalized health management.

Objectives and Study Overview

This white paper aims to describe a comprehensive workflow—from breath collection through data analysis—leveraging the ReCIVA™ Breath Sampler, Breath Biopsy Cartridges and Q Exactive™ GC Orbitrap mass spectrometer. A case study comparing breath VOC profiles of current smokers, ex-smokers and never-smokers illustrates the platform’s ability to discover and quantify smoking-related markers. Key goals include demonstrating:

• Standardized, reproducible breath sampling
• High-resolution accurate mass (HRAM) detection of VOCs over a wide dynamic range
• Parallel untargeted discovery and targeted quantitation workflows
• Data processing strategies for confident feature deconvolution and compound identification

Methodology

Subjects provided exhaled breath into the ReCIVA sampler, which monitors CO₂ and pressure to capture specific breath fractions (alveolar, upper airway or total). VOCs were pre-concentrated on sorbent-based Breath Biopsy Cartridges and thermally desorbed into a TRACE™ 1310 GC configured with a 30 m × 0.32 mm × 3 µm column. The Q Exactive GC Orbitrap acquired full-scan data (m/z 35–350) under electron ionization (EI) and chemical ionization (CI) at 60,000 resolving power, ensuring sub-1 ppm mass accuracy. Compound Discoverer™ 3.2 performed peak deconvolution, statistical analysis and high-resolution filtering; Chromeleon™ software enabled routine targeted quantitation and spectral confirmation.

Instrumentation

• ReCIVA™ Breath Sampler with CASPER™ Portable Air Supply for controlled inhalation
• Breath Biopsy Cartridges (Tenax TA/Carbograph sorbents)
• TD100-xr Thermal Desorption Autosampler (Markes International)
• Thermo Scientific™ TRACE™ 1310 Gas Chromatograph with TriPlus™ RSH Headspace Autosampler
• Thermo Scientific™ Q Exactive™ GC Orbitrap Mass Spectrometer
• Thermo Scientific™ Compound Discoverer™ 3.2 Software
• Thermo Scientific™ Chromeleon™ Chromatography Data System

Main Results and Discussion

The platform achieved consistent mass accuracy (<1 ppm) across peak profiles and a dynamic range exceeding six orders of magnitude, enabling reliable detection from ultra-trace to high-abundance VOCs. In the smoker study (n=12), untargeted analysis generated >500 features per sample, streamlined by HRAM filtering and retention index matching. Targeted quantitation of a six-compound smoking library revealed strong correlations for 2,5-dimethylfuran and toluene in current smokers, while other markers showed low fold change versus controls. The combination of full-scan HRAM data and retrospective analysis allows in-depth mining of metabolic fingerprints and flexible hypothesis testing.

Benefits and Practical Applications

• Non-invasive, pain-free sampling without biohazardous waste
• Detection of current metabolic state rather than genetic predisposition
• High sensitivity and specificity for early-stage disease biomarkers
• Simultaneous untargeted discovery and targeted screening from the same sample
• Retrospective data interrogation and expansion of compound panels
• Robust workflow supporting clinical research, QA/QC and precision medicine

Future Trends and Opportunities

Advances in softer ionization (e.g. variable C-Trap voltages, positive/negative CI) will enhance molecular ion detection for improved identification of isobaric compounds. Ongoing stability studies will refine breath sample storage protocols at –80 °C to widen deployment in multi-site trials. Integration of machine learning for feature selection and biomarker panel optimization can accelerate discovery of disease-specific signatures. Miniaturized, field-deployable GC-Orbitrap systems may enable point-of-care breath diagnostics. Expanding breathomics beyond smoking to respiratory, metabolic, oncological and infectious diseases holds promise for broad clinical impact.

Conclusion

The Breath Biopsy workflow combining ReCIVA sampling, TD-GC separation and Orbitrap HRAM detection delivers a powerful, standardized approach for breath VOC analysis. Its ability to uncover ultra-trace biomarkers with high confidence, coupled with flexible data mining and routine screening capabilities, paves the way for early non-invasive disease detection and personalized monitoring.

Reference

1. ERS Task Force on Exhaled Biomarkers in Lung Disease Technical Standard, European Respiratory Society, 2017.
2. Harshman SW et al., “Storage stability of exhaled breath on Tenax TA,” Journal of Breath Research, 2016, 10(4):046008.
3. Kang S, Thomas CLP, “Stability of VOCs trapped onto a mixed Tenax:Carbograph trap at –80 °C,” Journal of Breath Research, 2016, 10(2):026011.