Novel functionalized magnetic ionic liquid green separation technology coupled with high performance liquid chromatography: A rapid approach for determination of estrogens in milk and cosmetics

Xue Feng, Xu Xu∗∗, Zhen Liu, Shan Xue, Lei Zhang∗
College of Chemistry, Liaoning University, 66 Chongshan Middle Road, Shenyang, 110036, China




Several magnetic ionic liquids (MILs), [P6,6,6,14+][FeCl4−], [P

Dispersive liquid-liquid microextraction

6,6,6,14 ]2[NiCl4 ] were synthesized and applied for the extraction of siX estrogens (estrone, estradiol, 17-α-

Milk and cosmetics

hydroXyprogesterone, chloromadinone 17-acetate, megestrol 17-acetate and medroXyprogesterone 17-acetate) in dispersive liquid-liquid microextraction (DLLME). The [CoCl42−]-based MIL was selected as extraction solvent for the separation and concentration of estrogens from milk and cosmetics due to its visual recognition, no sign of hydrolysis, solution acquisition easier and the highest extraction capacity. In addition, the [CoCl42−]-based MIL with low UV absorbance allows direct analysis of the extraction solvent by HPLC-UV. The influence of the mass of MIL, extraction time, salt concentration, and the pH of the sample solution was investigated to obtain optimized extraction efficiency. Besides, extraction conditions including salt concentration, mass of MIL and extraction time were further optimized by the BoX-Behnken design through the response surface method. Under optimized conditions, the limits of detection (LODs) of all estrogens were ranged from 5 ng mL−1 to 15 ng mL−1. The recoveries ranging from 98.5% to 109.3% in milk and from 96.3% to 111.4% in cosmetics were also studied, respectively. Furthermore, the proposed method were statistically compared with the reported conventional IL- DLLME method and the National standard methods of food safety and cosmetics. The experimental results showed that the functionalized MIL could successfully applied for extraction, separation and pretreatment of estrogens in milk and cosmetics.

1. Introduction
As the most endocrine disruptors (EDCs), estrogen regulates the development and growth of animals and humans in the natural en- vironment and controls reproductive cycle as well as pregnancy [1–3]. They are widely concerned for their high risk of human and wildlife health due to their widespread presence and low environmental con- centrations. For example, the adverse effects of estrogen on wildlife and human reproduction are significant and many studies indicates that they are associated with many types of hormone-dependent cancers [4–6]. Recently, in many reports, estrogen has been detected in food samples such as milk [7] and chicken [8]. However, massive con- sumption of milk which contains naturally occurring estrogens such as estradiol may have an tremendous influence on human health, espe- cially on endocrine system [9]. Estrogen is also extensively used as cosmetic additive to firm and whiten skin. According to Cosmetic

technical safety regulations of China, estradiol, estrone and medroX- yprogesterone 17-acetate is not allowed more than 70 μg g−1, 800 μg g−1 and 8 μg g−1 in cosmetics. Meanwhile, the Standard of European Union requires the existence of estrogens clearly free in commercial cosmetic samples [10–13]. Hence, in order to protect human health, it is a challenge to detect trace estrogen in milk and cosmetics samples due to the complexity of the sample matriX and the low concentrations of estrogen residue.
In order to address above these challenges, a rapid and sensitive analytical method should be developed. Up to date, sample preparation is one of the most important step in the analysis of samples, since the analytes are with complex matrices and the compatibility between chemical structures and analytical instruments [14–16]. Hence, it is of great urgency to improve the sample pretreament technology [17]. Currently, dispersive liquid-liquid microextraction (DLLME) has been widely used in microextraction of organic pollutants due to its
Corresponding author. College of Chemistry, Liaoning University, 66 Chongshan Middle Road, Shenyang, 110036, PR China.
Corresponding author. College of Chemistry, Liaoning University, 66 Chongshan Middle Road, Shenyang, 110036, PR China.
E-mail addresses: [email protected] (X. Xu), [email protected] (L. Zhang).

Received 16 July 2019; Received in revised form 30 October 2019; Accepted 3 November 2019
0039-9140/©2019ElsevierB.V.Allrightsreserved.X. Feng, et al. Talantaxxx(xxxx)xxxx

advantages of simplified extraction and rapid preconcentration [18,19]. In the DLLME system, ionic liquids (ILs) with environmentally com- patibility have been applied as extraction solvents or sorbents [20]. To

Table 1
Chemical structures and properties of the studied analytes.
Analytes Chemical structure Retention time (min) pKa

further simplify the cumbrous centrifugation step in phase separation,

magnetic ionic liquids (MILs) have been proposed and reported in 2004 [21,22].
MILs are a subclass of ILs that inherit all advantages of ILs and can also respond to external magnetic fields [23,24]. Up to date, MILs containing anions based on Fe (III) [25,26], Mn (II) [25], Co (II) [27], and Ni (II) [26] have also been reported. Most studies have been con-

E1 11.965 (280 nm) 10.27
E2 15.486 (280 nm) 10.25

ducted with MILs containing the [FeCl −] anion, such as [N +]
to extract phenolic and acidic pharmaceuton with acceptable extraction efficiencies [28,29]. However, these [FeCl4−]-based MILs exhibits high absorption in the ultraviolet (UV) region and limits their application in high-preformance liquid chromatography (HPLC) when coupled with UV detection [30–33]. To solve the defect originating from MILs con- taining the [FeCl −] anion, anions based on Mn (II), Co (II) and Ni (II) are used as alternative extraction solvents.
During the past decades, MILs containing the manganese tetra-
chloride anion [MnCl 2−] were widely applied in DLLME due to the

CMA 21.825 (280 nm) n.a.4 2−low UV absorbance. Anderson et al. first used [MnCl4 ]-based MIL as

extraction solvent in DLLME [24]. There were subsequent studies fo- cusing on MILs containing the [MnCl 2−] anion extraction of organic micropollution form environmental samples. In 2018, MILs containing the [MnCl 2−] were used as extraction solvents coupled with HPLC to detect estrogens from human urine [34]. However, the color of [MnCl 2−]-based MILs are light yellow, which is difficult to observe separate situation. In contrast, the color of MILs containing anions based on Co (II) and Ni (II) are much more distinct, bring easier visual recognition and solution acquisition. These two anions based on MILs are rarely used as DLLME extraction solvents. Recently, Juan L. Benedé et al. used MIL based on Ni (II) anions to extract polycyclic aromatic hydrocarbons from environmental water [35]. And Stalikas et al. used

MPA 22.776 (250 nm) n.a.
MIL based on Co (II) anions to extract pesticides from vegetables [36].
However, these MILs have not been fully studied and used in extracting

estrogens from milk and cosmetics samples.

(tetradecyl) phosphonium chloride [P +][Cl−] (93.0%) was pur-

In this study, four MILs based on the trihexyl (tetradecyl) phospho- nium cation and different magnetic anions, [P6,6,6,14+][FeCl −],

chased from J&K Chemical Ltd (Beijing, China). Analytical reagent-
grade cobalt chloride hexahydrate (CoCl2·6H2O, ≥99.0%), manganese

chloride tetrahydrate (MnCl ·4H O, ≥99.0%), ferric chloride hexahy-

were synthesized and used as the extraction solvents, then DLLME coupled with HPLC-UV analytical method for th rapid extraction and determi- nation of siX estrogens (estrone, estradiol, 17-α-hydroXyprogesterone,

drate (FeCl3·6H2O, ≥99.0%) and nickel chloride hexahydrate (NiCl2·6H2O, ≥99.0%) were obtained from Sinopharm Chemical Re- agent Co Ltd (Shenyang, China). Milli-Q water system (Millipore, Bill-

chloromadinone 17-acetate, megestrol 17-acetate and medroX-

erica, MA, USA) was applied to obtain pure water. All solutions were

yprogesterone 17-acetate) in milk and cosmetics was presented. The parameters affecting DLLME were optimized by univariate and multi- variate methods, and the quality analysis parameters of each analyte were determined under optimal conditions. In addition, the proposed method was systematically compared with the reported conventional IL-DLLME method and the National standard method (GB 29698). Finally, the pre- sent method was successfully used for the determination of estrogens in milk and cosmetics samples.
2. Experimental
2.1. Chemicals and materials
Estrone (E2, 99.5%), estradiol (E1, 99.5%), chloromadinone 17-
acetate (CMA, 98.0%), megestrol 17-acetate (MGA, 97.5%), 17 α-hy- droXyprogesterone (HP, 97.0%), and medroXyprogesterone 17-acetate (MPA, 98.0%) were purchased from National Institute for the Control of

passed through 0.45 μm nylon filter prior to use.
2.2. Instrumentation
The HPLC of Shimadzu LC-16 system equipped with a SPD-16 UV–vis detector was employed. The C18 column (5.0 μm,
4.6 mm × 150 mm, Japan) was applied for separation of analytes, while it keeping at 30 °C. The mobile phase A and B were water and acetonitrile, respectively. The gradient separation was started and held at 45% B for 3 min, and increased to 60% B over 5 min, followed by an immediate increase to 80% B for 7 min, then held on 3 min. The total flow rate was set at 0.5 mL min−1. MPA and HP were detected at 250 nm and others estrogens were detected at 280 nm.
2.3. Synthesis and characterization of four magnetic ionic liquids
All MILs were synthesized based on previously published studies

Pharmaceutical and Biological Products (Beijing, China). The stock [26]. Four MILs, [P+] [CoCl 2−], [P +] [MnCl 2−],

standard solutions of each estrogen were made in methanol at a con- [P +

centration of 1000 mg L−1, and working standard solutions were pre- pared daily by appropriate dilution of stock standard solution. The chemical structure of each estrogen is shown in Table 1. Trihexyl

CoCl2·6H2O (0.5 equiv.), MnCl2·4H2O (0.5 equiv.), NiCl2·6H2O (0.5 equiv.) and FeCl3·6H2O (1 equiv.) were respectively added to di- chloromethane solution containing trihexyl tetradecyl phosphonium

chloride (1 equiv). The reaction was carried out at room temperature for 24 h under continuously agitation. Subsequently, the rotary eva- poration was used to remove dichloromethane. The acquired MIL was dried at 60 °C in vacuum oven.
The Raman spectroscopy, UV/Vis spectroscopy, Fourier-transform infrared spectroscopy (FT-IR) spectra and Thermogravimetric Analysis (TGA) were applied for characterization of the synthesized MILs and precursor ([P6,6,6,14+][Cl−]). Renishaw Raman Spectrometer equipped with an Ar-ion laser operated at 532 nm and a charge coupled device (CCD) detector was applied to obtain the Raman spectroscopy. TU-1900 UV/Vis spectrophotometer (PERSEE, Beijing) was applied to acquire the UV/Vis spectroscopy. FT-IR spectra were acquired on an Avatar 330 FT-IR spectrometer (Nicolet Co., USA) in a KBr pellet. The thermal properties of MILs were studied by using Thermogravimetric Analysis (TGA), which was carried out by a SDT Q600 thermoanalyzer (TA, USA) in a nitrogen (N2) atmosphere at temperatures ranging from 300 K to 873.15 K with a heating rate of 5 K min−1.

2.4. Procedures
2.4.1. Sample preparation
Milk sample was purchased from a local supermarket (Shenyang, China) and stored at 4 °C before analysis. The milk sample (5 mL) and perchloric acid (100 μL, 10% v/v) were added to a 10 mL polypropylene tube [37]. The homogenized milk centrifuged at 10 000 rpm for 3 min. The supernatant was collected, and adjusted to the pH of 4 with NaOH solution (1 mol L−1). The resulting solution was filtered through
0.45 μm filters to remove the denatured proteins, and then stored at 4 °C.
Lotion sample was purchased from a local supermarket (Shenyang, China). Lotion sample was centrifuged at 10 000 rpm for 10 min, the

Table 2
Parameters of BBD.
Analytes E2 E1 MGA CMA HP MPA
β0 41.29 13.29 38.54 48.64 63.64 37.51
β1 1.79 2.69 1.55 1.92 0.61 1.79
β2 5.12 11.47 6.33 4.03 2.5 6.84
β3 2.87 5.84 5.20 2.42 2.74 3.45
β12 3.70 −0.03 −0.01 −3.70 0.02 −7.41
β13 −8.77 −0.03 −5.26 9.65 −4.39 4.39
β23 0.02 0.170 5.84 0.04 0.11 0.02
β11 −0.03 −0.03 −0.01 −0.02 −7.78 −0.02
β22 −0.48 −1.16 −0.52 −0.39 −0.31 −0.57
β33 −0.11 −0.22 −0.21 −0.11 −0.13 −0.15
P-value of the mode 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001
Lack of fit value 0.1171 0.1345 0.0607 0.8358 0.0612 0.2138
R2 0.9931 0.9800 0.9982 0.9857 0.9913 0.9899

analytes was dissoloved in 500 μL of acetonitrile and injected 20 μL of solution into HPLC.

2.4.3. Box-Behnken design
BoX-Behnken design (BBD) is a set of rotatable or nearly rotatable second-order design based on three-level incomplete factorial design [38]. In this study, BBD was used to optimize the experimental condi- tions in DLLME. The BBD including three variables, which are mass of MIL (X1), extraction time (X2), and ionic strength (X3), respectively. The actual design experiments were shown in Table 2. So as to predict the optimal experimental condition, all the experimental results were ap- plied for the computer simulation programming with the quadratic (second degree) polynomial equation. For three significant independent variables, the equation is as follow:

supernatant was collected, and adjusted to the pH of 4 with HCl
(1 mol L−1). Then the solution was filtered through 0.45 μm filters, and

2.4.2. MIL-based DLLME method

The procedure of MIL-based DLLME coupled with HPLC was illu- strated in Fig. 1. Firstly, 5 mL of the aqueous sample (with or without spiking estrogens, depending on the experiment) was placed into 10 mL polypropylene tube. The pH of sample solution was adjusted to 4 with HCl (1 mol L−1). Subsequently, 5 mL of sample solution was added to
10 mL polypropylene tube with 40 mg MIL and 0.275 g NaCl. The

3. Results and discussion
3.1. Characterization of the MILs
Four MILs were characterized by using FTIR spectrum, Thermogravimetric Analysis, Raman and UV/Vis spectroscopy. As
shown in Fig. 2a, the Raman spectra of the [CoCl 2−]-based MIL and

miXture was vigorously shaken using a vortex agitator at 1500 rpm for

[NiCl442−]-based MIL satisfactorily exhibit characteristic of absorption

5 min. All analytes could be extracted into the MILs by this process. Then the MIL was isolated from the solution with a magnet positioned at the side of the polypropylene tube, while the supernatant was aspi- rated with a needle. Finally, the MIL together with the extracted

bands at 260 cm−1 and 266 cm−1 respectively, [MnCl42−]-based MIL and [FeCl −]-based MIL exhibits a characteristic peak at 250 cm−1 and 330 cm−1, which are unique characteristic peak for these anions com- pared to the chloride anion. Moreover, Raman characteristic peaks of
Fig. 1. Scheme of the MIL as extraction solvent in DLLME coupled to HPLC.

Fig. 2. (a) Raman spectrum. (b) UV/Vis spectrum of [P6,6,6,14+][Cl−], [P6,6,6,14+][FeCl4−] [P

 droplets in water (left) and response towards a magnet of (right).
these MILs are consistent with the reported results [27,39–43]. The UV/

darker. Droplets of the [P +] [CoCl 2−] can be added to a vial of

Vis spectra of the tetrahedral-coordinated CoCl 2− anion, as shown in

Fig. 2b, there were two sets of characteristic absorption observed in 425–550 nm region and two sets of characteristic absorption in 350–500 nm region of the tetrahedral-coordinated MnCl 2− anion [27,42]. As shown in Fig. 2c, the FTIR spectrum of four synthesized MIL

water, with which they are immiscible. The droplets can then be quite easily manipulated with the application of an external strong magnetic field (Fig. 2f).
3.2. Comparison of extraction efficiencies of different MILs

was compared with that of [P +][Cl−], the four spectra are almost
6,6,6,14 + −

identical, showing that the precursor [P6,6,6,14 ][Cl ] and MILs con-

The extraction efficiency of the each analyte is compared for four MILs

tain the same cationic structure. The spectroscopic data can safely lead to the conclusion that four intended MILs are synthesized successfully. As shown in Fig. 2d, it is indicated that these MILs were relatively stable with the temperature lower than 600 K, and suggested that these MIL
has good thermal stabilities. Appearance of four MILs are shown in

in the same extraction conditions. The UV wavelength region of the MIL for detection of estrogens should exhibit observably lower absorbance to allow sensitive analysis of estrogens and eliminate background. Compared to other three MILs, the [FeCl −]-based MIL exhibited considerably high

Concentration of MIL: 100 μg mL ; Solvent: Acetonitrile. (b) Comparison of extraction efficiencies using the four different MILs: [P6,6,6,14+][Cl−], [P6,6,6,14+]
[FeCl4−], [P +] [MnCl 2−], [P +] [CoCl 2−] and [P +] [NiCl 2−].

Fig. 4. (a) Effect of the mass of MIL ([P +] [CoCl 2−]). (b) Effect of extraction time. (c) Effect of concentration of salt (%). (d) Effect of pH on the extraction siX analytes. Moreover, it can be observed from Fig. 2e that the color of MIL based on [MnCl 2 -] is light yellow, which is difficult to observe and
affected by the mass of MIL. Different amounts of the MILs ranging from 20 to 60 mg were added in sample solution for extracting analytes. The separate. Based on the above statements, [P6,6,6,14 ]2[CoCl4 ] MIL was

extraction recovery increased with the increasing of MIL mass up to

chosen for consecutive studies to investigate the analytical performance of the proposed method.

40 mg. After that, there is no significant improvement (Fig. 4a) when MIL quality continues to increase. The phenomenon indicates that the

EXperimental results based on BBD.
Run Codedlevels Response estrogens recoveries (%)

1 −1(10.00) −1(1.00) 0(10.50) 81 79 82 85 88 80
2 0(40.00) −1(1.00) −1(1.00) 89 87 95 82 82 82
3 +1(70.00) +1(10.00) 0(10.50) 75 88 93 100 94 87
4 0(40.00) 0(5.50) 0(10.50) 130 110 120 104 118 118
5 0(40.00) 0(5.50) 0(10.50) 132 112 114 103 120 116
6 0(40.00) 0(5.50) 0(10.50) 130 112 118 103 116 115
7 0(40.00) −1(1.00) +1(20.00) 93 93 103 80 93 93
8 0(40.00) +1(10.00) +1(20.00) 103 97 105 97 97 100
9 +1(70.00) 0(5.50) +1(20.00) 85 85 97 88 88 89
10 −1(10.00) +1(10.00) 0(10.50) 81 81 85 90 90 85
11 +1(70.00) −1(1.00) 0(10.50) 92 84 92 84 100 86
12 −1(10.00) 0(5.50) +1(20.00) 112 83 83 89 92 82
13 0(40.00) 0(5.50) 0(10.50) 129 110 113 102 120 113
14 0(40.00) +1(10.00) −1(1.00) 70 87 90 81 85 85
15 0(40.00) 0(5.50) 0(10.50) 138 110 119 103 120 116
16 +1(70.00) 0(5.50) −1(1.00) 75 87 82 82 80 80
17 −1(10.00) 0(5,50) −1(1.00) 72 75 79 78 78 78

Fig. 5. Response surface obtained from the design expert using the [P +] [CoCl 2−] MIL for the optimization of (a) MIL mass and ionic strength; (b) MIL mass

and extraction time; (c) Ionic strength and extraction time.
6,6,6,14 2 4

Fig. 6. (a) Variation of adsorption capacity of MIL ([P +] [CoCl 2−]) with time for estrogen. (b) Pseudo-second-order kinetic.
extraction has reached saturation. The increase of extraction efficiency with increase of the MIL mass may make the formation of more droplets during the extraction and the hydrophobic MILs were applied to minimize solubility of the droplets. Similar research findings were reported in previous DLLME studies [44–46]. So mass of 40 mg MIL was Effect of extraction time. EXtraction time is crucial for DLLME system because extraction equilibration between MIL and the sample solution may occur when contact time is long. And extraction efficiency dependent on the degree of miXing of the sample solution and MIL. Therefore, the influence of the extraction time from 1 to 12 min for the

selected as consecutive extraction.

recoveries of siX analytes were studied. The experimental results

Analytical performances.

Sample Analytes Linear range (ng mL−1) Linear equation LOD (ng mL−1)

LOQ (ng mL−1)

R2 Precision n = 5 (%), 250 ng mL−1

intraday interday

Milk E2 40.0–1000.0 y=(3.06 ± 0.05) × 104X+(6.18 ± 2.09) × 102 15.0 35.0 0.9989 8.3 10.3
E1 40.0–1000.0 y=(7.91 ± 0.14) × 104X-(0.04 ± 0.05) × 103 15.0 35.0 0.9982 7.5 9.8
CMA 20.0–1000.0 y=(7.97 ± 0.04) × 105X-(1.94 ± 1.76) × 103 5.0 15.0 0.9997 7.4 9.3
MGA 30.0–1000.0 y=(4.58 ± 0.04) × 105X-(1.96 ± 1.47) × 103 8.0 25.0 0.9995 6.5 8.8
HP 30.0–1000.0 y=(9.99 ± 0.04) × 105X-(2.73 ± 1.73) × 103 8.0 25.0 0.9998 6.3 9.5
MPA 40.0–1000.0 y=(4.03 ± 0.05) × 105X-(0.90 ± 2.32) × 103 15.0 35.0 0.9990 3.9 7.4
Lotion E2 40.0–1000.0 y=(3.09 ± 0.05) × 104X-(1.48 ± 2.22) × 102 15.0 35.0 0.9988 6.9 10.1
E1 40.0–1000.0 y=(7.10 ± 0.12) × 104X+(6.25 ± 5.73) × 102 15.0 35.0 0.9985 4.3 8.2
CMA 20.0–1000.0 y=(7.12 ± 0.04) × 105X-(1.22 ± 1.68) × 103 5.0 15.0 0.9997 6.3 9.6
MGA 30.0–1000.0 y=(4.99 ± 0.03) × 105X-(1.58 ± 1.39) × 103 8.0 25.0 0.9996 7.8 10.5
HP 30.0-1000.0 y=(9.25 ± 0.05) × 105X+(3.75 ± 1.97) × 103 8.0 25.0 0.9998 3.9 8.4
MPA 40.0-1000.0 y=(3.97 ± 0.05) × 105X+(2.43 ± 2.24) × 103 15.0 35.0 0.9987 4.9 7.2

Fig. 7. HPLC chromatograms for the blank (black) and samples spiked with 50 ng mL−1 (red) and 250 ng mL−1 (blue). (A) Milk (B) Lotion. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
signified that recoveries of analytes increased when increasing the extraction time from 1 to 3 min, until rapidly reaching equilibrium in around 5 min (Fig. 4b). Therefore, 5 min was selected as the optimal extraction time. Effect of ionic strength. The extraction efficiency was influence by the ionic strength (expressed as NaCl content), as shown in Fig. 4c. Obviously, elevation of the NaCl content can make increase of the extraction efficiencies of all estrogens. And the addition of salt in the aqueous solution may increase the salting out effect and thereby improve the extraction efficiency. Moreover, the increase of salt concentration in the aqueous solution leads to the orderly arrangement of water molecules, reduces the solvation of the analyte, and facilitates the transfer of organic matter from the aqueous phase to the organic phase. Hence, 10% (w/v) NaCl was selected as the optimal concentration of salt. Effect of pH. As estrogens are amphoteric compounds, pH has a great influence on their molecular status. Besides, pH also strongly affects the stability of the MIL in the style. Therefore, the pH value of sample solution plays an important role in extraction process [47]. EXtraction recoveries were studied considering the effect of pH value of the samples solution in the wide range from 1 to 12. The results were shown in (Fig. 4d). In a weakly acidic solution, the estrogens can be protonated which is beneficial to produce an estrogen-MIL complex [48]. The protonated estrogens can be extracted easily by the MIL anion. Analytes recoveries approXimately decreased with pH values
ranging from 4 to 12. And the recoveries of most analytes at pH = 4 are 80–100%. With concentration of OH‾ excessively high, the more

interaction of OH‾ with the MIL may cause a decrease of extraction efficiency. Therefore, pH = 4 was selected as the following study.
3.3.2. Multivariate optimization for the [P +] [CoCl 2−] MIL
Three response surfaces obtained based on the results in the BBD are illustrated in Table 3. It shows the influence of different factors on the CMA recovery. CMA was selected as a model analyte for the estrogens since the obtained results from the siX estrogens were similar (see in Fig. 5). With increase of the MIL mass, the extraction efficiency in- creases firstly and then decreases. It was difficult to elute the estrogens completely when the mass of extraction solvent was too high, so the extraction efficiency decreased. The ionic strength, which is beneficial to extraction efficiency, has a positive effect on estrogens response. The 3D response surface is arched and the results predict that the interac- tion between the parameters is significant. In view of the resulting surface response plot, the optimal experimental conditions are the concentration of the salt was 5.5%, the extraction time was 10.5 min and the mass of MIL was 40 mg.
3.4. Extraction kinetics
To better understand the extraction rate and saturation extraction time which are meaningful in this study, the extraction of estrogens in the initial concentration of 10 mg L−1 as functions of time is shown in Fig. 6 (a). Fast extraction process could be obtained in the first 3 min for siX estrogens because of the abundant available sites in the initial stage. Then extraction rate becomes slow until reaching the extraction equi- librium in 10 min.(b) shows the experimental data of the extract studied can be well fitted to a pseudo-second-order kinetic model with a good

Table 5
The precision and recoveries of the assay.
Sample Analytes Spiked (ng mL−1) Recovery (%) RSD (n = 5, %)
linearity with correlation coefficients varying from 0.9982 to 0.9998 (milk) and 0.9985 to 0.9998 (lotion) were obtained. And the method LODs (S/N = 3) and LOQs (S/N = 10) for milk and lotion were shownin Table 4, and the results indicated that the present method was sui-table for determination of trace estrogens in milk and cosmetics.

3.5.2. Analysis of samples
In order to investigate the applicability of the present method, milk
and lotion were selected as actual samples and applied for analysis. The typical chromatograms of the blank and spiked samples are shown in Fig. 7. It can be seen that estrogens in the samples are not detected, and
no significant interference peaks are found at the retention positions of
estrogens. To investigate accuracy and precision of the proposed

method, the spiked samples were analyzed and the analytical results are
shown in Table 5. The recoveries varied from 98.5% to 109.3% in milk and from 96.3% to 111.4% in lotion, respectively. And acceptable recoveries are obtained for both sample matrices at the LOQ level. All in all, the results indicate that the established analytical method with the

The reported conventional IL-DLLME method (method Ⅰ) and the National standard methods of food safety and cosmetics (method Ⅱ) was statistically compared with this new method to evaluate the ac- curacy and precision and the results were shown in the Table 6 [50]. As revealed by Student’s t-test and variance ratio F-test, there is no sig- nificant difference between the results obtained by these methods. But the proposed method of MIL-DLLME saves cumbersome centrifugation steps, reduces the use of organic solvents and reduces processing time than Method 1 and Method 2. Therefore, these results indicated that the present method was convenient, time saving, and environmental friendly, which was suitable for the determination of estrogens in milk and cosmetics.

fit coefficient (R2 = 0.999), which means that the extraction with the nature of chemisorbed is than main process rather than diffusion [49] (see Table 3).

3.5. Method evaluation
3.5.1. Analytical performances
SiX different concentrations of analytes were added to the sample solution to obtain the working curves under the optimal conditions. The linear range, correlation coefficient (R2), limits of detection (LODs), limits of quantitation (LOQs) and relative standard deviation (RSD) were all derived from the working curves. As shown in Table 4, good
Table 6

The proposed method was also compared with other MILs-based microextraction methods, which have been applied for extraction of organic pollution compounds from environmental water, urine and vegetables listed in Table 7. These MILs can extract analytes from samples solution in less than 10 min, and enable rapid recovery of ex- traction solvent by aid of magnetic fields. The achieved performances by these methods were satisfactory. In this study, [CoCl 2−]-based MIL was selected to extract siX estrogens simultaneously in milk and lotion samples. Furthermore, compared with pale yellow [MnCl42−]-based MIL, dark blue [CoCl42−]-based MIL made visual discrimination and separate from aqueous solution easier. Due to complex matriX and low concentrate of estrogens in milk and cosmetics, cumbersome processing operations generally required more analysis time [48]. Therefore,

Assay results for the determination of estrogens in milk and lotion by the proposed and comparison methods.
Samples Analytes Proposed method n = 5 Method Ⅰ n = 5 Method Ⅱ n = 5

Recoveries (%) ± SD Recoveries (%), ± SD t* (2.31) F* (6.39) Recoveries (%) ± SD t*

E1 101.6 7.5 95.7 9.7 1.89 1.29 99.2 7.9 0.87 1.05
CMA 101.1 9.6 97.2 8.6 1.20 1.12 99.5 7.3 0.53 1.32
MGA 103.8 8.8 97.8 11.8 1.62 1.34 101.8 6.4 0.72 1.38
MPA 102.2 6.3 96.5 13.3 1.53 2.11 98.7 7.9 1.38 1.25
HP 99.4 8.9 95.5 12.9 0.98 1.45 96.9 8.8 0.87 1.01
*The figures between parentheses are the tabulated t and F values at P = 0.05.

acquired extraction time, extraction efficiency, ppb level of LODs and
more analytes by using [P +] [CoCl 2−] MIL were acceptable,
and the MIL could successfully be applied for extraction estrogen from milk and lotion samples.
4. Conclusion
Four different anions of magnetic ionic liquids containing
[P +] [CoCl 2−], [P +] [NiCl 2−], [P +][FeCl −] and
[P +] [MnCl 2−] were synthesized, then four MILs were used as
extraction solvents of the developed DLLME coupled with HPLC to se- parate effectively and determine trace amount of estrogens in milk and
cosmetics. The [P +] [CoCl 2−] MIL shows the optimal extraction
efficiencies for most estrogens and it has low chromatographic back- ground. Under the optimal experimental conditions, a wide linear range, low detection limit and good recovery were obtained. The method is fast, accurate and precise for separation and analysis of siX estrogens. In addition, the present method was statistically compared with the previously reported methods, and there was no significant difference with the other methods in accuracy and precision. In sum- mary, the method proposed in this paper is effective, economic and environmentally friendly that can be successfully applied to the de-
termination of trace estrogens in complex samples and
[P +] [CoCl 2−] MIL has significant application potential and
prospect in the extraction, separation and pretreatment of various samples.
Compliance with ethical standards
The authors declare no conflicts of interest.
This work was supported by the Science and Technology Foundation of Ocean and Fisheries of Liaoning Province (No. 201408, No. 201406), Liaoning Provincial Doctor Startup Fund Program (No. 201601092), the General project of scientific research of the Education Department of Liaoning Province (No. LQN201707), the Foundation for Young Scholars of Liaoning University (No. 2013LDQN13) and the Foundation for National Advance declaration of Liaoning University (No. LDGY201406).

[1] R.J. Letcher, J.O. Bustnes, R. Dietz, B.M. Jenssen, E.H. Jorgensen, C. Sonne,
J. Verreault, M.M. Vijayan, G.W. Gabrielsen, EXposure and effects assessment of persistent organohalogen contaminants in arctic wildlife and fish, Sci. Total Environ. 408 (2010) 2995–3043.
[2] S. Sarajari, M.M. Oblinger, Estrogen effects on pain sensitivity and neuropeptide expression in rat sensory neurons, EXp. Neurol. 224 (2010) 163–169.
[3] L. Storgaard, J.P. Bonde, J. Olsen, Male reproductive disorders in humans and prenatal indicators of estrogen exposure. A review of published epidemiological studies, Reprod. ToXicol. 21 (2006) 4–15.
[4] D. Ganmaa, A. Sato, The possible role of female sex hormones in milk from pregnant
cows in the development of breast, ovarian and corpus uteri cancers, Med. Hypotheses 65 (2005) 1028–1037.
[5] P.G.a.G.S. POPE, OESTROGENS IN MILK, .I. steroid, Biochem 19 (1983) 877–882.
[6] V. Pacakova, L. Loukotkova, Z. Bosakova, K. Stulik, Analysis for estrogens as en- vironmental pollutants–a review, J. Sep. Sci. 32 (2009) 867–882.
[7] L. Yuan, J. Ma, M. Ding, S. Wang, X. Wu, Y. Li, K. Ma, X. Zhou, F. Li, Preparation of
estriol–molecularly imprinted silica nanoparticles for determining oestrogens in milk tablets, Food Chem. 131 (2012) 1063–1068.
[8] S. Wang, Y. Li, X. Wu, M. Ding, L. Yuan, R. Wang, T. Wen, J. Zhang, L. Chen,
X. Zhou, F. Li, Construction of uniformly sized pseudo template imprinted polymers coupled with HPLC-UV for the selective extraction and determination of trace es- trogens in chicken tissue samples, J. Hazard Mater. 186 (2011) 1513–1519.
[9] L.-Q. Qin, P.-Y. Wang, T. Kaneko, K. Hoshi, A. Sato, Estrogen: one of the risk factors
in milk for prostate cancer, Med. Hypotheses 62 (2004) 133–142.
[10] J. Hosogi, H. Tanaka, K. Fujita, T. Kuwabara, S. Ikegawa, N. Kobayashi, N. Mano,
J. Goto, LC-MS/MS coupled with immunoaffinity extraction for determination of estrone, 17beta-estradiol and estrone 3-sulfate in human plasma, J Chromatogr B Analyt Technol Biomed Life Sci 878 (2010) 222–227.
[11] J.A. van Meeuwen, O. van Son, A.H. Piersma, P.C. de Jong, M. van den Berg, Aromatase inhibiting and combined estrogenic effects of parabens and estrogenic effects of other additives in cosmetics, ToXicol. Appl. Pharmacol. 230 (2008) 372–382.
[12] X. Zhang, Y. Peng, J. Bai, B. Ning, S. Sun, X. Hong, Y. Liu, Y. Liu, Z. Gao, A novel electrochemical sensor based on electropolymerized molecularly imprinted polymer and gold nanomaterials amplification for estradiol detection, Sens.
Actuators B Chem. 200 (2014) 69–75.
[13] S.S. Mingqin Kang, Na Li, Daihui Zhang, Mingyan Chen, Hanqi Zhang, EXtraction and determination of hormones in cosmetics by homogeneous ionic liquid micro- extraction high-performance liquid chromatography, J. Sep. Sci. 35 (2012)
[14] C.D. Stalikas, Y.C. Fiamegos, Microextraction combined with derivatization, Trac. Trends Anal. Chem. 27 (2008) 533–542.
[15] X. Liu, X. Qi, L. Zhang, 3D hierarchical magnetic hollow sphere-like CuFe2O4 combined with HPLC for the simultaneous determination of Sudan I–IV dyes in preserved bean curd, Food Chem. 241 (2018) 268–274.
[16] X. Liu, Y. Tong, L. Zhang, Tailorable yolk-shell Fe3O4@graphitic carbon sub- microboXes as efficient extraction materials for highly sensitive determination of
trace sulfonamides in food samples, Food Chem. 303 (2020) 125369.
[17] S. Jahan, H. Xie, R. Zhong, J. Yan, H. Xiao, L. Fan, C. Cao, A highly efficient three- phase single drop microextraction technique for sample preconcentration, Analyst 140 (2015) 3193–3200.
[18] M. Rezaee, Y. Assadi, M.R. Milani Hosseini, E. Aghaee, F. Ahmadi, S. Berijani,
Determination of organic compounds in water using dispersive liquid-liquid mi- croextraction, J. Chromatogr. A 1116 (2006) 1–9.
[19] L. Wang, D. Zhang, X. Xu, L. Zhang, Application of ionic liquid-based dispersive liquid phase microextraction for highly sensitive simultaneous determination of three endocrine disrupting compounds in food packaging, Food Chem. 197 (2016) 754–760.
[20] T.D. Ho, A.J. Canestraro, J.L. Anderson, Ionic liquids in solid-phase microextrac-
tion: a review, Anal. Chim. Acta 695 (2011) 18–43.
[21] S. Hayashi, H.-o. Hamaguchi, Discovery of a magnetic ionic liquid [bmim]FeCl4, Chem. Lett. 33 (2004) 1590–1591.
[22] Z.-G. Shi, H.K. Lee, Dispersive liquid-liquid microextraction coupled, Anal. Chem. 82 (2010) 1540–1545.
[23] K.D. Clark, O. Nacham, H. Yu, T. Li, M.M. Yamsek, D.R. Ronning, J.L. Anderson, EXtraction of DNA by magnetic ionic liquids: tunable solvents for rapid and selec- tive DNA analysis, Anal. Chem. 87 (2015) 1552–1559.
[24] H. Yu, J. Merib, J.L. Anderson, Faster dispersive liquid-liquid microextraction methods using magnetic ionic liquids as solvents, J. Chromatogr. A 1463 (2016)
[25] O. Nacham, K.D. Clark, H. Yu, J.L. Anderson, Synthetic strategies for tailoring the physicochemical and magnetic properties of hydrophobic magnetic ionic liquids, Chem. Mater. 27 (2015) 923–931.
[26] E. Santos, J. Albo, A. Rosatella, C.A.M. Afonso, Á. Irabien, Synthesis and char- acterization of magnetic ionic liquids (MILs) for CO2separation, J. Chem. Technol. Biotechnol. 89 (2014) 866–871.
[27] S. Pitula, A.V. Mudring, Synthesis, structure, and physico-optical properties of manganate(II)-based ionic liquids, Chemistry 16 (2010) 3355–3365.
[28] T. Chatzimitakos, C. Binellas, K. Maidatsi, C. Stalikas, Magnetic ionic liquid in stirring-assisted drop-breakup microextraction: proof-of-concept extraction of phenolic endocrine disrupters and acidic pharmaceuticals, Anal. Chim. Acta 910
(2016) 53–59.
[29] N. Deng, M. Li, L. Zhao, C. Lu, S.L. de Rooy, I.M. Warner, Highly efficient extraction of phenolic compounds by use of magnetic room temperature ionic liquids for en- vironmental remediation, J. Hazard Mater. 192 (2011) 1350–1357.
[30] K.D. Clark, M.M. Yamsek, O. Nacham, J.L. Anderson, Magnetic ionic liquids as PCR- compatible solvents for DNA extraction from biological samples, Chem. Commun. 51 (2015) 16771–16773.
[31] M. Döbbelin, V. Jovanovski, I. Llarena, L.J. Claros Marfil, G. Cabañero,
J. Rodriguez, D. Mecerreyes, Synthesis of paramagnetic polymers using ionic liquid chemistry, Polym. Chem. 2 (2011) 1275–1278.
[32] A.S. NSSON, Iron(III) hydrolysis and solubility, Environ. Sci. Technol. 41 (2007) 6117–6123.
S.S. Satoshi Hayashi, Hiro-o Hamaguchi, A new class of magnetic fluids: bmim [FeCl4] and nbmim[FeCl4] ionic liquids, IEEE Trans. Magn. 42 (2006) 12–14.
[34] J. Merib, D.A. Spudeit, G. Corazza, E. Carasek, J.L. Anderson, Magnetic ionic liquids as versatile extraction phases for the rapid determination of estrogens in human urine by dispersive liquid-liquid microextraction coupled with high-performance liquid chromatography-diode array detection, Anal. Bioanal. Chem. 410 (2018) 4689–4699.
[35] J.L. A, Juan L. Benedéa, Alberto Chisvert, Trace determination of volatile polycyclic aromatic hydrocarbons in natural waters by magnetic ionic liquid-based stir bar dispersive liquid microextraction, Talanta 176 (2018) 253–261.
[36] T.G. Chatzimitakos, J.L. Anderson, C.D. Stalikas, MatriX solid-phase dispersion based on magnetic ionic liquids: an alternative sample preparation approach for the
extraction of pesticides from vegetables, J. Chromatogr. A 1581–1582 (2018) 168–172.
[37] J. Ding, Q. Gao, X.S. Li, W. Huang, Z.G. Shi, Y.Q. Feng, Magnetic solid-phase ex- traction based on magnetic carbon nanotube for the determination of estrogens in milk, J. Sep. Sci. 34 (2011) 2498–2504.
[38] E. Martendal, D. Budziak, E. Carasek, Application of fractional factorial experi- mental and BoX-Behnken designs for optimization of single-drop microextraction of
2,4,6-trichloroanisole and 2,4,6-tribromoanisole from wine samples, J. Chromatogr. A 1148 (2007) 131–136.
[39] N. Banic, M. Vranes, B. Abramovic, J. Csanadi, S. Gadzuric, Thermochromism, stability and thermodynamics of cobalt(II) complexes in newly synthesized nitrate based ionic liquid and its photostability, Dalton Trans. 43 (2014) 15515–15525.
[40] Y. Kawazu, H. Hoke, Y. Yamada, T. Umecky, K. Ozutsumi, T. Takamuku, Complex formation of nickel(ii) with dimethyl sulfoXide, methanol, and acetonitrile in a TFSA(-)-based ionic liquid of [C2mim][TFSA], Phys. Chem. Chem. Phys. 19 (2017) 31335–31344.
[41] Y. Kohno, M.G. Cowan, M. Masuda, I. Bhowmick, M.P. Shores, D.L. Gin, R.D. Noble, A cobalt(II) bis(salicylate)-based ionic liquid that shows thermoresponsive and se- lective water coordination, Chem. Commun. 50 (2014) 6633–6636.
[42] E.R.S. Melissa, S. Sitze, Eric V. Patterson, R. Griffith Freeman, Ionic liquids based on FeCl3 and FeCl2. Raman scattering and ab initio calculations, Inorg. Chem. 40 (2001) 2298–2304.
[43] S. Saikia, P. Gogoi, A.K. Dutta, P. Sarma, R. Borah, Design of multifaceted acidic
1,3-disulfoimidazolium chlorometallate ionic systems as heterogeneous catalysts for the preparation of β-amino carbonyl compounds, J. Mol. Catal. A Chem. 416 (2016) 63–72.
[44] L.M. Ravelo-Perez, J. Hernandez-Borges, M. Asensio-Ramos, M.A. Rodriguez- Delgado, Ionic liquid based dispersive liquid-liquid microextraction for the ex- traction of pesticides from bananas, J. Chromatogr. A 1216 (2009) 7336–7345.
[45] Y. Wang, Y. Sun, B. Xu, X. Li, X. Wang, H. Zhang, D. Song, MatriX solid-phase dispersion coupled with magnetic ionic liquid dispersive liquid–liquid micro- extraction for the determination of triazine herbicides in oilseeds, Anal. Chim. Acta 888 (2015) 67–74.
[46] M. Yang, X. Wu, Y. Jia, X. Xi, X. Yang, R. Lu, S. Zhang, H. Gao, W. Zhou, Use of magnetic effervescent tablet-assisted ionic liquid dispersive liquid-liquid micro- extraction to extract fungicides from environmental waters with the aid of experi-
mental design methodology, Anal. Chim. Acta 906 (2016) 118–127.
[47] Y. Tong, X. Liu, L. Zhang, Green construction of Fe3O4@GC submicrocubes for highly sensitive magnetic dispersive solid-phase extraction of five phthalate esters in beverages and plastic bottles, Food Chem. 277 (2019) 579–585.
[48] M.Q.K.L. Lei, N. Li, a X. Yang, Z.L. Liu, Z.B. Wang, L.Y. Zhang, H.Q. Zhang, a.Y. Yu,
Determination of sex hormones in cosmetic products by magnetically stirring ex- traction bar liquid–liquid microextraction coupled with high performance liquid chromatography, Anal. Methods 6 (2014) 3674–3681.
[49] L. Yang, Y. Zhang, X. Liu, X. Jiang, Z. Zhang, T. Zhang, L. Zhang, The investigation
of synergistic and competitive interaction between dye Congo red and methyl blue on magnetic MnFe2O4, Chem. Eng. J. 246 (2014) 88–96.
[50] B. Socas-Rodríguez, J. Hernández-Borges, M. Asensio-Ramos, A.V. Herrera-Herrera,
J.A. Palenzuela, M.Á. Rodríguez-Delgado, Determination of estrogens in environ- mental water samples using 1,3-dipentylimidazolium Medroxyprogesterone  hexafluorophosphate ionic liquid as extraction solvent in dispersive liquid-liquid microextraction, Electrophoresis 35 (2014) 2479–2487.