Detection of an extended human volatome with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry

Abstract

Background: Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GCxGC-TOF MS) has been proposed as a powerful new tool for multidimensional analysis of complex chemical mixtures. We investigated GCxGC-TOF MS as a new method for identifying volatile organic compounds (VOCs) in normal human breath.

Methods: Samples of alveolar breath VOCs and ambient room air VOC were collected with a breath collection apparatus (BCA) onto separate sorbent traps from 34 normal healthy volunteers (mean age = 40 yr, SD = 17 yr, male/female = 19/15). VOCs were separated on two serial capillary columns separated by a cryogenic modulator, and detected with TOF MS. The first and second dimension columns were non-polar and polar respectively.

Results: BCA collection combined with GC×GC-TOF MS analysis identified approximately 2000 different VOCs in samples of human breath, many of which have not been previously reported. The 50 VOCs with the highest alveolar gradients (abundance in breath minus abundance in ambient room air) mostly comprised benzene derivatives, acetone, methylated derivatives of alkanes, and isoprene.

Conclusions: Collection and analysis of breath VOCs with the BCA-GC×GC-TOF MS system extended the size of the detectable human volatile metabolome, the volatome, by an order of magnitude compared to previous reports employing one-dimensional GC-MS. The size of the human volatome has been under-estimated in the past due to coelution of VOCs in one-dimensional GC analytical systems.

Conflict of interest statement

Competing Interests: Michael Phillips is President and CEO of Menssana Research, Inc. LECO Corporation subsidized the lease of the system to Menssana Research. ML performed this work while employed by LECO.

Figures

Figure 1. Chromatogram displaying analysis of breath VOCs in a typical normal human subject.

The x-axis (horizontal) displays retention time (sec) on the non-polar primary column, and the z-axis (front to rear) displays retention time (sec) on the secondary polar column. The y-axis (vertical) represents the intensity of the peak and varies with the abundance of a VOC and the molecule specific (but not currently described) sensitivity of the method to each analyte. Panel 1: Zero rotation about x-axis. In this view, the z-axis is not visible, and the chromatogram resembles a conventional 1D GC MS chromatogram displaying approximately 150-200 peaks. Panels 2 and 3: 30 and 60 degrees rotation about x-axis. As the chromatogram rotates, peaks that appeared apparently single on the x-axis in Panel 1 are resolved into several subsidiary peaks on the z-axis. Panel 4: 90 degrees rotation about x-axis. Each dot represents an individual VOC in the chromatogram. TOF-MS identified approximately 2,000 different VOC peaks in this chromatogram. This provides a more sensitive depiction of the chromatographic data because it displays VOCs whose peaks are too small to be visible in the other panels. Several different categories of chemical species were observed, including terpenes, alcohols, ketones, alkanes, alkenes, esters, aldehydes, furans, benzene derivatives, and sulfides. Contour plot displays of GC×GC peaks can potentially separate breath VOCs into “chemical islands”. For example, alkanes constitute the majority of the VOCs in the oval areas outlined in the figure. Groups of similar VOCs, differing by a methyl group for example, are resolved by this technique.

Figure 2. Topographical view of subtractive chromatogram displaying alveolar gradients in a typical normal human subject.

Background air VOCs have been subtracted from the breath VOCs. In this view, rotated in comparison to Figure 1, the x-axis (lower right) displays retention time (sec) on the non-polar primary column, and the z-axis (lower left) displays retention time (sec) on the secondary polar column. Generally, positive peaks represent endogenous VOCs synthesized in the body and negative peaks represent ambient room air VOCs that have been cleared from the body by catabolism and/or renal excretion.

Figure 3. Heat maps of breath VOCs.

The abundance of VOCs with an alveolar gradient greater than zero is shown in four randomly selected subjects (Figure 3). Color coding indicates the number of standard deviations by which the abundance of a VOC in an individual differs from the mean abundance in all subjects.

Figure 4. Comparison of 1D and 2D chromatograms containing hexane.

The bottom panel shows the 1D chromatogram of breath VOCs in a single subject (inset) with detail around the hexane peak at 7.23 minutes (peak a). The ion fragmentation spectrum of the peak is displayed on the right in red. The top panel shows six peaks (b-h) in the 2D chromatogram that coeluted with hexane (c) on the non-polar column but with different retention times on the polar column. These peaks were identified by Chroma-TOF and the NIST library as (b) 1,3-pentadiene (c) hexane, (d) dimethyl selenide, (e) 4H-pyrazole, 3-tert-butylsulfanyl-4,4-bistrifluoromethyl- (f) butanal, (g) methyl vinyl ketone and (h) 3,5-dihydroxybenzamide. The ion fragmentation spectrum of each peak is displayed on the right. All intensities in this figure are plotted on a logarithmic scale.