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Evaluation of fresh, frozen, and lyophilized fecal samples by SPME and derivatization methods using GC×GC-TOFMS

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Abstract

Introduction

Feces is a highly complex matrix containing thousands of metabolites. It also contains live bacteria and enzymes, and does not have a static chemistry. Consequently, proper control of pre-analytical parameters is critical to minimize unwanted variations in the samples. However, no consensus currently exists on how fecal samples should be stored/processed prior to analysis.

Objective

The effects of sample handling conditions on fecal metabolite profiles and abundances were examined using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS).

Methods

Solid-phase microextraction (SPME) and derivatization via trimethylsilylation (TMS) were employed as complementary techniques to evaluate fresh, frozen, and lyophilized fecal samples with expanded coverage of the fecal metabolome. The total number of detected peaks and the signal intensities were compared among the different handling conditions.

Results

Our analysis revealed that the metabolic profiles of fecal samples depend greatly on sample handling and processing conditions, which had a more pronounced effect on results obtained by SPME than by TMS derivatization. Overall, lyophilization resulted in a greater amount of total and class-specific metabolites, which may be attributed to cell lysis and/or membrane disintegration.

Conclusions

A comprehensive comparison of the sample handling conditions provides a deeper understanding of the physicochemical changes that occur within the samples during freezing and lyophilization. Based on our results, snap-freezing at -80 °C would be preferred over lyophilization for handling samples in the field of fecal metabolomics as this imparts the least change from the fresh condition.

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Funding

Authors would like to thank MITACS, DNA Genotek, Inc., The Natural Sciences and Engineering Research Council of Canada (NSERC) for support. The support of The Canada Foundation for Innovation (CFI), Genome Canada, and Genome Alberta to The Metabolomics Innovation Center (TMIC) is also acknowledged. The authors also wish to thank GC for her help in securing funding for this research.

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Authors and Affiliations

  1. Department of Chemistry, University of Alberta, T6G 2G2, Edmonton, Alberta, Canada

    Seo Lin Nam, Kieran Tarazona Carrillo, A. Paulina de la Mata & James Harynuk

  2. DNA Genotek, Ottawa, Ontario, Canada

    Olle M. de Bruin & Evgueni Doukhanine

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  1. Seo Lin Nam
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  2. Kieran Tarazona Carrillo
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  3. A. Paulina de la Mata
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  4. Olle M. de Bruin
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  5. Evgueni Doukhanine
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  6. James Harynuk
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Contributions

All authors contributed to the study conception and design. Sample preparation and data collection were performed by KTC; data processing and analysis were performed by SLN. The first draft of the manuscript was written by SLN and all authors commented on previous versions of the manuscript. All authors have read and agreed to the final manuscript.

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Correspondence to James Harynuk.

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Ethical approval

This study was approved by the University of Alberta Research Ethics Board, under approval number Pro00071285.

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Informed consent was obtained from all individual participants included in this study.

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Nam, S.L., Tarazona Carrillo, K., de la Mata, A.P. et al. Evaluation of fresh, frozen, and lyophilized fecal samples by SPME and derivatization methods using GC×GC-TOFMS. Metabolomics 18, 25 (2022). https://doi.org/10.1007/s11306-022-01881-z

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