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: (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
I. Pizarro et al. /Analytica Chimica Acta 495 (2003) 85–98 95
values (Table 2). The As species concentration found
in Dorm-2 (AsB: 15.84 ± 0.5 ␮gg

−1
As; DMA:
0.66 ± 0.04 ␮gg
−1
As) was close to those certified
values (AsB: 16.4 ± 0.5 ␮gg
−1
As). The As species
concentration for NIST 1558 (As(III) 75␮gg
−1
,
As(V) 12␮gg
−1
, DMA 180␮gg
−1
, MMA 9␮gg
−1
As) were close to the values reported in literature [15]
(inorganic As 92, DMA 174, MMA 8␮gg
−1
As).
Furthermore, the recovery test with known amounts
of As species (at an appropriate concentration level)
Fig. 5. HPLC–ICP-MS chromatograms (anionic column) for soils. (a) Soil 1 and (b) soil 2.
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 knowl-

edge of species stability in both raw material and
extracts.
96 I. Pizarro et al. /Analytica Chimica Acta 495 (2003) 85–98

0 100 200 300 400 500 600 700
800
1.0E7
2.0E7
sec->
As(III)
As(V)
Signal /Counts
Time/s
(b)
Fig. 5. (Continued ).
In order to evaluate how long the extracts could be
stored without the risk of any species loss or transfor-
mation, an evaluation was performed of the stability
of the As species in the different extracts (water,
methanol:water mixtures and phosphoric acid). The
extracts were stored at 4
◦
C in the dark and were
analyzed immediately and up to three months af-
ter preparation. Although the 1:1 methanol:water
mixture was initially chosen as the most appropri-
ate extract mixture for the As species from rice,
chicken, and fish, and 1M H
3
PO

4
for soil, the stabil-
ity study was carried out in each extractant previously
evaluated.
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) concen-
tration and a decrease in As(III) due to a gradual ox-
idation of As(III) to As(V). This fact was previously
0
1
2
3
4
5
6
7
8
9
01234
Time (months)
x ± s.d (10-2)mg/kg
DMA water
AsBet water
DMA m:w 1:1

AsBet m:w 1:1
Fig. 6. Stability of arsenic species in chicken extracts.
I. Pizarro et al. /Analytica Chimica Acta 495 (2003) 85–98 97
0
1
2
3
4
5
6
01234
Time (months)
x ± s.d (10-2) mg/kg
As (III) w ater
As (V) w ater
As (III) m:w 1:1
As(V)m:w 1:1
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 un-
known arsenic species detected was stable in all ex-
tracts for at least three months. However, DMA and
AsB were less stable in water than in methanol:water
mixtures (Fig. 7). The concentration of AsB de-
creased slightly after two months’ storage in all
methanol:water mixtures tested. On the other hand,

the concentration of DMA species increased after two
months.
0123
0
50
400
450
500
550
As(III) water
As(III) phosphoric acid 1M
As(V) water
As(V) phosphoric acid 1M
x s.d (mg/kg)
Time (months)
+
-
4
Fig. 9. Stability of arsenic species in soil extracts.
0
10
20
30
40
50
60
024
Time (months)
x ± s.d (mg/kg)
AsBet water

U. Peak water
AsBet m:w 1:1
U.Peak m:w 1:1
AsBet m:w 9:1
U.Peak m:w 9:1
Fig. 8. Stability of arsenic species in fish extracts.
4.3.3. Fish
The stability study of As species in fish tissue ex-
tracts showed a general tendency in AsB to transform
into the unknown species detected (possible TMAO).
This transformation is more acute as the methanol
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, 1M H
3
PO
4
is the most ef-
fective extractant (99% extraction efficiency) for As
species in soil. It has been observed that DMA and
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 one month (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 differ-
ent 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 the most adequate mixture
for all cases.
Three consecutive extractions provided a quantita-
tive recovery of As species from all matrices tested,
except for chicken (75%). However, for rice an ac-
ceptable recovery can be achieved with only one
extraction step, whereas for fish or chicken two or
three extraction steps, respectively, are recommended.
1MH
3
PO
4
is recommended as an extractant for soil
providing an 82% recovery with only one extraction
run.
The sum of the As species found represents 100%
of 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 mixtures. After two
months, a slight conversion of 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), com-
pared to the 1:1 methanol:water mixture. The transfor-
mation 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 extractants for
at least the three-month storage period, while As(III)
is gradually oxidized to As(V).
Acknowledgements
This research was conducted under DGICYT
Project PB98-07-68.
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