Speciation of arsenic in fish tissue using microwave-assisted extraction followed by HPLC-ICP-MS† Kathryn L. Ackley, Clayton B’Hymer, Karen L. Sutton and Joseph A. Caruso* Department of Chemistry, University of Cincinnati, P.O. Box 0172, Cincinnati, OH 45221–0172, USA Received 24th September 1998, Accepted 22nd February 1999 The use of microwave-assisted extraction for the extraction of arsenic species from fish tissue is described. Quantitative extraction of arsenic from spiny dogfish muscle (CRM, DORM-2) was achieved using methanol–water (80+20, v/v) with microwave heating at 65 °C in a closed-vessel microwave system. Extractions were performed with a variety of solvents including water, two diVerent methanol–water mixtures, and a 5% tetramethylammonium hydroxide solution. Extracted arsenic species were separated using both ion-exchange and ion-pair chromatography with ICP-MS detection.The DORM-2 along with three diVerent varieties of fish purchased from a local market were analyzed for arsenic. In all samples, the majority of arsenic present was in the form of arsenobetaine, a nontoxic arsenic species.arsenic species of interest in seafood samples. Inductively Introduction coupled plasma mass spectrometry (ICP-MS) has been used The determination of total arsenic is not suYcient to assess extensively as a detector for arsenic speciation.12,15–19,21 the risks associated with consumption of arsenic-containing ICP-MS oVers high sensitivity but is hindered in the detection foodstuVs since the toxicity of arsenic is highly dependent on of arsenic by a polyatomic interference caused by ArCl+.its chemical form with inorganic arsenic (arsenite and arsenate) However, this interference may be minimized by using being more toxic than monomethylarsonic acid (MMA) and HPLC.16,24 Chromatography may separate the chloride ions dimethylarsinic acid (DMA). Arsenobetaine and arsenocho- from the analytes of interest.Therefore, the chloride ions line are relatively non-toxic. As a result, much attention has would elute in a single peak instead of being present continubeen given to the elemental speciation of arsenic in environ- ously in the background and leading to ArCl+ in the plasma. mental and biological samples, and the subject has been No matter how eVective the separation technique is or how reviewed by Burguera and Burguera.1 sensitive the detector, the quality of the analysis is limited by Arsenobetaine has been identified to be the major arsenical the sample preparation step.Extraction of the analytes from in a variety of seafood products including several species of the tissue sample is usually achieved using a methanol– clams2 and many species of fish.3–6 Arsenobetaine is thought water–chloroform system.5,25,26 Although these solvent extracto be the final metabolite of arsenic in marine food chains,7 tion systems have been shown to extract arsenic from fish although arsenobetaine is not present in all species of fish,8 tissue quantitatively, the procedure is time consuming.In and the transformations that arsenic undergoes in the marine addition, extraction of the analytes from certain types of food chain are still being studied.7–10 samples such as mussels25 may be less eYcient. To perform arsenic speciation analyses, extraction methods Extraction of arsenic using a conventional solvent extraction must be capable of quantitatively extracting arsenic from the method is time consuming.Microwave-assisted extraction sample while not altering the individual arsenic species in any (MAE) is another alternative to conventional solvent extraction. way. Typically, arsenic species are extracted from seafood In MAE, microwave energy is used to heat solvents that are in samples using a standard solvent extraction method. Methanol, contact with solid samples so that analytes of interest will water and sometimes chloroform are used as the extracting partition from the sample into the solvent.27 Open focused solvents, and the procedure is performed at ambient tempera- microwave systems have been utilized for the dissolution, tures and pressures.Branch et al.3 detailed a typical method extraction and derivatization of organotin compounds in biomafor the extraction of arsenic from fish tissue. The sample and terials28–30 and sediments30,31 prior to speciation. Open focused extracting solvents were sonicated for 1 h.The solution was microwave-assisted sample preparation procedures have also centrifuged, the supernatant collected and the process repeated. been investigated for the application to speciation of mercury The methanol–water layer was separated from the chloroform in environmental samples.30 All these papers report that analytes layer. The solvent was removed by rotary evaporation, and were eVectively extracted, while the species information the sample dissolved in water.remained intact. Also, the overall analysis time was reduced The approaches to arsenic speciation in seafood have been dramatically with the use of the microwave systems. varied. Separation of the analytes is usually achieved through Microwave digestion has been used to prepare seafood32 high-performance liquid chromatography (HPLC). Ion-pair and certified marine samples33,34 for the determination of total chromatography11–15 as well as ion-exchange chromatogra- arsenic.However, in both cases, the digestion conditions were phy15–23 have successfully been employed for separating the harsh and no attempt was made to retain the integrity of each individual species. Two reports of the use of MAE for the preparation of †Presented at the Ninth Biennial National Atomic Spectroscopy Symposium (BNASS), Bath, UK, July 8–10, 1998. samples for arsenic speciation were found. Demesmay and J. Anal. At. Spectrom., 1999, 14, 845–850 845Olle�35 utilized microwave digestion to prepare sediment obtained from the United States Environmental Protection Agency (US EPA), Cincinnati, OH, USA.The compounds samples for the speciation of arsenic. The sediment samples were exposed to microwave power in the presence of a were used without further purification. MMA (ChemServices, West Chester, PA, USA), DMA (as cacodylic acid, Sigma, St. hydrochloric and nitric acid mixture. Of the four species investigated, MMA, DMA and As(V) were all stable during Louis, MO, USA), sodium arsenite and sodium arsenate (Matheson, Coleman and Bell, Norwood, OH, USA) were the microwave digestion.Arsenite [As(III)] was quantitatively oxidized to As(V). Larsen et al.36 utilized microwave heating also used without further purification. MMA and DMA were in the form of free acids. for the extraction of arsenic species from dried mushroom samples. Methanol–water (1+9, v/v) was used as the Samples extracting solvent.The purpose of the work reported here was to develop a The certified reference material was DORM-2 (National microwave extraction procedure capable of quantitatively Research Council of Canada), which has a certified arsenic extracting arsenic from seafood samples using extraction conconcentration of 18.0±1.1 mg kg-1. The market fish samples ditions that are mild enough to prevent a conversion of the were purchased as fresh filets from a local grocery store.original arsenic species. A variety of extracting solvents were Where required, the outer scales and skin of the fish were investigated. The eVect of varying the exposure time and removed with plastic utensils. The samples were then lyophil- temperature was also investigated. ized and homogenized. All samples were analyzed as freeze-dried powders. Experimental Extraction procedure Equipment Samples of DORM-2, ranging in mass from 0.10 to 0.13 g, The ICP-MS instrument was a VG PQ2 STE (VG Elemental, were accurately weighed into the reaction vessels.The Franklin, MA, USA) equipped with a concentric nebulizer extracting solvent (10 mL) was added to each sample. The and a double pass spray chamber, water-cooled to 5 °C. The microwave system was programmed to heat the sample to a rf power was 1350 W, and the nebulizer gas flow was optimized specified temperare, and the temperature was then main- prior to analysis with a 10 mg L-1 In solution. tained until 2 min had elapsed.Thus, if a sample was to be An MES 1000 (CEM, Matthews, NC, USA) microwave heated for 6 min, the 2 min sequence was initiated three times. extraction system was used. This is a closed-vessel system After microwave heating, the samples were allowed to cool capable of heating 12 samples at one time. The temperature and were then transferred into centrifuge tubes. The samples was monitored in a control vessel by an armored fiber-optic were centrifuged for 5 min at 3500 rpm.The supernatants were temperature control probe, and the pressure was monitored transferred into sample vials using Pasteur pipets (Fisher by an internal pressure control system. Samples were heated Scientific). Before injection, the samples were filtered with a to a set temperature by the microwave system, and microwave 0.2 mm Nylon disposable syringe filter (Fisher Scientific). energy was applied to the sample when its temperature fell below the set temperature.The reaction vessels were Advanced Flow injection analysis (FIA) Composite Vessels supplied by CEM. The liner and liner covers of these vessels were Teflon PFAA and have a maximum For flow injection work, the mobile phase consisted of 2% v/v operating temperature and pressure of 200 °C and 200 psi, nitric acid solution prepared with ACS plus nitric acid (Fisher respectively. Vessel liners were cleaned first using soap and Scientific) and filtered prior to use. The mobile phase was water followed by soaking for 1 h in a 50% nitric acid solution.pumped at a rate of 1 mL min-1. All standards were prepared The liners were rinsed with distilled, de-ionized water, then in the same extracting solvent as the samples to be analyzed. 2% nitric acid, and finally, more distilled, de-ionized water. Data were collected using time-resolved software, so both m/z An ISCO Model 2350 HPLC pump and an ISCO Model 2360 75 (75As and 40Ar35Cl ) and m/z 77 (77Se, 40Ar37Cl ) could be gradient programmer (ISCO, Lincoln, NE, USA) were used monitored.Both masses were monitored to determine the to deliver the mobile phase for both the flow injection and extent of ArCl+ formation. All calibration graphs and subchromatographic work. Samples were injected for flow injec- sequent sample concentrations were determined using peak tion and chromatography using a Rheodyne Model 4396 areas. The arsenic concentrations were determined from three injector (Rheodyne, Cotati, CA, USA) equipped with a 20 mL replicate samples, each analyzed in triplicate. sample loop.A 47 cm length of PEEK tubing (0.020 id, 0.062 od) was used to connect the injector to the nebulizer Chromatography during flow injection experiments. The same tubing was used Anion-exchange separations were performed using a Hamilton to connect the chromatographic column to the nebulizer during PRP X100 column (250×4.1 mm id) with a 10 mm particle HPLC-ICP-MS experiments. size (Phenomenex, Torrance, CA, USA).The mobile phase consisted of a 30 mmol ammonium carbonate (J.T. Baker, Reagents and standards Phillipsburg, NJ, USA) buVer. The pH of the solution was The water used was distilled and de-ionized to a resistance of adjusted with ammonia solution (Fisher Scientific) until a pH 18 MV cm. Arsenic standards for the total arsenic determi- of 9 was reached. The mobile phase flow rate was 1 mL min-1. nations were prepared using a 1000 mg mL-1 As stock solution The ion-pair separation was performed with a C18 column (Spex CertiPrep, Metuchen, NJ, USA).The diVerent extrac- (15 cm×2.0 mm id) with a 5 mm particle size (Phenomenex). tion solvents were prepared with distilled, de-ionized water, The mobile phase was an aqueous 25 mM citric acid solution HPLC-grade methanol (Fisher Scientific, Fair Lawn, NJ, (Fisher Scientific) that was also 10 mM in 1-pentanesulfonic USA), and 25% tetramethylammonium hydroxide solution acid sodium salt (Eastman Kodak, Rochester, NY, USA). (Alfa Aesar, Ward Hill, MA, USA).Solutions of 1000 mg L-1 The mobile phase flow rate was 0.15 mL min-1. (as As) of each of the arsenic species investigated were prepared. These standards were stored at ambient temperature in Discussion the dark. The arsenobetaine and arsenocholine were synthesized by Dr. William R. Cullen (University of British Detection of arsenic with quadrupole-based ICP-MS can be problematic if chloride ions are present in the sample since Columbia, Vancouver, British Columbia, Canada) and were 846 J.Anal. At. Spectrom., 1999, 14, 845–850Table 1 Comparison of extraction solvents for MAE. Samples were At first, these results seem counter intuitive, and one would heated to 50 °C for 4 min. Arsenic concentrations are reported expect more arsenic to be extracted at the higher temperature in mg kg-1. DORM-2 has a certified As value of 18.0±1.1 mg kg-1 of 80 °C.One possible explanation for this phenomenon may be derived from the boiling-point of the extraction solvent. At Determined As 50 °C, the extraction solvent may not be suYciently heated to Extraction solvent concentration/mg kg-1a RSD (%) produce convection currents that agitate the tissue sample and Distilled, de-ionized water 13.4±0.4 2.9 aid in the liberation of the arsenic species. Most conventional 5% Tetramethylammonium 17.1±0.9 6.6 solvent extraction techniques involve sonication or some form hydroxide solution of mechanical agitation.The samples prepared using MAE do Methanol–water (50+50) 15.5±0.8 6.4 not receive the same type of agitation. Samples were placed in Methanol–water (80+20) 13.4±1.2b 10.6 a carousel that slowly revolved to promote even heating. aConfidence limits were determined at a 95% probability interval. However, the speed of rotation was not high and would not be bAverage value based on eight measurements. The ninth value was expected to agitate the sample within the microwave vessel.At rejected by Q-test with >95% confidence. 65 °C, the sample is close enough to the boiling-point so that suYcient agitation occurs to extract the arsenic species from the tissue quantitatively. At the higher temperature of 80 °C, ArCl+ has the same mass-to-charge ratio as As+ (m/z 75). To the solution boils vigorously when observed in an open vessel. ascertain if the presence of ArCl+ would interfere with the Hence, one would expect a greater portion of the extracting detection of arsenic, mass 77 was also monitored since approxisolvent to reside in the gas phase at this elevated temperature, mately one quarter of all the ArCl+ would be expected to leaving less liquid to eVect the extraction.This may explain have a mass-to-charge ratio of 77. No appreciable signal was why the amount of arsenic extracted is lower at 80 °C than obtained at m/z 77. Therefore, all the counts obtained at m/z at 65 °C. 75 were attributed to arsenic ions. Fig. 1 shows the results from an experiment to compare the A comparison of extraction solvents was performed, and amount of arsenic extracted from samples exposed to micro- these results are reported in Table 1. All samples were heated waves and from those not exposed to microwaves. The samples to 50 °C and exposed to microwave power for 4 min. At this were prepared in the same manner as described previously. In temperature and exposure time, the highest recovery was both cases, samples were placed in the microwave carousel for obtained with an extraction solvent of 5% tetramethylamthe same period of time.However, only one set of samples monium hydroxide (TMAH). A TMAH solution was initially was exposed to microwave power. While a large portion of investigated because other investigators have used TMAH to the arsenic is recovered from the tissue using methanol–water solubilize tissue in an open focused microwave system.29 The (80+20, v/v) without microwave exposure, microwave heating high recovery is not unexpected since the TMAH solution ensured that all the arsenic was extracted from the sample.digests and solubilizes the tissue. Only a very small amount of Slightly more arsenic is extracted without microwave heating tissue residue was visible after the sample had been centrifuged. using the methanol–water solution than with microwave heat- This extraction solvent was not investigated further since other ing at 50 °C, illustrating that no real advantage is seen with neutralized TMAH solutions were found to shift peak retention MAE until the sample is heated to a temperature at the times when injected onto the C18 column.In addition, the boiling-point of the extracting solvent. Similarly, no real retention times in subsequent analyses were also shifted even diVerence was seen between microwave-heated and unheated if the sample was prepared in a diVerent solvent.samples when water was used as the extracting solvent at 65 °C. A univariate approach was used to optimize the temperature Pure water extracted the same amount of arsenic as methand exposure times. The first extraction solvent investigated anol–water (80+20, v/v) with greater precision at 50 °C. Since was methanol–water (80+20, v/v) since methanol–water solu- the analysis of arsenicals in distilled, de-ionized water would tions are commonly used to extract arsenicals from tissue at be ideal for introduction into chromatographic systems, an ambient temperature and pressure.Table 2 shows the arsenic investigation of the amount of arsenic extracted by water at recovered from DORM-2. various temperatures and exposure times was performed. The The largest variation in the amount of arsenic extracted is data are presented in Table 3. observed when samples are heated to 65 °C. About 80% of the The trends that were prominent in the data obtained using arsenic is extracted from the samples heated to 50 °C.The methanol–water as the extraction solvent were not visible in samples heated to 80 °C show slightly better recoveries. the data reported in Table 3. Average values increased slightly However, samples heated to 65 °C eVectively extract 100% of with increasing exposure time at 50 and 65 °C but decreased the arsenic in DORM-2. The pressure inside the reaction vessel slightly at 80 °C. Moreover, the average amount of arsenic fluctuates slightly at 65 °C but typically is below 10 psi.At extracted increased slightly with increasing temperature for 80 °C, the pressure remains above 10 psi and may fluctuate as high as 20 psi when microwave power is being applied. Table 2 Arsenic recovered from DORM-2 using MAE with methanol –water (80+20, v/v) as the extraction solvent. Arsenic concentrations are reported in mg kg-1. DORM-2 has a certified As value of 18.0±1.1 mg kg-1 Determined As concentration (mg kg-1)a at: Exposure time 50 °C 65° C 80°C 2 min 13.9±0.6 20.5±0.5 17.6±0.8 4 min 13.4±1.2b 19.4±0.5 14.2±0.8 6 min 11.9±0.4 18.7±0.4 13.8±0.6 aConfidence limits were determined at a 95% probability interval.bAverage value based on eight measurements. The ninth value was Fig. 1 Comparison between the amount of arsenic extracted from rejected by Q-test with >95% confidence. DORM-2 with and without microwave heating. J. Anal. At. Spectrom., 1999, 14, 845–850 847Table 3 Arsenic recovered from DORM-2 using MAE with distilled, de-ionized water as the extraction solvent.Arsenic concentrations are reported in mg kg-1. DORM-2 has a certified As value of 18.0±1.1 mg kg-1 Determined As concentration (mg kg-1)a at: Exposure time 50 °C 65° C 80° C 2 min 12.7±0.3 13.6±0.3 15.1±0.2 4 min 13.4±0.4 13.8±0.2 14.5±0.7 6 min 13.8±0.2 14.7±0.2 14.4±0.3 aConfidence limits were determined at a 95% probability interval. Fig. 2 Chromatogram of arsenic species separated on the Hamilton PRP-X100 anion-exchange column. Each species is present at a concentration of 17 ng mL-1 As, and the sample was prepared in most samples.This trend was more prominent for samples methanol–water (80+20, v/v). Peak identification: (1) arsenocholine, (2) arsenobetaine, (3) arsenite, (4) dimethylarsinic acid, (5) monome- heated for only 2 min. It is possible that 2 min is not a thylarsonic acid, (6) arsenate. suYcient length of time for the arsenic species to be totally extracted from the fish tissue. As a result, samples heated for only 2 min show more dramatic increases in the amount of arsenic extracted with increasing temperature.heated to 100 °C as well as the increased variation between the replicate samples. Results from other workers3,15,34 (ref. 3 and 15 refer to DORM-1 while ref. 34 refers to DORM-2) as well as those After total arsenic concentration information had been obtained with FIA-ICP-MS, speciation information was gath- presented here (working with DORM-2) show that the arsenic in DORMexists almost exclusively in the form of arsenobetaine.ered using HPLC-ICP-MS. Two diVerent chromatographic separations were used to identify the arsenic species. Anion- Hence, the MAE method with water, which extracts 70–80% of the arsenic in the certified reference material, is capable of exchange chromatography was utilized for the identification of the most toxic forms of arsenic (arsenite, arsenate, MMA, extracting arsenobetaine but not extracting it quantitatively.This phenomenon suggests that an equilibrium process of some and DMA). Arsenobetaine and arsenocholine co-elute in the void volume. A sample chromatogram is shown in Fig. 2. The sort may be taking place, in which case the use of a larger volume of extracting solvent may be advisable if the concen- use of a gradient between buVers of low and high ionic strength was investigated for the separation of these species. tration of arsenic in the sample is large enough so that excessive dilution will not hinder detection.The other possible explanation While using gradient elution provides better resolution between the first three peaks, gradient elution alters the plasma charac- is that the arsenobetaine may be incorporated within the sample matrix in more than one way. However, no reliable studies of teristics and necessitates re-equilibration between runs.Hence an isocratic separation, which provides suYcient resolution arsenobetaine binding in fish have been performed. The amount of arsenic recovered with water under the for the identification of each anionic species, was employed. A mobile phase with a pH of 9 was selected since, at this pH, conditions investigated is not as large as that obtained with a mixture of methanol and water. One possible explanation is the anionic species of interest have diVerent apparent charges and are more easily separated on the anion-exchange column that, since water has a higher boiling-point, the temperatures investigated did not produce adequate convection currents used in this work.37 To identify the species eluting in the void volume, samples were also separated using ion-pair chromatog- within the sample to provide suYcient agitation to aid in extraction.raphy. While reversed-phase techniques such as ion-pair chromatography tend to be more sensitive to matrix eVects than An investigation was made to see if more arsenicals could be extracted with water at higher temperatures.Samples of anion-exchange separations, preparing standards in the same extracting solvent as the samples to be analyzed allows the DORM-2 were prepared using water as the extracting solvent. The samples were heated for 1 min to 65 °C and then for 4 min identification of organoarsenicals by their retention times. Any sample preparation method to be used in conjunction to 100 °C.The amount of arsenic extracted in the three replicate samples was determined to be 16.4±1.8 mg kg-1. with speciation must not alter the individual species within the sample. To determine if this criterion was met, standard Although the average arsenic concentration for the three replicates is lower than the certified value, they cannot be solutions of each species were prepared in methanol–water (80+20, v/v). The test standards were placed in the microwave distinguished at a 95% confidence level. This result seems to support the idea that extractions performed near the boiling- system and exposed to microwave power for 4 min at 65 °C.The control standards were not exposed to microwave power, point of the extracting solvent extract arsenicals from the fish tissue more completely. and the solutions were analyzed using the anion-exchange chromatographic method with ICP-MS detection. The chrom- The variation in the amount of arsenic extracted between the three samples prepared in water at 100 °C is larger than atograms indicate that no sample degradation occurred, and the integrity of the species remained intact. While this result the variation obtained for the other samples prepared with water at lower temperatures.The appearance of these samples was expected for the organoarsenic species, the fate of the inorganic forms of arsenic was unknown since Demesmay and is also diVerent from that of samples heated at lower temperatures.Samples prepared at lower temperatures essentially Olle�35 reported the conversion of arsenite to arsenate in certain extraction/digestion media. However, the extraction conditions appeared the same as they did prior to microwave heating. Samples heated to 100 °C appeared much darker and had a in our work were not severe enough for oxidation to occur. Once the method had been shown to maintain species integ- much stronger odor. In addition, large portions of the sample were found to be adhered to the upper inside walls of the rity, the fish tissue extracts were analyzed using HPLC-ICP-MS.Anion-exchange chromatography was the first separation tech- TeflonA microwave vessel liners. The temperature may be high enough at 100 °C to go beyond heating the sample and may nique utilized, and the resulting chromatograms are shown in Fig. 3(a)–(d). All the fish extract chromatograms exhibited very actually begin to ‘cook’ the sample.This phenomenon would account for the diVerent odor and appearance of the samples large peaks where arsenobetaine and arsenocholine co-elute. 848 J. Anal. At. Spectrom., 1999, 14, 845–850Fig. 4 (a) Ion-pair chromatogram of arsenic species in water. Peak identification: (1–3) arsenite, arsenate, and MMA, (4) DMA, (5) arsenobetaine, (6) arsenocholine. (b) Ion-pair chromatogram of arsenic species in 80% methanol. Peaks elute in the same order as in (a).Fig. 4(a). The retention times are shifted when the species are introduced in methanol–water (80+20, v/v) as shown in Fig. 4(b). However, since arsenobetaine was expected to be the primary arsenic species in each fish sample, the separation was adequate for species confirmation. The ion-pair chromatograms for the fish samples are shown in Fig. 5(a)–(d). These chromatograms confirm that arsenobetaine accounts for most of the arsenic in DORM-2 while it accounts for all of the arsenic in the ocean whitefish, black tip shark and steelhead salmon samples.A small peak matching the retention time of DMA was observed in the anion-exchange chromatogram for DORM-2. The chromatogram is consistent with work previously reported in the literature. Goessler and co-workers34 (working with DORM-2) reported an arsenobetaine concentration of 16.0±0.7 mg kg-1, a DMA concentration of 0.28±0.01 mg kg-1, and inorganic arsenic, MMA and arsenocholine concentrations were all reported to be lower than 0.03 mg kg-1. Peaks for inorganic arsenic, MMA and arsenocholine were not observed in this work, which may be a result of diVerences in sensitivity between this method and other methods reported in the literature.An attempt was made to quantify the arsenobetaine using Fig. 3 (a) Anion-exchange chromatogram of DORM-2 extract in 80% HPLC-ICP-MS. The results are presented in Table 4. However, methanol. (b) Anion-exchange chromatogram of ocean whitefish these values should not be taken as an absolute quantification extract in 80% methanol.(c) Anion-exchange chromatogram of black since the arsenobetaine standard solutions used to generate tip shark extract in 80% methanol. (d) Anion-exchange chromatogram of steelhead salmon extract in 80% methanol. the calibration graph were prepared from arsenobetaine of unknown purity. Less than 1 g of arsenobetaine was available for this analysis, so further purification and standardization was not feasible. Solutions or arsenobetaine and arsenocholine Inorganic forms of arsenic were not detected while the DORM-2 chromatogram indicated a small peak where DMA elutes.were analyzed by HPLC-ICP-MS, and only one peak was observed for each standard. The presence of other substances, Any uncharged or positively charged organoarsenical would be expected to be unretained on the anion-exchange column, such as waters of hydration, was not known, making the preparation of a standard solution for quantification diYcult.so to confirm the presence of arsenobetaine, an alternative chromatographic technique was utilized. When the arsenic As the demand for accurate arsenic speciation information in food and environmental samples grows, so does the need for species are injected onto the C18 column as an aqueous solution, adequate resolution is obtained between DMA, commercially available standards of the major arsenic species of interest. arsenobetaine, and arsenocholine.The separation is shown in J. Anal. At. Spectrom., 1999, 14, 845–850 849Acknowledgements The authors gratefully acknowledge the US EPA for providing arsenic samples, lyophilizing fish samples and for funding through grant number CX826144–01–0. We also thank the CEM Corporation for the use of the MES 1000 unit. Although this work has been funded with EPA funds, it has not been subjected to the Agency’s peer and policy review and does not reflect the views of the EPA.References 1 M. Burguera and J. L. Burguera, Talanta, 1997, 44, 1581. 2 W. R. Cullen and M. Dodd, Appl. Organomet. Chem., 1989, 3, 79. 3 S. Branch, L. Ebdon and P. O’Neill, J. Anal. At. Spectrom., 1994, 9, 33. 4 E. H. Larsen, G. Pritzl and S. H. Hansen, J. Anal. At. Spectrom., 1993, 8, 1075. 5 D. Beauchemin, M. E. Bednas, S. S. Berman, J. W. McLaren, K. W. M. Siu and R. E. Sturgeon, Anal. Chem., 1988, 60, 2209. 6 E. H. Larsen, G. A. Pedersen and J.W. McLaren, J. Anal. At. Spectrom., 1997, 12, 963. 7 W. Goessler, W. Maher, K. J. Irgolic, D. Kuehnelt, C. Schlagenhaufen and T. Kaise, Fresenius’ J. Anal. Chem., 1997, 359, 434. 8 J. S. Edmonds, Y. Shibata, K. A. Francesconi, R. J. Rippingale and M. Morita, Appl. Organomet. Chem., 1997, 11, 281. 9 K. Hanaoka, T. Motoya, S. Tagawa and T. Kaise, Appl. Organomet. Chem., 1991, 5, 427. 10 W. R. Cullen, L. G. Harrison, H. Li and G. Hewitt, Appl. Organomet. Chem., 1994, 8, 313. 11 X.C. Le, M.Ma and N. A. Wong, Anal. Chem., 1996, 68, 4501. 12 X. C. Le and M. Ma, J. Chromatogr. A, 1997, 764, 55. 13 P. Boucher, M. Accominotti and J. J. Vallon, J. Chromatogr. Sci., 1996, 34, 226. 14 X.-C. Le, W. R. Cullen and K. J. Reimer, Talanta, 1994, 41, 495. 15 D. Beauchemin, K. W. M. Siu, J. W. McLaren and S. S. Berman, J. Anal. At. Spectrom., 1989, 4, 285. 16 C. Demesmay, M. Olle and M. Porthault, Fresenius’ J. Anal. Chem., 1994, 348, 205. 17 S. Saverwyns, X. Zhang, F.Vanhaecke, R. Cornelis, L. Moens and R. Dams, J. Anal. At. Spectrom., 1997, 12, 1047. 18 E. H. Larsen, Fresenius’ J. Anal. Chem., 1995, 352, 582. 19 P. Terasahde, M. Pantsar-Kallio and P. K. G. Manninen, J. Chromatogr. A, 1996, 750, 83. Fig. 5 (a) Ion-pair chromatogram of DORM-2 extract in 80% 20 K. J. Lamble and S. J. Hill, Anal. Chim. Acta, 1996, 334, 261. methanol. (b) Ion-pair chromatogram of ocean whitefish extract in 21 S. Branch, K. C. C. Bancroft, L. Ebdon and P. O’Neill, Anal. 80% methanol. (c) Ion-pair chromatogram of black tip shark extract Proc., 1989, 26, 73. in 80% methanol. (d) Ion-pair chromatogram of steelhead salmon 22 Z. Mester and P. Fodor, J. Anal. At. Spectrom., 1997, 12, 363. extract in 80% methanol. 23 J. Mattusch and R.Wennrich, Anal. Chem., 1998, 70, 3649. 24 B. S. Sheppard, W.-L. Shen, J. A. Caruso, D. T. Heitkemper and F. L. Fricke, J. Anal. At. Spectrom., 1990, 5, 431. 25 M. B. Amran, F. Lagarde and M. J. F. Leroy,ikrochim.Acta, Table 4 Quantification of arsenic species in fish samples. Arsenic 1997, 127, 195. concentrations are reported in mg kg-1.a Extractions were performed 26 M. Ochsenkuhn-Petropulu, J. Varsamis and G. Parissakis, Anal. at 65 °C for 4 min using methanol–water (80+20, v/v)b Chim. Acta, 1997, 337, 323. 27 H. M. Kingston and S. J. Haswell, Microwave-Enhanced Sample Arsenobetaine Total arsenic determined by FIA Chemistry, American Chemical Society, Washington, DC, 1997, p. 772. DORM-2 24.6±1.4 19.4±0.5 28 I. R. Pereiro, V. O. Schmitt, J. Szpunar, O. F. X. Donard and Steelhead salmon 2.9±2.0 2.6±0.1 R. Lobinski, Anal. Chem., 1996, 68, 4135. Black tip shark 19.9±1.9 14.5±0.5 29 V. O. Schmitt, J. Szpunar, O. F. X. Donard and R. Lobinski, Can. Ocean whitefish 7.9±4.5 7.2±0.1 J. Anal. Sci. Spectrosc., 1997, 42, 41. aConfidence limits were determined at a 95% probability interval. 30 V. O. Schmitt, A. d. Diego, A. Cosnier, C. M. Tseng, J. Moreau bFor chromatographic determinations, n=2–3. and O. F. X. Donard, Spectroscopy, 1996/1997, 13, 99. 31 O. F. X. Donard, B. Lale�re, F. Martin and R. Lobinski, Anal. Chem., 1995, 67, 4250. 32 B. S. Sheppard, D. T. Heitkemper and C. M. Gaston, Analyst, 1994, 119, 1683. 33 G. Damkro� ger, M. Grote and E. Janssen, Fresenius’ J. Anal. Conclusions Chem., 1997, 357, 817. Microwave-assisted extraction is an eVective method for the 34 W. Goessler, D. Kuehnelt, C. Schlagenhaufen, Z. Slejkovec, and K. J. Irgolic, J. Anal. At. Spectrom., 1998, 13, 183. extraction of arsenic species from fish tissue samples. When 35 C. Demesmay and M. Olle�, Fresenius’ J. Anal. Chem., 1997, 357, methanol–water (80+20, v/v) was used as the extracting 1116. solvent, each of the species investigated remained intact when 36 E. H. Larsen, M. Hansen and W. Go� ssler, Appl. Organomet. exposed to microwave power for 4 min at 65 °C. Under these Chem., 1998, 12, 285. conditions, 100% of the arsenic in DORM-2 was extracted. In 37 J. Gailer and K. J. Irgolic, Appl. Organomet. Chem., 1994, 8, 129. all of the fish samples investigated, the arsenic existed almost exclusively in the form of arsenobetaine. Paper 8/07466F 850 J. Anal. At. Spectrom., 1999, 14, 845&n