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Deep eutectic solvent based liquid-phase microextraction for the

Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
determination of pharmaceuticals and personal care products in
fish oil
Chang Liu, Donghui Liu, Xueke Liu, Xu Jing, Fulin Zong, Peng Wang, Zhiqiang Zhou*
Pharmaceuticals and personal care products (PPCPs) as the emerging pollutants, persistently entered into the aqueous
environment and could be accumulated by aquatic organism, which caused the concern whether fish oil as a kind of fish
products usually for the infant would have the risk of being contaminated by PPCPs. In this work, we focused on
developing a simple and effective method for the determination of four common PPCPs in fish oil by using deep eutectic
solvent (DES) as the green solvent in liquid-phase microextraction (LPME) with the aid of sonication. Effects of extraction
method and DES types were studied by traditional one-factor-at-a-time experiment. Influences of extraction time, volume
of DES and extraction cycles on the recovery were investigated and optimized by Box-Behnken design of experiment. The
results showed that DES was good alternatives for the extraction of PPCPs in fish oil with sonication, which was a robust
method for dispersing this viscous liquid. Under the optimal condition, sonication-assisted LPME based on DES showed
good linearity in the range of 0.1-20 g L-1 for sulfamethazine, sulfamethoxazole as well as triclocarban, and 0.05-20 g L-1
for carbamazepine. The recoveries of these four PPCPs were in the range of 87.0-93.8 % and the RSDs were varied from
1.6 % to 6.0 %. Moreover, the limits of detection (LODs) were ranged from 16.7 to 33.0 ng L-1. In general, the sonication-
assisted LPME based on DES was a green, sensitive, and convenient method for the determination of PPCPs in fish oil.

Introduction
Pharmaceuticals and Personal Care Products (PPCPs) as
pollutants have aroused general concern over the past few
1 monitor PPCPs in fish oil from the respective of public health.
decades. Many studies have reported that PPCPs widely exist
in the water environment, including surface water,
underground water and sediment, etc. The surface water and
treated water samples of Louisiaana, USA and Ontario,
Cannada had been analyzed ,and some common PPCPs could
-1 2
be detected with the concentration of 10-106 ng L . In
addition, 54 % of the 72 analyzed PPCPs were detected in the
3 emerging contaminants . In 2003, deep eutectic solvent (DES),
biosolids samples from 32 states and the District of Columbia.
On account of their broad and persistent input to the
environment, another concern about PPCPs is their potential
4,5
bioaccumulation in aquatic ecosystem. Some researchers
have observed that PPCPs could accumulate in fish. For
example, it was found that steroidal and phenolic endocrine
6 components to form a eutectic mixture. The current DES is a
disrupting chemicals were accumulated in carp . Besides, some
wild fish from five effluent-dominated rivers was observed the
accumulation of various PPCPs7. Thus, for human, it was
potentially exposed to PPCPs by eating fish and fish products.
Fish oil, a kind of fish processed products, is taken as dietary
supplement for the infants and the old whom are sensitive to
the pollutants. As far as we known, limited research has
developed the method for the determination of PPCPs in fish
oil. Thus, it is necessary to establish analytical methods to
To establishing an efficient and environmentally friendly
analytical method, solvent always plays the most important
role. As one of typical green solvents, ionic liquid (IL) was
8,9
widely applied in analytical chemistry. For example, IL was
used as extraction solvent in liquid-phase microextraction
10
(LPME) of polycyclic aromatic hydrocarbons (PAHs) and 14
11
similar to IL, was developed by Abbott using choline chloride
12
(ChCl) and urea . Nowadays, DES has been used in many
areas, including gas adsorption, electrochemistry,
nanomaterial synthesis, extraction and analytical chemistry.
5,13-19
Unlike IL, DES can be obtained by simply mixing two safe
20
kind of polar and water-miscible solvent, which can just serve
21
as extraction solvent for the nonaqueous matrix . For
example, DES composed of different alcohols and ChCl was
applied as a new type of green solvent to extract flavonoids
22
from Chamaecyparis obtuse leaves . Then, DES (ethylene
glycol/ChCl, glycerol/ChCl and urea/ChCl) was also used for
21
extracting phenolic compounds from model oil . Moreover,
DES was applied for extracting and preconcentrating heavy
23
metals, such as lead and cadmium . In general, DES are easy
16
to prepare, biodegradable and inexpensive makes it become

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a great alternative green solvent in analytical chemistry.
However, the viscosity of DESs is much higher than that of
most common organic solvents, which hinders their
application in extraction. Recent researches have applied
different approaches to contribute to extraction, such as
24 25 26 27
sonication , vortex , air , effervescence , etc. Thus, a robust
dispersing way was needed to assist the extraction efficiency
of this viscous liquid.
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In this study, ultrasound-assisted LPME based on DES
combined with ultra-high performance liquid chromatography-
tandem mass spectrometry (UHPLC-MS/MS) was developed
for the analysis of PPCPs in fish oil. Two factors, types of DES
and extraction methods, were studied by one-factor-at-a-time
experiment. Other factors, such as DES volume, extraction
cycles and extraction time were systematically studied by Box-
Behnken design (BBD) of experiment. Under the optimal
conditions, the ultrasound-assisted LPME based on DES was
validated and applied for the real samples.
Experiment
Reagents
Glycerol (99%), ethylene glycol (>99%), urea (99%), malonic
acid (99.5%), choline chloride (98%), sulfamethoxazole (98%),
sulfamethazine (99%) and triclocarban (98%) were purchased
from Aladdin. Carbamazepine (97%) was purchased from J&K
Chemical. Water was purified by a Milli-Q system (Millipore,
Billerica, MA, USA). All other chemicals with analytical grade
were purchased from common commercial suppliers. A stock
-1
solution of each analyte was initially prepared at 1000 mg L in
methanol, and was stored at 4 in the dark. Working
standard solutions were prepared by the appropriate dilution
of the stock solutions in methanol.
The fish oil samples were both purchased from commercial
suppliers. Samples for optimization and validation were
previously analyzed and no target analyte was observed. The
spiked samples containing PPCPs were prepared by adding the
mixed working standard solution into samples followed by well
mixing. Then, the samples were equilibrated for 1 h at room
temperature.
Instrument
The determination was carried out by ThermoFisher TSQ
Quantum Access MAX system (Tewksbury, Massachusetts,
USA). Chromatographic separation was performed by a
DionexTM UltiMateTM 3000 Open Sampler XRS UHPLC system
with a Hypersil GOLD C18 column (100 mm 2.1 mm i.d., 3 m
particle size). The mobile phase was acetonitrile/water
(containing 0.1% formic acid) (70/30,v/v) at a flow rate of 0.3
-1
mL min . And the injection volume was 3 L. A triple
quadrupole mass spectrometer with an electrospray ionization
source was applied for analyzing. The parameters of tune
method were as follows: spray voltage 3000 V, capillary
temperature 350 vaporizer temperature 200 , sheath
gas pressure 35 psi, aux valve flow 15 arb. The collision gas
was Argon (99.999%) with the pressure at 1.5 mTorr. All
analytes were ionized in positive mode. The MS was operated
2 | J. Name., 2012, 00, 1-3
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in selected reaction mode (SRM). Collision energy voltages and
tube lens of analytes are listed in Table S1.
Preparation of DES
DES was obtained by simply mixing ChCl and hydrogen bond
donors (HBDs) at a proper molar ratio with magnetic agitation
at 80-100 until a clear and colorless liquid formed. Table 1
New Journal of Chemistry Accepted Manuscript
shows the produced DESs abbreviations, composition of salt
and HBDs and the ratio.
Table 1 Composition of four DESs and the ratio
Abbreviation Salt HBDs Ratio (salt : HBD)
DES1 urea
DES2 glycerol
ChCl 1:2
DES3 ethylene glycol
DES4 malonic acid
LPME procedure
In the LPME, two grams of fish oil was diluted with 1 mL of n-
hexane in 15 mL centrifuge tube to reduce the viscosity of the
sample. Then, 75 L of DES was added into the tube and the
mixture was manually shaken for 30 s for premixing. After
shaking, the mixture was ultrasonicated for 3 min, which could
assist the extraction efficiency. Subsequently, the mixture was
centrifuged (4000 rpm) for 2 min to accelerate the stratifying.
The lower DES was aspirated by microsyringe prudently and
put into a 1.5 mL centrifuge tube. The extraction was repeated
and the DES was also merged into the tube. In order to
decrease its viscosity and interference to instrument signal,
the DES was diluted 3 times by water and filtered through 0.22
m syringe filter prior to UHPLC-MS/MS analysis.
Optimization strategy
Five factors (types of DES, extraction methods, extraction time,
volume of DES and extraction cycles) were selected to be
28,29
studied. Among them, types of DES and extraction
methods were optimized by one-factor-at-a-time experiment.
BBD was applied to get the optimal conditions for the other
three factors. The design and data analyses of Box-Behnken
expeirment were performed with Design-Expert 8.0 software.
Results and discussion
Effect of types of DES and extraction methods
In the experiment, four different DESs with the composition of
ChCl and different HBDs were used because of their lower
freezing point. The recoveries of four analytes for different
DESs are shown in Figure 1A. DES 2 showed better overall
extraction efficiency than the other three DESs. The reason
might be attributed to their viscosity which could hinder the
solvent dispersing in the matrix. The viscosities of different
types of DESs decreased in the following order: DES 4 > DES 1 >
DES 2 > DES 3.
20, 22
As shown in Figure 1A, the recoveries of
DES 2 and DES 3 were obviously higher than DES 1 and DES 4,
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which was in accordance with the order of viscosities.
Although both DES 2 and DES 3 were of low viscosity, the
polarity of DES 2 was higher than it of DES 3, which might be
the reason that extraction efficiency of DES 2 was higher than
22
that of DES 3. Thus, DES 2 consisting of ChCl and glycerol was
selected as the extraction solvent. In addition, three common
extraction methods were applied to study its effect on the
oscillators, respectively, and their rotational speed are
constant at 2000 rpm and 160 rpm. Whereas, compared with
ultrasonic, these two methods were not ideal for the
extraction. The reason might be that the DES was too sticky to
be dispersed in the sample by the oscillation and vortex.
Furthermore, it was proved the power ultrasound could
enhance the dispersion of the samples and improve the mass

New Journal of Chemistry Accepted Manuscript

21
recovery. The results of optimization of extraction methods transfer of the analytes for DES-based extraction . Thus, in
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are shown in Figure 1B. The vortex and oscillation extraction consideration of extraction efficiency and simplicity, ultrasonic
were conducted by the vortex agitator and water bath extraction was selected as the optimal extraction method.

Figure 1 Effect of A. types of DESs on the recoveries of four PPCPs (ultrasonic extraction, volume of DES: 200Lextraction time:
5min, extraction cycles: 2 times) and B. extraction method on the recoveries of four PPCPs (DES 2, volume of DES: 200L
extraction time: 5min, extraction cycles: 2 times)

Optimization of the extraction condition This design of experiment including 12 tests and 5 central
Three variables and three levels BBD was conducted to get the points were given by Design-Expert 8.0. Because of the minor
best extraction condition, which are shown in Table 2. diversity of recoveries of four analytes, sulfamethazine was
chosen for the optimization randomly. Based on the results of
Table 2 The variables and levels of the Box-Behnkendesgin data analysis, the quadratic equation was obtained.
y = a + bA + cB + dC + eA2 + fB2 + gC2 + hAB + iAC +
Level jBC
Variable Key
Low High Where y was the response, A, B and C were volume of DES,
Volume of DES(L) A 50 200 extraction time and extraction cycles, individually. The
coefficient of determination (R2) was 99.37%, which indicated
Extraction time(min) B 1 5 that the model fitted well the variations. The analysis of
Extraction cycles C 1 3 variance table for the model is shown in Table S3 which
includes the coefficient, F values and P values. Furthermore,
the 3D response surfaces represents as graphical
demonstration of the regression equation, which are Figure 2.

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Figure 2 Response surface for sulfamethazine using the Box-Behnken design obtained by plotting (a) volume of DES versus
extraction time, (b) volume of DES versus extraction cycles and (c) extraction time versus extraction cycles

As shown in table S3, the volume of DES was a significant
factor for the extraction efficiency because its P value was
much less than 0.05. Thus, as shown in Figure 2(a) and Figure
2(b), the recovery increased obviously with the increasing of
volume of DES in the range of 50 to 200 L. However, the
method would reduce the sensitivity with more volume of DES
because of the direct injection of DES phase. In order to
simultaneously ensure the miniaturization and recovery of
analysis method, 75 L was chosen as the optimal volume of
DES. Similar to volume of DES, the effect of extraction cycles
was positive to the recovery, which also needed to be
balanced at the limit of detection and recovery (Figure 2(b)
and Figure 2(c)). Thus, 2 times was selected as the extraction
cycles. As for the extraction time, the polar analytes could
quickly transfer from oil phase to the DES phase with the aim
of ultrasound. However, the recovery increased firstly and
then decreased slightly when extraction time varied 1 to 5 min.
The reduction of recovery might result from the dissolution of
the extractant in the sample matrix when the extraction time
lasted. Therefore, 3 min would be the best extraction time for
the recovery.
According to the above experiment results, the optimum
conditions were as follows: extraction solvent: DES 2 consisting
of ChCl and glycerol, extraction method: ultrasound, volume of
DES: 75 L, extraction time: 3 min, extraction cycles: 2 times.
Validation of the method
Under the optimal condition, calibration curves were
established in the concentration ranges of 0.1-20 g L-1 for the
three of analytes (sulfamethazine, sulfamethoxazole and
triclocarban) and in the concentration ranges of 0.05-20 g L-1
for carbamazepine. As shown in Table 3, good linearity was
obtained with the correlation coefficients ranging from 0.9979
to 0.9996. The LODs and LOQs varied from 16.7 to 33.0 ng L-1
and 55.6 to 109.9 ng L-1 based on signal-to-noise (S/N) of 3 and
10, respectively. The accuracy and the precisions of the
method were studied by five parallel experiments at
concentration of 0.4 g L-1 and 10 g L-1 and the recoveries and
RSDs were in the range of 87.0-93.8% and 1.6-6.0%. This
highlighted the precision and accuracy of the method.

Table 3 results of method validation

0.4 10
Linearity Correlation LOD LOQ
Analyte Regression equation (g/L) (g/L)
(g/L) coefficient (r) (ng/L) (ng/L) 1 1
R (%) RSD(%) R (%) RSD(%)
sulfamethazine 0.1-20 A = 8699.4C - 911.22 0.9994 26.9 89.6 92.6 3.1 93.0 1.6
sulfamethoxazole 0.1-20 A = 3429.9C + 531.14 0.9996 33.0 109.9 88.8 6.0 91.4 3.3
carbamazepine 0.05-20 A = 39508C + 3040 0.9979 16.7 55.6 89.7 5.3 93.8 3.9
triclocarban 0.1-20 A = 4653.1C +2308.7 0.9984 30.8 102.6 87.0 4.1 91.4 6.3
1
R means recovery.

Oil samples analysis

The method was applied in the other two fish oil products
purchased from local market. The results showed that the
concentration of four PPCPs were both below the limits of
detection.
Conclusions
In this study, sonication-assisted LPME based on DES followed
by UHPLC-MS/MS was established for the determination of
PPCPs in fish oil. Under the optimal condition, low LODs, good

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linearity and precision were obtained. On the one hand, this
method used the DES as extraction solvent with the aid of
ultrasound instead of using conventional organic solvent to
make LPME more environmentally friendly. On the other hand,
as far as we know, limited research had established analytical
methods of PPCPs in fish oil. Thus, this sonication-assisted
LPME based on DESs followed by UHPLC-MS/MS method could
21 T. Gu, M. Zhang, T. Tan, J. Chen, Z. Li, Q. Zhang and H. Qiu,
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New Journal of Chemistry Accepted Manuscript

25 E. Yiantzi, E. Psillakis, K. Tyrovola and N. Kalogerakis, Talanta,
fill in the gap and serve as the tool to monitor the PPCPs in fish 2010, 80, 2057-2062.
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oil. 26 M. A. Farajzadeh and M. R. A. Mogaddam, Anal. Chim. Acta,

Acknowledgements
This work was supported by a fund from the National Natural
Science Foundation of China (Contract Grants 21307155 and
21337005); Supported by Beijing Higher Education Young Elite
Teacher Project (YEPT0323).
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The analytical method of PPCPs in fish oil was firstly developed based on green solvent DES.