Chemical Papers 66 (10) 914–924 (2012)DOI: 10.2478/s11696-012-0207-6

ORIGINAL PAPER

Speciation of arsenic metabolites in the free-living mouse from Donana National Park used as a bio-indicator

for environmental pollution monitoring‡

a,b,cMiguel Ángel García-Sevillano, a,b,cMacarena González-Fernández,a,b,cRocío Jara-Biedma, a,b,cTamara García-Barrera*, dAmalia Vioque-Fernández,

dJuan López-Barea, dCarmen Pueyo, a,b,cJosé Luis Gómez-Ariza*

aDepartment of Chemistry and Material Science, Faculty of Experimental Sciences, cResearch Centre of Health and

Environment (CYSMA), University of Huelva, Campus El Carmen, 21007-Huelva, Spain

bInternational Agrifood Campus of Excellence, CIA3, CIDERTA Building, University of Huelva, Huelva Business Park,

21007-Huelva, Spain

dDepartment of Biochemistry and Molecular Biology, Campus de Rabanales, University of Córdoba,

Ed. Severo Ochoa, 1407-Córdoba, Spain

Received 29 October 2011; Revised 12 April 2012; Accepted 14 April 2012

A speciation approach based on orthogonal chromatographic systems coupled to inductively cou-pled plasma mass spectrometry (ICP-MS) was used to characterise the biological response of free-living mice Mus spretus to environmental pollution caused by arsenic in different areas of theDonana National Park (south-west Spain). The relative presence of inorganic and organic formsof arsenic was studied in cytosolic extracts from high metabolic activity organs of Mus spretusmice: kidneys, liver, and brain. An instrumental coupling of size-exclusion chromatography withUV and collision/reaction cell-ICP-MS detectors (SEC-UV-ICP-ORC-MS) both in analytical andpreparative scale was used for this purpose. The results showed the presence of low molecular mass(LMM) molecules linked to arsenic in these tissues especially in the kidneys, where the presenceof these arsenic metabolites was higher. On the other hand, the presence of these arsenicals var-ied from one area to the other, which can be related to a different occurrence of contaminants.These low molecular mass fractions were collected by preparative SEC chromatography for laterstudy with ion exchange chromatography and detection by ICP-ORC-MS, using both anionic andcationic columns. The results showed the higher presence of MMA and DMA in kidneys of micecaught in contaminated areas and the existence of small amounts of unidentified arsenicals whencation-exchange chromatography was used, which could be related to the presence of dimethylarsi-noylethanol (DMAE), thioarsenic species, or arsenocholine (AsC).c© 2012 Institute of Chemistry, Slovak Academy of Sciences

Keywords: Mus spretus, arsenic speciation, arsenic metabolites, environmental pollution, DonanaNational Park

Introduction

Arsenic is one of the most important global en-vironmental toxic elements present in the terrestrial

crust (medium concentration about 3 mg kg−1) in theform of arsenopyrite (FeAsS) and represents the 20thmost abundant element in our environment (Lobinski& Marczenko, 1997). Soils contain 0.05–0.2 mg kg−1

*Corresponding author, e-mail: ariza@uhu.es; tamara@dqcm.uhu.es‡Presented at the 5th Meeting on Chemistry & Life 2011, Brno, 14–16 September 2011.

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of this element, but marine sediments can accumulateup to 40 mg kg−1 of As (Francesconi & Edmonds,1994). Arsenic is commonly found in several chem-ical forms; the toxicity, environmental mobility, ac-cumulation in living organisms, and involvement inliving-organisms’ metabolism usually depend on theform in which the element is present (Vahter, 1999;Hirano et al., 2003). Inorganic forms of arsenic arehighly toxic, but this toxicity decreases in the methy-lated forms (monomethylarsonic acid (MMAV) anddimethylarsinic acid (DMAV)), which are consideredmoderately toxic; however, other organic species suchas arsenobetaine (AsB), arsenocholine (AsC), and ar-senosugars are regarded as innocuous (Geiszinger etal., 2002; Fattorini & Regoli, 2004; Fattorini et al.,2006). However, monomethylarsonous acid (MMAIII)formed in vivo is more toxic than arsenite in hamsters(Petrick et al., 2001; Thomas et al., 2007).

Arsenobetaine, arsenocholine, arsenosugars, andarsenolipids are usually present in aquatic organismsand marine animals (Edmonds & Francesconi, 1981,1987). In mammals, highly toxic inorganic arsenic ismetabolised in the liver, after absorption from the gas-trointestinal tract, to produce methylated species suchas MMAV and DMAV which are excreted in the urine(Del Razo et al., 1997; Hughes et al., 2003). How-ever, in recent years it has been demonstrated thatbiomethylation of inorganic arsenic is not necessar-ily a detoxification process, because the intermediatesand products formed may be more reactive and toxicthan inorganic arsenic (Thomas et al., 2007).

The environmental relevance of arsenic is due toits toxicity to mammals in which it leads to irre-versible damage to the central nervous system, bones,red blood cells, and kidneys. An excessive intake ofarsenic may cause skin lesions and peripheral vascu-lar diseases, in addition to cancer of the lungs, liver,skin, and bladder (Fadrowski et al., 2010; Bates etal., 1992). Oxidative stress associated with arsenic hasbeen related to carcinogenic processes and this ele-ment was reported as having the capacity to crossthe blood-brain barrier (BBB) (García-Chávez et al.,2003; Piao et al., 2005).

Free-living organisms have been used in envi-ronmental pollution assessment since they providemore unequivocal information about the biological re-sponses to contaminants than the direct analysis ofcontaminants. The mouse Mus spretus has been usedfor this purpose in the Donana National Park (SWSpain) and surrounding areas since it is not a pro-tected species and it feeds on plants, seeds, and in-sects around its burrow, hence it duly reflects the ac-tion of contaminants in the area in which it is caught(González-Fernández et al., 2011). This free-living or-ganism has not been yet genetically sequenced al-though it exhibits a close genetic homology with theconventional laboratory mouse Mus musculus, whichhas been sequenced. This facilitates proteomic studies

of Mus spretus using the database from Mus muscu-lus in order to monitor the action of contaminants onterrestrial ecosystems (Montes-Nieto et al., 2007).

The area of study, the Donana National Park, hasbeen declared a UNESCO World Heritage Site due toits ecological importance. However, this area suffersfrom contamination threats caused by adjacent agri-cultural activities, mining, and industrial activities(Funes et al., 2006; Vioque-Fernández et al., 2009).Therefore, monitoring of the environmental quality ofthis area is necessary and Mus spretus as an unpro-tected species living in the park that reaches a higherdensity of population is an appropriate bio-indicatorfor this purpose.

The present work explores a speciation approachto compare the biological response ofMus spretus col-lected from five areas of the Donana Park with differ-ent levels of contamination. The differences in levels ofarsenic metabolites can be used for unequivocal envi-ronmental contamination assessment. Several organsfrom Mus spretus were studied and the highest con-centration of arsenic was found in the kidneys. Themetallo-metabolites profiles in the kidneys from Musspretus were studied using SEC with the ICP-ORC-MS detection. The results show the presence of lowmolecular mass (LMM) molecules linked to arsenic inthese tissues, the intensity of which varies from onearea to the other, which can be related to differentlevels of contaminants. These low molecular mass frac-tions were collected for later study by ion-exchangechromatography and ICP-ORC-MS detection, usingboth anionic and cationic columns.

Experimental

Standard solutions, reagents, and instrumen-tation

All reagents used for sample preparation were ofthe highest available purity. Phenylmethanesulpho-nyl fluoride (PMSF) and reduced L-glutatione wereobtained from Sigma–Aldrich (Steinheim, Germany).Helium of high-purity grade (> 99.999 %) was used ascollision gas in ICP-ORC-MS experiments.

The standards used for mass calibration of theSEC columns were: bovine serum albumin (67 kDa)(purity 96 %), metallothionein I containing Cd, Cu,and Zn (7 kDa) (purity > 95 %), vitamin B12 (1.35kDa) (purity > 96 %), reduced glutathione (307 Da)(purity 98–100 %), and arsenobetaine (179 Da). Allthese reagents were purchased from Sigma–Aldrich(Steinheim, Germany). Standard stock solutions witha concentration of 10 mg mL−1 were prepared by dis-solving the respective compound in ultrapure water.Sodium arsenite and sodium arsenate, used for prepa-ration of arsenic(III) and arsenic(V) standards, re-spectively, by dissolution in water, and arsenobetaine(AsB) were purchased from Sigma–Aldrich (Stein-

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heim, Germany). Monomethylarsonic acid (MMAV)and dimethylarsinic acid (DMAV) were purchasedfrom Supelco (Bellefonte, USA). Tetramethylarson-ium ion (TMA+) and trimethylarsine oxide (TMAO)were supplied by Dr. Vélez (Instituto de Agroquímicay Tecnología de Alimentos (IATA-CSIC), Valencia,Spain). Individual working solutions for HPLC-ICP-MS analyses were prepared with a concentration of1 mg mL−1 of each of the compounds. Working solu-tions were prepared on the day of use by appropriatedilution of the stock solutions with water. The stocksolutions were stored at 4C in darkness.

The mobile phase used in SEC was 20 mM ofammonium acetate (pH 7.4) (Suprapur grade) pur-chased from Merck (Darmstadt, Germany). The voidvolume of the SEC column was determined by extrap-olation of the retention time of bovine serum albumin(BSA). The mobile phase was prepared on the day ofuse in ultrapure water (18 MΩ cm) obtained from aMilli-Q system (Millipore, Watford, UK) and the pHwas adjusted to pH 7.4 with ammonia solution (20mass %) (Suprapur, Merck, Darmstadt, Germany).The mobile phase used in the anion-exchange chro-

matography (AEC) was 30 mM of sodium dihydro-gen phosphate (p.a.) purchased from Sigma–Aldrich(Steinheim, Germany) and the pH adjusted to 6.0 byaddition of the appropriate volume of 20 mass % NH3aqueous solution. The mobile phase for the cation-exchange chromatography was 20 mM aqueous so-lution (Milli-Q water) of pyridine purchased fromSigma–Aldrich (Steinheim, Germany), with pH ad-justed to 2.5 with formic acid (> 98 %, Fluka, puriss,p.a.).

For total element determination, nitric acid (65mass %) and hydrogen peroxide (30 mass %) ofSuprapur grade (Merck, Darmstadt, Germany) wereused for mineralisation of the samples.

A cryogenic homogeniser SPEX SamplePrep, Free-zer/Mills 6770 (Metuchen, NJ, USA) was used to pre-pare a sample homogenate. Cytosolic extracts wereobtained from this homogenate using a Teflon/glasshomogeniser in a cold-chamber at 4C.

The extraction was followed by ultracentrifugation(ultracentrifuge Beckman model L9-90 K; rotor 70 Ti)using polycarbonate bottles of 10 mL with cap as-sembly (Beckman Coulter). Elemental detection was

Table 1. Operating conditions of HPLC-ICP-ORC-MS

SEC conditions

Column SuperdexTM-Peptide (300 mm × 10 mm × 13 µm)Resolution range < 10 kDa

Analytical columnMobile phase Ammonium acetate, 20 mmol L−1 (pH 7.4)

Flow-rate 0.7 mL min−1Injection volume 20 µL for liver and kidney extracts and 100 µL for brain extracts

Visible wavelength 254 nm

Column Hiload 26/60 Superdex 30 Prep (600 mm × 26 mm × 34 µm )Resolution range < 10 kDa

Preparative columnMobile phase Ammonium acetate, 20 mmol L−1 (pH 7.4)

Flow-rate 2 mL min−1Injection volume 500 µL

Visible wavelength 254 nm

IEC conditions

Column Hamilton PRP X-100 (250 mm × 4.6 mm × 5 µm)

Anion-exchange chromatographyMobile phase Sodium dihydrogen phosphate, 30 mM (pH 6)

Flow-rate 1 mL min−1Injection volume 20 µL

Column Supelcosil SCX (250 mm × 4.6 mm × 5 µm)

Cation-exchange chromatographyMobile phase Pyridine, 20 mM (pH 2.5)

Flow-rate 1.2 mL min−1Injection volume 20 µL

ICP-MS conditions

Forward power 1500 W He flow 3.9 mL min−1Plasma gas flow-rate 15.0 L min−1 Qoct –18 VAuxiliary gas flow-rate 1.0 L min−1 Qp –16 VCarrier gas flow-rate 0.9 L min−1 Dwell time 0.3 per isotopeSampling depth 8 mm Isotopes monitored 75AsSampling and skimmer cones Nickel – –

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Fig. 1. Sampling area in Donana National Park and its surroundings (SW Spain).

performed using an Agilent 7500ce ICP-MS spectrom-eter (Agilent Technologies, Tokyo, Japan) equippedwith an octopole collision cell. Chromatographic sep-arations were performed using a Model 1100 HPLCpump as the delivery system with a UV detector (Ag-ilent, Wilmington, DE, USA). The ICP-MS condi-tions (Table 1) in He mode were optimised using anaqueous solution of 1µg mL−1 of 75As with 2 vol. %HCl. The flow-rate of He collision gas was set at3.9 mL min−1 in order to avoid the interference of40Ar35Cl+. An AKTA-Prime system (pump and UVdetector at 254 nm) (Amersham Biosciences, Upp-sala, Sweden) was used as the eluent delivery system,equipped with a 2 mL sample loop.

Analytical SEC was performed with a SuperdexTM-Peptide (GE Healthcare, Uppsala, Sweden) column(10 mm × 300 mm, 13 µm) with an exclusion limit of10 kDa (with an effective separation range of 0.1–10kDa). Preparative SEC was carried out using a Hiload26/60 Superdex 30 Prep for separation range < 10kDa (low molecular mass (LMW)) (Amersham Bio-sciences, Uppsala, Sweden). Anion-exchange HPLCseparations were carried out using a Hamilton PRPX-100 (250 mm × 4.6 mm × 5 µm) column. Cation-exchange separations were carried out using a Supel-cosil LC-SCX (250 mm × 4.6 mm × 5 µm) column.

A MARS microwave-accelerated reaction system(CEM, Matthews, NC, USA) was used for minerali-sation of the samples.

Sampling area, animals, and sample prepara-tion

Free-living mice (Mus spretus) were collected be-tween November 10th and December 11th (2009) infive sampling areas from the Donana National Park(DNP) and its surroundings with different levels ofcontamination (Fig. 1). The sampling point site “Lu-

cio del Palacio” (LDP) was considered as the controlarea because it is located in the centre of the parkwhere the contaminants’ input is lower. In 2007, theWorld Wildlife Fund warned that strawberry farmssurrounding the park, where 95 % of Spanish straw-berries were produced, threatened to cause catas-trophic damage to the park by depleting the surround-ing groundwater, as well as creating considerable pes-ticide pollution; in addition, plastic cover waste fromthe crops was accumulated in the “La Rocina” stream(ROC). The other areas studied were: upstream anddownstream on the Partido (PAR and AJO, respec-tively) affected by citrus fruit, strawberry, and grapecrops; the “La Rocina”(ROC) was also affected byagricultural activities, and also by diffused pollutionfrom the petrochemical and chemical activities fromthe industrial belt of Huelva and mining activitiesfrom the north (Riotinto mine); and the “El Ma-tochal” (MAT) site next to the Guadiamar river withinputs from rice fields and metals from mining spillagecaused by the disaster in the Aznalcóllar mine, inwhich about 4 million cubic metres of acidic waterand 2 million cubic meters of mud containing toxicmetals, mainly zinc, lead, copper, arsenic, and aro-matic amines were released (Fernandez et al., 1992).The sampling sites are summarised in Table 2, whichincludes the codes of the samples, UTM coordinates,type of sample (reference/problem), and number ofmice of each sex sampled. Mus spretus mice werecaught using Sherman live-traps baited with hazel-nut cream over bread, which were mounted during theevenings and checked the next morning. Adult animalswere taken alive to a laboratory at Donana BiologicalReserve, and data on site/date of collection, sex, mass,and external measurements were recorded.

The mice were individually killed by cervical dislo-cation and dissected. Individual organs were excised,weighed in Eppendorf vials, cleaned with 0.9 mass %

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Table 2. Sites under study

UTM coordinates Number of miceArea (code) Type

X Y Males Females

Lucio del Palacio (LDP) 193800 4099515 Negative control 18 15La Rocina (ROC) 178653 4119937 Problem 7 4Ajolí (AJO) 192352 4124977 Problem 21 11Matochal (MAT) 208681 4102207 Problem 16 9Partido (PAR) 191173 4124977 Problem 9 8

NaCl solution, frozen in liquid nitrogen, and stored at–80C until they were used for extract preparation.Mice were handled in accordance with the standardsstipulated by the European Community. The investi-gation was performed after approval by the EthicalCommittee of the University of Huelva (Spain).

Samples preparation and determination of to-tal metal concentration

Livers, kidneys, and brains from 5 different malemice were pooled and treated following a proceduredescribed elsewhere (González-Fernández et al., 2011).Briefly, the samples were disintegrated by cryogenichomogenisation in a 6770 freezer/mill apparatus (1min at maximum rate). Then, the metal-biomoleculeswere extracted with a solution (3 mL per g of sam-ple) containing ammonium acetate buffer solution (20mM) at pH 7.4, GSH (1 mM), and phenylmethane-sulphonylfluoride (PMSF, 1 mM) using a glass/teflonhomogeniser. Finally, the extracts were centrifuged at120000g for 1 h at 4C and stored at –80C until anal-ysis.

The extracts were precisely weighed (0.200 g) in5-mL teflon microwave vessels and 640 µL of nitricacid and 160 µL of hydrogen peroxide was added. Themineralisation was carried out at 400 W from ambi-ent temperature increased to 160C within 15 min andheld at this temperature for 40 min. Then, the solu-tions were made up to 2 mL and analysed by ICP-MSusing rhodium (1 µg mL−1) as the internal standard.All the analyses were repeated three times.

Analysis of extracts with SEC coupled to ICP-MS

First, the extracts were filtered through Iso-Discpoly(vinylidene difluoride) filters (25 mm diameter,0.2 µm pore-size) to avoid column overloading or clog-ging and ultrafiltered with AMICON 30K (Millipore)by centrifugation at 10000g at 4C for 30 min. Analyti-cal SEC-ICP-ORC-MS online coupling was performedby connecting the outlet of the chromatographic col-umn to the Micromist nebuliser inlet (Glass Expan-sion, Switzerland) of the ICP-MS by means of a 30 cmPEEK tubing (0.6 mm i.d.). Preparative SEC-ICP-ORC-MS was performed with an analogous coupling

Table 3. Concentration of arsenic (ng g−1) in the extracts ofmice organs

Organ LDP AJO PAR MAT ROC

Liver < LOD < LOD 1.2 ± 0.2 1.6 ± 0.2 1.8 ± 0.3Brain < LOD < LOD < LOD < LOD < LODKidney < LOD 3.2 ± 0.5 2.4 ± 0.7 5.4 ± 1.1 4.3 ± 0.5

using a Babington nebuliser inlet (Agilent Technolo-gies, Japan). Collection of the fraction containing ar-senic metabolites was performed on the latter chro-matographic system without atomic detector, moni-toring the molecules output from the chromatographicsystem with a non-destructive UV detector.

Analysis of kidney extracts by IEC coupled toICP-MS

The fraction isolated by preparative SEC-ICP-ORC-MS was lyophilised and 10-times pre-concentra-ted with ultrapure water. Finally, this fraction wascentrifuged at 10000g at 4C for 10 min and stored at–80C until analysis. The IEC-ICP-ORC-MS onlinecoupling was performed by connecting the outlet ofthe chromatographic column to the Micromist nebu-liser inlet (Glass Expansion, Switzerland) of the ICP-ORC-MS by means of a 30 cm PEEK tubing (0.15 mmi.d.).

Results and discussion

Presence of arsenic in organ extracts

The amounts of arsenic present in the extractsfrom the different organs (liver, brain, and kidneys)of Mus spretus are shown in Table 3. Recovery exper-iments were performed by spiking the extracts with10 µg L−1 of arsenic. In all the samples, recoveriesare in the range of 80–110 %. Instrumental detectionlimit (LOD) was 0.046 µg L−1 As. The most signif-icant differences between the areas of study and theorgans were observed in the kidneys.

The greater presence of arsenic in the kidneys isremarkable. In the brain, the element was always un-der the limit of detection in the amount of extract

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Fig. 2. Arsenic metabolites in kidney and liver from Mus spretus recorded by SEC-ICP-ORC-MS: LDP kidney (A), LDP liver (B),AJO kidney (C), AJO liver (D), PAR kidney (E), PAR liver (F), MAT kidney (G), MAT liver (H), ROC kidney (I), ROCliver (J). For chromatographic conditions, see Table 1 (analytical column).

920 M. Á. García-Sevillano et al./Chemical Papers 66 (10) 914–924 (2012)

Fig. 3. Arsenic metabolites in brain from Mus spretus recorded by SEC-ICP-ORC-MS: LDP (A), AJO (B), PAR (C), MAT (D),ROC (E). For chromatographic conditions, see Table 1 (analytical column).

analysed (0.2 g). The highest concentration of arsenicwas found in the kidneys of mice caught in the areasof MAT, AJO, and ROC.

SEC profiles of arsenic compounds in cytosolicextracts from Mus spretus

The relative abundance of As-species in livers andkidneys from the sites under study was evaluated inthe cytosolic fraction of these organs by analyticalSEC-ICP-ORC-MS using a SuperdexTM-Peptide col-umn with effective range of separation < 10 kDa. Theresults are shown in Fig. 2.

The intensity of the signals of arsenic is higher

in the kidneys than in the liver. Two peaks relatedto low molecular mass metabolites can be observedin the liver and kidney extracts at 26 min and 27.5min of retention time. It is well known that inorganicarsenic in the liver of mammals goes through sev-eral stages of a biotransformation process that pro-duces organic arsenic species (mainly MMAV andDMAV) (Naranmandura & Suzuki, 2008), althoughAs-containing proteins are also present. These speciesof arsenic are finally excreted through the urine aspentavalent methylated arsenic forms (Mandal et al.,2001; Suzuki et al., 2002). This can explain the higherconcentration of arsenic metabolites in the kidneys inrelation to other organs.

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Fig. 4. Arsenic metabolites in kidneys from Mus spretuscaught in MAT recorded by SEC-ICP-ORC-MS. Forchromatographic conditions, see Table 1 (preparativecolumn).

The presence of two peaks is also observed in thebrain extracts from mice caught in the ROC and MATareas (Fig. 3). These signals can be observed when anextract’s volume of 100 µL is injected into the chro-matographic systems; a lower volume in a range of10–20 µL was used for analyses of liver and kidneyextracts. The presence of arsenic species in the brainother than DMAV can be explained by the presenceof other As metabolites in blood that cross the blood-brain barrier (BBB) when the capacity of methylationin the liver is exceeded (Vahter, 1981; Vahter & Norin,1980; Hughes et al., 2003). Experiments performedwith rats as a model of mammals revealed that Ascould cross the BBB and produce an increased quan-tity of reactive oxygen species (ROS) as well as causeoxidative stress (Piao et al., 2005).

The extracts of kidney from Mus spretus caughtin MAT were analysed by SEC using both typesof columns (analytical and preparative). The chro-matograms obtained by in-series UV and ICP-ORC-MS detection are shown in Fig. 4. The arsenic fraction-ation profile obtained with the LMM column showsone peak using the ICP-ORC-MS detection, whichis associated with the molecular mass range of 100–400 Da according to mass calibration. However, con-firmation of specific arsenic-metabolites is not possibleusing only a SEC separation (Michalke & Schramel,2004) and a complementary IEC method was appliedfor further purification of the arsenic SEC fraction.

Arsenic speciation by IEC-ICP-ORC-MS

The As-peak with the highest intensity in theSEC-ICP-ORC-MS chromatogram corresponds to thefraction with retention time of 100–123 min. Thisfraction was collected from 2 mL of the kidney ex-tract of mice caught in each area of study and sub-sequently submitted to a multi-dimensional orthogo-nal chromatographic separation based on anion- andcation-exchange interactions, monitoring the presenceof As in the eluent by ICP-ORC-MS. Chromatogramsof the arsenic standards used for calibration withtwo columns are shown in Fig. 5. Fig. 6 shows thechromatograms obtained from the kidney extracts ofmice from MAT spiked with 50 µg L−1 of each ar-senic species, using either an anion-exchange column(Fig. 6C) or a cation-exchange column (Fig. 6D).

The results obtained by IEC show that methylatedmetabolites (MMA and DMA) are the predominantspecies in the kidneys. The higher intensity of MMAand DMA in the kidneys of mice from contaminated

Fig. 5. Chromatogram of As standards with PRP-X100 column (iAsIII, iAsV, DMAV, and MMAV). For chromatographic con-ditions, see Table 1 (anion-exchange chromatographic column) (A); chromatogram of As standards with Supelcosil SCXcolumn (iAsIII, iAsV, AsB, DMAV, MMAV, TMAO, and TMA). For chromatographic conditions, see Table 1 (cation-exchange chromatographic column) (B).

922 M. Á. García-Sevillano et al./Chemical Papers 66 (10) 914–924 (2012)

Fig. 6. Speciation of arsenic metabolites in kidney from Mus spretus caught in different areas of study by IEC-ICP-ORC-MS:kidney (- -) and kidney MAT (—) extracts from Mus spretus spiked with 50 µg L−1 of each of the following arsenicals:iAsIII, iAsV, DMAV, and MMAV). For chromatographic conditions, see Table 1 (anion-exchange chromatographic column)(A); kidney (- -) and kidney MAT (—) extracts from Mus spretus spiked with 50 µg L−1 of each of the following arsenicals:iAsIII, iAsV, AsB, DMAV, MMAV, TMAO, and TMA. For chromatographic conditions, see Table 1 (cation-exchangechromatographic column) (B); As-metabolites in kidney extracts from Mus spretus under anion-exchange conditions (C);As-metabolites in kidney extracts from Mus spretus under cation-exchange conditions (D); for C and D: LDP (– – –), AJO(---), PAR (- - -), MAT (—) (black), ROC (—) (grey).

sites such as ROC, MAT, and AJO (Fig. 6C) contrastswith the lower content of these metabolites in the ar-eas with low contamination (LDP and PAR). Thiscan be related to the significant amount of arsenic inROC, affected by the diffuse contamination of this ele-ment transported in lixiviates from pyrite mines wastelocated in the north-east of Huelva province (Riot-into mine). In MAT, the proximity of the Guadiamarstream introduces arsenic and other metals from theAznalcóllar mine tailing pond where rupture of the up-stream dam occurred in 1998 (Grimalt et al., 1999).In addition, the concentration of arsenic in interstitialwaters from Guadiamar sediments is very remarkable(Tovar-Sanchez et al., 2006). These issues explain thehigher presence of arsenic species in these samplingsites. In PAR and LDP (uncontaminated areas), anal-ogous peaks are observed but with lower intensity.

When cation-exchange chromatography is used

(Fig. 6D), a peak at 4 min can be observed, whichis not associated with the presence of arsenobetaine,although its retention time closely matches that ofthe corresponding standard of this arsenic species be-cause this species is frequently found in aquatic organ-isms and marine mammals (Edmonds & Francesconi,1981, 1987). However, other arsenic species can alsobe related to this peak due to their similar chromato-graphic behaviour, such as dimethylarsinoylethanol(DMAE), a metabolite present in human urine and inurine from experimental animals (Francesconi et al.,2002), and thioarsenic species such as monomethyl-monothioarsonic acid (MMMTAV), dimethylmonoth-ioarsonic acid (DMMTAV), and dimethyldithioarsonicacid (DMDTAV) (Suzuki et al., 2010). In addition,an unidentified peak at about 7 min can be relatedto the presence of arsenocholine (AsC) according tothe retention time using the same column and under

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the identical chromatographic conditions reported byChatterjee (2000). This metabolite is present at thehighest concentration in mice from MAT and AJO(Fig. 6D).

Conclusions

The use of SEC-ICP-MS coupling is a good choiceto assess changes in the regulation of metal-bindingbiomolecules in environmental bio-indicators (e.g.Musspretus) caused by contamination episodes. The firstapplication of this arsenic speciation approach to thebio-indicator Mus spretus has provided promising re-sults in the assessment of environmental stress re-sulting from pollution. However, further research isneeded to separate SEC peaks containing arsenic inthe liver, brain and plasma and to unequivocally iden-tify unidentified peaks by organic mass spectrometry(Qq-TOF-MS).

Acknowledgements. This work was supported by the pro-jects: CTM-2009-12858-C02 01 (Ministerio de Ciencia eInnovación-Spain), P08-FQM-03554 and P09-FQM-04659(Consejería de Innovación, J. A). M. A. García-Sevillanowishes to thank Ministerio de Educación for a PhD scholar-ship (FPU). M. González-Fernández wishes to thank Ministe-rio de Ciencia e Innovación for a pre-doctoral scholarship. R.Jara-Biedma wishes to thank Consejería de Innovación Cien-cia y Empresa (Junta de Andalucía) for a pre-doctoral schol-arship. We are grateful to Dr. Vélez (Instituto de Agroquímicay Tecnología de Alimentos (IATA-CSIC), Valencia, Spain) forsupplying the arsenic standards.

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