Online vs offline breath analysis: the differences

Aug 11, 2021

Exhaled breath contains a matrix of biomarkers, many of which provide relevant information about a person’s health status to the clinician and researcher. This unlimited resource can be easily collected without burden to the test person, making it highly appealing. Different techniques are available for breath analysis and they all have their value depending on the specific goal or application [i].

Volatile Organic Compounds (VOCs) comprise less than 1% of the total breath volume and offer an insight into a person’s metabolism and health status [i]. The many different approaches for breath analysis can be divided into online and offline methods. For online analysis breath is sampled directly by the instrument and measured immediately, allowing rapid, near patient sample analysis without the need for sample storage or manipulation. Offline analysis collects the samples for transfer and subsequent measurement by the analytical instrument, which require sample preparation or manipulation [ii, iii].

Online breath analysis

Online techniques will analyse the breath samples in only a few minutes directly during or immediately after exhalation by the test person. Furthermore, it has the possibility to provide a direct result output. These advantages make real-time breath analysis an excellent choice for disease diagnosis and personalized medicine. Available techniques range from very small and low-cost chemical sensors used in electronic noses (eNoses) to optical spectroscopy and mass spectrometry (MS) based techniques combined with soft chemical ionization [i]. eNoses analyse the pattern of the complete mixture of exhaled VOCs or the “breath profile” using cross-reactive sensor arrays without identification of individual VOCs. Powerful pattern recognition techniques are used to recognize different “breath profiles” linked to disease states, in much the same way the mammalian olfactory system recognizes different smells. Real-time MS based techniques (e.g. SIFT-MS, PRT-MS, SESI-MS) acquire breath profiles based on the VOCs fragments resulting from different ionization methods that can also be analysed using pattern recognition methods. These devices, however, are not easily portable and less user-friendly than eNoses.

Breathomix offers BreathBase® as a complete solution for online breath analysis. It allows for online analysis of breath VOCs by means of the cloud-connected eNose, the SpiroNose®, which is linked to the online BreathBase platform. Based on advanced signal processing, powerful pattern recognition and an extensive online reference database, BreathBase® can differentiate between “breath profiles” from individuals with and without disease. The technically and clinically validated SpiroNose® [iv] has been shown to be useful in diagnosing and subtyping (aka phenotyping) diseases such as asthma, chronic obstructive pulmonary disease, interstitial lung disease, lung cancer and respiratory infections [iv-viii]. In addition, the technology has the potential to prevent ineffective immunotherapy in patients with non-small cell lung cancer [vii]. BreathBase® offers the complete breath analysis workflow within 1 minute and provides immediate feedback at the point-of-care.

Offline breath analysis

Offline measurements of exhaled breath involve a two-step process of sample collection and storage followed by sample analysis in a specialized laboratory [i]. Breath VOCs collected using breath sampling bags or other containers are usually transferred to an adsorbent material for storage and transport. Heating the adsorbent material releases the VOCs into the gaseous phase for analysis. This allows to increase the concentration of the sampled VOCs prior to analysis, which is an advantage when VOCs of interest are present in very low concentrations. However, there are some complications with chemical stability during storage that need to be considered (e.g.: degradation, sample losses) [i]. Additionally, many types of adsorbent materials are available and the choice of the adsorbent can impact the obtained results. Therefore, a proper combination of the adsorbent material and the sample is necessary to increase the robustness and reproducibility of biological studies [ix].

The specialized laboratories for breath analysis are often equipped with gas chromatography (GC) and mass spectrometry (MS). Different VOCs in the sample are separated by GC, while MS can indicate which VOC is present based on their ionization fragments. The ability to detect individual VOCs is considered the biggest advantage of offline GC-MS, as it can be useful for pathophysiologic research in combination with other omics technologies (e.g. genomics, proteomics etc.) [ii]. However, it is rather time consuming with 45 to 60 minutes analysis time per sample and there is no direct output of results. Technical operators are required because the instrument is not easy-to-use and frequent calibration of the system is needed.  All these make the clinical implementation of the GC-MS in daily practice rather complex. Although GC-MS is considered the gold standard for the analysis of complex volatile mixtures and according to many breath researchers also for breath analysis [ii], replication of research findings have been proved difficult to achieve for many years [iii, x]. Therefore, one might start to wonder if it should be.

What to choose?

Both the online and offline approaches for breath analysis have their advantages and drawbacks. Depending on the research question, the appropriate method can be selected. For individual VOC detection, offline GC-MS analysis would still the method of choice because real-time methods based on pattern recognition will not allow identification of individual VOCs. On the other hand, when the entire “breath profile” is of interest it can be reliably measured by online methods, which can allow rapid assessment with the possibility of continuous monitoring [ii]. Furthermore, when looking for VOC profiles, the real-time approach often is the method of choice. Within Breathomix we focus on high-quality real-time breath measurements using the online BreathBase® solution with a cloud-connected SpiroNose® which allows for the generation and validation of diagnostic models and composite biomarkers that can be implemented at point-of-care.


[i] T. Brudere et al. “On-line analysis of exhaled breath” Chemical Reviews, 2019.

[ii] M.D. Davis et al. “Exhaled breath testing – A tool for the clinician and researcher” Paediatr Respir Rev, 2019.

[iii] J. Beauchamp and W. Miekisch. “Breath sampling and standardisation” in Breathborn Biomarkers and the Human Volatilome, 2020, pp. 23-41.

[iv] R. de Vries et al. “Integration of electronic nose technology with spirometry: validation of new approach for exhaled breath analysis” J Breath Res, 2015.

[v] R. de Vries et al. “Clinical and inflammatory phenotyping by breathomics in chronic airway diseases irrespective of the diagnostic label” Eur Respir J, 2018.

[vi] C.C. Moor  et al. “Exhaled breath analysis by use of eNose technology: a novel diagnostic tool for interstitial lung disease” Eur Respir J, 2020.

[vii] R. de Vries et al. “Prediction of response to anti-PD-1 therapy in patients with non-small-cell lung cancer by electronic nose analysis of exhaled breath”, Annals of Oncology, 2019.

[viii] R. de Vries et al. “Rulling out SARS-CoV-2 infection using exhaled breath analysis by electronic nose in a public health setting” medRxiv, 2021.

[ix] F.A. Franchina et al. “Impact of the adsorbent material on volatile metabolites during in vitro and in vivo bio-sampling” Talanta, 2021.

[x] B. Henderson et al. “A benchmarking protocol for breath analysis: the peppermint experiment” J Breath Res, 2020.

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Front-end Developer

Front-end Developer Breathomix is a fast-growing, internationally operating Medical Tech Scale-Up. Our core business is breath analysis for diagnosing a multitude of conditions (Asthma, COPD, lung cancer, and many more) and enabling precision medicine. Our team is...

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Functional Designer Breathomix is a fast-growing, internationally operating Medical Tech Scale-Up. Our core business is breath analysis for diagnosing a multitude of conditions (Asthma, COPD, lung cancer, and many more) and enabling precision medicine. Our team is...

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Backend Azure Developer Breathomix is a fast-growing, internationally operating Medical Tech Scale-Up. Our core business is breath analysis for diagnosing a multitude of conditions (Asthma, COPD, lung cancer, and many more) and enabling precision medicine. Our team is...