Journal of Chromatography A, 1391 (2015) 917

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Mercury speciation in seawater by liquid chromatography-inductively

coupled plasma-mass spectrometry following solid phase extraction
pre-concentration by using an ionic imprinted polymer based on
methyl-mercuryphenobarbital interaction

Mara Pilar Rodrguez-Reino, Roi Rodrguez-Fernndez, Elena Pena-Vzquez,

Raquel Domnguez-Gonzlez, Pilar Bermejo-Barrera, Antonio Moreda-Pineiro

Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Santiago de Compostela, Avenida das Ciencias, s/n,
15782 Santiago de Compostela, Spain

a r t i c l e

i n f o

Article history:
Received 19 December 2014
Received in revised form 23 February 2015
Accepted 24 February 2015
Available online 3 March 2015
Keywords:
Ionic imprinted polymer
Mercury speciation
Seawater
Solid phase extraction
High performance chromatography
Inductively coupled plasma-mass
spectrometry

a b s t r a c t
Trace levels of inorganic mercury, methyl-mercury and ethyl-mercury have been assessed in seawater
by high performance liquid chromatography (HPLC) hyphenated with inductively coupled plasma-mass
spectrometry (ICP-MS) after solid phase extraction (SPE) pre-concentration with a novel synthesized
ionic imprinted polymer. The adsorbent material was prepared by trapping a non-vinylated chelating
ligand (phenobarbital) via imprinting of a ternary mixed ligand complex of the non-vinylated chelating
agent, the template (methyl-mercury), and the vinyl ligand (metacrylic acid, MAA). Ethylene dimetacrylate (EDMA) and 2,2 -azobisisobutyronitrile (AIBN) were used as cross-linker and initiator reagents,
respectively; and the precipitation polymerization technique was used in a porogen of acetonitrile/water
(4:1). The best retention properties for methyl-mercury, inorganic mercury and ethyl-mercury species
from seawater were obtained when loading 200 mL of sample adjusted to pH 8.0 and at a ow rate of
2.0 mL min1 on a column-packed with 200 mg of the material. Quantitative mercury species recoveries
were obtained using 4 mL of an eluting solution consisting of 0.8% (v/v) 2-mercaptoethanol and 20% (v/v)
methanol (pH adjusted to 4.5) pumped at a ow rate of 2.0 mL min1 . Mercury species separation was
achieved on a Kinetex C18 column working under isocratic conditions (0.4% (v/v) 2-mercaptoethanol,
10% (v/v) methanol, pH 2.5, ow rate 0.7 mL min1 ). ICP-MS detection was performed by monitoring the
mercury mass to charge ratio of 202. The limits of quantication of the method were 11, 6.7, and 12 ng L1 ,
for inorganic mercury, methyl-mercury and ethyl-mercury, respectively (pre-concentration factor of 50);
whereas, analytical recoveries ranged from 96 to 106%. The developed method was successfully applied
to several seawater samples from unpolluted areas.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Although the use of mercury (in the chloralkali industry and,
previously, in the wood pulping industry) is decreasing, high mercury concentrations are still present in sediments associated with
the industrial applications of this metal. In addition, the burning of
fossil fuels and pollution of water by mine tailings can also be signicant sources of mercury [1]. However, low levels of dissolved
mercury are expected in the marine environment [2]. Concentrations range from 0.5 to 3.0 ng L1 in open ocean; whereas, values

Corresponding author. Tel.: +34 881814375; fax: +34 981 547141.

E-mail address: antonio.moreda@usc.es (A. Moreda-Pineiro).

http://dx.doi.org/10.1016/j.chroma.2015.02.068
0021-9673/ 2015 Elsevier B.V. All rights reserved.

within the 215 ng L1 range can be found in coastal seawater [1].

Nevertheless, local variations from these values can be observed in
coastal seawater and inland waters where near to anthropogenic
sources, and where mercury associated with suspended material
may also contribute to the total load [1]. Even at low concentrations,
mercury species can drive bio-accumulation and bio-magnication
to levels of this toxic metal in sh, posing human and ecological
health risks [24]. Mercury is thus included in the Annex I (Environmental Quality Standards for Priority Substances and Certain
Other Pollutants) of the Directive 2013/39/UE of the European Parliament and of the Council (12/08/2013), and a maximum allowable
concentration (MAC) of mercury and its compounds of 70 ng L1 in
inland surface waters and other surface waters has been established
[5].

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M.P. Rodrguez-Reino et al. / J. Chromatogr. A 1391 (2015) 917

Despite the better performances (lower limit of detection and

better separation) obtained when using gas chromatographyinductively coupled plasma-mass spectrometry (GC-ICP-MS), the
need of derivatization of mercury species is drawback. HPLC-ICPMS method, although less sensitive allows, however a direct liquid
sample introduction. Pre-concentration procedures are therefore
needed when using HPLC-ICP-MS because the expected mercury
concentrations are within the ng per liter range, and complex
samples, such as seawater, contain high salt levels. Solid phase
extraction (SPE) has been demonstrated to be a suitable preconcentration technique for extracting mercury species, allowing
quantitative recoveries, efcient salt removal, and guarantying
minimal/negligible mercury species changes [6]. Recent applications for mercury speciation include anion (quaternary amine type
[7], and SAX [8]), and cation (SCX, benzenesulfonic acid-type) [9,10]
exchange resins; and also polymeric resins, mainly reverse phase
C18 [1118]. The latter solid supports require mercury species complexation to enhance analyte interaction with the sorbent [1113],
or the immobilization of the complexing reagent on the C18 particles [1418].
Current trends in SPE are mainly focused on the development of
new sorbents such as nanometer-sized materials, egg-shell membranes, modied silica beads, molecularly/ion imprinted polymers,
and mesoporous materials. These new solid supports are prepared mainly to improve selectivity [6]. As reported, molecularly
imprinted polymers (MIPs) and ionic imprinted polymers (IIPs)
have been proposed for achieving efcient pre-concentration of
both organic and inorganic analytes, and also for clean-up purposes [1922]. In contrast to organic molecules, the absence of
functional groups in most ions difcults a templatemonomer
interaction, and the typical binary (template/vinylated monomer)
pre-polymerization mixtures when synthesizing MIPs must be
changed to ternary pre-polymerization mixtures in which an auxiliary non-vinylated reagent (a ligand exhibiting afnity for the
metal ion and adequate functional groups) is mixed with the template and the monomer. After polymerization, the non-vinylated
chelating ligand is therefore trapped via imprinting [22]. On other
occasions, bifunctional reagents (a ligand exhibiting afnity for the
metal ion and containing vinyl groups for polymerization after cross
linking) can be used, and the complexing ligand is therefore chemically immobilized in the polymeric matrix. The main drawback
of the latter approach is the fact that complexing ligands showing vinyl groups are scarce and are not commercially available,
so the synthesis of these complexing monomers must be performed in the laboratory. Although some bifunctional monomers
have been used for preparing IIPs for mercury recognition [23,24],
most of the synthesized IIPs used non-vinylated ligands trapped
in the polymeric matrix after ternary templatenon-vinylated
ligandvinylated monomer interactions when using Hg(II) [2530]
and methyl-mercury (Me-Hg) [31] as templates.
The aim of the current work was the application of the precipitation polymerization approach for synthesizing an IIP for mercury
species [Hg(II), Me-Hg, and ethyl-mercury (Et-Hg)] retention
against major components in seawater (mainly sodium and chloride). A strategy based on using a ternary template/non-vinylated
ligand/vinylated monomer (Me-Hg/phenobarbital/metacrylic acid
(MAA) was adopted. Phenobarbital has not previously been proposed for IIP synthesis. However, phenobarbital was recently
demonstrated to exhibit adequate functional groups for interacting with vinylated monomers such as MMA [32]. Mercury
speciation (Hg(II), Me-Hg, and Et-Hg) was performed by high
performance liquid chromatography (HPLC)-inductively coupled
plasma-mass spectrometry (ICP-MS). The chromatographic separation was attained in 5 min, a shorter chromatographic time
than those reported in other published applications (within the
1025 min range) [3336]). Because of the short analysis time

(5 min) when comparing to those previously reported applications,

and because of the high sensitivity inherent in the use IIP-SPE
procedure, the developed method allows the assessment of mercury species in seawater at levels lower than the MAC established
in the Directive 2013/39/UE for environmental quality standards
(70 ng L1 ) [5].
2. Experimental
2.1. Apparatus
A Flexar LC HPLC (LC pump, column oven, and LC autosampler) from Perkin Elmer (Waltham, MA, USA) was used for mercury
species separations. The HPLC system was coupled to a Perkin Elmer
Nex-Ion 300X ICP-MS, instrument which was also used for total
mercury determinations [in such case, a SeaFast SC2 DX autosampler from Elemental Scientic (Omaha, NB, USA) was used]. Reverse
phase chromatographic separations were performed on a Kinetex
C18 100 A (100 mm length 2.10 mm i.d., 5.0 m particle diameter) analytical column (Phenomenex, Torrance, CA, USA) connected
to a C8 guard column (4 mm length 3.0 mm i.d.) from Phenomenex. Other tested analytical columns were Kinetex C18 100 A
(100 mm length 4.60 mm i.d., 2.6 m particle diameter) from
Phenomenex, and Zorbax Eclipse XDB-C8 (4.6 150 mm) from Agilent (Santa Clara, CA, USA). Polymerization was performed by using
a temperature-controlled incubation camera (Stuart Scientic, Surrey, UK) equipped with a low-prole roller (Stovall, Greensboro,
NC, USA). IIPs were packed into 5 mL syringes (Braun, Wertheim,
Germany) between replacement Teon frits (Supelco, Bellefonte,
PA, USA). SPE was performed by using a Miniplus 3 peristaltic pump
equipped with a head for delivering low pulse ows of uids in 8
channel standard ow rate (Gilson, Middleton, WI, USA), and PVC
2-stop tubing (3.175 mm i.d.) from SCP Sciences (Quebec, Canada).
Seawater samples were ltered through Durapore 0.45 m lters
(Millipore, Billerica, MA, USA) by using a vacuum pump (Millipore).
Synthesized IIP material was ltered through Durapore 0.22 m
lters (Millipore). IIP characterization was performed by using a
scanning electron microscope EVO LS 15 from Zeiss (Oberkochem,
Germany), and an energy dispersive X-ray uorescence spectrometer (molybdenum-based anode) laboratory-made by RIAIDT (Rede
de Infraestruturas de Apoio Investigacin e ao Desenvolvemento
Tecnolxico) at the University of Santiago de Compostela. Other
laboratory devices were a Basic20 pH meter with a glasscalomel
electrode (Crison, Barcelona, Spain) for pH measurements, a Reax
2000 (Heidoph, Niederbayern, Germany) for vortexing solutions,
and an oven model 207 from Selecta (Barcelona, Spain) for IIP drying.
2.2. Reagents
Ultrapure water of resistivity 18 M cm obtained from a
Milli-Q purication device (Millipore) was used to prepare all the
solutions. Hg(II) stock standard solution (1000 mg L1 ) was from
Panreac (Barcelona, Spain). Methyl-mercury stock standard solution (1000 mg L1 ) was prepared from methyl-mercury chloride
from Sigma (Steinhelm, Germany) by dissolving the reagent in a
small volume of hot methanol, and further dilution with ultrapure
water. Ethyl-mercury stock standard solution (1000 mg L1 ) was
prepared from ethyl-mercury chloride from Alfa Aesar (Ward Hill,
MA, USA) by reagent dissolution in a small volume of 5% (v/v)
sulfuric acid in methanol before dilution with ultrapure water.
Mercury (I) stock standard solution (1000 mg L1 ) was prepared
by dissolving mercury (I) chloride from Sigma. Multi-element
calibration standard 3 (10 mg L1 of Al, Ca, Cd, Cr, Cu, Fe, Mg,
Mn, Ni, Pb, Sr, V, and Zn), and Y (10 mg L1 ) used as an internal

M.P. Rodrguez-Reino et al. / J. Chromatogr. A 1391 (2015) 917

11

Fig. 1. Scanning electron microscopy pictures for IIP (A) and NIP (B).

standard were from Perkin Elmer (Shelton, CT, USA). HPLC grade
methanol, high purity nitric acid (96% (v/v)), sulfuric acid (69%
(v/v)), hydrochloric acid (37% (v/v)), and analytical grade sodium
chloride and sodium hydrogencarbonate were purchased from
Panreac. Metacrylic acid (MAA), ethylene dimetacrylate (EDMA),
phenobarbital, thiourea, and 2-mercaptoethanol were supplied
by Sigma. 2,2 -Azobisisobutyronitrile (AIBN) was purchased from
Fluka (Buchs, Switzerland). This reagent was puried by crystallization at 20 C after dissolving the reagent in methanol
(Panreac) at 5060 C. After purication, this reagent was stored at
4 C. High purity ammonia (25% (v/v)), ethanol, sodium hydroxide,
ammonium chloride, and hydroxylamine hydrochloride were purchased from Merck (Darmstadt, Germany). HPLC grade acetonitrile
was obtained from Scharlab (Barcelona, Spain). A synthetic seawater sample of low salinity (34.2) was prepared by dissolving
sodium chloride and sodium hydrogencarbonate from Panreac,
and heptahydrate magnesium sulfate from Merck (32, 0.15, and
14 g in 1 L of ultrapure water, respectively) [37]. ORMS-5 (Elevated
mercury in river water) certied reference material was from
National Research Council of Canada (Ottawa, Canada).
All glass and plastic material were rigorously cleaned and kept
into 10% (m/m) nitric acid for at least 48 h. The material was then
rinsed three times with ultrapure water before being used.

polymerization was nished, the synthesized material was vacuum

ltered, washed with acetonitrile/water (4:1), and then oven-dried
overnight at 40 C. Masses of polymer ranging from 1.5 to 1.7 g were
obtained.
Blank polymer particles (non-imprinted polymers, NIPs) were
also prepared in the same way as IIPs, using the same masses
and volumes as for IIP synthesis, but without adding the template
(methyl-mercury chloride). The NIPs were then subjected to the
same washing pre-treatment described above.

2.3. Seawater collection

2.6. Characterization of the synthesized IIP

Surface seawater samples were collected from several estuaries in Galicia (northwestern Spain) in pre-cleaned high density
polyethylene bottles. After collection, seawater samples ltered
through 0.45 m lters and subjected to analysis. When not immediately subjected to analysis, ltered seawater samples were kept
at 20 C.

Scanning electron microscopy (SEM) pictures were taken from

IIP and NIP (Fig. 1) to study the morphology and the size of the
synthesized materials. Both IIP and NIP consisted of agglomerates
of spherical particles (mean diameter lower than 10 m). Particles agglomeration in NIPs (Fig. 1B) appears to be lower, a fact
that can be attributed to the absence of the template during the
polymerization process as shown when preparing other IIPs/NIPs
[3840].
Energy dispersive X-ray uorescence (EDXRF) was used for conrming the presence of mercury in the synthesized IIP (and the
absence of mercury in NIP), and also to prove an efcient MeHg removal from IIPs after leaching with thiourea. Patterns for IIP
material before and after leaching (Fig. 2A and B, respectively), as
well as for NIP (Fig. 2C) were obtained, showing that mercury is only
present in the unleached IIP particles, and it is totally removed after
the described template removal procedure.

2.4. Synthesis of ionic imprinted polymer particles

Solid methyl-mercury chloride (18.3 mg) and phenobarbital
(61.0 mg) were mixed with 51.2 L of MAA and 12 mL of porogen
(acetonitrile/water 4:1) into 15 mL glass test tubes. This implies
a template/non-vinylated reagent/vinylated reagent molar ratio
of 1:2:2 in the polymerization mixture. Other molar ratios (1:1:1
and 1:2:4) led to IIPs of poor retention capacity. The mixture was
then stirred for 5 min and was kept in the dark overnight. EDMA
(365 L), and puried AIBN (43.3 mg), according with previous
studies [3840], were added to the test tubes placed in an ice-bath,
and after stirring for 1 min, the mixture was purged with argon
for 10 min at low temperature (test tubes in an ice-bath), and was
immediately sealed, and placed in a low-prole roller (33 r.p.m
about its long axis) inside a temperature-controllable incubator
camera (the temperature was ramped from room temperature to
60 C for 2 h, and then maintained at 60 C for a further 24 h). Once

2.5. Template removal procedure

Several portions (200 mg) of the material were packed into 5 mL
syringes between two Teon frits. The template (methyl-mercury)
must be removed from the polymer particles, leaving free cavities
complementary in size, shape, and functionality ready for analyte
recognition. This procedure was performed by pumping a thiourea
solution in an acid medium (1 M thiourea in 1 M HCl) at a ow rate of
1 mL min1 . Negligible mercury concentrations (ICP-MS measurements) were found in the washing/ltrate solutions after passing
200 mL of acidic thiourea. After complete template removal, the
packed syringes were extensively washed by pumping ultrapure
water for thiourea residues removal.

2.7. IIPs solid phase extraction

SPE syringes containing the polymer were conditioned by
pumping 10 mL of a 0.1 M/0.1 M NH3 /NH4 Cl buffer solution (pH 8.0)
at a ow rate of 2 mL min1 . Previous to sample loading, 200 mL
of seawater (pH approximately 6.7) was mixed with a 0.1 M/0.1 M
NH3 /NH4 Cl buffer solution (pH 8.0) to obtain a pH of 8.0 0.5

12

M.P. Rodrguez-Reino et al. / J. Chromatogr. A 1391 (2015) 917

Fig. 2. Energy dispersive X-ray uorescence spectra for the unleached IIP (A), NIP (B), and leached IIP (C).

(2540 mL of the buffer solution were needed). Seawater samples

adjusted to a pH of 8.0 were then pumped through the syringe at a
ow rate of 2 mL min1 . The syringes were then rinsed with 20 mL
of the NH3 /NH4 Cl (pH 8.0) buffer solution (2 mL min1 ), and the
retained mercury species were then subsequently eluted with four
1.0 mL aliquots of an eluting solution consisting of 0.8% (v/v) 2mercaptoethanol and 20% (v/v) methanol (pH 4.5) at a ow rate of
2.0 mL min1 . A pre-concentration factor of 50 was achieved under
these operating conditions. After elution, the IIPs were treated with
10 mL of ultrapure water (2 mL min1 ) and then conditioned by
passing 10 mL of the 0.1 M/0.1 M NH3 /NH4 Cl buffer solution at pH
8.0 (2 mL min1 ). Eight samples can simultaneously be treated in
approximately 100 min because of the use of a peristaltic pump
with 8 channels.

ndings reported by Chang et al. [33], increased background at mercury mass m/z 202 (ICP-MS detection) was observed in the presence
of l-cysteine in our preliminary studies when using the selected
analytical column (Kinetex C18 100 A, 100 mm length 2.10 mm
i.d., 5.0 m particle diameter, Table 1), and also when using the
Kinetex C18 100 A (100 mm length 4.60 mm i.d., 2.6 m particle
diameter), and the Zorbax Eclipse XDB-C8 analytical columns. In
addition, the presence of l-cysteine in the mobile phase resulted in
a partial reduction of Hg(II) species to Hg(I) (two chromatographic
signals were observed when injecting Hg(II) standards when using
both Kinetex C18 columns). Therefore, RP separations based on
using 2-mercaptoethaol/methanol mixtures were further considered for optimization. A study of the effect of the mobile phase
composition on the retention times and chromatographic signals

2.8. ICP-MS and HPLC-ICP-MS measurements

Total mercury, as well as other trace elements such as Al, Cd, Cr,
Cu, Fe, Mn, Ni, Pb, Sr, and V (assessment of the interferences from
other toxic metals on SPE) were determined by ICP-MS (Table 1).
Yttrium (25 g L1 ) was used as an internal standard, and calibration was performed within the 0100 g L1 range. Inorganic
mercury (Hg(II)), MeHg, and Et-Hg in the extracts after IIP-SPE
were separated by reverse phase (RP) HPLC and detected by ICPMS. Operating conditions are listed in Table 1. Chromatograms of
a pre-concentrated seawater sample under optimized IIP-SPE conditions (2.30 0.13, 0.62 0.035, and 0.45 0.03 g L1 for Hg(II),
Me-Hg and Et-Hg, respectively), and a 5.0 g L1 standard solution
of Hg(II), Me-Hg, and Et-Hg is shown in Fig. 3 as an example.

Table 1
Instrumental parameters for ICP-MS and HPLC-ICP-MS measurements.
ICPMS
Ar ow rate
(L min1 )
O2 ow rate
(L min1 )
KED mode: He ow
rate (mL min1 )
Mass-to-charge
ratio

RP-HPLC

3. Results and discussion

3.1. Optimization of operating RP conditions
Mobile phases based on acidic aqueous l-cysteine [41], lcysteine/methanol [10,34], and acidic aqueous 2-mercaptoethanol
solutions with [35] and without [8] methanol have been proposed when separating mercury species by RP. In accordance with

Mobile phase
composition
Mobile phase ow

Radiofrequency power (W)

Peristaltic pump speed (rpm)
Plasma/auxiliary/nebulizer

1600
2.5
18.0/1.2/0.89
0.01

Al, Cr, Mn, Cu, Cd, Hg, and Pb

Mg, Ca, V, Fe, Ni, Zn, and Sr
Y (internal standard)
Mg, Al, Ca, V, Cr, Mn, Fe, Ni, Cu, Zn, Sr,
Cd, Hg, and Pb

Kinetex C18 100 A (100 mm

length 2.10 mm i.d., 5.0 m particle
diameter) analytical column connected
to a C8 guard column (4 mm
length 3.0 mm i.d.)
Injection volume/L
Isocratic: 0.4% (v/v)
2-mercaptoethanol, 10% (v/v)
methanol, pH 2.5
0.7 mL min1

1
4
89
24, 27, 43, 51,
52, 55, 57, 60,
65, 66, 88, 114,
202, and 208

50

M.P. Rodrguez-Reino et al. / J. Chromatogr. A 1391 (2015) 917

The loading ow rate (0.5, 1.0, 1.5, and 2.0 mL min1 ) was also
studied by using seawater samples spiked with 2.0 g L1 of each
analytes, and treated with 0.1 M/0.1 M NH4 Cl/NH3 buffer solutions
(pH 8.0). As shown in Figure S1B (Supplementary electronic information), lower analytes retention was observed when loading at
0.5 mL min1 ; whereas, analyte retention increased when loading
at ow rates within the 1.02.0 mL min1 range. Therefore, a loading ow rate was nally set at 2.0 mL min1 to obtain a faster SPE
process.

4500
5.0 g L-1 standard

4000

Me-Hg

Seawater SW1

Intensity/ cps

3500
3000
Hg(II)

2500

Et-Hg

2000

13

1500
1000
500
0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Time/ min
Fig. 3. Reverse phase chromatograms for a 5.0 g L1 Hg(II), Me-Hg, and Et-Hg
standard, and for a pre-concentrated seawater sample (2.30 0.13, 0.62 0.035,
and 0.45 0.03 g L1 for Hg(II), Me-Hg, and Et-Hg, respectively).

(integrated area and shape) of mercury species led to the selection

of a mobile phase consisting of 0.4% (v/v) 2-mercaptoethanol and
10% (v/v) methanol (pH of 2.5) as listed in Table 1.
3.2. Preliminary evaluation of IIP-SPE conditions
Since mercury species are retained by binding to phenobarbital residues, elution must lead to mercury speciesphenobarbital
bonding destruction. First attempts for eluting the retained analytes by using a 0.1 M/0.1 M thiourea/hydrochloric acid solution
[23,25,27,28,31] led to an incomplete elution, as well as a partial
reduction of Hg(II) into Hg(I) and bad chromatographic proles for Me-Hg. The previously optimized mobile phase (0.4%
(v/v) 2-mercaptoethanol, 10% (v/v) methanol, pH 2.5) was tried
as an eluting solution, and similar chromatograms to those
obtained when injecting aqueous standards were obtained. However, signal intensities were low after eluting with two successive
2 mL aliquots. Therefore, a solution consisting of 0.4% (v/v) 2mercaptoethanol, 10% (v/v) methanol, and pH 2.5 (non-optimized
concentrations) was adopted for further experiments. In order to
avoid cross contamination and to guarantee that the IIP was free of
analytes when studying experimental loading/elution conditions,
after elution with the proposed solution, the IIP-SPE syringes were
cleaned by pumping 10 mL of 0.1 M/0.1 M thiourea/hydrochloric
acid solution.
3.3. Optimization of mercury species IIP-SPE from seawater
3.3.1. Loading conditions: effect of sample pH and loading ow
rate
Seawater samples (100 mL) spiked with 2.0 g L1 of each analytes were treated with 0.1 M/0.1 M NH4 Cl/NH3 buffer solutions of
pHs 7.0, 7.5, 8.0, and 9.0. In addition, a fortied seawater sample
without pH adjustment (pH of 6.7) was also tested. After analytes assessment by RP-HPLC-ICP-MS (calibration matched with
the mobile phase, and covering analyte concentrations between
0 and 100 g L1 ) analytical recoveries (three replicates) plotted
in Figure S1A (Supplementary electronic information) showed that
Hg(II) is conveniently retained at any pH (recoveries of approximately 80%); whereas Me-Hg and Et-Hg retention is favored when
working at higher pHs (analytical recoveries for Me-Hg were close
to 60% at pHs of 8.0 and 9.0, and approximately 25% for Et-Hg at a
pH of 8.0). These low analytical recoveries must be attributed to an
inefcient elution rather than bad retention (un-optimized elution
conditions were used). Therefore, a pH of 8.0 was nally chosen.

3.3.2. Eluting conditions: effect of the composition of the eluting

solution
The percentage of 2-mercaptoethanol was rst studied within
the 0.20.8% (v/v) range. Results (mean analytical recoveries of
three replicates after mobile phase matched calibration between
0 and 100 g L1 ) plotted in Figure S2A (Supplementary electronic information) showed that analytes release was increased
when using higher percentages of 2-mercaptoethanol. Analytical
recoveries close to 100% were observed for inorganic mercury
when using the highest percentages of 2-mercaptoethanol (0.6
and 0.8% (v/v)). In addition, Me-Hg recoveries were also increased
up to approximately 80% (0.6 and 0.8% (v/v) 2-mercaptoethanol);
whereas, the highest Et-Hg recovery (48%) was obtained when
eluting with solutions containing 0.8% (v/v) 2-mercaptoethanol.
However, it must be mentioned that recoveries plotted in Figure
S2A are obtained after subtracting the analyte concentrations found
in non-spiked seawater samples subjected to the same conditions.
This was signicant mainly for inorganic mercury (analytical recoveries closed to 120% for spiked seawater samples); whereas, it was
negligible for organic mercury species.
After xing the 2-mercaptoethanol percentage at 0.8% (v/v), the
pH was studied within the 2.05.0 range (the methanol percentage
was xed at 10% (v/v)). Results in Figure S2B (Supplementary electronic information) showed that quantitative recoveries for Me-Hg
were achieved when using extracting solutions adjusted at pHs
higher than 4.0. In addition, Et-Hg recoveries were also increased
up to 60% when using higher pHs. Regarding inorganic mercury,
quantitative recoveries were attained within the whole pH range
studied. Finally, the effect of the percentage of methanol on the
analyte elution was tested. As shown in Figure S2C (Supplementary electronic information), higher methanol percentages (15 and
20% (v/v)) led to higher Et-Hg recoveries.
The composition of the eluting solution was therefore xed at
0.8 and 20% (v/v) for 2-mercaptoethanol and methanol, respectively, and a pH of 4.5 (pH adjusted by using 0.1 M hydrochloric
acid).
3.3.3. Eluting conditions: effect of the eluting ow rate and
volume of the eluting solution
Experiments performed by pumping the eluting solution at
a ow rate of 0.5, 1.0, 1.5, and 2.0 mL min1 showed quantitative mercury species recoveries (mobile phase matched calibration
between 0 and 100 g L1 ) for all cases. The highest eluting ow
rate (2.0 mL min1 ) was therefore chosen for further experiments.
Regarding the volume of the eluting solution, quantitative
recoveries for the three mercury species were observed when using
4.0 and 5.0 mL (Figure S3, Supplementary electronic information).
In addition, the best elution was achieved after pumping successive
1 mL aliquots. Since both eluting volumes have led to good analytical recoveries, an eluting volume of 4.0 mL was chosen to obtain
the highest pre-concentration factor.
3.3.4. Effect of the sample volume (breakthrough volume)
Experiments were performed by increasing the volume of
spiked (2.0 g L1 of each analyte) seawater samples from 100 mL

14

M.P. Rodrguez-Reino et al. / J. Chromatogr. A 1391 (2015) 917

Table 2
Extraction efciency (%), distribution ratios (D) and selectivity coefcients (SMe-Hg/M )
of for IIP and NIP.

110

Analytical recovery (%)

100
90

Extraction
efciency (%)a

80
70

Me-Hg
Hg(II)
Et-Hg

60
50
40
30
20
10
0
100

150

200

250

Sample volume (mL)

Fig. 4. Effect of seawater sample volume on the analytical recovery of mercury
species by IIP-SPE.

to 250 mL. Results in triplicate (Fig. 4) showed that higher sample volumes (up to 250 mL) can be used when assessing organic
mercury species (Me-Hg and Et-Hg) since quantitative recoveries
are attained for all cases. However, inorganic mercury recoveries
decreased to 87 1% when loading seawater volumes of 250 mL.
Therefore, the breakthrough volume can be established at 200 mL.
Taking into account the volume of the eluting solution (4.0 mL),
and the maximum volume of seawater sample without reaching
the breakthrough volume (200 mL), a pre-concentration factor of
50 can be established.

Ionic imprinted polymer (IIP)

97
Me-Hg
94
Hg(II)
98
Et-Hg
Al(III)
39
Cd(II)
80
2.0
Cr(III)
75
Cu(II)
73
Fe(III)
1.4
Mn(II)
Ni(II)
20
Pb(II)
57
Sr(II)
3.4
1.3
V(III)
Non-ionic imprinted polymer (NIP)
47
Me-Hg
43
Hg(II)
47
Et-Hg
Al(III)
20
79
Cd(II)
Cr(III)
5.5
65
Cu(II)
65
Fe(III)
0.4
Mn(II)
22
Ni(II)
Pb(II)
59
1.6
Sr(II)
V(III)
0.85

Distribution
ratio (D)b

Selectivity coefcient
(SMe-Hg/M )c

32
16
49
0.27
6.6
0.01
3.7
2.7
0.012
0.21
1.6
0.033
0.012

2.0
0.65
119
4.8
3200
8.6
12
2667
152
20
970
2667

0.89
0.75
0.89
0.25
3.8
0.058
1.9
1.9
0.004
0.28
1.7
0.017
0.009

1.2
1.0
3.6
0.24
15
0.48
0.48
222
3.2
0.52
52
103

% = (A2 /AT ) 100.

D = (A2 /A1 ).
SMe-Hg/M = DMe-Hg /DM .
A1 = amount of metal ion in aqueous solution at equilibrium.
A2 = amount of metal ion enriched by IIP/NIP at equilibrium.
AT = total amount of metal ion used in extraction.
DMe-Hg = distribution ratio for Me-Hg (template)
DM = distribution ratio for M (M = Hg(II), Et-Hg, Al(III), Cd(II), Cr(III), Cu(II), Fe(III),
Mn(II), Ni(II), Pb(II), Sr(II), and V(III)).
a

3.3.5. Assessment of the interferences from other toxic metals on

IIP-SPE
Different experiments were performed to evaluate the selectivity of the synthesized material for interacting with mercury
species. Several 200 mL aliquots of a synthetic seawater sample
(low salinity, 32) [37] spiked Hg(II), Me-Hg, and Et-Hg (1 g L1 )
and other ions (2 g L1 ) as listed in Table 2, were subjected to
the optimized IIP-SPE procedure. After ICP-MS measurement for
trace element assessment and RP-HPLC-ICP-MS for mercury speciation, parameters such as extraction efciency (analytical recovery),
distribution ratio (D) and selectivity coefcient (SMe-Hg/M ), dened
as shown in Table 2, were calculated. High extraction efciencies, as well as high distribution ratios, were obtained for Me-Hg
(the template when synthesizing the IIP material), and also for
Hg(II) and Et-Hg. In addition, low selectivity coefcients (Table 2)
were observed for mercury species. However, low extraction efciencies and distribution ratios (and therefore high selectivity
coefcients) were obtained for other ions. These results indicate
that the prepared material is selective for Me-Hg (template) and
other mercury species; whereas, other transition metals are not
retained.
Similar experiments were performed by using the synthesized
NIP. As shown in Table 2, lower extraction efciencies and distribution ratios were obtained for mercury species as well as for other
ions. It can therefore be concluded that IIP offers imprinting properties for Me-Hg (the template) and also for Hg(II) and Et-Hg; because
the NIP material shows low afnity (extraction efciencies lower
than 50%), indicating that those specic cavities formed for Me-Hg
during the IIP polymerization are responsible for mercury species
retention. The relative high extraction efciencies for Cd(II), Cu(II),
and Fe(III) (Table 2) when using both IIP and NIP materials indicate that these ions are retained by adsorption (the IIP does not
offer imprinting properties for these ions because similar extraction
efciencies are observed when using the NIP).

3.4. Analytical performances

3.4.1. Calibration. Evaluation of matrix effect
Five external calibrations matched with mobile phase and covering analyte concentrations of 0.0, 5.0, 7.5, 15, and 30 g L1
were obtained in different days. Similarly, ve standard addition calibrations were also obtained in ve different days by
spiking seawater samples in duplicate with 0.0, 0.10, 0.15,
0.30, and 0.60 g L1 of each analyte (concentration within
the 030 g L1 after a pre-concentration factor of 50). The
mean and standard deviation for the slopes of ve standard
addition graphs were 35 104 2.8 104 , 52 104 1.9 104 ,
and 42 104 4.5 104 cps L g1 for Hg(II), Me-Hg, and EtHg, respectively; whereas, mean and standard deviation for
the slopes of ve matched calibration were 40 104 2.2 104 ,
48 104 3.6 104 , and 39 104 3.1 104 cps L g1 for Hg(II),
Me-Hg, and Et-Hg, respectively. The application of CochranC and
Bartletts tests (comparison of variances), and the ANOVA test
(comparison of means) at a 95.0% condence interval led to
matched calibration and standard addition graphs were statistically comparable, and matrix effect due to the high salt content in
seawater was found to be unimportant. This fact offers a practical
advantage so that a tedious and time consuming standard addition
technique is not necessary.
3.4.2. Limit of detection and limit of quantication of the method
The limit of detection (LOD) and the limit of quantication
(LOQ) dened through the 3/10 criterion as LOD = 3/m and

M.P. Rodrguez-Reino et al. / J. Chromatogr. A 1391 (2015) 917

15

Table 3
Comparison of LODs and pre-concentration factor for mercury speciation by SPE.
LOD (ng L1 )

Off-line phenobarbital IIP

Off-line dithizone-functionalized C18
Off-line sodium diethyldithiocarbamate
immobilized in polyurethane foam
Off-line 1,3-bis(2-cyanobenzene)triazene
modied C18
On-line ammonium
pyrrolidinedithiocarbamate modied C18
Off-line C60 fullerene (analytes are previously
complexed with sodium
diethyldithiocarbamate)
Off-line dithizone-functionalized C18
Off-line 2-mercaptoethanol modied C18
On-line strong anion-exchange column
On-line cation-exchange column
On-line C18
a

Pre-concentration factor

Hg(II)

Me-Hg

Et-Hg

HPLC-ICP-MS
HPLC-ICP-MS
HPLC-ICP-MS

3.4
3.0
4.6

2.0
3.0
5.2

2.1
3.0

HPLC-UV

1.3

1.0

HPLC-MS

0.09a

0.37a

0.28a

GC-MS

1.5

1.2

1.5

HPLC-UV
HPLC-CV-AFS
HPLC-ICP-MS
HPLC-ICP-MS
HPLC-ICP-MS

0.54a
800
0.016
0.042
0.07

0.58a
4300
0.01
0.016
0.02

1400
0.009
0.008

50
23.831.2
100

Ref.

This work
[16]
[18]

25

[17]

2500

[12]

100

[13]

200
1000
1025
1250

[14]
[15]
[8]
[10]
[11]

Absolute LOD (expressed as ng).

Table 4
Inter-day precision and inter-day analytical recovery (n = 14) at two mercury species concentration levels.
Concentration level (g L1 )

Hg(II)

0.1
0.6

Me-Hg

Et-Hg

Analytical recovery (%)

RSD (%)

Analytical recovery (%)

RSD (%)

Analytical recovery (%)

RSD (%)

96 12
99 7

12
7

98 8
100 6

9
6

106 8
101 5

8
5

LOQ = 10/m ( is the standard deviation of eleven measurements

of a blank, and m is the mean slope of the calibration) were calculated. By assuming a pre-concentration factor of 50, LOD values
were 3.4, 2.0, and 2.1 ng L1 for Hg(II), Me-Hg, and Et-Hg, respectively. Similarly, values of 11, 6.7, and 7.0 ng L1 were obtained as
LOQ for Hg(II), Me-Hg, and Et-Hg, respectively. As summarized in
Table 3, LODs achieved are similar than those previously obtained
by other authors when using other adsorbents for off-line SPE pretreatment before HPLC-ICP-MS detection [16,18], HPLC-UV [17],
and GC-MS [13]. LOD achieved are lower than those reported when
using UV spectophotometry [14], mass spectrometry [12], and cold
vapour atomic uorescence spectrometry (CV-AFS) [15] as detection systems for HPLC (Table 3). However, values are higher than
those obtained when using on-line SPEHPLC-ICP-MS [8,10,11]
(Table 3). LOQs achieved by applying the current development are
low enough to assess mercury species in seawater since the values
are lower than the MAC of 70 ng L1 established in the Directive
2013/39/eu of the European Parliament and of the Council [5].
3.4.3. Repeatability and accuracy of the method
Table 4 shows good inter-day precision and inter-day analytical
recovery (seawater samples spiked with a low and a high analytes
concentration, 0.1 and 0.6 g L1 , respectively, and experiments
performed in two different days). Accuracy was also evaluated

through the analysis of the CRM ORMS-5 (elevated mercury in

river water). After applying the developed IIP-SPE-HPLC-ICP-MS
method (pre-concentration factor of 2.5) non quantitative results
were obtained for inorganic mercury when using aqueous calibration and the standard addition technique. This fact can be
attributed to the presence of bromine monochloride (BrCl) at 0.5%
(v/v), added as a preserver in the CRM. The negative effects of the
BrCl matrix and/or the presence of BrCl-Hg(II) complexes on Hg(II)
interaction with the IIP were partially reduced by treating the CRM
with hydroxylamine hydrochloride [42]. However, non quantitative results (inorganic mercury concentration of approximately 60%
of the certied concentration) were obtained, and more drastic
conditions may be required for destroying the BrCl matrix/BrClHg(II) complex so that an efcient Hg(II) interaction with the IIP
can occur. Total mercury (inorganic mercury) determination in the
CRM was therefore assessed by direct analysis of the untreated CRM
by ICP-MS. A total mercury concentration of 25.5 0.5 pg g1 (ve
determinations) was found, which agrees with the certied value
(26.2 1.3 pg g1 ).
3.5. Application to seawater samples
The developed method was applied to eight seawater samples collected from several estuaries along the Galician coast

Table 5
Mercury species concentration in some seawater samples.
[Hg(II)] (ng L1 )
SW1
SW2
SW3
SW4
SW5
SW6
SW7
SW8
a
b

45.9
12.7
15.8
16.6
11.5
13.1
16.4
21.1

2.6
0.4
0.9
0.3
0.3
0.5
1.1
1.3

[Me-Hg] (ng L1 )

[Et-Hg] (ng L1 )

[Hg] (ng L1 )a

12.4 0.7
<6.7
<6.7
<6.7
<6.7
<6.7
<6.7
<6.7

9.0 0.6
<7.0
<7.0
<7.0
<7.0
<7.0
<7.0
<7.0

67.3
12.7
15.8
16.6
11.5
13.1
16.4
21.1

Concentration expressed as the sum of mercury species concentrations.

Total mercury concentration assessed by ICP-MS.

2.9
0.4
0.9
0.3
0.3
0.5
1.1
1.3

[Hg] (ng L1 )b
66.1
13.4
15.6
17.0
17.1
19.8
16.3
22.3

1.7
0.5
0.4
0.7
0.9
0.6
0.4
0.7

16

M.P. Rodrguez-Reino et al. / J. Chromatogr. A 1391 (2015) 917

(north-western Spain). Each seawater sample was subjected in

triplicate to the proposed IIP-SPE method, and each eluate was
measured twice by the proposed RP-HPLC-ICP-MS procedure. The
eluates after IIP-SPE were also analyzed in triplicate for total mercury by ICP-MS. Table 5 lists the obtained concentrations for each
analyte, and also for total mercury in seawater. Most mercury
occurs as inorganic mercury; whereas, Me-Hg and Et-Hg was only
quantied in one seawater sample. In addition, Me-Hg concentration was between the LOD and LOQ in two other samples. It can
also be seen that total mercury concentrations are similar to the
assessed inorganic mercury concentrations (and to the mercury
concentration expressed as the sum of mercury species concentrations in seawater samples SW1, SW5 and SW6), which means that
there are not other mercury species unresolved by the RP-HPLC
method. Finally, all concentrations were lower than the MAC of
70 ng L1 established in the Directive 2013/39/eu of the European
Parliament and of the Council [5].
4. Conclusions
Mercury speciation has been performed by a fast RP chromatography (chromatographic run of 5 min) working under isocratic
conditions, and with a mobile phase quite similar to the eluate matrix composition used in the IIP-SPE procedure (formally
2-mercaptoethanol and methanol in water). The phenobarbital
trapping in the polymeric matrix (IIP adsorbent) prepared in the
presence of MAM and EDMA as vinylated monomer and crosslinker, respectively, has generated a IIP exhibiting recognition
capacities for Me-Hg, Hg(II) and Et-Hg ions, as well as quantitative
separation/pre-concentration of mercury species from seawater
samples. The synthesized polymeric material has not offered afnity for major elements in seawater samples, removing efciently
the salt matrix of seawater. This fact has been veried after statistical comparison of matched calibration with mobile phase and
standard addition calibrations (calibration through the IIP-SPE procedure). The developed IIP-SPE allowed a pre-concentration factor
of 50, and the achieved LODs/LOQs resulted low enough for monitoring mercury species in seawater samples (values lower than the
MAC of 70 ng L1 ).
Acknowledgements
The authors wish to acknowledge the Ministerio de Ciencia e
Innovacin (project number CTQ2012-38091-C02-02) for nancial
support.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.chroma.
2015.02.068.
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