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

[Narrator] In this video we will demonstrate the process for the extraction and detection of geosmin and 2-methylisoborneol from both water and fish samples using high-capacity sorptive extraction probes and gas chromatography-mass spectrometry, or GC-MS. Geosmin and 2-methylisoborneol are volatile organic compounds of microbial origin that occur in streams, ponds, wells, or even recirculating aquaculture systems. These odorant compounds can confer unpleasant odors and flavors to water and fish grown therein even at extremely low concentrations. Here we see a scientist collecting water from a recirculating aquaculture system. Geosmin and 2-methylisoborneol water samples are collected in brown glass vials rated for VOCs with no air left in the vial. To do this, submerge and cap the vial underwater so no air bubbles are remaining. We begin by preparing our calibration and internal standards. First, prepare a stock solution labeled as C1 by adding 10 microliters of geosmin and 2-methylisoborneol solution at 100 micrograms per liter concentration to a 100-milliliter volumetric flask. Fill the flask to the volume mark with the LC/MS grade methanol. This results in a solution containing each analyte at a concentration of 10 micrograms per liter. Next, calibration standards are prepared by adding specified volumes of the C1 stock solution to the 100-milliliter volumetric flasks and filling to volume with deionized water. It is important to prepare fresh calibration standards daily, as these solutions are extremely perishable. For the internal standards, create stock solution I1 by adding 10 microliters of 2-isopropyl-3-methoxypyrazine, also known as IPMP, into a one-liter volumetric flask filled with LC/MS grade methanol, making a 10 milligram per liter stock solution. Then prepare a second solution I2 by adding a hundred microliters of the I1 stock solution to a hundred-milliliter volumetric flask and filling with deionized water to achieve a 10 microgram per liter concentration. Begin by placing one 20-milliliter vial water sample into the sample tray. Weigh out 2.5 grams of sodium chloride and add it to each sample vial. Adding salt enhances the transfer of geosmin and MIB from the water to the headspace. Add five milliliters of the water sample to each vial and spike it with 50 microliters of the internal standard solution I2. Cap and seal each vial immediately after preparation to minimize analyte loss. The next step is the analyte extraction. Place the sample tray into the autosampler tray holder and prepare the instrument method with the following key settings: Pre-sampling agitation time at 10 minutes. Sampling parameters: Incubate at 65 degrees Celsius for 30 minutes with an agitation speed of 400 RPM. Ensure the Wash HiSorb probe option is enabled to prevent cross-contamination between samples. Probe desorption: Set for 15 minutes at 270 degrees Celsius. Trap settings: A trap load temperature of 25 degrees Celsius and a split flow rate of eight milliliters per minute. Once these settings are in place, start the autosampler in conjunction with the GC-MS method for water sample analysis. For fish tissue analysis, place one 20-milliliter vial per fish sample into the tray. Homogenize the fish tissue using a food processor and weigh one gram of the homogenized tissue into each vial. Add five milliliters of a saturated sodium chloride solution and spike each sample with 100 microliters of the internal standard solution I2. Cap and seal each vial immediately. The extraction method for fish samples is similar to that for water but with key differences due to the lipid-rich nature of fish tissue. These differences include pre-sampling agitation time: Set for 20 minutes. Sampling incubation temperature: 80 degrees Celsius for 30 minutes with an agitation speed of 400 RPM. Ensure the Wash HiSorb probe function is enabled. Once these steps are complete, run the autosampler in conjunction with the GC-MS method for fish sample analysis, The same GC-MS parameters apply for both water and fish tissue methods. The GC is equipped with five MS 30 meter by 0.25 millimeter by 0.25 micron column. An ultrapure helium is used as the carrier gas, with a flow rate of two milliliters per minute. The oven program starts with an initial temperature of 60 degrees Celsius held for three minutes, followed by a temperature ramp of 10 degrees Celsius per minute to 100 degrees Celsius, then 20 degrees Celsius per minute to 190 degrees Celsius, and 30 degrees Celsius per minute to 280 degrees Celsius with a final hold time of two minutes. The total runtime is approximately 16.5 minutes. For the transfer line between the GC and the MS, set the temperature to 280 degrees Celsius with the ion source at 250 degrees Celsius and the quadruple at 200 degrees Celsius. In the MS for scan mode, use a range of 50 to 350 mass-to-charge ratio. For selected ion monitoring, or SIM, use the following ions for quantification and confirmation: IPMP, 152 and 137 mass-to-charge ratio. MIB, 95 to 107 mass-to-charge ratio. And geosmin, 112 and 55 mass-to-charge ratio. Lastly, always manually review peak selections in your chromatography software to avoid errors. This protocol outlines the steps for the detection of geosmin and 2-methylisoborneol in water and fish samples utilizing high-capacity sorptive extraction probes and GC-MS. By carefully following these instructions, researchers can ensure accurate and reliable results in analyzing the presence of these compounds. Figure One shows the effect of probe desorption temperature on carryover. A laboratory standard containing geosmin and 2-methylisoborneol at 20 nanograms per liter was analyzed at three different probe desorption temperatures. Panel A provides the mean peak areas for geosmin and MIB. Each probe was subsequently desorbed twice more at the same initial temperature to assess carryover. The results for geosmin and MIB are shown in panels B and C respectively. Carryover is expressed as the peak area as a percentage of the initial peak area. The error bars represent the standard deviation across three replicates. The results indicate that a higher desorption temperature minimizes carryover without compromising sensitivity. Figure Two represents a comparison of corrected versus uncorrected data recovery for analytical standards at 15 and 40 nanograms per liter using PDMS probes for extraction. The samples were corrected with the use of 2-isopropyl-3-methoxypyrazine, or IPMP, as an internal standard. Without correction, recovery was significantly lower, with corrections at approximately 60% of the known spiked amounts. Once corrected, recovery was near 100%, demonstrating the importance of using internal standards for accurate quantification. Figure Three compares the recovery data for geosmin on the left and 2-methylisoborneol on the right at 15 nanograms per liter using two types of probes: PDMS-only and triple-phase probes, which include PDMS, divinylbenzene, and carbon wide range. Both probe types show good performance, though PDMS-only probes had slightly better recovery rates. Given this result and the cost savings, future analysis were conducted with PDMS-only probes. Figure Four shows the linear regression of calibration curves for geosmin and MIB. The solid range represents geosmin with an R-squared value of 0.9999, and the dashed line represents MIB, also with an R-squared value of 0.9999. Both curves demonstrate excellent linearity with minimal error, validating the method's quantitative accuracy over the tested concentration range. Figure Five illustrates the concentrations of geosmin and 2-methylisoborneol in water samples taken from seven different aquaculture systems. Panel A shows the geosmin concentrations, and panel B shows the MIB concentrations. Each system had at least three replicates, and in most cases, concentrations of these compounds were above the method detection limits. One system, labeled as system one, exceeded the human odor threshold for geosmin, highlighting a potential issue with water quality in that system. Figure Six provides data from two fish samples, labeled F1 and F2, which were spiked with geosmin and 2-methylisoborneol. Both compounds were analyzed in triplicate to determine recovery and precision. The concentrations of geosmin and MIB and the spiked fish samples are plotted, demonstrating the method's capability to accurately detect these compounds even in lipid-rich fish tissues. The methods outlined today provide a robust framework for the sensitive detection of geosmin and 2-methylisoborneol. Through the integration of high-capacity sorptive extraction probes and precise GC-MS analysis, we've demonstrated a technique that significantly enhances our detection capabilities. This methodological advancement not only improves the throughput but also reduces the risk of human error and increases the reproducibility of results. As we continue to refine these techniques, they will undoubtedly become indispensable tools in the field of environmental monitoring and food-quality assessment.