Arsenic speciation in environmental and biological samples extraction and stability studies

Arsenic speciation in environmental and biological samples extraction and stability studies

Analytica Chimica Acta 495 (2003) 85–98 Arsenic speciation in environmental and biological samples Extraction and stability studies I. Pizarro, M. Gómez, C. Cámara, M.A. Palacios ∗ Departamento de Qu´ımica Anal´ıtica, Facultad de C.C. Qu´ımicas, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain Received 7 March 2003; received in revised form 14 July 2003; accepted 5 August 2003 Abstract Our study evaluated the efficiency of consecutive extraction using several individual extractants or solvent mixtures: water, methanol:water (1:1, 9:1, 1:1–9:1 in two consecutive steps) and phosphoric acid for arsenic species extraction from rice, fish and chicken tissue, and soil samples. Arsenic species were quantified by HPLC (anionic and cationic chromatographic column) coupled to ICP-MS. The presence of As(III), As(V), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) was quantified in rice and soil whereas AsB, DMA and an unknown arsenic species were quantified in chicken tissue. AsB (major component) and one non-identified arsenic species were quantified in fish tissue. The sum of the arsenic species (as As) found in each extract for all matrices studied was equivalent to its total arsenic content. The best extraction efficiency and easiest handling were provided by the 1:1 methanol:water mixture for rice, fish and chicken tissue, and by 1M phosphoric acid for soil. Three consecutive extractions provided quantitative recovery of As species from all matrices tested. It was demonstrated that arsenic species in rice extracts remained stable during the three-month test period, whereas in fish and chicken tissue extracts, AsB was transformed into DMA over time. MMA and DMA were stable in the 1M phosphoric acid extracts from soils whereas As(III) gradually oxidised to As(V). As species from chicken and fish (higher protein content than rice and/or soil) became more stable as the methanol content increased in the extractant mixture used. © 2003 Elsevier B.V. All rights reserved. Keywords: Arsenic speciation; Extraction; Stability; Biological samples; Sediments; HPLC–ICP-MS; HG-AFS 1. Introduction Arsenic is an analyte of high concern in the scien- tific community due to its toxic properties. It is very well known that toxicity depends not only on the to- tal concentration but also on the chemical species in ∗ Corresponding author. Tel.: +34-913944318; fax: +34-913944329. E-mail address: palacor@quim.ucm.es (M.A. Palacios). which this analyte is present. Of the inorganic forms, arsine is highly toxic, and arsenite is accepted as being more toxic than arsenate [1]. The methylated organic species monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) are less toxic than the inorganic forms, and organoarsenicals, arsenobetaine and arsenocholine are generally considered to be non-toxic [2]. Arsenic may enter the environment as inorganic arsenic from pesticides and fertilizers used in agriculture, or from industrial processes such as 0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2003.08.009 86 I. Pizarro et al. /Analytica Chimica Acta 495 (2003) 85–98 the production of alloys and glass [3]. The presence of arsenic in fish, shellfish and crustaceans has been known for many years [4]. Inorganic arsenic can be methylated in the environment forming MMA, DMA, AsB, AsC, arsenosugars, etc. and thus enter the food chain in different forms. It is of paramount impor- tance to monitor the arsenic content and its chemical species distribution in soils, in high-consumption food (representative food items for humans), such as fish, chicken and rice, and in environmental samples. It is well known that fish and shellfish have the ability to bioaccumulate the non-toxic AsB while other food, such as rice, is a bioaccumulative plant of the most toxic As species (inorganic As, MMA and DMA). On the other hand, few data on the As content in chicken (in spite of its high consumption worldwide) have been reported. The As level in soils [5] has a considerable effect on the use of land for housing and agriculture. As the arsenic species content in such samples used to be rather low (␮gkg −1 ), the coupling of liquid chromatography with inductively coupled plasma mass spectrometry (HPLC–ICP-MS) and hy- dride generation atomic fluorescence spectrometry (HG-AFS) are the most highly-recommended tech- niques for speciation and total As determination, respectively. The quantitative and reproducible extraction of As species, especially from solid samples, is the weakest link in the sequence of analytical operations. Extrac- tion recoveries depend on the matrix, species present, types of solvents and extraction time–temperature. Several extractant mixtures and extraction techniques including mechanical shaking, microwave-assisted extraction (MAE) or sonication for arsenic extrac- tion have been employed [4,6]. The used of MAE with HNO 3 and H 2 O 2 reduces the extraction time, but not the risk of As species interconversion. At present, the methanol:water mixture at different ratios is the most widely-used extractant for fish and veg- etable samples [7–10], usually requiring more than one extraction step to achieve a quantitative recovery [11]. Phosphoric [12] and hydrochloric acids [13] have been proposed as efficient As species extrac- tants for soil using MAE and MAE plus sonication, respectively [14]. The use of ␣-amilase overnight as a step prior to As species extraction using sev- eral solvent mixtures has been reported to increase the As species extraction efficiency for some veg- etables [6,14]. The quantitative extraction of species is sometimes not easy to achieve, and some authors have applied recovery factors to compensate for the lack of quantitative recovery in the extraction step [15]. However, as the extraction efficiency may not be the same for all species, the knowledge of each arsenic species recovery in order to apply an appro- priate correction factor is of paramount importance [16,17]. On the other hand, it is also important to know the arsenic species stability in the extracts under several storage conditions, due to the possible high lapse of time that may occur between sample preparation and analysis. Thus, this work has mainly two objectives: (a) yield evaluation of As species extraction with several ex- tractants (under mild conditions to avoid species inter- conversion) in a variety of samples such as vegetables, meat, fish and soils; (b) evaluation of arsenic species stability in the extractants selected. 2. Experimental 2.1. Instrumentation For the determination of total arsenic concentra- tion, a flow injection hydride generation atomic fluo- rescence spectrometer, FI-HG-AFS (Excalibur, PSA, UK), was used. Polytetrafluoroethylene tubing (i.d. 1.6 mm) was used for all connections. An ICP-MS (HP-4500, Agilent Technologies, Analytical System, Tokyo, Japan), equipped with a Babington-type nebulizer, a Fassel torch and a double-pass Cott-type spray chamber cooled by a Peltier system was used as a detector after HPLC species separation. Single ion monitoring at m/z 75 was used to collect the data. The analytical peaks were integrated as a peak area using ICP-MS software. For chromatographic separations, a high-pressure pump (Milton Roy LDC Division, Riviera Beach, FL, USA) equipped with an injection valve (Rheo- dyne, 9125, USA) was used as the sample delivery system. All the connections were made of polyte- trafluoroethylene tubing (i.d. 0.5mm). The chromato- graphic conditions used for As species separation and quantification and the instrumental parameters used I. Pizarro et al. /Analytica Chimica Acta 495 (2003) 85–98 87 Table 1 Instrumentation parameters HG-AFS NaBH 4 concentration 1% m v −1 HCl concentration 1.5 M NaBH4 flow rate 1.0 mlmin −1 HCl flow rate 1.5 mlmin −1 Flow rate of sample 0.8 mlmin −1 H 2 flow to feed diffusion flame 60 mlmin −1 Ar carried gas flow 200 mlmin −1 Ar auxiliary gas flow 100mlmin −1 Lamp Arsenic 197.26 nm Primary current 27.5 mA Boost current 35 mA ICP-MS rf power Forward 1350W Reflected: 2.2 W Ar flow rate Coolant: 14 lmin −1 Nebulizer: 1.0lmin −1 Auxiliary: 0.9 lmin −1 Measurement mode Peak area of 75 As Integration time 0.1 s (spectrum) per point Points per peak 3 Internal standard 72 Ge 10 ppb HPLC Anionic column Hamilton PRP-X100 (10 ␮m, 250mm × 4.1mm) Guard column Hamilton PRP-X100 4.6 mm Mobile phase 10 mM PO 4 −3 ,pH6.0 Cationic column Hamilton PRP-X200 (10 ␮m, 250mm × 4.1mm) Guard column Hamilton PRP-X200 4.6 mm Mobile phase 4 mM pyridine/formiate, pH 2.8 Injection volume 100 ␮l Flow rate 1.5 mlm −1 Mode Isocratic for FI-AFS and HPLC–ICP-MS are summarized in Table 1. Sample mineralization and species extraction were carried out using PTFE reactors of 90 ml ca- pacity (Reactor Savillex Corporation 6138, Min- neuka, USA) in an oven. An I.R. distiller (Berghof, BSB-9391R) was used for HNO 3 and HCl purifica- tion. The supernatants were evaporated using a Centrivap Evaporator and Cold Trap system (Labconco, Kansas City, MO, USA). The samples were sonicated in a focused ultrasonic bath (Bandelin Sonopuls HD-2200, Fungilab S.A., USA). 2.2. Materials and reagents Stock solutions of 100mg l −1 arsenic were pre- pared from CH 3 AsO 3 Na 2 (MMA), Merck, 98%, C 2 H 6 AsNaO 2 ·3H 2 O (DMA), Fluka, 98%, NaAsO 2 (As(III)) and Na 2 HAsO 4 ·7H 2 O (As(V)) Sigma– Aldrich, 100%, C 3 H 6 AsCH 2 COOH (AsB), Tri Chemical Laboratory INC, Japan, 99%. High-purity demonized water (Milli-Q system, Millipore, USA) was used for sample preparation. The stock solutions were kept at 4 ◦ C in the dark and the working solutions were prepared daily. The extractant solutions were prepared from deion- ized water and HPLC-grade methanol (Merck, Darm- stadt, Germany). High-purity nitric and hydrochloric acids were obtained by distillation of reagents grade (Merck). HF acid was Suprapur grade Merck. K 2 S 2 O 8 (Fluka, 99.5%) was prepared in NaOH (Suprapur, Merck). H 2 SO 4 (Suprapur, 96%, Merck) and NaBH 4 (Fluka, 98%) in NaOH, used for reduction, were pre- pared daily. H 3 PO 4 and HClO 4 were obtained from Merck. The chromatographic mobile phase was 10 mM am- monium dihydrogen phosphate (Merck), adjusted to pH 6.0 with 0.1% NH 4 OH (Fischer certified ACS grade) when the anionic column was used, and 4 mM pyridine formiate at pH 2.8 when the cationic column was used. Both phases were filtered through a 0.45 ␮m nylon membrane and degassed in an ultrasonic bath. 2.3. Samples Arsenic compounds were determined in four pow- der candidate reference materials (prepared within the framework of a European project) of environmental and biological origin: rice, chicken, fish and soil sam- ples. Sample preparation of lyophilized pool was car- ried out at the IRMN Institute in Geel (Belgium) and the samples were kept frozen (−20 ◦ C) for further analysis. No unstability was demonstrated of the As species in the lyophilized samples studied during the six-month test period. Soils 1 and 2 having reductant and oxidant charac- teristics, respectively, were used. They were obtained from an As contaminated area. Two CRMs, NIST 1568a (rice flour) and NRCC DORM-2 (dogfish muscle), were used to validate the 88 I. Pizarro et al. /Analytica Chimica Acta 495 (2003) 85–98 total arsenic determination and for total As species characterization and/or validation. 3. Procedures 3.1. Mineralization for total arsenic determination 3.1.1. Fish, rice and chicken samples About 0.5g of the sample was placed in a PTFE reactor, 10ml of concentrated HNO 3 were added and the reactor was covered and pre-digested overnight. Next, 20mg of Na 2 S 2 O 8 and 3 ml of HClO 4 (or 0.3 ml of concentrated HF in rice) were added and heated to 150 ◦ C for 3h in an oven. After cooling, 0.5 ml of concentrated H 2 SO 4 was added and the digested sample was heated by refluxing for about 2 h until the final volume was about 2 ml. Next, the sample was diluted to 10ml with 0.5 M HCl. For analysis, three sub-samples and blanks were prepared in parallel and each one was analyzed in triplicate. 3.1.2. Soil sample Approximately, 0.5g of the sample was placed in a PTFE reactor and 10 ml of 1:1 HNO 3 :HCl mixture and 0.5 ml HF were added. The mixture was maintained at 150 ◦ C for 2 h. After cooling, the digested samples were heated until total elimination of the nitric acid, and finally diluted to 25 ml with 0.5 M HCl. 3.2. Extraction of arsenic species 3.2.1. Fish, rice and chicken samples Approximately, 1.0 g of the test materials was placed in a Teflon reactor and 10ml of 1:1 methanol: water were added following a similar treatment per- formed by Shibata et al. [18]. The mixture was maintained at 55 ◦ C for 10 h and then treated in an ultrasonic focalized bath for 5 min. The samples were centrifuged for 15 min at 6000 rpm, the ex- tract was then removed using a Pasteur pipette and the residue was re-extracted following the former procedure. The two combined extracts were mixed, evaporated to dryness using a centrivap evaporator and cold trap system, diluted with deionised water and filtered through a 0.45 ␮m nylon syringe filter. The same procedure was followed for extraction in degasified deionised water, in 9:1 methanol:water and 1:1–9:1 (1:1 followed by 9:1) mixtures. Each residue was dissolved in adequate water volumes, filtered (0.45 ␮m) and kept frozen (−20 ◦ C) prior to analysis. Three extracts were prepared from each sample. 3.2.2. Soil sample Approximately, 0.3g of the test material was placed in a Teflon reactor and 10ml of 1M of phosphoric acid were added. The mixture was heated at 150 ◦ C for 3h and the resultant extract evaporated to dryness. The residue was dissolved with 25ml of 10mM phosphate solution at pH 6. Three extracts were prepared from each sample. 3.3. Total arsenic determination Total arsenic concentration was determined in each raw material and extracts after their mineralization by FI-HG-AFS. The operating parameters used are given in Table 1. The analytical signals were evaluated as peak height, and quantification was carried out by the standard addition method. 3.4. Determination of arsenic species The As species were separated by HPLC following a method similar to that proposed by Beauchemin et al. [19] under the conditions given in Table 1. The arsenic species were quantified by measurement of the peak area by ICP-MS. 10 ␮gl −1 of Ge was used as the internal standard to correct any drift in the response of the ICP-MS. Since the results achieved on speciation by external calibration and standard additions matched well, it was no longer necessary to apply the standard addition. The detection limits for freeze-dried tissue of fish, chicken, rice and soil were within the 1.1–1.8, 1.6–4.5, 1.6–5.4, 1.7–4.5 and 2.1–2.4 ranges for As(III), As(V), MMA, DMA and AsB, respectively. The maximum RSD achieved was about 4%. It has been demonstrated by monitoring both 40 Ar 35 Cl and 40 Ar 37 Cl (m/z 75 and 77) that the pres- ence of chloride does not interfere because of its low concentration in all the extracts. I. Pizarro et al. /Analytica Chimica Acta 495 (2003) 85–98 89 Table 2 Extraction efficiency of total arsenic in rice, chicken, fish and soil. Expressed as percent ¯x ± s for three consecutive extractions Sample (total content, mg kg −1 ) Extraction efficiency number of extraction Water Methanol:water 1 M H 3 P0 4 1:1 9:1 1:1–9:1 Rice (0.182 ± 0.031) 1st 77.0 ± 2.0 80.0 ± 4.0 62.0 ± 2.0 80.0 ± 3.0 – 2nd 9.1 ± 1.0 10.2 ± 1.5 13.9 ± 1.7 7.6 ± 2.1 – Extractions n = 3 93.0 ± 3.0 96.02 ± 4.0 86.0 ± 4.0 92.0 ± 3.0 – Chicken (0.168 ± 0.002) 1st 60.0 ± 3.0 42.0 ± 2.0 55.0 ± 2.7 42.0 ± 1.5 – 2nd 6.8 ± 1.5 26.0 ± 2.0 7.3 ± 2.0 17.1 ± 1.0 – Extractions n = 3 73.0 ± 3.0 75.0 ± 2.9 70.0 ± 2.3 73.0 ± 2.9 – Fish (68.3 ± 1.9) 1st 53.0 ± 2.0 58.0 ± 2.2 56.0 ± 2.1 58.0 ± 2.8 – 2nd 25.0 ± 1.7 27.0 ± 2.0 24.0 ± 1.9 16.6 ± 1.1 – Extractions n = 3 90.0 ± 3.1 92.0 ± 3.0 85.0 ± 2.8 90.0 ± 3.0 – Soil (631.8 ± 3.0) 1st 50.0 ± 3.0 50.0 ± 2.9 46.0 ± 2.2 50.0 ± 3.0 82.0 ± 3.0 2nd 28.0 ± 1.9 18.0 ± 2.0 12.2 ± 2.5 20.0 ± 2.2 17.0 ± 2.0 Extractions n = 3 85.0 ± 3.7 80.0 ± 3.5 68.0 ± 2.9 89.0 ± 3.5 99.0 ± 3.0 Certified values of NIST 1568a (0.29 ± 0.03 ␮gg −1 As) and DORM-2 (18.0 ± 1.1 ␮gg −1 As). 4. Results and discussion 4.1. Extraction efficiency of total arsenic for chicken, rice, fish and soil In order to increase the extraction efficiency achieved, three consecutive extractions were car- ried out for each extractant tested: degasified water; methanol:water (1:1, 9:1, 1:1–9:1) and 1 M H 3 PO 4 (only for soil samples). Table 2 shows the total arsenic content found in rice, chicken, fish and soil, after acid digestion of raw samples and determination by HG-AFS, and the percentage of total arsenic in each extract. The total arsenic in each extract was determined by HG-AFS after mineralization in similar conditions as those used for the raw sample. The extracts were conve- niently digested to form species capable of generating arsine in the presence of borohydride. The arsenic content in rice and chicken is of the same order of magnitude and about three and two orders of magnitude lower than that of soil and fish, respectively. The 9:1 methanol:water mixture for arsenic extrac- tion from rice is not adequate, providing the worst recoveries (62% in the first extraction). Analogous results were obtained for water and 1:1 and 1:1–9:1 methanol:water extracts. About 80% of the total ar- senic in rice was extracted in 1:1 methanol:water in the first run, which means that one extraction might be sufficient to identify and quantify the arsenic species present in this matrix. An almost quantitative recov- ery was achieved with three extractions from water and the 1:1 and 1:1–9:1 methanol:water mixtures. However, the 1:1 methanol:water mixture provided clearer extracts and the procedure was faster than that required for the 9:1 and 1:1–9:1 methanol:water mixtures. Thus, the 1:1 methanol:water mixture was chosen as the most appropriate extractant, providing the highest extraction efficiency (96%) for the three consecutive extractions. Arsenic extraction efficiency for chicken in the first extract ranged from 42 to 60% of total ar- senic present in the raw material, and in the three consecutive extracts from 70 to 75% [18]. The 1:1 methanol:water mixture was chosen since the 9:1 and 1:1–9:1 methanol:water mixtures provide similar re- coveries, although no solid residues were detected in the former. Extraction recovery for fish was far from being quantitative in the first extraction for all extractants tested (53–58%). However, three consecutive extrac- tions provided about 90% recovery for all of them, except the 9:1 methanol:water mixture (86%). The ex- tractant 1:1 methanol:water was chosen. This extrac- tant for fish was also proposed by Shibata et al. [18]. It is important to mention that the efficiency of H 3 PO 4 as an As extractant for soil is much higher 90 I. Pizarro et al. /Analytica Chimica Acta 495 (2003) 85–98 than for water or the different methanol:water mix- tures. About 82% of arsenic was recovered in only one extraction run. An almost quantitative recovery was achieved in two consecutive extraction steps, 92 and 99% within three consecutive extraction runs. Similar results were obtained for soil 2 (containing 1800 mg kg −1 of As). 4.2. Arsenic species extraction for chicken, rice, fish and soil Six non-volatile species (arsenite, arsenate, MMA, DMA, AsB and AsC) were considered for arsenic spe- ciation by HPLC–ICP-MS in these matrices. Fig. 1. HPLC–ICP-MS chromatograms for a mixture of As species containing 15 ␮gl −1 of AsB and 5 ␮gl −1 of the other species in: (a) anionic column and (b) cationic column. In our chromatographic conditions using the an- ionic chromatographic column, As(III) and AsB coelute (Fig. 1a). Therefore, a cationic chromato- graphic column (which resolves As(III) and AsB peaks, Fig. 1b) was used to identify and/or quantify both species under the conditions detailed in Table 1. We evaluated whether there was any difference in the extraction efficiency between arsenic species for the different extractants checked. We also checked whether the second or third extraction could preferen- tially extract any species not extracted in the first one. As shown in Fig. 2a, the main arsenic species in chicken were AsB, DMA, and one non-identified peak (which elutes before AsB in the anionic col- I. Pizarro et al. /Analytica Chimica Acta 495 (2003) 85–98 91 Fig. 1. (Continued ). umn). When the cationic column was used (Fig. 2b) only two peaks were obtained corresponding to AsB and DMA + unknown species. The concentration of this unknown species is quite high and its ap- proximate concentration was determined by refer- ring its peak area to the AsB peak in the anionic column. Table 3 shows the extraction efficiency of each As species in chicken in the three consecutive extractions for all extractants. The recovery of each As species 92 I. Pizarro et al. /Analytica Chimica Acta 495 (2003) 85–98 Fig. 2. HPLC–ICP-MS chromatograms for chicken: (a) anionic column and (b) cationic column. (as As) is given as a percentage of the total arsenic in the extract. The extraction efficiencies of each arsenic species in the three consecutive extractions for all extractants tested were similar to those achieved for the first ex- traction (in both cases the results are expressed as a percentage of each As species with respect to the total As content in the extract). This fact indicates that each As species behaved in a similar way in the different conditions tested. Since total As in the extracts and the sum of each As species quantified were in good agreement, we concluded that no loss took place on the column. Similar conclusions were reached from the studies performed in parallel for rice and fish. The arsenic species detected in rice are As(III), fol- lowed by DMA and As(V), while MMA is present in a low content (Fig. 3). For fish, only AsB (main As species) and one unknown arsenic species (Fig. 4a and b) were detected in the extracts. The unknown peak does not overlap in any column with any of the As species evaluated. Table 4 shows the efficiency of species extraction in soil 1. The predominant arsenic species in this soil is As(V) (80%) and, in a much lower content (3–7%), I. Pizarro et al. /Analytica Chimica Acta 495 (2003) 85–98 93 Table 3 Efficiency of species extraction in chicken ± expressed as percent ¯x ± s) referring to total content in the corresponding extract Extraction Species Water Methanol:water 1:1 9:1 1:1–9:1 First extraction AsB 15.4 ± 2.0 16.5 ± 2.1 14.6 ± 2.0 15.3 ± 2.3 DMA 48.7 ± 3.0 49.5 ± 3.0 49.4 ± 3.0 48.7 ± 3.0 Unknown peak 33.8 ± 2.8 33.0 ± 2.0 34.0 ± 2.5 33.4 ± 2.0 As species 97.9 ± 4.5 99.0 ± 4.2 98.0 ± 4.4 97.4 ± 4.3 Extractions n = 3 AsB 13.7 ± 1.0 19.5 ± 2.0 15.0 ± 1.6 18.4 ± 1.0 DMA 48.7 ± 2.4 51.0 ± 3.0 48.1 ± 2.5 50.2 ± 3.0 Unknown peak 35.0 ± 2.0 28.0 ± 1.6 35.0 ± 2.0 28.4 ± 2.0 As species 97.4 ± 3.3 98.5 ± 3.9 98.1 ± 3.6 97.0 ± 3.7 As(III), DMA and MMA (Fig. 5a). Fig. 5b shows that As(V) and As(III) are the only species present in soil 2. The sum of the arsenic species concentration (as As) agrees with the total As content in the extract Fig. 3. HPLC–ICP-MS chromatogram (anionic column) for rice. using 1 M phosphoric acid as an extractant [14].No significant differences among species extraction were found for both soils as had occurred inthe rice, chicken and fish samples. 94 I. Pizarro et al. /Analytica Chimica Acta 495 (2003) 85–98 Fig. 4. HPLC–ICP-MS chromatograms for fish: (a) anionic column and (b) cationic column. No species transformation was detected for the samples tested during the extraction procedure, when analyzing the extracts after different extraction times, the same As species were detected although efficiency decreased with time. Table 4 Efficiency of species extraction in soil (expressed as percent ¯x ± s) referring to total content in the corresponding extract Extraction Species Water Methanol:water 1 M H 3 PO 4 1:1 9:1 1:1–9:1 First extraction As(III) 3.0 ± 0.9 3.2 ± 1.0 2.9 ± 0.8 3.2 ± 1.0 3.1 ± 1.0 DMA 7.9 ± 1.0 8.1 ± 1.1 8.3 ± 1.3 8.3 ± 1.2 8.2 ± 2.0 MMA 7.0 ± 1.0 7.3 ± 1.0 7.1 ± 1.0 7.2 ± 1.3 7.3 ± 2.0 As(V) 80.7 ± 3.1 80.4 ± 3.2 80.4 ± 3.2 80.1 ± 3.0 80.0 ± 3.2 As species 98.6 ± 3.4 99.0 ± 3.6 98.7 ± 3.7 98.8 ± 3.6 98.6 ± 4.2 Extractions n = 3 As(III) 3.0 ± 0.8 3.3 ± 1.0 3.1 ± 1.1 3.0 ± 0.9 3.0 ± 1.0 DMA 8.1 ± 1.0 8.1 ± 1.4 8.0 ± 1.6 7.9 ± 1.3 8.0 ± 0.8 MMA 7.1 ± 1.0 7.2 ± 1.1 7.0 ± 1.3 7.0 ± 1.0 7.0 ± 2.0 As(V) 80.0 ± 3.6 80.2 ± 3.3 79.1 ± 3.0 80.0 ± 3.0 80.0 ± 3.2 As species 98.2 ± 3.9 98.8 ± 3.9 97.2 ± 3.8 97.9 ± 3.5 98.0 ± 4.0 To validate the analytical methodology, the total As content and As species were quantified in the CRMs used, NIST 1558a (rice flour) and NRCC Dorm-2 (dogfish muscle). The total As content for both ma- terials was in good agreement with their certified [...]... spiked to the raw samples was higher than 90% for each arsenic species in all cases 4.3 Stability of arsenic species in rice, chicken, fish and soil extracts One of the main problems in speciation analysis, apart from the sample treatment, is the lack of knowledge of species stability in both raw material and extracts Fig 5 HPLC–ICP-MS chromatograms (anionic column) for soils (a) Soil 1 and (b) soil 2... (Continued ) 4.3.1 Rice The As species stability study in the extracts for the 1:1, 9:1 and 1:1–9:1 methanol:water mixtures did not show any significant difference in the concentration of each As species during the three-month storage period tested However, the situation changed when degasified deionized water was used as an extractant, as shown in Fig 6, with an increase in As(V) concentration and a... Stability of arsenic species in fish extracts Fig 7 Stability of arsenic species in rice extracts reported for waste water samples [20] Thus, it is clear that the presence of methanol in the extract prevents the oxidation of As(III) and consequently stabilizes the As(III) species 4.3.2 Chicken A stability study on extracts showed that the unknown arsenic species detected was stable in all extracts for at... However, DMA and AsB were less stable in water than in methanol:water mixtures (Fig 7) The concentration of AsB decreased slightly after two months’ storage in all methanol:water mixtures tested On the other hand, the concentration of DMA species increased after two months 4.3.3 Fish The stability study of As species in fish tissue extracts showed a general tendency in AsB to transform into the unknown... (months) Fig 9 Stability of arsenic species in soil extracts 3 4 98 I Pizarro et al / Analytica Chimica Acta 495 (2003) 85–98 MMA can be considered to have been stable during the three months tested for all the extractants used However, a gradual oxidation of As(III) to As(V) was detected in each case since the beginning of the storage period, except in cases where both the species remained stable for... the As content in all the extracts Water is not recommended as an extractant from the stability point of view, except for soil The stability of the As species is affected by the matrix and the nature of the extractant As species (As(III), As(V), MMA and DMA) in rice extracts remain stable for at least three months As species extracted from chicken tissue remain stable for up to two months in all methanol:water... AsB into DMA was detected As species (AsB and one unknown species) from fish were more stable in the extractant with a higher methanol content, 9:1 and 1:1–9:1 (two months), compared to the 1:1 methanol:water mixture The transformation of AsB into the unknown arsenic species was detected in all the fish extractants As(V) is the most abundant As species found in soil (80%) DMA and MMA are stable in all... phosphoric acid) The extracts were stored at 4 ◦ C in the dark and were analyzed immediately and up to three months after preparation Although the 1:1 methanol:water mixture was initially chosen as the most appropriate extract mixture for the As species from rice, chicken, and fish, and 1 M H3 PO4 for soil, the stability study was carried out in each extractant previously evaluated 9 8 7 6 5 4 3 2 1 0... (Fig 9) In case soil extracts have to be stored for analysis, water could be an appropriate extractant, although an extraction efficiency of only 85% was achieved 5 Conclusions All the solutions tested (water and the different methanol:water mixtures) were adequate for As species extraction from rice, fish and chicken tissue The efficiency related to the nature of the extractant, 1:1 methanol:water being... concentration decreases, reaching its peak when only water is used as an extractant (Fig 8) It can be considered that all the As species in the 9:1 and 1:1–9:1 methanol:water extracts are stable up to two months of storage 4.3.4 Soil As previously stated, 1 M H3 PO4 is the most effective extractant (99% extraction efficiency) for As species in soil It has been observed that DMA and 550 x + s.d (mg/kg) - . Analytica Chimica Acta 495 (2003) 85–98 Arsenic speciation in environmental and biological samples Extraction and stability studies I. Pizarro, M. Gómez, C. Cámara,. reserved. Keywords: Arsenic speciation; Extraction; Stability; Biological samples; Sediments; HPLC–ICP-MS; HG-AFS 1. Introduction Arsenic is an analyte of high concern in