Name: Extraction and Detection of Geosmin and 2-Methylisoborneol in Water and Fish using High-Capacity Sorptive Extraction Probes and GC-MS (Video) | JoVE Journal
Uploaded: 2025-07-03T13:45:00.000+00:00
Duration: 11 min 59 s
Description: Learn to extract and detect geosmin and 2-methylisoborneol in water and fish using high-capacity sorptive extraction probes and GC-MS. This automated protocol enables early detection of odorant compounds for aquaculture quality control.

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5 minutes

We would like to thank the United States Department of Agriculture, Agricultural Research Service (USDA-ARS) for the funding to acquire the equipment used and continuing funding to complete all of this research. We would also like to thank Dr. Heather Hamlin, Dr. Amalia Harrington, Dr. Deborah Bouchard, Dr. Sarah Turner, Gareth Dobson, Cameron Coulombe, and Abriah James for their assistance and support throughout this research.

The fish samples were collected from animals that were already slaughtered for human consumption. Therefore, no ethical clearance was required.
1. Preparation of calibration and internal standards
<ol>
	<li>Calibration standard preparation
	<ol>
		<li>Prepare stock solution C1 by adding 10 &#181;L of geosmin and 2-methylisoborneol solution (100 &#181;g/mL) to a 100 mL volumetric flask and filling to volume with LC-MS grade methanol. The resulting solution will contain each analyte at 10 &#181;g/L (ppb).</li>
		<li>Prepare calibration standards by adding the C1 stock solution in the volume indicated in Table 1 to a 100 mL volumetric flask and filling to volume with deionized water.</li>
		<li>Make fresh calibration standards daily, as these are extremely perishable. 
		NOTE: It is important to prepare these stock solutions fresh every day.</li>
	</ol>
	</li>
	<li>Internal standard preparation
	<ol>
		<li>Prepare stock solution (I1) by adding 10 mg&#160;of 2-isopropyl-3-methoxypyrazine (IPMP) (&#62;98% pure) to a 1 L volumetric flask filled to volume with LC-MS grade methanol making a 10 mg/L stock.</li>
		<li>Pipette 100 &#181;L of solution I1 to a 100 mL volumetric flask and fill to volume with deionized water to give solution I2 (10 &#181;g/L). Spike 50 &#181;L into every sample vial to reach a final concentration of 100 ng/L.</li>
	</ol>
	</li>
</ol>
2. Water analysis
<ol>
	<li>Sample preparation
	<ol>
		<li>Place one 20 mL vial for each water sample being analyzed in the sample tray.</li>
		<li>Using a calibrated scale weigh out and place 2.5 g of NaCl into each 20 mL crimp top sample vial. 
		NOTE: Adding salt enhances the transfer of the MIB and geosmin from the water to the headspace.</li>
		<li>Using a pipette or bottle top dispenser, add 5 mL of the sample water to each 20 mL crimp top sample vial.</li>
		<li>Pipette 50 &#181;L of the internal standard solution I2 to each sample, calibration standard, and any QA/QC samples, including blanks.</li>
		<li>Place the cap on top of the vial and seal it with a vial crimping tool to minimize analyte loss.</li>
		<li>Only process 1-2 samples at a time and quickly seal each vial once complete to minimize analyte loss of volatile analytes.</li>
	</ol>
	</li>
	<li>Analyte extraction (water)
	<ol>
		<li>Place the sample tray on the autosampler tray holder.</li>
		<li>To prepare an instrument method, open the instrument program, and click on the Method Editor.
		<ol>
			<li>Create a new method by entering the following parameters into the method editor (leaving other parameters at their default values):</li>
			<li>In &#39;Pre-sampling&#39;, set the Vial Agitation Time to 10 min to allow analytes to partition from the water to the headspace.</li>
			<li>In &#39;Sampling&#39;, set the Incubation Temperature to 65 &#176;C, incubation time to 30 min, and Agitator speed to 400 rpm.</li>
			<li>Ensure &#39;wash HiSorb probe&#39; is Enabled; otherwise, the residual sample matrix may contaminate the system.</li>
			<li>In &#39;Probe desorption&#39;, set the Desorption time to 15 min and the Desorption temperature to 270 &#176;C.</li>
			<li>In the Trap settings, set the Trap low temperature to 25 &#176;C and &#39;split flow&#39; to 8 mL/min.</li>
		</ol>
		</li>
		<li>Run the autosampler method in conjunction with the GC-MS method by pressing the Start button on each of the respective programs.</li>
	</ol>
	</li>
</ol>
3. Fish tissue analysis
<ol>
	<li>Sample preparation
	<ol>
		<li>Place one 20 mL vial for each fish sample being analyzed in the sample tray.</li>
		<li>Homogenize the fish tissue using a commercially available food processor.</li>
		<li>Using a calibrated scale, weigh 1.00 g of homogenized fish tissue into each vial.</li>
		<li>Pipette 5 mL of saturated NaCl solution into each vial.</li>
		<li>Pipette 100 &#181;L of the internal standard solution I2 to each sample, calibration sample, and any QA/QC samples.</li>
		<li>Place a cap on top of the vial and seal with the vial crimping tool.</li>
		<li>Prepare only 1-2 samples at a time to reduce the loss of semi-volatile and volatile compounds.</li>
	</ol>
	</li>
	<li>Analyte extraction (fish)
	<ol>
		<li>Place the sample tray on the autosampler tray holder.</li>
		<li>Prepare an instrument method as follows (leaving other parameters at their default values).&#160; 
		NOTE: pre-sampling vial agitation time and incubation temperature differ from Section 2.2.2.
		<ol>
			<li>In &#39;Pre-sampling&#39;, set vial agitation time to 20 min.</li>
			<li>In &#39;Sampling&#39;, set incubation temperature to 80 &#176;C, Incubation time to 30 min, and Agitator speed to 400 rpm.</li>
			<li>Ensure &#39;wash HiSorb probe&#39; is enabled, otherwise, residual sample matrix may contaminate the system.</li>
			<li>In &#39;Probe desorption,&#39; set the desorption time to 15 min and the Desorption temperature to 270 &#176;C.</li>
			<li>In trap settings, set the trap low temperature to 25 &#176;C and split flow to 8 mL/min. 
			NOTE: A longer, hotter pre-sampling step is required here due to the higher affinity of MIB and GSM for lipid-rich fish tissues versus water.</li>
		</ol>
		</li>
		<li>Run the autosampler method in conjunction with GC-MS method by pressing the Start button on each of the respective programs.</li>
	</ol>
	</li>
</ol>
4. Separation and detection using GC-MS
<ol>
	<li>Use the same GC-MS parameters for water and fish tissue methods.</li>
	<li>Ensure the GC is fitted with a 5MS, 30 m x 0.25 mm x 0.25 &#181;m column and is supplied with ultra-pure helium carrier gas. Set the carrier flow rate to 2 mL/min.</li>
	<li>Prepare a GC oven program as follows: An initial temperature of 60 &#176;C, held for 3 min, then a temperature ramp of 10 &#176;C/min to 100 &#176;C, then 20 &#176;C/min to 190 &#176;C, then 30 &#176;C/min to 280 &#176;C, with a final hold time of 2 min. The total run time is set to 16.5 min.</li>
	<li>Set the transfer line between GC and MS to 280 &#176;C and the ion source and the quadruple to 250 &#176;C and 200 &#176;C, respectively.</li>
	<li>For MS detection in scan mode, use a scan range of m/z 50-350.</li>
	<li>For selected ion monitoring (SIM) operation, use quantification and confirming ions respectively of m/z 95 and 107 for MIB, m/z 137 and 152 for IPMP, and m/z 112 and 55 for GSM. 
	NOTE: Many chromatography software packages feature auto peak selection, but it is highly recommended to manually review the peak selection for errors.</li>
</ol>

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<li>Hearn, L., Cole, R., Spadafora, N. D., Szafnauer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Volatile+and+semi-volatile+compounds+in+flavoured+hard+seltzer+beverages%3A+Comparison+of+high-capacity+sorptive+extraction+%28HiSorb%29+methods%5BTitle%5D)+AND+%22Adv+Sample+Prep%22%5BJournal%5D)">Volatile and semi-volatile compounds in flavoured hard seltzer beverages: Comparison of high-capacity sorptive extraction (HiSorb) methods.</a> Adv Sample Prep. 3, 100032(2022).</li>
<li>Bu, N., Yang, Q., Chen, J., Li, Y., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterised+and+discrimination+of+volatile+compounds+in+chilled+Tan+mutton+meat+during+storage+using+HiSorb-TD-GC-MS+and+E-nose%5BTitle%5D)+AND+%22Molecules%22%5BJournal%5D)">Characterised and discrimination of volatile compounds in chilled Tan mutton meat during storage using HiSorb-TD-GC-MS and E-nose.</a> Molecules. 28, 4993(2023).</li>
<li>Fella, P., Stylianou, M., Agapiou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+green+sorptive+extraction+method+%28HiSorb-TD-GC-MS%29+for+determining+the+extra+virgin+olive+oil+%28EVOO%29+aroma+profile%5BTitle%5D)+AND+%22Pure+Appl+Chem%22%5BJournal%5D)">A green sorptive extraction method (HiSorb-TD-GC-MS) for determining the extra virgin olive oil (EVOO) aroma profile.</a> Pure Appl Chem. 95, 595-610 (2023).</li>
<li>Fenn, D., Lilien, T. A., Hagens, L. A., Smit, M. R., Heijnn, N. F. L., Tuip-de Boer, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Validation+of+volatile+metabolites+of+pulmonary+oxidative+injury%3A+a+bench+to+bedside+study%5BTitle%5D)+AND+%22ERJ+Open+Res%22%5BJournal%5D)">Validation of volatile metabolites of pulmonary oxidative injury: a bench to bedside study.</a> ERJ Open Res. 9, (2023).</li>
<li>Schincaglia, A., Purcaro, G., Beccario, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unlocking+the+aroma+compounds+of+pistachios+using+the+power+of+multi-dimensional+gas+chromatography%5BTitle%5D)+AND+%22Poster+presented+at+Advances+in+Separation+Science%22%5BJournal%5D)">Unlocking the aroma compounds of pistachios using the power of multi-dimensional gas chromatography.</a> Poster presented at Advances in Separation Science. , (2023).</li>
</ol>

Authors in this publication employed at Markes International Ltd or Markes International GmbH Assisted with system training, system protocols, experimental designs and writing. To avoid bias or perceived bias in our research, all laboratory research and data analysis was conducted at the University of Maine under the direct supervision of the United States Department of Agriculture- Agriculture Research Service (USDA-ARS) and the University of Maine&#39;s Aquaculture Research Institute.

GSM and MIB are off-flavor compounds occurring in a wide range of water monitoring settings, and demand for testing capacity is high. Furthermore, GSM and MIB are perceptible to humans at trace levels. An ideal analytical system must, therefore, combine high sensitivity with high throughput to produce high volumes of quality data. We have demonstrated in previous sections that extraction with HCSE probes provides sensitivity that is in excess of what is required.
The robust design of HCSE probes precludes any breakages, an issue that can interrupt SPME workflows. Following method optimization, we experienced no difficulties with system contamination or carryover, which are common issues with P and T methodologies. The biggest contribution to this method&#39;s high throughput, however, undoubtedly came from the autosampler platform used. With this system, analysis continued around the clock, with manual intervention required to supply samples to the sample tray only. This is in contrast to other high-capacity sorptive extraction methods, such as SBSE, which require manual operation. While SPME is commonly fully automated, throughput is limited in that incubation of the next sample can, typically, only begin once the previous sample has been injected into the GC column. Assuming that the GC cycle time is short, the GC may, therefore, sit idle while the next sample completes extraction. With our method, multiple samples can undergo pre-sampling and extraction simultaneously such that GC downtime is minimized. Hence, we were able to process 1.8 samples per hour per system.
We have demonstrated MDLs of 1.2 and 1.1 ng/L for GSM and MIB, respectively, thus far surpassing the sensitivity required. While other techniques such as P and T, LLE, SBSE, and SPME may be able to reach lower MDLs under certain circumstances11, these increases in sensitivity are of limited practical value and do not justify losing the advantages of HCSE probes outlined above. Further, some of these techniques require large sample volumes, with attendant increases in waste generation and transport costs. For LLE, sample volumes are typically in the range of 200 - 500 mL15,16,17, while for SPME, SBSE, and P and T, typical values are 10 - 100 mL18,19,20,21,22. Excellent reproducibility was observed with RSD values of 6.34% and 4.82% for GSM and MIB, respectively. These values are broadly similar to those found in other methods11, and, in particular, surpass the RSDs in published SPME methods23,24. Again, our low RSD values were achieved with sample volumes of just 5 mL, lower than the vast majority of published extraction techniques. Thus, we are confident that HCSE probes provide sufficient sensitivity and reproducibility along with a wide suite of benefits that combine the best aspects of all prominent alternatives into a single simple, fully automated and sustainable method.
Geosmin and 2-methylisoborneol are lipophilic compounds, which presents a challenge to scientists trying to extract those compounds from the lipid-rich tissue for analysis. We, therefore, adjusted our pre-sampling conditions, using a longer and hotter pre-sampling procedure, to drive more analytes from the sample matrix to the headspace. Having made these adjustments, we determined that the method worked well for analyzing GSM and MIB in fish tissues. It should be noted&#160;that for samples with a very high lipid content, it may be necessary to reduce the mass of sample used, e.g., to 0.50 g. This is to avoid lipids forming a complete seal on top of the water that prevents analytes from reaching the headspace.
This method uses equilibrium principles to extract analytes from the headspace of a sample. Although we use water and fish samples from aquaculture here, we believe that the method is applicable to odorant extraction from any solid or liquid matrix that can be homogenized and accommodated within a 20 mL sampling vial. Example applications include monitoring of environmental waters and soils, testing of drinking water, and testing of foods such as fruits and vegetables, or non farm-grown aquatic life. In farming, GSM and MIB are often found in damp soils and can impart off-flavors to crops grown therein2, therefore both soils and crops could be tested via our method to monitor odorant levels as a form of quality control and/or to diagnose the source of malodorous complaints. Many environmental waters, such as rivers and lakes experience seasonal spikes in odorant concentration due to algal blooms25, and this method offers a high-throughput, highly sensitive means of monitoring the extent of these spikes. This method is also suitable for academic research, for example, to measure the rate of odorant production by microorganisms26 or to examine the relationship between odorant concentrations in water and in animal tissues27. Other research groups have used HCSE process to extract from a range of sample types such as alcoholic drinks28, mutton meat29, olive oil30, human cell culture media31and pistachios32. These studies targeted other analytes or are non-targeted profiling studies and therefore were not optimized for the analysis of GSM and MIB, but are presented here as they offer general insights on preparing various sample types for HCSE probes extraction.
While the water method described is likely to be applicable to all water or water-based samples with minimal modification, application to other solid or semi-solid samples may require some matrix-specific alteration to sample preparation (section 3.1) and analyte extraction (section 3.2). While it would not be possible to cover every sampling scenario here, we offer the following considerations. (1) Solid samples should be homogenized to a fine powder via grating, grinding or blending to ensure representativeness and to provide the maximum possible surface area for egress of analytes. (2) Solid samples with a low lipid content may not require the addition of water or salt - i.e. it may be sufficient to weigh 1.00 g into a vial and cap. This will allow for a more direct transfer of analytes from the sample to the headspace, without an intermediate transfer from the sample matrix to the added water. However, this will reduce the efficacy of stirring. Analysts should perform experiments to determine the optimum sample preparation technique for their sample matrix. (3) Different matrices will have different affinities for GSM and MIB. Therefore, different incubation temperatures will be required to reach analyte equilibrium in a timely manner. However, some matrices may be chemically altered by heat such that interferences are produced by chemical reactions at high temperatures. Again, experiments are required to determine the optimum incubation temperature for a particular sample type (note: Samples with a high moisture content should never be incubated above 90 &#176;C&#160;to avoid vials shattering from the vapor pressure of boiling water).
We have developed methods for the detection of geosmin and 2-methylisoborneol in water and fish tissue. Tests on laboratory standards confirm that the methods are quantitative and reproducible, while application to real-world samples (water from recirculating aquaculture systems, fish tissues derived from such systems) demonstrate performance even in the context of complex, dirty samples. Although we have applied our methods to samples from aquaculture, we suggest that they are suitable for GSM and MIB monitoring in a wide range of sample types, including water and solid or semi-solid materials.

Geosmin (GSM) and 2-methylisoborneol (MIB) occur naturally in water and soils as by-products of microbial metabolism1,2. While not generally hazardous to environmental or human health, they impart unpleasant 'earthy' and 'muddy' odors and flavors to water, which are perceptible even at extremely low concentrations, as low as 10 and 20 ng/L for GSM and MIB, respectively3,4. As such, the presence of these odorants reduces the perceived quality of water and is highly undesirable in most contexts. In inland waterways, odorants reduce the appeal to visitors and may elicit complaints to environmental authorities5, while in drinking water, odorants impart an unpleasant smell and taste that consumers find unacceptable6. Indeed, many countries have imposed regulatory limits on odorant levels in drinking water, with China stipulating 10 ng/L as the maximum GSM concentration permissible7.
Recirculating aquaculture systems (RASs) are becoming increasingly important in the aquaculture industry as a means of fish production. These systems operate on land and continuously reuse water, thereby reducing environmental impact, but extensive filtering of RAS water is required to maintain optimal conditions for fish rearing. Moving bed biofilters become colonized by microbes due to the abundance of nutrients in fish feces and uneaten feed8. These microbes are beneficial for the removal of nitrogenous waste and excess nutrients; however, some species may produce GSM, MIB, and other odorants4. These odorants then leach into the water and are absorbed into the flesh of the fish, where they can impart unpleasant odor and flavor characteristics that consumers find objectionable. Odor threshold values in fish flesh are species-dependent, but values for Atlantic salmon, a common species in RASs, are 44 - 500 ng/kg for GSM and &#62;900 ng/kg for MIB4. Hence, RAS operators should regularly monitor GSM and MIB concentrations in their systems to ensure that these do not reach problematic levels4. Affected fish can be purged of off-flavor compounds pre-harvest to recover the quality of the fillet; however, this is an expensive process requiring the facility to stop recirculation of water and instead flow new water through the system for 10 -14 days9. Given that GSM and MIB present a persistent and economically costly problem across a wide range of industries, it makes sense to reassess and improve analytical methods for detection. Ideally, RAS operators should be able to monitor both RAS water to identify increasing GSM and MIB concentrations before they become problematic, and fish tissues to determine whether and to what extent GSM and MIB levels have affected product quality.
Most methods for the detection of GSM and MIB use gas chromatography (GC) with either a mass spectrometer (MS) or flame ionization detector (FID) for separation and detection, but how odorants are initially extracted from the water matrix varies greatly, with many methods published10. That such a variety of methods have been trialed and, indeed, are still in routine use speaks to the fact that each has significant drawbacks. These drawbacks include a requirement for large sample size and large volumes of solvent as in liquid-liquid extraction (LLE), low sensitivity and water interference as in headspace-based methods, or extensive issues with carryover and system contamination as in purge and trap (P and T). 
Sorptive extraction covers a range of extraction techniques wherein a solid sorptive phase can be used to extract odorants from the headspace above a sample or from the sample itself via direct immersion10. These techniques are compatible with smaller sample volumes than other techniques, do not require solvents, and are not typically severely impacted by water interference. However, the most common form of sorptive extraction, solid-phase microextraction (SPME), uses only a small volume of sorptive phase with low capacity for analytes, which limits extraction efficiency. Additionally, the brittle fibers involved can become damaged or broken11, interrupting workflows. Stir-bar sorptive extraction (SBSE) overcomes these difficulties with a larger phase volume and more durable design but is not readily automatable, requiring manual transfer of stir-bars into samples and from samples into thermal desorption tubes12. In the context of routine water screening, where high throughput is crucial, this lack of automation is a significant barrier. 
The goal of our method is to improve upon the aforementioned methods with respect to sample throughput and the overall sorptive extraction capacity. Thus, we developed a technique using high-capacity sorptive extraction (HCSE) probes, combining the high analyte capacity and robustness of SBSE with the phase selection and automation of SPME for the quantitative, high-throughput analysis of GSM and MIB. With some modifications, the technique can be applied both to water samples and to samples of homogenized fish tissue. Here, we describe the protocol in full and present validation data demonstrating that it is reproducible, linear, and highly sensitive. Although aquaculture samples are used for demonstration purposes here, the protocol is suitable for monitoring odorants in a wide range of sample types, including waters, soils, and various foodstuffs. Thanks to full automation, the protocol is particularly beneficial where sample numbers are expected to be high.

<table><tbody><tr><td>0.5-10uL pipette</td><td>Nichipet Air</td><td>A21405581</td><td></td></tr><tr><td>1000uL pipette tips</td><td>VWR</td><td>76322-154</td><td></td></tr><tr><td>100-1000uL pipette</td><td>eppendorf</td><td>285612</td><td></td></tr><tr><td>100mL glass flask</td><td>VWR</td><td>76008-704</td><td></td></tr><tr><td>10-100uL pipette</td><td>Nichipet Air</td><td>A20814031</td><td></td></tr><tr><td>10-100uL Pipette tips</td><td>mega</td><td>24502-18</td><td></td></tr><tr><td>10xL Pipette tips</td><td>VWR</td><td>76322-132</td><td></td></tr><tr><td>20mL Glass beakers</td><td>VWR</td><td>10754-698</td><td></td></tr><tr><td>2-Isopropyl-3-methoxypyrazine</td><td>TCI</td><td>I0577</td><td>Internal standard</td></tr><tr><td>40 ml Amber type-1 (Borosilicate) Glass vial</td><td>Environmental sampling company</td><td>0040-0400-QC</td><td>Water Sampling vial</td></tr><tr><td>5mL Pipette tip</td><td>VistaLab technologies</td><td>4058-5102</td><td></td></tr><tr><td>Balance</td><td>VWR</td><td>VWR-124B2</td><td></td></tr><tr><td>Centri 360</td><td>Markes</td><td>U-CENTRI-1691</td><td>Autosampler</td></tr><tr><td>Chromeleon Version 7.3</td><td>Thermo Scientific</td><td></td><td>GC-MS Software</td></tr><tr><td>Focusing trap</td><td>Markes</td><td>U-T12ME-2S</td><td></td></tr><tr><td>Gas Chromatograph, Trace 1310</td><td>Thermo Scientific</td><td>14800403</td><td></td></tr><tr><td>GC Column</td><td>Thermo Scientific</td><td>26098-1420</td><td>TG-5MS</td></tr><tr><td>Geosmin and 2-Methylisoborneol solution</td><td>SIGMA-ALDRICH or MilliporeSigma</td><td>CRM47525</td><td></td></tr><tr><td>Headspace vial, 20 mL</td><td>Markes</td><td>C-VCC20</td><td></td></tr><tr><td>HiSorb handle</td><td>Markes</td><td>C-HH-6</td><td></td></tr><tr><td>HiSorb injector cap</td><td>Markes</td><td>C-HSPCCS</td><td></td></tr><tr><td>HiSorb injector liner</td><td>Markes</td><td>U-LINER-HISORB</td><td></td></tr><tr><td>HiSorb probe, PDMS, short version</td><td>Markes</td><td>H1-XXABC</td><td></td></tr><tr><td>HiSorb vial caps</td><td>Markes</td><td>C-HSPCCS</td><td></td></tr><tr><td>Laboratory metal scoop</td><td></td><td></td><td></td></tr><tr><td>Mass Spectrometer, ISQ 7000</td><td>Thermo Scientific</td><td>ISQ7K-NOVPI</td><td></td></tr><tr><td>Methanol</td><td>VWR chemicals</td><td>BDH1135-4LP</td><td></td></tr><tr><td>Methanol, Optima LC-MS grade</td><td>Fisher-scientific</td><td>A456-4</td><td></td></tr><tr><td>O-rings size 201</td><td>Markes</td><td>U-COV201</td><td>For HiSorb injector cap and HiSorb storage</td></tr><tr><td>Ovation automatic pipettor (100-5000uL)</td><td>VistaLab technologies</td><td>213626</td><td></td></tr><tr><td>Purple Nitrile-Xtra Powder Free Exam Gloves</td><td>Kimtech</td><td>50602-54</td><td></td></tr><tr><td>Sodium chloride (salt)</td><td>VWR chemicals</td><td>BDH9286-2.5K</td><td></td></tr><tr><td>Switching adapter (automatic pipettor)</td><td>CUI INC</td><td>SMI10-5</td><td></td></tr><tr><td>Vial crimping tool</td><td>Markes</td><td>U-HSVCR</td><td></td></tr><tr><td>Vial decapper</td><td>Markes</td><td>U-HSVDC</td><td></td></tr><tr><td>Weigh boats</td><td>Grainger</td><td>8AG69</td><td></td></tr></tbody></table>

extraction and detection of geosmin and 2-methylisoborneol in water and fish using high-capacity sorptive extraction probes and gc-ms

Extraction and Detection of Geosmin and 2-Methylisoborneol in Water and Fish using High-Capacity Sorptive Extraction Probes and GC-MS

Geosmin and 2-methylisoborneol are volatile organic compounds of microbial origin that frequently occur in recirculating aquaculture systems and confer unpleasant odors and flavors to fish therein, even at extremely low concentrations. Existing analytical methods for these compounds in fish struggle to achieve enough sample throughput and are oftentimes not automated, requiring extensive hands-on preparation. We have developed methods for analyzing geosmin and 2-methylisoborneol from water and fish tissues, respectively, using a metal probe bearing a high-capacity sorptive extraction phase. A robust and quantitative detection was achieved from both water and fish tissues, with minimum detection limits of 1.2 ng/L for geosmin and 1.1 ng/L for 2-methylisoborneol from water and 18.1 ng/kg and 13.6 ng/kg, respectively, from fish tissue. These concentrations were well below human detection thresholds. Hence, we concluded that these methods could detect geosmin and 2-methylisoborneol before they became perceptible to humans. In contrast to other high-capacity sorptive extraction techniques, such as stir-bar sorptive extraction, this technique is fully automatable, dramatically improving throughput and precluding manual handling errors, while the durable design of the probe precludes breakage, such as is common with solid phase microextraction (SPME) fiber. This method is suitable for odorant monitoring in all water quality settings, as well as for the detection of off-flavor compounds in solid and semi-solid matrices.

Method optimization
Various experiments have been conducted to ensure the method is optimized, including a carryover assessment, optimization of probe desorption time, an internal standard assessment, a probe phase assessment, an assessment of linearity and repeatability, and calculation of the method detection limit.
Carryover assessment
We began our experiment with a probe desorption temperature of 250 °C. Calibration standards were prepared, as shown in Table 1. To assess carryover, we analyzed GSM and MIB standards at 20 ng/L and then proceeded with, in order, a run of the GC-MS parameters without injecting anything to the column (column blank), a desorption of the focusing trap without transferring anything from the injector (trap blank), a heating of the injector without inserting a probe (injector blank), and a desorption of the probe without prior sampling (probe blank). Less than 1% carryover was observed in the column blank, trap blank, and injector blank, but in the probe blank, carryover was at 9.35% and 3.77% for GSM and MIB, respectively (Table 2). Hence, we determined that there was a significant carryover of GSM on the PDMS probe under the conditions used.
Desorption time optimization
Having identified carryover on the PDMS probe, we sought to resolve this by adjusting the probe desorption temperature. A higher probe desorption temperature would be expected to increase the rate of analyte desorption from the probe such that desorption would be more complete by the end of the 15 min probe desorption time. However, a higher temperature could also promote analyte breakdown and, therefore, negatively affect sensitivity. Higher temperatures can also damage sorptive materials, so temperatures over 280 °C must be avoided. Carryover of GSM was significant at the 250 °C probe desorption temperature, but this reduced substantially to well below the 5% acceptability threshold when 270 °C was used instead (Figure 1). This improvement was achieved with no loss of sensitivity, with peak areas produced for the initial analysis being no smaller at 270 °C than for lower probe desorption temperatures (Figure 1). This indicates that any increase in analyte breakdown is negligible.
Use of internal standard
The compound 2-isopropyl-3-methoxypyrozine has been widely employed as an internal standard during the detection of GSM and MIB13. We compared the calculated concentrations of GSM and MIB with the known spiked concentrations, both with and without internal standard correction. Recovery without internal standard correction was poor, with calculated concentrations in 60% of the known spiked concentrations (Figure 2). In contrast, with internal standard correction applied, recovery was very good, in the region of 100%. Thus, we applied internal standard correction in all future analyses.
Phase selection
In SPME analyses, carbon wide range (CWR) and/or divinyl benzene (DVB) are often added to PDMS to improve sensitivity, but this increases cost. We sought to compare PDMS-only probes with triple-phase (DVB/CWR/PDMS) probes, considering that the increased phase volume of the probe is likely to improve sensitivity versus SPME fibers. Recovery data for PDMS and triple-phase probes (Figure 3) illustrate broadly similar performance between the two probe types, with recoveries slightly closer to 100% with the PDMS probes. Thus, given the slightly superior performance and reduced costs, we performed all future analyses with PDMS probes. It should be noted that since the probe collects analytes from the headspace and not via direct contact with the sample matrix, the kinetics of GSM / MIB partitioning to the phase material should not differ between water and fish samples.
Method validation
A calibration series covering six concentrations of analytes in water from 1 to 100 ng/L was analyzed (Figure 4). Excellent linearity was achieved, with r2 of 0.9999 for GSM and 0.9999 for MIB. The standard error of regression was 0.43 and 0.37 for GSM and MIB, respectively. This indicates that the method is suitable for quantitative analyses over the range assessed. We determined repeatability from eight replicates of laboratory standards containing GSM and MIB at 40 ng/L in water. RSD values were 6.3% for GSM and 4.8% for MIB, demonstrating remarkable reproducibility of results. These replicates were analyzed in three separate batches on different days, further demonstrating the precision of the method. Hence, HCSE with GC-MS analysis is a quantitative and reliable method.
Minimum detection limits (MDL)
The MDL was calculated as per the U.S. EPA protocol14 using spiked water (n=10) or spiked fish samples (n=11). The method for extraction and detection of GSM and MIB in water produced detection limits of 1.2 ng/L for GSM and 1.1 ng/L for MIB. These MDLs were far below the human odor threshold values of approximately 10 and 20 ng/L, respectively. Thus, the proposed method can confidently and quantitatively detect the accumulation of these odorous molecules before they reach human sensory thresholds. Detection limits for the extraction and detection of GSM and MIB from fish produced limits of 18.1 ng/kg for GSM and 13.6 ng/kg for MIB.
Analysis of water samples from aquaculture
We analyzed water samples taken from seven recirculating aquaculture systems, with five replicates (system 3) or three replicates (all other systems) assessed for each. All but one of the recirculating aquaculture systems analyzed contained geosmin and MIB at concentrations above our MDL (Figure 5), indicating a potential issue with water quality. However, we noted that in all cases, MIB was well below the human odor threshold of 20 ng/L, while only one system exceeded the GSM odor threshold of 10 ng/L (system 1, GSM concentration = 12.69 ng/L). While we did detect GSM and MIB in system 3, we note that the concentration for MIB falls below our calculated MDLs, and therefore, the concentration cannot be confidently quantified. The method demonstrated impressive precision across all systems despite the use of real-world water samples with very little variation in calculated concentration among replicate samples in each system (Figure 5).
Analysis of fish tissues
Fish tissue, rich in lipids, imparts a strong matrix effect that impedes extraction of GSM and MIB during this method. For that reason, subtle changes are made in the method to increase the extraction efficiency, including a higher incubation temperature (80 °C) and longer incubation time (20 min) compared to the water extraction method. Fish tissues were previously analyzed to determine background concentrations GSM and MIB. Those same samples were then spiked with a known amount of GSM and MIB and analyzed in triplicate to determine percent recovery and precision (Figure 6).
<img alt="Desorption temperature effects on GSM, MIB carryover; table, bar charts, analytical chemistry data." class="xfigimg" src="/files/ftp_upload/67280/67280fig1v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Effect of probe desorption temperature on carryover. (A) A laboratory standard containing GSM and MIB at 20 ng/L was analyzed at one of three probe desorption temperatures in triplicate, with mean peak areas. Each probe was subsequently desorbed twice more at the initial temperature to assess carryover with results in (B) (GSM) and (C) (MIB). Carryover is expressed as peak area as a percent of initial peak area. Error bars represent standard deviation across three replicates. <a href="https://www.jove.com/files/ftp_upload/67280/67280fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Box plot comparison of recovery rates at 15 ng/L and 40 ng/L; data analysis results." class="xfigimg" src="/files/ftp_upload/67280/67280fig2v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Internal standard usage. Comparison of corrected versus uncorrected data recovery data for analytical standards at 15 or 40 ng/L, with extraction by PDMS probes only. Sample concentrations were corrected with the use of IPMP as an internal standard. <a href="https://www.jove.com/files/ftp_upload/67280/67280fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Box plot showing recovery rates of GSM and MIB using PDMS and 3-phase methods in chromatography." class="xfigimg" src="/files/ftp_upload/67280/67280fig3v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Analyte recovery. Recovery data for analytical standards of GSM (left) and MIB (right) at 15 ng/L, with extraction by PDMS-only or triple-phase (PDMS, DVB, CWR) probes. <a href="https://www.jove.com/files/ftp_upload/67280/67280fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chromatography results graph, GSM and MIB concentration vs. area counts analysis." class="xfigimg" src="/files/ftp_upload/67280/67280fig4v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Analytical calibration curves. Linear regressions of calibration curves for GSM (solid line, r2 of 0.9999; S=0.37) and MIB (dashed line, r2 of 0.9999; S=0.37). <a href="https://www.jove.com/files/ftp_upload/67280/67280fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Geosmin and MIB concentration bar charts; water analysis; ng/L; experimental data analysis." class="xfigimg" src="/files/ftp_upload/67280/67280fig5v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Aquaculture water application. Concentrations of (A) GSM and (B) MIB in water samples taken from seven aquaculture systems (n ≥ 3 for each system). <a href="https://www.jove.com/files/ftp_upload/67280/67280fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar chart of GSM and MIB concentration levels in samples, showing comparative data analysis results." class="xfigimg" src="/files/ftp_upload/67280/67280fig6v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 6"> 
Figure 6: Fish tissue application. Concentrations of GSM and MIB from two fish samples spiked with GSM and MIB (F1 and F2). <a href="https://www.jove.com/files/ftp_upload/67280/67280fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution name</td>
			<td>Stock C1 volume added</td>
			<td>Total volume</td>
			<td>Resultant concentration</td>
		</tr>
		<tr>
			<td>Stock C1</td>
			<td>10ul</td>
			<td>100ml</td>
			<td>10 ug/ml</td>
		</tr>
		<tr>
			<td>Cal 1</td>
			<td>10 ul</td>
			<td>100 ml</td>
			<td>1 ng/L </td>
		</tr>
		<tr>
			<td>Cal 2</td>
			<td>20 ul</td>
			<td>100 ml</td>
			<td>2ng/L</td>
		</tr>
		<tr>
			<td>Cal 5</td>
			<td>50 ul</td>
			<td>100 ml</td>
			<td>5 ng/L</td>
		</tr>
		<tr>
			<td>Cal 10</td>
			<td>100 ul</td>
			<td>100 ml</td>
			<td>10 ng/L</td>
		</tr>
		<tr>
			<td>Cal 20</td>
			<td>200 ul</td>
			<td>100 ml</td>
			<td>20 ng/L</td>
		</tr>
		<tr>
			<td>Cal 50 </td>
			<td>500 ul</td>
			<td>100 ml</td>
			<td>50 ng/L</td>
		</tr>
		<tr>
			<td>Cal 100</td>
			<td>1000 ul</td>
			<td>100 ml</td>
			<td>100 ng/L</td>
		</tr>
		<tr>
			<td>Cal 200 </td>
			<td>2000 ul</td>
			<td>100ml</td>
			<td>200 ng/L</td>
		</tr>
	</tbody>
</table>
Table 1: Calibration standards. Geosmin and 2-MIB standards all made from CRM47525 geosmin and 2-methylisoborneol solution (100 µg/mL) in methanol that was diluted into a stock solution C1 (10 µg/ml) in methanol.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Analysis</td>
			<td colspan="2">Carryover (%)</td>
		</tr>
		<tr>
			<td>GSM</td>
			<td>MIB</td>
		</tr>
		<tr>
			<td>GC Run</td>
			<td>0.04</td>
			<td>0.08</td>
		</tr>
		<tr>
			<td>Trap desorption</td>
			<td>0.09</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Inlet desorption</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Probe redesorption</td>
			<td>9.35</td>
			<td>3.77</td>
		</tr>
	</tbody>
</table>
Table 2: Probe carryover assessment. Carryover following extraction, desorption, and GC-MS analysis of a laboratory standard containing GSM and MIB at 20 ng/L, with internal standard IPMP at 30 ng/L. Descriptions of analyses are given in 2.2.1.

This protocol presents a fully automated workflow for the extraction of geosmin and 2-methylisoborneol from water and lipid-rich fish tissues. The method allows for the early detection of these molecules before they reach odor thresholds. Representative data from an aquaculture setting are provided.

Geosmin and 2-methylisoborneol are volatile organic compounds of microbial origin that frequently occur in recirculating aquaculture systems and confer ...

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Research

JoVE Journal

Chemistry

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

A protocol about the characterization and application of five different passive sampling devices is presented.

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

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.

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

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.

Sampling and Analysis of Animal Scent Signals

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

A fast-multidisciplinarystrategy for early detection of cyanobacterial blooms and associated cyanotoxins is described here. It allows for the detection of cyanobacteria and related cyanotoxins in water samples and in organic matrices, such as bivalve samples, in 24 h.

Early Detection of Cyanobacterial Blooms and Associated Cyanotoxins using Fast Detection Strategy

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.

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

This study demonstrates an approach to measure methane gas concentrations in aqueous samples using portable optical analyzers coupled to an injection chamber in a closed loop. The results are similar to conventional gas chromatography, presenting a practical and low-cost alternative particularly suitable for remote field studies.

Measuring Dissolved Methane in Aquatic Ecosystems Using An Optical Spectroscopy Gas Analyzer

This protocol presents a validated liquid chromatography-ion mobility-high resolution mass spectrometry method to determine the presence of ergot alkaloids in food in compliance with the recently released Commission Regulation (EU) 2023/915.

Detection of Regulated Ergot Alkaloids in Food Matrices by Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometry

Learn to extract and detect geosmin and 2-methylisoborneol in water and fish using high-capacity sorptive extraction probes and GC-MS. This automated protocol enables early detection of odorant compounds for aquaculture quality control.

Extraction and Detection of Geosmin and 2-Methylisoborneol in Water and Fish using High-Capacity Sorptive Extraction Probes and GC-MS (Video) | JoVE Journal

Extraction and Detection of Geosmin and 2-Methylisoborneol in Water and Fish using High-Capacity Sorptive Extraction Probes and GC-MS

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