Solid-Phase Microextraction and the Human Fecal VOC Metabolome

Emma Dixon,Larry Ammann,Pat Gillevet,Nazia Kazmi,Huzefa Rangwala,Sara Pittman,Cynthia Clubb,Robin D Couch,Ali Keshavarzian,Zeehasham Rasheed,Hassan Ashktorab

doi:10.1371/journal.pone.0018471

Abstract

The diagnostic potential and health implications of volatile organic compounds (VOCs) present in human feces has begun to receive considerable attention. Headspace solid-phase microextraction (SPME) has greatly facilitated the isolation and analysis of VOCs from human feces. Pioneering human fecal VOC metabolomic investigations have utilized a single SPME fiber type for analyte extraction and analysis. However, we hypothesized that the multifarious nature of metabolites present in human feces dictates the use of several diverse SPME fiber coatings for more comprehensive metabolomic coverage. We report here an evaluation of eight different commercially available SPME fibers, in combination with both GC-MS and GC-FID, and identify the 50/30 µm CAR-DVB-PDMS, 85 µm CAR-PDMS, 65 µm DVB-PDMS, 7 µm PDMS, and 60 µm PEG SPME fibers as a minimal set of fibers appropriate for human fecal VOC metabolomics, collectively isolating approximately 90% of the total metabolites obtained when using all eight fibers. We also evaluate the effect of extraction duration on metabolite isolation and illustrate that ex vivo enteric microbial fermentation has no effect on metabolite composition during prolonged extractions if the SPME is performed as described herein.

Highlights

Volatile organic compounds (VOCs) are a large and highly diverse group of carbon-based molecules, generally related by their volatility at ambient temperature
A typical headspace solid-phase microextraction (SPME) analysis involves the extraction of the VOCs via partitioning into a polymeric coating adhered to a fused silica rod, subsequent desorption of the VOCs by heating the fiber in the injection port of a gas chromatograph, separation of the VOCs by gas-liquid partition chromatography, and detection of the VOCs via flame ionization and/or mass spectrometry
Extraction duration To ascertain the effect of extraction duration on metabolite isolation from the headspace above human feces, we used three different SPME fibers (a 50/30 mm CAR-DVB-PDMS, 85 mm PA, and a 60 mm polyethylene glycol (PEG) fiber) in conjunction with GC-MS to identify and quantify the volatile metabolites

Summary

Introduction

Volatile organic compounds (VOCs) are a large and highly diverse group of carbon-based molecules, generally related by their volatility at ambient temperature. Specialized headspace sampling methods, such as solid-phase microextraction (SPME), have greatly facilitated the isolation of VOCs from biological specimens [10,11,12,13]. The duration of extraction and choice of polymeric coating are two important considerations when performing a SPME analysis. A quantitative analysis is performed when the analyte distribution is in equilibrium between the sample and the SPME fiber coating, such that small deviations in extraction duration do not significantly alter analyte titers [14]. While the polarity of the analyte of interest is typically used to guide the selection of a particular SPME fiber [13], metabolomic analyses generally strive to isolate and identify numerous, chemically diverse types of analyte molecules. The multifarious nature of biological sample composition complicates the SPME procedure, as compounds with higher affinity for the fiber may compete with and displace those with lower affinity [15]

Methods

Results

Conclusion