This research was supported by the National Natural Science Foundation of China (32002094, 32102444), the China Agriculture Research System of MOF and MARA (CARS-19), and the Innovation Project for Chinese Academy of Agricultural Sciences (CAAS-ASTIP-TRI).

1. Preparation of the internal standard and tea infusion
<ol>
	<li>Stock solution: Dissolve 10.0 mg of paraxylene-d10 (see Table of Materials) in 10.0 mL of anhydrous ethanol to prepare a 1,000 ppm stock solution of the internal standard.</li>
	<li>Working solution: Dilute 1 mL of the stock solution (step 1.1) to 100 mL with pure water to prepare a 10 ppm working solution of the internal standard. 
	NOTE: The working solution must be prepared on the same day as the analysis.</li>
	<li>Place 3 g of tea leaves (both for green tea and black tea, see Table of Materials) into an Erlenmeyer flask, and add 150 mL boiling water. Cover the flask with a glass stopper.</li>
	<li>After 5 min, quickly filter out the tea infusion through a 300-mesh sieve.</li>
	<li>Wash the spent tea leaves twice with 30 mL of water, and combine the wash solution with the tea infusion.</li>
	<li>Cool the tea infusion to room temperature quickly in an ice water bath.</li>
	<li>Add 1.00 mL of working solution (step 1.2) into the tea infusion, and mix them well.</li>
</ol>
2. Distillation of the tea infusion by SAFE and liquid-liquid extraction of the distillate
<ol>
	<li>Prepare the SAFE assembly following the steps below.
	<ol>
		<li>Install the SAFE assembly (Figure 1), and connect the distillation bottle at the lower left (Figure 1[3]) and the collection bottle at the lower right (Figure 1[4]). Connect the circulating water tube at the rear of the SAFE glass assembly. Install the cold trap (Figure 1[5]), and connect the tube to the vacuum pump (see Table of Materials) at the upper right of the glass assembly. 
		NOTE: Check the connection of the circulating water tube; ensure that the inlet enters the top and the outlet exits from the bottom. Use deionized water for the circulation to prevent the scale from blocking the white tube in the SAFE assembly, which would result in poor circulation of the circulating water and the eventual explosion of the SAFE assembly.&#160;The distillation bottom (Figure 1[3]) can be stirred by a stirring bar to facilitate the evaporation of the sample.</li>
		<li>Set the temperature of the circulating water to 50 &#176;C and that of the water bath for the sample flask to 40 &#176;C. Close the vacuum valve (Figure 1[2]).</li>
	</ol>
	</li>
	<li>Perform the vacuum pump operation.
	<ol>
		<li>Power on the vacuum pump.</li>
		<li>Gradually increase the speed to the maximum speed of 100%. 
		NOTE: If the speed does not reach 100%, check whether the system is airtight and whether there is solvent residue inside the system.</li>
		<li>After reaching a high vacuum (preferably 10-3 Pa) 
		NOTE: The vacuum will improve when the liquid nitrogen is added to the cold trap.</li>
	</ol>
	</li>
	<li>Perform sample distillation.
	<ol>
		<li>Start the water circulation.</li>
		<li>Add liquid nitrogen to the cold trap to cover the outside of the collecting bottle.</li>
		<li>Pour the tea infusion into the sample funnel at the top left (Figure 1[1]), and then cover it with a glass stopper.</li>
		<li>Introduce the sample into the distillation flask dropwise. Control the sample drop speed so that the vacuum is kept in the proper range of around 10&#8722;3 Pa. 
		NOTE: Add liquid nitrogen during the process to ensure that the right collecting bottle is always submerged in liquid nitrogen. Try to avoid condensate formation in the cold trap.</li>
	</ol>
	</li>
	<li>Turn off the vacuum pump after the distillation is completed.
	<ol>
		<li>Press the power switch. When &#34;STOP&#34; flashes, press the Enter key to confirm.</li>
		<li>Unplug the power cord when the speed of the molecular pump decreases to &#34;0&#34;. 
		NOTE: Restart only when the speed decreases to &#34;0&#34;.</li>
	</ol>
	</li>
	<li>Restore the system to atmospheric pressure.
	<ol>
		<li>Remove the grinding plug above the sampling bottle.</li>
		<li>Unscrew the knob of the vacuum valve slowly to restore the system to atmospheric pressure.</li>
	</ol>
	</li>
	<li>Take down the collecting bottle with the sample.
	<ol>
		<li>Remove the liquid nitrogen outside the collection bottle after recovering the system to atmospheric pressure.</li>
		<li>Unscrew the collection bottle slowly. Take down the collecting bottle with the sample carefully.</li>
		<li>Close the circulating water.</li>
	</ol>
	</li>
	<li>Perform liquid-liquid extraction of the SAFE distillate.
	<ol>
		<li>Let the SAFE distillate in the bottle warm to room temperature.</li>
		<li>Extract the SAFE distillate thrice with 50 mL of dichloromethane (see Table of Materials).</li>
		<li>Combine the dichloromethane layers. Dry the extract with anhydrous sodium sulfate (see the Table of Materials). 
		NOTE: The anhydrous sodium sulfate in the solvent is considered dry enough when it is no longer cemented and can flow freely.</li>
		<li>Concentrate the extract to about 2 mL using a gentle nitrogen stream.</li>
		<li>Transfer to a sample vial of 1-2 mL, and further concentrate to 200 &#181;L using a gentle nitrogen stream.</li>
	</ol>
	</li>
</ol>
3. GC-MS analysis and data processing
<ol>
	<li>Analyze the aroma concentrates prepared in protocol section 2 using a GC-MS system (Figure 2) equipped with fused silica capillary columns (see the Table of Materials).</li>
	<li>Use helium as carrier gas with a linear velocity of 40 cm/s.</li>
	<li>Inject 3 &#181;L of the concentrate in splitless injection mode.</li>
	<li>Set the GC oven temperature program: (1) hold at 40 &#176;C for 5 min; (2) increase to 200 &#176;C at 5 &#176;C/min; (3) increase to 280 &#176;C at 10 &#176;C/min; (4) hold at 280 &#176;C for 10 min.</li>
	<li>Operate the mass selective detector in positive EI mode13 with a mass scan range from 30 m/z to 350 m/z at 70 eV.</li>
	<li>Deconvolute the GC-MS data using the Automated Mass Spectral Deconvolution and Identi&#64257;cation System (AMDIS, see the Table of Materials).</li>
	<li>Match and qualify the data after deconvolution using the NIST (National Institute of Standards and Technology) 17 mass spectrometer search program3.</li>
	<li>Calculate the retention index of the compounds14 based on the result of a set of n-alkanes (C5-C25, see the Table of Materials) under the same GC conditions.</li>
	<li>Identify the GC peaks using the NIST mass spectrometry library and the retention index database based on the simultaneous matching of the mass and retention indexes.</li>
	<li>Calculate the concentration of each volatile component in the SAFE sample relative to the internal standard using the TIC (total ion chromatography) peak area.</li>
	<li>Repeat the analysis three times, starting from the tea infusion preparation.</li>
</ol>

Unveiling Tea Aroma Complexity with Solvent-Assisted Flavor Evaporation Method

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+volatile+aroma+compounds+from+five+types+of+Fenghuang+Dancong+tea+using+headspace-solid+phase+microextraction+combined+with+GC-MS+and+GC-olfactometry.">Analysis of volatile aroma compounds from five types of Fenghuang Dancong tea using headspace-solid phase microextraction combined with GC-MS and GC-olfactometry.</a> International Food Research Journal. 28 (3), 612-626 (2021).</li><li>Dong, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbivore-induced+volatiles+from+tea+(Camellia+sinensis)+plants+and+their+involvement+in+intraplant+communication+and+changes+in+endogenous+nonvolatile+metabolites.">Herbivore-induced volatiles from tea (Camellia sinensis) plants and their involvement in intraplant communication and changes in endogenous nonvolatile metabolites.</a> Journal of Agricultural and Food Chemistry. 59 (24), 13131-13135 (2011).</li><li>Acena, L., Vera, L., Guasch, J., Busto, O., Mestres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+two+extraction+techniques+to+obtain+representative+aroma+extracts+for+being+analysed+by+gas+chromatography-olfactometry:+Application+to+roasted+pistachio+aroma.">Comparative study of two extraction techniques to obtain representative aroma extracts for being analysed by gas chromatography-olfactometry: Application to roasted pistachio aroma.</a> Journal of Chromatography A. 1217 (49), 7781-7787 (2010).</li><li>Kumazawa, K., Wada, Y., Masuda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+epoxydecenal+isomers+as+potent+odorants+in+black+tea+(Dimbula)+infusion.">Characterization of epoxydecenal isomers as potent odorants in black tea (Dimbula) infusion.</a> Journal of Agricultural and Food Chemistry. 54 (13), 4795-4801 (2006).</li><li>Wu, H. 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The authors have nothing to disclose.

This article describes an efficient method for analyzing volatile compounds in tea infusions using SAFE and GC-MS analysis.
Tea infusions have a complex matrix with a high content of non-volatile components. Several methods have been described in the literature for isolating the volatile components from tea infusions. A common method is simultaneous distillation extraction (SDE)15,16. However, it is not suitable for the analysis of tea aromas because the tea leaves must be boiled with water for the entire distillation/extraction process, which results in the tea components undergoing a chemical reaction and, thus, yields an odor profile very different from the original sample17. SAFE distillates the tea infusion at a low temperature under a high vacuum, thus minimizing changes in the analytes and allowing the original aroma composition to be preserved.
Solid-phase microextraction (SPME) is another method commonly used for the aroma analysis of tea18,19. Its advantages lie in the simple and solvent-free procedure. However, the selectivity of the fiber adsorption of the aroma components makes it difficult to obtain a quantitative profile reflecting the aroma characteristics of the sample, which limits the application of this method for tea aroma analysis20.
The high-vacuum transfer (HVT) technique was developed to reduce the chance of artifacts being formed in aroma analyses21. However, HVT has a low extraction yield for substances with high boiling points and strong polarity, which limits its scope of use.
Unlike with the above custom methods, the SAFE distillate of a tea infusion is free of any non-volatile components22,23,24. The aroma in the distillate can be quantitatively extracted using organic solvents, meaning that an extract with an odor profile close to the original sample can be obtained. Engel et al.11 distilled mixtures of n-alkanes using HVT or SAFE distillation to check the efficiency. The distillate yields using the SAFE system were found to be significantly higher than those of HVT for each alkane. Additionally, alkanes with boiling points below 285 &#176;C could be completely recovered by SAFE.
Close attention needs to be paid to the experimental details for further successful analyses. (1) The vacuum pressure during the SAFE distillation can affect the recovery of the volatile components and must be maintained at a high level, such as by slowing down the sample addition. (2) It is necessary to ensure that the collection bottle is immersed in liquid nitrogen before the system returns to atmospheric pressure to avoid solvent volatiles being condensed by the upper-right cold trap or entering the vacuum pump. (3) One should ensure that the circulating water is turned on first and turned off last. The circulating water should only be turned off after the liquid nitrogen has been removed; otherwise, it will freeze the device. (4) The water bath should be stirred with a magnet to aid the heat transfer.
In this study, SAFE distillation was performed before the solvent extraction. A reversed procedure is also feasible, and this would be especially advantageous if a large volume of tea infusion is extracted first and the obtained extract is then distilled by SAFE. The challenge of infusion extraction using an organic solvent is the possible formation of an emulsion. In this case, additional steps are needed to recover the organic layer, such as centrifugation or choosing different solvents. After the experiment, the SAFE glass assembly must be cleaned. Ethanol or acetone can be used as the cleaning solvent. The parts should be dried before use.
In summary, this protocol proposes a method to obtain an aroma concentrate with an odor profile close to the original tea sample using SAFE distillation followed by solvent extraction. This method can be applied to all types of tea samples, including, for example, instant tea powders and tea concentrates, and is well suited for molecular sensory studies of tea.

Tea is a preferred beverage of many people all over the world1,2. The aroma of the tea is a quality criterion as well as a price-determining factor for tea leaves3,4. Thus, the analysis of the aroma composition and content of tea is of great significance for molecular sensory studies and the quality control of tea. As a result, aroma composition analysis has been an important topic in tea research in recent years5,6,7.
The content of aroma components in tea is very low, as they generally only account for 0.01%-0.05% of the dry weight of the tea leaves8. Furthermore, the large amount of non-volatile components in the sample matrix significantly interferes with analysis by gas chromatography9,10. Therefore, a sample preparation procedure is essential to isolate the volatile compounds in tea. The key consideration for the isolation and enrichment method is minimizing the matrix interference and, at the same time, maximizing the preservation of the original odor profile of the sample.
Solvent-assisted flavor evaporation (SAFE), originally developed by Engel, Bahr, and Schieberle, is an improved high-vacuum distillation technique used to isolate volatile compounds from complex food matrixes11,12. A compact glass assembly connected to a high-vacuum pump (under a typical operating pressure of 5 x 10&#8722;3 Pa) can efficiently collect volatile compounds from solvent extracts, oily foods, and aqueous samples.
This article described a method that combines the SAFE technique with solvent extraction to isolate volatile substances from a black tea infusion, followed by analysis using GC-MS.

<table><tbody><tr><td>Alkane mix (C10-C25)</td><td>ANPEL</td><td>CDAA-M-690035</td><td></td></tr><tr><td>Alkane mix (C5-C10)</td><td>ANPEL</td><td>CDAA-M-690037</td><td></td></tr><tr><td>AMDIS</td><td>National Institute of Standards and Technology</td><td>version 2.72</td><td>Gaithersburg, MD</td></tr><tr><td>Analytical balance</td><td>OHAUS</td><td>EX125DH</td><td></td></tr><tr><td>Anhydrous ethanol</td><td>Sinopharm</td><td></td><td></td></tr><tr><td>Anhydrous sodium sulfate</td><td>aladdin</td><td></td><td></td></tr><tr><td>Black tea</td><td>Qianhe Tea</td><td></td><td>Huangshan, Anhui province, China</td></tr><tr><td>Concentrator</td><td>Biotage</td><td>TurboVap</td><td></td></tr><tr><td>Data processor</td><td>Agilent</td><td>MassHunter</td><td></td></tr><tr><td>Dichloromethane</td><td>TEDIA</td><td></td><td></td></tr><tr><td>GC</td><td>Agilent</td><td>7890B</td><td></td></tr><tr><td>GC column</td><td>Agilent</td><td>DB-5MS</td><td></td></tr><tr><td>Green tea</td><td>Qianhe Tea</td><td></td><td>Huangshan, Anhui province, China</td></tr><tr><td>MS</td><td>Agilent</td><td>5977B</td><td></td></tr><tr><td>p-Xylene-d10</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>SAFE</td><td>Glasbl&amp;auml;serei Bahr</td><td></td><td></td></tr><tr><td>Ultra-pure deionized water</td><td>Milipore</td><td>Milli-Q</td><td></td></tr><tr><td>Vacuum pump</td><td>Edwards</td><td>T-Station 85H</td><td></td></tr></tbody></table>

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.

The analytical procedure described above is illustrated in this section using the example of the aroma analysis of black tea and green tea samples.
A representative GC-MS chromatogram is shown in Figure 3. Figure 3A shows a set of n-alkanes, and Figure 3B shows the profile of an internal standard. The evaluation results for the extracts from the green tea and black tea samples are shown in Figure 3C and Figure 3D, respectively. By analyzing the internal standards, one definitive peak with a stable baseline can be detected (Figure 3B). The GC chromatogram shows the complete GC profiles obtained from the green tea and black tea infusion extracts after the total ion counting.
A total of 104 aroma compounds were identified in the green tea and black tea samples by mass spectrometry matching combined with the retention index. The relative quantification was calculated by the peak area of the compound relative to the internal standard. The heat map, drawn according to the qualitative and quantitative results, shows the aroma compound contents relative to the internal standard for the green tea and black tea samples (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65522/65522fig01v2.jpg" /> 
Figure 1: Schematic diagram of the SAFE system. (1) The sample bottle for the sample collection. (2) The vacuum valve; the system must be kept closed before adding samples, and the drop flow of the sample should be adjusted appropriately. (3) The distillation bottle for the sample distillation. (4) The collection bottle for the collection of the distilled sample. (5) The cold trap for the recovery of samples not collected by the collection bottle and for preventing the solvent from entering the vacuum pump. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65522/65522fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65522/65522fig02.jpg" /> 
Figure 2: Schematic diagram of the GC-MSD system. The GC/MS system is equipped with (1) a multi-mode injection port, (2) a flow control module (PCM) that controls the helium carrier flow, (3) a 60 m x 0.25 m x 0.25 m 5 ms capillary column, and (4) a GC column oven. The tea extracts in the injected sample are separated in the GC column, through which the carrier gas flows and the oven temperature increases. The components are ionized by an EI ion source and then analyzed in a mass analyzer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65522/65522fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65522/65522fig03.jpg" /> 
Figure 3: Typical total ion chromatogram from a successful GC-MS analysis. (A) The chromatogram of the n-alkanes. All the n-alkane peaks are assigned to the corresponding carbon number. (B) The chromatogram of the internal standard (paraxylene-d10). (C) Representative aroma profile of the green tea infusion. (D) Representative aroma profile of the black tea infusion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65522/65522fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65522/65522fig04.jpg" /> 
Figure 4: Heatmap of 104 aroma compounds identified in the black tea (BT) and green tea (GT) samples. The number next to the color note on the right side of the heat map indicates the content of the compound (relative to the internal standard). The color depth indicates the level of matter content; the deeper the color, the higher the relative content. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65522/65522fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Exploring Tea Aroma Using Solvent-Assisted Flavor Evaporation Technique at JoVE.com

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 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 ...

tea-aroma-analysis-based-on-solvent-assisted-flavor-evaporation

Nanyang Technological University

Research

JoVE Journal

Chemistry

3.0K Views.  Tea Research Institute Chinese Academy of Agricultural Sciences. We onderzoeken de moleculaire basis van smaak en aroma, met de nadruk op theeproducten afgeleid van de Camellia sinensis-plant. Ons doel is om de smaakmoleculen te karakteriseren en de biochemische processen te onderzoeken die de sensorische eigenschappen van thee beïnvloeden. Het analyseren van theearoma vereist de scheiding en verrijking van vluchtige stoffen die in zeer lage concentraties aanwezig zijn uit de complexe matrix van theemonsters.De monstervoorbereidingsmethode vormt va...

Video: Auteur in de schijnwerpers: theearoma verkennen met behulp van oplosmiddelondersteunde smaakverdampingstechniek 

PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that belongs to the Direct-Injection Mass Spectrometric (DIMS) technologies. These techniques allow the rapid determination of volatile organic compounds (VOCs), assuring high sensitivity and accuracy. In general, PTR-MS requires neither sample preparation nor sample destruction, allowing real time and non-invasive analysis of samples. PTR-MS are exploited in many fields, from environmental and atmospheric chemistry to medical and biological sciences. More recently, we developed a methodology based on coupling PTR-ToF-MS with an automated sampler and tailored data analysis tools, to increase the degree of automation and, consequently, to enhance the potential of the technique. This approach allowed us to monitor bioprocesses (e.g. enzymatic oxidation, alcoholic fermentation), to screen large sample sets (e.g. different origins, entire germoplasms) and to analyze several experimental modes (e.g. different concentrations of a given ingredient, different intensities of a specific technological parameter) in terms of VOC content. Here, we report the experimental protocols exemplifying different possible applications of our methodology: i.e. the detection of VOCs released during lactic acid fermentation of yogurt (on-line bioprocess monitoring), the monitoring of VOCs associated with different apple cultivars (large-scale screening), and the in vivo study of retronasal VOC release during coffee drinking (nosespace analysis).

Proton Transfer Reaction Time of Flight Mass Spectrometry allows high-sensitivity, rapid and non-invasive analysis of volatile organic compounds. To demonstrate its potential, we give three examples: lactic acid fermentation of yogurt (on-line bioprocess monitoring), different apple genotypes (large-scale screening), and retronasal space after drinking coffee (nosespace analysis).

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that ...

Fizzy Extraction of Volatile Organic Compounds Combined with Atmospheric Pressure Chemical Ionization Quadrupole Mass Spectrometry

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to the gas phase, and efficiently transferred to a detection system. Fizzy extraction takes advantage of the effervescence phenomenon. First, a carrier gas (here, carbon dioxide) is dissolved in the sample by applying overpressure and stirring the sample. Second, the sample chamber is decompressed abruptly. Decompression leads to the formation of numerous carrier gas bubbles in the sample liquid. These bubbles assist the release of the dissolved analyte species from the liquid to the gas phase. The released analytes are immediately transferred to the atmospheric pressure chemical ionization interface of a triple quadrupole mass spectrometer. The ionizable analyte species give rise to mass spectrometric signals in the time domain. Because the release of the analyte species occurs over short periods of time (a few seconds), the temporal signals have high amplitudes and high signal-to-noise ratios. The amplitudes and areas of the temporal peaks can then be correlated with concentrations of the analytes in the liquid samples subjected to fizzy extraction, which enables quantitative analysis. The advantages of fizzy extraction include: simplicity, speed, and limited use of chemicals (solvents).

Fizzy extraction is a new laboratory technique for analysis of volatile and semivolatile compounds. A carrier gas is dissolved in the liquid sample by applying overpressure and stirring the sample. The sample chamber is then decompressed. The analyte species are liberated to the gas phase due to effervescence.

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to ...

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.

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 ...

Biochemistry

Employing Pressurized Hot Water Extraction (PHWE) to Explore Natural Products Chemistry in the Undergraduate Laboratory

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products research has also found applications as an effective teaching tool. Specifically, this technique has been used to introduce second- and third-year undergraduates to aspects of natural products chemistry in the laboratory. In this report, two experiments are presented: the PHWE of eugenol and acetyleugenol from cloves and the PHWE of seselin and (+)-epoxysuberosin from the endemic Australian plant species Correa reflexa. By employing PHWE in these experiments, the crude clove extract, enriched in eugenol and acetyleugenol, was obtained in 4-9% w/w from cloves by second-year undergraduates and seselin and (+)-epoxysuberosin were isolated in yields of up to 1.1% w/w and 0.9% w/w from C. reflexa by third-year students. The former exercise was developed as a replacement for the traditional steam distillation experiment providing an introduction to extraction and separation techniques, while the latter activity featured guided-inquiry teaching methods in an effort to simulate natural products bioprospecting. This primarily derives from the rapid nature of this PHWE technique relative to traditional extraction methods that are often incompatible with the time constraints associated with undergraduate laboratory experiments. This rapid and practical PHWE method can be used to efficiently isolate various classes of organic molecules from a range of plant species. The complementary nature of this technique relative to more traditional methods has also been demonstrated previously.

Employing Pressurized Hot Water Extraction PHWE to Explore Natural Products Chemistry in the Undergraduate Laboratory

Here, we employ a pressurized hot water extraction (PHWE) method, which utilizes an unmodified household espresso machine to introduce undergraduates to natural products chemistry in the laboratory. Two experiments are presented: PHWE of eugenol and acetyleugenol from cloves and PHWE of seselin and (+)-epoxysuberosin from the Australian plant Correa reflexa.

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products ...

Source and Route of Pyrrolizidine Alkaloid Contamination in Tea Samples

Toxic pyrrolizidine alkaloids (PAs) are found in tea samples, which pose a threat to human health. However, the source and route of PA contamination in tea samples have remained unclear. In this work, an adsorbent method combined with UPLC-MS/MS was developed to determine 15 PAs in the weed Ageratum conyzoides L., A. conyzoides rhizospheric soil, fresh tea leaves, and dried tea samples. The average recoveries ranged from 78%-111%, with relative standard deviations of 0.33%-14.8%. Fifteen pairs of A. conyzoides and A. conyzoides rhizospheric soil&#160;samples and 60 fresh tea leaf samples were collected from the Jinzhai tea garden in Anhui Province, China, and analyzed for the 15 PAs. Not all 15 PAs were detected in fresh tea leaves, except for intermedine-N-oxide (ImNO) and senecionine (Sn). The content of ImNO (34.7 &#181;g/kg) was greater than that of Sn (9.69 &#181;g/kg). In addition, both ImNO and Sn were concentrated in the young leaves of the tea plant, while their content was lower in the old leaves. The results indicated that the PAs in tea were transferred through the path of PA-producing weeds-soil-fresh tea leaves in tea gardens.

The present protocol describes the contamination of pyrrolizidine alkaloids (PAs) in tea samples from PA-producing weeds in tea gardens.

Toxic pyrrolizidine alkaloids (PAs) are found in tea samples, which pose a threat to human health. However, the source and route of PA contamination in ...

Environment

Derivatization and Separation of Barley Flour Metabolites into Four Fractions

Metabolomic Analysis of Barley by Gas Chromatography/Mass Spectrometry

Climate change increases drought risk to agriculture and impacts both food nutrient content and overall food security. Metabolomics is one way to observe and quantify the impacts of drought on grain and other agricultural products. The identified metabolites may allow for the identification of the biochemical response that allows the plant to tolerate stressful environments. The methodology presented herein allowed for the total metabolomic analysis of barley flour using gas chromatography/mass spectrometry (GC/MS). 
Barley flour metabolite extracts were fractionated into four fractions based on polarity. To allow for analysis by GC/MS, metabolites were derivatized to increase volatility and metabolite separation: fatty acids esters were derivatized into fatty acid methyl esters; sugars were oximated into their straight chain form; and metabolites with hydroxyl groups were converted to their corresponding silyl ethers. The derivatized samples were injected into the GC/MS and the generated mass spectra were used for metabolite identification by comparing the generated spectra to the National Institute of Standards and Technology (NIST) Tandem Mass Spectra library. The method described here can also be used to examine the total metabolome for other plants, furthering our understanding of the biochemical responses of stressed plants.

A method for metabolomic analysis of barley is presented. The method entails fractionation and derivatization of metabolites and analysis thereof by gas chromatography/mass spectrometry (GC/MS). Metabolomic analysis can be used to determine the effect of intercropping and drought stress on the grain.

Climate change increases drought risk to agriculture and impacts both food nutrient content and overall food security. Metabolomics is one way to observe ...

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 ...

Trimethylamine Detection in Animal-Derived Medicine by Headspace GC-MS

An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the poor compliance of clinical patients. Trimethylamine (TMA) is one of the key fishy odor components in animal-derived medicine. It is difficult to identify TMA accurately using the existing detection method due to the increased pressure in the headspace vial caused by the rapid acid-base reaction after the addition of lye, which causes TMA to escape from the headspace vial, stalling the research progress of the fishy odor of animal-derived medicine. In this study, we proposed a controlled detection method that introduced a paraffin layer as an isolation layer between acid and lye. The rate of TMA production could be effectively controlled by slowly liquefying the paraffin layer through thermostatic furnace heating. This method showed satisfactory linearity, precision experiments, and recoveries with good reproducibility and high sensitivity. It provided technical support for the deodorization of animal-derived medicine.

Author Spotlight: An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry  

Here, a headspace gas chromatography-tandem quadrupole mass spectrometry (HS-GC-MS/MS) method suitable for the determination of trimethylamine (TMA) in animal-derived medicines is described. The protocol includes sample pretreatment, headspace treatment, analysis conditions, methodological validation, and the determination of TMA in animal-derived medicines.

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the ...

Fruit Volatile Analysis Using an Electronic Nose

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit aroma. Maturity at harvest is one of the key factors influencing the flavor quality of fruits and vegetables1. The validation of robust methods that rapidly assess fruit maturity and aroma quality would allow improved management of advanced breeding programs, production practices and postharvest handling. 
Over the last three decades, much research has been conducted to develop so-called electronic noses, which are devices able to rapidly detect odors and flavors2-4. Currently there are several commercially available electronic noses able to perform volatile analysis, based on different technologies. The electronic nose used in our work (zNose, EST, Newbury Park, CA, USA), consists of ultra-fast gas chromatography coupled with a surface acoustic wave sensor (UFGC-SAW). This technology has already been tested for its ability to monitor quality of various commodities, including detection of deterioration in apple5; ripeness and rot evaluation in mango6; aroma profiling of thymus species7; C6 volatile compounds in grape berries8; characterization of vegetable oil9 and detection of adulterants in virgin coconut oil10. 
This system can perform the three major steps of aroma analysis: headspace sampling, separation of volatile compounds, and detection. In about one minute, the output, a chromatogram, is produced and, after a purging cycle, the instrument is ready for further analysis. The results obtained with the zNose can be compared to those of other gas-chromatographic systems by calculation of Kovats Indices (KI). Once the instrument has been tuned with an alkane standard solution, the retention times are automatically converted into KIs. However, slight changes in temperature and flow rate are expected to occur over time, causing retention times to drift. Also, depending on the polarity of the column stationary phase, the reproducibility of KI calculations can vary by several index units11. A series of programs and graphical interfaces were therefore developed to compare calculated KIs among samples in a semi-automated fashion. These programs reduce the time required for chromatogram analysis of large data sets and minimize the potential for misinterpretation of the data when chromatograms are not perfectly aligned.
We present a method for rapid volatile compound analysis in fruit. Sample preparation, data acquisition and handling procedures are also discussed.

A rapid method for volatile compound analysis in fruit is described. The volatile compounds present in the headspace of a homogenate of the sample are rapidly separated and detected with ultra-fast gas chromatography (GC) coupled with a surface acoustic wave (SAW) sensor. A procedure for data handling and analysis is also discussed.

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit ...

Biology

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

Tea Aroma Analysis Based on Solvent-Assisted Flavor Evaporation Enrichment

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