Article pubs.acs.org/ac
In Vivo Solid-Phase Microextraction with in Vitro Calibration: Determination of Off-Flavor Components in Live Fish Ziwei Bai,† Alexandre Pilote,‡ Pallab Kumer Sarker,‡ Grant Vandenberg,‡ and Janusz Pawliszyn*,† †
Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada Department des Sciences Animales, Université Laval, Pavillon Paul-Comtois, Québec, Québec G1K 0A6, Canada
‡
ABSTRACT: The presence of off-flavor compounds in fish represents a significant economic problem encountered in aquaculture production. The off-flavor compounds are due to the absorption of substances produced by a range of microorganisms. Currently, a number of strategies have been used to prevent or limit the growth of these microorganisms. Therefore, it is important to evaluate the effectiveness of strategies via monitoring the concentrations of off-flavor compounds in the recirculating aquaculture system. In vivo solid-phase microextraction (SPME), a rapid and simple sample preparation method, will allow monitoring the concentration of off-flavor compounds in live fish. In this research, geosmin and 2-methylisoborneol (2-MIB) produced by cyanobacteria and actinomycetes, which are the major sources for “earthy” and “muddy” flavors in fish, were selected as representatives. In order to accurately quantify these compounds in fish muscle, two kinetic calibration methods, on-fiber standardization and measurement using predetermined sampling rate, were used as quantification methods, which were both validated by traditional methods. The detection limit of in vivo SPME in fish muscle was 0.12 ng/g for geosmin and 0.21 ng/g for 2-MIB, which are both below the human sensory thresholds. ecirculating aquaculture systems (RAS) are fish culture systems in which water is largely reused after undergoing treatment.1 Currently, commercial RAS production systems typically recirculate over 99% of their water usage, thus significantly reducing water consumption.2 In addition, RAS improves opportunities for waste management and nutrient recycling,3 thus providing better hygiene and disease management,4 as well as biological pollution control.5 However, one major disadvantage of producing fish in RAS is the presence of off-flavor compounds. Among those flavors, the “earthy” and “muddy” odors constitute more than 80% of the off-flavor problems found in farm-raised fish.6 Such flavors come from the absorption by fish of substances including geosmin and 2methylisoborneol (2-MIB), which are produced by a broad group of microorganisms in water.7−9 The presence of undesirable off-flavors in products raised in RAS may cause a major reduction in the consumption of the products, or make them unsuitable for sale. The presence of off-flavors therefore represents a major hurdle for the wide-scale adoption of RAS technologies as a production technique. Currently in RAS research, there is focus on developing strategies to prevent or limit the development of microorganisms that produce the off-flavor compounds found in fish. Therefore, in order to evaluate the effectiveness of these strategies on microorganisms, a method needs to be implemented that can monitor the level of target compounds by repeatedly sampling the individual living fish in RAS over time. Recently, solid-phase microextraction (SPME) has been
R
© 2013 American Chemical Society
widely used as a rapid, inexpensive, and solvent-free sample preparation method, which combines sampling, analyte isolation, and enrichment into one step.10 Successful use of in vivo SPME depends on the selection of an appropriate calibration method. The on-fiber standardization method is one of the most widely used calibration methods for SPME in vivo sampling,11−15 which uses the symmetry between the desorption of deuterated standards from the extraction phase to the sample and the absorption of analytes from sample to the extraction phase.16 Recently, another calibration method using predetermined sampling rates of the analytes has been reported by Ouyang et al.17 It assumes that the rate of mass transfer or sampling rate remains constant throughout the duration of sampling within the linear range. The relationship between the concentration of target analytes in the sample matrixes (C0) and the extracted amount of analytes at time t(n) can be expressed with eq 1: C0 = n/R st
(1)
where Rs is the sampling rate for the target analyte and t is the sampling time. Use of eq 1 assumes that the intersample matrix differences in semisolid tissues (such as fish muscle) are slight between individuals of the same species. Consequently, the Received: November 15, 2012 Accepted: January 18, 2013 Published: January 18, 2013 2328
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sampling rate of the SPME fiber can be predetermined under laboratory conditions and directly used for real sample analysis.18 With this method, no distribution coefficient (K) determination and deuterated standard uploading prior to sampling are needed any longer. The objective of this study was to develop an effective and simple method to determine geosmin and 2-MIB in fish using in vivo sampling technique. In order to accomplish this goal, two kinetic calibration methods were investigated and verified: (1) on-fiber standardization and (2) measurement using predetermined sampling rates of analytes. Finally, the developed methods were applied for on-site determination of off-flavors. These results were compared to those obtained from the traditional methods of analysis using lethal sampling and microwave distillation prior to SPME.
the desorption time profile was drawn by preloading same amounts of standards and desorbing in the samples for different times. In Vivo Sampling System Setup. For this study, in vivo sampling experiments were performed at Alma Aquaculture Research Station, University of Guelph. The source of water in the station was from the underground wells nearby and was proved to contain no detectable 2-MIB or geosmin by using a headspace SPME analysis technique.19 The water temperature was kept constant at 8.5 °C. Rainbow trout (Oncorhynchus mykiss) were of marketable table size at 900 ± 154 g (n = 48). Before experiments, fish were acclimated with the diet in a flowthrough system 1 week prior to experimentation. The system was located indoors and consisted of 16 (729 L) tanks. Each tank held four fish, three for in vivo sampling and one for lethal sampling. Before exposure to tainted water, the fish were deprived of food for 48 h. This was in order to minimize variations in metabolic rates and ventilation rates between individual fish with different feeding histories.20 During the exposure time, the fish were deprived of food as well. The uptake of off-flavor compounds from contaminated water by fish as well as the in vivo sampling experiments were both performed in a static, albeit oxygen offered system (no water flow rate). The water inside each static system (round tank) was transferred from flow-through system, and the volume was 90 L. The procedure for operating in vivo sampling was as follows: First, fibers were prepared by uploading deuterated geosmin standard from the headspace of pump oil solution. Before each SPME sampling, fish were anesthetized in a 80 μg/mL MS-222 water solution for the duration of approximately 1 min. After that, an 18 gauge needle was used to pierce the fish skin, where it is below the anterior point of the dorsal fin (as shown in Figure 1), and a 21 gauge long stainless steel wire was
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EXPERIMENTAL SECTION Chemicals and Materials. Geosmin and 2-MIB were purchased from Wako Chemicals U.S.A., Inc. Deuterated MIB (MIB-d3) was obtained from CDN Isotopes Inc. (Pointe-Claire, Quebec, Canada), and deuterated geosmin (geosmin-d3) was purchased from Sigma-Aldrich (Oakville, Ontario, Canada), with the purity of 99%. All chemicals purchased were of the highest possible purity and were used without further purification. Chloroform for fat content measurement was purchased from EMD Chemicals Inc. (Damstadt, Germany). HPLC (high-performance liquid chromatography) grade methanol was purchased from Fisher Scientific (Unionville, Ontario, Canada). HPLC grade acetone was obtained from Caledon Laboratories Ltd. (Georgetown, Ontario, Canada). Pump oil was purchased from Varian Vacuum Technologies (Lexington, MA). Nanopure water was obtained using the Barnstead Nanopure water system. The 1 cm metal core commercial PDMS fibers were obtained from Supelco (Bellefonte, PA). Taint-free rainbow trout fillets were purchased from Sobeys Inc. (Ontario, Canada). Rainbow trout fillets contaminated with geosmin and 2-MIB were obtained from Alma Aquaculture Research Station (University of Guelph, Ontario, Canada). All fish tissue experiments were performed in accordance with protocols approved by our institutional Animal Care Committee (AUP no. A-12-01) in the Chemistry Lab Facility at the University of Waterloo. All in vivo fish experimental procedures were approved by the Animal Care Committee at University of Guelph (AUP no. 12R066). Symmetry of Geosmin and 2-MIB on SPME under in Vitro Condition for the On-Fiber Standardization Calibration Method. Desorption time profiles of deuterated standards and absorption time profiles of analytes were performed simultaneously to verify the symmetry. Deuterated standards preloaded onto the fiber prior to the extraction were prepared by using headspace sampling from deuterated standards pump oil solution under 25 °C. Both homogenized and nonhomogenized fish tissue samples were utilized. Contaminated homogenized fish muscle was obtained by spiking specific amounts of geosmin and 2-MIB methanol solution into taint-free homogenized fish tissue. Nonhomogenized fish tissue with off-flavors was prepared by cutting each sample in 4 g size. Sampling was conducted by directly inserting the SPME fibers in the homogenized and nonhomogenized fish. All the experiments were performed under 8.5 °C, the same temperature as in vivo sampling. The absorption time profile was obtained by extracting from geosmin and 2-MIB contaminated samples for different sampling times. Meanwhile,
Figure 1. SPME fiber inserting position for in vivo sampling in fish muscle.
introduced into the same perforation and penetrated to the same length as the SPME fiber, because the needle was not as long as the SPME fiber. Then the SPME fiber was inserted, and the hub was pushed to the end. Following fiber placement, fish were placed back in the static tank. After 30 min of SPME extraction, the fish was anesthetized again. At last, the fiber was removed, gently washed with Nanopure water, wiped with a Kimwipe, sealed with a Teflon cap, wrapped with aluminum foil, and stored in the dry ice until gas chromatography mass spectrometry (GC/MS) analysis. 2329
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Table 1. Time Constant Results and Conclusions for 2-MIB and Geosmin under Both in Vitro and in Vivo Conditionsa 2-MIB homogenized fish tissue nonhomogenized fish tissue live fish a
geosmin
desorption a (min−1) (R2)
absorption a (min−1) (R2)
desorption a (min−1) (R2)
absorption a (min−1) (R2)
0.012 ± 0.002 (0.946) 0.012 ± 0.003 (0.844) 0.010 ± 0.003 (0.852)
0.011 ± 0.001 (0.984) 0.012 ± 0.006 (0.780) 0.013 ± 0.006 (0.790)
0.008 ± 0.001 (0.987) 0.007 ± 0.001 (0.884) 0.012 ± 0.003 (0.747)
0.008 ± 0.001 (0.984) 0.010 ± 0.004 (0.837) 0.011 ± 0.007 (0.839)
Correlation coefficients are given in parentheses.
Instrumentation. A Varian 3800 GC coupled with a Varian 4000 electron ionization ion-trap mass spectrometer (GC/EIIT-MS) (Varian, Inc. Walnut Creek, CA, U.S.A.) was used for analysis. The GC was equipped with a capillary column (RTX5, 30 m × 0.25 mm i.d., 0.25 μm film thickness) (Restek Corporation, Bellefonte, PA, U.S.A.). The oven temperature program was 60 °C (0.5 min) increasing at 40 °C/min to 110 °C, increasing at 1 °C/min to 117 °C, increasing at 5 °C/min to 156 °C, increasing at 40 °C/min to 250 °C (10 min). The split/splitless injector of the GC was equipped with a Merlin microseal injector adapter (Merlin Instrument Company, Half Moon Bay, CA, U.S.A.) for metal fiber desorption. For both liquid injection and SPME, splitless mode was used, and split was opened after 5 min of desorption. The temperature of the transfer line was 280 and 240 °C for the ion trap. The column flow rate of the helium carrier gas was 1 mL/min. The 1079 injector was equipped with a programmed temperature vaporizer. The temperature of the injector was 250 °C. When using selected ion storage (SIS) scan mode, m/z 112, m/z 150, m/z 115, and m/z 138 were selected for quantification of geosmin, 2-MIB, geosmin-d3, and MIB-d3, respectively. As qualifier ions, m/z 126 and 182 were for geosmin, m/z 168 was to represent 2-MIB, m/z 129 and 185 were for geosmin-d3, and m/z 171 were selected for MIB-d3. When performing the in vivo sampling analysis, 2-MIB required using MS/MS mode, and geosmin kept using SIS mode. Under MS/MS mode, the parent ion of 2-MIB m/z 95 yielded daughter ions m/z 67 and m/z 91. The excitation amplitude was set as 1.0 V applied with resonant waveform. The emission current and maximum ionization time were set as 80 μA and 65 000 μs, respectively.
Fouling issues due to the adsorption of macromolecular compounds on the SPME coating surface, such as lipids and protein,23 was found after the fiber was desorbed in the GC in the form of 2-MIB and geosmin peak intensity enhancement. To address this, following the desorption, the fiber was gently wiped with an acetone-soaked Kimwipes and rinsed with large amounts of water, followed by conditioning in the GC injector for 10 min at 250 °C. As a result, the repeatability of the fiber was kept acceptable. Symmetry of Desorption and Absorption Verification under in Vivo and in Vitro Conditions. The kinetics of absorption and desorption was performed in homogenized fish tissue, nonhomogenized fish tissue, and live fish. Time profiles were used for this verification. Because this calibration method could not be used to determine a low concentration of 2-MIB in fish tissue, the concentrations of 2-MIB in all these three systems were high. The details about the concentration for both analytes were as follows: homogenized fish tissue 0.5 μg/g for 2-MIB and 0.025 μg/g for geosmin; nonhomogenized fish tissue and live fish were around 0.3 μg/g for both analytes in these two systems. The time constants of absorption and desorption were very similar for both 2-MIB and geosmin in all three systems, which means the symmetry was kept all through the desorption and absorption processes (as shown in Table 1). Different Concentrations of Analytes in Homogenized Fish Tissue. A various range of concentrations (0.5, 2.5, 5.0, 12.5, 25.0 ng/g) of 2-MIB and geosmin in homogenized fish tissue were prepared and followed by a 30 min extraction. Equation 2 describes the entire absorption kinetics of the analyte from the sample matrixes to SPME liquid, in which n is the amount of extracted analyte at time t, ne is the amount of analyte extracted at equilibrium, and a is a absorption time constant.24 According to eq 2, the extraction amount should be proportional to the initial concentration, if all the parameters keep constant. The linearity displayed in Figure 2 indicates that the extraction time constant for both analytes remained consistent in this concentration range. In addition, the value of the extraction time constant was obtained in Table 1. This discovery was important for both calibration methods. For the on-fiber standardization calibration method, it needs the
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RESULTS AND DISCUSSION Preliminary Experiments. Selected ion storage mode was utilized for symmetry determination. To determine geosmin, geosmin-d3 was used as an internal standard. In this case, the EI spectrum of the labeled compound produced a base peak at m/ z 115, which contained three deuterium atoms, in the agreement with the fragmentation proposed by Lloyd and Grimm.21 The quantification of geosmin and geosmin-d3 was achieved using the ion pair at m/z 112 and 115, and qualification was established by using the ions m/z 126 and 182 for geosmin and m/z 129 and 185 for geosmin-d3. However, for 2-MIB and MIB-d3, the base peak ion at m/z 95 for both was not a deuterated fragment. Finally, only m/z 150 and 138 could be decided to represent 2-MIB and MIB-d3. However, in that case, the limit of detection for 2-MIB was as high as 10 ng/g in fish tissue, which was more than 10 times higher than the human sensory threshold.22 Consequently, the on-fiber standardization calibration method is not suitable for determination of low contamination of 2-MIB in fish. Therefore, measurement using the predetermined sampling rates method, which does not require the use of the internal standard, was placed.
Figure 2. Different concentrations of analytes in homogenized fish tissue. 2330
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symmetry of desorption and absorption during the sampling time. For the calibration method using a predetermined sampling rate, it requires the constant sampling rate.
Table 2. Results Comparison between MD-SPME and in Vivo SPME 2MIB (ng/ g)
K VV n = [1 − exp(−at )]ne = [1 − exp(−at )] fs f s C0 K fs + Vs (2)
Determination of Sampling Rate for Calibration Using a Predetermined Rate. Different concentrations (0.5, 2.5, 5.0, 12.5, 25.0 ng/g) of 2-MIB and geosmin in homogenized fish tissue were prepared to obtain the extraction amount n under 30 min of sampling time, and all the other conditions were performed the same as in vivo sampling SPME. Plotting C0t versus n, the slope obtained was Rs (as shown in Figure 3). As a result, the Rs value was determined to be 1.6 × 10−4 g/min for 2-MIB and 7.8 × 10−5 g/min for geosmin.
fish no.
mass (g)
1 2 3 4 5 6 7 8 9
1013.0 883.3 855.4 1003.2 894.1 985.6 772.6 958.3 896.0
sex
fat content (%)
F M M M M M M M M
3.4 3.0 2.7 3.7 1.6 2.1 2.9 2.7 3.7
geosmin (ng/g)
PDSR
MDSPME (ng/g)
OFS
PDSR
MDSPME (ng/g)
3.4 3.9 2.8 2.6 3.3 3.4 4.9 3.8 4.5
3.0 3.9 3.6 4.4 3.3 4.6 3.7 5.4 4.4
2.6 3.0 2.1 2.0 3.4 2.2 6.1 3.0 5.2
2.6 3.3 2.2 1.6 5.0 2.6 5.9 3.9 5.2
3.0 3.7 2.9 3.0 4.6 3.7 5.9 5.1 5.0
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CONCLUSION AND FUTURE DIRECTIONS An in vivo SPME sampling technique to measure the concentration of off-flavor compounds in fish was developed. When compared with traditional methods, it has the advantages of time efficiency, simplicity of procedure, and most important, the capability of monitoring target compound concentration changes when sampling from the same individual fish. Kinetic calibration methods were used for quantification. The on-fiber standardization method was used to measure geosmin, while predetermined kinetic rates were employed to determine the concentration of both 2-MIB and geosmin in live fish. The symmetry character of desorption of internal standards from fiber and absorption of analytes onto fiber for these two compounds was confirmed. The future application of this method could assist in realtime, nonlethal monitoring of key sensory qualities and drug/ pesticide or other contaminants residues of fish and other food products prior to harvesting. To facilitate the on-site analysis, a portable GC or other instrumentation could be utilized.
Figure 3. Determination the Rs of 2-MIB and geosmin in homogenized fish tissue.
Validation of in Vivo SPME Sampling Using Microwave Distillation SPME (MD-SPME). Nine fish were exposed to a low concentration of geosmin and 2-MIB (0.05 ng/mL) in water. Preloading was performed by headspace extracting from 50 μg/g geosmin-d3 pump oil solution with agitation, and the extraction time was 10 s under 25 °C. The on-fiber standardization (OFS) calibration method was applied for the quantification of geosmin, and the predetermined sampling rate (PDSR) approach was used to determine both 2-MIB and geosmin. Fat content of rainbow trout was assessed according to Zhang et al.’s method.25 According to the results, there is no obvious relationship between mass, fat, and concentration of analytes. Fish from different tanks do not exhibit a significant difference. To confirm the accuracy of results, a traditional method, microwave distillation SPME, was used for validation. Microwave distillation and SPME headspace extraction procedures were executed in accordance with Lloyd and Grimm’s publication.21 The same instrument parameters were used as in vivo sampling analysis of fish. Table 2 displays the results of off-flavor compounds concentrations of nine lowcontaminated fish using two sample preparation methods. The Student t test was applied to confirm that the on-fiber standardization calibration method and the measurement using a predetermined sampling rate calibration method or in vivo SPME and MD-SPME yield the same results, which validates the use of the in vivo sampling SPME technique with both calibration methods.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +1-519-888-4641. Fax: +1-519-746-0435. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the Natural Sciences and Engineering Research Council (NSERC) of Canada for financial support. The authors acknowledge Alma Research Station of University of Guelph for their support in providing the in vivo sampling equipment.
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