Background: Plants constantly produce primary and secondary metabolites, and a significant fraction of these are volatile organic compounds (VOCs). Factors including the life stage of the plant, temperature, environment, and stress influence the abundance and types of VOCs emitted. The analysis of VOCs released by plants during different stages or with different conditions provides insight into plant metabolism and stress responses. Collecting the VOC profiles of plants in vivo makes it possible to obtain a representative sample of the entire plant volatilome under controlled conditions with minimal invasiveness. In addition, in vivo sampling can also be used to compare the impacts of different environmental conditions or stressors on plants, i.e., the presence/absence of a pest or amount of nitrogen in soil. Methods: In this study, an in vivo plant sampling technique is introduced and validated using active sampling and thermal desorption (TD) tubes with comprehensive two-dimensional gas chromatography coupled to a time-of-flight mass spectrometer (TD-GC×GC-TOFMS). The purpose of this work is to highlight a novel technique to analyze headspace secondary plant metabolites with a minimal invasiveness. Results: It was concluded that in vivo active sampling onto TD tubes provides a wider global coverage of compounds and larger peak areas when compared to extraction by solid-phase microextraction (SPME). Additionally, the Horwitz ratio of active sampling onto TD tubes was 0.893, demonstrating this technique to be a reliable and reproducible method. Lastly, a variety of plants were sampled to assess the versatility of this technique across various plant species with different sizes and volatile profiles. Hundreds of compounds were measured with this analysis, including terpenes, aldehydes, ketones, terpenoids, and alcohols. Conclusions: This novel in vivo active sampling method provides an additional technique for extracting and analyzing volatile secondary plant metabolites.

Stool is a mixture of excrement, microbiota, enzymes, undigested material, and small molecules. Fecal metabolomics has gained interest recently, owing to advances in metabolomics and growing research into both the host’s physiology and the gut microbiome. One challenge with fecal metabolomics is preserving the sample integrity from collection until analysis, as the microbiota and enzymes continue to alter the metabolome following defecation. Currently, flash-freezing or lyophilization are utilized to minimize post-collection metabolome changes; however, this requires complex equipment and immediate processing, precluding the possibility for at-home sampling. Commercial devices containing stabilizing solvents have been developed to facilitate at-home collection, ambient transport, and sample storage. Here, we explore the efficacy of a commercially available stool collection device with a stabilization reagent tailored to fecal metabolomics. Stool samples from six donors were either processed shortly post-collection or stored at room temperature for seven days in the tube, with and without the stabilization reagent. Comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS)-based untargeted metabolomics was utilized for analyzing extracted metabolites. Chemometric analysis was used to evaluate the performance of the device. We found that the device with the stabilization reagent minimized changes in the metabolite profile relative to unstabilized stool left at room temperature for one week.