USDA-ARS Research Project number 6036-22000-028-00D. The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agriculture Research Service of any product or service to the exclusion of others that may be suitable. Additionally, the University of Florida Biology Department-Lewis and Varina Vaughn Fellowship in Orchid Biology (2017), and a University of Florida Graduate Research Fellowship (2014-2018) provided funding as well. We also thank Cindy Bennington from Stetson University for the orchid plant used during filming of this video.

NOTE: Perfumes or scented lotions and products must not be worn during any of these procedures.
1. Flower selection
NOTE: Flowers used can be either naturally growing in the environment or kept under artificial environmental conditions. Temperature, humidity, and light level during collection can vary based on the specific flower species used, and what type of data is being collected. For example, data has been collected during the day and at night for the same flower to determine if fragrance varies over time of day, and collected from both in situ and greenhouse flowers.
<ol>
	<li>Select a flower that is initially unopened, to standardize sample collection time. This controls for a flower changing fragrance over time.</li>
	<li>Depending on the duration of the blooming time, if possible, wait at least 24 hours after blooming to collect the sample, setting a standard time for all samples.</li>
	<li>If there are multiple blooming flowers on a plant, mark the one that will be used with flagging tape or something similar to ensure repeated sampling of the same flower.</li>
</ol>
2. Material preparation
<ol>
	<li>Use oven bags (approximately 40.5 cm &#215; 44.5 cm) and corrugated PTFE tubing.</li>
	<li>Initially, boil oven bags in water for ~30 min to remove residual plastic compounds. To dry, bake in an oven at 175 &#176;C.</li>
	<li>Once the bags have dried, add a polypropylene bulkhead union to each corner of the closed end of the oven bags. These attachments allow connection of tubes to push charcoal filtered air in and pull fragrance out of the headspace.</li>
	<li>Rinse all bags and tubes with 75% ethanol. Let both air dry after rinsing.</li>
	<li>After the oven bags have dried, bake bags and tubes in an oven at low heat, approximately 74-85 &#176;C for 30 min.</li>
</ol>
3. Volatile collection
NOTE: Sterile neoprene gloves need to be worn throughout this process, as contacting the bag or filter cartridges can contaminate the samples.
<ol>
	<li>Cover the selected flower with a baked oven bag. Cinch the bag together tightly with a plastic zip tie below the flower to prevent unwanted airflow into the bag.</li>
	<li>Attach a tube from the air outlet of the collection equipment and connect it to one of the bulkhead unions on the oven bag.</li>
	<li>On the other bulkhead union, attach a glass filter cartridge containing porous polymer adsorbent.</li>
	<li>Attach a second tube to the collection equipment on the vacuum input. Connect end of the second tube onto the glass volatile collection filter cartridge.</li>
	<li>Turn on both the air pump and vacuum at the same time set at ~0.05 L/min. The headspace around the flower will fill with air, but not become overinflated. The system will pull air from the bag through the filter, trapping the floral volatiles.</li>
	<li>Allow the machine to run for 10 min and then turn off both the air pump and vacuum. 
	NOTE: Flower species that produce/emit a smaller amount of fragrance may need to be sampled for a longer period of time.</li>
	<li>Disassemble the tubes and the glass filter cartridge. Place the filter into a glass vial with a screw-on cap. Once the cap is on, seal the vial with PTFE pipe thread tape.</li>
	<li>Store samples in a freezer until analyzed using GC-MS.</li>
	<li>Repeat this process with a clean oven bag and glass filter, this time with an empty oven bag, to collect a blank air sample as a control. This allows any background volatiles collected to be identified. 
	NOTE: Repeating sample collection needs to be done at approximately the same time each day, as some flowers produce varying fragrance levels over the course of a day.</li>
</ol>
4. GC-MS
<ol>
	<li>Remove the glass filter cartridge from the freezer and place into a GC-MS in the injector port.</li>
	<li>Release headspace volatiles collected on porous polymer adsorbent from the adsorbent by heating in the thermal collection trap (TCT) to 220 &#176;C for 8 min within a flow of helium gas (rate: 1.2 mL/min).</li>
	<li>Collect desorbed compounds in the TCT cold trap unit at -130 &#176;C. The cold trap temperature is regulated by the GC-MS program.</li>
	<li>Flash heat the TCT cold trap unit to inject the compounds into the capillary column of the gas chromatograph to which the TCT cold trap unit was connected. The method for the TCT begins at -20 &#176;C and ends at 150 &#176;C.</li>
	<li>Program the GC-MS to rise from 40 &#176;C to 280 &#176;C at 15 &#176;C/min, with a 5 min hold at 40 &#176;C.</li>
</ol>
5. Data analysis
<ol>
	<li>For identification, compare the mass spectra of the sample to those from mass spectra libraries (NIST and Department of Chemical Ecology, Goteborg University, Sweden11), as well as retention times of the volatiles to times of authentic compound standards12.</li>
	<li>Compare chromatograms of collected volatiles to identify common reoccurring peaks.</li>
	<li>After identifying the peak volatiles, use Pherobase (online database of semiochemicals and pheromones) to determine if they have been previously described in floral fragrances10.</li>
</ol>

<ol>
	<li>Knudsen, J. T., Tollsten, L., Bergstrom, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+scents-+A+checklist+of+volatile+compounds+isolated+by+head-space+techniques.">Floral scents- A checklist of volatile compounds isolated by head-space techniques.</a> Phytochemistry. 33, 253-280 (1993).</li><li>Dudareva, N. A., Pichersky, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of floral scent. , (2006).</li><li>Altenburger, R., Matile, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythms+of+fragrance+emission+in+flowers.">Rhythms of fragrance emission in flowers.</a> Planta. 174, 242-247 (1988).</li><li>Dodson, C. H., Dressler, R. L., Hills, H. G., Adams, R. M., Williams, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologically+active+compounds+in+orchid+fragrances.">Biologically active compounds in orchid fragrances.</a> Science. 164, 1243-1249 (1969).</li><li>Theis, N., Raguso, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+pollination+on+floral+fragrance+in+thistles.">The effect of pollination on floral fragrance in thistles.</a> Journal of Chemical Ecology. 31 (11), 2581-2600 (2005).</li><li>Heath, R. R., Manukian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+systems+to+collect+volatile+semiochemicals+from+insects+and+plants+using+a+charcoal-infused+medium+for+air+purification.">Development and evaluation of systems to collect volatile semiochemicals from insects and plants using a charcoal-infused medium for air purification.</a> Journal of Chemical Ecology. 18, 1209-1226 (1992).</li><li>Cancino, A., Damon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+floral+fragrance+components+of+species+of+Encyclia+and+Prosthechea+(Orchidaceae)+from+Soconusco,+southeast+Mexico.">Comparison of floral fragrance components of species of Encyclia and Prosthechea (Orchidaceae) from Soconusco, southeast Mexico.</a> Lankesteriana. 6, 83-139 (2006).</li><li>Cancino, A., Damon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragrance+analysis+of+euglossine+bee+pollinated+orchids+from+Soconusco,+south-east+Mexico.">Fragrance analysis of euglossine bee pollinated orchids from Soconusco, south-east Mexico.</a> Plant Species Biology. 22, 129-134 (2007).</li><li>Sadler, J. J., Smith, J. M., Zettler, L. W., Alborn, H. T., Richardson, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragrance+composition+of+Dendrophylax+lindenii+(Orchidaceae)+using+a+novel+technique+applied+in+situ.">Fragrance composition of Dendrophylax lindenii (Orchidaceae) using a novel technique applied in situ.</a> European Journal of Environmental Science. 1, 137-141 (2011).</li><li>Ray, H. A., Stuhl, C. J., Gillett-Kaufman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+fragrance+analysis+of+Prosthechea+cochleata+(Orchidaceae),+an+endangered+native,+epiphytic+orchid,+in+Florida.">Floral fragrance analysis of Prosthechea cochleata (Orchidaceae), an endangered native, epiphytic orchid, in Florida.</a> Plant Signaling and Behavior. , (2018).</li><li>. National Institute of Standards and Technology. U.S. Department of Commerce Available from: <a target="_blank" href="https://www.nist.gov/">https://www.nist.gov/</a> (2019)</li></ol>

The authors declare no conflicts of interest.

Though this technique is incredibly valuable for its sampling speed and portability, one limitation is using it for epiphytic species, or those growing on trees and not from the ground. In the original study10, one of the flowers sampled was epiphytic. Because the machine is too heavy to hang freely, a stable, elevated base must be made for sampling. Additionally, the machine can either be plugged in to an electrical outlet or battery powered, so if there is prolonged field sampling, there must be...

Flowers typically produce a fragrance used to attract pollinators. These fragrances are made up of many chemical compounds all acting together as a floral blend1,2,3. Without these fragrances, flowers would be less likely to pass on their genetic information using pollinators. Floral fragrance has been documented in many flowering plant families, with Orchidaceae being one of the more common families studied4. To understand the role of floral fragrance in pollination, it is important to nondestructively collect and analyze the chemical compounds being emitted from the flowers at different times of the day and during the several days to weeks the flower blooms are open, as fragrance can vary over time5.
An early protocol for this kind of sampling was developed by Heath and Manukian6. The goal of their sampling methods was to reduce stress on the specimen (e.g., plants, insects) being studied. Earlier papers documented that destructive procedures to the plant were required, such as removing blooming flowers in order to collect the fragrance. More recent floral fragrance publications by Cancino and Damon7,8 used similar methods. This study put the flowers in glass chambers and passed purified air over them; then fragrance compounds from the chamber were absorbed onto porous polymer adsorbents in clear Pasteur pipettes. The fragrances were collected for at least two hours during this study. Sadler et al.9 carried out floral fragrance studies on an epiphytic orchid in south Florida, much like the original study10. Again, this study required the flowers to be sampled for over two hours to collect the fragrance volatiles, with fragrance collected onto the porous polymer adsorbent. The paper here presents a non-destructive method that allows for much quicker sampling, lasting only 10 minutes. Also, instead of using a glass chamber oven baking bags are used, which allow for more flexible movement of the chamber and reduce the chances of damage to the flowers. These bags come in several sizes allowing the option to select the size of bag that will easily fit individual samples without damaging the sample or the surrounding material. The adsorbent used in this study was Tenax Porous Polymer Adsorbent. This differs from Porapak, because the sample can be thermally desorbed onto the GC-MS column for analysis, eliminating the use of a chemical solvent.
The methods in this study provide a way to quickly sample fragrance volatiles produced by flowers and could be used to sample volatiles from other specimens as well, such as insect pheromones, or mushroom volatiles. The reduced time for sampling means there is less stress on the sample and the ability to collect many samples in a short period of time. For example, in Sadler et al.9, the flower was only fragrant at night, so only two or three samples could be collected each night. With the method here, samples could be taken all night at 15-20-minute intervals from the same flower. Additionally, by using bags instead of glass chambers, the headspace can be suspended easier for sampling in the field for in situ collection on endangered or threatened plant species. Using the method presented here, we were able to sample flowers 1.5 to 2 meters above ground. These methods are incredibly useful for fragrance collection in the laboratory and the field, and provides researchers with a sampling technique that is fast and non-destructive to the sample.

<table><tbody><tr><td>Bulkhead Union</td><td>Cole-Palmer</td><td>UX-06390-10</td><td></td></tr><tr><td>FEP tubing</td><td>Cole-Palmer</td><td>UX-06407-60</td><td></td></tr><tr><td>Gas Chromatography</td><td>Hewlett Packard</td><td>6890</td><td></td></tr><tr><td>Glass Wool, Silanized</td><td>Sigma-Aldrich</td><td>20411</td><td></td></tr><tr><td>Inlet liner</td><td>Agilent</td><td>5062-3587</td><td></td></tr><tr><td>Mass Spectrometer</td><td>Hewlett Packard</td><td>5973</td><td></td></tr><tr><td>Reynolds oven bag</td><td>Reynolds Consumer Products</td><td>Turkey size</td><td></td></tr><tr><td>Tenax Porous Polymer Adsorbent</td><td>Sigma-Aldrich</td><td>11982</td><td></td></tr></tbody></table>

rapid collection of floral fragrance volatiles using a headspace volatile collection technique for gc-ms thermal desorption sampling

Fragrances of many flower families have been sampled and the volatiles analyzed. Knowing the compounds that make up the fragrances can be an important step to conservation of flowers that are threatened or endangered. Because floral fragrance is critical for attracting pollinators, this method could be used to better understand or even enhance pollination. We present a protocol using a portable charcoal air filter and vacuum to collect floral fragrance volatiles, which are then analyzed by a GC-MS. By using this method, fragrance volatiles can be sampled using a non-destructive method with a machine that is easily transported. This methodology uses a rapid sampling procedure, cutting sampling time down from 2-3 hours to approximately 10 minutes. Using GC-MS, the fragrance compounds can be identified individually, based on authentic standards. The steps used for collecting fragrance and control data are presented, from material setup to collecting the data output.

Representative data from the GC-MS are shown as a chromatogram in Figure 1. In addition to the chromatogram, a data file of results is also provided (Supplementary File 1). This data file provides the retention time for each peak (RT), and an identification of what compound that peak is (Library/ID). Peaks between 10:00 and 15:00 minutes are floral volatiles, due to the molecular weight of the compounds10. The numbers above the peaks signify the reten...

Watch this Scientific Journal Video about Rapid Collection of Floral Fragrance Volatiles using a Headspace Volatile Collection Technique for GC-MS Thermal Desorption Sampling at JoVE.com

Rapid Collection of Floral Fragrance Volatiles using a Headspace Volatile Collection Technique for GC-MS Thermal Desorption Sampling

Here, we present a protocol for collecting the floral fragrance volatiles from blooming flowers, using a non-destructive sampling procedure.

Fragrances of many flower families have been sampled and the volatiles analyzed. Knowing the compounds that make up the fragrances can be an important ...

rapid-collection-floral-fragrance-volatiles-using-headspace-volatile

Research

JoVE Journal

Biochemistry

6.8K Views.  University of Florida. Omdat bloemengeur is van cruciaal belang voor het aantrekken van bestuivers, deze methode kan worden gebruikt om beter te begrijpen of zelfs te verbeteren bestuiving van inheemse bloemsoorten. Met deze methode worden geurvolstelingen bemonsterd met behulp van een niet-destructieve methode met een gemakkelijk te vervoeren machine. Deze methode maakt gebruik van snelle bemonstering, het snijden van bemonstering tijd van twee tot drie uur tot 10 minuten.Deze methoden bieden een manier om ...

Video: Rapid Collection of Floral Fragrance Volatiles using a Headspace Volatile Collection Technique for GC-MS Thermal Desorption Sampling

Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.

Trace explosive vapors of TNT and RDX collected on sorbent-filled thermal desorption tubes were analyzed using a programmed temperature desorption system coupled to GC with an electron capture detector. The instrumental analysis is combined with direct liquid deposition method to reduce sample variability and account for instrumentation drift and losses.

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive ...

Chemistry

Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' (Formicidae) for Volatilome Analysis by GC-MS and MetaboliteDetector

The aim of this manuscript is to present a protocol describing the metabolomic analysis of Bornean &#39;exploding ants&#39; belonging to the Colobopsis cylindrica (COCY) group. For this purpose, the model species C. explodens is used. Ants belonging to the minor worker caste possess distinctive hypertrophied mandibular glands (MGs). In territorial combat, they use the viscous contents of their enlarged mandibular gland reservoirs (MGRs) to kill rival arthropods in characteristic suicidal &#39;explosions&#39; by voluntary rupture of the gastral integument (autothysis). We show the dissection of worker ants of this species for the isolation of the gastral portion of the wax-like MGR contents as well as listing the necessary steps required for solvent-extraction of the therein contained volatile compounds with subsequent gas chromatography-mass spectrometry (GC-MS) analysis and putative identification of metabolites contained in the extract. The dissection procedure is performed under cooled conditions and without the use of any dissection buffer solution to minimize the changes in the chemical composition of the MGR contents. After solvent-based extraction of volatile metabolites contained therein, the necessary steps for analyzing the samples via liquid-injection-GC-MS are presented. Lastly, data processing and putative metabolite identification with the use of the open-source software MetaboliteDetector is shown. With this approach, the profiling and identification of volatile metabolites in MGRs of ants belonging to the COCY group via GC-MS and the MetaboliteDetector software become possible.

Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' Formicidae for Volatilome Analysis by GC-MS and MetaboliteDetector

Minor workers of the ant species Colobopsis explodens are dissected to isolate the wax-like content stored in their hypertrophied mandibular gland reservoirs for subsequent solvent-extraction and analysis by gas chromatography-mass spectrometry. The&#160;annotation and identification of volatile constituents using the open-source software MetaboliteDetector is also described.

The aim of this manuscript is to present a protocol describing the metabolomic analysis of Bornean &#39;exploding ants&#39; belonging to the Colobopsis ...

Environment

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

Biology

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

Neuroscience

Sampling and Analysis of Animal Scent Signals

We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry, to understand how they may be used in animal communication. This technique allows the semi-quantitative analysis of the volatile components of odor secretions by enabling the separation and tentative identification of the components in the sample, followed by the analysis of peak area ratios to look for trends that could signify compounds that may be involved in signaling. The key strengths of this current approach are the range of sample types that can be analyzed; the lack of need for any complex sample preparation or extractions; the ability to separate and analyze the components of a mixture; the identification of the components detected; and the capability to provide semi-quantitative and potentially quantitative information on the components detected. The main limitation to the methodology relates to the samples themselves. Since the components of specific interest are volatile, and these could easily be lost, or their concentrations altered, it is important that the samples are stored and transported appropriately after their collection. This also means that sample storage and transport conditions are relatively costly. This method can be applied to a variety of samples (including urine, feces, hair and scent-gland odor secretions). These odors consist of complex mixtures, occurring in a range of matrices, and thus necessitate the use of techniques to separate the individual components and extract the compounds of biological interest.

We have developed an effective methodology for sampling and analysis of odor signals in order to understand how they may be used in animal communication. Particularly, we use headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry to analyze the volatile components of animal odors and scent-markings.

We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas ...

Behavior

Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars with enhanced organoleptic characteristics and thus, to increase consumer acceptance. High-throughput metabolomic platforms have been recently developed to quantify a wide range of metabolites in different plant tissues, including key compounds responsible for fruit taste and aroma quality (volatilomics). A method using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) is described here for the identification and quantification of VOCs emitted by ripe blackcurrant fruits, a berry highly appreciated for its flavor and health benefits.
Ripe fruits of blackcurrant plants (Ribes nigrum) were harvested and directly frozen in liquid nitrogen. After tissue homogenization to produce a fine powder, samples were thawed and immediately mixed with sodium chloride solution. Following centrifugation, the supernatant was transferred into a headspace glass vial containing sodium chloride. VOCs were then extracted using a solid-phase microextraction (SPME) fiber and a gas chromatograph coupled to an ion trap mass spectrometer. Volatile quantification was performed on the resulting ion chromatograms by integrating peak area, using a specific m/z ion for each VOC. Correct VOC annotation was confirmed by comparing retention times and mass spectra of pure commercial standards run under the same conditions as the samples. More than 60 VOCs were identified in ripe blackcurrant fruits grown in contrasting European locations. Among the identified VOCs, key aroma compounds, such as terpenoids and C6 volatiles, can be used as biomarkers for blackcurrant fruit quality. In addition, advantages and disadvantages of the method are discussed, including prospective improvements. Furthermore, the use of controls for batch correction and minimization of drift intensity have been emphasized.

A headspace solid-phase microextraction-gas-chromatography platform is described here for fast, reliable, and semi-automated volatile identification and quantification in ripe blackcurrant fruits. This technique can be used to increase knowledge about fruit aroma and to select cultivars with enhanced flavor for the purpose of breeding.

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars ...

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host&#39;s microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples.
Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the ...

Gas Chromatography-Mass Spectrometry Paired with Total Vaporization Solid-Phase Microextraction as a Forensic Tool

Gas Chromatography &#8211; Mass Spectrometry (GC-MS) is a frequently used technique for the analysis of numerous analytes of forensic interest, including controlled substances, ignitable liquids, and explosives. GC-MS can be coupled with Solid-Phase Microextraction (SPME), in which a fiber with a sorptive coating is placed into the headspace above a sample or immersed in a liquid sample. Analytes are sorbed onto the fiber which is then placed inside the heated GC inlet for desorption. Total Vaporization Solid-Phase Microextraction (TV-SPME) utilizes the same technique as immersion SPME but immerses the fiber into a completely vaporized sample extract. This complete vaporization results in a partition between only the vapor phase and the SPME fiber without interference from a liquid phase or any insoluble materials. Depending upon the boiling point of the solvent used, TV-SPME allows for large sample volumes (e.g., up to hundreds of microliters). On-fiber derivatization may also be performed using TV-SPME. TV-SPME has been used to analyze drugs and their metabolites in hair, urine, and saliva. This simple technique has also been applied to street drugs, lipids, fuel samples, post-blast explosive residues, and pollutants in water. This paper highlights the use of TV-SPME to identify illegal adulterants in very small samples (microliter quantities) of alcoholic beverages. Both gamma-hydroxybutyrate (GHB) and gamma-butyrolactone (GBL) were identified at levels that would be found in spiked drinks. Derivatization by a trimethylsilyl agent allowed for conversion of the aqueous matrix and GHB into their TMS derivatives. Overall, TV-SPME is quick, easy, and requires no sample preparation aside from placing the sample into a headspace vial.

Total Vaporization Solid Phase Microextraction (TV-SPME) completely vaporizes a liquid sample whilst analytes are sorbed onto a SPME fiber. This allows for partitioning of the analyte between only the solvent vapor and the SPME fiber coating.

Gas Chromatography &#8211; Mass Spectrometry (GC-MS) is a frequently used technique for the analysis of numerous analytes of forensic interest, including ...

Unveiling Tea Aroma Complexity with Solvent-Assisted Flavor Evaporation Method

Tea Aroma Analysis Based on Solvent-Assisted Flavor Evaporation Enrichment

Tea aroma is an important factor in tea quality, but it is challenging to analyze due to the complexity, low concentration, diversity, and lability of the volatile components of tea extract. This study presents a method for obtaining and analyzing the volatile components of tea extract with odor preservation using solvent-assisted flavor evaporation (SAFE) and solvent extraction followed by gas chromatography-mass spectrometry (GC-MS). SAFE is a high-vacuum distillation technique that can isolate volatile compounds from complex food matrices without any non-volatile interference. A complete step-by-step procedure for tea aroma analysis is presented in this article, including the tea infusion preparation, solvent extraction, SAFE distillation, extract concentration, and analysis by GC-MS. This procedure was applied to two tea samples (green tea and black tea), and qualitative as well as quantitative results on the volatile composition of the tea samples were obtained. This method can not only be used for the aroma analysis of various types of tea samples but also for molecular sensory studies on them.

Author Spotlight: Exploring Tea Aroma Using Solvent-Assisted Flavor Evaporation Technique 

Presented here is a method for enriching and analyzing the volatile components of tea extracts using solvent-assisted flavor evaporation and solvent extraction followed by gas chromatography-mass spectrometry, which can be applied to all types of tea samples.

Tea aroma is an important factor in tea quality, but it is challenging to analyze due to the complexity, low concentration, diversity, and lability of the ...

Rapid Collection of Floral Fragrance Volatiles using a Headspace Volatile Collection Technique for GC-MS Thermal Desorption Sampling

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