Test tubes in a laboratory

Headspace VOC analysis of in vitro, ex vivo and biofluid samples

Identify VOC biomarkers relevant to key disease processes through in vitro analysis


Linking breath and biology

Discovering and validating biomarkers for disease is a key stage in developing valuable and effective diagnostic tests for clinical applications. Do you have a detailed understanding of the biology of your disease of interest? Headspace sampling underpins the biological understanding of disease, supporting the translation of clinically useful biomarkers.

Our headspace sampling pipeline, supported by our Breath Biopsy® Services, can help you to identify volatile organic compounds (VOCs) produced by your laboratory samples that have the potential to be effective biomarkers for non-invasive clinical breath testing. We provide an optimized detection process supported by expert VOC analysis and identification that provides high confidence candidate biomarker discovery for clinical translation.

in vitro pipeline

Our approach is designed to bridge the gap in biomarker research. Some existing studies have investigated metabolic processes involved in disease but have not identified clinically viable biomarkers. While others have taken an untargeted approach to discovering clinical biomarkers without insight from the underlying biology.

Many studies show that VOCs on breath have the potential to be valuable biomarkers for a range of diseases. The majority of these have hypothesized biological origins for biomarkers but relatively few have directly investigated the relationship between disease biology and biomarker abundance.

Analyzing VOCs produced by in vitro models of disease or drug response can demonstrate mechanistic links between disease biology and biomarker candidates. These can subsequently be verified in clinical studies sampling the same VOCs on breath, providing vital evidence that could bring diagnostic breath tests into clinical practice.


Why do headspace sampling?

  • DISCOVERY: Identify clinically relevant candidate breath biomarkers
  • DISEASE RELEVANCE: Directly relate disease biology to breath biomarkers
  • MECHANISMS: Gain insight into mechanisms underlying disease and drug response
  • CAUSE & EFFECT: Dissect markers related to causes and effects of disease
  • VALIDATION: Validate the chemical identities of your biomarkers
  • REGULATORY APPROVAL: Generate evidence to support your case for clinical approval
  • RELEVANCE: Relate your findings to other published literature

Why is understanding the biological origins of VOC biomarkers important?

Read the Blog
Cells and VOCs
In Vitro flowchart in the Breath Biopsy Laboratory

Breath Biopsy headspace sampling

Our headspace sampling capability integrates seamlessly with Breath Biopsy Services. The Breath Biopsy Laboratory is equipped with HiSorb probes, which are analysed on a Centri device, for the automated collection of VOCs produced by in vitro cell cultures, macromolecular isolations, ex vivo tissues, biofluids and fecal samples.

This is coupled to our high-resolution gas chromatography mass spectrometry (GC-MS) biomarker analysis platform. The Breath Biopsy Platform, built around GC-Orbitrap devices, has been optimized for the detection of volatile compounds found on breath and provides high sensitivity VOC detection, even for low-abundance compounds in the parts per trillion (ppt) range.

Key features:

  • VOC collection using automated HiSorb probes dynamic sampling

  • Direct from sample collection to HRAM GC-MS

  • Suitable for study of cell cultures, ex vivo tissues, fecal headspace, biofluids and/or macromolecular isolations

  • Compare disease site VOCs to breath samples on the same platform

Contact us to discuss integrating Breath Biopsy into your research:


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Identifying Translational Biomarkers using Breath Biopsy in vitro Headspace Analysis

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In Vitro Webinar

Case Studies: In vitro studies at Owlstone Medical

Lung cancer

Aldo-keto reductases (AKRs) are upregulated in some cancers as an adaptation to oxidative stress and lipid peroxidation. We used in vitro cell cultures of lung cancer cell lines to try and target AKRs in these cells and monitor the metabolic processes they are involved in on breath. To do that we modulated the catalytic activity of AKRs using small compounds and also developed knock-out (KO) cells for target AKR genes.

AKR Study Methodology

We treated our cell cultures with a range of substrates then monitored substrate and product levels using the same GC-MS workflow used for the detection of VOCs in clinical breath samples (OML Breath Biopsy® workflow). This allowed us to demonstrate that the target enzymes were responsible for the metabolization of the substrates and appeared to confirm that in vitro cell cultures, coupled with VOC analysis can return information about genomic manipulations and drug treatments.

View the lung cancer results

Liver disease

We have used in vitro sampling to identify alterations of metabolic pathways induced by a chronic liver disease. By treating cell cultures of human primary hepatocytes with substrates we were able to use VOC analysis to find alterations in the bioproduct generation of healthy hepatocytes compared to hepatocytes in which a non-alcoholic steatohepatitis (NASH) phenotype was induced.

In Vitro Liver Sampling

Using pre-clinical models, like this one, to evaluate the relationship between substrates and metabolism alterations represents a step-forward for the identification and assessment of prospective biomarkers for breath diagnosis of disease.

Read our scientific poster

Inflammatory disease

Lipid peroxidation is a biological mechanism associated with many diseases as well as inflammatory immune responses.

We performed in vitro analysis of lipid peroxidation of various polyunsaturated fatty acids to understand the VOCs produced by each lipid species, finding that each fatty acid produces a different complement of VOCs which can be detected and quantified using the Breath Biopsy Platform.


We have also investigated the VOCs associated with bronchial inflammation.

Using headspace sampling of the air liquid interface assay of in vitro bronchial mucosa samples, VOCs were collected from cell media headspace and bronchial mucus headspace. Inflammation was induced via bleomycin or lipopolysaccharide (LPS) treatment.

Both bleomycin and LPS were shown to produce distinct changes in VOC profiles across two days in both cell media and bronchial mucus. While some of these changes were shared across each treatment there are also differences, likely indicative of differences in inflammation mechanisms.

Analysis of cell medium yielded more detectable VOCs, with a greater distinction between groups and more consistent results across repeats. Significant detected VOCs such as alkanes and aldehydes appeared to relate to well established inflammation responses such as lipid peroxidation.

Further work could verify VOC identities and confirm their origins.

About Lipid Peroxidation

Gastrointestinal (Fecal Headspace Analysis)

The analysis of VOCs collected from fecal headspace is already a well-established practice in gastrointestinal disease research. Conducting analysis of fecal headspace VOCs in parallel to investigations into novel breath-based VOC biomarkers for gastrointestinal disease can be illuminating. Performing cross-matrix data comparisons could help suggest key VOCs that are worth focusing efforts on. Identifying prospective biomarkers that can be strongly corroborated across both matrices can help build confidence for further study.

Fecal headspace VOCs can be collected on sorbent tubes in the same way as breath VOCs are collected and then analyzed using our Breath Biopsy Workflow.

If you’re interested in conducting an in vitro study as part of your breath research, we can help.

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