Anal. Method Environ. Chem. J. 3 (3) (2020) 5-17
Research Article, Issue 3
Analytical Methods in Environmental Chemistry Journal
Journal home page: www.amecj.com/ir
AMECJ
Speciation of selenium (IV, VI) in urine and serum of thyroid
patients by ultrasound-assisted dispersive liquid-liquid
microextraction
Elham Mosafayian Jahromy
a
and Negar Motakef Kazemi
a,*
a
Department of Medical Nanotechnology, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences,
Islamic Azad University, Tehran, Iran
ABSTRACT
In-vitro speciation of inorganic selenium (Se
IV
and Se
VI
) in serum
blood and urine of hyperthyroidism and hypothyroidism patients
based on isopropyl 2-[(isopropoxycarbothiolyl) disulfanyl] ethane
thioate (IICDET) as a complexing agent were studied by ultrasound-
assisted dispersive liquid-liquid bio-microextraction procedure (USA-
DLLMBE). In rst stage, 100 μL (≈0.1 g) of hydrophobic ionic liquid
of [C
8
MIM][PF
6
] mixed with IICDET ligand and 100 μL of acetone.
Then, the mixture injected to 10 mL of human samples at pH=4. After
shacking, the Se (IV) ions were complexed by IICDET and extracted
to IL at pH=4 (R-S: …Se). The IL phase was separated from sample
by centrifuging and inorganic selenium (Se
IV
) in remained samples
was determined by electro thermal atomic absorption spectrometry
(ET-AAS) after back extraction of Se (IV). As speciation, the Se (VI)
reduced to Se (IV) in acidic pH (HCl, 130
O
C) and the total Se(T-
Se) was obtained at pH=4. Therefore, the Se (VI) was calculated
by difference of T-Se and Se (IV). After optimized conditions, the
enrichment factor (EF), Linear range and limit of detection (LOD)
for inorganic Se (IV) were obtained 20.1, 0.75- 20 μg L
-1
and 0.18
μg L
-1
in serum and urine samples respectively. The results showed
us, the concentration of selenium was decreased in thyroid patients
as compared to healthy peoples. The validation of methodology was
achieved by certied reference material (CRM) and ICP-MS.
Keywords:
Selenium,
Inorganic speciation,
Serum and urine,
Isopropyl [(IsopropoxyCarbothiolyl)
Disulfanyl] Ethane Thioate,
Ultrasound-assisted dispersive liquid-
liquid bio-microextraction,
ARTICLE INFO:
Received 22 May 2020
Revised form 17 Jul 2020
Accepted 11 Aug 2020
Available online 26 Sep 2020
*Corresponding Author: Negar Motakef Kazemi
Email: negar.motakef@gmail.com
https://doi.org/10.24200/amecj.v3.i03.109
------------------------
1. Introduction
Selenium is an essential trace element in humans.
The soluble selenium compounds can be easily
absorbed through the lungs and the gastrointestinal
tract. Selenium is mainly excreted in human urine
[1]. When the exposure is very high it can also be
excreted in exhaled air as dimethylselenide vapor
(DMSe). Normal selenium concentrations in serum
and urine are dependent on daily intake, which may
vary considerably in different parts of the world but
are usually below 15 μg per 100 mL
-1
and 25 μg
g
-1
creatinine, respectively [2-5]. The concentration
of selenium in urine is mainly a reection of recent
exposure. The relationship between the intensity of
exposure and selenium concentration in urine has not
been established yet. It seems that the concentration
in plasma (or serum) and urine mainly reects to
short-term exposure, whereas the selenium content
of erythrocytes reects more long-term exposure
6
Anal. Method Environ. Chem. J. 3 (3) (2020) 5-17
[6]. Measuring selenium in blood or urine gives
some information on selenium status. Currently it is
more often used to detect a deciency rather than an
overexposure. Since the available data concerning the
health risk of long-term exposure to selenium and the
relationship between potential health risk and levels
in biological media are too limited. So, the biological
threshold value for Se wasn’t reported [7]. The thyroid
is the organ with the highest selenium content per gram
of tissue because it expresses specic selenoproteins.
The value of selenium supplementation in
autoimmune thyroid disorders has been emphasized.
Most authors attribute the effect of supplementation
on the immune system to the regulation of the
production of reactive oxygen species and their
metabolites [8-10]. The mechanism and role of
selenium in inammation, immunity and hepatocytes
was reported [11, 12] In patients with Hashimoto’s
disease and in pregnant women with anti-TPO
antibodies, selenium supplementation decreases anti-
thyroid antibody levels and improves the ultrasound
structure of the thyroid gland [13]. Although clinical
applications still need to be dened for Hashimoto’s
disease, they are very interesting for pregnant
women given that supplementation signicantly
decreases the percentage of postpartum thyroiditis
and denitive hypothyroidism. In Graves’ disease,
selenium supplementation results in euthyroidism
being achieved more rapidly and appears to have a
benecial effect on mild inammatory orbitopathy
[14]. A risk of diabetes has been reported following
long-term selenium supplementation, but few data
are available on the side effects associated with such
supplementation and further studies are required.
One of the diseases that affect the thyroid gland is
subclinical hypothyroidism, which is characterized
by elevated serum levels of thyroid-stimulating
hormone (TSH) at a concentration recommended
for prohormone thyroxine (T4) and active hormone
triiodothyronine (T3). The decompensated levels of
thyroid hormones may contribute to atherosclerotic
events and an increase in cardiovascular-related
mortality [15]. Also, observational longitudinal
studies have shown an inverse association between
selenium exposure and risk of some cancer types
but still to be conrmed [16]. It is estimated that
subclinical hypothyroidism affects 3–8% of the
general population and is more common in women
than in men. In Brazil, an epidemiological study
in elderly reported that prevalence of subclinical
hypothyroidism was 6.5%. The thyroid gland
contains high levels of selenium (Se) and expresses
a variety of selenoproteins that are involved in
protection of oxidative stress and metabolism of
thyroid hormones (TH) [17]. Selenium deciency
impairs regular synthesis of selenoproteins and
adequate TH metabolism. Therefore selenium species
in serum and urine must be evaluated and determined
by favorite techniques. The different methods such
as, ame atomic absorption spectrometry [18],
electrothermal atomic absorption spectrometry [19],
liquid chromatography and liquid chromatography
inductively coupled plasma mass spectrometry (LC
and LC-ICP- MS) [20-22] and high-performance
liquid chromatography coupled to hydride generation
atomic uorescence spectrometry [23,24] were used
for Se determination in different human and water
samples. A sample preparation is required to extract
metals ions in different biological samples. The sample
preparation such as microextraction techniques [25],
suspended dispersive solid phase microextraction
[26], ultrasonic assisted dispersive liquid-liquid
microextraction method [27] and ultrasound assisted-
ionic liquid-solid phase microextraction [28] were used
for extraction metals in human samples. In this study,
the mixture of hydrophobic ionic liquid of [C
8
MIM]
[PF
6
], IICDET ligand and acetone was used for
selenium speciation /extraction based on ultrasound-
assisted dispersive liquid-liquid bio-microextraction
procedure (USA-DLLMBE) and determined by ET-
AAS. The Se (IV) ions were complexed by IICDET
and extracted to IL at pH=4. Then speciation of Se
was obtained by total determination of selenium.
Validation methodology was conrmed by spiking of
standard samples and ICP-MS.
2. Experimental
2.1. Instrument and Reagents
The electrothermal atomic absorption
spectrophotometer (ET-AAS, GBC 932 plus, Australia)
7
Speciation of selenium (IV, VI) in human urine and serum Elham Mosafayian Jahromy et al
equipped with a graphite furnace (Pal GF3000) were
used for the validation and determination of selenium
(Se) in samples (Wavelength 349.9 nm; slit 0.2 nm;
current 10 mA). The working range as peak area and
height was obtained 15- 400 μg L
-1
and 15-210 μg L
-1
,
respectively. The linear range was achieved for peak
area of 0.3 Abs for sample injection. Based on the
manual book of ET-AAS, the Se determination was
achieved by injecting 20 µL of sample to graphite tube
with auto-sampler in three steps of drying, ashing,
and atomization for Se. The ICP-MS (Perkin Elmer,
USA) as ultra-trace analysis with high sensitivity was
used for determining of Se(IV) and Se(VI) in human
blood and water samples (1100 W; 15 L min
-1
; 1.5 sec
per mass; auxiliary gas 1.12 L min
-1
). The Metrohm
pH meter based on the glass electrode was used for
measuring pH in serum, urine and blood samples
(E-744, Switzerland). The vortex mixer were used
for shaking of human samples based on 300 rpm
speeds and centrifuged by Falcon accessory by 4000
rpm speeds (Thermo, USA). An ultrasonic bath was
used for blood and urine samples with heat controller
between 30- 120
o
C (Thomas, USA). The standard
solution of Se (VI, VI) was purchased from Merck
CO. (Germany) with a concentration of 1000 mg L
-1
in 1 % HNO
3
. The different concentration of Selenium
was prepared by dilution of deionized water (DW)
and ultrapure water was purchased from Millipore
Company. The 1-Hexyl-3-methylimidazolium
hexauorophosphate as hydrophobic ionic liquid was
purchased from Sigma Aldrich ([HMIM][PF
6
], CAS
N: 304680-35). Isopropyl 2-[(isopropoxycarbothioyl)
disulfanyl] ethane thioate was synthesized and puried
by Azad university laboratories (IICDET; (CH3)
4
(CO)
2
S
4
). The acetate (CH
3
COOH/ CH
3
COONa) and
phosphate buffer was used to adjust the pH between
2.8–6.2 and 6.2–8.2, respectively. The analytical grade
of reagents such as polyoxyethylene octyl phenyl
ether (TX-100) as the anti-sticking agent, HNO
3
, HCl,
acetone, and ethanol were purchased from Sigma
Aldrich, Germany.
2.2. Preparation of human samples
All glass or PCV tubes were cleaned with a 1.0
mol L
-1
HNO
3
solution for at least one day and
then washed for ten times with ultrapure water.
As low concentrations of Se(IV) and Se(VI) in
human serum, blood and urine samples, the cations
or anions contamination at any stage of sample
preparation, saving and analytical processes can be
affected on the results accuracy. Heparin was used
as anticoagulants for human blood samples into
Eppendorf (5 mL) tubes and kept at -20
O
C for two
weeks. Each blood samples were prepared by 10
μL of pure heparin (free Se) to blood sample. The
serum, blood and urine samples were collected from
hypothyroidism patients (50) and healthy peoples
(50) with aged between 25 - 60 years, Tehran
(IRAN). In this study, the world medical association
declaration of Helsinki (WMADH) based on
guiding physicians in human body research was
considered by project of Azad university (AZAD
UN. SPN: 960250673). The human samples were
prepared based on WMADH law and absolutely
protected the life and health of the human subject.
2.3. Synthesis of IICDET ligand
The 2.0×10
-3
mol of potassium O-isopropyl
(ditiocarbomate) was dissolved in 20 mL of DW
and cooled in an ice bath for 10 minutes. The
2.0×10
-3
mol of iodine solution and potassium
iodide as drop wise was added to 20 ml of DW. After
stirring of mixture for 1h, the aqueous phase was
extracted with CH
2
Cl
2
and washed with 30 mL of
aqueous Na
2
S
2
O
3
(10%) and DW. The organic phase
was dried and evaporated with powder anhydrous
calcium chloride Ca Cl
2
(99.99%; CAS Number:
10043-52-4). The purication was obtained by
recrystallization in hexane. A pale yellowish crystal
of isopropyl 2-[(isopropoxycarbothiolyl) disulfanyl]
ethane thioate with a yield of 95% was achieved. The
structure and isopropyl 2-[(isopropoxycarbothiolyl)
disulfanyl] ethane thioate were conrmed by NMR
spectroscopic methods (Fig. 1).
1
H NMR (CDCl
3
).
δ (ppm): 1.43 (d, 12H, CH
3
), 5.63 (m, 2H, CH).
13
C NMR (CDCl
3
). δ (ppm): 22.2, 80.6, 207.1.
IR (KBr). νmax (cm−1): 2979.8 (s), 2869.9 (w),
1463.9 (s), 1442.7 (s), 1373.0 (s), 1271.1 (s, b),
1145.6 (s), 1082.2 (s), 1048.0 (s, b) 898.8 (s), 796.
5 (s), 690. 5 (m).
8
Anal. Method Environ. Chem. J. 3 (3) (2020) 5-17
Fig.1. NMR spectroscopic for isopropyl 2-[(isopropoxycarbothiolyl) disulfanyl] ethane thioate
2.4. Extraction Procedure
The Se (IV) based on IICDET ligand was extracted
by ultrasound-assisted dispersive liquid-liquid bio-
microextraction procedure (USA-DLLMBE). By
procedure, 100 μL (≈0.1 g) of hydrophobic ionic
liquid of [C
8
MIM][PF
6
] mixed with 0.35×10
-6
mol
L
-1
of IICDET solution and 100 μL of acetone at
pH of 4. The mixture based on Triton X-100, an
emulsier and anti-sticking agent was injected to 5
mL of blood, serum and urine samples which was
diluted with 5 mL of DW. For optimizing, 1.0 μg
L
-1
and 20 μg L
-1
of standard solution of Se (IV) as
LLOQ and ULOQ was used instead of the blood
and serum samples. After shacking, the Se (IV)
ions were complexed by IICDET and extracted
to IL at pH=4 (R-S: …Se). By centrifuging (3
min), the IL phase was separated from sample and
inorganic selenium (Se
IV
) was back- extracted
from IL phase in basic pH (0.25 mL of 1.0 mol
L
-1
NaOH). Finally, the remained samples was
determined by electro thermal atomic absorption
spectrometry (ET-AAS). the Se (VI) reduced to Se
(IV) in acidic pH (HCl, 130
O
C) and the total Se(T-
Se) was obtained at pH=4. Therefore, the Se (VI)
was calculated by difference of T-Se and Se (IV)
amount (Fig.2).
3. Results and Discussion
The human blood, serum and urine samples based
on IICDET was used for selenium speciation with
high accuracy by USA-DLLMBE procedure.
The results showed us, the mean concentrations
of Se (IV and VI) in human biological samples in
hypothyroidism patients (50) were signicantly
higher than the healthy peoples (50). As linear
range of selenium between 0.75-20 μg L
-1
, the
human samples can be diluted before using by
proposed procedure.
Based on results, the mean
concentration of total selenium in urine and serum
of hypothyroidism patients was obtained 11.8 μg
g
-1
creatinine and 52.6 μg L
-1
, respectively which
was less than 25 μg g
-1
creatinine for urine samples
and 150 μg L
-1
for serum samples as TLVs in
standard references.
9
Speciation of selenium (IV, VI) in human urine and serum Elham Mosafayian Jahromy et al
3.1. Effect of ETAAS
The effect of pyrolysis temperature on the
absorbance of Se was studied up to 1000
º
C. The
maximum absorbance was achieved within a range
of 600–800
º
C. Therefore, 700
º
C was selected as
the working pyrolysis temperature for selenium by
nickel nitrate as modier at concentration of 0.05 to
0.1% Ni. In addition, a drying as a 25 s was chosen
for water evaporation, and a long ramp time of 40
s was chosen as it allowed gradual elimination of
trace ionic liquid solution in liquid phase (350
º
C)
and avoided Se loss in pyrolysis temperature. The
effect of atomization temperature on chromium
signal was studied within the range of 2000–2500
º
C, and the maximum signal was obtained at
approx. 2400
º
C. Cleaning time and temperature
were ordered at 2 s and 2500
º
C, respectively, and
argon ow rate was 350 mL min
-1
3.2. Optimization of pH
The sample pH for extraction Se(IV) ions based
on IICDET ligand was studied and optimized in
different pH ranges between 2-11 for 0.75μg L
-1
as a lower limit of quantication (LLOQ) and 20
μg L
-1
selenium as upper limit of quantication
(ULOQ). The complexation Se with sulfur of
IICDET ligand was strongly depended on the pH
of serum, blood and urine samples and caused to
increase the recovery of extraction by ligand. Based
on experimental results, the extraction efciency
of Se(IV) ions was perfectly achieved at pH =3.5-
4.5. Therefore, the USA-DLLMBE procedure
was used to speciation of selenium at pH=4 by
IICDET ligand. The mechanism of Se extraction
was obtained based on the complex formation of
IICDET ligand between Se(IV) ions and sulfur
covalence bonding of IICDET at optimized pH.
The sulfur groups can be deprotonated (SH
-
) at pH
range of 3.5-8 and pH=4 was used as favorite pH
for extraction of Se(IV) from human biological
samples which was shown in Figure 3. As hydroxyl
form of Se(OH)
4
in above pH (more than 6), the
extraction capacity of Se(IV) in basic pH may be
attributed to the afnities of OH groups.
Fig. 2. Separation of selenium (IV) by ultrasound-assisted dispersive liquid-liquid bio-microextraction
procedure (USA-DLLMBE)
10
Anal. Method Environ. Chem. J. 3 (3) (2020) 5-17
3.3. Optimization of IICDET ligand
The concentration of IICDET ligand as important
parameters must be studied and optimized by
USA-DLLMBE procedure. For optimizing, the
concentration of 0.1×10
-6
-1.0 ×10
-6
mol L
-1
of
IICDET ligand was used for evaluation. Due
to results, the more concentration of 0.33×10
-6
mol L
-1
of IICDET, has no effect on recoveries.
So, the concentration of 0.35×10
-6
mol L
-1
of IICDET was selected as optimum ligand
concentration for high extraction efficiency.
The signal remained constant from 0.35×10
-6
mol L
-1
up to at least 1.0 ×10
-6
mol L
-1
IICDET
for 0.75μg L
-1
Se as a LLOQ range
.
Therefore
0.35×10
-6
mol L
-1
of IICDET concentration
was used for further works. As 20 μg L
-1
of
Se
as a ULOQ range, the signal remained constant
from 0.4×10
-6
mol L
-1
up to at least 1.0×10
-6
mol L
-1
and 0.4×10
-6
mol L
-1
was selected
as an optimized IICDET concentration.
By adjusting pH, the best performance of
the Se extraction was achieved between
0.3-0.4 μmol L
-1
(Fig. 4).
3.4. Optimization of volume and ionic liquid
amount
The sample volume as main parameters for Se
extraction based on IICDET ligand and must
be optimized at pH=4. So, the different volume
of sample urine, blood and serum from 1-20
mL was used for extraction of Se ions by USA-
DLLMBE procedure as 0.75 μg L
-1
and 20 μg L
-1
of selenium (IV). Perfect extraction more than 95%
was achieved by sample volume of 1 - 10 mL.
By increasing of sample volumes, the extraction
efciency was reduced. On the other hand, in
high sample volumes, the partially solubilized the
ionic liquid phase was increased and decreased
accuracy and precision of results. So, a sample
volume of 5 mL was selected as optimum volume
for Se(IV) extraction based on ICDET ligand by
USA-DLLMBE procedure (Fig. 5). Furthermore,
the amount of ionic liquid effected on extraction
recovery of Se in serum, blood, urine samples.
Therefore, the different amount of ([C
8
MIM][PF
6
]
as hydrophobic ionic liquid was studied from
the range of 0.05–0.35 g. Quantitative extraction
was observed at higher than 0.08 g. So, 0.1 g of
Fig.3. The effect of pH on Extraction Se(IV) based on IICDET ligand by USA-DLLMBE procedure