Rapid and quantitative release, separation and determination of inorganic arsenic [As(III )+As(V )] in seafood products by microwaveassisted distillation and hydride generation atomic absorption spectrometry Ociel Mun�oz,a Dinoraz Ve� lez,a Marý�a Luisa Cerverab and Rosa Montoro*a aInstituto de Agroquý�mica y Tecnologý�a de Alimentos (CSIC), Apdo. Correos 73, 46100 Burjassot, Valencia, Spain. E-mail: Rmontoro@iata.csic.es bDepartamento de Quý�mica Analý�tica, Universidad de Valencia, Dr.Moliner 50, 46100 Burjassot, Valencia, Spain. E-mail: M.Luisa.Cervera@uv.es Received 22nd June 1999, Accepted 29th July 1999 A precise, simple and rapid method is described for the determination of inorganic arsenic [As(III)+As(V)] in seafood products. The inorganic species were isolated from the matrix by microwave-assisted distillation and determined by hydride generation atomic absorption spectrometry (HGAAS). The microwave and chemical parameters were optimized in order to obtain quantitative inorganic arsenic recoveries.The analytical features of the method are as follows: detection limit 10 ng g-1 (dry mass) or 2 ng g-1 (fresh mass); precision (RSD) 4%; recoveries 106±3% for As(III ) and 113±4% for As(V). Under the optimized conditions, arsenobetaine, arsenocholine and tetramethylarsonium ion added to samples of seafood were not distilled; however, minor species were distilled and were detected in various percentages: 109% monomethylarsonic acid; 11% dimethylarsinic acid; 0.2% trimethylarsine oxide.The concentrations expressed as arsenic contributed by these species would imply a mean overestimate of inorganic arsenic of about 24 ng g-1 dry mass (dm). The concentrations of inorganic arsenic found in natural seafood samples ranged between 0.053 and 1.145 mg g-1 (dm) (mean moisture content 78%). The procedure was compared with an alternative methodology in which acid digestion-solvent extraction-HGAAS was used for the determination of inorganic arsenic.A Student’s t-test for paired samples of the data obtained by the two methodologies showed no significant diVerences (P-value 0.66). In fact, most of the studies that quantify inorganic arsenic in Introduction biological matrices after extraction by means of organic sol- The concentrations of arsenic found in seafood products vary vents do not provide recovery values for these arsenic over a wide range [values expressed in mg g-1, fresh mass species,5–7 or else the recoveries are low for the two species,8 (fm)]: fish 0.06–45; lamellibranchs 0.1–14.4; cephalopods especially for As(III).9 This is because the organic solvent is 0.1–49; gasteropods 1.0–40; crustaceans <0.04–91.1 This total unable to break the bond of As(III ) to thiol groups in proteins, arsenic is contributed by diVerent forms of arsenic which diVer and to release the As(III).For this reason it is necessary to considerably in their toxicological connotations.For example, use an attack with a non-oxidizing acid (HCl), which the most toxic species are As(III ) and As(V), the sum of which completely denatures the proteins in the sample without alterconstitutes the inorganic arsenic, whereas no harmful eVects ing the chemical structure of the organic arsenic species.9 have been attributed to arsenobetaine (AB).2 For the methyl- Procedures for determining inorganic arsenic based on attackated species such as monomethylarsonic acid (MMA), ing the sample with acid have previously been reported in the dimethylarsinic acid (DMA) and trimethylarsine oxide literature.10–12 Our laboratory has recently established a meth- (TMAO), with the exception of tetramethylarsonium ion odology based on solubilization with HCl, reduction and (TMA+), the toxicity diminishes as the degree of methylation subsequent extraction with chloroform, back-extraction into increases. The International Agency for Research on Cancer dilute HCl, dry-ashing and quantification by HGAAS (IARC) has evaluated arsenic ingested or inhaled as a Group which allows quantitative recovery of inorganic arsenic.9 I carcinogen to humans.3 The scientific community attributes Other organoarsenic species [DMA, AB, arsenocholine (AC), the majority of cancerous pathologies to inorganic arsenic.4 TMA+] are not co-extracted or degraded.MMA is quantitat- This situation creates a need for methodologies capable of ively recovered and TMAO is recovered to a small extent quantifying inorganic arsenic in order to make a real evaluation (3–10%).of the health risk involved in the consumption of seafood As an alternative to the extraction procedures cited above, products that contain arsenic. procedures based on distillation appear in the literature. For The procedures for determining inorganic arsenic reported example, Lunde13 developed a methodology that obtained in the literature include some that are performed by means of quantitative recoveries for As(III) and As(V), using conextraction with organic solvents.5–8 However, in a previous ventional distillation that combined release and separation paper we showed that extraction of inorganic arsenic by means in a single stage employing HCl and a reducer [Fe(II)]. Subsequently, Flanjak14 modified this methodology, using HBr of organic solvents does not provide quantitative recoveries.9 J.Anal. At. Spectrom., 1999, 14, 1607–1613 1607as the reducing agent, and obtained inorganic arsenic recover- with a Perkin-Elmer Model 5000 atomic absorption spectrometer (Perkin-Elmer, Norwalk, CT, USA) equipped with ies of the order of 94–106%.None of the procedures mentioned included a study of co-distillation or degradation of the organic a Perkin-Elmer FIAS 400 system operating as a hydride generator in continuous flow mode. An electrothermally heated arsenic species present in seafood products.In order to simplify and speed up the determination of inorganic arsenic proposed quartz cell was employed. Other equipment used included a lyophilizer equipped with by Lunde,13 our laboratory has developed a methodology that employs microwave-assisted distillation as an alternative to a microprocessor controlling the lyophilization process (FTS Systems, New York, USA) and connected to a computer; a conventional distillation, with subsequent determination of inorganic arsenic in the distillate by HGAAS.This method- PL 5125 sand bath (Raypa, Scharlau, Barcelona, Spain); a K 1253 muZe furnace equipped with a Eurotherm Controls 902 ology reduces the distillation process, improves the reduction of As(V) to As(III ) and proves the non-degradability of AB.15 control program (Heraeus, Madrid, Spain); a KS 125 Basic mechanical shaker (IKA Labortechnik, Merck Farma y In the present study, starting from the methodology of Lo�pez et al.,15 we have redesigned the system of the distillation Quý�mica, Barcelona, Spain) and an Eppendorf 5810 centrifuge (Merck Farma y Quý�mica, Valencia, Spain).collector flask, optimized the physical and chemical parameters, replaced HGAAS batch determination of arsenic by Reagents HGAAS determination in continuous flow mode, and studied the possible interference that could be caused by organoarsenic De-ionized water, of 18 MV cm resistivity, obtained with a species frequently found in fish (MMA, DMA, AC, TMAO Milli-Q water purification system (Millipore, Millipore Ibe�rica, and TMA+).The methodology developed improves on the Madrid, Spain), was used for the preparation of reagents and methodology of Lo�pez et al. in terms of speed and oVers standards. All chemicals including standards and reagents better analytical characteristics and absence of interference. It were of pro analysi quality or better. was applied to natural seafood samples and the results were The stock standard solution of As(III ) (1000 mg l-1) was compared with those obtained by acid digestion-solvent prepared by dissolving 1.320 g of arsenic trioxide (Riedel extraction-HGAAS.9 de-Hae�n, Hannover, Germany) in 25 ml of 20% m/v KOH solution, neutralizing with 20% v/v H2SO4, and diluting to 1 l Experimental with 1% v/v H2SO4.The standarmg l-1) was prepared by dilution of the Titrisol Instrumental standard (Merck, Darmstadt, Germany).The standard solutions (1000 mg l-1) of MMA, DMA, TMAO and TMA+ The microwave-assisted distillation was performed with a were prepared by dissolving appropriate amounts of domestic Moulinex Micro-Chef 700 microwave oven CH3AsO(ONa)2·6H2O (Carlo Erba, Milan, Italy), (Moulinex, Valencia, Spain). Microwave emission was (CH3)2AsO2Na·3H2O (Fluka, Madrid, Spain), (CH3)3AsO unpulsed, using maximum power (700 W) and an operating (Hot Chemical Co., Tokyo, Japan) and (CH3)4As+I- frequency of 2450 MHz.The sample was placed inside a (Hot Chemical Co.), respectively, in water. Solutions of poly(tetrafluoroethylene) (PTFE) vessel with an internal AB [(CH3)3As+CH2COO-] (973 mg l-1) and AC volume of 120 ml (4.0 cm id, height 9.5 cm) and 10 mm wall [(CH3)3As+CH2CH2OH] (1000 mg l-1) were obtained from thickness and with a tight-fitting screw-cap lid designed in our the Service Central d’Analyse du CNRS (Vernaisson, France). laboratory. The lid was provided with an outlet so that the Ashing aid suspension was prepared by stirring 20 g of distillate could be emptied from the vessel by means of a Mg(NO3)2·6H2O and 2 g of MgO in 100 ml of water until PTFE tube (1.0 mm id).The tube was passed through the homogeneous. The following reducing solutions were used: vent holes of the oven. The distillate was collected in a 63 ml 5% m/v KI+5% m/v ascorbic acid, aqueous solution; 1 ml of gas collection flask (2.0 cm id, height 20.0 cm) which had a 1.5% m/v hydrazine sulfate aqueous solution+2 ml (48%) glass tube in the centre (0.3 cm id, length 35.0 cm) connected HBr; 1 ml of 1.0% m/v hydrazine sulfate aqueous solution+ to the PTFE tube through which the distillate from the PTFE 1 ml (48%) HBr.vessel passed. A porous glass plate was cemented to the funnel- As reducing solution for hydride generation, sodium tetra- shaped lower end of the glass tube to allow uniform diVusion hydroborate(III) solution (1% m/v) was prepared by dissolving of the distillate in the collecting liquid (Fig. 1). NaBH4 powder in 0.7% m/v NaOH solution and filtering Determination of inorganic and total arsenic was performed through Whatman No. 42 paper. Fresh NaBH4 solution was prepared daily. All glassware was treated with 10% v/v HNO3 for 24 h, and rinsed three times with de-ionized water before being used. The reference materials employed, DORM-1 and DORM-2 (Dogfish Muscle, Squalus acanthias) and TORT-2 (Lobster Hepatopancreas), were purchased from the National Research Council Canada (Ottawa, Ontario, Canada).Sample preparation Inorganic arsenic was determined in various fresh, frozen and canned products purchased at local retail outlets. The fresh samples were prepared as they are normally eaten. The brine or sauce in the canned seafood products was removed by the method for determining the drained mass of canned foods.16 The drained samples of fresh and canned products were cut into pieces and frozen at -20 °C and afterwards freeze-dried for 48 h at a chamber pressure of 0.225 Torr.Sublimation heat Fig. 1 Diagram of microwave-assisted distillation system. A: Collector was supplied by conduction from heating plates at 20 °C. The gas tube (2.0 cm id, height 20.0 cm); B: PTFE tube (1.0 mm id); C: lyophilized samples were crushed and homogenized to a fine PTFE vessel (4.0 cm id, height 9.5 cm); D: security load; E: microwave oven; and F: poly(propylene) container. powder in a mill.The resulting powder was stored in previously 1608 J. Anal. At. Spectrom., 1999, 14, 1607–1613decontaminated twist-oV flasks and kept in the refrigerator at inorganic arsenic in the chloroform phase by agitating for 3 min with 10 ml of 1 mol l-1 HCl. Separate the phases by 4 °C until analysis. centrifugation, aspirate the aqueous phase and pour it into a beaker; repeat this stage again and combine the back- Determination of total arsenic extraction phases obtained.Weigh 0.25±0.01 g of dry sample in a 250 ml beaker. Add The determination of inorganic arsenic is performed by 1 ml of ashing aid suspension and 5 ml of 50% v/v HNO3. means of the following procedure: Add 2.5 ml of ashing aid After evaporation to total dryness on a sand-bath, mineralize suspension and 10 ml of concentrated HNO3 to the combined in accordance with the programme described in a previous back-extraction phases. Evaporate and treat in the same way paper.17 Dissolve the ash from the mineralized samples in 5 ml as for total arsenic.of 50% v/v HCl, and pre-reduce the extract with 5 ml of With samples that, in the back-extraction phase, generate KI+ascorbic acid. After 30 min, dilute to volume with water emulsions that cannot be broken up by centrifugation at over and filter through Whatman No. 1 filter-paper into a 25 ml 2000 rpm, transfer the emulsion into a beaker. After adding calibrated flask. The instrumental conditions used for the the ashing aid suspension and HNO3 and heating gently in determination of arsenic by HGAAS in continuous-flow mode the sand-bath for not more than 30 s, break up the emulsion are shown in Table 1.and remove the chloroform phase created by aspiration. Determination of inorganic arsenic Results and discussion Microwave-assisted distillation (proposed method). Weigh Microwave oven parameters 0.50±0.01 g of lyophilized sample in the PTFE vessel. Add 2 ml of H2O and 1 ml of reducing solution (KI+ascorbic For optimization of the analytical method a domestic microacid). Agitate, wait 5 min until the sample is completely moist, wave oven was used, with the rotating platform removed.The and add 12 ml of 8.25 mol l-1 HCl. Cover the PTFE vessel. PTFE vessel was placed inside a poly(propylene) container in In samples of products conserved in oil perform the wetting the lid of which a hole was made for the PTFE tube from the process for 10 min.Add 8 ml of H2O to the collector flask. PTFE vessel. This container protects the microwave oven from Connect the PTFE vessel to the collector flask and radiate at acid corrosion if there is leakage during distillation. The maximum power (700 W) until bubbling stops in the collector diVerent positions in the microwave cavity are not identical flask (4–6 min). Dilute the distillate to 50 ml, take an aliquot from the microwave radiation point of view. Therefore, the of 10 ml and add 1 ml of KI+ascorbic acid.Leave to react methodology of Yba�n� ez et al.17 was used to determine the for 30 min and determine inorganic arsenic by HGAAS, area of maximum radiation in which to place the PTFE vessel. applying the operating conditions described in Table 1. In This position was 13 cm from the door and 13 cm order to avoid cross-contamination between samples, clean from the magnetron. Similarly, the security load required to the inside of the PTFE tube after each distillation by con- avoid damage to the magnetron through overheating was necting it to a peristaltic pump and passing 20 ml of de-ionized established (25 ml ).water through it in the opposite direction to that of the distillation. Distillation parameters This study was aimed, in the first instance, at determining Solvent extraction.9 Weigh the lyophilized sample As(III ) and As(V) separately in seafood samples. The following (0.50±0.01 g) into a 50 ml screw-top centrifuge tube.Add strategy was proposed: distillation of As(III) during a first 4.1 ml of water and agitate until completely moistened. Then stage, and addition of a reducer and distillation of As(V) in a add 18.4 ml of concentrated HCl and agitate again for 1 h. second stage. With this in mind, the acidity conditions and Leave to stand for 12–15 h (overnight). collection of distillate proposed by Lo�pez et al.15 were tested Add the reducing agent (1 ml of 1.5% m/v hydrazine sulfate with each of the standards [As(III ), As(V), MMA or DMA], solution+2 ml of HBr) and agitate for 30 s.Add 10 ml of without a reducing agent, and the reagent volumes were CHCl3 and agitate for 3 min. Separate the phases by centrifuadjusted so that the volume of distillate was in keeping with gation at 2000 rpm for 5 min. Separe chloroform phase the capacity of the collector flask. Accordingly, 1 ml of by aspiration and pour it into another tube. Repeat the 1 mg ml-1 of each standard solution (expressed as As), 4 ml extraction process two more times.Combine the chloroform of H2O and 10 ml of 9.9 mol l-1 HCl (final concentration phases and centrifuge again. Eliminate the remnants of the 6.6 mol l-1 HCl) were placed in the PTFE vessel. The distil- acid phase completely by aspiration (acid phase remnants in lates were collected in 15 ml of water. The analytical determi- the chloroform phase cause substantial overestimates of nation by HGAAS of the arsenic present in the distillates was inorganic arsenic).Eliminate possible remnants of organic performed immediately after the process had ended. The material in the chloroform phase by passing it through a recoveries obtained for the various species determined were: Whatman GD/X syringe filter with a 25 mm PTFE membrane As(III ) 104%, As(V) 0–26%, MMA 0% and DMA 0%. The (Merck Farma y Quý�mica, Barcelona, Spain). Back-extract the variability in the distillation of As(V) suggests the possible non-quantitative formation of arsenic pentachloride (AsCl5).Table 1 Operating conditions for HGAAS This species is unstable and is completely dissociated to AsCl3 Hydride generation continuous-flow mode— and Cl2.18 Cell temperature 900 °C A study was made of whether the distillation of As(III ) took Sample solution 1 ml min-1 flow rate place throughout radiation or during a shorter period. It was Reducing agent 1.0% m/v NaBH4 in 0.7% m/v found that the entire volume in the PTFE vessel had to be NaOH; 1.0 ml min-1 flow rate distilled in order to obtain quantitative recoveries.The process HCl solution 1.5 mol l-1; 2.5 ml min-1 flow rate takes place within a single stage, providing a considerable Carrier gas Argon; 45 ml min-1 flow rate reduction in distillation time (4–6 min) by comparison with Atomic absorption spectrometer— the methodology of Lo�pez et al.15 Wavelength 193.7 nm The variation in the distillation percentage of the As(V) Spectral bandpass 0.7 nm standard calls into question the viability of the working Lamp power As EDL system 2; 400 mA conditions employed previously for the independent J.Anal. At. Spectrom., 1999, 14, 1607–1613 1609determination of As(III) and As(V). However, despite the HGAAS. Greater concentrations of L-cysteine cause interference in the hydride generation, reducing the sensitivity results obtained with standards, it was considered useful to study the overall eVect of the matrix on the distillation of drastically.The optimized conditions for standards were then tested As(III ) and As(V). Accordingly, applying the procedure described earlier, recovery tests of As(III), As(V) and with natural samples. Lyophilized sardine (1 and 0.5 g) was spiked with 1 ml of 1 mg ml-1 (as As) of standard [As(III), [As(III)+As(V)] were made on natural seafood samples (sardines and small squid). In each assay 1 g of lyophilized sample As(V), MMA or DMA], 5 ml of 0.1% m/v L-cysteine and 10 ml of 9.9 mol l-1 HCl.Quantitative recoveries were was placed in the PTFE vessel together with 1 ml of 1 mg ml-1 of each standard solution (expressed as As), 4 ml of water and obtained for As(III ), As(V) and MMA, and recoveries of 78% for DMA, showing that the methylated species are also pre- 10 ml of 9.9 mol l-1 HCl. The distillate obtained was quantified in the same way as for the standards. It was shown that the reduced to their trivalent state, forming organosulfur derivatives. 21 Taking as a basis the mean concentrations of MMA As(III ) added to the two samples was recovered quantitatively (sardine 118%; small squid 113%), as was the As(V) (sardine and DMA found by us in a wide range of seafood products [MMA: 6 ng g-1 dm; DMA: 167 ng g-1 dm, both expressed 103%; small squid 87%). Combined addition of the two standards also provided quantitative recoveries (sardine 97%; as As (dm=dry mass)],22 we evaluated the significance of the recoveries obtained for MMA and DMA on the level of small squid 100%).Comparison of these results with those obtained in the distillation of the standards [As(III ) 104%; inorganic arsenic detected when this reducer is used. The concentration of DMA, higher than that of MMA, causes the As(V) 0–26%] revealed a diVerence in the behaviour of As(V), which was not distilled quantitatively without the matrix but high recovery percentage in the distillate to lead to a substantial mean overestimate of the inorganic arsenic (136 ng g-1 dm), was distilled quantitatively when added to a natural sample.This suggests the existence of reducing agents in the samples which rules out the use of this reducer. Also, although the use of L-cysteine at concentrations of 0.1% does not cause inter- tested, which transform the As(V) into As(III) and make its distillation possible. A similar phenomenon was reported by ference in the determination of standards, in natural samples it triggers the formation of volatile sulfurized compounds us in a previous paper dealing with the existence of reducing agents in a natural sample which was able to reduce As(V) which interfere in hydride generation.during the extraction stage of inorganic arsenic.9 Consequently, Use of the reducing mixture HBr–hydrazine sulfate it was decided to add to the sample a reducer which, irrespective of the type of sample, would ensure quantitative reduction In view of the previous use of HBr as a reducing agent in of As(V) to As(III).Under these conditions it is only possible conventional distillation of inorganic arsenic,14 and the use of to make a quantification of the two species combined. the reducing mixture HBr–hydrazine sulfate for the reduction of arsenate,9,23 it was decided to test the validity of this Use of the reducer KI reducing mixture under the following conditions: 1 ml of concentrated HBr, 1 ml of 1.0% m/v hydrazine sulfate and First, a study was made of the individual behaviour of each of the arsenic species [As(III), As(V), MMA or DMA] in the 12 ml of 8.25 mol l-1 HCl (final concentration 6.6 mol l-1 HCl) were added to 1 ml of 1 mg ml-1 (as As) of standard presence of KI as reducer.Accordingly, 1 g of sample was placed inside the PTFE vessel together with 1 ml of 1 mg ml-1 [As(III ), As(V), MMA or DMA]. The distillate was collected in 8 ml of water and an aliquot of the distillate (10 ml ) was (as As) of each standard [As(III ), As(V), MMA, DMA], 5 ml of 30% m/v KI, and 10 ml of 9.9 mol l-1 HCl.In the collector then reduced with 1 ml of KI+ascorbic acid and quantified by HGAAS. The distillation of this mixture showed that the flask 15 ml of a 0.25% m/v solution of hydroxylamine hydrochloride were placed, this being the solution used previously arsenic species As(III), As(V) and MMA are recovered quantitatively, whereas the recovery of DMA is 81%.The recovery by Lo�pez et al.15 to maintain reducing conditions in the collecting flask. It was observed that As(III ) and As(V) distilled of these species suggests that the As(V) in the methylated species has been reduced to the corresponding species of quantitatively, whereas MMA and DMA were determined non-quantitatively (56 and 10%, respectively). The distillation As(III ), and that the bromides of these methylated species of As(III ) co-distil with the inorganic arsenic, as for reduction of the methylated species shows that the As(V) in these species has been reduced to the corresponding species of As(III), and with KI described above.Use of the HBr–hydrazine sulfate mixture was rejected the iodides of these methylated species of As(III) co-distil with inorganic arsenic. The existence of these species of As(III) because of the high recovery percentage of DMA, since, as with L-cysteine, it would cause a substantial overestimate of (MMAI2 and DMAI) was described by Suzuki et al.19 The use of 30% m/v KI as a reducing agent generates a the inorganic arsenic. considerable amount of I3- in the distillate, which cannot be Use of the reducing mixture KI–ascorbic acid completely reduced by the hydroxylamine hydrochloride. Contly, the presence of I3- creates interference in the The reducing mixture KI+ascorbic acid has been extensively subsequent hydride generation, making it necessary to use the used for reduction of arsenic.24 The reducing agent (I-) is method of additions or high dilutions which increase the limits constantly regenerated each time the As(V) is reduced to of detection (LODs).Given the low levels at which inorganic As(III ), since the I3- produced in the reaction is reduced by arsenic is found in some seafood products, the search for the ascorbic acid. This increases the eYciency of the iodide as methodologies with low LODs makes it advisable to test a reducer during distillation and eliminates interference due alternative reduction conditions.to excess of I3-. This reducing mixture was first tested on arsenic standards. Use of the reducer L-cysteine In the PTFE vessel 1 ml of water, 1 ml of the reducing mixture (KI+ascorbic acid) and 12 ml of 8.25 mol l-1 HCl (final The use of cysteine in hot acidic medium reduces As(V) quantitatively to As(III ).20 Therefore, it was decided to test concentration 6.6 mol l-1 HCl) were added to 1 ml of 1 mg ml-1 (as As) of each standard [As(III), As(V), MMA, this substance as a reducing agent.The optimum conditions required for quantitative distillation of 1 ml of 1 mg ml-1 of DMA, AB, AC, TMAO or TMA+]. The distillate was collected in 8 ml of water and made up to 50 ml with water. As(III ) and As(V) were: 10 ml of 9.9 mol l-1 HCl and 5 ml of 0.1% m/v L-cysteine (final concentration 6.2 mol l-1 HCl). The An aliquot of 10 ml of the distillate was pre-reduced with KI+ascorbic acid and quantified by HGAAS.The recoveries distillate was collected in water and immediately quantified by 1610 J. Anal. At. Spectrom., 1999, 14, 1607–1613Table 3 Analytical characteristics of the method Table 2 Recovery of distilled standards of arsenic species. Quantification by HGAAS or dry-ashing HGAAS LOD/ng g1 Asa 10 (dm)&2 (fm) Precision (RSD %)b Sardine 4% [104 (dm)&25 (fm)] Species Arsenic recovery (%) Cockle 4% [678 (dm)&163 (fm)] Small squid 3% [93 (dm)&22 (fm)] Distillation, Distillation, HGAAS dry-ashing HGAAS Mean 4% Recovery (%)c As(III ) Sardine 103¡À4% (25, 48) Cockle 109¡À5% (163, 168) As(III ) 108 107 As(V) 108 101 Mean 106¡À3% As(V) Sardine 109¡À2% (25, 48) MMA 96 96 DMA 6 65 Cockle 116¡À5% (163, 168) Mean 113¡À4% AB 0 0 AC 0 0 MMA Sardine 106¡À2% (25, 48) Cockle 112¡À3% (163, 168) TMAO 0.2 12 TMA+ 0 0 Mean 109¡À3% DMA Sardine 8¡À2% (25, 48) Cockle 13¡À3% (163, 168) Mean 11¡À3% were quantitative for As(III) (98%), As(V) (99%) and MMA aNine reagent blanks were employed; (dm) dry mass; (fm) fresh mass.(101%). For the remaining organoarsenic species the recoveries bRelative standard deviation from six independent analyses. Values in obtained ranged from 0 to 6%: AB (0%), DMA (6%), AC square brackets are the mean inorganic arsenic concentrations for the (0%), TMAO (0.2%) and TMA+ (0%) (Table 2). In order to samples analysed, expressed in ng g1 As dm and fm. cPercentage recoveries from three independent analyses expressed as verify whether the recovery percentages obtained previously mean¡Àstandard deviation.Values in parentheses are the mean inor- were due to distillations of the various species or to the ganic arsenic concentrations of the unspiked samples (first value) and numerous factors that aVect hydride generation (type and concentration added (as arsenic) of each arsenic species (second value) concentration of reagents, reduction temperature, oxidation in ng g1 As (fm).state, chemical form, sample matrix),25 the following experiment was performed: an aliquot of 25 ml of distillate was mineralized by dry-ashing and the arsenic was quantified by HGAAS (Table 2). It was seen that for As(III ), As(V) and MMA the results obtained by direct reading of the distillate (106¡À3%) and As(V) (113¡À4%) demonstrate the validity of and readings after dry-ashing coincided. The results obtained this methodology. These analytical characteristics are an by dry-ashing HGAAS show that the distillation of DMA was improvement on the LOD (68 ng g1 dm) and precision (9%) 64%, but in the distillate read directly only 6% was detected.obtained in the methodology proposed by Lo¡äpez et al.15 The behaviour of TMAO was similar: the distillation was 12%, although in the direct reading by HGAAS only 0.2% Application to reference materials was detected. The other arsenic species (AB, AC and TMA+) were not detected in the direct reading of the distillate, as was The proposed methodology was applied to reference materials with a certified total arsenic content: DORM-1, DORM-2, to be expected, since they are not hydride-generating species, nor were they detected in the mineralized distillate, since they TORT-2. In these samples total arsenic was quantified by dryashing HGAAS.For all the samples the results obtained were are not volatile species. The distillation conditions were then applied to natural within the certified concentration ranges: value found/value certified (mg g1, dm): DORM-1: 16.2¡À0.4/17.7¡À2.1; samples of sardine and cockle.The use of this reducer does not cause negative interference during instrumental reading; DORM-2: 17.9¡À0.5/18.0¡À1.1; TORT-2: 22.3¡À0.2/21.6¡À1.8. As there are no certified values for the concentrations of under these conditions it does not reduce analytical sensitivity and it is possible to use samples of 0.5 g.In the PTFE vessel inorganic arsenic in DORM-1, DORM-2 and TORT-2, the results obtained were compared with those provided by other 1 ml of 1 mg ml1 (as As) of each standard [As(III), As(V), MMA, DMA, AB, AC, TMAO or TMA+], 1 ml of water, workers. For the matrices mentioned, Table 4 shows the concentrations of As(III)+As(V)+MMA, and also As(III)+As(V) 1 ml of the reducing mixture (KI+ascorbic acid) and 12 ml of 8.25 mol l1 HCl were added to 0.5 g of sample. As in the and MMA found by various workers,5,9,26 together with the concentrations of As(III )+As(V)+MMA+11% DMA found tests with standards, the distillation percentages were quantitative for As(III), As(V) and MMA and remained at 0.2% for in the present work.DORM-1 is the sample that has been most analyzed in TMAO, and there was no distillation of AB, AC and TMA+. However, for DMA the mean distillation percentage in the studies of arsenic speciation. Its mean concentration of DMA, obtained from data in the literature,27,28 is 0.500 mg g1(dm), samples increased from 6 to 11%.Given the mean values of MMA and DMA in seafood products reported previously,22 of which 11% (0.055 mg g1, dm) is detected with the methodology developed in this work. If we subtract from the concen- the distillation of these methylated species would lead to a mean overestimate of the concentration of inorganic arsenic tration of As(III)+As(V)+MMA+11% DMA obtained in this work (0.196 mg g1, dm) the arsenic contributed by the of 6 ng g1 by co-distillation of MMA (100% of 6 ng g1 dm) and of 18 ng g1 by co-distillation of DMA (11% of percentage of DMA quantified with the present methodology (0.055 mg g1, dm), it is possible to compare the 167 ng g1 dm).In the light of the results obtained we consider that the most suitable reducer is the mixture of KI+ascorbic mean value resulting (0.141 mg g1) with the values of As(III )+As(V)+MMA reported by other workers. Those acid, given the insignificant mean overestimate of inorganic arsenic that it produces (24 ng g1 dm) and the fact that it values (0.180 mg g1 Beauchemin et al.,5 0.165 mg g1 S¡¦ lejkovec et al.,26 0.124 mg g1 Muno¡ä z et al.9) are in reasonable does not cause interference under the working conditions employed. agreement with the result obtained in this work (0.141 mg g1).The concentrations of inorganic arsenic obtained for the In order to determine whether the methodology developed is suitable for the determination of inorganic arsenic in seafood other CRMs with the proposed methodology (DORM-2: 0.102¡À0.029 mg g1; TORT-2: 0.506¡À0.031 mg g1) are products, its analytical characteristics were evaluated (Table 3).The values obtained for LOD (10 ng g1 dm) and close to those reported by Munoz et al.9 (DORM-2: 0.145¡À0.011 mg g1; TORT-2: 0.581¡À0.055 mg g1). precision (4%) and the satisfactory recoveries of As(III) J. Anal. At. Spectrom., 1999, 14, 1607�C1613 1611Table 4 Concentrations of inorganic arsenic in certified reference materials CRM Arsenic species/mg g.1 (dm) Reference DORM 1 As(III)+As(V)+MMA 0.180¡¾0.040 Beauchemin et al.5 As(III)+As(V) 0.129¡¾0.003 S¢§ lejkovec et al.26 MMA 0.036¡¾0.010 S¢§ lejkovec et al.26 As(III)+As(V)+MMA 0.124¡¾0.004 Mun.oz et al.9 As(III)+As(V)+MMA+11% DMA 0.196¡¾0.004 This work DORM-2 As(III)+As(V)+MMA 0.145¡¾0.011 Mun.oz et al.9 As(III)+As(V)+MMA+11% DMA 0.102¡¾0.029 This work TORT-2 As(III)+As(V)+MMA 0.581¡¾0.055 Mun.oz et al.9 As(III)+As(V)+MMA+11% DMA 0.506¡¾0.031 This work Table 5 Total arsenic and moisture content in seafood.Inorganic arsenic content of samples obtained by solvent extraction and microwave distillation Sample Moisture (%) Total Asa/ Inorganic Asb/mg g.1 (dm) mg g.1 (dm) Solvent extraction Microwave distillation Fresh. Anchovy 76 18.78¡¾0.45 0.176¡¾0.005 0.123¡¾0.006 Clam 86 16.12¡¾0.56 1.009¡¾0.090 1.145¡¾0.012 Cockle 78 20.71¡¾0.43 0.688¡¾0.021 0.678¡¾0.029 Mussel 83 11.52¡¾0.03 0.223¡¾0.004 0.265¡¾0.009 Mussel 82 11.37¡¾0.11 0.220¡¾0.022 0.202¡¾0.069 Mussel 79 12.03¡¾0.18 0.335¡¾0.009 0.270¡¾0.013 Mussel 84 9.14¡¾0.56 0.212¡¾0.007 0.211¡¾0.051 Mussel 75 9.42¡¾0.08 0.245¡¾0.007 0.280¡¾0.001 Mussel 83 14.79¡¾0.09 0.238¡¾0.005 0.285¡¾0.033 Mussel 82 17.48¡¾0.10 0.400¡¾0.019 0.305¡¾0.030 Mussel 83 11.83¡¾0.24 0.224¡¾0.003 0.277¡¾0.047 Mussel 84 11.42¡¾0.04 0.196¡¾0.007 0.151¡¾0.012 Sardine 76 16.72¡¾0.41 0.137¡¾0.008 0.099¡¾0.004 Small squid 80 9.85¡¾0.30 0.063¡¾0.002 0.053¡¾0.005 Squid 86 1.90¡¾0.09 0.074¡¾0.003 0.104¡¾0.006 Canned.Cockle 77 9.09¡¾0.01 0.749¡¾0.031 0.894¡¾0.064 Langostillo 72 6.97¡¾0.09 0.477¡¾0.025 0.385¡¾0.027 Octopus 69 9.47¡¾0.23 0.319¡¾0.007 0.313¡¾0.008 Razor clam 69 1.98¡¾0.14 0.201¡¾0.019 0.249¡¾0.023 Shrimp 73 3.18¡¾0.02 0.247¡¾0.013 0.246¡¾0.029 Frozen. Shrimp 82 6.00¡¾0.16 0.384¡¾0.028 0.396¡¾0.042 aTotal arsenic determined in duplicate (mean¡¾standard deviation).bInorganic arsenic determined in triplicate (mean¡¾standard deviation). Application to natural samples; comparison of methodologies methodology described here oVers the performance necessary for use in routine analyses with the instrumentation available The optimized methodology was applied to 21 natural seafood in many control laboratories. samples. Table 5 shows the concentrations of total arsenic and inorganic arsenic and the moisture content for each sample.It also shows the values of inorganic arsenic obtained for the Acknowledgements same samples by applying the methodology of extraction with The authors gratefully acknowledge the financial support organic solvents developed by Mun.oz et al.9 The Student¡�s tof the Comisio¢¥n Interministerial de Cienc©¥¢¥a y Tecnolog©¥¢¥a test for paired samples was used to compare the values of (CICyT), Project ALI96.0511, for which they are deeply inorganic arsenic found by the two methodologies.The Pindebted. O. Mun.oz received a Research Personnel Training value obtained (0.66) indicates that there are no significant Grant from the Instituto de Cooperacio¢¥n Iberoamericana diVerences between the two methodologies with a confidence (ICI). level of 95%. 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