Selective Extraction of Trace Levels of Polychlorinated and

Anal. Chem. 2005, 77, 2259-2265

Selective Extraction of Trace Levels of Polychlorinated and Polybrominated Contaminants by Supercritical Fluid-Solid-Phase Microextraction and Determination by Gas Chromatography/Mass Spectrometry. Application to Aquaculture Fish Feed and Cultured Marine Species R. Rodil, A. M. Carro,* R. A. Lorenzo, and R. Cela Torrijos

Dpto. de Quı´mica Analı´tica, Nutricio´ n y Bromatologıá, Facultad de Quı´mica, Instituto de Investigacio´ n y Ana´ lisis Alimentarios, Universidad de Santiago de Compostela, Avda, de las Ciencias, s/n 15782 Santiago de Compostela, Spain

The persistence, ubiquity, and toxicity of polyhalogenated compounds, together with their presence in fish feed, make it necessary to monitor these organic pollutants in the routine quality assurance programs of aquaculture activities, as this food chain is a source of these toxic compounds for human consumers. A new approach based on simultaneous supercritical fluid extraction-sample cleanup, followed by solid-phase microextraction-gas chromatography/mass spectrometry (SFE-SPME-GC/MS/ MS) has been developed as an advantageous analytical tool for the determination of 15 organohalogenated compounds (including pesticides, polychlorinated and polybrominated biphenyls, and polybrominated diphenyl ethers) in aquaculture feed at very low levels. The influence of several parameters in the efficiency of the SPE/ SPME combination was systematically investigated by chemometric approaches. In the optimal conditions, the developed procedure provides an excellent linearity, detection, and quantification limits (below 10 pg/g) for most of the analytes investigated, being at the same time advantageous in terms of rapidity, convenience, and avoiding the need of toxic organic solvents. The procedure was applied to the analysis of aquaculture feed and cultured marine species and tested for accuracy against IAEA 406 reference material. Worldwide and in particular the European aquaculture industry has experienced a steady growth and for some species even an impressive growth in production over the last years and decades. According to available data, the European Union is the world leader for most of the species farmed on its territory, such as trout, turbot, mussel, gilthead, and seabass.1 At a global level, the Galician (Spain) aquaculture, amounting to 200 000 tonnes/year, * To whom correspondence should be addressed. E-mail: [email protected]. (1) Communication from the Commission to the Council and the European Parliament. Strategies for the sustainable development of European aquaculture. Commission of the European Communities. Brussels, COM 2002 511, 19/09/2002. 10.1021/ac048994p CCC: $30.25 Published on Web 02/18/2005

© 2005 American Chemical Society

represents ∼35% of total European Union fisheries sector production.2 Having regard to the importance firmly attached today to conservation and environmental protection, interactions between aquaculture and the environment are subject to increasingly strict control and regulation. A recent study suggests that consumption of farmed Atlantic salmon may result in exposure to a variety of persistent bioaccumulative contaminants with the potential for an elevation in attendant health risks.3 This study used the approach of the U.S. Environmental Protection Agency (EPA) to assess the comparative health risks of consuming farmed and wild salmon.4 Individual contaminant concentrations in farmed and wild salmon do not exceed U.S. Food and Drug Administration (FDA) action or tolerance levels for polychlorinated biphenyls (PCBs) and dieldrin.5 However, further studies of contaminant sources, particularly in feeds used for farmed carnivorous species such as salmon, are needed. Monitoring the presence of PCBs, polybrominated biphenyls (PBBs), and polybrominated biphenyl ethers (PBDEs) in aquaculture systems at very low concentration levels is becoming more and more important, because they can be considered as sources of contamination present in mixed feed.6 Recently, the significance to human exposure to halogenated compounds has been the subject of extensive discussions, and the European Commission has established daily intake limits.1,7 Restrictions of poly(2) Consellerıá de Pesca e Asuntos Marı´timos. Xunta de Galicia (Spain). http:// www.xunta.es/conselle/pe/sector_acui.htm. (3) Hites, R. A.; Foran, J. A.; Carpenter, D. O.; Hamilton, M. C.; Knuth, B. A.; Schwager, S. J. Science 2004, 303, 226-229. (4) U.S. EPA, Guidance for Assessing Chemical Contaminant Data for Use in Fish Advisories. Volume 2: Risk Assessment and Fish Consumption Limits (U.S. EPA, Washington, DC, ed. 3, 2000 (available at www.epa.gov/ost/ fishadvice/volume2/index.html) (5) U.S. FDA, Center for Food Safety and Applied Nutrition, Fish and Fisheries Products Hazards and Controls Guidance, ed. 3, Chapter 9 (available at www.cfsan.fda.gov/∼acrobat/haccpc09.pdf). (6) Certain brominated flame retardants-polybrominated diphenyl ethers, polybrominated biphenyls, hexabromo cyclodecane. OSPAR Commission, ISBN 0 946956 70 7, 2001.

Analytical Chemistry, Vol. 77, No. 7, April 1, 2005 2259

halogenated compounds in fish feed reflects concerns about the limitations and provides recommendations for further study to improve the scientific relevance and accuracy of future environmental risk assessments of these compounds.8 Continued improvements of various sample preparation techniques such as accelerated solvent extraction and microwaveassisted extraction allow the rapid obtaining of sample extracts.9-11 However, the selective extraction of trace analytes from complex matrixes with high fat content is more difficult because of coextraction of matrix components.12 Commonly, to improve this situation, additional cleanup steps (using solid-phase extraction)13 are applied to the sample extract enabling the separation of coextractants from the analytes. Alternatively, in the case of supercritial fluid extraction (SFE), it is possible to achieve enhanced selectivity in a single SFE step placing one or several adsorbents directly into the extraction cell.14,15 The application of solid-phase microextraction (SPME) to water samples can be easily achieved, but SPME from solid samples is more difficult except in cases of samples previously extracted by means of solvents or Soxhlet extraction.16 Additionally, for complex environmental sample extracts, very selective detection (mass spectrometry or preferably MS/MS) is usually needed.17 The aim of the present work was to take advantage of the efficiency, speediness, and selectivity offered by SFE with simultaneous cleanup, in combination with the increase of sensitivity and selectivity provided by SPME. The SFE followed by SPME is a newly developed combination for sample preparation in aquaculture feed and biological solid samples for a wide range of organohalogenated pesticides, PCBs, PBBs, and PBDEs. The GC/ MS/MS method was developed and applied for the determination of analytes. Because a CRM of fish feed is not currently available, the accuracy of the SFE-SPME-GC-MS-MS method was tested for several organohalogenated compounds in a multispecies biological reference material (IAEA-406). EXPERIMENTAL SECTION Reagents and Materials. Pesticide grade n-hexane, sulfuric acid 96%, isooctane, and silica gel 60-Å pore size (0.040-0.063 mm, 230-400 mesh) were purchased from Merk (Darmstadt, Germany). Aluminum oxide activated basic (150 mesh) was obtained from Aldrich (Steinheim, Germany). (7) Commission Recommendation of 4 March 2002 on the reduction of the presence of dioxins, furans and PCBs in feedingstuffs and foodstuffs. Official J. L067, 09/03/2002, 2002; pp 69-73, (8) Wenning, R. J.Chemosphere 2002, 46, 779-796. (9) Bordet, F.; Inthavong, D.; Fremy, J. M. J. AOAC Int. 2002, 85, 13981409. (10) Martens, D.; Gfrerer, M.; Wenzl, T.; Zhang, A.; Gawlik, B. M.; Schramm, K. W.; Lankmayr, E.; Kettrup, A. Anal. Bioanal. Chem. 2002, 372, 562568. (11) Zhu, L. Y.; Hites, R. A. Anal. Chem. 2003, 75, 6696-6700. (12) Aguilera, A.; Brotons, M.; Rodrı´guez, M.; Valverde, A. J. Agric. Food Chem. 2003, 51, 5616-5621. (13) Ahmed, F. E. TrAC, Trends Anal. Chem. 2003, 22, 170-185. (14) Rodil, R.; Carro, A. M.; Lorenzo, R. A.; Cela, R., Proceedings of the VIII International Symposium on Analytical Methodology in the Environmental Field. A Corun ã, Spain, October 21-24, 2003. (15) Ja¨remo, M.; Bjo ¨rklund, E.; Nilsson, N.; Karlsson, L.; Mathiasson, L. J. Chromatogr., A 2000 877, 167-180. (16) Fidalgo-Used, N.; Centineo, G.; Blanco-Gonza´lez, E.; Sanz-Medel, A. J. Chromatogr., A 2003, 1017, 35-44. (17) Hyo ¨tylaïnen, T.; Hartonen, K. TrAC, Trends Anal. Chem. 2002, 21, 1329.

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Pesticides (R-BHC, 1000 µg/mL in MeOH; γ-BHC, 1000 µg/ mL in MeOH; heptachlor, 1000 µg/mL in MeOH; and 4,4′-DDT, 98.4% as solid) were supplied by Supelco (Bellefonte, PA). A mixture of PCBs at 10 µg/mL in ethanol, PCB-10 (2,6-dichlorobiphenyl, 100%), PCB-28 (2,4,4′-trichlorobiphenyl, 100%), PCB-52 (2,2′,5,5′-tetrachlorobiphenyl, 100%), PCB-138 (2,2′,3,4,4′,5′-hexachlorobiphenyl, 100%), PCB-153 (2,2′,4,4′,5,5′-hexachlorobiphenyl, 100%), and PCB-180 (2,2′,3,4,4′,5,5′-heptachlorobiphenyl, 100%) was obtained from Supelco. A mixture of PBDEs at 10 µg/mL in cyclohexane, PBDE-47 (2,2′,4,4′-tetrabromodiphenyl ether, 42.5%), PBDE-99 (2,2′,4,4′,5-pentabromodiphenyl ether, 10.9%), and PBDE100 (2,2′,4,4′,6-pentabromodiphenyl ether, 39.3%), was supplied by Dr. Ehrenstorfer (Augsburg, Germany). PBB-15 (4,4′-dibromobiphenyl, 99.8%) and PBB-49 (2,2′,4,5′-tetrabromobiphenyl, 97%) were purchased from Supelco. Stock solutions of PBBs and PBDEs were prepared in isooctane. A mixture of 13C-labeled PCBs, 5 µg/mL in nonane, PCB-28, PCB-52, PCB-101, PCB-138, PCB153, PCB-180, and PCB-209, was supplied by Cambridge Isotope Laboratories (Andover, MA). Sample Preparation. Samples. Optimization experiments were carried out on an stock of a 100-g homogenate of turbot feed spiked with 50 µL of PCBs 10 µg/mL solution, 100 µL of PBDEs 10 µg/mL solution, 50 µL of PBB-15 22 µg/mL solution, 35 µL of PBB-49 15 µg/mL solution, and 335 µL of R-HCH, γ-HCH heptachlor, and 4,4′-DDT 1.6 µg/mL solution. The spiking of the turbot feed was performed with 150 mL of n-hexane containing the above-mentioned amounts of pesticides, PCBs, PBDEs, and PBBs. The hexane was removed by evaporation to air-dry. The homogeneity of the spiked material was evaluated on sample masses from 0.5 to 2 g. Relative standard deviations lower than 16% for all the 15 studied analytes were obtained when the amount of sample taken for analysis was above 1 g. Thus, 1 g was fixed as the minimum sample intake in all experiments in order to prevent the variability between sample portions from masking the influence of experimental variables. Fish feed samples were commercially available. Muscle of farmed turbot was acquired from local farms. Farmed shellfish (clam, mussel, and cockle) were taken in the estuary of Ria de Arousa, Pontevedra, Spain, from sites of marine culture industry. All the real samples were triturated and homogenized in a grinder and freeze-dried before processing. Also, the reference material, IAEA 406 obtained from the International Atomic Energy Agency (Vienna, Austria) was analyzed. SFE Procedure. A supercritical fluid extraction method, employing different adsorbents as lipid retainers inside the extraction chamber has been applied.14 A 60-g aliquot of acidic silica gel, previously dried at 104 °C, was prepared adding by 40 g of H2SO4. Basic alumina and silica gel were stored in a sealed bottle until analysis. After optimization, a procedure was adopted of filling the extraction cells in sandwich mode, first placing 1.0 g of sample at the bottom of the thimble, followed by a layer of 1.5 g of aluminum oxide activated basic, and finally, 1.5 g of acidic silica, on the top, using wads of filter paper and disks of filter paper at both the bottom and the top of the cell to prevent small particles reaching the restrictor and avoiding dead volume.

Figure 1. Comparison of responses in different SPME modes for the 15 selected polyhalogenated compounds.

Figure 2. SPME extraction time profiles of polyhalogenated compounds. (A) PCBs, (B) pesticides, (C) PBBs, and (D) PBDEs.

A pressure of 165 bar and a temperature of 60 °C with a flow of 2 mL/min CO2 for 5 min of static extraction and then 27 min of dynamic extraction were the optimal conditions applied to the extraction module obtained by experimental designs.18 The adsorption and desorption temperature in the collection module (ODS packed trap) was 25 °C using 2 mL of n-hexane as eluting solvent. The final extract was concentrated to dryness under a nitrogen stream.

SPME Procedure. Fused-silica fibers coated with 100 µm of poly(dimethylsiloxane) were selected for this study. Fibers were handled in SPME manual holders supplied by Supelco and conditioned before use as recommended by the manufacturer. (18) Rodil, R.; Lorenzo, R. A.; Carro, A. M.; Cela, R. Proceedings of the Eighth International Symposium on Hyphenated Techniques in Chromatography and Hyphenated Chromatographic Analyzers. Brugge, Belgium. February 4-6, 2004.


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Figure 3. Response surface for global desirability in the SFE extraction of a group of 15 polyhalogenated compounds as a function of pressure and dynamic extraction time.

Parameters affecting the SPME process were optimized by experimental design and extraction kinetics evaluated. In the optimal operational conditions, SPME was carried out in GC sampler vials of 1.8-mL nominal capacity containing the dry SFE extract. Vials were thermostated at 75 °C, and the fiber was placed in the vial allowing the sampling process for 60 min. Once finished with the exposition period, the fiber was immediately inserted into GC injector and the chromatographic analysis by GC/MS/MS carried out. Desorption time was set at 5 min, and desorption temperature was optimized at 274 °C. Possible carryover was prevented by keeping the fiber in an injector port for an additional period of time of 5 min. SFE Instrumentation. All the extraction experiments were performed on a Hewlett-Packard (Wilmington, DE) 7680A SFE module. Hewlett-Packard standard 7-mL extraction thimbles were used in all experiments. The analytes were collected on a HewlettPackard standard trap packed with octadecylsilane. GC/MS/MS Instrumentation. A Varian (Walnut Creek, CA) 3900 gas chromatograph equipped with an ion trap mass detector Varian Saturn 2100T was used. Gas chromatography was carried out on a 30 m × 0.25 mm i.d. HP-5 ms (5% polydiphenylsiloxane; Agilent Technologies) fused-silica column (0.25-µm film thickness). Injections with SPME fiber were made in splitless mode (2 min). Split flow was set at 50 mL/min. The initial temperature was 70 °C, held for 2 min; ramped at 20 °C/min up to 170 °C, and held for 2 min; a second rate at 4 °C/min up to 250 °C; a third rate at 10 °C/min up to 300 °C and held for 5 min. Helium (purity 99.999%) was employed as carrier gas with a constant column flow of 1 mL/min. RESULTS AND DISCUSSION Optimization of SFE/SPME Sample Treatment. In the optimization of a sample processing procedure comprising two combined techniques, many factors have to be taken into account. 2262


Some of them are related to the practical feasibility of the combination of both techniques while others correspond to the particular optimization of each technique. Thus, a chemometric strategy was applied in which the several factors were studied and optimized sequentially while maintaining the number of experiments to a minimum. First, the variables that may affect technique compatibility were explored. Among these variables, the most critical is the fat fraction extracted from samples. Fat content in extracts strongly affect SPME performance so SFE procedure must be developed with the objective of reducing as much as possible the level of extracted fat. Because exploratory experiments indicated that most of fat in samples is extracted by SFE, the extraction procedure was developed to include a simultaneous cleanup process. To do this, several adsorbents, including basic alumina, Florisil, acidic silica, and C18, were assayed. Layers of these adsorbents were placed in the extraction chamber and the mass of fat in the extracts as well as other interferences was evaluated. Results in this study indicated that most of the adsorbents are efficient for fat removal, thus producing extracts ready for SPME. Best results were achieved by layering together acidic silica and basic alumina, so the procedure described under Experimental Section was adopted for SFE stage in the SFE/SPME combination. Second, a sampling mode in SPME (direct immersion or headspace) must be adopted. In both modes, hexane in SFE extracts must be evaporated and the residue may be sampled as such or redissolved into water containing a small amount (5%) of methanol. Headspace sampling can be carried out on both types of processed extracts. Figure 1 compares the results obtained in these experiments carried out by fixing the sampling time (60 min) and vial’s volume (10 mL). It is apparent that for all considered compounds the higher extraction efficiency is attained when dry extracts are processed so this was the operational procedure followed in all the remaining studies. This result has the additional advantage of avoiding the solvent exchange stage. Once the compatibility between techniques was granted, the particular variables affecting each technique were studied. In the case of SFE, the extraction temperature (in the range 60-120 °C), pressure (between 140 and 320 bar), static and dynamic extraction times (5-20 and 10-60 min, respectively) and carbon dioxide flow rate (1-2.5 mL/min) were considered. First, a fractional factorial 25-1 design in 16 experiments19 was applied to screen the really significant factors among the five considered. The conclusions of this study were that the extraction cell temperature, pressure, and dynamic extraction time were the main significant factors in the SFE procedure. Also some two-factor interactions appeared significant, suggesting that main factors are not independent of each other. These conclusions were confirmed by means of a Doehlert design20 developed to study in detail the two more significant factors (the dynamic extraction time and pressure). Results of this study indicated that effectively both factors are significant as well as their interaction, although the influence of factors is different as a function of the considered analytes, so compromise operational settings have to be used. Something similar occurs with the SPME operating variables. In that case, the extraction time (in the 5-90-min range) and (19) NemrodW 2000, LPRAI, University of Aix-Marseille III, Marseille. (20) Doehlert, D. H. Appl. Statistics 1970, 19, 231-239.

Table 1. Retention Times and MS/MS Detection Parameters for the Polyhalogenated Compounds retention time (min)

m/z range

parent ion (m/z)

PCB-10 R-BHC γ-BHC PCB-28 heptachlor PCB-52 PBB-15 PCB-153 PBB 49 4,4′-DDT PCB-138 PCB-180 PBDE-47

9.70 10.89 11.89 13.86 14.38 15.16 16.30 22.27 23.10 23.29 23.44 26.35 26.45

110-240 100-240 100-240 120-280 110-300 120-310 140-320 180-380 100-480 100-480 100-480 160-500 160-500

222 219 219 258 272 292 312 360 470 235 360 396 486

PBDE-100 PBDE-99

30.11 30.97

300-580 300-580

566 566

PCB-28 C13 PCB-52 C13 PCB-101 C13 PCB-153 C13

13.88 15.15 18.50 22.26

120-280 120-310 180-350 180-380

270 304 338 372

PCB-138 C13

23.45

100-480

372

PCB-180 C13

26.35

160-500

408

compound

excitation storage level (m/z)

excitation amplitude (V)

152 + 187 181 + 183 181 + 183 186 + 221 235 + 237 255 + 257 152 290 + 325 389 + 391 165 290 + 325 359 + 361 324 + 326 + 328 404 + 406 404 + 406

110 120 120 130 140 150 140 180 220 110 180 220 220

1.40 1.30 1.30 1.20 1.70 1.80 2.80 2.30 1.80 1.90 2.30 2.30 2.10

240 240

1.90 1.90

198 + 233 267 + 269 301 + 303 302 + 335 + 337 302 + 335 + 337 371 + 373

140 160 180 190

1.20 1.80 1.40 2.29

190

2.29

230

2.29

quantification ions (m/z)

Table 2. Performance of the SFE-SPME-GC-MS/MS Method for Polyhalogenated Compounds in Aquaculture Related Samples

compound

spiked feed (ng/g)

correl coeff

repeatblty (RSD, %, n ) 6)

recovery (%)

method LOD (pg/g)

method LOQ (pg/g)

PCB-10 R-BHC γ-BHC PCB-28 heptachlor PCB-52 PBB-15 PCB-153 PBB-49 4,4′-DDT PCB-135 PCB-180 PBDE-47 PBDE-100 PBDE-99

13.56 16.75 15.86 18.16 10.09 18.49 13.16 16.62 19.40 10.47 15.33 21.32 7.93 1.69 6.07

0.993 0.994 0.997 0.997 0.999 0.997 0.997 0.997 0.998 0.991 0.996 0.993 0.991 0.995 0.991

12 7 7 8 9 4 7 7 9 8 9 7 8 16 17

78 ( 11 86 ( 9 89 ( 12 92 ( 5 65 ( 7 100 ( 3 77 ( 10 89 ( 5 99 ( 11 69 ( 6 77 ( 7 101 ( 8 101 ( 14 82 ( 16 91 ( 14

0.3 0.7 1.2 1.3 1.8 0.2 2.1 0.9 1.2 2.6 0.5 0.8 8.9 1.8 2.9

1.0 2.5 4.0 4.4 6.0 0.5 6.9 3.1 3.9 8.7 1.7 2.8 29.7 5.9 9.5

temperature (25-75 °C) as well as the temperature in the GC injector (250-280 °C) and desorption time (1-12 min) were considered for optimization purposes using factorial19 and Doehlert20 designs as in the SFE case. SPME extraction profiles are depicted in Figure 2, indicating that equilibrium may be attained for most compounds in 60 min. Also in that case, results of experimental designs indicated that the significant factors affect the considered compounds differently requiring compromise conditions. To find a compromise set of operational conditions in so complex an experimental system, we have resorted to multicriteria decision-making strategies using desirability function optimization.21 In this approach, each response may be associated with its (21) Lewis, G. A.; Mathieu, D.; Phan-Tan-Luu, R. Pharmaceutical Experimental Design; Marcel Dekker: New York, 1999; p 265.

own nonlinear partial desirability function. The experimenter may penalize response values near the thresholds, below which the results are not acceptable and, correspondingly, favor those near targets, above which the experimenter is totally satisfied. The individual desirability functions are combined together, usually as the geometric mean, to obtain the overall desirability function for the system whose maximum value can be looked for within domain. In our case, desirability for responses requiring left unilateral functions22 was considered providing the operational conditions described under the Experimental Section. It should be noted that this strategy operates on the results produced by the factorial or Doehlert designs, without requiring additional experiments. As an example of these results, Figure 3 show a two-dimensional plot of the isodesirability curves corresponding (22) Derringer, G.; Suich, R. J. Qual. Technol. 1980, 12, 214-219.


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Table 3. Validation of the SFE-SPME-GC-MS/MS Method Using Reference Material IAEA 406 and Analysis of Real Samples of Aquaculture Fish Feeds and Cultured Species validation of the method

compound PCB-10 R-BHC γ-BHC PCB-28 heptachlor PCB-52 PBB-15 PCB-153 PBB-49 4.4′-DDT PCB-135 PCB-180 PBDE-47 PBDE-100 PBDE-99 a

IAEA 406 ref value (ng/g)

confidence interval (ng/g)

IAEA 406 obtained value (ng/g)

0.79 0.27 0.57 0.32 1.3

0.23-1.7 0.11-0.80 0.43-1.3 0.23-0.46 1.0-2.2

0.26 ( 0.02 0.69 ( 0.11 0.60 ( 0.06 0.36 ( 0.04 1.12 ( 0.09

3.7

2.9-6.0

3.47 ( 0.26

3.0 4.0 1.2

1.8-5.6 2.5-6.3 1.0-1.2

3.48 ( 0.25 3.69 (0.15 1.14 ( 0.13

concentrations (ng/g) of polyhalogentad compoundsd in real samples (n ) 3) large small trout trout turbot feed feed feed turbot cockle clam mussel nda nd nd 0.2 nd 0.3 nd 5.7 nd 0.3 3.5 1.7 nd nd nd

nd nd nd 1.1 nd 0.8 nd 2.8 nd 1.0 1.5 0.9 1.2 nd nd

nd nd nd 0.03 nd 0.2 nd 1.4 nd 0.8 1.2 0.4 0.5 nd nd

nd nd nd 0.5 nd 0.6 0.04 1.5 nd 0.4 0.9 0.2 1.1 nd nd

0.2 nd nd nd nd nd 0.2 4.1 nd 4.9 6.9 3.4 nd nd nd

0.4 nd 3.5 4.7 nd 16.3 nd 13.4 nd 4.5 2.2 2.1 1.7 nd nd

0.3 nd 15.9 5.1 nd 13.2 nd 33.2 nd 8.8 9.4 1.2 1.0 nd nd

nd, nondetected.

Figure 4. SFE-SPME-GC-MS/MS chromatogram for a clam sample including MS/MS spectra of target compounds.

to the study of SFE main critical factors. Regions in gray correspond to null desirability and, thus, to factor settings not suitable at all. Optimum conditions were obtained by numerical optimization and in the case of Figure 3 correspond to the optimal compromise values adopted in the SFE stage of the SFE/SPME combination. Optimization of the GC/MS/MS Method. The mass spectrometer was operated in electron ionization mode at 70 eV. The mass range scanned from 90 to 650 m/z at 0.80 s/scan for full2264


scan mode. The trap, manifold, and transfer line temperatures were maintained at 250, 50, and 280 °C, respectively. General parameters were as follows: multiplier offset +100, emission current 90 µA, A GC target value 2000 counts. For MS/MS, all compounds were analyzed using a resonant waveform type. Specific MS/MS conditions for each analyte are listed in Table 1. Quantitation was accomplished by relative areas versus 13C PCBs used as internal standards, which were added to samples just before the supercritical fluid extraction.

Performance of the SFE-SPME-GC-MS/MS Procedure. The whole analytical procedure using SFE/SPME combined with GC/MS/MS was tested for linearity in the range 0.2-40 ng/g, recovery, repeatability, and detection and quantification limits using fortified feed samples. Results are shown in Table 2. Quantification was accomplished by the standard addition method with internal standard (13C-labeled PCBs mixture). The recoveries for fish feed fortified sample were between 65 and 102% (n ) 6). Repeatability of the SFE-SPME-GC-MS/MS method was studied using a spiked concentration of 200 pg/g. Limits of detection were below 9 pg/g (from 0.3 to 8.9 pg/g) and limits of quantification were below 9.5 pg/g except for PBDE-47 (29.7 pg/g). The accuracy of the proposed method was tested by the analysis of a multispecies biological reference material (IAEA 406) (n ) 6). The obtained concentrations are in good agreement with reference values. and all of them are inside the confidence interval for IAEA 406 material, as can be seen in Table 3. Relatively large variability in results can be attributed to inhomogeneity of this material, which is only guaranteed for homogeneity using sample intakes greater than 5 g, while the maximum allowable amount of sample in our SFE procedure using standard 7-mL thimbles is 2 g. The optimized procedure was also applied to the analysis of several real samples of different origin, including commercial aquaculture fish feeds and cultured marine species as turbot muscle, cockle, clam, and mussel. Table 3 shows the results obtained for n ) 3, using 1.0 g of sample intake. Fish feed samples analyzed contained appreciable amounts of 4,4’-DDT, PBDE-47, and some of the PCB congeners studied. On the other hand, PBB-15 was detected at low nanogram per gram levels in cultured turbot and cockle samples. The highest concentrations corresponded to γ-BHC, PCB-52, and PCB-153 found in clam and mussel samples. In addition, 4,4’-DDT, PCB138, PCB-153, and PCB-180 were found in all samples evaluated. As an example, Figure 4 shows the selected extracted ion GC/MS/ MS chromatograms obtained after the application of the developed procedures to the analysis of a clam sample. As can be observed, the spectrum of each peak obtained by MS/MS confirms the identity of the polyhalogenated compounds detected in the sample. These findings are in concordance with other published data showing that higher chlorinated PCB congeners are the most frequently detected pollutants in biological samples.23,24 The

detection of several of the investigated compounds in real samples confirms the need for monitoring these pollutants in aquaculture samples.

(23) Merone, M. L.; Aizpuń de Moreno, J. E.; Moreno, V. J.; Lafranchi, A. L.; Metcalfe, C. D. Arch. Environ. Contam. Toxicol. 2000, 38, 202-208. (24) Andersen, G.; Kovacs, K. M.; Lydersen, C.; Skaare, J. U.; Gjertz, I.; Jenssen, B. M. Sci. Total Environ. 2001, 264, 267-281

Received for review July 9, 2004. Accepted January 18, 2005.

CONCLUSIONS A simple two-step (supercritical fluid extraction with built-in cleanup, followed by concentration using solid-phase microextraction) sample preparation method for the determination of 15 polyhalogenated compounds including pesticides, PCBs, PBBs, and PBDEs in aquaculture fish feed samples and cultured marine species by GC/MS/MS has been developed. Quantification limits in the low picogram per gram level were achieved for all compounds. Conversely to conventional solvent extraction strategies, which require the concentration of large volumes of sample, simultaneous SFE cleanup reduces both solvent usage and solvent waste generation. When combined with SPME of dry extracts and analysis by GC/MS/MS, a highly sensitive and selective analytical procedure is developed for multiresidue screening of polyhalogenated pollutants in complex biological matrixes including aquaculture feed and farmed fish and molluscs. Additional advantages derived from rapidity, easy removal of fat, and extraction solvents, thus providing enhanced protection to the GC injector, are also characteristic of the proposed procedure. Although the method has been tested only on fish-related samples, it may be potentially applicable to other kind of food matrixes with high fat content. Our data in real samples indicate the importance of analytical control methods for the aquaculture industry and the need of further studies of contaminant sources in cultured fish and shellfish and particularly in feeds used for farmed carnivorous species. General prospects for the future development of the aquaculture industry show on one hand a promising further growth and on the other significant market changes and increasing pressures on the industry in relation to food safety, the protection of the environment, and the management of coastal zones and aquatic resources. ACKNOWLEDGMENT The Spanish MCYT-DGI (Research Project BQU2002-01944) and the Xunta of Galicia (PGIDIT03PXIC23701PN) are thanked for financial support. R.R. acknowledges her doctoral grant from the Xunta de Galicia.

AC048994P


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Selective Extraction of Trace Levels of Polychlorinated and

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