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.
Keywords: comprehensive two-dimensional gas chromatography; in vivo active sampling; plant volatiles; thermal desorption (TD); volatile organic compounds (VOCs).