GC Inlet temperatures per fiber type.

<p>GC Inlet temperatures per fiber type.</p

Fiber-dependent GC-MS identification and analysis of metabolites present in the human fecal VOC metabolome.

<p>A heat map presents the identified metabolites extracted by each SPME fiber and their relative chromatographic peak areas. Each of the eight columns in the heat map represents a different fiber. Metabolites are indicated on each row and are organized by functional group. Each extraction was performed in duplicate and replicates combined by averaging peak area values. Fiber legend: A - 75 µm CAR-PDMS, B - 85 µm CAR-PDMS, C - 50/30 µm CAR-DVB-PDMS, D - 85 µm PA, E - 65 µm DVB-PDMS, F - 7 µm PDMS, G - 100 µm PDMS, H - 60 µm PEG.</p

Extraction duration and headspace SPME of human feces.

<p>A) A plot of identified analytes as a function of extraction time. Nonlinear regression fitting the hyperbolic extraction curve yields a Y_max of 114+/−3 for the CAR-DVB-PDMS fiber (R² = 0.9937) and 94+/−4 for the PA fiber (R² = 0.9791). The PEG plot could not be extended beyond 20 min due to an unknown analyte overwhelming the MS detector. Also shown in this plot is the number of identified analytes obtained from a sample that was pretreated by boiling for 5 min before extraction. Essentially no difference in analyte number or composition is observed with the pretreated sample (CAR-DVB-PDMS+boiled sample) relative to an untreated sample (CAR-DVB-PDMS). All samples were analyzed in duplicate. See text for further discussion. B) A plot of area under the chromatographic curve as a function of time for the indicated analytes obtained using the CAR-DVB-PDMS fiber. Differences in extraction rates for the indicated metabolites are apparent.</p

Heat sterilization of human feces.

<p>A) Human fecal aliquots, dispensed in vials, were either autoclaved or placed in a boiling water bath then used as inoculum for liquid cultures incubated aerobically or anaerobically, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018471#s2" target="_blank">Materials and Methods</a>. While untreated (not autoclaved or boiled) fecal samples display growth in all three media compositions (LB – Luria Bertani media, TSB-cys – tryptic soy broth +0.1% cysteine media, Chamberlain – Chamberlain media) and culturing conditions, autoclaving or boiling the samples abolishes growth. Reported values are average of duplicates. B) Aliquots of a human fecal sample were incubated at 60°C for the indicated duration then used as inoculum for LB-agar plates, as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018471#s2" target="_blank">Materials and Methods</a>. A plot of colony forming units (cfu) as a function of incubation duration illustrates the loss of enteric microbial viability over the first hour of 60°C incubation, with no growth observed after 2 hours and beyond. The experiment was performed in duplicate.</p

Binary plot illustrating the GC-MS chromatographic peaks (metabolites) associated with each SPME fiber.

<p>Columns represent the fibers while rows indicate peak retention times. Each extraction was performed in duplicate and replicates combined. Fiber legend: A - 75 µm CAR-PDMS, B - 85 µm CAR-PDMS, C - 50/30 µm CAR-DVB-PDMS, D - 85 µm PA, E - 65 µm DVB-PDMS, F - 7 µm PDMS, G - 100 µm PDMS, H - 60 µm PEG.</p

Top fiber combinations.

a<p>based on maximal coverage of the total metabolites identified.</p>b<p>bracketed values are the percentage of total metabolites obtained using all eight fibers.</p

Recommended fiber combinations.

a<p>percentage of total metabolites obtained using all eight fibers.</p>b<p>substitution with the 85 µm PA fiber results in 92, 92, and 97% coverage, respectively.</p

Fiber-dependent extraction of metabolites.

a<p>bracketed values are the percentage of total metabolites identified.</p>b<p>number of metabolites exclusively associated with the fiber type.</p