Microchemical Journal 159 (2020) 105592

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Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

A universal method for the speciation analysis of arsenic in various seafood

based on microwave-assisted extraction and ion
chromatography-inductively coupled plasma mass spectrometry
Yaohui Lin a, Ying Sun a, Xusheng Wang a, Shilong Chen a, Yongning Wu b, FengFu Fu a, *
a
Key Laboratory for Analytical Science of Food Safety and Biology of MOE, Fujian Provincial Key Lab of Analysis and Detection for Food Safety, College of Chemistry,
Fuzhou University, Fuzhou, Fujian 350116, China
b
NHC Key Lab of Food Safety Risk Assessment, Food Safety Research Unit (2019RU014) of China Academy of Medical Science, China National Center for Food Safety
Risk Assessment, Beijing 100021, China

A R T I C L E I N F O A B S T R A C T

Keywords: The speciation analysis of arsenic in seafood is of great significance for objectively and scientifically assessing
Arsenic arsenic health risks in seafood. In general, different types of seafood contain a variety of different arsenic species
Speciation analysis and have quite different matrix. Thus, the detection of arsenic species in different types of seafood required
Fish
different extracting and analytical methods. In this study, a universal microwave-assisted extraction was
Shellfish
Seaweed
developed to extract each arsenic species including As(V), As(III), monomethylarsonic acid (MMA), dimethy­
larsinic acid (DMA), arsenobetaine (AsB), arsenosugars and so on in various seafood with a extraction efficiency
> 95% and without altering their original species, and a universal cation exchange IC-ICP-MS (ion
chromatography-inductively coupled plasma mass spectrometry) method was developed for the detection of
arsenic species in various seafood (seaweed, fish, shellfish and shrimp) with a method detection limit of 8.0 ng
As/g dried seafood–12.0 ng As/g dried seafood. Especially, the extract of seafood obtained with our microwave-
assisted extraction can be directly used for the following IC-ICP-MS detection without any additional pretreat­
ment. By using the extraction method and IC-ICP-MS method, we have successfully detected each arsenic species
including As(V), As(III), MMA, DMA, AsB, arsenosugars and so on in seaweed, fish, shellfish and shrimp samples
with a recovery of 92%–104% and a relative standard deviation (RSD, n = 5) < 5%. The developed microwave-
assisted extraction method and IC-ICP-MS detection method is simple, has broad applicability, high efficiency,
excellent accuracy and strong resistibility to the complicated matrix, which promising a reliable approach for
objectively and scientifically assessing arsenic health risks in various seafood to ensure the consumption safety of
seafood.

1. Introduction generally contained As(III), As(V), arsenobetaine (AsB), dimethylarsinic

Arsenic (As) is a deadly toxic substance widely existing in various
foods. With the rapid development of industry, the arsenic pollution of
coastal seawater became more serious in developing countries in recent
years, and thus resulted in a higher level of arsenic in seafood, especially
in edible seaweeds, due to bioaccumulation [1–4]. Marine organisms
can convert inorganic arsenic into various organic arsenic after they
intake or ingest inorganic arsenic from environment [4–7]. Thus, all
seafood contained a variety of arsenic species including inorganic and
organic arsenic, and different types of seafood generally contained
different arsenic species [1–10]. For example, shellfish and fish
acid (DMA) and monomethylarsonic acid (MMA), with AsB as pre­
dominant species [10–12]. Whereas, seaweeds usually contained
(DMA), As(V), AsB and arsenosugars, with arsenosugars as predominant
species [2,13,14]. It was well known, the toxicity of arsenic greatly
depends on its chemical species, and the bio-accessibility/bio-
availability of arsenic depends on both its species and food’s matrices
[7–10]. Thus, it is of great significance to accurately detect the con­
centrations of each arsenic species in various seafood, in order to
objectively and scientifically assess arsenic health risks in seafood and
ensure consumption safety of seafood.
Speciation analysis of arsenic in seafood faces two challenging tasks:

* Corresponding author.
E-mail address: fengfu@fzu.edu.cn (F. Fu).

https://doi.org/10.1016/j.microc.2020.105592
Received 27 July 2020; Received in revised form 27 September 2020; Accepted 28 September 2020
Available online 2 October 2020
0026-265X/© 2020 Elsevier B.V. All rights reserved.
Y. Lin et al.
1) each arsenic species in seafood should be completely extracted out
without altering their original chemical species, and the best is the
extract can be directly used for next speciation analysis of arsenic
without any additional pretreatment; 2) to develop a universal method,
which is applicable to various seafood samples, for the accurate and
sensitive analysis of each arsenic species. The ultrasonic and microwave-
assisted extractions are the two main techniques used to extract arsenic
species in seafood at present. They were widely used to extract out
arsenic species in edible seaweeds with a satisfied recovery (>90%) and
no species change by using methanol-water as extracting solvent
[2,13–15]. However, the extraction used for seaweeds is not suitable for
fish and shellfish samples since fish and shellfish samples have quite
different matrix [11,16–18]. In fact, different types of seafood have
quite different matrix and contain quite different arsenic species.
Therefore, corresponding to different types of seafood, a variety of
extracting methods have been developed to extract out arsenic species in
different types of seafood. For example, two-step ultrasonic-assisted
extraction (using two different solvents) was developed for sea snail and
mussels [15,19], enzyme-assisted ultrasonic extraction was developed
for fish, mussels and oyster [12,20], and microwave-assisted extraction
using gastric solution as solvent was developed for fish and shellfish [9].
So far, there is no other reported universal extraction method which can
be simultaneously used to extract arsenic species in various seafood such
as fish, shellfish, seaweed and shrimp samples. Therefore, it is extreme
essential to develop a universal extraction method for the detection of
arsenic species in various seafood samples.
The techniques used for the speciation analysis of arsenic mainly are
high performance liquid chromatography (HPLC) coupled with ICP-MS
(inductively coupled plasma mass spectrometry) (HPLC-ICP-MS), ion
chromatography (IC) coupled with ICP-MS (IC-ICP-MS), and capillary
electrophoresis (CE) coupled with ICP-MS (CE-ICP-MS)
[2,8–12,18,20–27]. Among these techniques, CE-ICP-MS has poorer
stability and lower sensitivity due to less sample injection volume
[2,25–27], and thus it has been few used for the speciation analysis of
arsenic in actual seafood samples. Actually, the techniques used for the
speciation analysis of arsenic in seafood are mainly HPLC-ICP-MS and
IC-ICP-MS. As mentioned above, different types of seafood contain
different arsenic species and have different matrix, and thus the speci­
ation analysis of arsenic in different types of seafood usually required
different HPLC-ICP-MS or IC-ICP-MS methods [8,9,11,12,18,20–24].
The HPLC-ICP-MS or IC-ICP-MS method, which was used for the
detection of arsenic species in fish and/or shellfish samples [9–11]. is
not applicable for the detection of arsenic species in seaweeds since
seaweeds contain high contents of pigment and multiple arsenosugars
[2,5,13,22,28–30]. The speciation analysis of arsenic in seaweeds is a
hard challenge by either using HPLC-ICP-MS or using IC-ICP-MS. So far,
there is no other reported universal analytical method which can be
simultaneously used to analyze each arsenic species in various seafood
such as fish, shellfish, seaweed and shrimp samples.
In this study, a universal microwave-assisted extraction method was
developed to completely extract arsenic species in all of fish, shellfish,
seaweed and shrimp samples without altering original arsenic species,
and a simple cation exchange IC-ICP-MS method was developed to
accurately analyze each arsenic species in all above extracts without any
additional pre-treatment, in order to provide a reliable approach for
scientifically assessing arsenic health risks in seafood and ensuring
consumption safety of seafood.
2. Materials and methods
2.1. Microwave-assisted extraction of arsenic species in various seafood
samples
The fresh seaweeds and the meat or tissue of fresh fish, shellfish and
shrimp were firstly dried under − 45 ◦ C by using vacuum freeze dryer,
and sequentially were grinded with agate mortar. Then, 0.10 g of dried
2
Microchemical Journal 159 (2020) 105592
seafood samples (seaweed, fish, shellfish and shrimp) were weighed and
put into a 100 mL Teflon jar, and then 6.0 mL of 20 mmol/L HNO3 so­
lution was added. In the case of fish, shellfish and shrimp samples, the
jar was then placed at room temperature for 12 h to previously soak the
samples. Sequentially, the jar was put into a microwave digestion/
extraction apparatus and the microwave digestion/extraction apparatus
was programmed to heat sample to 120 ◦ C within 10 min and hold for
30 min at 120 ◦ C. After the sample was cooled to room temperature, the
extract was separated and collected by centrifugation, and the residue
was repeatedly extracted for once again with 4.0 mL of 20 mmol/L
HNO3 solution by immediately heating sample to 120 ◦ C within 10 min
and hold for 30 min at 120 ◦ C. Two extracts were combined and the
whole extract was diluted 0 to 5 times (according to total arsenic con­
centration in sample) with pure water, and the final solution was used
for IC-ICP-MS detection of arsenic species.
2.2. Cation exchange IC-ICP-MS detection of arsenic species in extract of
various seafood samples
All arsenic species in the extract of various seafood samples were
separated and detected with cation exchange IC-ICP-MS, which used a
Dionex IonPac TMCS12A cation exchange column as separation column.
Each arsenic species including As(V), As(III), MMA, DMA, AsB, arsen­
osugars and unidentified As species were qualitatively analyzed based
on their retention time in IC separation and were quantitatively deter­
mined based on peak areas with standard curve method. The detailed IC
parameters are: iso-elution mode, sample injection volume is 10 µL,
temperature of separation column is 30 ◦ C, eluent is 5.0 mmol/L HNO3-
5.0 mmol/L EDTA (sodium ethylenediamine tetraacetate), and flow rate
of eluent is 0.7 mL/min. The detailed parameters of IC-ICP-MS system
are also shown in Table S1 in electronic Supplementary information
(ESI). The seafood samples spiked with different concentrations of As
(V), As(III), MMA, DMA and AsB standards were prepared by directly
adding standard solutions of arsenic species into dried seafood samples,
and then the seafood samples were dried again by using vacuum freeze
dryer. The various seafood samples, which previously spiked with
different concentrations of As(V), As(III), MMA, DMA and AsB stan­
dards, were then extracted and detected with the same manner to obtain
recoveries.
3. Results and discussions
3.1. Optimization of microwave-assisted extraction for extracting arsenic
species in different types of seafood
As we mentioned above, several microwave-assisted extraction
methods have been developed for the extraction of arsenic species in
different seafood such as seaweed, shellfish and fish [2,9,11,16]. How­
ever, some of previous microwave-assisted methods could not be used
for fish, shellfish and shrimp samples [2], and some of them have a lower
extraction efficiency when they were used to extract arsenic species in
some shellfish with more complex matrix such as Razor clam and fish
samples [11,16]. In addition, the extract of seafood obtained with some
of previous extraction methods required a pre-purification and pre-
concentration treatment before the following HPLC-ICP-MS measure­
ment [9]. To date, there is no other reported universal and simple
extraction method which can be simultaneously used to extract arsenic
species in seaweed, fish, shellfish and shrimp samples.
By referring to previous studies [2,16,18,19], the mixture of meth­
anol and water (CH3OH-H2O) and the diluted nitric acid (HNO3) were
used as potential solvent for the microwave-assisted extraction of
arsenic species in different types of seafood in this study, and the effect
of extracting solvents on arsenic extraction efficiency (=extracted As ×
100%/total As, see Table 1) of various seafood samples was investigated
in detail. As Table 1 showed, when CH3OH-H2O (CH3OH/H2O = 1/1 or
3/1, v/v) was used as extracting solvent, arsenic in fish, shellfish and
Y. Lin et al. Microchemical Journal 159 (2020) 105592

Table 1
The effect of solvent on arsenic extraction efficiency of different types of seafood under different microwave-assisted extraction.
Types of Sample †
Total As ‡
Extracted As (µg/g)/Extraction efficiency of As (%)
seafood name (µg/g) §
No pre-soaking for 12 h *Pre-soaking for 12 h

CH3OH- CH3OH- 5 mM 15 mM 20 mM CH3OH- CH3OH- 5 mM 15 mM 20 mM

H2O (1:1) H2O (3:1) HNO3 HNO3 HNO3 H2O (1:1) H2O (3:1) HNO3 HNO3 HNO3

Seaweed Sarcodia 55.1 47.4/86% 52.9/96% 46.9/ 54.5/ 54.0/ 48.3/88% 52.3/95% 47.4/ 53.4/ 55.7/
ceylanica 85% 99% 98% 86% 97% 101%
Sea mustard 28.6 23.7/83% 27.2/95% 23.5/ 27.5/ 29.2/ 25.2/88% 28.0/98% 23.7/ 28.0/ 27.7/
82% 96% 102% 83% 98% 97%
Fish Pagrus 15.6 7.60/49% 11.2/72% 12.3/ 12.9/ 13.0/ 8.10/52% 11.4/73% 13.3/ 15.8/ 15.1/
major 79% 83% 83% 85% 101% 97%
Sand pike 25.6 11.0/43% 17.9/70% 20.7/ 21.0/ 21.8/ 12.3/48% 18.4/72% 22.0/ 26.4/ 24.8/
81% 82% 85% 86% 103% 97%
Shellfish Mussel 11.2 6.50/58% 7.60/68% 7.90/ 8.60/ 8.80/ 6.40/57% 7.50/67% 9.40/ 10.1/ 10.8/
71% 77% 79% 84% 90% 96%
Razor clam 9.65 5.40/56% 6.90/71% 6.66/ 7.24/ 7.72/ 5.79/60% 7.04/73% 7.62/ 8.49/ 9.17/
69% 75% 80% 79% 88% 95%
Shrimp Chinese 1.59 0.80/50% 1.03/65% 1.11/ 1.43/ 1.51/ 0.87/55% 1.08/68% 1.19/ 1.51/ 1.53/
prawns 70% 90% 95% 75% 95% 96%
†
The As concentrations in seafood samples detected with ICP-MS after samples were completely decomposed with 7.0 mol/L HNO3.
‡
The concentration of extracted As in seafood samples detected with ICP-MS, and extraction efficiency of As = extracted As × 100%/total As.
§
Seafood samples were directly extracted for two times under microwave-assistance.
*
Seafood samples were firstly soaked with 6.0 mL of 20 mmol/L HNO3 for 12 h and then was extracted for two times microwave-assistance.

shrimp can be only partly extracted out with a extraction efficiency of
43%–72% (see No pre-soaking for 12 h), although the arsenic in sea­
weeds can be extracted out with a satisfied extraction efficiency
(>95%). In other hand, when diluted nitric acid was used as extracting
solvent, the extraction efficiency of arsenic in various seafood increased
with the increasing of HNO3 concentration. However, too high HNO3
concentration may alter arsenic species during extraction, and thus
HNO3 solution with a highest concentration of 20 mmol/L was used in
this study. By using 20 mmol/L HNO3 solution as extracting solvent,
arsenic in seaweeds and shrimp can be extracted out with a satisfied
extraction efficiency (>95%), whereas arsenic in fish and shellfish still
was only partly extracted out (79–85%) even sample was repeatedly
extracted for 3 times with conventional microwave-assisted extraction
(see No pre-soaking for 12 h in Table 1). This should be attributed to the
more complicated matrix of fish and shellfish.
In the experiment, we find that arsenic in fish and shellfish samples
can be effectively released by previously soaking samples with extract­
ing solvent (20 mmol/L HNO3), and finally greatly improve the
extraction efficiency of arsenic. As results shown in Table 1 (Pre-soaking
for 12 h), under the same conditions and 20 mmol/L HNO3 of extracting
solvent, in comparison with microwave-assisted extraction without
previous soaking, the microwave-assisted extraction efficiency of
arsenic in fish and shellfish can be increased to > 95% from 79 to 85% by
previously soaking sample for 12 h with 20 mmol/L HNO3 under room
temperature. This means that arsenic in all of seaweed, fish, shellfish and
shrimp samples may be completely extracted out via microwave-assisted
extraction by using 20 mmol/L HNO3 as extracting solvent together with
12 h previous soaking of sample, which satisfied the requirement of
arsenic speciation analysis. In order to shorten the soaking time, we tried
to previously soak sample with 20 mmol/L HNO3 at 40 ◦ C together with
agitation, and the results indicated that higher temperature do not
improve the releasing rate of arsenic. Thus, 12 h previous soaking at
room temperature was employed in this study.
The extracting temperature, ratio of sample mass to extracting sol­
vent volume and extracting time was also optimized under pre-soaking
microwave-assisted extraction. Higher extracting temperature is favor of
improving the extraction efficiency and increasing extraction speed,
however, too high extracting temperature may lead to altering of arsenic
species during microwave-assisted extraction. By referring to previous
studies [10,16], 120 ◦ C was selected as the optimal extracting temper­
ature in this study. More extracting solvent can improve the extraction
efficiency of arsenic, but also increase reagent blank and produce
excessive dilution at the same time, which will lead to a lower sensitivity
finally. To obtain the optimal ratio of sample mass to extracting solvent
volume, shellfish (Razor clam), whose arsenic is more difficult to be
extracted, was used as representative of seafood to optimize the
extracting solvent volume. The experimental results (Table S2 in ESI)
revealed, for 0.1 g of Razor clam sample, 10 mL of 20 mmol/L HNO3 (6.0
mL for previous soaking and first extraction, 4.0 mL for second extrac­
tion) is the optimal selection.
Extracting time of each extraction will affect the extraction efficiency
of arsenic. In the experiment, we find that the arsenic extraction effi­
ciency of each extraction reaches the maximum value when the
extracting time is 30 min and then keeps almost no change when
extracting time is longer than 30 min. Thus, 30 min was selected as
optimal extracting time of each extraction. Summary, the optimal
microwave-assisted extraction for the simultaneous extraction of arsenic
species in seaweed, fish, shellfish and shrimp samples is: 0.1 g dried
seafood sample was previously soaked with 6.0 mL of 20 mmol/L HNO3
for 12 h, and then the mixture was extracted under microwave-
assistance for 30 min at 120 ◦ C. After the extract was separated and
collected, the residue was repeatedly microwave-assisted extracted once
again for 30 min with 4.0 mL of 20 mmol/L HNO3 under 120 ◦ C and
without previous soaking. By using above optimal microwave-assisted
extraction, arsenic species in seaweed, fish, shellfish and shrimp sam­
ples can be completely extracted out with a extraction efficiency > 95%.
Especially, the extract of seafood obtained with above microwave-
assisted extraction can be directly used for the following IC-ICP-MS
detection without any additional pretreatment, since the extracting
solvent is diluted (20 mmol/L) HNO3.
To confirm whether the arsenic species were altered during
microwave-assisted extraction, a mixed standard of As(III), As(V), MMA,
DMA and AsB, which had a final concentration of 5.0 ng/mL, was pre­
treated with the same microwave-assisted extraction and then was
analyzed by IC-ICP-MS, and the results were compared with that of the
mixed standard without microwave-assisted extraction pretreatment. As
Fig. S1 (see ESI) showed, the mixed standard pre-treated with
microwave-assisted extraction showed the same results as that of orig­
inal standard, indicating that each arsenic species do not happen change
during microwave-assisted extraction.
All above facts indicated that the microwave-assisted extraction
developed in this study has high extraction efficiency and excellent
universality, it can be used to completely extract out arsenic species in
different types of seafood including seaweed, fish, shellfish and shrimp

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Y. Lin et al. Microchemical Journal 159 (2020) 105592

without altering their original species, which provided a guarantee for
the speciation analysis of arsenic in seafood.
3.2. Optimization of IC-ICP-MS parameters for speciation analysis of
arsenic in different types of seafood
As we mentioned above, different types of seafood samples have
quite different matrix and contain quite different arsenic species. In
comparison with fish, shellfish and shrimp samples, the speciation
analysis of arsenic in seaweeds is the most difficult since seaweed
samples have complicate matrix and contain multiple arsenosugars.
Especially, the concentrations of arsenosugars in seaweeds are much
higher than that of other arsenic species such as As (III) and As (V).
Therefore, the HPLC-ICP-MS and IC-ICP-MS method previously reported
for arsenic species detection in fish and shellfish samples can not be used
for the detection of arsenic species in seaweed [9–11]. In order to detect
arsenic species in seaweeds, the extracts of seaweeds are usually
detected for twice by using CE-ICP-MS under different pH [2]. In this
study, cation exchange IC-ICP-MS was used for the speciation analysis of
arsenic, and seaweed (Sarcodia ceylanica) was used as the representative
of seafood to optimize the analytical parameters in order to develop a
universal IC-ICP-MS method for the detection of arsenic species in all
seaweed, fish, shellfish and shrimp samples.
When cation exchange IC-ICP-MS was used for the speciation anal­
ysis of arsenic, generally, diluted HNO3 (2 mmol/L or higher concen­
tration) was used as mobile phase [11]. However, when cation exchange
IC-ICP-MS with diluted HNO3 as mobile phase was used for arsenic
speciation analysis in seaweed (Sarcodia ceylanica), the huge peak of
arsenosugar, which was concluded to be DMAsSugarMethoxy based on
infusion electrospray ionization-triple quadrupole mass spectrometry
(ESI-3Q-MS) analysis (Fig. S2 in ESI) and previous literature [2,31],
completely overlapped with that of As(V) (Figs. S3 and S4 in ESI) and
thus make the detection of trace As(V) is impossible. To resolve this
problem, we tried to alter IC retention times of arsenic species via adding
EDTA into mobile phase to chelate them and finally alter their charges.
species in seaweed (Sarcodia ceylanica) including As(V), As(III), arsen­
osugar (DMAsSugarMethoxy), DMA and AsB can be base-line separated
and accurately detected by using IC-ICP-MS (Fig. 1). The same IC-ICP-
MS method was also used to detect arsenic species in other types of
seafood including fish, shellfish and shrimp to validate the adaptability
of our method. As Figs. 2 and 3 showed, arsenic species in fish (Red eye
silver carp and Sea crucian carp), shellfish (Mussel) and shrimp (Chinese
prawns) including As(V), As(III), MMA, DMA, AsB and two unidentified
arsenic species were base-line separated and accurately detected, indi­
cating that our method has excellent adaptability and can be used for
arsenic speciation analysis in various seafood.
Figs. 2 and 3 in here
3.3. Analytical performance of the method and detection of actual
seafood samples
Under the optimal parameters of IC-ICP-MS shown in Table S1 (see
ESI), a series of mixed standards of As(V), As(III), MMA, DMA and AsB
were detected to evaluate the performance of the method in the range of
0.0 ng/mL–50 ng/mL. As Table S3 (see ESI) showed, the IC-ICP-MS
signal (peak area) of each arsenic species showed a good linear rela­
tionship with the concentrations of each arsenic species with a corre­
lation coefficient (R) > 0.9991. The instrumental detection limit (LOD,
3σ/S, σ is the counts of reagent blank (20 mmol/L HNO3), which was
calculated based on 10 measurements of reagent blank, and S is sensi­
tivity) was calculated to be 0.08 ng/mL for As(V), 0.10 ng/mL for As(III),
0.12 ng/mL for MMA, 0.09 ng/mL for DMA and 0.09 ng/mL for AsB. The
instrumental limit of quantitation (LOQ, 10σ/S) was calculated to be
0.26 ng/mL, 0.33 ng/mL, 0.40 ng/mL, 0.30 ng/mL and 0.30 ng/mL for
8000
Arsenosugar c
6000 As(V) As(III)
Co u n t s / s

As Fig. S4 (see ESI) showed, upon the addition of EDTA, the IC retention 4000
DMA
time of As(III) obviously prolonged and the retention time of As(V) only
slightly increased. At the same time, the retention time of DMA almost 2000 AsB
keeps no change and that of AsB greatly decreased. As a result, the
separation of the As(V), arsenosugar (DMAsSugarMethoxy) and As(III) 0
was obviously improved (Fig. S3-b). When EDTA concentration in mo­ 5000
bile phase increased to 5.0 mM, there is a maximum increase in reten­ As(III) b
tion time of As(III), and thus the separation of As(V), 4000
DMAsSugarMethoxy and As(III) is the best (Fig. S3-c). Thus, 5.0 mmol/L
3000
Counts/s

of EDTA was added into mobile phase in this study.

DMA
Under 5.0 mmol/L EDTA, the effect of HNO3 (mobile phase) con­ 2000 As(V)
centration on the separation of As(V), DMAsSugarMethoxy and As(III)
was investigated in the range of 2.0 mmol/L to 10.0 mmol/L. From 1000 AsB
Fig. S5 (see ESI), we clearly observed that the separation of As(V),
arsenosugar (DMAsSugarMethoxy) and As(III) was improved with the 0
3000
increasing of HNO3 concentration in the range of 2.0 mmol/L to 5.0
Arsenosugar
mmol/L, and then the separation of As(V), DMAsSugarMethoxy and As 2500 a
(III) turn to become bad when HNO3 concentration is bigger than 5.5
2000
mmol/L. Therefore, the mixture of 5.0 mmol/L EDTA and 5.0 mmol/L
Counts/s

HNO3 was used as IC mobile phase in this study. 1500

By using 5.0 mmol/L EDTA–5.0 mmol/L HNO3 solution as mobile
1000
phase, the flowing rate of mobile phase was also optimized in the range
of 0.7 mL/min–1.0 mL/min. As Fig. S6 (see ESI) revealed, in the range of 500 As(V) DMA
0.7 mL/min to 1.0 mL/min, the lower flowing rate is favor of increasing 0
the retention times of each arsenic species and thus improving the 0 200 400 600 800 1000
separation of As(V), DMAsSugarMethoxy and As(III). However, too low Time/s
flowing rate will broaden the peaks of each arsenic species and thus Fig. 1. The IC-ICP-MS chromatograms for determining the extract of seaweed
worsen the resolution of each arsenic species. By considering the anal­ (Sarcodia ceylanica) under Table S1 optimal parameters. (a) seaweed (Sarcodia
ysis time and the resolution of each arsenic species of seaweed, 0.7 mL/ ceylanica); (b) 5.0 ng/mL mixed standards of As(V), As(III), DMA and AsB; (c)
min was selected as optimal flowing rate. seaweed (Sarcodia ceylanica) spiked with 5.0 ng/mL of As(V), As(III), DMA
Under all above optimal IC parameters (see Table S1 in ESI), arsenic and AsB.

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Y. Lin et al. Microchemical Journal 159 (2020) 105592

10000 As(V), As(III), MMA, DMA and AsB, respectively. As we mentioned

AsB C above, the 10 mL extract of 0.1 g seafood can be directly used for IC-ICP-
8000
Response (counts/s)

MS detection without any additional pretreatment, thus the method

6000 As(III) detection limit (LOD) was calculated to be 8.0 ng As/g dried seafood for
4000 As(V), 10.0 ng As/g dried seafood for As(III), 12.0 ng As/g dried seafood
MMA
for MMA, 9.0 ng As/g dried seafood for DMA and AsB.
2000 As(V)
DMA To confirm the applicability of our method to actual seafood samples,
0 arsenic species in different types of seafood including seaweed (Sarcodia
10000 ceylanica), fish (Sea crucian carp), shellfish (Mussel) and shrimp (Chinese
B
8000 prawns) were firstly extracted out with the developed microwave-
Response (counts/s)

assisted extraction, and the concentrations of each arsenic species in

6000
the extracts were then detected with the developed IC-ICP-MS method.
4000 The samples of seaweed, fish, shellfish and shrimp, which previously
2000 spiked with different concentrations of As(V), As(III), MMA, DMA and
AsB standards, were also analyzed with the same manner to obtain re­
0
coveries. The analytical results were shown in Tables 2 and 3 and their
10000
A chromatograms were shown in Figs. 1–3. Three arsenic species namely
8000 As(V), DMA and one arsenosugar, which was identified to be DMAsSu­
Response (counts/s)

garMethoxy by using infusion ESI-3Q-MS (Fig. S2 in ESI) and referring to

6000
previous study [2,31], were detected in seaweed (Sarcodia ceylanica)
4000 with a concentration of 0.851 µg/g for As(V), 1.248 µg/g for DMA and
2000 52.97 µg/g for DMAsSugarMethoxy. In fish (Sea crucian carp) sample,
0 five arsenic species, namely As(III), As(V), MMA, DMA and AsB, were
0 200 400 600 800 1000 1200 detected with a concentration of 0.111 µg/g, 0.169 µg/g, 0.062 µg/g,
Time/s 0.433 µg/g and 8.541 µg/g respectively. In shrimp (Chinese prawns)
sample, three arsenic species, namely As(V), DMA and AsB were
Fig. 2. The IC-ICP-MS chromatograms for determining the extract of fish under
detected with a concentration of 0.063 µg/g, 0.072 µg/g and 1.391 µg/g
the Table S1 parameters. (A) fish sample (Red eye silver carp); (B) fish sample
(Sea crucian carp); (C) Sea crucian carp spiked with 1.0 ng/mL of As(V), As(III),
respectively. For shellfish (Mussel) sample, seven arsenic species, namely
MMA, DMA and 5.0 ng/mL of AsB. As(III), As(V), MMA, DMA, AsB and two unidentified arsenic species
(AsL1 and AsL2) were detected with a concentration of 0.239 µg/g,
0.043 µg/g, 0.352 µg/g, 0.987 µg/g, 7.991 µg/g, 1.478 µg/g and 0.772
3000 µg/g respectively. As Tables 2 and 3 showed, arsenic species in various
seafood samples have been accurately detected by our method with a
2500 AsB c
recovery > 92% and a relative standard deviation (RSD, n = 5) < 5%,
2000 respectively. From Tables 2 and 3, we also observed that the sum of the
1500
concentrations of each arsenic species in each sample consisted well
Counts/s

MMA
As(III) Unidentified
Unknown As 22
As species with the total concentration of arsenic (the concentration determined
1000
As(V) DMA Unidentified As 1 1
Unknown As species
with ICP-MS after sample was completely decomposed with 7 moL/L
500 HNO3), further verified that each arsenic species in each seafood sample
had been completely extracted out by using our extracting method. The
0 approximately 100% of recovery for each arsenic species in various
0 400 800 1200 1600 2000
2500 seafood samples further indicated that each arsenic species kept no
b altering during extracting, since the recovery of at least one arsenic
2000
species should excessively deviated from 100% if any arsenic species
1500 was changed during extracting. All above results verified that the
microwave-assisted extraction and IC-ICP-MS method developed in this
Counts/s

1000 study has broad applicability, excellent accuracy and strong resistibility
to the complicated matrix, which makes the method can be used to
500
detect each arsenic species in various seafood with a satisfied accuracy.
0
The success of this work offers a reliable and universal approach for
0 400 800 1200 1600 2000 more objectively assessing arsenic health risks in seafood and ensuring
1000
safety of seafood.
a
800
4. Conclusions
600
Counts/s

In summary, a universal microwave-assisted extraction method and

400
IC-ICP-MS detection method was developed for the speciation analysis
200 of arsenic in various seafood samples. The developed extraction method
has broad applicability and high efficiency, it can be simultaneously
0 used to extract arsenic species in various seafood with an extraction
0 400 800 1200 1600 2000
Time/s
efficiency > 95% and without altering arsenic original species. Espe­
cially, the extract of seafood obtained with our microwave-assisted
Fig. 3. The IC-ICP-MS chromatograms for determining the extract of shrimp extraction can be directly used for the following IC-ICP-MS detection
and shellfish under the Table S1 parameters. (a) Shrimp (Chinese prawns); (B) without any additional pretreatment. The developed IC-ICP-MS detec­
Shellfish (Mussel); (C) Shellfish (Mussel) spiked with 1.0 ng/mL of As(V), As(III), tion method has broad applicability and strong resistibility to matrix
MMA, DMA and 2.0 ng/mL of AsB. interference, and it can be used to detect each arsenic species in various

5
Y. Lin et al. Microchemical Journal 159 (2020) 105592

Table 2
Analytical results of Seaweed (Sarcodia ceylanica) and Fish (Sea crucian carp) samples.
Seaweed (Sarcodia ceylanica) Fish (Sea crucian carp)
†
Total As Con. 55.57 µg/g 10.01 µg/g

Arsenic species ‡
Added (µg/g) §
Detected (µg/g) RSD (n = 5, %) Rec. (%) ‡
Added (µg/g) §
Detected (µg/g) RSD (n = 5, %) Rec. (%)

As(III) 0.000 – – – 0.000 0.111 4 –

0.500 0.511 3 102 0.100 0.206 5 95
As(V) 0.000 0.851 2 – 0.000 0.169 4 –
0.500 1.331 4 96 0.100 0.265 3 96
MMA 0.000 – – – 0.000 0.062 5 –
0.500 0.519 3 104 0.100 0.154 4 92
DMA 0.000 1.248 3 – 0.000 0.433 3 –
0.500 1.733 3 97 0.100 0.527 3 94
AsB 0.00 – – – 0.000 8.541 2 –
0.500 0.491 2 98 0.500 9.011 3 94
DMAsSugarMethoxy 0.000 52.97 4 – 0.000 – – –
†
The As concentrations in seafood detected with ICP-MS after samples were completely decomposed with 7.0 mol/L HNO3.
‡
The concentrations of arsenic species added in seafood.
§
The concentrations of arsenic species in seafood obtained with our method.

Table 3
Analytical results of Shrimp (Chinese prawns) and Shellfish (Mussel) samples.
Shrimp (Chinese prawns) Shellfish (Mussel)
†
Total As Con. 1.592 µg/g 11.19 µg/g

Arsenic species ‡
Added (µg/g) Detected (µg/g)
§
RSD (n = 5, %) Rec. (%) ‡
Added (µg/g) §
Detected (µg/g) RSD (n = 5, %) Rec. (%)

As(III) 0.000 – – – 0.000 0.239 4 –

0.100 0.103 4 103 0.100 0.335 3 96
As(V) 0.000 0.063 5 – 0.000 0.043 5 –
0.100 0.159 4 96 0.100 0.137 4 94
MMA 0.000 – – – 0.000 0.352 3 –
0.100 0.093 5 93 0.100 0.449 4 97
DMA 0.000 0.072 3 – 0.000 0.987 4 –
0.100 0.167 3 95 0.100 1.090 2 103
AsB 0.00 1.391 4 – 0.000 7.991 3 –
0.500 1.886 2 99 0.500 8.471 3 96
AsL1 0.000 – – – 0.000 1.478 5 –
AsL2 0.000 – – – 0.000 0.772 5 –
†
The As concentrations in seafood detected with ICP-MS after samples were completely decomposed with 7.0 mol/L HNO3.
‡
The concentrations of arsenic species added in seafood.
§
The concentrations of arsenic species in seafood obtained with our method.

seafood with a method detection limit of 8.0 ng As/g dried seafood–12.0
ng As/g dried seafood. By using the developed microwave-assisted
extraction method and IC-ICP-MS detection method, we have success­
fully detected each arsenic species including As(III), As(V), MMA, DMA,
AsB, DMAsSugarMethoxy and two unidentified As species in seaweed,
fish, shellfish and shrimp samples with a recovery > 92% and a RSD (n
= 5) < 5%. The developed microwave-assisted extraction and IC-ICP-MS
detection method is simple, has broad applicability, excellent accuracy
and strong resistibility to the complicated matrix, which promising a
reliable and universal approach for more objectively assessing arsenic
health risks in seafood and ensuring safety of seafood.
Acknowledgements
The authors gratefully acknowledge The National Key Research and
Development Program of China (2017YFC1600500), NSFC (21677034,
21976029) and Fujian Provincial Department of Science and Technol­
ogy (2019Y0002) for financial support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.microc.2020.105592.

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