Abstract
IntroductionDisturbance 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.ObjectivesTo compare a sample preparation method for extracting volatile organic compounds (VOCs) from equine faeces.MethodsVolatile 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.ResultsThe 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.ConclusionsAn 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.
Highlights
Disturbance to the hindgut microbiota can be detrimental to equine health
It has previously been demonstrated that vial volume, SPME fibre coating and mass of faecal material have an effect on the number and abundance of volatile organic compounds (VOCs) in human and murine faecal samples (Reade et al 2014)
The coefficient of variation (CV) of VOC peak area was calculated for VOCs shared between replicated of each sample mass
Summary
The equine hindgut contains a complex and diverse community of microorganisms (microbiota). The low molecular weight and high vapour pressure of VOCs allows them to enter the gaseous phase at room temperature; contributing to the odour of faeces, urine, breath, saliva, blood and sweat These compounds may be generated by physiological processes from the host or by its microbiota (Amann et al 2014). It has previously been demonstrated that vial volume, SPME fibre coating and mass of faecal material have an effect on the number and abundance of VOCs in human and murine faecal samples (Reade et al 2014). This is not surprising as faecal VOC profiles differ between species (Saric et al 2008). The SPME fibres were pre-conditioned in accordance with manufacturer’s instructions before use
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