High sensitivity detection of deuterium-labelled volatile compound probes with Orbitrap-MS: from in-vitro headspace to Breath Biopsy for lung cancer.

Martin¹, J. Mizen¹, D. Otero¹, I. Banda¹, R. Stallard¹, B. Taylor¹, C. Clarke¹, N. Shaju¹, M. Alkaseem¹, K. Joyce¹ M. Ferreira Leal¹, A. Smolinska^1,2, B. Boyle¹, M. Allsworth¹.

¹Owlstone Medical, Cambridge, United Kingdom.

²Department of Pharmacology and Toxicology, Maastricht University, Maastricht, The Netherlands.

Poster PDF

Introduction

Exogenous volatile organic compounds (eVOCs) can be used to explore metabolic pathways dysregulated by lung cancer. Higher expression of aldoketoreductase (AKR) in response to lipid peroxidation, characteristic of cancer, can be monitored with aldehyde and alcohol detection. Additionally, exposure of β-glucuronidase to the extracellular tumour microenvironment, converse to healthy intracellular expression, can be targeted with specific substrates linked to cleavable volatiles.

To demonstrate the potential for eVOCs to be used as probes within a breath test for early cancer detection, we require a sensitive method to accurately detect multiple eVOCs in biological matrices. Here, we show the development of a thermal-desorption gas-chromatography mass-spectrometry (TD-GC-MS) method for multiple deuterium-labelled eVOCs in different matrices for translation from pre-clinical to clinical breath studies.

Methods

The eVOC panel comprised deuterium-labelled aldehydes and alcohols. Sensitivity was demonstrated with standards in cell culture media, blood and animal chambers, down to 3.5 ppb. A549 and H460 lung cancer cell lines were used to monitor AKR activity by headspace analysis in-vitro. AKR activity was modified chemically with inhibitors (Tolrestat and JF0064) and biologically with CRISPR knockouts. AKR activity was monitored by aldehyde and bioproduct alcohol detection. Headspace eVOCs were sampled with HiSorb probes. In-vivo breath samples were collected through an animal chamber onto sorbent tubes.

HiSorb probes and sorbent tubes were thermally desorbed onto a focussing trap, desorbed onto a Stabilwax GC column for separation before detection by Orbitrap MS in electron ionisation mode with single ion monitoring (SIM).

Preliminary data

In-vitro headspace TD-GC-MS analysis demonstrated that conversion of multiple aldehydes into alcohols was dependent on AKR activity in A549 and H460 lung cancer cell lines. Inhibition with Tolrestat showed dose-dependent production of alcohols, and CRISPR knockouts showed reduced production of alcohols, demonstrating lower AKR activity. The results indicate AKR activity can be probed with administration of aldehydes and monitoring for aldehyde and alcohol content on breath. The improvement of the analytical sensitivity with deuterium-labelling and SIM provides a robust platform to use eVOCs to target AKRs for diagnosis of lung cancer.

probe an alternative and complementary route to lung cancer detection, β-glucuronidase was targeted with the labelled EVOC probe, D5-ethyl-βD-glucuronide. In-vitro headspace analysis confirmed the release of the D5-ethanol reporter upon hydrolysis of the probe by β-glucuronidase and its dependency on enzyme concentration. Conversion of the EVOC probe to D5-ethanol was also monitored in whole blood with and without administered β-glucuronidase, demonstrating sensitivity in the blood matrix for use as an orthogonal pre-clinical measure.

This result was replicated in-vivo with LL2 Lewis Lung Cancer model mice in our novel device for mouse breath collection, with capability of 3.5 ppb analyte detection. The device was employed to determine the optimum dosing strategy to distinguish healthy mice from mice with lung cancer. The analytical TD-GC-MS method proved robust to unavoidable background contaminants from the in-vivo laboratory and moisture collected in breath sampling. The optimised sensitivity allowed for detection even at the lowest dosing strategy, minimising clinical eVOC requirements.

Furthermore, intravenous administration of the eVOC probe to human subjects with lung cancer and controls revealed an excellent safety profile. The cleavage product D5-ethanol could be detected on breath in a subset of participants and the optimised sensitivity for VOC detection is compatible with low eVOC probe dosing required for clinical studies.

Novel Aspect

Sensitive targeted method robust to backgrounds associated with diverse sample matrices applicable to workflow from pre-clinical studies to clinical diagnostics.