Headspace sorptive extraction-gas chromatography-mass spectrometry method to measure volatile emissions from human airway cell cultures - PubMed (original) (raw)

Headspace sorptive extraction-gas chromatography-mass spectrometry method to measure volatile emissions from human airway cell cultures

Mei S Yamaguchi et al. J Chromatogr B Analyt Technol Biomed Life Sci. 2018.

Abstract

The human respiratory tract releases volatile metabolites into exhaled breath that can be utilized for noninvasive health diagnostics. To understand the origin of this metabolic process, our group has previously analyzed the headspace above human epithelial cell cultures using solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). In the present work, we improve our model by employing sorbent-covered magnetic stir bars for headspace sorptive extraction (HSSE). Sorbent-coated stir bar analyte recovery increased by 52 times and captured 97 more compounds than SPME. Our data show that HSSE is preferred over liquid extraction via stir bar sorptive extraction (SBSE), which failed to distinguish volatiles unique to the cell samples compared against media controls. Two different cellular media were also compared, and we found that Opti-MEM® is preferred for volatile analysis. We optimized HSSE analytical parameters such as extraction time (24 h), desorption temperature (300 °C) and desorption time (7 min). Finally, we developed an internal standard for cell culture VOC studies by introducing 842 ng of deuterated decane per 5 mL of cell medium to account for error from extraction, desorption, chromatography and detection. This improved model will serve as a platform for future metabolic cell culture studies to examine changes in epithelial VOCs caused by perturbations such as viral or bacterial infections, opening opportunities for improved, noninvasive pulmonary diagnostics.

Copyright © 2018 Elsevier B.V. All rights reserved.

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Conflict of interest statement

Conflict of interest disclosure

The authors have no conflicts to declare.

Figures

Figure 1

Figure 1

A typical cell culture jar. Magnets hold PDMS-coated stir bars in place for headspace extraction. Transwells contain an immortalized epithelial cell line, which lives as a confluent layer on top of the media.

Figure 2

Figure 2

Raw chromatograms of media control samples. Background VOCs from the two media types were very similar; Opt-MEMS (OPT) was chosen as the optimum medium as it provides more nutrients to cells compared to the phosphate-buffer solution (PBS).

Figure 3

Figure 3

PCAs of headspace extraction (HSSE, A) and liquid extraction (SBSE, B) for cell cultures and media control samples. HSSE was able to distinguish epithelial VOCs from the media along the first principal component whereas SBSE was not.

Figure 4

Figure 4

Number of discrete compounds detected from epithelial cells and media controls at three HSSE extraction times. The 24 h samples extracted the highest number of cellular VOCs while still being distinguishable from media controls.

Figure 5

Figure 5

PCA comparing desorption temperatures. Nine samples were taken per jar and were split into three desorption temperatures. The highest, 300 °C, gave the best separation between media and cell culture samples.

Figure 6

Figure 6

Raw chromatograms of SPME versus Twister® samples from the same jar of cells. Twisters® extracted more cellular VOCs due to the larger amount of sorbent compared to the SPME fibers.

Figure 7

Figure 7

Figure 8

Figure 8

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