Metabolomics: Developing and optimizing a robust HRAM GC-MS pipeline for on-breath global biomarker analysis

Importance of the Topic

Breathomics leverages the analysis of volatile organic compounds (VOCs) in exhaled breath as non-invasive biomarkers for early disease detection and precision medicine. Over a thousand VOCs of endogenous and exogenous origin can be measured, offering a window into systemic metabolic processes and potential clinical applications across a range of conditions.

Objectives and Study Overview

The study aimed to overcome the historical limitations of clinical breath testing by establishing a robust, reproducible high-resolution accurate mass (HRAM) GC-MS pipeline. Key goals included:

• Maximizing the number of detectable breath VOCs.
• Minimizing analytical variability to enhance sensitivity to biological signals.
• Validating a workflow using the Thermo Scientific™ Orbitrap Exploris™ GC 240 MS with thermal desorption.

Methodology

Human tidal breath samples were collected via the Breath Biopsy Collection Station onto four thermal desorption tubes, selectively sampling the alveolar fraction. Blank samples were acquired to assess background VOC levels. Thermal desorption released analytes onto a thick-film GC column (30–280 °C gradient) with full-scan EI analysis (m/z 30–450) on the Orbitrap Exploris GC 240 MS. A feature extraction and data processing workflow used Thermo Chromeleon™ CDS and Compound Discoverer™ software. Breath-or-blank (BoB) studies standardized method development by comparing true breath signals against ambient contamination.

Used Instrumentation

• Breath Biopsy Collection Station (Owlstone Medical).
• CASPER portable air supply for blank correction.
• Thermal desorption tubes and TD system.
• Thick-film GC column (30–280 °C ramp).
• Thermo Scientific™ Orbitrap Exploris™ GC 240 Mass Spectrometer.
• Electron ionization at 70 eV and variable electron voltage (35 eV).
• Thermo Chromeleon™ CDS and Compound Discoverer™ software.

Main Results and Discussion

The optimized pipeline achieved:

• Excellent linearity (R2 = 0.9988) over 0.5–200 ng using automated gain control with sub-nanogram detection capability and quantitation up to ~500 ng within the same retention window.
• Sub-1 ppm mass accuracy across chromatographic peaks, improving deconvolution and ion-ratio stability even at high analyte loads.
• Detection of a median of 1,454 VOC features per sample, with 517 confirmed on-breath compounds per individual through BoB studies.
• Enhanced molecular ion recovery at reduced EI voltage (35 eV) and potential for complementary chemical ionization (CI) to differentiate closely related compounds.

Benefits and Practical Applications

The validated HRAM GC-MS pipeline provides a high-throughput, non-invasive approach for reliable breath VOC profiling. It supports early disease screening, monitoring of treatment efficacy, and large-scale clinical studies by ensuring reproducible quantitation and identification of trace biomarkers.

Future Trends and Opportunities

Advancements likely to enhance breathomics include:

• Integration of variable ionization techniques and expanded spectral libraries for improved compound annotation.
• Standardized BoB workflows across laboratories to facilitate cross-study comparability.
• Machine learning-driven data interpretation for personalized biomarker discovery.
• Miniaturized or portable HRAM instruments enabling point-of-care breath testing.

Conclusion

This work demonstrates a robust end-to-end HRAM TD-GC-MS workflow for breathomics, achieving exceptional sensitivity, dynamic range, and mass accuracy. The standardized pipeline and quality-controlled procedures pave the way for clinical application of breath biomarkers in early disease detection and precision medicine.

References

• Holden KA, Ibrahim Q, Salman D et al. Use of the ReCIVA device in breath sampling patients with acute breathlessness: a feasibility study. ERJ Open Research. 2020;6(4).