Analysis of Volatile Organic Components in Human Breath by GCxGC-TOFMS

Importance of the Topic

Exhaled human breath contains a multitude of volatile organic compounds (VOCs) that reflect physiological and metabolic states. Non-invasive breath analysis enables monitoring of biomarkers linked to diseases such as diabetes, hypercholesterolemia, and oxidative stress. However, the low concentration of many VOCs and the high humidity of breath demand advanced analytical methods capable of high sensitivity and comprehensive separation.

Objectives and Study Overview

This study aimed to profile changes in breath VOCs following ingestion of orange juice and a sugar-free energy drink. By sampling alveolar breath before and after consumption, the authors sought to detect trace compounds and compare relative abundance through sample subtraction techniques.

Methodology and Instrumentation

• Sample Collection: Subjects exhaled slowly for 7 seconds to exclude tidal breath, then fully into 3 L Tedlar bags at 50 mL/min. Alveolar breath was isolated by nose closing and timed exhalation.
• Water Removal: After loading PDMS-foam filled thermal desorption tubes (Gerstel TDU), a 100 mL dry nitrogen purge removed residual moisture.
• Thermal Desorption/Inlet: A Gerstel TDU/CIS 4 system desorbed analytes from PDMS foam. TDU ramped from 30 °C to 300 °C; cold CIS trap at −120 °C focused compounds before fast transfer to the column.
• GC×GC-TOFMS Analysis: A LECO Pegasus 4D system with two capillary columns (RTX-5 first dimension, DB-17 second dimension) separated compounds under a 40–240 °C temperature program, 5 s modulation, and 1.5 mL/min flow. Mass spectra were acquired at 200 spectra/s over 5–1000 m/z.
• Data Processing: ChromaTOF software conducted contour plotting and sample-to-sample subtraction to reveal positive and negative changes in VOC profiles.

Major Results and Discussion

• Orange Juice Ingestion: Post-consumption breath exhibited increases in terpenes (limonene, pinene, myrcene) absent or minimal in baseline samples. Siloxane artifacts from PDMS desorption were identified and disregarded.
• Energy Drink Ingestion: Subtraction of pre- and post-consumption profiles highlighted new peaks and shifts in terpene-related compounds, demonstrating the method’s sensitivity to dietary VOC intake.
• Method Robustness: GC×GC-TOFMS effectively resolved hundreds of coeluting trace compounds in highly humid samples. The “Subtract Second Sample” feature expedited identification of differential biomarkers.

Benefits and Practical Applications

• Non-invasive diagnostics: Enables monitoring of disease biomarkers without blood draws.
• High sensitivity: Detects trace compounds at low ppb levels in breath.
• Comprehensive profiling: Two-dimensional separation resolves complex breath mixtures for greater metabolomic insight.

Future Trends and Opportunities

• Advanced sampling media: Development of coatings for enhanced polar analyte capture and reduced condensation losses.
• On-line analysis: Integration of real-time breath sampling with portable GC×GC-TOFMS for clinical point-of-care applications.
• Machine learning: Automated pattern recognition to link complex VOC profiles with specific disease states.

Conclusion

Comprehensive two-dimensional GC×GC-TOFMS combined with PDMS-foam thermal desorption offers a powerful tool for breath VOC analysis. The approach achieves high sensitivity, detailed separation, and rapid comparison of pre- and post-consumption breath samples, supporting its potential for clinical and metabolic research.

Used Instrumentation

• Gerstel TDU 4 Thermal Desorption Unit and CIS 4 cooled inlet system
• LECO Pegasus 4D GC×GC-TOFMS
• Columns: RTX-5 (10 m×0.18 mm×0.2 µm) and DB-17 (1.0 m×0.10 mm×0.1 µm)
• ChromaTOF software version 4.20