Volatile Biomarkers in Biomedical Research

Martin Perkins

25th July 2017

Alveolar, Biomarkers, Biomedical Research, Breath Analysis, Endogenous, Exogenous, GC-MS, IMS, Ion Mobility Spectrometry, LC-MS, Metabolites, Microbiome, Selected Ion Flow Tube Mass Spectrometry, SIFT-MS, VOCs, Volatile, Volatile Organic Compounds, Volatolome,

Why are Volatile Biomarkers Important in Biomedical Research?

The pattern of individual’s volatile metabolites (volatolome) can be used to understand the biochemical processes associated with metabolism and disease.

An individual’s volatolome changes, both at the cellular and tissue level and within their microbiome, as a result of endogenous and exogenous stimuli.  Such changes are observed in cancer, infection, auto-immune and inflammatory disease, metabolic disease, infection and pathogenesis, environmental pollution, surgery, medication, lifestyle and nutrition.

Volatile compounds are present in breath, bio-fluids & secretions, stools and tissue samples and consistent, clinically diagnostic profile changes may be detected at the earliest stages of disease.  These changes can facilitate minimally invasive (breath, saliva, urine, stools) diagnosis and early treatment, improve both prognosis and patient outcomes and reduce costs to the healthcare system.

Furthermore, since volatile compounds in perfused tissues are transported throughout the bloodstream, and blood gases are in equilibrium with the (alveolar) portion of the breath, they may be used as diagnostic biomarkers of localised disease in tissues or organs.

Consequently, both volatile organic compounds (VOCs) and inorganic gases may be used as diagnostic disease biomarkers, to monitor individual patient responses to treatment (including therapy selection and stratification), compliance with medication regimes and aid with differential diagnosis (e.g. between cancer types and stages).

Current Tests Using Volatile Biomarkers

There have been many biomedical research studies demonstrating that volatile compounds can be selective and specific biomarkers for disease diagnosis and many clinical trials have either been done or in progress.

The FDA has however, only approved three diagnostic breath tests in the last 20 years:

  • Breath hydrocarbons to monitor for heart transplant rejection
  • Urea breath test for H.pylori infection
  • NO test for asthma therapy

The successful development of these tests is the result of several common factors:

  • Single (individual or class) biomarker of disease
  • Point-of-care sampling (or testing) by healthcare staff with minimal additional training
  • Biomarkers can either be tested with low cost point-of-care diagnostic device or are stable for transport for laboratory analysis
  • Test is clinically selective and specific for (differential) diagnosis of disease
  • Test provides earlier disease diagnosis and/or improved patient outcomes than the previous test

Challenges in Volatile Biomarker Analysis

The main advantage of using volatile biomarkers lies in the fact that samples can be obtained from patients of all ages (neonates to geriatric patients) and conditions (mobile outpatient to ventilated in-patient).

The nature of volatile compounds in biological samples present several unique challenges in the field of biomarker discovery and validation including:

  • Volatile profiles respond rapidly to physiological or environmental stimuli
  • Volatile metabolites are trace components, ppm(v) to ppt(v), in high humidity matrices
  • Contamination with exogenous (dietary or environmental) VOCs is a problem
  • For breath samples the alveolar (in equilibrium with blood) fraction of the breath is normally sampled
  • Many volatile metabolites are reactive species (e.g. carbonyl compounds) exhibiting limited stability or low/differential recoveries from sampling devices
  • There are few single, specific volatile biomarkers which are best suited to point of care or home testing
  • Clinically relevant differences are normally observed as a change in the pattern (fingerprint) of volatile metabolites
  • Sample pre-concentration (e.g. on adsorbent tubes) is required for GC-MS analysis.

Many of these challenges are met through the routine standardisation applied to clinical studies to ensure that study results are not biased however, additional precautions such as ensuring subjects are relaxed, breathing normally and acclimatised to humidified medical grade air before samples may be required, especially for breath sampling studies.

In clinical studies exogenous contaminants can be managed through the study design, the parallel collection of procedural blanks or by acclimatising subjects with humidified medical grade air before sampling.  Common environmental contaminants are, naturally, excluded as candidate biomarkers.

Representative sampling can be done for most sample types using standard protocols and specimen tubes.  For tissue, stool and adsorbed breath samples, temperature controlled transport and storage are essential to manage sample stability.

For breath sampling specialist collection devises, (such as that developed by the Breath Free Consortium), are used to sample and concentrate volatile metabolites onto (single or multiple) adsorbent tubes for subsequent GC-MS analysis.

With real-time instrumental analysis (e.g. SIFT-MS) direct sampling may be done using spirometers or samples may be collected in specimen containers (Gas Sampling bags, Exetainer® tubes, Thermal Desorption (TD) tubes) for offline analysis.

What are the Current Approaches?

Clincial biomarker studies, using LC-MS, typically use different analytical platforms and approaches in the biomarker discovery and verification/validation stages: with dual (LC-MS & NMR) or multi- (LC-MS, NMR, GC-MS) platform studies to cross validate the results.

For most volatile biomarker studies, untargeted volatile biomarker discovery is normally done by TD-GC-MS for breath samples, and headspace Solid Phase Micro-Extraction (HS-SPME) for other biological samples.

Whilst many studies repeat GC-MS on a larger scale at the validation/verification stage, relatively few include parallel targeted analysis with a real-time platforms such as Selected Ion Flow Tube – Mass Spectrometry (SIFT-MS) or Ion Mobility Spectrometry (IMS).

This is largely because the widespread adoption of these techniques has been limited by the lack of commercially available instruments that combine a wide dynamic range, analytical selectivity, specificity, sensitivity and resolution and are also capable of quantitatively determining trace level VOCs in high humidity samples.

How Are Anatune Solutions Different?

Following on from last year’s Seminar on the Analysis of Biomarkers in Breath and Body Fluids by SIFT-MS, we actively engaged with the Breath and Volatile Biomarker research community in the UK.

Having taken the time to listen, we identified a ‘wish list’ of features for volatile biomarker analysis platforms that includes:

  • Flexibility to sample from a range of sampling ‘devices’:
    • 3½ x ¼” stainless steel Thermal Desorption tubes
    • Gas sampling bags
    • Exetainer® tubes
    • Commercially available specimen tubes (blood, urine)
  • Dynamic Headspace (DHS) for comprehensive volatile profiling from bio-fluids, tissues and microbial/cell cultures
  • Temperature controlled sample storage to maintain sample integrity
  • Multi-format barcode reading for traceability and sample tracking
  • High capacity Thermal Desorption (>100 samples) for large scale clinical studies
  • Integrated software tools for processing and interpreting volatolomics data
  • Automate analysis of samples by SIFT-MS
  • Common, scalable platforms that can grow as research translates from biomarker discovery to verification and validation
  • We’re aiming for a home test of point-of-care diagnostic device but need the backup of a routine lab test at price that the NHS/NICE can afford.

We recently completed the kit list for our toolbox and can now fulfil the complete wish list with the new GERSTEL MultiPurpose Sampler MPS Robotic Series.

If you would like to learn more about our Enabling Technology solutions for metabolic phenotyping of volatiles then please call us on 01223 279210 or email enquiries@anatune.co.uk now.