Arsenic speciation in environmental and biological samples extraction and stability studies

Arsenic speciation in environmental and biological samples extraction and stability studies

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 1 M 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 1 M 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

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 (␮g kg−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 HNO3 and H2O2 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 0.5 mm) The chromato-graphic conditions used for As species separation and quantification and the instrumental parameters used

Table 1

Instrumentation parameters

HG-AFS

NaBH 4 concentration 1% m v −1

HCl concentration 1.5 M

NaBH4 flow rate 1.0 ml min −1

HCl flow rate 1.5 ml min −1

Flow rate of sample 0.8 ml min −1

H 2 flow to feed

diffusion flame

60 ml min −1

Ar carried gas flow 200 ml min −1

Ar auxiliary gas flow 100 ml min −1

Primary current 27.5 mA

Boost current 35 mA

ICP-MS

Reflected: 2.2 W

Ar flow rate Coolant: 14 l min −1

Nebulizer: 1.0 l min −1

Auxiliary: 0.9 l min −1

Measurement mode Peak area of75As

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, 250 mm × 4.1 mm)

Guard column Hamilton PRP-X100 4.6 mm

Mobile phase 10 mM PO 4 −3, pH 6.0

Cationic column Hamilton PRP-X200

(10␮m, 250 mm × 4.1 mm)

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 ml m −1

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 HNO3 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 100 mg l−1 arsenic were pre-pared from CH3AsO3Na2 (MMA), Merck, 98%, C2H6AsNaO2·3H2O (DMA), Fluka, 98%, NaAsO2 (As(III)) and Na2HAsO4·7H2O (As(V)) Sigma– Aldrich, 100%, C3H6AsCH2COOH (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 K2S2O8 (Fluka, 99.5%) was prepared in NaOH (Suprapur, Merck) H2SO4(Suprapur, 96%, Merck) and NaBH4 (Fluka, 98%) in NaOH, used for reduction, were pre-pared daily H3PO4 and HClO4 were obtained from Merck

The chromatographic mobile phase was 10 mM am-monium dihydrogen phosphate (Merck), adjusted to

pH 6.0 with 0.1% NH4OH (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

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 0.5 g of the sample was placed in a PTFE

reactor, 10 ml of concentrated HNO3were added and

the reactor was covered and pre-digested overnight

Next, 20 mg of Na2S2O8 and 3 ml of HClO4 (or

0.3 ml of concentrated HF in rice) were added and

heated to 150◦C for 3 h in an oven After cooling,

0.5 ml of concentrated H2SO4 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 10 ml 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, 0.5 g of the sample was placed in a

PTFE reactor and 10 ml of 1:1 HNO3: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 10 ml 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, 0.3 g of the test material was placed

in a Teflon reactor and 10 ml of 1 M of phosphoric acid were added The mixture was heated at 150◦C for 3 h and the resultant extract evaporated to dryness The residue was dissolved with 25 ml of 10 mM 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

inTable 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 inTable 1 The arsenic species were quantified by measurement of the peak area by ICP-MS 10␮g l−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

40Ar35Cl and40Ar37Cl (m/z 75 and 77) that the

pres-ence of chloride does not interfere because of its low concentration in all the extracts

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 efficiencynumber of extraction Water Methanol:water 1 M H3P04

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 –

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 –

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 –

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

Certified values of NIST 1568a (0.29 ± 0.03 ␮g g−1 As) and DORM-2 (18.0 ± 1.1 ␮g g−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 H3PO4

(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 H3PO4 as an As extractant for soil is much higher

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−1of 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 ␮g l −1 of AsB and 5␮g l −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 inTable 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-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 3shows the extraction efficiency of each As species in chicken in the three consecutive extractions for all extractants The recovery of each As species

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%),

Table 3

Efficiency of species extraction in chicken± expressed as percent ¯x ± s) referring to total content in the corresponding extract

Unknown peak 33.8 ± 2.8 33.0 ± 2.0 34.0 ± 2.5 33.4 ± 2.0

Unknown peak 35.0 ± 2.0 28.0 ± 1.6 35.0 ± 2.0 28.4 ± 2.0

As(III), DMA and MMA (Fig 5a).Fig 5bshows 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 in the rice, chicken and fish samples

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

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

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

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