MSACL 2019 EU Abstract

Determination of Aldehydes, Ketones, Short Chain Fatty Acids and Alkanes in Breath and Biofluids with GcxGC-TOF-MS-FID

Antonis Myridakis, Sung-Tong Chin and George Bushra Hanna
Imperial College London, Department of Surgery and Cancer, St. Mary’s Hospital, London, UK

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Antonis Myridakis (Presenter) Imperial College London

Presenter Bio: I am a postdoctoral research associate at the Division of Surgery, Imperial College London. My current research at Imperial College is focused on GC(xGC)-MS method development and application for the discovery of cancer biomarkers in breath and biofluids. Furthermore, I have extensively applied MS metabolomics in order to investigate the link between gut microbiome metabolites and the pathogenesis of cardiometabolic and neurodevelopmental diseases.
In the past, my main research activities were focused on the environmental exposure and risk assessment of endocrine disrupting and neurotoxic chemicals in pregnant women and their children.
I studied at the University of Crete, Greece. I was awarded a PhD in “Environmental and Analytical Chemistry” (2015), a MSc in “Environmental Science and Engineering” (2011) and a BSc in Chemistry (2009).

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

Introduction: Volatolomics is the study of the volatile organic compounds (VOC) and one of the most fast-growing fields in non-invasive diagnostics. Several prospective clinical series have shown exhaled aldehydes, ketones, alkanes and short chain fatty acids to accurately identify patients with cancer. GC-MS primarily and online-MS, as proton transfer reaction-MS or selected ion flow tube-MS, are the most widely used platforms for VOC profiling. They have showed high coverage of the volatolome, low detection limits and high-throughput. However, they lack selectivity in several categories of chemical compounds (e.g. structural isomers). In these cases, multidimensional GC combined with high resolution MS can offer the required resolving power for the robust separation and determination.
In this study, we aim to develop a quantitative method for the concurrent determination of alkanes, short chain fatty acids, ketones and aldehydes in human breath, biofluids and tissue. The key-steps for the optimisation of the method are I) the sample-preparation procedure, II) the derivatisation protocol and III) a chromatographic method which will enable the concurrent separation of the wide-polarity range compounds and V) the application to a human cohort.

Methods: All samples are spiked with isotopically labeled internal standards. Breath samples are collected in tenax/carbograph thermal desorption tubes. Human tissue and biofluids are homogenized with bead beading, acidified and cleaned-up with liquid-liquid extraction. Finally, ketones and aldehydes are derivatized to oxime analogues. The samples are analyzed in a Markes Bench-TOF Select GCxGC system, equipped with a SepSolve flow modulator, a liquid autosampler and a Markes TD-100-XR thermal desorber. The primary column is an Agilent DB-WAX UI (20m x 0.180mm, 0.18μm) and the secondary an SGE BPX5 (5m x 0.25mm, 0.1μm). Data are acquired and processed with ChromSpace (SepSolve).

Results and Discussion: Liquid/liquid extraction with acidification is highlighted as the most effective clean-up technique and demonstrated high recoveries for most of the target compounds. Oximation with O-(2,3,4,5,6-Pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA·HCl) allows the baseline separation of ketones and aldehydes and greatly reduces their detection limits while the reaction takes place in room temperature. Short chain fatty acids are separated and detected without derivatization however their methylation is evaluated as an extra derivatization step to further increase their chromatographic performance and the detection of the higher molecular weight ones. Furthermore, blank contaminations, linearity range, recoveries, intra- and inter- day reproducibility are evaluated for each target analyte.