Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018;14(2):19.
doi: 10.1007/s11306-017-1315-7. Epub 2018 Jan 4.

A comparison of sample preparation methods for extracting volatile organic compounds (VOCs) from equine faeces using HS-SPME

Rachael Hough  1 , Debra Archer  2 , Christopher Probert  3
Affiliations

A comparison of sample preparation methods for extracting volatile organic compounds (VOCs) from equine faeces using HS-SPME

Rachael Hough et al. Metabolomics. 2018.

Abstract

Introduction: Disturbance to the hindgut microbiota can be detrimental to equine health. Metabolomics provides a robust approach to studying the functional aspect of hindgut microorganisms. Sample preparation is an important step towards achieving optimal results in the later stages of analysis. The preparation of samples is unique depending on the technique employed and the sample matrix to be analysed. Gas chromatography mass spectrometry (GCMS) is one of the most widely used platforms for the study of metabolomics and until now an optimised method has not been developed for equine faeces.

Objectives: To compare a sample preparation method for extracting volatile organic compounds (VOCs) from equine faeces.

Methods: Volatile organic compounds were determined by headspace solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GCMS). Factors investigated were the mass of equine faeces, type of SPME fibre coating, vial volume and storage conditions.

Results: The resultant method was unique to those developed for other species. Aliquots of 1000 or 2000 mg in 10 ml or 20 ml SPME headspace were optimal. From those tested, the extraction of VOCs should ideally be performed using a divinylbenzene-carboxen-polydimethysiloxane (DVB-CAR-PDMS) SPME fibre. Storage of faeces for up to 12 months at - 80 °C shared a greater percentage of VOCs with a fresh sample than the equivalent stored at - 20 °C.

Conclusions: An optimised method for extracting VOCs from equine faeces using HS-SPME-GCMS has been developed and will act as a standard to enable comparisons between studies. This work has also highlighted storage conditions as an important factor to consider in experimental design for faecal metabolomics studies.

Keywords: Equine; Faeces; GC-MS; SPME; Volatile organic compounds.

PubMed Disclaimer

Conflict of interest statement

Compliance with ethical standardsRH, DA and CP declare that they have no conflict of interest.Ethical approval was granted by the University of Liverpool ethics committee (ref: VREC279). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Figures

Fig. 1
Fig. 1
A PCA of the VOC profiles of 100, 1000 and 2000 mg of horse faeces analysed by HS-SPME-GCMS. Grouped samples are contained within a 95% confidence interval
Fig. 2
Fig. 2
An overlay of chromatograms generated from the HS-SPME-GCMS analysis of faeces of horse 2 (H2). The black trace represents the DVB-CAR-PDMS fibre and the red is CAR-PDMS
Fig. 3
Fig. 3
A dendrogram constructed using the elucidean distance and ward clustering algorithm. Technical replicates of 1000 mg of faeces from four horses, analysed by HS-SPME-GCMS. VOCs were extracted using a DVB-CAR-PDMS SPME fibre
Fig. 4
Fig. 4
a A PCA of the VOC profiles of an equine faecal sample analysed by HS-SPME-GCMS 2 h post collection, after storage for 1 week, 6 and 12 months at − 20 and − 80 °C. 3 b A stack plot of the chemical classes of compounds found in each storage condition
See this image and copyright information in PMC

References

    1. Achá SJ, Kühn I, Mbazima G, Colque-Navarro P, Möllby R. Changes of viability and composition of the Escherichia coli flora in faecal samples during long time storage. Journal of Microbiological Methods. 2005;63:229–238. doi: 10.1016/j.mimet.2005.04.024. - DOI - PubMed
    1. Aggio R, Villas-Bôas SG, Ruggiero K. Metab: An R package for high-throughput analysis of metabolomics data generated by GC-MS. Bioinformatics. 2011;27:2316–2318. doi: 10.1093/bioinformatics/btr379. - DOI - PubMed
    1. Amann A, Costello BDL, Miekisch W, Schubert J, Buszewski B, Pleil J, et al. The human volatilome: Volatile organic compounds (VOCs) in exhaled breath, skin emanations,urine, feces and saliva. Journal of Breath Research. 2014;8:34001. doi: 10.1088/1752-7155/8/3/034001. - DOI - PubMed
    1. Arthur CL, Pawliszyn J. Solid phase microextraction with thermal desorption using fused silica optical fibers. Analytical Chemistry. 1990;62:2145–2148. doi: 10.1021/ac00218a019. - DOI
    1. Bergman EN. Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Physiological Reviews. 1990;70:567–590. doi: 10.1152/physrev.1990.70.2.567. - DOI - PubMed

LinkOut - more resources