# Analysis of _In Vivo_ Plant Volatiles Using Active Sampling and TD-GC\(\times\)GC-TOFMS
## 1 Introduction  
As plants grow, they produce many primary metabolites with a variety of functions including growth regulation, development, and reproduction [1]. Plants also produce a variety of secondary metabolites for purposes such as defense from biotic and abiotic stressors, plant communication, and the attraction of pollinators [1; 2; 3; 4]. Volatile organic compounds (VOCs) are one of the most important groups of plant metabolites; as plants can emit them, they represent one of the few ways in which a plant can influence the behavior of other nearby organisms. For example, maize (_Zea mays_) seedlings exposed to defense VOCs from neighboring plants will produce a higher amount of volatile sesquiterpenes to protect themselves from a potential stressor, demonstrating the importance of signaling between plants [5]. In tomato (_Solanum_ spp.) plants, it has been observed that some varietiesrelease zingiberene and curcuminene, which are sesquiterpenes that reduce infestation by whiteflies [6]. Sesquiterpenes are considered juvenile hormone analogs in insects that are important in insect development and reproduction [7]. There are several chemical families of plant VOCs including terpen, ketones, aldehydes, hydrocarbons, aromatics, and thiols [8]. Understanding the VOC profile of plants provides valuable insight into plant biology, specifically in terms of plant defense to abiotic and biotic stressors, intra- and interspecies plant communication, and pollination. There are several extraction techniques that have been used to study plant metabolites, including solvent extraction, Soxhlet extraction, and supercritical fluid extraction (SFE) [9; 10; 11; 12; 13]. Extractions can be performed on whole plants or parts of plant organs or using a sorbing matrix that has collected headspace plant VOCs [13; 14; 15]. Unfortunately, these methods require the use of solvents, are destructive to plants, and are time-consuming, and volatiles may be lost during the extraction process [16]. A popular and powerful approach for studying plant VOCs is using solid-phase microextraction (SPME) as the extraction mechanism [9; 14; 16; 17; 18]. SPME is a simple, solvent-free, fast, and sensitive sampling technique that can be conducted statically (no movement or flow in the headspace) or dynamically (movement/flow in the headspace), with dynamic sampling enhancing the extraction of trace compounds [19]. Although SPME has improved the analysis of plant VOCs over previous methods, this technique does have several disadvantages including complex quantification (SPME is an equilibrium method and the plant fiber partition coefficients of analytes are generally unknown), and the sorbent with its limited surface area greatly influences the quantity and type of compounds being extracted [15]. An alternative to SPME is active sampling onto a thermal desorption (TD) tube. This has been frequently used to analyze environmental, human breath, and human blood samples [20; 21; 22; 23]. In this mode of operation, a pump pulls an air sample through a TD tube containing a bed of one or more sorbents. A larger range of chemistries, higher masses of sorbent, and a concomitant increase in surface area increase the range of analyte chemistries that can be collected and the absolute mass of analyte extracted, when compared to SPME [24; 25]. Volatiles are desorbed from the TD tube by heating it while a flow of inert gas transports sorbed compounds to a smaller cold trap for focusing. Following focusing, the rapid heating of the smaller trap facilitates desorption directly into a gas chromatograph (GC) for the separation of the volatile mixture. Coupling GC with TD, as the sample introduction method, significantly improves the sensitivity, reproducibility, and recovery compared to solvent extractions and SPME [26; 27]. Additionally, TD tubes can be reused approximately 100 times, samples can be stored long-term, the transportation of samples between laboratories is simplified, and the automation of TD is readily available and solvent-free [26; 28; 29; 30].