Microchemical Journal 97 (2011) 78–84

Contents lists available at ScienceDirect

Microchemical Journal
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i c r o c

Development of analytical method for determination of Sb(V), Sb(III) and TMSb(V)

in occupationally exposed human urine samples by HPLC–HG-AFS
Waldo Quiroz a,⁎, Helen Arias a, Manuel Bravo a, Marcela Pinto a, María Gabriela Lobos b, Marisol Cortés a
a
Laboratorio de Química Analítica y Ambiental, Instituto de Química, Pontificia Universidad Católica de Valparaíso, Avenida Brasil 2950, Valparaíso, Chile
b
Universidad de Valparaíso, Facultad de Ciencias, Departamento de Química y Bioquímica, Avenida Gran Bretaña 1111, Playa Ancha, Valparaíso, Chile

a r t i c l e i n f o a b s t r a c t

Article history: In the present paper, we develop a methodology for antimony speciation in occupationally exposed human
Received 8 January 2010 urine samples by high-performance liquid chromatography with hydride generation atomic fluorescence
Received in revised form 31 May 2010 spectrometry (HPLC–HG-AFS). The methodology was applied to the determination of Sb(V), Sb(III) and
Accepted 19 June 2010
(CH3)3SbCl2 (TMSb(V)). Retention time of Sb(V), Sb(III) and TMSb(V) species were 0.88, 2.00 and 3.61 and
Available online 26 June 2010
the detection limits were 0.18, 0.19 and 0.12 μg L− 1, for 100 μL loop injection respectively which is
considered useful for elevated/occupationally exposed urine samples. Studies on the stability of antimony
Keywords:
Antimony speciation
species in urine samples on the function of the elapsed time of preservation (4 °C) and storage (− 70 °C)
Urine were performed. Results revealed that antimony species are highly unstable at −70 °C, probably due to co-
HPLC–HG-AFS precipitation reaction. In this kind of matrix transformation during preservation time may occur, such as
oxidation of Sb(III) to Sb(V) and transformation into species that do not elute from the column. EDTA shows
that it is able to stabilize Sb(III) for more than one week of preservation time at 4 °C avoiding co-
precipitation during storage at − 70 °C. Finally the methodology was applied to occupationally exposed
human urine samples. 25% of specimens present antimony levels (Sb(V)) of more than 5 μg L− 1.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction According to literature, antimony in humans is mainly excreted

Antimony is a cumulative toxic element which can be present in
environmental, biological and food samples in two oxidation states:
Sb(III) and Sb(V). Inorganic compounds of antimony are more toxic
than its organic forms and the toxicity of Sb(III) ions is 10 times higher
than of Sb(V) ions [1].
The potentially harmful effects of Sb have been recognized by
health authorities to such an extent that this metalloid has been listed
as a priority pollutant by the US Environmental Protection Agency and
the German Research Community [2].
This element is categorized as non-essential for plants, animals or
humans and in environmental matrices it is mainly present at trace
levels. In spite of this, environmental samples such as airborne
particulate matter [3] and soils located near major traffic routes [4]
have been highly contaminated with antimony, concentrations which
may pose a risk for human health. Recently, our group reported high
blood antimony levels as a result of similar anthropogenic activities
[5], however similar studies are scarce. For these reasons it became
mandatory to dispose reliable information about the content of this
metalloid in human biological fluids.
⁎ Corresponding author.
E-mail address: waldo.quiroz@ucv.cl (W. Quiroz).
through urine [6]. Biomonitoring of trace elements in urine of healthy
humans has been evaluated for many elements at different geo-
graphical locations [7]. Concentrations of Sb in urine as reported in the
literature are in the range 0.10–1.8 μg L− 1 [8]. Besides, this element
has been detected in urine samples for exposed and non-exposed
workers to anthropogenic activities [9].
Most of the analytical methodologies for antimony determination
are based on total antimony quantification. However, it is widely
accepted that the impact of a toxic element cannot be established by
measuring only its total concentration, and environmental risk linked
to the presence of antimony depends to a great extent on the chemical
form on which it is present [10]. This is due to the fact that different
species of an element present differences in its physical, chemical and
toxicological properties [11,12].
For antimony speciation many analytical methods have been
reported in the literature. The most commonly employed analytical
techniques for the separation and detection of antimony species are
high-performance liquid chromatography coupled to powerful ele-
ment specific detectors like ICP-MS [13,14] or HG-AAS [15] and HG-
AFS[16,17]. Besides, due to the low concentrations of Sb in enviro-
mental samples, some authors have incorporated the use of a
preconcentration step as necessary [2,18].
On the other hand, few studies have addressed the problem of the
reliability of the results for antimony speciation in urine samples.
Lindenmann et al. studied the stability of antimony species in this

0026-265X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.microc.2010.06.015
 W. Quiroz et al. / Microchemical Journal 97 (2011) 78–84
matrix [19]. Besides, Krachler and Emons reported the first study on
the determination of Sb species in urine as an effective way of
monitoring the administration of Sb(V) in patients with leishmaniasis
[15]. Miekeley et al. studied the transformation of N-methyl
meglumine antimoniate after intramuscular administration detecting
Sb(III) and Sb(V) [20]. In these investigations, it is clear that stability
of antimony species during the whole analytical procedure was the
main analytical problem. In the case of environmental monitoring,
sampling is normally followed by two steps before analysis: first
sample preservation until laboratory arrival which normally can be
performed in cooler systems at 4 °C and second storage which in the
case of urine, frozen at −70 °C is industry standard. Thus, stability
must be evaluated in both cases.
On our previous works, we presented the optimization of ana-
lytical methodologies to carry out the speciation of antimony in sea
water [21], marine sediments [22], and marine biota [23], demon-
strating that in order to assure the quality of analytical results the
stability of these species must be controlled. Following our research
about antimony speciation, the aim of the present work was to
develop an analytical method for determination of Sb(V), Sb(III) and
TMSb(V) in occupationally exposed human urine samples by HPLC–
HG-AFS.
2. Material and methods
2.1. Instrumentation
The chromatographic separation of antimony species was per-
formed with a Hewlett Packard HPLC system 1050 model equipped
with quaternary pumps, degassers, autosampler, injector with 100 μL
loop and a short Hamilton PRP-X100 column (100 mm × 4.1 mm)
with small particle size (5 μm).
Hydride generation of volatile stibines was carried out by the on-
line addition of 3 mol L− 1 HCl (0.4 mL min− 1) and 3% (w/v) NaBH4
(0.25 mL min− 1) solutions at the outlet of the column by means of the
two peristaltic pumps of the HG-AFS system (PS Analytical Excalibur
Millennium System). An argon flow of 300 mL min− 1 was fixed to
carry the volatile stibines generated to the detector. Before detection,
a secondary argon flow of 40 mL min− 1 and a supplementary hydro-
gen flow of 40 mL min− 1 were injected to maintain stable the argon/
hydrogen diffusion flame, optimization of NaBH4, and HCl, and
supplementary hydrogen was already reported [24]. The gas flow
was dried through a hygroscopic membrane drying tube (Perma
Pure product, dryer model MD-110-12 FP). A boosted discharge
antimony hollow cathode lamp (Sb BDHCL Super lamp, Photon,
Victoria, Australia) was used as a radiation source of the atomic
fluorescence detector.
The K2S2O8 solution, added using a T-joint at the outlet of the
chromatographic column, was delivered by the peristaltic pump of the
HG-AFS system at a flow rate of 0.93 mL min− 1. For photo-oxidation a
PTFE tube was wrapped around a Philips TUV-15 lamp (253.7 nm,
15 W and 44 cm long). Samples were digested in a microwave
oven (Microdigest A 301 PROLABO, France). Instrumental operating
conditions are given in Table 1.
Table 1
Microwave program applied for the digestion of urine samples for the determination of total antimony.
Program Step
1 2 3 4 5 6
Reagent HNO3 H2SO4 – – – H2O2
Volume (mL) 8 3 – – – –
Power (W) 10 45 55 65 75
Time (min) 2 4 2 4 6 8
79
2.2. Reagents
All chemicals and reagents used in this study were analytical grade
or higher purity. De-ionized water (18.2 MΩ cm− 1) was obtained
from a Nanopure system (Barnstead, Dubuque, IA, USA). Glass and
polyethylene ware were cleaned by soaking for one day in 10% v/v
nitric acid (analytical grade) and were rinsed several times with de-
ionized water before use.
Individual stock solutions of antimony species were prepared from
potassium hexahydroxy–antimoniate KSb(OH)6 (99.95%), potassium
antimonyl tartrate K(SbO)C4H4O6H2O (99.95%) and trimethylanti-
mony dichloride (CH3)3SbCl2 (TMSb) (96%) purchased from Sigma
Aldrich (USA), and are termed as Sb(V), Sb(III) and TMSb(V),
respectively. Stock solutions of Sb (V) (500 mg L− 1) and TMSb
(500 mg L− 1) were prepared by dissolving the appropriate amount of
the respective compounds in de-ionized water and were stored in the
dark at 4 °C until use.
Standard solution of Sb(III) was prepared freshly before use by
dissolving potassium antimonyl tartrate in de-ionized water. Working
antimony standards of lower concentration (individual and/or mixed
species) were prepared daily, as required, by an appropriate dilution
of the stock solution with de-ionized water or with the mobile phase.
The mobile phases were freshly prepared and filtered on 0.45 μm
membrane filters, (Millipore, type HA) and degassed by sonication
before use. K2S2O8 1% (w/v) (99.9%, Merck), in 1% (w/v) NaOH
(Merck) solution was used for photo-oxidation.
2.3. Sample collection
The urine samples corresponded to the first urination of the mid-
dle stream in the morning; the samples were collected in polyeth-
ylene clean bottles of 250 mL previously washed with potable water,
de-ionized water and diluted nitric acid and later were stored in a
refrigerator at 4 °C. Urine samples of 8 adult men, inhabitants of
Puchuncaví commune were analyzed. Puchuncaví valley, an agricul-
tural zone at the north of Valparaíso city, Chile, receives the impact of
the industrial complex: a Smelter-Electrorefinery plant and a
thermoelectric power plant. This valley limits to north with Zapallar
commune, to the south with the municipalities of Quillota and
Quintero, to the east with the municipalities of Nogales and La Cruz
and the west by the Pacific Ocean. Its surface is 306.5 km2 and its
population density is at 49.13 inhabitants/km2.
Prior to analysis of speciation, the human urine samples were
filtered on 20–25 μm filters (Sartorius) and then through a 0.22 μm
pore size membrane of cellulose ester (GS Millipore). All plastic- and
glassware were decontaminated for 24 h in 10% v/v HNO3, rinsed
several times with high purity water, and were then stored in a
refrigerator at 4 °C until analysis is performed.
2.4. Total antimony determination
The urine samples were transferred into the digestion vessels with
8 mL of nitric acid and conserved at room temperature, overnight. On
the following day, the samples were digested in an oven microwave
applying the heating program presented in Table 1. After reaching
7 8 9 10 11 12 13 14
– – K2S2O8 – K2S2O8 –
6 – – – 10 – 10 –
65 75 75 – 10 70 10 65
2 2 2 10 30 2 30 2
80 W. Quiroz et al. / Microchemical Journal 97 (2011) 78–84

room temperature, the clear digested solutions were quantitatively

transferred into 50 mL polyethylene flask and filled to the mark with
de-ionized water. The blank were prepared with the same reagents,
without the samples, undergoing a similar digestion procedure.
For total antimony determination, aliquots of sample solutions
were placed in 50 mL volumetric flasks. The solutions were reduced to
Sb(III) in KI (1.5% w/v) and L-ascorbic acid C6H8O6 (0.6% w/v) in HCl
1.5 mol L− 1 media. The solutions were analyzed through HG-AFS after
30 min of pre-reduction time. The measuring conditions of the HG-
AFS system are shown in Table 2.

2.5. Antimony speciation

The determination of Sb(III), Sb(V) and TMSb(V) in urine samples

was carried out by anion exchange high-performance liquid chroma-
tography (HPLC–HG-AFS), and a post-column photo-oxidation step was
included (HPLC–UV–HG-AFS) [22]. The chromatographic separation
was performed using a Hamilton PRP-X100 column (100 mm × 4.1 mm)
with a gradient elution program between 20 mol L− 1 EDTA + 2 mol L− 1
KHP, pH 4.5 as the first mobile phase and a 50 mol L− 1 phosphate
solution, pH 8.3 as the second one. Aliquots of 100 μL were injected into
the HPLC system. The determination of each antimony species was
performed by the standard addition method, using peak area measure-
ments. Detailed instrumental parameters and the optimization of the
chromatographic separation for antimony species have been described
in recent literature [22,23].

3. Results and discussion

3.1. Speciation methodology

Determination of antimony species in urine samples was carried

out by anion exchange high-performance liquid chromatography
(HPLC–HG-AFS) for separation and detection, respectively. For spe-
ciation analysis, methodology applied was based on our previous
studies [21–23]. It is worth mentioning that modification for hydride
generation conditions was necessary since the values for hydrochloric
Fig. 1. Chromatogram of antimony species (10 μg Sb L− 1) in (A) 20 mmol L− 1 EDTA +
acid and sodium borohydride concentration (3 mol L− 1 and 3% w/v, 2 mmol L− 1 KHP solution, pH 4.5; and (B) urine matrix. (Y axis corresponds to arbitrary
respectively) proposed in those studies raised the background noise units (UA) of the interface HPLC–HG-AFS proportional to fluorescence counts.)
unnecessarily. Final experimental conditions are presented in Table 2.
Fig. 1A shows a chromatogram of a synthetic solution of Sb
species prepared in the same mobile phase EDTA + KHP media, while Fig. 1B shows a chromatogram obtained of urine sample spiked with
10 μg L− 1 of Sb(V), Sb(III) and TMSb. Retention time of Sb(V), Sb(III),
and TMSb species were 0.87, 1.87 and 3.57 min respectively and
Table 2
Summary of conditions for HPLC coupled to HG-AFS detection system. chromatographic resolutions (Rs) were 1.5 and 1.9.
On the other hand, the retention times of Sb species in the urine
HPLC (Hewlett Packard HPLC system, 1050 model)
samples were 0.88, 2.00 and 3.61 min respectively, while resolutions
Column Hamilton PRP-X100 (100 × 4.1 mm id, particle size 5 μm) were 1.6 for Sb(V)–Sb(III) and 1.5 for Sb(III)–TMSb, which implied
Mobile phases First mobile phase that the chromatographic separation is robust enough for its
20 mmol L− 1 EDTA + 2 mmol L− 1 KHP (potassium
hydrogen phthalate), pH = 4.5
application in this kind of samples.
Second mobile phase Comparing both chromatograms of Fig. 1, a highest background
50 mmol L− 1 (NH4)2HPO4 solution, pH = 8.1 noise can be observed for spiked urine sample, probably caused by the
Flow rate 1.5 influence of sample matrix. Higher signal was obtained for Sb (III),
(mL min− 1)
since it presented better kinetics of stibine formation than the signals
Injection volume (μL) 100
for Sb(V) and TMSb. Chromatographic signal of TMSb is not com-
Detection HG-AFS (PS Analytical Ltd, Millennium Excalibur system) pletely symmetrical, since it presents a tailing effect, which probably
Sb BDHCL
could be caused by molecular re-arrangement of methyl species
Primary current(mA) 15 (CH3)3Sb, (CH3)2SbH, and CH3SbH2 during hydride generation reac-
Boosted (mA) 18 tion such as was described by Craig et al. [25] or during the chroma-
HCl 1.5 mol L− 1 (9 mL min− 1) tographic process such as was described by Krachler and Emons [15].
NaBH4 0.35% w/v (in 0.4% NaOH, 0.35 mL min− 1)
Argon flow (primary) 300
(mL min− 1) 3.2. Determination of analytical parameters for the antimony speciation
(secondary) 20
(mL min− 1) A significant matrix effect has been detected. Thus the determi-
Hydrogen (auxiliary) 60 nation of Sb(III), Sb(V) and TMSb was performed by calibrating with
(mL min− 1)
one standard addition curve for each antimony species. Table 3
 W. Quiroz et al. / Microchemical Journal 97 (2011) 78–84 81

Table 3
Analytical characteristics for antimony speciation in human urine.

Characteristics Sb(V) Sb(III) TMSb

Working linear range (μg Sb L− 1) 0.5–100 0.5–100 0.5–100

Correlation coefficient 0.9984 0.9974 0.9970
Detection limit (μg L− 1) 0.18 0.19 0.12
Quantification limit (μg L− 1) 0.60 0.62 0.59
Repeatability (%CV 0.8 μg L− 1) 5.8 6.1 5.9

presents the figures of merit for determination of antimony species in

urine. Detection (LOD) and quantification (LOQ) limits were calculated
according to IUPAC criteria as 3 × sb / m and 10 × sb / m respectively,
where sb is standard deviation of the signal of a spiked urine sample
with 0.4 μg/L of each Sb species and m is the slope of standard addition
curve. Same solution was used for repeatability determination.
As it can be seen in Table 3, repeatability of the methodology was
found in about 6% relative, which is quite satisfactory for trace level
analysis in spite of matrix complexity. Comparing LOD values with
those obtained in the literature it is possible to see that the reported
values were about 3 times higher than those reported by Krachler and
Emons by using ICP-MS as detector system. However HG-AFS
detection system is much cheaper than ICP-MS; on the other hand,
in the proposed methodology no dilution of urine sample was neces-
sary and LODs are low enough considering total antimony levels in
urine samples of exposed persons reported in literature [26] which
is about 0.4 μg L− 1 for non occupational exposed workers and 0.43 μg
L− 1 for 95th percentile for total Sb in the US population [27].
According to this information our detection limits for each antimony
species were low enough for the monitoring of occupational exposed
workers to antimony emissions.

3.3. Stability study of antimony species in human urine

Stability study was performed considering two steps. First sample

preservation until laboratory arrival which normally can be per-
formed in cooler systems (about 4 °C) and second storage which in
the case of urine, frozen at −70 °C is industry standard.
Due to the lack of reference materials for antimony species in
urine, it was not possible to validate the speciation methodology and
for this reason recovery essays in both situations (4 °C and −70 °C)
were performed. One major problem on antimony speciation analysis
is the stability of Sb(III). About this topic, Lindemann et al. studied the
stability of inorganic antimony species during five days by spiking
reference material NIST 2670 for urine with 20 μg L− 1 of Sb (III)
and 5 μg L− 1 of Sb (V). Results show that urine samples should be
analyzed immediately after their collection to avoid errors [19].
Therefore, prior to our speciation analysis in urine samples,
stability study of Sb(III), Sb(V) and TMSb was performed. For this
purpose, two groups of spiked urine samples were prepared and
analyzed; one in the presence and another group in the absence of
EDTA 20 mmol L− 1 + KHP 2 mmol L− 1. All samples were kept in a
refrigerator in two groups, one at 4 °C, which is the temperature
generally used just for preservation until laboratory arrival, and the
second group at −70 °C which is normally performed on arsenic
speciation [28].
EDTA was chosen because it is well known that Sb (III) can be
stabilized by formation of complex SbEDTA(OH)− 2
3
[29] such as was
reported previously by sea water speciation analysis [21]. An aliquot
of morning middle stream urine sample – in which antimony species
were not detected – was filtered and spiked with 20 μg L− 1 of Sb(III),
Sb(V) and TMSb (final volume of 50 mL). Antimony species in the
samples were determined every two days for a period of nine days by
the experimental conditions previously reported in Table 2; the re-
sults are presented in Fig. 2. As can be observed the stability of
antimony species in Fig. 2A and B showed a clear influence of EDTA +
Fig. 2. Recovery of Sb(V), Sb(III) and TMSb in urine (20 μg L− 1 each one) on the function
of elapsed storage times. (A) Samples without EDTA and (B) samples treated with EDTA
20 mmol L− 1–KHP 2 mmol L− 1.
KHP. Sb(III) recovery decrease during the storage period, phenomena
which did not occur with Sb (V) (Fig. 2A). In contrast, in the urine
sample stabilized with EDTA + KHP no Sb (III) loss was detected
(Fig. 2B).
In general, there was a decrease in the initial antimony con-
centration added of species in the two types of samples studied, being
significant the time-dependent stability of Sb(III) in the presence of
EDTA + KHP in the urine sample and of Sb(V) in the sample without
additional reagent.
According to the results without EDTA–KHP, Sb(V) is recovered
almost 100% until a week after storage in contrast to 80% for Sb(III)
and TMSb was recovered after two days of conservation. Based on
these results, a maximum period of two days could be considered for
speciation analysis of antimony in urine sample, not exceeding 20%
error in the quantification of the concentration of species. About the
same subject Lindemann et al. [30] reported about 30% recovery for Sb
(V) on the fifth day of analysis in a urine sample without addition of
reagents and stored at 3 °C.
For antimony species in urine treated with EDTA–KHP, it was
observed for Sb (III), around 100% recovery in the period of a week of
study. Meanwhile, the recovery rate was around 60% for Sb(V) and
TMSb, at the same storage time. These results show clearly that the
presence of EDTA–KHP stabilizes Sb(III), probably due to the for-
mation of a negatively charged complex SbEDTA(OH)− 2
3
inhibiting its
82 W. Quiroz et al. / Microchemical Journal 97 (2011) 78–84

oxidation to Sb(V). The formation of this complex was evidenced by

Sb(III) behavior in a urine sample without mobile phase (Fig. 2A),
where loss of the species cannot be completely justified by oxidation
in the matrix, since there was no proportional increase in the recovery
of Sb(V). In order to explain the systematic loss of Sb(V) and TMSb in
the presence of EDTA–KHP, the first hypothesis was the formation of
some kind of complex between antimony species — EDTA and the
urine matrix constituents, which may be retained in column or by the
formation of compound that did not generate hydrides. This
phenomenon has been reported for arsenic species such as arseno-
betaine [31] and for Sb(V)–Citrate [22].
The loss of TMSb, either in the presence or absence of EDTA–KHP,
cannot be related with the interconversion to Sb(III) or Sb(V) since no
increase on the recovery of both species was detected. In order to
explain these results we believe that TMSb could be involved in a
precipitation reaction by molecular crowding, which could be caused
by the decrease of pH in the presence of EDTA–KHP (pH of the sample
decreases by about two units), as it recovers over 10% TMSb at the end
of the study period.
Another hypothesis is that the decrease of the concentration of
antimony species could be explained by the formation of volatile
compounds by microbial activity. It should be noted that the results
were similar for three urine samples from different people, spiked and
treated equally.
In order to test whether the decrease in the concentrations of
antimony species during the storage time could be due to any of the
hypothesis previously proposed, we proceeded to determine the total
antimony concentration in spiked urine samples that had been
preserved in a refrigerator at 4 °C.
The results for total antimony concentration in urine sample
without EDTA–KHP were 56 ± 2 μg L− 1, while for urine samples with
EDTA–KHP the concentration was 55 ± 2 μg L− 1. Considering that
total antimony added spiked was originally 60 μg L− 1 we can con-
clude that the formation of volatile compounds is not significant, since
the recovery was almost quantitative.
Considering these last results it was possible to postulate that Sb
(V) and TMSb were transformed during the storage of urine samples
Fig. 3. Chromatograms (HPLC–UV–HG-AFS) of spiked urine samples (A) without EDTA
into species that did not generate hydrides. For the purpose of
and (B) with EDTA–KHP, after one month of storage at 4 °C. (Y axis corresponds to
detecting these hypothetical species, antimony speciation in urine arbitrary units (UA) of the interface HPLC–HG-AFS proportional to fluorescence
was performed with a post-column photo-oxidation step assisted by counts.)
UV radiation (HPLC–UV–HG-AFS) using peroxodisulfate (solution 2%
w/v in NaOH 2% w/v). According to literature, this system has been
widely used for degradation of arsenobetaine molecules, as well as On the other hand, stability studies on frozen condition (−70 °C)
other arsenic species that cannot be volatilized on the HG-AFS system. were performed for 5 days. Results are shown on Table 4.
The experimental conditions for HPLC–UV–HG-AFS have been Results show that for antimony speciation analysis it is not ade-
previously reported by our group [22]. quate to storage urine at −70 °C without EDTA. As can be seen after
Fig. 3A clearly shows that after one month of preservation Sb(III) 5 days, Sb(V) were not detected and TMSb(V) is recovered less than
in samples not treated with EDTA–KHP was not detected. Meanwhile 50%. In order to explain these results it is important to mention that a
Sb(V) signal becomes higher concluding that a small fraction of Sb(III) yellow solid material was obtained in urine samples before being
was oxidized to Sb(V) which is consistent with our previous studies frozen, which could not be re solubilized at room temperature and
where Sb(III) is oxidized to Sb(V) in sediment extract [22] and sea this solid material was retained on 0.2 μm filter. Therefore the most
water matrix [21]. On the other hand post-column photo-oxidation plausible hypothesis to explain the loss of Sb(V) and TMSb(V) is the
chromatogram of Fig. 3A and B indicates that this decrease in precipitation of both species during the freezing process, with results
concentration was not due to the formation of molecules that did not consistent with those obtained at 4 °C for TMSb(V).
generate hydrides since unknown signals were not detected. About Sb(III), it is interesting to note that during storage at − 70 °C
In order to explain the loss for the rest of Sb(III) spiked in urine the no oxidation of Sb(III) was detected; however, it is clear that the
only plausible explanation for us is by an hydrolysis reaction of Sb(III)
present in the urine, with formation of Sb(OH)3, insoluble species that
Table 4
could be irreversibly retained on the column. Stability studies of antimony species during a 5 day storage time at − 70 °C with and
It is worth mentioning that in Fig. 3A and B there are no chroma- without mobile phase as stabilizing agent.
tographic peaks that could be attributed to species other than those
Species Sb μg L− 1
generated by antimony standards. This fact indicated that although
the concentration of Sb(V) decreased with time, this species remains Urine matrix Urine + mobile phase

in solution after having reached the equilibrium state in urine sample Sb(V) N.D. N.D.
in the presence of EDTA. Similar behavior was observed for TMSb in Sb(III) 4±1 8.3 ± 0.7
TMSb(V) 2.3 ± 0.4 8.1 ± 0.3
the absence and presence of EDTA.
 W. Quiroz et al. / Microchemical Journal 97 (2011) 78–84 83

Table 5
Urine sample analysis.
Sample Sbtotal (μg L− 1)
A b LOD
B b LOD
C 6±2
D b LOD
E b LOD
F b LOD
G 6.3 ± 0.5
H b LOD
LOD 0.1 μg/L.
that only Sb(V) is present in the urine samples analyzed. This is
consistent with the chromatogram of Fig. 4 where only Sb(V) was
μg Sb g− 1 Sb(V) μg Sb(V) g− 1 detected. The presence of Sb(V) in urine could involve either direct
creatinine creatinine exposure to pentavalent antimony compounds or exposure to Sb(III)
– – – and further oxidation to Sb(V); however the oxidation reactions have
– – – not been well documented in human fluids.
2.4 ± 0.3 4.9 ± 0.8 1.9 ± 0.2 Sb(V) levels obtained in this study could be compared with those
– – –
of Krachler et al. [26] with concentration of Sb(V) in urine of two
– – –
– – – individuals exposed of 5.9 ± 0.2 μg L− 1 and 2.0 ± 0.1 μg L− 1, respec-
9.40 ± 0.01 6.2 ± 0.5 9.3 ± 0.1 tively. Furthermore, these authors determined concentrations of Sb
– – – (III) and TMSb, which were an average of around 0.15 ± 0.05 μg L− 1
and 0.49 ± 0.08 μg L− 1, respectively. However in the actual study no
Sb(III) or TMSb were detected in the urine samples analyzed.

recovery of Sb(III) in urine matrix was lower than that obtained in
urine with mobile phase. Our explanation of this result is that com-
plexation reaction between EDTA–Sb(III) avoids the precipitation of
this species.
In summary, we believe that the freezing of urine is a source risk of
loss of analyte by precipitation or co-precipitation of antimony
species. Frozen at − 70 °C as industry standard for storage of urine
samples cannot be applied to antimony speciation analysis unless this
problem is solved.
3.4. Urine sample analysis
The methodology developed in this work was applied to urine
samples from residents or workers in Puchuncaví area a site highly
contaminated by mining activities in Chile [32,33]. Eight samples of
urine were collected and filtered for subsequent analysis (pH
measurements, creatinine, Sb(V), Sb(III), TMSb and total antimony
content); the analysis was performed before 48 h of sampling. The pH
values of urine samples ranged between 4.5 and 6.7, concentrations of
creatinine in the range recommended by ACGIH (0.5 to 3.0 g L− 1).
Finally, results for total antimony concentration and speciation
analysis are summarized in Table 5.
Speciation analysis was performed over a period of less than two
days from the date of sample collection and by adding EDTA 20 mmol
L− 1–KHP 2 mmol L− 1 as stabilizing agents. Finally speciation analysis
was performed only on those samples where antimony was found in
quantifiable levels. Chromatograms obtained for urine samples are
presented in Fig. 4.
In Table 4 by comparing Sb(V) and total antimony concentrations
in samples C and G – which were statistically similar – we can deduce
4. Conclusions
In this work, we have developed a methodology for antimony
speciation in urine samples. The methodology developed represents a
useful tool for antimony monitoring in occupationally exposed human
urine samples.
The methodology described represents an improvement on the
stabilization of antimony species during the entire analytical pro-
cedure including storage time by using EDTA 20 mmol L− 1 + KHP
20 mmol L− 1 as stabilizing agent. The developed method offers good
resolution, detection limits and enough stabilization time to perform
an antimony speciation analysis without transformation of species.
According to our results, we must take special care in the storage
step of urine samples. It is not advisable to freeze the sample at
−70 °C without EDTA due to analyte loss attributed to precipitation of
species during storage time, specially for Sb(V) and TMSb(V).
Regarding the stability of antimony species in urine samples, it can
be concluded that samples must be preserved at 4 °C and stabilized
with EDTA–KHP to avoid the oxidation of Sb(III) to Sb(V) and precip-
itation of TMSb(V) during storage step at –70 °C in urine samples.
In order to evaluate the risk of human exposure to atmospheric
environments contaminated with antimony such as airborne partic-
ulate material, future investigations will be focused on the develop-
ment of antimony speciation methodologies on other biological
human fluids.
Acknowledgements
The authors gratefully acknowledge the financial support of
FONDECYT (project 11080084). We especially thank our teachers
Dr. Ida De Gregori and Dr. Hugo Pinochet for the high quality science
education that they gave us and we wish them a happy retirement.

References
Fig. 4. Chromatograms of (A) urine sample and (B) urine sample + 10 μg L− 1 of Sb(V),
Sb(III) and TMSb. (Y axis correspond to arbitrary units (UA) of the interface HPLC–HG-
AFS proportional to fluorescence counts.)
[1] T. Gebel, Arsenic and antimony: comparative approach on mechanistic toxicology,
Chemico-Biological Interactions 107 (3) (1997) 131–144.
[2] P.H. Pacheco, R.A. Gil, L.D. Martinez, G. Polla, P. Smichowski, A fully automated
system for inorganic antimony preconcentration and speciation in urine,
Analytica Chimica Acta 603 (1) (2007) 1–7.
[3] J. Zheng, M. Ohata, N. Furuta, Antimony speciation in environmental samples by
using high-performance liquid chromatography coupled to inductively coupled
plasma mass spectrometry, Analytical Sciences 16 (1) (2000) 75–80.
[4] S. Amereih, T. Meisel, E. Kahr, W. Wegscheider, Speciation analysis of inorganic
antimony in soil using HPLC–ID–ICP-MS, Analytical and Bioanalytical Chemistry
383 (7) (2005) 1052–1059.
[5] Waldo Quiroz, Ida De Gregori, Paola Basilio, Manuel Bravo, Marcela Pinto, Maria
Gabriela Lobos, Heavy weight vehicle traffic and its relationship with antimony content
in human blood, Journal of Environmental Monitoring 11 (2009) 1051–1055.
[6] Ronland H.R. Suchenwirth, Thomas W. Gebel, Claudia Bolten, H. Hartmut Dunkel-
berg, Human biomonitoring of arsenic and antimony in case of an elevated geogenic
exposure, Environmental Health and Perspectives 106 (1) (1998) 33–39.
[7] P. Heitland, H.D. Köster, Biomonitoring of 30 trace elements in urine of children
and adults by ICP-MS, Clinica Chimica Acta 365 (1–2) (2006) 310–318.
[8] A.A.S. Caroli, E. Coni, F. Petrucci, O. Senofonte, N. Violante, The assessment of
reference values for elements in human biological tissues and fluids: a systematic
review, Critical Reviews in Analytical Chemistry 24 (1994) 363–398.
84 W. Quiroz et al. / Microchemical Journal 97 (2011) 78–84
[9] I. Iavicoli, S. Caroli, A. Alimonti, F. Petrucci, G. Carelli, Biomonitoring of a worker
population exposed to low antimony trioxide levels, Journal of Trace Elements in
Medicine and Biology 16 (1) (2002) 33–39.
[10] F.A., Douglas M. Templeton, Rita Cornelis, Lars-Göran Danielsson, Herbert
Muntau, Herman P. van Leeuwen, Ryszard Lobinski, Guidelines for terms related
to chemical speciation and fractionation of elements. Definitions, structural
aspects, and methodological approaches (IUPAC Recommendations 2000), Pure
Applied Chemistry 72 (2000) 1453–1470.
[11] A.K. Das, M. de la Guardia, M.L. Cervera, Literature survey of on-line elemental
speciation in aqueous solutions, Talanta 55 (1) (2001) 1–28.
[12] M. Filella, N. Belzile, M.-C. Lett, Antimony in the environment: a review focused
on natural waters. III. Microbiota relevant interactions, Earth-Science Reviews 80
(3–4) (2007) 195–217.
[13] J. Lintschinger, I. Koch, S. Serves, J. Feldmann, W.R. Cullen, Determination of
antimony species with high-performance liquid chromatography using element
specific detection, Fresenius' Journal of Analytical Chemistry 359 (6) (1997)
484–491.
[14] M. Krachler, H. Emons, Speciation analysis of antimony by high-performance
liquid chromatography inductively coupled plasma mass spectrometry using
ultrasonic nebulization, Analytica Chimica Acta 429 (1) (2001) 125–133.
[15] H.E. Michael Krachler, Potential of high performance liquid chromatography
coupled to flow injection hydride generation atomic absorption spectrometry for
the speciation of inorganic and organic antimony compounds, Journal of
Analytical Atomic Spectrometry 15 (2000) 281–285.
[16] R.B. Ana Sayago, José L. Gómez-Ariza, Hydride generation atomic fluorescence
spectrometry (HG-AFS) as a sensitive detector for Sb(III) and Sb(V) speciation in
water, Journal of Analytical Atomic Spectrometry 15 (2000) 423–428.
[17] H. Sousa Ferreira, S.L. Costa Ferreira, M.L. Cervera, M. de la Guardia, Development
of a non-chromatographic method for the speciation analysis of inorganic
antimony in mushroom samples by hydride generation atomic fluorescence
spectrometry, Spectrochimica Acta Part B: Atomic Spectroscopy 64 (6) (2009)
597–600.
[18] T. Madrakian, E. Bozorgzadeh, Spectrophotometric determination of Sb(III) and
Sb(V) in biological samples after micelle-mediated extraction, Journal of Hazardous
Materials 170 (2–3) (2009) 809–813.
[19] T. Lindemann, A. Prange, W. Dannecker, B. Neidhart, Stability studies of arsenic,
selenium, antimony and tellurium species in water, urine, fish and soil extracts
using HPLC/ICP-MS, Fresenius' Journal of Analytical Chemistry 368 (2) (2000)
214–220.
[20] N. Miekeley, S. Mortari, A. Schubach, Monitoring of total antimony and its species
by ICP-MS and on-line ion chromatography in biological samples from patients
treated for leishmaniasis, Analytical and Bioanalytical Chemistry 372 (3) (2002)
495–502.
[21] I.D. Gregori, W. Quiroz, H. Pinochet, F. Pannier, M. Potin-Gautier, Simultaneous
speciation analysis of Sb(III), Sb(V) and (CH3)3SbCl2 by high performance liquid
chromatography-hydride generation-atomic fluorescence spectrometry detec-
tion (HPLC–HG-AFS): application to antimony speciation in sea water, Journal of
Chromatography A 1091 (1–2) (2005) 94–101.
[22] M. Potin-Gautier, F. Pannier, W. Quiroz, H. Pinochet, I. de Gregori, Antimony
speciation analysis in sediment reference materials using high-performance
liquid chromatography coupled to hydride generation atomic fluorescence
spectrometry, Analytica Chimica Acta 553 (1–2) (2005) 214–222.
[23] I. De Gregori, W. Quiroz, H. Pinochet, F. Pannier, M. Potin-Gautier, Speciation
analysis of antimony in marine biota by HPLC–(UV)–HG-AFS: extraction
procedures and stability of antimony species, Talanta 73 (3) (2007) 458–465.
[24] I. De Gregori, W. Quiroz, H. Pinochet, F. Pannier, M. Potin-Gautier, Simultaneous
speciation analysis of Sb(III), Sb(V) and (CH3)(3)SbCl(2)by high performance
liquid chromatography–hydride generation-atomic fluorescence spectrometry
detection (HPLC–HG-AFS): application to antimony speciation in sea water,
Journal of Chromatography A 1091 (1–2) (2005) 94–101.
[25] J. Craig, D. Miller, R.O. Jenkins, An analytical method for the detection of
methylantimony species in environmental matrices: methylantimony levels in
some UK plant material, Analyst 124 (1999) 1243–1248.
[26] H.E. Michael Krachler, Urinary antimony speciation by HPLC–ICP-MS, Journal of
Analytical Atomic Spectrometry 16 (2001) 20–25.
[27] Fourth National Report on Human Exposure to Environmental Chemicals, USA:
Department of Health and Human Services, Centers for Disease Control and
Prevention, 2009, p. 176.
[28] C. Verdon, K. Caldwell, M. Fresquez, R. Jones, Determination of seven arsenic
compounds in urine by HPLC–ICP–DRC-MS: a CDC population biomonitoring
method, Analytical and Bioanalytical Chemistry 393 (3) (2009) 939–947.
[29] D.D. Perrin (Ed.), International Union of Pure and Applied Chemistry IUPAC
Chemical Data Series No. 22, Pergamon Press, 1979, p. 770.
[30] T. Lindemann, A. Prange, W. Dannecker, B. Neidhart, Simultaneous determination
of arsenic, selenium and antimony species using HPLC/ICP-MS, Fresenius' Journal
of Analytical Chemistry 364 (5) (1999) 462–466.
[31] Z. Slejkovec, J.T. van Elteren, A.R. Byrne, Determination of arsenic compounds in
reference materials by HPLC–(UV)–HG-AFS, Talanta 49 (3) (1999) 619–627.
[32] A. Neaman, L. Reyes, F. Trolard, G. Bourrié, S. Sauvé, Copper mobility in con-
taminated soils of the Puchuncaví valley, central Chile, Geoderma 150 (3–4) (2009)
359–366.
[33] R. Ginocchio, Effects of a copper smelter on a grassland community in the
Puchuncaví Valley, Chile, Chemosphere 41 (1–2) (2000) 15–23.