Talanta 210 (2020) 120646

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Talanta
journal homepage: www.elsevier.com/locate/talanta

Simultaneous speciation analysis of inorganic arsenic and methylmercury in T

edible oil by high-performance liquid chromatography–inductively coupled
plasma mass spectrometry
Tomohiro Narukawaa,∗, Takahiro Iwaib, Koichi Chibac
a
National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8563,
Japan
b
Forensic Science Group, Photon Science Research Division, RIKEN Spring-8 Center, Kouto 1-1, Sayo-cho, Sayo-gun, Hyogo, 679-5178, Japan
c
Department of Environmental and Applied Chemistry, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo, 669-1337, Japan

ARTICLE INFO ABSTRACT

Keywords: A simultaneous speciation method of arsenic and mercury species by inductively coupled plasma mass spec-
Speciation analysis trometry with a reversed-phase C18 ODS column was developed. The separation of inorganic arsenic (iAs) from
Arsenic species As species and inorganic mercury (iHg) from methylmercury (MeHg) were achieved by HPLC with a single
Mercury species mobile phase containing ion-pair reagent and L-cysteine. The limits of detection of iAs and MeHg were
Edible oil sample
0.05 ng g−1 as As and 0.09 ng g−1 as Hg, respectively. The simultaneous extraction, i-As and MeHg from in
Extraction condition
HPLC-ICP-MS
edible oil were achieved using 2% (w/w) tetramethylammonium hydroxide (TMAH) solution. The proposed
method was successfully applied to the food matrix type CRMs. When the simultaneous speciation was applied to
several kinds of edible oil, iAs and MeHg were founded in the concentration range of 0.001 mg kg−1 to
0.010 mg kg−1 and 1.21 ng g−1 to 10.18 ng g−1, respectively.

1. Introduction hand, MeHg is commonly contained in marine organisms, although the

The presence of toxic elements in diets possesses obvious human
health risks. Arsenic (As) and mercury (Hg) are very typical examples
and their toxicities vary depending on their chemical forms. Inorganic
As [iAs: As(III)+As(V)] is much more toxic than organic As, but in the
contrary methylmercury (MeHg) is much more harmful (dangerous)
than inorganic Hg (iHg). The intake of the elements from food is a main
concern in the public health and safety [1–4]. Edible oils and fats are
one of the most important diets containing essential nutrition of lipids,
and therefore, the contents of certain metals, such as As, Hg, Cd, Pb,
and Cr, are regulated in terms of food safety. Especially, iAs and MeHg
are widely monitored in markets in many countries.
The Codex Alimentarius Committee recommends that total As in
edible fats and oils, fat spreads, and blended spreads should be less than
0.1 mg kg−1 and iAs in polished (white) and unpolished (brown or
husked) rice should be less than 0.2 and 0.35 mg kg−1, respectively
[5,6]. Arsenic and iAs in rice products and apple juice are regulated in
the EU and US [7–9]. Furthermore, some countries regulate total As
and/or iAs in marine products according to their own reference values,
since As is abundantly found in marine organisms [10]. On the other
concentration is not high. The EU sets the allowable limits of Hg to
0.5 mg kg−1 for fish in general and 1.0 mg kg−1 for some larger pre-
datory species including shark, swordfish, marlin, tuna, and orange
roughly, based on the Codex Alimentarius guidelines [11,12].
A large number of studies on the analytical techniques for As and Hg
species have already been reported. For example, high-performance
liquid chromatography–inductively coupled plasma mass spectrometry
(HPLC–ICP-MS) and liquid chromatography–tandem mass spectrometry
(LC–MS/MS) are usually applied to the speciation of As species
[13–25], and gas chromatography–mass spectrometry (GC-MS) and
HPLC–ICP-MS including an isotope dilution method are widely used for
the speciation of Hg species [26–35]. In addition, several HPLC-ICP-MS
methods was also applied to edible oil analysis [36,37], and simulta-
neous analysis of As and Hg speciation has been achieved in several
works [38,39].
As for the extraction of As species, water [40,41], acid [42] and
enzyme extractions [15] are widely used for water-soluble As species,
and organic solvent extraction [13] for organoarsenic species such as
arsenolipids. For Hg species, the alkaline extraction [43,44] and the
acidic leaching [45,46] are commonly used. However, almost of

∗
Corresponding author. .
E-mail address: tomohiro-narukawa@aist.go.jp (T. Narukawa).

https://doi.org/10.1016/j.talanta.2019.120646
Received 24 October 2019; Received in revised form 6 December 2019; Accepted 13 December 2019
Available online 14 December 2019
0039-9140/ © 2019 Elsevier B.V. All rights reserved.
 T. Narukawa, et al.

Table 1
Recoveries of As species spiked in fish oil samples under different extraction conditions.
Solvent Technique Temperature As(V) As(III) MMA DMA AsB TMAO TeMA AsC iAsa Sum** Recovery (%)***

Spiked concentration (ng g−1)

10.19 10.07 9.93 10.09 9.97 10.00 9.89 9.91 20.26 80.05

water ultrasonication room **** 10.47 ± 0.22 9.69 ± 0.28 10.86 ± 0.51 10.59 ± 0.17 9.70 ± 0.32 9.81 ± 0.44 9.88 ± 0.46 10.40 ± 0.26 20.17 81.40 102%
water heating 60 deg. 10.63 ± 0.21 9.74 ± 0.30 10.44 ± 0.50 11.89 ± 0.19 10.57 ± 0.41 9.76 ± 0.44 9.09 ± 0.43 10.27 ± 0.23 20.37 82.39 103%
water heating 80 deg. 10.02 ± 0.22 10.38 ± 0.27 10.26 ± 0.51 10.52 ± 0.18 10.51 ± 0.44 10.61 ± 0.48 10.49 ± 0.49 10.60 ± 0.22 20.40 83.38 104%
water heating 100 deg. 10.39 ± 0.23 9.17 ± 0.26 10.41 ± 0.46 11.21 ± 0.17 10.18 ± 0.40 10.28 ± 0.46 9.57 ± 0.45 11.93 ± 0.33 19.57 83.15 104%
0.15 M HNO3 ultrasonication room **** 10.13 ± 0.23 9.11 ± 0.28 9.32 ± 0.53 10.48 ± 0.21 10.48 ± 0.35 9.66 ± 0.43 9.20 ± 0.43 10.03 ± 0.27 19.24 78.42 98%
0.15 M HNO3 heating 60 deg. 10.92 ± 0.11 9.69 ± 0.29 10.85 ± 0.54 11.44 ± 0.21 10.82 ± 0.42 10.00 ± 0.45 10.67 ± 0.50 11.19 ± 0.30 20.61 85.58 107%
heating 80 deg. 10.91 ± 0.22 10.02 ± 0.28 10.96 ± 0.52 10.91 ± 0.19 11.03 ± 0.21 11.17 ± 0.38 11.04 ± 0.52 11.54 ± 0.31 20.93 87.58 109%

2
0.15 M HNO3
0.15 M HNO3 heating 100 deg. 10.12 ± 0.19 9.62 ± 0.29 11.65 ± 0.57 10.21 ± 0.16 10.71 ± 0.46 10.46 ± 0.42 10.62 ± 0.48 11.43 ± 0.38 19.74 84.81 106%
0.28 M HNO3 ultrasonication room **** 10.75 ± 0.15 9.98 ± 0.26 10.60 ± 0.57 10.43 ± 0.17 10.13 ± 0.42 10.35 ± 0.38 10.12 ± 0.47 9.99 ± 0.27 20.73 82.35 103%
0.28 M HNO3 heating 60 deg. 10.69 ± 0.29 9.11 ± 0.26 10.79 ± 0.52 10.16 ± 0.20 11.09 ± 0.35 10.43 ± 0.38 10.02 ± 0.55 11.39 ± 0.35 19.80 83.69 105%
0.28 M HNO3 heating 80 deg. 13.59 ± 0.32 7.04 ± 0.20 10.29 ± 0.51 11.38 ± 0.11 11.87 ± 0.37 11.82 ± 0.42 11.69 ± 0.53 9.20 ± 0.23 20.63 86.88 109%
0.28 M HNO3 heating 100 deg. 15.45 ± 0.44 5.33 ± 0.15 10.55 ± 0.51 11.12 ± 0.25 11.10 ± 0.41 11.38 ± 0.53 11.24 ± 0.54 11.43 ± 0.31 20.78 87.60 109%
2%(w/w) TMAH ultrasonication room **** 20.54 ± 0.42 0.21 ± 0.004 10.73 ± 0.45 10.84 ± 0.17 10.60 ± 0.44 11.84 ± 0.41 11.49 ± 0.51 11.62 ± 0.31 20.75 87.86 110%
2%(w/w) TMAH heating 60 deg. 19.82 ± 0.42 0.20 ± 0.002 10.45 ± 0.48 10.70 ± 0.18 9.02 ± 0.46 10.03 ± 0.42 10.76 ± 0.51 10.92 ± 0.29 20.02 81.91 102%
2%(w/w) TMAH heating 80 deg. 20.21 ± 0.42 0.20 ± 0.003 10.31 ± 0.43 10.50 ± 0.18 10.51 ± 0.38 10.18 ± 0.39 10.93 ± 0.49 10.76 ± 0.27 20.41 83.61 104%
2%(w/w) TMAH heating 100 deg. 20.15 ± 0.42 0.20 ± 0.001 10.44 ± 0.38 10.38 ± 0.17 10.23 ± 0.27 11.87 ± 0.25 10.34 ± 0.53 10.64 ± 0.29 20.36 84.27 105%
10%(w/w) TMAH ultrasonication room **** 20.24 ± 0.45 0.20 ± 0.006 9.82 ± 0.37 10.43 ± 0.18 9.77 ± 0.35 9.04 ± 0.43 9.21 ± 0.41 8.81 ± 0.24 20.44 77.52 97%
10%(w/w) TMAH heating 60 deg. 19.94 ± 0.44 0.20 ± 0.006 10.28 ± 0.29 10.87 ± 0.16 9.55 ± 0.37 3.28 ± 0.15 16.70 ± 0.15 7.55 ± 0.20 20.14 78.36 98%
10%(w/w) TMAH heating 80 deg. 21.05 ± 0.39 0.21 ± 0.006 11.16 ± 0.36 11.00 ± 0.11 9.57 ± 0.37 13.37 ± 0.60 23.26 ± 0.60 0.00 21.26 89.61 112%
10%(w/w) TMAH heating 100 deg. 20.53 ± 0.37 0.21 ± 0.005 10.57 ± 0.44 10.92 ± 0.15 8.54 ± 0.33 14.23 ± 0.62 24.43 ± 0.64 0.00 20.74 89.42 112%

a
iAs: As(III)+As(V), **Sum of all As species, ***Recovery = Sum of the speciation results/Concentration of additional As species × 100, ****Room temperature 20 °C ± 10 °C.
Talanta 210 (2020) 120646
T. Narukawa, et al.
20
18
16
Retention time (min)
14
12
10
8 tions of methylmercury (MeHg), ethylmercury (EtHg), and phe-
6
4 (C2H5HgCl), and phenylmercury(II) (C6H5HgOCOCH3) reagents
2
0 and then, diluted to proper concentrations with pure water. The total
2.3 2.4 2.5 2.6 2.7
pH
As(V) As(III) MMA DMA
TMAO TeMA AsC i-Hg
Fig. 1. Effect of the eluent pH on the retention times of As and Hg species.
extraction procedures for As and Hg species have been developed as an
independent analytical method each other, so they require different
sample pretreatments and different measurement conditions for each
other.
In this study, the simultaneous extraction and speciation of iAs and
MeHg in edible fish oil were developed, since a rapid and simple
monitoring method is highly required in the food sanitation field. The
method developed here was applied to the certified reference materials.
2. Material and methods
2.1. Instruments
A 7500c ICP-MS system (Agilent, Tokyo, Japan) equipped with a
MicroMist quartz nebulizer (0.1 mL min−1; Glass Expansion, Australia)
and a Scott spray chamber (2 °C) was used. The operating parameters
were set as follows: incident radio-frequency power was 1600 W, and
outer, intermediate, carrier, and makeup Ar gas flow rates were
15 L min−1, 0.9 L min−1, 0.8 L min−1, and 0.4 mL min−1, respectively.
The ICP-MS system was operated in the He collision mode (He
3 mL min−1) to reduce polyatomic molecular interferences. An HPLC
system equipped with an ODS L-column 2 (length 150 mm, internal
diameter 4.6 mm; Chemicals Evaluation and Research Institute, Tokyo,
Japan) was used for the separation of the As and Hg species; the column
exit was directly connected to the nebulizer of the ICP-MS system via
polyetheretherketone tubing.
A heating block system (Digi PREP, SCP Science Inc., Quebec,
Canada) was used for the heat-assisted extraction. A microwave MLS-
1200 mega apparatus (Milestone Inc, Italy) was used for sample di-
gestion.
2.2. Reagents
The Japan Calibration Service System (JCSS) As standard solution
(1000 mg L−1 As, made from high-purity As2O3) was used as the As
(III) source of the standard solutions (Kanto Chemical Industries, Ltd.,
Tokyo, Japan). Arsenic (V) certified reference material (NMIJ CRM
7912-a), dimethylarsinic acid (DMA) certified reference material (NMIJ
CRM 7913-a), and arsenobetaine (AsB) certified reference material
(NMIJ CRM 7901-a) disseminated from the National Metrology
Institute of Japan/National Institute of Advanced Industrial Science and
Technology (NMIJ/AIST, Ibaraki, Japan) were used as sources of the
standard solutions. All of them are SI traceable.
Talanta 210 (2020) 120646
Standard solutions for other organoarsenic species such as mono-
methylarsonic acid (MMA), trimethylarsine oxide (TMAO), tetra-
methylarsonium (TeMA), and arsenocholine (AsC) were prepared from
commercially available reagents (Tri-Chemical Laboratories Inc.,
Yamanashi, Japan) after their purities were evaluated. Each standard
solution contained 1000 mg As kg−1.
The JCSS mercury standard solution (1000 mg L−1, Kanto) was used
as the source of inorganic Hg (iHg) standard solutions. Standard solu-
nylmercury (phenHg) (1000 mg Hg kg−1) were prepared from com-
mercially available methylmercury(II) (CH3HgCl), ethylmercury(II)
(< 95.0%, Kanto). Appropriate amounts of MeHg, EtHg, and phenHg
were dissolved in a small amount of methanol solution in glass vials,
2.8 2.9 3.0
Hg concentration of each solution was determined by ICP-MS according
to the JCSS standard solution, and MeHg, EtHg, and phenHg were as-
AsB signed by evaluating the impurity of Hg species such as iHg with HPLC-
ICP-MS.
MeHg
Working As and Hg mixture standard solutions (0–50 ng g−1, cali-
bration linear R2 > 0.999) were prepared daily from the stock solu-
tions by diluting with 1%(w/w) tetramethylammonium hydroxide
(TMAH) solution containing 2.5 mM L-cysteine.
The nitric acid, methanol, and acetone used were of ultrapur®, HPLC
grade, and ultrapure grade (Kanto), respectively. L-cysteine, malonic
acid and ammonium dihydrogen phosphate of special grade (Wako
Pure Chemical Industries, Ltd., Osaka, Japan), sodium 1-butanesulfo-
nate (Ace grade; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), and
TMAH (Tamapure-AA 25%; Tama Chemicals Co., Ltd., Kanagawa,
Japan) were used without further purification. Ultrapure water was
obtained with a Milli Q-Labo filter (Nippon Millipore, Ltd., Tokyo,
Japan) and used throughout the experiments.
2.3. Standard reference materials
The following certified reference materials (CRMs) were used for
the validation of the proposed method: NMIJ CRM 7402-a cod fish
tissue [total As (36.7 ± 1.8) mg kg−1: AsB (35.5 ± 1.8) mg kg−1 as
As: total Hg (0.61 ± 0.02) mg kg−1: MeHg (0.58 ± 0.02) mg kg−1 as
Hg, expanded uncertainty k = 2], NMIJ CRM 7503-b white rice flour
[total As (0.164 ± 0.005) mg kg−1: iAs (0.153 ± 0.010) mg kg−1:
DMA (0.0111 ± 0.0005) mg kg−1 as As. k = 2], NMIJ CRM 7533-a
brown rice flour [total As (0.63 ± 0.02) mg kg−1: iAs
(0.530 ± 0.016) mg kg−1: DMA (0.092 ± 0.004) mg kg−1 as As,
k = 2]. In addition, NMIJ CRM 7405-a hijiki seaweed [total As
(35.8 ± 0.9) mg kg−1: As(V) (10.1 ± 0.5) mg kg−1 as As. k = 2] was
used for preparation of the artificial sample containing arsenolipid
species.
2.4. Oil and fat materials
Raw sardine oil, krill oil, fish liver oil, and whale oil and fat were
analyzed. Two kinds of artificial fish oil samples, sample A and B, were
prepared. The artificial sample A was prepared as follows; Aliquot
amounts of eight As and two Hg species standard solutions were dis-
solved in an acetone solution and it was added to the sardine oil to
prepare an artificial oil sample containing 10 ng g−1 each of As species
and Hg species. The artificial oil sample B, which contained arsenolipid
species, was prepared as follows; oreganoarsenic species were extracted
from the hijiki seaweed CRM by the dichloromethane and methanol
(2:1) mixture, and the mixture was dissolved in an acetone solution,
which was added to the sardine oil [47].
2.5. Sample digestion procedure
A portion (ca. 1 g) of each oil and fat sample was accurately
3
T. Narukawa, et al. Talanta 210 (2020) 120646

and 2 and 10%(w/w) TMAH. A HPLC with a reversed-phase C18 ODS

Fig. 2. Chromatogram of As species with the optimized eluent.
Fig. 3. Chromatogram of Hg species under the optimized eluent.
weighed in a 15 mL perfluoroalkoxy tube, and then 2 g of 2%(w/w)
TMAH (8 g of 25% TMAH in 100 g of water) was added to it. The tube
was capped and placed in a heating block system at 80 °C for 1 h,
followed by centrifugation at 4000 rpm for 5 min. A portion (about
0.5 g) of the aqueous phase was weighed into 15 mL polypropylene
tube and 0.5 g of 5 mM L-cysteine solution was added to it. The ex-
tractant with 2.5 mM L-cysteine was analyzed by HPLC-ICP-MS just
after the preparation.
column was applied with the eluent containing 10 mM sodium 1-bu-
tanesulfonate, 4 mM malonic acid, 4 mM TMAH and 0.05% methanol
(pH 3.0) for As speciation analysis [48] and the eluent containing
0.5 g L−1 L-cysteine and 0.1% methanol mixture (pH 2.3) for Hg spe-
ciation [49]. The results of As species are shown in Table 1.
Water-soluble As species were extracted almost 100% under the any
extraction conditions applied. However, the water extractant emulsi-
fied, the resulting aqueous solution phase. The HNO3 extractant did not
emulsify it, but the As(V) peak slightly broadened with the tailing due
to the strong acidity. When the extraction was performed with TMAH,
most of the As(III) was oxidized to As(V). The retention of TMAO,
TeMA, and AsC shortened with increase in the alkalinity of the mea-
surement solution and they overlapped each other to hinder their
quantification when 10%(w/w) TMAH was used. AsB split into two
peaks with the application of 10%(w/w) TMAH. However, iAs was
extracted almost completely under any extraction conditions applied.
Fish oils generally contain fat-soluble As species such as arsenoli-
pids, and there is a possibility that a part of them are dissociated to iAs
during the extraction under the strong acid or alkaline conditions.
Therefore, their extraction behavior was investigated using the simu-
lated oil sample B containing arsenolipids extracted from the hijiki
seaweed CRM. As a result, no peak was found corresponding to the iAs
peaks, although several additional peaks were observed besides the
eight water-soluble As species.
When iHg and MeHg were extracted with water and HNO3, their
extraction efficiencies were 70%–80% and the deviations were about
5%. On the other hand, TMAH extracted 100% of MeHg, iHg peak split
from 1 to 3 peaks depending on the extraction or the storage conditions.
It means that a part of the iHg extracted might change into different
forms in solution.
Consequently, the water-soluble species of As and Hg were ex-
tracted by 100% with TMAH under the alkaline extraction conditions.
The heat extraction with 2%(w/w) TMAH at 80 °C for 1 h was finally
employed as an optimum condition, considering the extraction effi-
ciency and measurement repeatability of iAs and MeHg.
3.2. Chromatographic conditions

3. Results and discussions
3.1. Extraction of As and Hg species in the simulated oil sample
The extraction behaviors of As and Hg species were investigated
using the artificial sample A. The ultrasonic extraction at room tem-
perature and the heat extraction at 60 °C, 80 °C and 100 °C were applied
with the extracting solvent such as water, 0.15 M and 0.28 M HNO3,
The eluent for the As species speciation, mentioned in the previous
section, was originally reported by Shibata et al. [50], and it has widely
been used together with the reversed-phase column. It is very effective
to separate water-soluble As species each other in acid and neutral
solutions [40–42]. On the other hand, the eluent for Hg speciation is
contained L-cysteine as an essential component. The optimum eluent
composition for the simultaneous measurement of iAs and MeHg was
developed based on the above two eluent conditions.

Table 2
Determination results (n = 3) for inorganic As and methylmercury in the certified reference materials.
7503-b White rice 7533-a Brown rice 7402-a Cod fish tissue

Certified value Result Certified value Result Certified value Result

Mean ± ua Mean ± ua Mean ± ua

(mg/kg) (k = 2) (mg/kg) (mg/kg) (k = 2) (mg/kg) (mg/kg) (k = 2) (mg/kg)

iAs [As(V)+As(III)] (as As) 0.153 ± 0.010 0.152 ± 0.005 0.530 ± 0.016 0.514 ± 0.001
DMA (as As) 0.0111 ± 0.0005 0.0111 ± 0.0002 0.092 ± 0.004 0.091 ± 0.002
AsB (as As) 35.5 ± 1.8 35.0 ± 0.5
unidentified As species 0.98 ± 0.01
total As 0.164 ± 0.005 0.164 ± 0.003b 0.63 ± 0.02 0.605 ± 0.002b 36.7 ± 1.8 36.0 ± 0.5b
iHg 0.012 ± 0.001 0.064 ± 0.001 0.046 ± 0.001
MeHg (as Hg) ND ND 0.58 ± 0.02 0.586 ± 0.01
total Hg 0.012 ± 0.001b 0.064 ± 0.001b 0.61 ± 0.02 0.632 ± 0.002b

a
Combined standard uncertainty.
b
Sum of the speciation analysis.

4
T. Narukawa, et al. Talanta 210 (2020) 120646

The effects of sodium 1-butanesulfonate, malonic acid, TMAH, L-

Recoveryc
cysteine, and pH on As and Hg species separation were studied when a

10%
24%

28%
C18 ODS column was used. The decrease of sodium 1-butanesulfonate in
(%)

2%
2%
the eluent made As species elute faster, and in particular, the orga-
noarsenic species overlapped with each other without sodium 1-buta-
(mg/kg)

nesulfonate. The decrease of malonic acid did not affect the retention
Sumb

0.61
1.18
0.06
0.04
0.45
times of the As species, but resulted in significant tailing of iAs peak.
The decrease of TMAH made the elution of TMAO, TeMA, and AsC
unidentified As species

slow, and it took more than 1200 s to elute the As species without
Mean ± u (mg/kg)

TMAH. L-cysteine is necessary to separate Hg species, but it caused no

0.0002
0.001
0.004
0.001

0.001

significant problem for the As species separation, even though As(III)

has high affinity to sulfur-containing compounds such as L-cysteine.
±
±
±
±
±

On the contrary, when sodium 1-butanesulfonate, malonic acid, and

0.047
0.444
0.045
0.023
0.119

TMAH were added to the L-cysteine eluent for the Hg speciation, the
sodium 1-butanesulfonate made the retention times of iHg and MeHg
longer, although malonic acid and TMAH had no significant effect. In
Mean ± u (mg/kg)

particular, it took about 1800 s to elute MeHg with the eluent con-
0.005 ± 0.0002
0.004 ± 0.0002
0.209 ± 0.002

taining 10 mM sodium 1-butanesulfonate. However, when L-cysteine

equivalent to 2.5 mM was added to the test solutions in advance, MeHg
eluted for about 720 s even with the eluent containing 10 mM sodium
AsB

ND

ND

1-butanesulfonate, because the L-cysteine forms the complex with the

Hg species and suppresses the ion-pair formation during elution.
Mean ± u (mg/kg)

The pH of the eluent was very effective to the elution time of each
0.0002
0.0002

species, when the eluent contained 10 mM sodium 1-butanesulfonate,

0.004
0.004

0.004

4 mM malonic acid, 4 mM TMAH, 5 mM L-cysteine, and 0.05% me-

±
±
±
±
±

thanol and when the test solutions contained L-cysteine. As can be seen
0.548
0.513
0.008
0.009
0.316
DMA

in Fig. 1, when the eluent pH increased from 2.3 to 3.0, the retention
time of each As species slightly lengthened and the overall separation
was slightly improved. In the contrary, the retention time of Hg species
Mean ± u (mg/kg)

considerably shortened, although they were still separated each other.

0.009 ± 0.0001
0.001 ± 0.0001

0.017 ± 0.001

As a result, simultaneous speciation of iAs and MeHg was achieved in

the pH range of 2.3–3.0, although the retention of species was affected
by pH .
MMA

Considering ICP-MS operation, an eluent containing low con-

ND
ND

centration of components is required to avoid the damage of an inter-

face and the interferences on a measurement. Finally, NH4H2PO4 was
Mean ± u (mg/kg)

used instead of malonic acid since it was more effective to suppress

tailing of i-As peak than malonic acid, and the concentration of sodium
1-butanesulfonate was reduced to 5 mM. The optimum eluent compo-
As(III)

sition consisted of 5 mM sodium 1-butanesulfonate, 2 mM NH4H2PO4,

ND
ND
ND
ND
ND

4 mM TMAH, 5 mM L-cysteine, and 0.1% methanol (pH 2.3) for the

simultaneous separation of As and Hg species.
Mean ± u (mg/kg)

The chromatograms obtained under the optimized condition are

0.001 ± 0.0001
0.010 ± 0.0008

0.002 ± 0.0003

shown in Figs. 2 and 3, after As and Hg species were extracted with the
0.003 ± 0.0003

Recovery = (Sum of the speciation analysis/Extracted total) × 100.

TMAH under alkaline conditions. The eight water-soluble As species

appeared at the retention time from 160 to 390 s, and iHg and MeHg
Determination results (n = 3) for As species in oil and fat samples.
As(V)

from 300 to 700 s. The water-soluble As species could be separated with

ND

each other although TMAO, TeMA and AsC were separated somewhat
inadequately, and iHg and MeHg could also be separated at the same
Mean ± u (mg/kg)

time.
Assuming the actual samples analysis, most As species including
0.02
0.02
0.02
0.02
0.01

unknown ones empirically appear within 900 s. As can be seen in Fig. 3,

extracted
total Asa

±
±
±
±
±

iHg and MeHg elute within 900 s, although EtHg and phenHg elute at
Sum of mean of the speciation analysis.
5.87
4.91
2.64
2.09
1.64

approximately 1500 and 2100 s, respectively. EtHg and phenHg are

unlikely contained in actual samples except the contaminated ones.
Consequently, the measurement time for simultaneous speciation of iAs
Mean ± u (mg/kg)

and MeHg was set to 900 s (15 min) in this study.

Sample digestion/ICP-MS.
0.02
0.02
0.01
0.01
0.01

3.3. Method validation and application

total Asa

±
±
±
±
±
5.88
4.95
2.51
2.13
1.65

The proposed measurement procedures were applied to the fish

tissue and the rice food CRMs, since there is no fish oil CRM with the
Fish liver oil

certified values of iAs and MeHg. The results obtained are shown in
Sardine oil
Whale fat
Whale oil

Table 2. As for the rice CRMs, iAs and DMA were determined, and their
Krill oil
Table 3

analytical results were in good agreement with the certified values.

b
a

Most part of iAs was detected as As(V), although the main As species is
c

5
T. Narukawa, et al.
Table 4
Determination results (n = 3) for Hg species in oil and fat samples.
Total Hga Extracted total Hga
Mean ± u (ng/g) Mean ± u (ng/g)
Fish liver oil 2.23 ± 0.11 2.10 ± 0.08
Krill oil 7.41 ± 0.17 7.65 ± 0.17
Whale oil 14.66 ± 0.03 14.46 ± 0.07
Whale fat 10.83 ± 0.51 10.76 ± 0.09
Sardine oil 1.63 ± 0.01 1.21 ± 0.05
a
Sample digestion/ICP-MS.
b
Sum of mean of the speciation analysis.
c
Recovery = (Sum of the speciation analysis/Extracted total) × 100.
Fig. 4. Chromatograms of As and Hg species in fish liver oil.
originally As(III) in the rice CRM. It means that As(III) was oxidized to
As(V) during the extraction with TMAH. In the case of the cod fish
tissue CRM, AsB was detected but iAs and DMA were not obssssserved.
The analytical value of AsB agreed well with its certified value. Some
unidentified As species were also detected, then the sum of the As
species agreed with the certified value of the total As.
MeHg in the fish tissue CRM was also determined and the result
obtained was in good agreement with the certified value.
3.4. Speciation analysis of oil and fat samples
The proposed method was applied to some fish oil and fat samples.
The results are shown in Table 3 for As species and Table 4 for iHg and
MeHg, and the chromatograms of As and Hg species of fish liver oil are
shown in Fig. 4.
The total As and the total Hg were determined by ICP-MS via a
calibration method and a standard addition method, after microwave
digestion, respectively, since the Hg concentration was too low to dilute
sample solutions further. The As and Hg in the TMAH extractant were
determined by ICP-MS similar to the total As and Hg. The contents of As
and Hg in samples and extractants were in good agreement with each
other. It suggests that As and Hg species in fish oil and fat samples could
be extracted to TMAH phase completely.
When the TMAH extraction phase was applied to the speciation
analysis, As(III), As(V), MMA, DMA, and AsB were determined, although
a few unidentified peaks were also detected. They were quantified from
the chromatogram peaks according to the As(V) concentration. The sums
of As species obtained by the speciation analysis were less than 30% of
the total As concentration, although all the As had been extracted to the
TMAH phase. It means that 70% of As species extracted were trapped in
the column and did not elute with the eluent for water-soluble As. But,
fortunately, the As species trapped did not deteriorate the chromato-
graphic performance even after 50 measurements.
Talanta 210 (2020) 120646
iHg MeHg Sumb Recoveryc
Mean ± u (ng/g) Mean ± u (ng/g) (ng/g) (%)
0.29 ± 0.01 1.85 ± 0.01 2.14 102%
ND 7.20 ± 0.08 7.20 94%
4.19 ± 0.02 10.18 ± 0.04 14.38 99%
6.36 ± 0.03 4.59 ± 0.06 10.95 102%
ND 1.21 ± 0.04 1.21 100%
As for the Hg speciation analysis, iHg and MeHg were found in the
fish oil and fat samples and their concentrations were individually de-
termined in the chromatograms according to the iHg concentration. The
sum of the Hg species concentrations agreed well with the total and
extracted concentrations.
Finally, when the spike and recovery tests were applied to the
samples, all the As and Hg peaks were measured at 100 ± 2% re-
coveries in the chromatograms.
4. Conclusions
The method for the simultaneous measurements of iAs and MeHg in
food samples, especially fish oils, was developed. The water-soluble As
such as iAs, MMA, DMA and AsB and Hg species such as iHg and MeHg
simultaneously are determined within 15 min using HPLC-ICP-MS with
a reversed-phase column, following the heat extraction with TMAH.
The simultaneous quantification method for iAs and MeHg could be
useful for monitoring and screening tests of food samples.
Credit author statement
Tomohiro Narukawa: Conceptualization, Methodology, Validation,
Writing - Original Draft. Takahiro Iwai: Validation, Investigation.
Koichi Chiba: Writing - Review & Editing.
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