Talanta 58 (2002) 7796

Arsenic speciation analysis

Zhilong Gong, Xiufen Lu, Mingsheng Ma, Corinna Watt, X. Chris Le *
En6ironmental Health Sciences Program, Department of Public Health Sciences, Faculty of Medicine,
10102 Clinical Sciences Building, T6G 2G3 Edmonton, Alberta, Canada
Received 8 February 2002; received in revised form 29 April 2002
Abstract
Nearly two dozen arsenic species are present in the environmental and biological systems. Differences in their
toxicity, biochemical and environmental behaviors require the determination of these individual arsenic species.
Considerable analytical progresses have been made toward arsenic speciation analysis over the last decade.
Hyphenated techniques involving a highly efcient separation and a highly sensitive detection have become the
techniques of choice. Methods based on high-performance liquid chromatography separation with inductively coupled
plasma mass spectrometry, hydride generation atomic spectrometry, and electrospray mass spectrometry detection
have been shown most useful for arsenic speciation in environmental and biological matrices. These hyphenated
techniques have resulted in the determination of new arsenic species, contributing to a better understanding of arsenic
metabolism and biogeochemical cycling. Methods for extracting arsenic species from solid samples and for stabilizing
arsenic species in solutions are required for obtaining reliable arsenic speciation information. 2002 Elsevier Science
B.V. All rights reserved.
www.elsevier.com/locate/talanta
1. Introduction
Arsenic is the twentieth most abundant element
in the earths crust. Many arsenic compounds are
present in the environment and in biological sys-
tems (Table 1). It has long been realized that the
determination of total arsenic concentration is
insufcient for clinical and environmental consider-
ations. The toxicity of arsenic is dependent on the
chemical species present. The improvements of
analytical techniques have continued to further our
understanding of arsenic biogeochemistry, toxicity,
and metabolism. These techniques have provided
information on biomarkers of exposure and arsenic
cycling in the natural environment. Arsenic specia-
tion studies can help make more accurate assess-
ments of environmental impact and health risks.
Knowledge of the speciation of arsenic in natural
water is important because the bioavailability and
the physiological and toxicological effects of ar-
senic depend on its chemical form. There is consid-
erable information regarding the speciation of
arsenic in water [14]. There are also reports of
unidentied arsenic species in aquatic systems [5
10]. The identication of these species may offer
important details regarding the arsenic speciation
in natural waters and biological production in
organisms.
* Corresponding author. Tel.: +1-780-492-6416; fax: +1-
780-492-7800
E-mail address: xc.le@ualberta.ca (X.C. Le).
0039-9140/02/$ - see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S0039- 9140( 02) 00258- 8
Z. Gong et al. / Talanta 58 (2002) 7796 78
In addition to drinking water, humans are ex-
posed to a considerable amount of arsenic
through food [1]. Food products from the marine
environment contain the highest concentration of
arsenic [13,6]. Many species of arsenic have been
detected (Table 1); however, there is concern that
the methods of extraction may not be efcient or
may destroy the original species present. Little is
known about arsenic speciation in most foods we
eat.
Urinary excretion is the major pathway for the
elimination of arsenic compounds from the body.
Arsenic concentrations in urine have a short half-
life and represent recent exposure. Arsenic specia-
tion in urine has been considered a biomarker of
exposure. In recent years, methylated trivalent
arsenic metabolites have been detected in human
urine [1114]. Some studies have shown that these
metabolites may be more toxic than inorganic
arsenic [1517]. The biomethylation of arsenic in
humans had previously been considered to be a
detoxication process. It is therefore important to
develop techniques that can determine all of the
arsenic methylation metabolites and intermedi-
ates. This information may provide clues to the
metabolism of certain arsenic species in humans.
Blood is a more difcult matrix than urine for
speciation analysis, and so until recently, only
total arsenic concentrations in whole blood were
reported [1]. Inorganic arsenic, dimethylarsinic
acid (DMA
V
) and arsenobetaine (AsB) have been
identied in serum. In the serum of uraemic pa-
tients, DMA
V
was the predominant arsenic spe-
cies detected [18].
The techniques used for the detection of arsenic
species in environmental and biological samples
should be sensitive and selective. The rapid analy-
sis of samples to prevent species conversion is also
important. Inductively coupled plasma-mass spec-
trometry (ICPMS) has become a favored detec-
tion technique in arsenic analysis [19]. It provides
ultra-sensitivity, multi-element capability and can
be combined with the separation techniques for
speciation analysis. The multi-element capability
allows for the simultaneous determination of dif-
ferent elements in addition to arsenic. Another
common technique used in arsenic speciation is
hydride generation (HG) [1,19]. HG allows for
extremely low detection limits. However, not all
arsenic species form hydrides, and decomposition
techniques are usually required.
A combination of analytical techniques is often
necessary to achieve both selectivity and sensitiv-
ity. The direct coupling of a separation device to
Table 1
Arsenic species commonly detected in the environmental and
biological systems
Name Abbreviation Chemical formula
As(OH)
3
Arsenite (arsenous As
III
acid)
As
V
AsO(OH)
3
Arsenate (arsenic
acid)
Monomethylarsonic CH
3
AsO(OH)
2
MMA
V
acid
Monomethylarsonous MMA
III
CH
3
As(OH)
2
acid
DMA
V
Dimethylarsinic acid (CH
3
)
2
AsO(OH)
Dimethylarsinous DMA
III
(CH
3
)
2
AsOH
acid
DMAE (CH
3
)
2
AsOCH
2
Dimethylarsinoyl
ethanol CH
2
OH
TMAO (CH
3
)
3
AsO Trimethylarsine
oxide
(CH
3
)
4
As
+
Me
4
As
+
Tetramethylarsonium
ion
Arsenobetaine (CH
3
)
3
As
+
CH
2
AsB
COO

(CH
3
)
3
As
+
CH
2
AsB-2 Arsenobetaine 2
CH
2
COO

Arsenochline (CH
3
)
3
As
+
CH
2
AsC
CH
2
OH
Trimethylarsine TMA
III
(CH
3
)
3
As
Arsines AsH
3
, MeAsH
2
, (CH
3
)
x
AsH
3x
(x=03) Me
2
AsH
Et
x
AsMe
3x
Ethylmethylarsines (CH
3
CH
2
)
x
As
(CH
3
)
3x
(x=03)
Phenylarsonic acid C
6
H
5
AsO(OH)
2
PAA
Arylarsenicals used as animal feed additi6es
p-ASA NH
2
C
6
H
4
AsO p-Arsanilic aicd
(OH)
2
NO
2
C
6
H
4
AsO 4-Nitrophenylarsonic 4-NPAA
acid (OH)
2
NO
2
(OH)C
6
H
4
3-NHPAA 4-Hydroxy-3-nitrophe
AsO(OH)
2
nylarsonic acid
p-Ureidophenylarson NH
2
CONHC
6
H
4
p-UPAA
AsO(OH)
2
ic acid
Arsenic-containing Arsenosugars See Scheme 1
XXVI ribosides
Z. Gong et al. / Talanta 58 (2002) 7796 79
various detection instruments enables improved
specicity and detection for individual arsenic
species. Hyphenated techniques allow for the pos-
sible separation of all soluble species in the sam-
ple and selective detection at low concentrations.
High-performance liquid chromatography
(HPLC) is frequently used as the separation tech-
nique in arsenic speciation [1,19,20].
The separation and detection techniques used in
arsenic analysis are only as reliable as the sample
procedure used. Species instability during sam-
pling, storage, and sample pretreatment are all
very important issues that must be considered.
The knowledge of the stability of species exam-
ined under different conditions is necessary. Spe-
cies may be converted from one form to another
or lost from the sample [21]. The optimal storage
conditions, the maximum length of storage with-
out a signicant risk of transformation of species,
and whether the extraction method produces any
transformation of the species present in solution
must be determined. If the original distribution of
the species in the sample is destroyed, the result of
speciation analysis is questionable. The extraction
of arsenic from solid samples is another area
where care must be taken. The methods for ex-
traction must be efcient and minimize the de-
struction of the arsenic species present in the solid
materials.
The instability of arsenic species in water sam-
ples is also very important. In groundwater, arse-
nate (As
V
) has commonly been reported as the
predominant water-soluble species in groundwa-
ter. However, the procedures used for sample
handling and analysis may result in the oxidation
of arsenite (As
III
) to As
V
. The changes of sample
conditions from the eld to the laboratory envi-
ronment can lead to alterations of chemical spe-
cies in the original sample. If no reliable sampling
techniques can be found, then on-site speciation
analysis may be necessary. For water analysis,
portable methods continue to be developed that
are able to analyze samples in the eld. Field
methods allow for rapid analysis and species
conservation.
There have been previous reviews on arsenic
speciation analysis and metal speciation analysis
in general [1,19,20,22,23]. This paper reviews re-
Scheme 1.
cent research on arsenic speciation analysis in
various environmental and biological samples.
The techniques of separation, detection and sam-
ple handling will be described.
2. Separation techniques
The most commonly used speciation techniques
often involve a combination of chromatographic
separation with spectrometric detection. This sec-
tion describes separation techniques for arsenic
speciation. HPLC is most commonly used, while
gas chromatography (GC), supercritical uid
chromatography (SFC) [24,25] and capillary elec-
trophoresis (CE) [25,26] have also been applied to
arsenic speciation analysis to a lesser extent. The
main forms of HPLC separation for arsenic speci-
ation analysis are described below, including ion-
pairing, ion exchange, and size exclusion.
2.1. Ion-pair chromatography
Ion-pair chromatography has been developed
for routine analysis of neutral and ionic arsenic
species. Recent publications on arsenic speciation
using ion-pair chromatography are summarized in
Table 2. Previous applications of ion-pair chro-
matography to the separation of arsenic species
can be found in an earlier review [20].
Both anion-pairing and cation-pairing chro-
matography techniques have been developed for
the separation of arsenic species. Tetrabutylam-
Z. Gong et al. / Talanta 58 (2002) 7796 80
T
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+
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m
)
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r
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V
6
.
2
8
1
.
2
0
P
h
e
n
o
m
e
n
e
x
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D
S
(
3
)
A
s
I
I
I
,
A
s
V
,
M
M
A
V
,
[
3
0
]
U
r
i
n
e
5
m
M
T
B
A
H
,
3
m
M
H
G
A
F
S
(
1
5
0

4
.
6
m
m
,
3
m
m
)
D
M
A
V
,
M
M
A
I
I
I
m
a
l
o
n
i
c
a
c
i
d
,
5
%
m
e
t
h
a
n
o
l
,
p
H
5
.
8
5
1
.
2
0
H
G
A
F
S
A
s
I
I
I
,
A
s
V
,
M
M
A
V
,
U
r
i
n
e
[
1
2
]
P
h
e
n
o
m
e
n
e
x
O
D
S
(
3
)
5
m
M
T
B
A
H
,
3
m
M
m
a
l
o
n
i
c
a
c
i
d
,
5
%
(
1
5
0

4
.
6
m
m
,
3
m
m
)
D
M
A
V
,
M
M
A
I
I
I
,
D
M
A
I
I
I
m
e
t
h
a
n
o
l
,
p
H
5
.
8
5
Z. Gong et al. / Talanta 58 (2002) 7796 81
monium (TBA, both hydroxide and phosphate) is
the common pairing cation for separating As
III
,
As
V
, MMA
V
and DMA
V
(Table 2). The elution
order is consistently As
III
, DMA
V
, MMA
V
and
As
V
, independent of the various reverse-phase
columns used for the separation. The resolutions
of these arsenic species depend on the concentra-
tion of the ion-pair reagent, the ow rate, ionic
strength, and pH of the mobile phase [27,28]. The
optimum pH range for separating the four arsenic
species is between 5.0 and 7.0. In this pH range,
As
III
(pK
a
=9.2) is a neutral species, which is
eluted in the void volume. As
III
becomes a nega-
tively charged species as the pH of the mobile
phase is increased above its pK
a
value of 9.2.
Using a resin-based column and a mobile-phase
pH of 9.0, As
III
is weakly retained and can be
separated from AsB. A zwitterion, AsB is not
retained under the anionic-pairing chromato-
graphic conditions. Thus, AsB was separated
from As
III
[29].
The speciation of arsenic metabolites was ex-
tended to include two key arsenic biomethylation
intermediates, monomethylarsonous acid
(MMA
III
) and dimethylarsinous acid (DMA
III
), in
human urine samples [12,30]. Ion-pair chromatog-
raphy separation was performed on a reversed-
phase column (ODS-3) using a mobile phase
containing 5 mM tetrabutylammonium hydrox-
ide, 3 mM malonic acid and 5% methanol at pH
5.85. MMA
III
, DMA
III
, and four other arsenic
species usually present in human urine were sepa-
rated and detected within 7 min. A post-column
HGatomic uorescence system was used for de-
tection. The method has been used routinely for
toxicological and epidemiological studies of ar-
senic [11,31,32].
For the speciation of a large number of envi-
ronmental and biological samples, a high-
throughput routine analytical method is needed.
The separation of As
III
, As
V
, MMA
V
and DMA
V
usually required 810 min when a conventional
30 cm column was used. However, near-baseline
resolution of the four species was achieved within
2 min by using two guard columns or 4 min by
using a 15 cm column (3 mm ODS-3) [33]. Using a
narrow bore column for ion-pair chromatogra-
phy, the four arsenic species were baseline-re-
solved within 2 min [27].
Another ion-pairing chromatographic system
using tetraethylammonium hydroxide (TEAH) as
the ion-pair reagent was developed for the separa-
tion of AsB and arsenosugars [34,35]. Applica-
tions included analysis of human urinary arsenic
metabolites following ingestion of seaweed [36]
and studies of arsenic species in the environment
[37].
The cationic arsenic species were separated us-
ing pentanesulfonate [38,39], hexanesulfonate
[40,41], heptanesulfonate [35,42] and dodecylsul-
fonate [43] as the pairing anions. A mixed ion-pair
mobile phase containing 10 mM hexanesulfonate
and 1 mM TEAH was used to separate As
III
, As
V
,
MMA
V
, DMA
V
, AsB, arsenocholine (AsC), and
tetramethylarsonium ion (Me
4
As
+
) on a single
reverse-phase C18 column [40,44]. These seven
arsenic species were detected using on-line mi-
crowave oven digestionHGatomic uorescence
[40,44].
2.2. Ion-exchange chromatography
Both anion and cation-exchange chromatogra-
phy techniques [14,4549] have been used for
arsenic speciation analysis (Table 3). Depending
on the ionic characteristics of the arsenic com-
pounds, anion exchange was the most commonly
used to analyze As
III
, As
V
, MMA
V
and DMA
V
,
whereas cation exchange was used to separate
AsB, AsC, trimethylarsine oxide (TMAO) and
Me
4
As
+
species. The arsenic species separated are
listed in Table 3 according to their elution order.
A polymeric anion-exchange column (Hamilton
PRP X100) that is stable under a wide range of
pH (from 1 to 13) has proven useful for arsenic
speciation. Similar to anion-pairing chromatogra-
phy, AsB and As
III
co-eluted at the void volume
under the neutral pH conditions. However, As
III
could form a complex when tartaric acid was used
as the mobile phase. As
III
formed an anionic
complex species, which could be separated from
AsB. Six arsenic species were separated within 15
min in the order of AsC, AsB, DMA
V
, MMA
V
,
As
III
and As
V
[50]. Increasing the pH to 9.0 also
allowed for the separation of AsB from As
III
using 30 mM ammonium carbonate as the mobile
phase. The whole separation required 20 min [39].
Z. Gong et al. / Talanta 58 (2002) 7796 82
T
a
b
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%
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,
p
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8
.
8
Z. Gong et al. / Talanta 58 (2002) 7796 83
T
a
b
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3
(
c
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Z. Gong et al. / Talanta 58 (2002) 7796 84
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Z. Gong et al. / Talanta 58 (2002) 7796 85
With a gradient elution using varying concentra-
tions of ammonium phosphate as the mobile
phase, a baseline resolution of AsB and As
III
was
achieved [51]. Alternatively, oxidation of As
III
to
As
V
prior to HPLC separation removed the inter-
ference of As
III
in the determination of AsB [52].
Separation of AsB, As
III
, As
V
, MMA
V
and
DMA
V
was achieved on another ion-exchange
column (Dionex IonPac AS 14) by gradient elu-
tion with 2 mM tetramethylammonium hydroxide
and 10 mM ammonium carbonate binary mobile
phase [45]. The ve species were baseline-resolved
within 10 min. For the analysis of urine samples,
a 1:5 dilution of the samples was needed to reduce
interference from the urine sample matrix [45]. In
another study, MMA
III
and DMA
III
were sepa-
rated within 10 min on a Shodex Asahipak ES-
502N 7C anion-exchange column by using citric
acid (pH adjusted to 2 with nitric acid) as the
mobile phase [14]. This method was also used to
determine arsenic species in over 400 human urine
samples from an arsenic-affected area in West
Bengal, India [14].
Cation-exchange chromatography has been
shown useful for the separation of AsB, AsC,
TMAO and Me
4
As
+
species [42,48,49,53,54]. Re-
cently, a cation-exchange method was developed
for the determination of DMA
III
[55]. Cation
exchange was also used to separate arsenosugars
and their metabolites [46,47].
2.3. Ion-exclusion and size-exclusion (gel
permeation) chromatography
Ion-exclusion chromatography involves the use
of strong anion- or cation-exchange resins for the
separation of weakly ionized or neutral com-
pounds. In this mode of chromatography, the
charge on the ion-exchange resin is the same as
that of the weakly ionized species [56]. Negatively
charged arsenic species are separated using resin-
containing anionic sulfonate functional groups.
Ion-exclusion chromatography has three types of
interactions, ion exclusion, ion exchange, and hy-
drophobic interaction, which are suitable to sepa-
rate various arsenic species [57]. Excellent
separation of As
V
, MMA
V
, DMA
V
, As
III
, and
AsB was achieved on a carboxylated methacrylate
resin [58]. TMAO and AsC were not resolved
from each other. A drawback of this method is
that each separation of the eight arsenic species
took more than an hour, although the rst ve
arsenic species eluted within 13 min.
Both low-pressure and high-pressure size-exclu-
sion chromatography techniques have been
demonstrated for arsenic speciation. Low-pressure
size exclusion was usually used to remove large
matrix molecules such as proteins from serum
[59,60] and biological extract [61]. Size-exclusion
HPLC was typically used in conjunction with
other separation techniques to identify organic
arsenic species in organisms [34,6163].
2.4. Multidimensional chromatography
Multiple columns and separation modes have
been combined to attempt the separation of a
range of arsenic species. The multidimensional
separations have been carried out either off-line
or on-line. Geiszinger et al. [64] determined AsB,
TMAO, AsC and Me
4
As
+
by using cation ex-
change and then determined As
III
, As
V
, MMA
V
and DMA
V
using anion exchange. Several arseno-
sugars were also determined by either cation- or
anion-exchange chromatography [64].
Several on-line column switching systems have
been described [57,61,64,65]. These typically in-
volved a cation-exchange column and an anion-
exchange column connected via a switching valve.
The combination allowed the separation of both
cationic and anionic arsenic species. Applications
were demonstrated for the determination of wa-
ter-soluble arsenic species in seafood samples
[61,66].
2.5. CE
CE has been tested repeatedly for elemental
speciation [67,68] including arsenic speciation
[6979] because of its high separation efciency.
As
III
, As
V
, DMA
V
, MMA
V
, AsB, and AsC were
separated by using capillary zone electrophoresis
(CZE) interfaced with ICPMS detection [76].
Buffer constituents, concentration and pH affect
the separation of arsenic species. As
V
either could
not be analyzed (at alkaline pH) or suffered from
long analysis time (at acidic pH) [70].
Z. Gong et al. / Talanta 58 (2002) 7796 86
Negative voltage separation mode could be
used when the electroosmotic ow (EOF) was
reversed or suppressed by using EOF modiers
[71] or by using a coated capillary [73]. In this
case, EOF and anions all migrated to the detec-
tion end. Van Holderbeke et al. described a
method to separate As
III
, As
V
, DMA
V
, MMA
V
AsB and AsC by using negative voltage at the
injection inlet [72]. The six species were separated
in 20 min. This analysis time was reduced to 10
min when a positive pressure was applied to the
inlet vial during the electrophoresis [72]. While a
positive pressure could shorten the migration
time, a loss of separation resolution was observed
[73].
Most of the CE methods for the separation of
arsenic deal with standard solutions. However,
when used for actual sample analysis, the shifts in
migration time were observed [74]. Although
some progress has been made to reduce matrix
interference, CE separation methods for arsenic
speciation have been mostly limited to pure stan-
dard solutions or simple matrix systems. In addi-
tion, concentration detection limits for CE
methods were inferior to the HPLC methods
when the same detection systems were used.
2.6. Other separation techniques
The HGcryogenic trapping GC method for
arsenic speciation was the rst environmental ar-
senic speciation method [80]. Several arsenic spe-
cies can form arsines upon treatment with sodium
borohydride in an acid medium: As
III
and As
V
give AsH
3
, MMA gives CH
3
AsH
2
, and DMA
gives (CH
3
)
2
AsH. Because the boiling points of
these arsines are different (C) AsH
3
, 55;
CH
3
AsH
2
, 2; and (CH
3
)
2
AsH, 35.6the arsenic
species can be differentiated by using HG with
cryogenic trapping and GC [81,82]. Arsines pro-
duced in a reaction vessel were swept into and
trapped in a U-shaped tube immersed in liquid
nitrogen. After complete trapping of arsines (usu-
ally took 20 min), liquid nitrogen was removed,
and the U-tube was warmed up. The hydrides
evaporated upon heating, and were transported to
a detection system, where AsH
3
, CH
3
AsH
2
, and
(CH
3
)
2
AsH were detected sequentially. Both triva-
lent and pentavalent arsenic species could form
hydride under acidic conditions (pHB1). When
the pH was increased to above six, only the
trivalent arsenic species formed hydride. There-
fore, by regulating the pH of the reaction
medium, for example pH 6, the trivalent arsenic
species (As
III
, MMA
III
, and DMA
III
) could be
selectively determined without interference from
the pentavalent arsenic species (As
V
, MMA
V
, and
DMA
V
) [13].
3. Detection techniques
Speciation of trace levels of arsenic in environ-
mental and biological samples requires high-sensi-
tivity detection. Atomic spectrometry provides the
best sensitivity for arsenic detection. This section
briey describes several spectrometric techniques
coupled with chromatography for arsenic specia-
tion analysis.
3.1. Atomic absorption and inducti6ely coupled
plasma optical emission spectrometry
Flame atomic absorption spectrometry (FAAS)
was used as an HPLC detector for the speciation
of arsenic in the 1980s, and its use has declined.
Because FAAS suffers from low sensitivity and
high background noise for arsenic determination,
most recent applications of AAS are combined
with HG [8386]. Research was also conducted to
use graphite furnace atomic absorption spec-
trometry (GFAAS) for HPLC detection. How-
ever, a direct coupling of HPLC to GFAAS is
difcult because it is necessary to use a long
analytical cycle, including drying and ashing the
sample prior to furnace atomization. Thus, te-
dious procedures involving the collection of chro-
matographic fractions followed by batch analysis
of each fraction by using GFAAS have often been
utilized [28,87].
Inductively coupled plasma atomic emission
spectrometry (ICPAES) has been successfully cou-
pled to HPLC for use in arsenic speciation [87
90]. The coupling is straightforward because the
usual ow rate under which an HPLC operates,
typically 1 ml min
1
, is compatible with the
Z. Gong et al. / Talanta 58 (2002) 7796 87
uptake ow rate of an ICPAES system. A number
of applications have been demonstrated, primarily
for samples containing high levels of arsenic. For
systems containing lower levels of arsenic,
HPLCICPAES does not provide sufcient sensi-
tivity for arsenic speciation. Several studies incor-
porated HG between HPLC and ICPAES to
enhance the sensitivity [9194].
3.2. ICPMS
The coupling of HPLC with ICPMS offers
several advantages because of the extremely high
sensitivity, multi-element capability, large dy-
namic range, and isotope ratio measurement ca-
pability that an ICPMS instrument can offer. A
wide range of applications of HPLCICPMS to
arsenic speciation has been demonstrated
[14,41,58,95100]. HPLCICPMS is now the
most effective tool in many arsenic research
laboratories.
Several groups have used ICPMS for CE detec-
tion, demonstrating various levels of success
[72,101,102]. Two major problems are the mis-
match of sample volume between CE and ICPMS
and the suction generated by nebulization. To
solve these problems, several interfaces between
CE and ICPMS have been described, most of
which involved some type of direct nebulization/
injection. Although some progress has been made,
the matrix effect and poor sensitivity limit the
CEICPMS techniques to standard solutions and
simple systems.
The use of HPLCICPMS also allowed for
simultaneous speciation of arsenic and other rele-
vant elements [41,49,103].
3.3. HG with spectrometry
HG is a chemical derivatization process that
produces volatile hydrides upon chemical treat-
ment of a sample with a reducing agent, typically
sodium borohydride. HG techniques coupled with
atomic absorption, atomic emission, atomic
uorescence, and mass spectrometry (MS) have
found wide applications in the determination of
trace levels of arsenic. As an efcient sample
introduction method, HG enhances sensitivity
normally by 10100-fold over the more com-
monly used liquid sample nebulization proce-
dures. Also, the target arsenic species can be
separated from almost all other accompanying
materials in the sample through the HG process.
Only gaseous hydrides are introduced to the de-
tector, and the sample matrix is left in the liquid
waste. Thus, spectral and chemical interferences
encountered in the detection systems are essen-
tially eliminated.
Several organoarsenic compounds do not form
volatile hydrides under the borohydride treat-
ment. Methods have been developed to convert
them to hydride-forming species. Microwave-as-
sisted oxidation [104107] and UV photo-oxida-
tion [83,108110] with potassium persulfate and
sodium hydroxide have proven successful to con-
vert AsB, AsC, Me
4
As
+
, arsenosugars, and aryl
arsenicals to hydride-forming species.
HG has been used as a sample introduction
interface between HPLC and AAS
[18,65,108,111113]. Because of the efcient and
fast reaction, there was little post-column band
broadening due to the HG process. Using
HPLCHGAAS, detection limits for As
III
, As
V
,
MMA
V
, and DMA
V
were at low mg l
1
levels
[40,83,108,112,114].
The detection limits were further improved with
atomic uorescence spectrometry (AFS) detec-
tion. One of the most attractive features of
uorescence methods is their inherent high sensi-
tivity. While single-molecule detection limit has
been achieved with molecular uorescence tech-
niques, the same was not achievable with conven-
tional atomic uorescence based on liquid sample
introduction. This has been primarily due to the
interference effects that occur in AFS when real
samples are analyzed. Light scattering and back-
ground due to the sample matrix are the main
problems. However, separating arsines from the
sample matrix through an HG process solved
these problems. Because only gaseous arsines are
introduced to the AFS detector and the sample
matrix is removed, spectral interference encoun-
tered in the detection system is essentially elimi-
nated. This is particularly benecial to AFS
detection where the interference had previously
been the major problem. Thus, in the absence of
Z. Gong et al. / Talanta 58 (2002) 7796 88
scattering and background interference from the
sample matrix, the detection limit by using AFS
can be dramatically improved [44,115,116]. HG
also enhanced sensitivity for laser-induced uores-
cence and laser-enhanced ionization spectrometry
detection of arsenic [118].
The coupling of HPLC with HGAFS takes
advantage of both the separation power offered
by HPLC and the good selectivity and sensitivity
obtainable by using HGAFS. Detection limits in
the order of sub-microgram per liter have been
achieved for arsenic speciation [12,33,119]. These
are comparable to those achieved by HPLC
ICPMS using pneumatic nebulization. HPLC
HGAFS techniques have been applied to the
speciation of arsenic in various environmental and
biological samples [12,22,30,33,44,116,119122].
The features of sensitivity improvement and
interference reduction by HG have also been ap-
plied to HPLCICPAES [9194], HPLCICPMS
[57,123,124], and CEICPMS [101]. The detection
limits for arsenic species using HPLCHG
ICPMS were as low as nanograms per liter.
3.4. MS
The identication of arsenic compounds has
been enhanced by mass spectral information. Fast
atom bombardment (FAB) tandem MS (MS
MS) was demonstrated for the characterization of
arsenosugars in partially puried algal extracts
[125]. More recently, electrospray ionization (ES)
MS has repeatedly shown to be well suited for
arsenic speciation, either used alone or in combi-
nation with HPLC [47,62,126130]. Unlike
ICPMS, ICPAES, AAS, and AFS, where elemen-
tal arsenic is detected, ESMS can provide molec-
ular information of arsenic compounds for
positive identication. Due to the availability of
the structural information provided by ESMS, a
number of studies have focused on the characteri-
zation and identication of organoarsenicals, such
as arsenosugars and new arsenic species
[47,63,66,124,127,131133].
Corr and Larsen rst demonstrated the use of
positive-ion ESMS for determining dimethylated
arsenosugars at trace levels [47]. Subsequently,
Pergantis et al. reported the determination of 10
organoarsenic compounds using HPLC coupled
with ESMSMS [127]. The selectivity achieved
by using tandem MS (MSMS) allowed for dif-
ferentiating arsenicals that co-eluted from the
HPLC column. The method was applied to the
analysis of a urine standard reference material in
which arsenobetaine was determined to be present
at the low micrograms per liter level.
A nanoelectrospray quadrupole time-of-ight
(TOF) MS technique was described for the iden-
tication of arsenosugars at the picogram level
[131]. Numerous product ions, suitable for char-
acterizing naturally occurring dimethylated ar-
senosugars, were generated in high abundance by
using negative-ion nanoelectrospray, low-energy
tandem MS. The method was applied to an algal
extract. It demonstrated the presence of a single
dimethylated arsenosugar. In the positive-ion
mode, characteristic tandem mass spectra were
obtained for four trimethylarsonioribosides, al-
lowing their identication without the need for
standards.
Both HPLCICPMS and HPLCESMS were
used to characterize and quantify arsenic species
in algal products [132]. A large-scale extract of the
brown alga Fucus serratus was found to contain
four arsenosugars together with traces of DMA
V
and As
V
. The identity of the arsenosugars was
conrmed by HPLCESMS. Pedersen and
Francesconi recently used variable fragmentor
voltages to obtain elemental and molecular mass
spectral data for arsenic compounds [130]. The
HPLCESMS method was applicable to the
determination of four arsenosugars, AsB, DMA
V
,
and dimethylarsinoylacetic acid. It was not suit-
able for the two inorganic arsenic species, As
III
and As
V
. The method was used to identify and
quantify the major arsenosugars in crude extracts
of two brown algae.
Anion-exchange HPLCICPMS and size-exclu-
sion HPLCESMS were used to study arsenic
species in 10 commercially available edible algal
food products, with an emphasis on arsenic-con-
taining ribosides [63]. A previously unreported
compound, 5-dimethylarsinoyl-b-ribofuranose,
was isolated and identied by ESMSMS [66].
Anion-exchange chromatography was optimized
to produce a chromatographically pure peak of
Z. Gong et al. / Talanta 58 (2002) 7796 89
AsB that was used to quantify this compound. The
ESMSMS technique was also used to demon-
strate the presence of AsB, trimethyl(2-car-
boxyethyl)arsonium, AsC, DMA
V
, Me
4
As
+
, As
V
and two arsenosugars in an oyster test reference
material.
Ion chromatography with ESMSMS and
membrane HG ICPMS techniques were used for
the determination and identication of arsenosug-
ars in kelp extracts [124,133].
Solid-phase microextraction was coupled to
HPLCESMS for arsenic speciation [134].
Polypyrrol-coated capillary in-tube solid-phase mi-
croextraction was carried out for the extraction
and concentration of arsenosugars from their
aqueous samples. Detection limits in the solution
ranged from 0.2 to 1.2 ng ml
1
. A certied
reference material (DORM-2) was analyzed for
AsB content.
3.5. Other detection techniques
Several electrochemical techniques have been
described for arsenic speciation analysis. A voltam-
metric stripping procedure was reported for the
determination of As
V
in a mannitol sulphuric acid
medium [135]. The detection limit was 0.5 ng ml
1
.
By varying the composition of the supporting
electrolyte, differentiation between As
III
and As
V
was possible. A ow-through stripping coulometric
method was used for the determination of As
III
and
total arsenic in contaminated water samples after
microwave-assisted reduction of As
V
[136]. The
detection limit was 0.2 ng ml
1
. A microfabricated
gold ultramicroelectrode array [137] has recently
been described for on-site analysis of arsenic in
groundwater. The sensor with a unique gold sur-
face created by electron beam evaporation was
demonstrated to be highly sensitive to As
III
using
square-wave anodic stripping voltammetry. The
detection limit for As
III
could reach 0.05 ng ml
1
.
A genetically engineered bacterium that pro-
duces the enzyme b-galactosidase in response to
As
III
has been applied to arsenic detection [138].
The activity of this enzyme was monitored electro-
chemically. The bacterial sensing system responded
selectively to As
III
and to a lesser extent As
V
.
4. Sample handling techniques
4.1. Species stability, preser6ation, and on-site
speciation
A crucial requirement for obtaining reliable
speciation information is to maintain the concen-
tration of the original chemical species in the
sample before analysis. As
V
and As
III
are the
dominant arsenic species in water. As
V
has com-
monly been reported as the predominant water-sol-
uble species in groundwater. However, studies now
show that As
III
may be present in higher concentra-
tions. The procedures used for sample handling
and analysis may result in the oxidation of As
III
to
As
V
[48,139141]. As the methods of sampling,
preservation and analysis improve, there is increas-
ing evidence that As
III
might be more prevalent
[48,139141].
Numerous methods have been used to attempt
to preserve the arsenic species distribution in sam-
ples. At room temperature, in Ottawa River and
deionized water samples spiked with As
III
and As
V
(0.520 mg l
1
), As
V
was reduced to As
III
within
a few days [142]. Edwards et al. used ascorbic acid
and HCl addition for preservation [143]. In spiked
synthetic water, both preservatives maintained the
concentration of As
III
and As
V
for 28 days. In
spiked natural water samples with ascorbic acid,
As
III
was slowly transformed to As
V
during the 8
day observation period. In spiked deionized water
containing humid substances with added HCl,
approximately 25% of the As
III
converted to As
V
within 8 days. In rainwater, As
V
was converted to
As
III
within 3 days in the presence of ascorbic acid.
Difculties arise when trying to preserve arsenic
species in iron-rich waters. In iron-rich (Fe
III
)
drinking waters, soluble arsenic species can form
insoluble iron precipitates. The formation of these
precipitates results in a loss of aqueous arsenic,
which causes inaccurate speciation analysis. The
preservation of As
III
in Fe
III
-containing samples
was not possible with only the addition of hydro-
gen chloride [144]. Gallagher et al. added EDTA to
water samples to prevent the formation of iron
precipitates [145]. Reagent water that was fortied
with As
III
, Fe
III
, and EDTA showed less than a 1
Z. Gong et al. / Talanta 58 (2002) 7796 90
mg l
1
change in the As
III
concentration in 16
days [145]. In three well waters, EDTA was able
to preserve the original As
III
+As
V
concentration
for 10 days. After 1427 days, approximately 23
mg l
1
of As
III
was converted to As
V
. Borho and
Wilderer added HCl (pHB2) and excess Fe(II) to
groundwater samples, and As
III
was stable in so-
lution for 1 week [144].
Samples that contained concentrations of As
III
and As
V
at 0.5 mg ml
1
or 1 mg ml
1
and were
stored at 4 C were stable for 21 days and showed
some transformation after 29 days of storage
[146]. At 25 C, it appeared that solutions with
the highest concentrations of arsenic species (20
mg ml
1
or higher) could be stored without sig-
nicant loss of species [146]. However, at lower
concentrations, species transformation was ob-
served by the end of the rst week. Some studies
recommend freezing aqueous samples at 20 C
as the best way to preserve the species [147].
Lindemann et al. tested the storage of arsenic
species at temperatures of 20, +3 and +
20 C [148]. The best storage results were ob-
tained at +3 C and the worst storage results at
20 C.
Methods of on-site separation of As
III
and As
V
immediately after water sample collection are par-
ticularly useful. The portability of the methods
allows for complete separation and no preserva-
tion of the samples is needed. Le et al. used
disposable solid-phase cartridges for the specia-
tion of particulate and soluble arsenic [117]. A
measured volume of a water sample is passed
through a 0.45 mm membrane lter and a silica-
based strong anion-exchange cartridge connected
in serial. Particulate arsenic is captured on the
lter, and the anion-exchange cartridge retains
As
V
. As
III
is not retained and is detected in the
efuent. The anion-exchange cartridge is subse-
quently eluted with 1 M hydrochloric acid (HCl),
and the eluent is analyzed for As
V
concentration.
Kim also used a portable ion-exchange method
with a strong anion-exchange resin in the eld
and found that neither As
V
nor As
III
changed its
oxidation state during the experiment [139]. How-
ever, upon examination of samples from private
wells in Genesee County, MI, the oxidation of
inorganic As
III
to As
V
occurred within 3 days of
storage in a refrigerator [139]. Immediately after
sampling, As
III
represented 8295% of the total
arsenic and after 3 days, 2530% of the original
As
III
was oxidized to As
V
[139]. As
III
dropped to
approximately 70% of its original value after 6
months of storage [140]. A eld-portable stripping
voltammetric instrument has also been used to
measure As
III
and As
V
[149].
A eld method that incorporated membrane
extraction with a sorbent interface-micro-GC sys-
tem was developed by Segal et al. [150]. Miller et
al. used an ion-exchange method to separate ar-
senic species in aqueous solution and found that
the organic arsenic species eluted with As
III
, and
so caution should be exercised in the analysis of
arsenic species or determining the toxicity of ar-
senic in drinking water [151]. Organic species and
particulate arsenic could be detected as As
III
using
this method. A microfabricated gold ultrami-
croelectrode array has also been used for the
on-site analysis of arsenic in groundwater [137].
Methylarsenicals are more stable than inorganic
arsenic both in water and in urine samples
[21,48,146148,152]. Larsen et al. observed that
the concentrations of DMA
V
, MMA
V
and AsB
were relatively constant [48]. Jokai et al. found
that at concentrations of 0.0110 ng ml
1
,
MMA
V
and DMA
V
were stable for 56 months
at room temperature [146]. Palacios et al. found
that As
V
, MMA
V
, DMA
V
, and AsB in urine were
stable for at least 67 days at 4 C [147]. In
another systematic study, the effects of different
storage temperatures, storage duration, and the
use of additives on the stability of ve arsenic
species were examined using normal and spiked
volunteer urine samples [152]. The urine samples
could be stored at low temperatures (4 and
20 C) for 2 months without substantial changes
of arsenic speciation. For longer storage times,
the stability of arsenic species varied with the
sample matrix. The use of additives, such as HCl,
sodium azide, benzoic acid, benzyltrimethylamm-
noium chloride and cetylpyridinium chloride, did
not improve the stability of arsenic speciation in
urine. The addition of dilute acid (0.1 M HCl) to
urine samples caused relative changes in inorganic
As
III
and As
V
concentrations [152].
Z. Gong et al. / Talanta 58 (2002) 7796 91
With the discovery of MMA
III
and DMA
III
in
human urine [1214,30], there is an increasing
interest in the determination of these arsenic spe-
cies in toxicological and environmental studies.
Gong et al. have studied the oxidative stability of
these trivalent arsenic species in water and urine
samples [21]. Low-temperature conditions (4 and
20 C) were recommended for the improved
stability of these arsenic species over the room
temperature. MMA
III
in deionized water was rela-
tively stable for almost 4 months when stored at 4
or 20 C. Less than 10% of the MMA
III
was
oxidized to MMA
V
. DMA
III
in deionized water
was stable for only 23 days and was rapidly
oxidized to DMA
V
. The conversion of DMA
III
to
DMA
V
in spiked urine samples was complete
after 17 and 12 h when stored at 20 C and
4 C, respectively. The oxidation of MMA
III
[to
MMA
V
] and DMA
III
[to DMA
V
] was found to be
matrix-dependent [21]. The matrix-dependent sta-
bility of MMA
III
and DMA
III
was also reported
by Del Razo et al. [13]. Mandal et al. put urine
samples in saltice mixture for a period of up to
72 h during the transportation and kept them
frozen at 28 C for about 2 months before
analyses [14]. They found 25% of MMA
III
and
421% DMA
III
in the urine samples from a
highly exposed group of people in India.
4.2. Extraction of arsenic species from solid
samples
Sample preparation for solid samples generally
may include procedures such as mincing, freeze
drying, milling, grinding, homogenization, and
sieving, followed by extraction. A desirable ex-
traction method should quantitatively extract all
arsenic species without altering their original spe-
ciation. Also, the solvent used to extract samples
should not interfere with the species analysis.
Traditional extraction methods involved solvent
extraction with the assistance of physical shaking
or sonication. A variety of solvents were used to
extract arsenic species from seafood. The
methanol water system is commonly used to ex-
tract arsenic species from food [65,66,130,133].
Acetonitrile was also tested as an extraction sol-
vent [153]. Lipids and fats in the sample extracts
may interfere with either arsenic separation or
detection, so a clean-up procedure involving a
C18 cartridge was used [133]. However, a signi-
cant retention of DMA
V
on the C18 cartridge was
observed [154].
A sequential extraction procedure (SEP) was
employed to extract arsenic species in sh tissue
[155]. First, acetone was used to extract fats and
lipids from sh tissue, and then a methanol and
water mixture (1:1 m/m) was used to extract the
polar compounds. It was found that less than 5%
of arsenic was extracted by acetone in the ve sh
tissue samples. The extraction efciencies of ar-
senic in the polar fraction were 84.987% by
using sonication [155]. Chloroform was another
solvent used to remove the lipids and fats from
the samples [156,157].
Gallagher et al. found that the extraction ef-
ciencies for two seaweeds were 25.6% and 50.5%
when an extraction method was optimized using
ribbon kelp as a model sample (extraction ef-
ciency 72.5% for kelp) [133]. Arsenic species de-
tected in ribbon kelp were three arsenosugars.
Considerable amounts of inorganic arsenic species
were found in the two other seaweed samples
[133]. A variety of extraction efciencies were also
studied by other researchers [46]. As
III
was found
in living organisms bound to SH groups of cyto-
solic proteins and micromolecular constituents
[158]. It was postulated that As
III
was selectively
retained by the presence of thiol groups that
remained active during the extraction procedure.
Methods based on solubilization with HCl and
microwave-assisted distillation were described for
the extraction of inorganic arsenic from seafood
products [113,159]. However, this method was not
suitable to determine As
III
and As
V
species be-
cause As
V
was converted to As
III
during the hy-
drolysis and extraction process. The conversion
between As
III
and As
V
was also observed by using
triuoroacetic acid to hydrolyze rice samples
[160].
Recently, accelerated solvent extraction (ASE)
was employed to extract arsenic species in solid
samples [133]. This semi-automated method, ap-
plying pressure and temperature during the ex-
traction, was faster and less labor-intensive than
the traditional extraction method. The procedure
Z. Gong et al. / Talanta 58 (2002) 7796 92
was validated by using kelp as a model sample.
Three arsenosugars present in the kelp were mon-
itored. No signicant changes of the arsenosugars
were observed except under high-temperature
conditions [133]. As
III
, As
V
, MMA
V
, DMA
V
, and
AsB spiked to a carrot matrix were quantitatively
recovered, indicating that there were no species
conversion during the extraction procedure [154].
However, compared with sonication extraction
method, the recovery of arsenic in terms of total
arsenic was 1020% lower [153].
Enzyme digestion in combination with extrac-
tion has been explored to improve extraction ef-
ciency for some biological samples [153,160,162].
Trypsin, pancreatin and a-amylase have been used
[156,160,162]. An increase of recovery by more
than 20% was observed when a-amylase was used
to assist the extraction of arsenic species from
freeze-dried apple samples [153].
Other extraction procedures, such as Soxhlet
extraction [161] and solid-phase extraction [134],
have also been demonstrated.
4.3. Leaching of arsenic species from soil
Arsenic speciation in soil samples is often based
on the use of leaching or extraction procedures,
which enables the assessment of the mobility of
arsenic. The mobility of arsenic depends on indi-
vidual arsenic species as well as physical and
chemical characteristics of the soil, such as tex-
ture, chemical composition, pH, and biological
characteristics. Several extraction procedures have
been studied to evaluate metal mobility in soils
[163165]. Wenzel et al. proposed SEP to frac-
tionate arsenic in soil [166]. It involved ve extrac-
tion steps, sequentially using 0.05 M (NH
4
)
2
SO
4
,
0.05 M NH
4
HSO
4
, 0.2 M ammonium oxalate
buffer at pH 3.25, 0.2 M ammonium oxalate and
ascorbic acid at pH 3.25, and HNO
3
H
2
O
2
.
These arsenic fractions were associated with non-
specically sorbed, specically sorbed, amorphous
and poorly-crystalline hydrous oxides of Fe and
Al, well-crystallized hydrous oxides of Fe and Al,
and residual phase, respectively [166]. Other SEP
protocols were also proposed [167,168]. However,
most SEPs needed long mechanical shaking,
which was very time-consuming. The extracts ob-
tained from some fractions may not be suitable
for speciation analysis because the solvents may
interfere with the subsequent HPLC separation.
Another in vitro method to determine arsenic
bioavailability in contaminated soil and mineral
wastes was developed by using a physiologically
based extraction test (PBET). Synthetic leaching
uids mimicking those of the human stomach and
small intestine were used to evaluate the
bioavailability of arsenic in soil. PBET provides a
potentially valuable mechanism for rening risk
assessments of arsenic contamination sites [169].
Orthophosphoric acid was used to extract ar-
senic species from soil based on the ion-exchange
reaction between phosphate and arsenic species
[171]. The stability of As
III
, As
V
, MMA
V
, and
DMA
V
during the extraction was examined. Al-
though high concentration of phosphate could
extract more arsenic species from the soil, the
oxidation of As
III
to As
V
was observed. As
V
and
As
III
were the main arsenic species in soil samples
[172,173].
5. Concluding remarks
Arsenic compounds are ubiquitous in the envi-
ronment. Humans are exposed to arsenic primar-
ily from the ingestion of food and water. Urinary
excretion of arsenic metabolites is the primary
pathway for the elimination of arsenic from hu-
man body. Speciation of arsenic in water, food,
urine, and biological tissues plays an important
role in understanding arsenic exposure,
metabolism, and health effects.
The combination of chromatographic separa-
tion with element-specic spectrometric detection
has proven to be most useful for the speciation of
trace levels of arsenic compounds. In particular,
HPLC separation with ICPMS, HG atomic spec-
trometry, and electrospray MS detection have
played important roles in chemical speciation
studies of arsenic. With good separation by
HPLC and sensitive detection by atomic and MS,
these methods have been applied to health science
and environmental science studies with respect to
the absorption, distribution, metabolism, excre-
tion, and environmental arsenic cycles. Much re-
Z. Gong et al. / Talanta 58 (2002) 7796 93
search continues on improving our understanding
of the health effects and environmental chemistry
of arsenic species. With the increase of sensitivity,
several new arsenic species have been detected,
but are yet to be identied. The hyphenated tech-
niques involving the use of electrospray tandem
MS are useful for characterization of these un-
known arsenic species.
A crucial requirement for obtaining reliable
speciation information is to maintain the concen-
tration of the original chemical species in the
sample prior to analysis. This is a special require-
ment for speciation analysis. For determining to-
tal element concentrations the main
considerations for sample collection and storage
are to prevent contamination and to minimize loss
of trace levels of analytes. In the case of specia-
tion analysis, the concentration of individual spe-
cies of the element must be unchanged by sample
handling and treatment. There is a need to de-
velop methods for stabilizing arsenic species in
liquid samples (water, urine, and blood) and for
extracting arsenic species from solid materials
(food, biological tissues).
Acknowledgements
This work was supported by the Natural Sci-
ences and Engineering Research Council of
Canada, the Canada Research Chair Program,
the Canadian Water Network National Centers of
Excellence (NCE), and Alberta Health and
Wellness.
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