Food Chemistry 113 (2009) 720–726

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Analytical Methods

Sequential extraction combined with HPLC–ICP-MS for As speciation in dry seafood products
Xuan Cao a, Chunli Hao b, Geng Wang c, Huanghao Yang d, Dengyun Chen e, Xiaoru Wang d,*

College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266003, PR China Fuzhou University of China, Fuzhou 350000, PR China c Shandong University of China, Ji’nan 250000, PR China d The First Institute of Oceanographic Institute, State Oceanographic Administration, Qingdao 266061, PR China e Agilent Technologies, USA

a r t i c l e

i n f o

a b s t r a c t
A sequential extraction procedure followed by HPLC–ICP-MS analysis was developed for the speciation and quantization of arsenic species in dry seafood products (DSPs). The extraction process involved three major steps, which produced respectively three As fractions: non-polar (Asnonpolar), polar (Aspolar) and inorganic arsenic species (Asinorganic). The extraction efficiency (EE%) is in the range of 87–115%. Asnonpolar is 0.6–10.2% of total arsenic in DSPs, which is served as the index of level of arsenolipid. For Aspolar and Asinorganic, a hyphenated HPLC–ICP-MS technique was established for the separation and quantification of six arsenic species including arsenobetaine (AsB), arsenocholine (AsC), arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). The results indicate that AsB and AsV are the two dominate species in DSPs, while all other species are present in relatively low concentrations. The recovery efficiency of 77–02.8% could be obtained with spike recovery test in this two-steps extraction. Ó 2008 Elsevier Ltd. All rights reserved.

Article history: Received 19 December 2007 Received in revised form 11 June 2008 Accepted 3 August 2008

Keywords: HPLC ICP-MS Sequential extraction Arsenic Speciation Dry seafood products

1. Introduction Dry seafood products (DSPs) are very popular in China and some Asian countries. The production of DSPs in the region is huge, and their manufacturing involves a widely diverse processes including braise, baking, drying, flavoring, etc. Just as fresh seafood, DSPs are also an important source of proteins, polyunsaturated fatty acids, and a wide range of vitamins (B, D, and A) and minerals (calcium, phosphorus, iron, etc.) in this part of the world. (FAO., 1998). In fact, compared with the fresh ones, DSPs are more convenient to store and cook, and are often sold commercially in instant food formulations. Both DSPs and the fresh seafood are sometimes contaminated with trace toxic elements such as arsenic. (Sikorski & Kolakowska, 1994). For safety evaluation, the speciation of these trace elements is just as important as the determination of their total concentration levels since toxicity is critically dependent on their exact chemical forms (Andreae, 1986; Penrose, 1974). For example, the LD50 values (50% lethal dose; mg/kg) of different arsenic species vary widely as: arsine 3, arsenite 14, arsenate 20, monomethylarsonic acid 700–1800, dimethylarsinic acid 700– 2600, arsenocholine 6500 arsenolipids >8000, and arsenobetaine >10,000 (Nriagu, 1994).
* Corresponding author. Fax: +86 532 88963253. E-mail address: (X. Wang). 0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2008.08.001

Unfortunately, same as their fresh seafood counterparts, in most countries, there is yet no well-defined, species-specific, legislative control of DSPs (Buchet & Pauwels, 1994; Francesconi & Edmonds, 1994; Phillips, 1994). One of the best cited As standards for arsenic is for drinking water and its value in China is 50 mg/l. The setting of this limit is largely based on toxicity data for inorganic arsenite and arsenate. If this same limit were to applied to DSPs, most of them would probably be banned from the market since the arsenic content in seafood typically exceeds that of the drinking water by a factor of 1000 (Cullen & Reimer, 1989; National Academy of Sciences, 1997). In these cases, it is obvious that the setting of limits based solely on total arsenic concentrations is scientifically unfounded, and instead, the identification and quantification of individual arsenic species in the sample would be a more reasonable approach. In this regard, the absence of effective analytical method has been the main obstacles. The subject of arsenic speciation analysis has been extensively reported in the literatures but those related to applications in DSPs are very scarce (Gomez-Ariza & Sanchez-Rodas, 2000; Larsen & Pritzi, 1993; Larsen & Quetel, 1997). In the present work, a sequential solvent extraction procedure has been developed for the fractionation of different arsenic species. These fractions were then analyzed by ICP-MS or hyphenated HPLC–ICP-MS for total As and arsenic speciation analysis, respectively.

Reno. Milli-Q ultra pure deionized (DI) water (Millipore. and the mixed standard solutions containing all arsenic species was prepared from the above single standard solutions. The samples were then sonicated for 20 min. The three supernatants were combined and filtered through a 0. and the resulting mixtures were centrifuged at the rate of 5000 rpm for 10 min at 4 °C.1 mm. 10 lm) 1. 1999). Sequential extraction procedures 4. The samples were collected in April 2006 from some large supermarkets in Qingdao. During the process DI . USA).10 L/min 0.2 g) and 5 ml of concentrated HNO3 were added together into a PTFE bomb.5) Time (min) A (%) B (%) 0 100 0 15 0 100 30 100 0 Agilent 7500a 1350 W 1. Japan) was used to evaporate the organic solvents and water used in the extraction process. NV. After extraction. a glass double path spray chamber and a standard quartz torch. Sample and sample treatment Reference material TORT-2 (lobster hepatopancreas) was purchased from National Research Council of Canada (Ottawa. and this samples extraction procedure was repeated for three times. A rotary evaporator (EYELA. In the procedure. 4. The interference correction equations were chosen from the library for EPA200. Experimental 2.10 L/min 0. 72Ge. USA) was used to freeze dry the samples to a constant weight. Ontario.X. and arsenate oxide (AsV) from Alfa Aesar (Ward Hill. equipped with a Babington nebulizer. Eight of the more popular dried seafood products in China were selected for real samples in this work. and then frozen dry a constant weight. After 10 min the bombs were sealed off and put into an oven at 80 °C for 4 h. and monomethylarsinic acid (MMA) were prepared by Tsinghua University (Beijing. arsenocholine (AsC). 4. 0. The rare earth element 89Y was used as an internal standard. the sample powders and acetone solvent were mixed thoroughly by reciprocal shaker for 5 min. and the final concentrate was digested by the procedure as described in EPA method 200. (McKiernan & Creed. Canada). 35Cl. 2. the solutions were filtered and then placed in an oven at 65 °C to remove the solvent mixture. Step I: non-polar arsenic compounds extraction The DSPs were first extracted by acetone to extract the relatively non-polar arsenic compounds.2.2.1. USA) was used for the separation of arsenic species.1 rps Gradient program ICP-MS RF power RF matching Carrier gas flow rate Peristaltic pump flow rate Monitored signals solution. This solution was diluted to a final mass of 25 g with deionized water for ICP-MS analysis. USA). The bombs were then placed back in the oven at 170 °C for 4 h. Cao et al. MA). Step II: extraction of polar arsenic compounds The solid residue from step I was dried in an oven at 50 °C to drive away the residue acetone. The bottom residue was then re-dispersed in the extractant solvent by shaking. Arsenobetaine (AsB).6 V 1. HPLC grade ammonium carbonate from Fischer Scientific (Fairlawn. The solvent in the filtrate was removed by rotary evaporation. Standards and reagents Stock solutions of individual arsenic compounds were prepared using the following standard: arsenite triiodide (AsIII). Determination of total Arsenic Dried seafood (0.2 g of powdered samples were accurately weighed into 10 ml centrifugal tubes followed by the addition of 4 ml of acetone. The drying process proceeded slowly to a final volume of about 3–5 ml. Germany). The arsenic concentration in this fraction.3. was determined by ICP-MS. After closing the lids securely. All samples were rinsed three times with deionized water to remove the salt on the surface. 77Se and 83Kr Agilent 7500a 1350 W 1. An Agilent 1100 series high performance liquid chromatography (HPLC) system consisting of a binary pump. and a manual-injector with a 20 ll sampling loop was used in HPLC analysis.5 mL/min 25 lL H2O 50 mM (NH4)2CO3 (pH = 9. A variable speed reciprocal shaker (Apparatyus. All the stock solutions were stored in a dark room at 4 °C.3 (Wahlen & McSheehy. They were grinded into powders and then (particle size: <2 mm) stored in the dark at À70 °C before analysis. USA). used for extraction were of high purity grade purchased from Merck (Darmstadt. The solvents acetone. The Arsenic determination was performed by an Agilent 7500a ICP-MS (Agilent Technologies. and dimethylarsinic acid (DMA) from Acros Organics (NJ. Detail information of the samples was summarized in Table 2.5 rps 75 As. 1 ml H2O2 was added onto the residue inside the bomb to form a clear Table 1 Instrumental parameters for total As determination and As speciation analysis Total arsenic ICP-MS RF power RF matching Carrier gas flow rate Peristaltic pump flow rate Arsenic speciation HPLC Analytical column Flow rate Injection volume Mobile phase A Mobile phase B Agilent 1100 Series Hamilton PRP-X100 (250 Â 4. A PRP-X100 anion exchange column (Hamilton. Instrumentation A lyophilizer (Labconco. USA) was used throughout the experiment. 2004). The supernatant and the residue were again separated by centrifugation. CA. The total arsenic content in the dry seafood samples was measured before and after extraction using inductively coupled plasma mass spectrometry (ICP-MS). was used to prepare the mobile phase used for the chromatographic separations. a vacuum degasser. NJ. China) was used for sample homogenization.8 to correct 75ArCl+ interference. / Food Chemistry 113 (2009) 720–726 721 2.6 V 1. and HPLC operating conditions are listed in Table 1. Germany) was used to separate the extract. denoted as Asnonpolar.22 lm membrane filter. and sonicated for another 10 min. China). After cooling down the bomb. USA). 2. Strong oxidants included 65% nitric acid (Merck) and 30% hydrogen peroxide (Merck) were used for DSP samples digestion. It was then extracted by 4 ml of a mixture of 50% methanol in deionized water (DI). 3. Afterwards the bombs were cooled down and the gas was released to discharge the high pressure in the bombs.1. The extraction procedure was the same as those described in step I. The Accelerated solvent extraction (ASE) instrumentation used for sample extraction was an ASE 100 from Dionex Corporation (Sunnyvale. Centrifugation (HERMLE. methanol and hydrochloric acid.

03 ± 0. DMA. 4.6 18.3 EE% 87. gradient elution with low initial concentration of mobile phase coupled with high elution rate were used. the concentrations in shrimp1 and oyster are higher or chose to algae.5 ± 0. and with a linear gradient elution from 100%A to 100%B in 18 min. the first step was to remove the fats from the samples. The extraction efficiency (EE%) is defined as the percentage of total acid digestion arsenic.2 0.6 ± 1. Fig.3.04 ± 0. It is obvious that the best separation was achieved when the flow rate was 1.01 ND ND ND 0. The results indicated that low (NH4)2CO3 concentration and high elution rate enhance the separation of AsB and AsC.1 EE% 2. The accuracy of arsenic determination was examined by running Certified Reference Material (CRM).6 10. Deionized water (A) and 50 mM (NH4)2CO3 (B) were used as the mobile phase.7 ± 0.9 ± 0.3 ± 0. a moderate flow rate (1. 1a compares the ion chromatograms observed under different gradient elution conditions. detailed Arsenic speciation analysis is deserved. The concentrations of arsenic in CRM by the microwave assisted digestion ranged from 90% to 100% of the certified results.16 ± 0. and no significant difference was found between the two methods.4 Asresidue 0. 2004).01 0.03 2. Anion exchange chromatography was used for the separation of the six arsenic species (AsIII. Step III: extraction of inorganic arsenic compounds The solid residue from the step II was dried in oven at 65 °C to drive away methanol. indubitably contains the high amount of arsenicals.3 6. Sealed bomb digestion was chosen finally as it could digest more samples at the same time.3 1.26 ± 0.42 ± 0. however.2 6.1 4.8 67.b Sequential extraction Asnonpolar Sardine Whitebait Squid1 Squid2 Oyster Clam Brown algae Shrimp1 Shrimp2 a b c d Total extractable arsenic Aspolar 1. AsV. 2002) was slightly modified to make it adoptable for our experiment. MMA. which.03 ± 0. Thus. Cao et al.97 ± 0.6 lg/g (n = 3). Canada).4 ± 0. 6. and show the absence of significant difference between the two sets of data.01 0.4 7.42 1. As a compromise. Total arsenic concentration in DSPs The total arsenic concentrations found in the DSP samples are given in Table 2.9 Asinorganic 0.3 35. The fraction was denoted as (Aspolar).10 13.28 1.0 7.30 Total EE%c EE% c Yellow Yellow Yellow Yellow Yellow Yellow Yellow Bo sea Yellow sea sea sea sea sea sea sea sea 2.1.3 13.2 0. / Food Chemistry 113 (2009) 720–726 Table 2 Total arsenic concentration in samples by acid digestion and sequential extractions (mg/kg dry weight. TORT-2 (NRC–CNRC.7 ± 1. The total arsenic in CRM (TORT-2) was determined by both the sealed bomb and the microwave assistant digestion methods.02 0. Compared with the fresh seafood (Li & Wei. 1a compares the different gradients used in this experiment and their separation efficiencies. The latter method followed the procedure reported by Li and wei (2003).33 ± 0.99 0. using the separation procedure listed in Table 1.02 5.01 18. All samples were analyzed in three independent replicates to ascertain the reproducibility of the analyses.44 1.5 ± 1.7 9.6 100. To solve this problem. is readily separated from the rests.9 6. . with its long retention time (15 min). 5. The extraction procedure was the same as those of step I.02 ND 0. where high solvent loading resulted in plasma instability.7 ± 0. the flow rate and the gradient elution time were optimized. The optimization process targeted five of the arsenical peaks except AsV.01 ND 2.05 0.6 ± 1.05 mM hydrochloric acid (HCl) was added.01 ± 0.7 ± 0.5 14 ± 1. According to the conditions in Brisbin’s method. MMA can be separated completely in an anion exchange column but not AsB and AsC.25 ± 0.5 ± 1.2.6 3.91 17. suggesting the validity of the method developed here.2 81.3. Samples including shrimp1.9 8.8 ± 0. Detailed results of As concentrations in individual fractions are listed on the top of Table 3.5 1. The final concentrate was then diluted and made up to the volume of 25 ml with the addition of DI. To optimize the extraction process.32 ± 0. 6.5 ml/min) and gradient elution conditions (0–15 min linear gradient from 100%A to 100%B) were chosen. then 0.1 0. was slowly added to solvent exchange with methanol.2 – 2.53 7.5 ± 0.27 7. in which results from this work are compared with those reported by Raimund Wahlen. The arsenic species in TORT-2 were extracted following the method described in Raimund Wahlen’s reference (Wahlen & McSheehy. AsV.02 2. The fraction was denoted as Asinorganic. and the separation achieved under the conditions is illustrated in Fig.3 97. The two aims of this step are: (1) to remove the fats along with other biological matrix interferences before chromatography (McKiernan & Creed.9 57.4 52. Optimization of arsenic separation The method of Judith A.6 2. AsC). The beneficial effects of high flow rate.4 ± 0.0 74.17 ± 0. The final solutions from this step were diluted and made up to the volume of 25 ml by DI. which agrees well with the certified value of 21. Total arsenic concentration were considered as acid digestion arsenic. the total arsenic concentration (dry weight basis) in DSPs is much higher. The measured total arsenic in the CRM material is 21.05 ± 0. Furthermore.1 ± 0. oyster and brown algae containing more arsenicals than those of others and algae. Interestingly.02 ± 0.7 0.4 49. 2003).4 0.36 1. Brisbin (Brisbin & Hymer.3 29. Results and discussion 6.8 lg/g.3 1. ND represents not detected.05 45.23 11.7 ± 1. DMA. Fig.4 10.9 ± 0. marked variation in total arsenic concentrations was observed among the different species of DSPs investigated in this report. while the extraction solutions were concentrated the procedure as described in step II. and baseline separations of individual peaks were achieved.2 14. AsIII.6 74.9 ± 0.27 ± 0.1 0.8 0.04 0.1 ± 0.1 1.07 4. Quality assurance and quality control (QA/QC) The sample extraction and analytical procedures were accompanied by the running of blanks and spiked standards.5 68.9 93 107 100 87 114 97 92 87 115 The precision is expressed as ± one standard deviation.722 X.3 ± 3. The two sets of data agree very well. is offset by operational problems in ICP-MS. AsB. Sequential extraction of DSPs A sequential extraction procedure was developed to fractionate the arsenic compounds in DSPs.2 NDd 0.8 ml/min.0 1. as a kind of ‘‘arsenic concentrator”. n = 3) Samples Source Total arsenica.02 ± 0. The two cationic species AsB and AsC co-elute together on an anion column without separation.05 ± 0. 1999) and (2) to find out the amount of non-polar 6.4 2.1 ± 0.7 1. 1b.17 ± 0.

It has been reported that oyster and algae contains high concentration of arsenolipid (Schmeisser & Goessler. / Food Chemistry 113 (2009) 720–726 Table 3 Speciation of arsenic compound in sample of CRMs.1 5. AsB and AsV were detected in all DSP samples studied. arsenicals reside in this extract. The results of extraction efficiency indicate the 1:1 MeOH/Water are suitable for DSPs extraction except for Oyster (57.01 ND 0. For the extractable arsenicals by polar solvent 1:1 MeOH/Water.08 14. universal solvency power and water miscibility.8 0.093 ± 0. The highest value occurred in oyster (0. methanol or its water solution was the best choice because of its high extraction efficiency (EE%).01 0.61 ± 0. Thus.02 2.3 0.6 ± 1.2 ± 1.1 4. and the value for fresh fish falls in the same range as the DSPs.2 – 0. indicating good recovery and material balance of the sequential extraction procedure total EE% was obtained.01 ND ND ND ND ND ND ND ND ND PE%c 8.6 2.1 – 0.1 35. respectively. As for the water-soluble arsenicals.3 ± 0.84 ± 0.6 27.4 ± 0.4 11. 1981).8 ND ND 0.5 1.45 ± 0. n = 3) Sample CRM TORT-2b TORT-2 Sample Step II Sardine Whitebait Squid1 Squid2 Oyster Clam Brown algae Shrimp1 Shrimp2 Step IIIg Sardine Whitebait Squid1 Squid2 Oyster Clam Brown algae Shrimp1 Shrimp2 a b c d e f g f 723 AsC AsB AsIII DMA MMA As V Total extractable arsenica 21.2 – 2. the samples were further extracted by dilute hydrochloric (0.3 – – – – – – – – – 14. 6.04 0. suggesting the absence of apparent differences between fresh seafood and DSPs.1 ND ND ND ND ND ND ND – 3.5 ± 0.2 11. Extracted by 0. PE% is defined as the percentage of total extractable arsenic.4 0.01 ± 0.1 ± 0.6 ± 1.3 0.1 mg/kg).1 0.2 1.6±1.26 ± 0.0 7. 1:1(V:V) MeOH/water and 0.3 2.2 6.2 ± 0.03 2.9 0.1 ND ND 0. step II and step El (mg/kg dry weight.17 ± 0.1 1.09 ± 0. Sum of arsenic speciation.01 ± 0.6 ± 0.01 0. would enhance the signals in ICP-MS because of the carbon effect (Allain & Jaunault.02 5.08 3.03±0.4 74.9 ± 0.7 ± 0.1 0.5 ND 0.01 0.0 ± 0. 2007).25 ± 1.9 ± 0.16 ± 0.02 ± 0.1 ND ND ND ND ND ND 0.04 11.03 0.01 0.3 6.9%.0 19. The results indicate that this sequential extraction procedure could extract quantitatively the arsenicals in DSPs. In our DSP samples.7 0.0 ± 0. These were reference value given by Raimund Wahlen (14).31 ± 0.04 ± 0.4 ± 1.2 2.2 Total extractable arsenic determined by ICP-MS with standard addition calibration in aqueous extracts (sonication). because AsB is not transformed further in humans and other mammals.3 0.2 ND 0. This portion of the arsenicals is often neglected by other researchers because of their low concentrations.7 27. has low EE% towards inorganic arsenicals species.17 ± 0.0 ND 0.6 Totald 1. The direct determination of arsenic concentration in extraction solution of step II and step III using ICP-MS.7 ± 0.7 ± 0.0 0. (McKiernan & Creed.1 0.3 6. the efficiencies of acetone (McKiernan & Creed.05 M HCl.2 DMA ND ND ND ND 1.5 ± 1.3 0.8 0.03 1.05 0.4. In this experiment.0371 ND AsIII ND 0. 2007).9 34.0 39.8 29.2 ± 0. Therefore. AsB could be excreted essentially unchanged.4 1. brown algae and whitebait samples in general contain high amounts of Asnonpolar.2 ± 0.9 14.2% of the total arsenicals.61 Astotale 1.04 1.01 AsV 0.1 AsB 1.3 35.7 61.9 6.02 MMA ND 0.1 PE% – 2.57 ± 0.2 1. may result from the incompletely evaporation of MeOH in this sample. The extraction efficiency.0 40.8 4. Thus.01 0.5 PE% 60.3 5.2 8. 57% extraction efficiency would be unacceptable.005 0.5 0.20 ± 0.2 ± 0.1 0.01 ± 0.9 ± 0.01 ± 0.14 ± 0.7 1.9 ± 0.3 0.41 ± 0.8 ± 1.9 – – – – – – – – – 0.06 ND ND ND ND ND ND ND ND ND PE% – – – – 7. the Asnonpolar can serve as the index of the level of arsenolipid in DSPs. The extraction yield and efficiency (EE%) of different extractants towards samples TORT-2 and shimp1 are shown in Table 4.1 PE% 20.05 0.7 ± 0.9 14.3 ± 1.03 0.1 0.5 ± 0.7 0. Acetone gave the best performance and was therefore chosen as the solvent in further experiments.010 ND AsC 0.2 2.05 ± 0.06 ± 0.3 11.9 20.5 9. which is above 100%.4 ± 0.4 3.1 1.5 2.5 14 ± 1.8 ± 0.3 0. 1999) and N-hexane (Schmeisser & Goessler.0 ± 1.3 ± 0.01 0.6 – – – – – – – ND ND ND ND 0. in the final step. In contrast to those of other seafood. however.7 0.3 ± 0.0371 0.1 0. even though the total arsenic concentration in seafood is high.6–10.5 ± 0.2 10.07 ND ND ND 0.X.6 0.01 ND PE% – 2.0%).4 ± 0.058 ± 0.1 3.4 0.5 26.04 0.2 0. Table 4 compares the amounts of arsenicals extracted by the two solvents used alone or in sequence.01 2.05 mM hydrochloric (HCl) acid were finally fixed as the extractants in threesteps.7 – – – – – – 0. and they represented the most abundant arsenic species among total extractable arsenicals in the two fractions.8 ± 0.3 31. AsB (arsenobetaine) represents the vast majority of total arsenic in marine organisms. the fractional yield of arsenicals ranged from 57–100. and relatively low arsenic concentration was observed. Cao et al. So dilute hydrochloric (HCl) acid is needed to enhance EE%. while brown algae (0. 1999).7 1.069 0.3 6.04 0. .03 1.09 ± 0.4 1.7 65.12 ± 0.9 – 1.1 ± 0.2 ± 0. The oyster.01 ND 0.3 ND 0.28 ± 0.02 5.1 – – – 8. 2005) were first evaluated and compared. Extracted by 1:1 MEOH/water.7 5.7 52.10 0.2 ± 0. (Table 2).1 1.3 1. such as MeOH.8 21.5 ± 0. The arsenic in the residues of extraction were also determined by acid digestion.09 0.1 mg/kg) also contains high concentration of Asnonpolar.58 ± 0.6 0.0928 ± 0.39 ± 0.03 0.024 ± 0. The MeOH.05 mM HCl) acid because of the its reported high EE% on inorganic arsenicals extraction (Mir & Rutter.6 ± 0. Arsenic speciation in DSPs Speciation analysis of arsenicals was performed on fractions obtained from step II (Aspolar) and step III (As inorganic) by hyphenated HPLC–ICP-MS technique (Table 3).6 – – 2.1 0.01 1.17 ± 0. acetone extraction accounted for 0.42 ± 0.6 ± 0.05 ± 0.9 ± 0. 1991).6 5.4 – 0.0928 ± 0. 2005).9 2. It has been proposed that AsB represents the ‘‘end point of the arsenic cycle in the marine ecosystem” (Borak & Hosgood. which can be extracted by acetone.35 ± 0. It is reported that carbon-containing compounds.01 ± 0.1 ± 0.25 ± 0.03 5. it is considered as a harmless arsenical (Cannon & Edmonds. As a method for food safety determination.7 ± 1. The combined yield after these threestep extraction range from 87 to 115%.1 5.7 9.

which were result from 75ArCl+ interference. the overall toxicity is low since AsB accounts for majority of the As species in these samples.7% of total extractable arsenic). arsenobetaine also represented the most abundant arsenicals in the samples. Cao et al. 1. After step I extraction. compared with AsB and AsVI. However. Gradient anion exchange ICP-MS chromatograms of As III. mixed spike arsenicals were added . The highest AsB concentration was found in the sample of shimp1 (step II + step III): 29. We did not take into account the species of arsenosugar in DSPs because of standard shortage. DMA. For the same sample. there is no obvious change between them except for some differences on peak area (Fig. whose amount exceeds 50% of total extractable arsenic in clam. baking. 1c). Since DSPs was made from fresh seafood. another dominating arsenicals found in DSPs was AsV. To eliminate the possibility of 75ArCl+ interference for arsenic in ICP-MS detection. followed by brown algae: 11. Other arsenicals (DMA. AsIII. an advanced type of ICP-MS (Agilent7000ce) with a reaction/collision cell was used to eliminate polyatomic interference. AsB and AsC. they were not the main component in amount. There is little report on this phenomenon. Further research would be needed to substantiate this claim. Recovery experiments were conducted using shimp2.3 mg/kg (64. AsC) were also found in some DSPs.6 mg/kg (67. MMA. Interestingly. / Food Chemistry 113 (2009) 720–726 a Aboudance (cps) 48000 40000 32000 24000 16000 8000 0 0 100 200 300 Time (sec) 400 500 600 1 2 5 (I) (II) (III) 3 4 b Abundance (cps) 3500 3000 2500 2000 1500 1000 500 0 0 AsB AsC DMA MMA As(III) As(V) 100 200 300 400 500 600 700 800 900 Time (sec) c Abandance (cps) 2400 2000 1600 1200 800 400 0 0 1 2 Agilent 7500a Agilent 7500ce 3 4 5 100 200 300 400 Time (sec) 500 600 700 800 Fig.8% of total extractable arsenic). etc). flavoring. This could explain the small differences observed between the sum of six species of arsenicals and the total extractable arsenic in each extraction step. we hypothesized that some arsenicals (AsIII or organic arsenicals) were transformed to inorganic forms during these process (braise. and inorganic arsenic was customarily considered as a kind of trace arsenicals in marine animals compared with organic arsenic. drying.724 X. MMA.

J. National Research Council. Regulatory Toxicology and Pharmacology. A.27 ± 0. 198–228).5 3 3 1. High extraction efficiency and high recovery (77–102. A. Gomez-Ariza. such as shrimp1.7 104. Analytical Chemistry.19 ± 0. 713–764. This value is obtained by acetone extraction after N-hexane extracted.04 – AcetoneN-hexanec ND – ND – ND – 0. 72(4).. An optimized HPLC procedure was developed. 1075–1084. 57(3–4). Biotransformation of arsenic in the marine environment. & Quetel. Conclusion Sequential extraction is an effective method to extract the arsenicals in DSPs.8% recoveries were obtained using these twostep extraction processes. A. W. Marine Chemistry. Huss. Australian Journal of Chemistry. suggesting the possible occurrence of As transformation processes during the manufacturing processes. 341–346. Weihua. J. Talanta. & Edmonds. G. Nriagu. P. J. 89(4). 58(1). Brisbin.. Borak.9 ± 0. (2007).8 ND 90.0 ± 0. Francesconi.3 90. & Hosgood. (1989). Larsen. R. P.1 64 12.4 ND 95. S.. New York: Wiley. .78 ± 0.01 59 10. J. It reaches 14.1 – 0.2 ND ND ND ND 94. Talanta. Table 5 Result of spike recovery experiment (mg/kg dry weight. 12).53 ± 0. A survey of arsenic species in Chinese seafood.0 AsC AsB AsIII DMA MMA As VI 1.0 ND ND 1. J. In El Pescado Fresco: su Calidady Cambios de su Calidad.. O. (1993). & Sanchez-Rodas. J. Arsenic in the environment: Human health and ecosystem effects. J. Craig (Ed. n = 3) Sample Asnonpolarb Acetone Shrimp1 EE%a TORT-2 EE% a b c d 725 N-hexane acetonned 100% MeH N-hexane ND – 0. J.03 ± 0. L. J.5 ± 0. Part 1: Cycling and Characterization (pp. Nriagu (Ed.8 ± 1. R. Allain.1 ND 10.77 ± 0. H. Extraction and speciation of arsenic in plants grown on arsenic contaminated soils. Larsen.09 ND ND ND ND ND 83. the dusky shark. Andreae. (1999). M. & Creed.39 ± 0. J. 221–263).7 AsIII ND ND 0. (1997). A comparison of automated and traditional methods for the extraction of arsenicals from fish. compared to extraction by single solvent extraction for which the efficiency is much lower. & Rutter. Seafood arsenic: Implications for human risk assessment. (1991). a constituent of the western rock lobster. 204–212. 1497–1498.. A comparison between ICP-MS and AFS detection for arsenic speciation in environmental samples. Mir. Li. Cullen..5 6 1.5 1. The results are listed in Table 5.83 ± 0. J. 1103–1110. R. in which baseline separation of AsB and AsC.. Washington. 7. 133–145. E. into the sample. AsB was observed as the most component in the DSPs. 77–102. Journal of Analytical Atomic Spectrometry. 51(2). (2007). 607–613. & Hymer. E.. ´ ´ FAO. extracted as steps II and III. 20–34. (2000).86 ± 0.1 ± 0. O.02 M HCl ND – 0. (2002).04 47 The extraction efficiency (EE%) is defined as the percentage of total acid digestion arsenic. Principles and Reactions (pp. Isolation. 34(4).7 ND ND 102. & Wei. Acknowledgments We gratefully acknowledge the financial support by the National Natural Science Foundation of China (No: 20675021).9 ± 0. & Reimer. 14(4).7% (clam) to 74.03 ± 0. The precision is expressed as ± one standard deviation. (1998).95 ± 0.). Chao (2003). Arsenic in the Environment.7 ± 0.01 62 8.01 57 1:1 MeOH/Water 1:2 MeOH/Water Water 0. Food and Chemical Toxicology.05 M HCl 0.7 ND ND ND ND ND 2.8%) were obtained using this method. D. 66(1). 47(2).2 AsB ND 9. B.2 ± 0. Assessment of exposure to inorganic arsenic following ingestion of marine organisms by volunteers. & Edmonds. C.6 AsVI ND ND ND ND 1. A. D. H.01 50 0.05 60 12. 8(8). J.28 ± 0. (1994). FAO: Rome. DC. McKiernan. Organoarsenic Compound in Environment. (1981).67 ± 0. (1986).9 ± 0. O. H. K. K. H.03 84 13. The method has been applied to the screening and speciation of As species in DSPs.1 Asinorganic Spike Recovey % Aspolar + Asinorganic Spike Recovery % a Spiked and recovered spike concentration (average ± 1 S. US. 41(8). Arsenic speciation in the environment.3 ND ND ND ND ND 77. (1997).a AsC Aspolar + AsC AsB AsIII DMA MMA As VI 1 4 1 2 2 1 0.1 ND ND ND ND 80.). L. 787–798. oyster and clam. Composicion quımica. Arsenic. Chemical Review. A gradient anion exchange chromatographic method for the speciation of arsenic in lobster tissue extracts. 257–268.4 ND ND ND ND 2. 1507–1518. ND ND ND 100 DMA ND ND ND 2. Arsenic speciation in seafood samples with emphasis on minor constituents: an investigation using high-performance liquid chromatography with detection by inductively coupled plasma mass spectrometry.8 ND ND 5 ND 3. K. Environmental Research.9 MMA ND ND ND ND 2. New York: Wiley. H. Buchet.D.. from three independent determinations) are reported in mg/kg (dry weight). 44–51.8 ± 1. Cannon.1 M HCl 0.88 ± 0. crystal structure and synthesis of arsenobetaine.8 ± 0. & Pritzi. Signal enhancement of elements due to the presence of carbon-containing compounds in inductively coupled plasma mass spectrometry. W. 27).69 ± 0.2 ± 0. In P.84 ± 0.3 ND ND ND ND 104. S. Arsenic speciation in shrimp and mussel from the Mid-Atlantic hydrothermal vents..07 – 0. AsV was also observed in a high concentration in some DSPs. J.03 67 14.01 54 10. indicating the validity of the method. and some samples of human urine. n = 3) Extraction procedure Spikes Spiked conc.1% (shimp2) of total extractable arsenic. In J.8 ± 0. (1994). References National Academy of Sciences. / Food Chemistry 113 (2009) 720–726 Table 4 Total arsenic in samples by sonication in different extraction solution(mg/kg dry weight. Advances in Environmental Science and Technology (Vol. Recovered spike conc. This value is obtained by N-hexane extraction after acetone extracted. New York: John Wiley & Sons (chap.. C. & Pauwels.. Journal of Analytical Atomic Spectrometry. (1994). T. & Jaunault. Cao et al. 63(14). Organoarsenic compounds in environment. Talanta.X.02 59 1.09 41 0.02 65 0.02 67 0.6 ND ND 101... Ed.

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