Abstract
Volatile organic compounds (VOCs) have been proposed in the last two decades as biomarkers for disease detection and therapeutic monitoring. Model in vitro experiments with established cell lines are fundamental to clarify whether given VOCs originate from normal human cells or pathogens, including transformed cancer cells. Due to the trace concentrations of target metabolites, adsorptive enrichment is needed before gas chromatography-mass spectrometry (GC-MS) analysis, with solid-phase microextraction (SPME) being perfectly suited for this purpose. Here, a modification of SPME, the thin-film microextraction (TFME) technique, is proposed for analysis of cellular VOCs, which utilizes a planar mesh coated with stationary phase to increase the extraction phase volume and active surface area. In this study, four different adsorbents were compared: carboxen, divinylbenzene, hydrophobic−lipophilic balanced and polydimethylsiloxane. Amongst them, HLB sheets using poly(divinylbenzene-co-N-vinyl-pyrrolidone) skeleton structure proved to be the most versatile, enabling the most sensitive analysis of VOCs with a broad polarity and volatility. For HLB, sampling type (internal static headspace, external bi-directional headspace), extraction temperature and extraction time were also examined. An established method was successfully applied to analyze metabolites produced by A549 cells revealing five volatiles at significantly higher (additionally benzaldehyde at lower) levels in cell culture medium compared to the cell-free reference medium headspace.
Highlights
Non-small cell lung cancer (NSCLC) is still one of the most common cancer diseases, with a five year survival rate varying from 92 to 0% [1] depending on the cancer stage at initial diagnosis
In the in vitro studies focused on the A549 volatilome, diverse sampling techniques were used, inter alia, an exhaustive technique with a packed sorption tube monolith monotrap, but in most cases volatile organic compounds (VOCs) were extracted on solid-phase microextraction (SPME) fibers coated either with carboxen-divinylbenzene [8,10,15], or a carboxen [16] phase
In ref [9,16] cultivation flasks were separated from the incubator atmosphere by turning caps to the gas tight position 1 h before sampling. Such an approach allows for a “cleaner” background, lack of CO2 may have an impact on cell growth
Summary
Non-small cell lung cancer (NSCLC) is still one of the most common cancer diseases, with a five year survival rate varying from 92 to 0% [1] depending on the cancer stage at initial diagnosis. Volatile organic compounds (VOCs) are receiving increasing interest as potential biomarkers related to lung cancer [3], but their trace concentrations require a preconcentration step prior to offline instrumental analysis. In this regard, solid-phase microextraction (SPME) is a widely used preparation technique that relies on obtaining equilibrium between analyte concentration in a sample and the amount extracted by the device [4]. SPME fibers have been proven to extract VOCs from diverse human body fluids including exhaled breath samples, tissues from stomach cancer patients [6] and from lung cancer patients [7,8]
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