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ZnFe2O4 nanocomposite as magnetic sorbents for enrichment of estrogens
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Facile preparation of reduced graphene oxide/ZnFe2O4 nanocomposite as magnetic sorbents for enrichment of estrogens Wenqi Lia, Jing Zhanga, Wenli Zhua, Peige Qina, Qian Zhoua, Minghua Lua,∗, Xuebin Zhangb, Wuduo Zhaoc, Shusheng Zhangc, Zongwei Caid,∗∗ a
Henan International Joint Laboratory of Medicinal Plants Utilization, School of Chemistry and Chemical Engineering, Henan University, Kaifeng, 475004, Henan, China Center for Multi-Omics Research, Institute of Plant Stress Biology, Henan University, Kaifeng, 475004, Henan, China c Center for Advanced Analysis and Computational Science, Zhengzhou University, Zhengzhou, 450001, Henan, China d State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong SAR, China b
ARTICLE INFO
ABSTRACT
Keywords: Magnetic solid-phase extraction Reduced graphene oxide Zinc ferrite Estrogens High performance liquid chromatography Environmental sample
Reduced graphene oxide/ZnFe2O4 (rGO/ZnFe2O4) nanocomposite was facile prepared and applied as magnetic sorbent for the extraction of estrogens including 17β-estradiol, 17α-estradiol, estrone and hexestrol from water, soil, and fish samples prior to HPLC analysis. The rGO/ZnFe2O4 nanocomposite was characterized by scanning electron microscope, Fourier transform-infrared spectroscopy, X-ray diffraction, and vibrating sample magnetometer. The experimental parameters affecting the efficiency of magnetic solid-phase extraction (MSPE) including the amount of material, extraction time, pH, temperature, desorption solvents, desorption time, and desorption solvent volume were investigated respectively. With the developed method, good linearity was observed in the range of 0.05–500 ng/mL with the correlation coefficients (R2) between 0.9978 and 0.9993. The limits of detection (S/N = 3) and limits of quantification (S/N = 10) were achieved at 0.01–0.02 ng/mL and 0.05 ng/mL, respectively. The enrichment factors were calculated as the range of 241–288. Using rGO/ZnFe2O4 nanocomposite as the sorbent, the developed MSPE followed by HPLC analysis, was applied to analysis of estrogens in river water, soil and fish samples. The method has the potential application in the extraction and preconcentration ultra trace compounds in complex matrices, such as environmental and biological samples.
1. Introduction Sample pretreatment is considered as the most crucial step in a complete analytical process for identification, confirmation and quantification of analytes, especially those in complicated samples, namely environmental, biological, industrial samples etc. [1–4]. In a traditional analytical process, sample pretreatment is time consuming and often devour about 60% of the total time, which does not meet the standard of high efficiency. Thus, considering the low content of analytes and complex interferents, the development of sample pretreatment techniques with high efficiency is urgent. Depending on the phase of extraction material, sample pretreatments can be defined as solvent-based and sorption-based extractions [5]. In the past few years, sorption-
based extraction plays a dominant role in environmental and biological sample pretreatment. For sorption-based extraction, such as solid-phase extraction (SPE) [6], dispersive solid-phase extraction (dSPE) [7], magnetic solid-phase extraction (MSPE) [8], solid-phase microextraction (SPME) [9] and so on, the key element is the sorbent material which determines the sensitivity and selectivity of the method. Therefore, the development of simple, highly efficient, and environmentfriendly sorbent for sample pretreatment is urgently needed. Graphene, a two-dimensional single-atom thick carbon nanomaterial which hexagonally arrayed with sp2-bonded carbon atoms in a closely packed honeycomb crystal lattice, has attracted considerable attention in varieties of applications since it was firstly discovered in 2004 [10]. Due to its large specific surface area, excellent mechanical
Abbreviations: Reduced graphene oxide/ZnFe2O4, rGO/ZnFe2O4; magnetic solid-phase extraction, MSPE; solid-phase extraction, SPE; dispersive solid-phase extraction, dSPE; solid-phase microextraction, SPME; 17β-estradiol, 17β-E2; 17α-estradiol, 17α-E2; estrone, E1; hexestrol, HEX; ethylene glycol, EG; polyethyleneglycol, PEG; graphene oxide, GO; scanning electron microscopy, SEM; Fourier transform-infrared spectroscopy, FT-IR; X-ray diffraction, XRD; vibrating sample magnetometer, VSM; high-performance liquid chromatography, HPLC ∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (M. Lu), [email protected] (Z. Cai). https://doi.org/10.1016/j.talanta.2019.120440 Received 11 June 2019; Received in revised form 18 September 2019; Accepted 3 October 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Wenqi Li, et al., Talanta, https://doi.org/10.1016/j.talanta.2019.120440
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and thermal stability, π delocalized electrons system, and hydrophobic property, graphene is considered as an ideal candidate for extraction and enrichment of carbon-based ring structures in sample preparation [11,12]. Graphene and graphene-based materials were mainly used as sorbents for SPE [13–15] and coating for SPME [16,17]. Compared with traditional column SPE method, dispersive extraction modes including dSPE and MSPE can provide better extraction efficiency because the enhanced contact area between sorbents and target analytes. On the other hand, dSPE and MSPE avoid the packing SPE column that has difficulty in non-professional laboratory [16]. In dSPE, it is often hard to completely retrieve graphene or graphene-based materials from the suspension owing to ultra-light property and extreme dispersity even with high-speed centrifugation [18]. To solve this problem, graphene-based magnetic sorbents are prepared that can be separated from the sample solutions by an external magnetic field. The Fe3O4 nanoparticle is the most commonly used magnetic source because of its good biocompatibility, superparamagnetic property, low toxicity and easy preparation [19]. However, the Fe3O4 nanoparticles usually showed low stability in acidic solution. Therefore, it is desirable to develop an efficient and stable material as magnetic source with a simple and rapid synthesized method. Recently, magnetic spinel ferrites [M(II)Fe(III)2O4, M = Ni, Co, Zn, Mn, etc.] with a face-centred cubic structure have attracted considerable attention because of their uniquely structural, electrical, and magnetic properties [20]. Noormohamadi et al. [21] prepared polyethyleneimine modified cobalt ferrite as magnetic sorbent for extraction of tartrazine from food and water samples. Pastor-Belda and coworkers [22] synthesized oleic acid coated cobalt ferrite nanoparticles as the sorbent of MSPE that coupled to gas chromatography-mass spectrometry for determination of alkylphenols in baby foods. However, the introduction of Co, Ni may lead to the heavy metal pollution to the environment. Due to unique physicochemical properties, good stability, nontoxicity, high specific surface area and excellent magnetism, zinc ferrite (ZnFe2O4) can be used as environmentally friendly magnetic source. Moreover, comparing with conventional Fe3O4 nanoparticle, ZnFe2O4 provide more stable property because it can't be dissolved in most acidic and alkaline medium [23–25]. Endocrine disrupting chemicals refer as exogenous substances are related to diverse health issues in intact organisms or able to alter the endocrine functions of the offspring consequently [25,26]. As a significant group of endocrine disrupting chemicals, estrogens have caught great attentions due to their existence in environmental samples. Study related to estrogens in surface waters has been reported since the early 1980s [27]. Environmental estrogens including endogenous estrogens (17β-estradiol, 17α-estradiol, estrone, estriol) and artificial estrogens (hexestrol, ethinylestradiol, diethylstilbestrol, dienestrol) are the main sources of natural and synthetic hormones which are the excrement of humans and animals. They can get into the aquatic environment through the sewage, agricultural, pharmaceutical and aquaculture waste system [28–30]. Abundant studies have demonstrated that estrogens have potentially adverse health effects to aquatic wildlife and human causing growth, development, and reproduction problems, such as feminization and hermaphroditism [31–34]. Once excessive estrogens get into the human body through food chain, they can interfere the normal functions of the endocrine systems leading to the risk of getting cancers such as prostate cancer and breast cancer [35,36]. Therefore, the development of analytical techniques to efficiently enrich and analyze estrogens at low concentrations is of great significance of science and health issues. Herein, reduced graphene oxide/ZnFe2O4 (rGO/ZnFe2O4) magnetic nanocomposite was synthesized by one-step hydrothermal method, and was used as the sorbent of MSPE for the extraction and preconcentration of estrogens (17β-estradiol (17β-E2), 17α-estradiol (17α-E2), estrone (E1), hexestrol (HEX), the chemical structure referring to Fig. S1 in supplementary material) from river water soil, and fish samples. Comparing with conventional Fe3O4 as magnetic source, ZnFe2O4
provides more stability in acidic solution. The prepared rGO/ZnFe2O4 exhibits excellent extraction efficiency for estrogens in real environmental samples, such as river water and soil samples. 2. Experimental section 2.1. Reagents and material The standard compounds including 17β-estradiol, estrone and hexestrol were purchased from Aladdin Chemistry Co., Ltd (Shanghai, China). 17α-estradiol was supplied by Marcklin Biochemical Co., Ltd (Shanghai, China). Methanol and acetonitrile with HPLC grade were provided by Tedia Company Inc. (Fairfield, USA). Acetone (HPLC grade) was obtained from Anaqua Chemicals Supply (Eldridge Parkway, Houston, USA). Graphite powder (99.95%, 8000 mesh), H2O2 (30%), FeCl3·6H2O (99%), NaOH (96%), isooctane (99.8%), ethyl acetate (99%), H3BO4 (≥99.5%), H2SO4 (98%), HCl (36%), NaNO3 (99%), ZnCl2 (98%) and KMnO4 (≥98%) were achieved from Aladdin Chemistry Co., Ltd (Shanghai, China). Ethylene glycol (EG), polyethyleneglycol (PEG), H3PO4 (≥99.5%), NaAC (99.0%) were purchased from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). Water used throughout the work was produced by a Milli-Q ultrapure water system (Millipore, Bedford, MA, USA). 2.2. Instruments, HPLC-DAD and UPLC-QTOF-MS conditions The morphology of rGO/ZnFe2O4 was characterized by scanning electron microscopy (SEM, JSM-7610F, Japan). Fourier transform infrared spectroscopy (FT-IR) was obtained by a VERTEX 70 spectrometer (Bruker, Germany). X-ray diffraction (XRD) measurement was carried out on Bruker D8 Advance (Bruker, Germany) using Cu Kα radiation. The magnetic property was analyzed using a vibrating sample magnetometer (VSM, Quantum Design, MPMS3). The chromatographic analysis was performed on an Agilent 1260 high-performance liquid chromatography (HPLC) equipped with a diode array detector (DAD). The optimum separation was obtained on a reversed phase Eclipse Plus C18 column (4.6 × 100 mm, 3.5 μm) with a binary mobile phase composed of 45% ultrapure water (solvent A) and 55% acetonitrile (solvent B). The flow rate, column temperature, injection volume and UV wavelength were set at 0.5 mL/min, 5.0 μL, 25 °C and 200 nm, respectively. The UPLC-QTOF-MS analysis was performed on a Waters ACQUITY UPLC/Synapt G2 MS system (Waters, USA) to further confirm the results of real samples that obtained by the developed HPLC-DAD method. The electrospray ionization (ESI) with full scan (m/z, 50–500) in negative ion mode was adopted. The electrospray capillary voltage was set as 3000 V, the source temperature and desolvation temperature was 150 °C and 450 °C, respectively. The cone gas flow was 50 L/h and desolvation gas flow was 450 L/h. The ion source nebulizing gas (N2) pressure was set at 6.5 bar and collision energy was 30 V. 2.3. Preparation of graphene oxide (GO) GO was synthesized from natural flake graphite by a modified Hummers' method [37]. At first, 1.0 g graphite and 0.5 g NaNO3 were dissolved in 23 mL H2SO4 under an ice bath (0–5 °C) for 30 min. 3.0 g KMnO4 was added to the above solution gradually with vigorous stirring and keep the temperature of the solution < 20 °C. The solution was stirred continuously in a water bath at 35 °C for 2 h until it became pasty brownish, then 186 mL ultrapure water was added and stirred for another 30 min, and then 2.5 mL 30 wt% H2O2 was slowly added to the mixture to reduce the residual KMnO4, after which the color of the mixture changed to brilliant yellow. The obtained mixture was centrifuged and was washed by HCl (10%) and ultrapure water until the pH was approached to 7. Finally, the obtained solid was freeze-dried in vacuum freeze dryer to achieve graphite oxide powder. 2
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Fig. 1. Characterization of as-prepared materials. SEM of GO with magnification of 3000 (a), SEM of rGO/ZnFe2O4 with magnification of 50000 (b) and 20000 (c), XRD patterns of GO and rGO/ZnFe2O4 (d), FT-IR spectra of GO and rGO/ZnFe2O4 (e), VSM magnetization curve of rGO/ZnFe2O4 (f).
2.4. Synthesis of rGO/ZnFe2O4
concentrations of estrogens in the initial solution and residual solution (mg/L), respectively. V (L) is the volume of estrogens. M (g) is the amount of adsorbent. The desorption ratio is calculated the following equation [41].
The magnetic rGO/ZnFe2O4 nanocomposite was prepared by a solvothermal method [38]. Briefly, 100 mg of GO was dispersed in 50 mL of EG with ultrasonication for 2 h, then 0.540 g (2 mmol) of FeCl3·6H2O and 0.197 g (1 mmol) of ZnCl2·4H2O were added into the GO dispersion with vigorous stirring. Subsequently, 3.60 g NaAc and 1.00 g PEG were added, followed by stirring for 1 h. The mixture was then transferred into a 100 mL Teflon-lined stainless steel autoclave and maintained at 200 °C for 8 h. During the solvothermal process, EG was used as not only solvent but also agent, meanwhile NaAc would provide an alkaline atmosphere, which could make GO reduce to rGO. The resultant black product was washed with deionized water and ethanol several times and was dried at 50 °C in a vacuum oven for further use.
Qd =
where Qd is the desorption ratio, M1 (g) is the amount of adsorbed target analytes and M2 (g) is the amount of released target analytes. 2.6. Preparation of standard solutions and real samples Stock solutions were prepared at a concentration of 100 μg/mL in methanol. The standard working solutions were stored at 4 °C and daily prepared by appropriate dilutions of the stock solutions with ultra-pure water to obtain the required concentrations prior to use. The environmental water samples were collected from Housi River (Gongyi, China). The water sample was filtered through 0.45 μm filter membrane and stored in the refrigerator at 4 °C. Prior to analysis, the pH of water samples were adjusted to 4, and 30 mL of prepared water sample was subjected to MSPE process before HPLC-DAD analysis. The soil sample was collected from the east suburb of Kaifeng. The soil sample was air dried at room temperature and sieved to a particle size of 0.45 mm before analysis. 10 g soil sample was homogenized in 10 mL of methanol, and was ultrasonicated for 30 min at room temperature. The homogenate was centrifuged at 8000 rpm for 10 min. The residue was extracted again as described above. The two supernatants were combined and dried under a N2 stream, subsequently, the dry residue was redissolved in 500 μL methanol, and 100 μL of the solution was added into 29.9 mL ultra-pure water which was adjusted pH to 4 for MSPE. The fish was purchased from local market (Kaifeng, Henan), which was freeze-dried and stored in the refrigerator at −18 °C 10 g freezedried fish sample was grounded into powder and homogenized in 10 mL of acetonitrile. The mixture was ultrasonicated for 30 min at room temperature. After ultrasonication, the homogenate was centrifuged at 8000 rpm for 15 min. The residue was extracted again as described above, and the subsequent procedures were the same as the preparation of the water and soil samples.
2.5. MSPE procedures In MSPE process, 10 mg of rGO/ZnFe2O4 magnetic composite was added into 30 mL mixed standard solution with a concentration of 200 ng/mL for each analyte. Then, the mixture was vortexed for 1 min in an oscillator and sonicated vigorously for 15 min to make the sorbents dispersed uniformly and fully adsorbed in the mixed standard solution. Subsequently, a strong magnet was placed at the bottom of the beaker to separate the sorbents from the solution. The suspension became limpid gradually and the supernatant was decanted until the supernatant became clear. Then the analytes were eluted from the rGO/ ZnFe2O4 with 3 mL of acetonitrile under ultrasonication for 5 min. After the magnetic separation, the supernatant was dried under a stream of nitrogen at 60 °C and redissolved in 100 μL methanol, afterwards filtered through disposable syringe filters (0.45 μm) into a sample vial and 5 μL of this solution was injected into the HPLC system for analysis. The performance of MSPE is evaluated by adsorption capacity and desorption ratio. The adsorption capacity is investigated by the following equitation [6,39,40].
Qa = [(Co
M2 × 100% M1
C ) × V ]/ M
where Qa (mg/g) is the amount of estrogens adsorbed onto the unit amount of the adsorbent; C0 (mg/L) and C (mg/L) are the 3
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Fig. 2. Factors affecting the adsorption capacity. (a) Adsorption temperature. Other conditions: adsorbent, 10 mg; adsorption time, 10 min; adsorption pH, 7; (b) Adsorption time. Other conditions: adsorbent, 10 mg; adsorption temperature, 30 °C; adsorption pH, 7; (c) pH. Other conditions: adsorbent, 10 mg; adsorption temperature, 30 °C; adsorption time, 15 min.
3. Results and discussion
ZnFe2O4 can be easily separated from the aqueous solution by an external magnetic field.
3.1. Characterization of sorbent
3.2. Optimization of MSPE
The morphology of GO and rGO/ZnFe2O4 composite were characterized by SEM and the results are presented in Fig. 1. The irregular flake-like GO sheets with wrinks are observed in Fig. 1a. It can be seen from Fig. 1b and c that ZnFe2O4 nanoparticles are homogeneously dispersed on the silk-like rGO surface. The size of the ZnFe2O4 nanoparticles on rGO is around 200 nm. The crystalline phase of the GO and rGO/ZnFe2O4 nanocomposites were measured by XRD and illustrated in Fig. 1d. The diffraction pattern of GO showed a strong peak at around 2θ = 10.2°, which was assigned to its (001) plane. In the diffraction patterns of rGO/ZnFe2O4 composites, it was obviously observed that the peak at 2θ = 10.2° was disappeared, which confirmed that oxygen groups have been removed and GO has been flaked and reduced to rGO nanosheets through the solvothermal method. The rGO has no apparent peaks, which may own to its amorphous state, suggesting that the crystal structures of ZnFe2O4 was not altered with the addition of rGO [42]. The other peaks at 2θ values of 29.9°, 35.3°, 42.8°, 53.1°, 56.6°, 62.2° can be indexed to (220), (311), (400), (422), (511), and (440) crystal planes of spinel ZnFe2O4 (JCPDS NO. 22–1012). Furthermore, the diffraction pattern confirmed the formation of the rGO/ZnFe2O4 composites. The FT-IR is another main tool to verify the functional groups of the synthesized rGO/ZnFe2O4. As shown in Fig. 1e, absorption bands of the GO sheets appeared at 3422 cm−1 (O–H stretching band), 1725 cm−1 (C]O stretching band), 1627 cm−1 (skeletal vibrations of aromatic domains), 1396 cm−1 (bending absorption of carboxyl group O]C–O), 1225 cm−1 (O–H bending vibrations), and 1031 cm−1 (C–O stretching vibrations), suggesting that GO was successfully formed from graphite. For rGO/ZnFe2O4 composites, the bands appeared at around 570 cm−1, 450 cm−1 can be attributed to the octahedral Fe3+ (Fe–O mode) stretching vibration and the tetrahedral Zn2+ (Zn–O mode) stretching vibration [43], the peaks corresponding to the oxygen containing functional groups become a slightly weaken, and the characteristic peak at around 1571 cm−1 could be assigned to the stretching vibrations of the conjugated carbon backbone, it was concluded that GO was successfully reduced to rGO during the process of one-pot solvothermal reaction [44]. The intensity of magnetism is another important property for magnetic materials. The magnetic property of the rGO/ZnFe2O4 was studied using a VSM method. The saturation magnetization value for rGO/ZnFe2O4 is 52.6 emu g−1. Fig. 1f shows the magnetic hysteresis loop measured at room temperature. The S-like magnetic hysteresis loop shows no apparent hysteresis, remanence and coercivity, indicating that the rGO/ZnFe2O4 is superparamagnetic and it is a desirable candidate for practical applications in sample pretreatment with great separability and recyclability. The inset of Fig. 1f shows that rGO/
The parameters affecting the extraction efficiency and recovery of analytes were optimized to achieve the best MSPE conditions for extraction of estrogens. 30 mL of the standard solution with a concentration of 200 ng/mL for each analyte was used for optimization. The adsorbents amount, pH, adsorption time, adsorption solvent as well as the desorption temperature, desorption time and eluant volume were systematically investigated as following. 3.2.1. Effect of the amount of rGO/ZnFe2O4 During the MSPE process, the sorbent amount is a major factor that affecting adsorption efficiency. The effect of rGO/ZnFe2O4 amount on adsorption capacity for estrogens was investigated between the ranges from 2 to 15 mg. The results indicated that the adsorption capacity increased rapidly with increase of the amount of rGO/ZnFe2O4 from 2 to 10 mg, demonstrated that the remarkable enrichment ability of rGO/ ZnFe2O4. However, the adsorption capacity was not significantly improved with further increasing the amount of the sorbent. Therefore, 10 mg was selected as the optimum amount of sorbent to ensure the extraction efficiency and quantitative recovery in the following experiments. 3.2.2. Effect of adsorption temperature Considering the fact that extraction efficiency can be influenced by the adsorption temperature as temperature controls the diffusion rate of the target analytes from the sample solution to adsorbent phase, adsorption temperature from 10 to 60 °C was investigated (Fig. 2a). As previously reported that the diffusion rate increase as the temperature rise. But, when the temperatures was higher than 30 °C, there was a significant decrease in adsorption efficiency, which may be due to the exothermic nature of this adsorption [45]. So, 30 °C was selected as the optimum adsorption temperature. 3.2.3. Effect of the adsorption time Adsorption time is another important parameter that influence the extraction efficiency which is directly related to the contact time between analytes and sorbents. Adsorption time ranging from 2 to 20 min was investigated and the result is shown in Fig. 2b. It can be observed that the adsorption capacity for target estrogens was close to the maximum level at 15 min. The adsorption capacity has a slight decrease with further prolonged adsorption time because the adsorption and release was a dynamic equilibrium during the ultrasonication process. Thus, the optimum adsorption time was chosen as 15 min for future experiment. 4
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3.2.4. Effect of pH Estrogens are ionizable compounds, thus, their existing forms in the aqueous solution can be affected by the pH value and the extraction efficiency. Therefore, the effects of solution pH on extraction efficiency were examined between the range of 2.0–9.0 (Fig. 2c). The highest adsorption capacity was observed at pH = 4.0, and the extraction efficiency under alkaline condition is significantly lower than that in acidic condition. The possible reason can be explained as the hydrophobic interaction between the analytes and sorbents was suppressed by the ionized form of estrogens when the pH higher than 7.0 due to the pKa values of target estrogens ranging from 9.80 to 10.34 [45]. On the other hand, the ionization of –OH in the structure of estrogen could be suppressed in acid condition. Therefore, estrogens could be protonated or ionized to form ions at lower or higher pH values, which are not beneficial for adsorption [46,47]. It can be concluded that the adsorption of estrogens with rGO/ZnFe2O4 sorbent was mainly based on hydrophobic interaction and π-π interaction, as well as the intermolecular hydrogen bond between adsorbents and estrogens. Therefore, in the following experiment, pH = 4.0 was chosen as the optimum solution pH.
3 mL. The desorption ratios for estrogens were found to be 92.91% (17β-E2), 93.24% (17α-E2), 95.18% (E1) and 98.54% (HEX), respectively. It is illustrated that 3 mL of acetonitrile is sufficient to obtain satisfactory desorption ratios. 3.3. Reusability of the sorbents The reusability of rGO/ZnFe2O4 sorbent for extraction of estrogens with MSPE was investigated under the optimal conditions and the results are shown in Fig. S2. The results showed that the rGO/ZnFe2O4 composite is still robust and durable after being reused for 15 times, which indicate that the rGO/ZnFe2O4 adsorbent has good reusability, and the extraction efficiency decreased significantly after 20 cycles, this may be due to the shedding of rGO during ultrasonic process. 3.4. Validation of the MSPE-HPLC method The analytical performance of the method including linear range, correlation coefficients (R2), limits of quantification (LOQs, S/N = 10), limits of detection (LODs, S/N = 3), and method precisions were investigated in the standard solutions under the optimized conditions. The enrichment factors were calculated from 241 to 288 according to equation S1 which is presented in supplementary materials. As listed in Table 1, a good linearity was obtained in the concentration range of 0.05–500 ng/mL for all estrogens with R2 between 0.9978 and 0.9993, The LOQs after treatment with the rGO/ZnFe2O4 adsorbent for the four estrogens were calculated to be 0.05 ng/mL. The LODs obtained were 0.01 ng/mL for 17α-estradiol and 0.02 ng/mL for 17β-estradiol, estrone and hexestrol. The repeatability was obtained by analyzing a sample five times during a working day, while the intermediate precision was determined by analyzing a sample once a day over five contiguous days. The RSDs of repeatability and intermediate precision were in the range of 1.24–2.58% and 2.75–7.22%, respectively. It was demonstrated that the developed method possessed reliable and repeatable properties. To verify the repeatability of as-prepared material as sorbent, five different batches of materials were selected for extraction of target analytes. The results demonstrated that the RSDs were not higher than 7.14%, illustrating good synthetic repeatability for preparation of the rGO/ ZnFe2O4. The spiked river water, soil and fish samples with three different concentrations (2.0, 20.0, and 200 ng/mL) were analyzed under the optimal conditions and the results are listed in Table 2. The recoveries for target estrogens were in the range of 73.50–104.13% with RSDs not higher than 8.33%. The results demonstrated that rGO/ZnFe2O4 sorbent had good recovery and intermediate precision, which could used as sorbent for extraction trace estrogens from environmental and biological samples.
3.2.5. Effect of desorption solvents The desorption solvent is another critical parameter during the desorption process. To completely elute the analytes from sorbent and to improve the desorption efficiency, the affinity of desorption solvent toward the target analytes should be higher than that of the sorbent. Different organic solvents including methanol, acetonitrile, acetone, isooctane, ethyl acetate were investigated and the result was shown in Fig. 3a. It can be seen that acetonitrile exhibited the best desorption ratios under the same extraction and desorption conditions. Under this conditions, the desorption ratios for estrogens were found to be 52.50% (17β-E2), 68.15% (17α-E2), 58.08% (E1) and 74.30% (HEX), respectively. Hence, acetonitrile was selected as the desorption solvent. 3.2.6. Effect of desorption time To complete desorption of the analyte from sorbents, the desorption time as an important parameter effect on desorption efficiency was investigated from 1 to 12 min. As shown in Fig. 3b, the desorption process basically reached desorption equilibrium after 5 min. The desorption ratios for estrogens were found to be 52.67% (17β-E2), 69.86% (17α-E2), 59.72% (E1) and 75.02% (HEX), respectively. Therefore, 5 min was selected as desorption time. 3.2.7. Effect of desorption volume To achieve the best desorption ratios for estrogens, an optimum volume of desorption solvent was optimized ranging from 1 to 7 mL. According to Fig. 3c, the desorption ratios were rapid increased upon increasing the volume of acetonitrile from 1 to 3 mL, and there was no obvious enhancement when the volume of acetonitrile was higher than
Fig. 3. Factors affecting the desorption efficiency. (a) Desorption solvents. Other conditions: desorption time, 10 min; desorption solvent volume, 1 mL; (b) Desorption time. Other conditions: desorption solvent, acetonitrile; desorption solvent volume, 1 mL; (c) Desorption solvent volume. Other conditions: desorption solvent, acetonitrile; desorption time, 5 min. 5
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Table 1 The Linear range, correlation coefficients, EFs, LOQs and LODs of the developed MSPE-HPLC method for the analysis of estrogens. Analytes
17β-estradiol 17α-estradiol estrone hexestrol
Linear range (ng/mL)
0.05–500 0.05–500 0.05–500 0.05–500
R2
0.9981 0.9978 0.9980 0.9993
LOQs (ng/mL)
LODs (ng/mL)
0.05 0.05 0.05 0.05
0.02 0.01 0.02 0.02
EFs
241 268 261 288
RSD (%, n = 5) Repeatability
Intermediate
Batch to batch
1.24 2.58 2.50 1.78
4.20 7.22 2.96 2.75
7.14 5.19 5.98 4.72
3.5. Real sample analysis The river water, soil and fish were selected as testing samples to verify the proposed method and the results were listed in Table 2. Among target analytes, 17β-estradiol and hexestrol were detected in river water sample and the content were calculated as 1.92 and 1.30 ng/mL. In the soil sample, the hexestrol was detected with a concentration of 15.9 ng/g. The target analyte was not detected in the fish sample. The chromatograms of water sample are shown in Fig. 4. It can be seen from graph a in Fig. 4 that the target estrogens were not detected in the river water sample by HPLC-DAD. With rGO/ZnFe2O4 as sorbent for extraction and preconcentration of target analytes, two target analytes (17β-estradiol and hexestrol) were detected. Graph C in Fig. 4 demonstrated the analysis of the spiked river water sample (20 ng/mL) by developed MSPE with rGO/ZnFe2O4 as sorbent. The typical chromatograms obtained by the analysis of soil sample and fish sample are presented as Figs. S3 and S4 in the supplementary material. To validate the results of real samples obtained with HPLC method, the extracted solution of water and soil samples with rGO/ZnFe2O4 nanocomposite as sorbent was further analyzed by UPLC-MS, respectively. As shown in Fig. S5, the molecular ion peaks of [M − H]- with m/z at 271.1688 and 269.1532 were observed in water sample, which could be identified as 17β-E2, and HEX. The molecular ion peak of [M − H]- with m/z at 269.1532 was observed in soil sample, which corresponding to HEX. The results obtained with UPLC-MS were same as that of HPLC-DAD.
Fig. 4. The typical chromatograms obtained by direct analysis of river water sample (a), river water sample pretreated with rGO/ZnFe2O4 as MSPE sorbent (b), and spiked river water sample (20 ng/mL) pretreated with developed MSPE procedures (c). Peaks identification: (1) 17β-E2, (2) 17α-E2, (3) E1, (4) HEX.
and Xu et al. [50] both reported magnetic sorbents for extraction of estrogens with LODs in the range of 4.3–7.5 ng/mL and 3.2–20.1 ng/L, respectively. The sorbent amounts were 500 and 200 mg in above two works [49,50]. Zhao and co-workers [51] prepared a molecularly imprinted polymer used to on-line extract four estrogens in milk sample with the LODs of 1–8 ng/g. Wang et al. [52] analyzed the estrogens in water sample using in-tube SPME coupled with HPLC and the LODs were found in the range of 0.05–0.3 ng/mL. Comparing with other reported method, the method developed in current study for analysis of estrogen in water, soil and fish samples displayed a range of advantages including better selectivity, relatively lower LODs, higher enrichment factors and fewer sorbent amount.
3.6. Comparison with other methods The developed MSPE coupled with HPLC-DAD method was compared with other reported methods (Table 3), and the calibrators and samples are subject to the same preparation. Jiang and co-workers [48] reported a method for the determination of estrogens in water samples with LODs ranging from 1.7 to 3.4 ng/mL using ionic liquid as the adsorbents of dispersive liquid-phase microextraction. Tian et al. [49] Table 2 Results for analysis of real samples and the recoveries of method. Samples
River water
Soil
Fish
a b
Analytes
17β-estradiol 17α-estradiol estrone hexestrol 17β-estradiol 17α-estradiol estrone hexestrol 17β-estradiol 17α-estradiol Estrone hexestrol
Found (ng/mL or ng/g)a
1.92 N.d.b N.d. 1.30 N.d. N.d. N.d. 15.9 N.d N.d. N.d N.d
Added 2 ng/mL
Added 20 ng/mL
Added 200 ng/mL
Recoveries (%)
RSD (%) (n = 3)
Recoveries (%)
RSD (%) (n = 3)
Recoveries (%)
RSD (%) (n = 3)
80.24 92.07 92.95 73.69 83.26 81.54 79.94 78.56 74.70 75.97 74.10 73.50
3.81 7.68 6.20 7.66 5.92 3.85 8.33 7.47 4.21 5.93 1.94 1.87
91.57 96.17 96.06 87.17 87.63 86.73 89.57 80.17 82.53 78.43 82.60 74.57
3.19 5.60 4.41 2.29 5.96 3.08 3.88 2.68 6.25 5.80 3.87 4.37
85.32 88.40 91.17 91.55 104.13 90.32 89.21 89.50 89.29 92.63 100.10 95.47
5.65 5.20 2.15 1.68 2.74 3.59 3.54 4.68 4.35 0.82 1.96 3.11
The units are ng/mL for liquid sample and ng/g for solid sample. Not detected. 6
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Table 3 Comparison with other methods for the determination of estrogens. Sorbents IL Fe3O4@TiO2/GO MC MIP Nano-CaCO3 rGO/ZnFe2O4
Samples Water Milk Water Milk Water Water, Soil
Extraction method DLPME MSPE MSPE On-line SPE IT-SPME MSPE
LODs
Linearity
1.7–3.4 ng/mL 4.3–7.5 ng/mL 3.2–20.1 ng/L 1.0–8.0 ng/g 0.05–0.3 ng/mL 0.01–0.02 ng/mL
3
5.0–1.0 × 10 ng/mL 5.0–5.0 × 103 ng/mL 0.1–100 mg/L 3–2500 ng/g 0.15–20 ng/mL 0.05–500 ng/mL
Sorbent amount
Refs.
60 μL 500 mg 200 mg 50 mg 166 mg 10 mg
[48] [49] [50] [51] [52] This work
IL: ionic liquid. MC: magnetic chitosan. MIP: molecularly imprinted polymer. DLPME: dispersive liquid-phase microextraction. IT-SPME: in-tube solid-phase microextraction.
3.7. Potential interferences and the selectivity of the method
References
The potential interferences of environmental samples include benzene compounds, phenolic compounds, nitrobenzene, chlorinated alkanes, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, as well as a variety of organic pesticides and so on. The adsorption mechanism of rGO/ZnFe2O4 mainly contained π-π interaction and hydrogen bonds interaction. Therefore, the rGO/ZnFe2O4 can absorb compounds that containing benzene rings with π-π interaction between rGO and benzene rings. Meanwhile, hydrogen bond interaction also play an important role for the adsorption because the hydroxyl and carboxyl groups on rGO surface tend to form hydrogen bonds with hydroxyl and carboxyl groups in the target analytes. However, the adsorption of the as-prepared material for the target analytes was not the selective adsorption. Therefore, the selectivity of the material was hoped to be improved with other techniques, such as molecular imprinting.
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4. Conclusion In this work, rGO/ZnFe2O4 magnetic composites were prepared by a simple hydrothermal method. The rGO/ZnFe2O4 was used as sorbents for MSPE with many advantages comparing with previously reported methods including lower cost and better precision. In addition, the preparation procedures are simple and environment friendly. Compared with conventional Fe3O4 nanoparticle, ZnFe2O4 is more stable because it doesn't dissolve in most acidic and alkaline medium. The developed MSPE with rGO/ZnFe2O4 as sorbents, coupled with HPLC-DAD, for the analysis of estrogens provided better linearity, lower detection limits, higher extraction efficiency, higher enrichment factors and better precision. The method was also practically applied to analyze target analytes in river water, soil and fish samples, and has great potential for the high efficiency extraction and preconcentration of trace estrogens in other environmental and biological samples. Acknowledgement This work was supported by the National Nature Science Foundation of China (21477033), Program for Science & Technology Innovation Talents in Universities of Henan Province (17HASTIT003), and Ministry of Education Key Laboratory for Analytical Science of Food Safety and Biology Open Fund (Fuzhou University). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120440. 7
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Facile preparation of reduced graphene oxide/ZnFe2O4 nanocomposite as magnetic sorbents for enrichment of estrogens Wenqi Lia, Jing Zhanga, Wenli Zhua, Peige Qina, Qian Zhoua, Minghua Lua,∗, Xuebin Zhangb, Wuduo Zhaoc, Shusheng Zhangc, Zongwei Caid,∗∗ a
Henan International Joint Laboratory of Medicinal Plants Utilization, School of Chemistry and Chemical Engineering, Henan University, Kaifeng, 475004, Henan, China Center for Multi-Omics Research, Institute of Plant Stress Biology, Henan University, Kaifeng, 475004, Henan, China c Center for Advanced Analysis and Computational Science, Zhengzhou University, Zhengzhou, 450001, Henan, China d State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong SAR, China b
ARTICLE INFO
ABSTRACT
Keywords: Magnetic solid-phase extraction Reduced graphene oxide Zinc ferrite Estrogens High performance liquid chromatography Environmental sample
Reduced graphene oxide/ZnFe2O4 (rGO/ZnFe2O4) nanocomposite was facile prepared and applied as magnetic sorbent for the extraction of estrogens including 17β-estradiol, 17α-estradiol, estrone and hexestrol from water, soil, and fish samples prior to HPLC analysis. The rGO/ZnFe2O4 nanocomposite was characterized by scanning electron microscope, Fourier transform-infrared spectroscopy, X-ray diffraction, and vibrating sample magnetometer. The experimental parameters affecting the efficiency of magnetic solid-phase extraction (MSPE) including the amount of material, extraction time, pH, temperature, desorption solvents, desorption time, and desorption solvent volume were investigated respectively. With the developed method, good linearity was observed in the range of 0.05–500 ng/mL with the correlation coefficients (R2) between 0.9978 and 0.9993. The limits of detection (S/N = 3) and limits of quantification (S/N = 10) were achieved at 0.01–0.02 ng/mL and 0.05 ng/mL, respectively. The enrichment factors were calculated as the range of 241–288. Using rGO/ZnFe2O4 nanocomposite as the sorbent, the developed MSPE followed by HPLC analysis, was applied to analysis of estrogens in river water, soil and fish samples. The method has the potential application in the extraction and preconcentration ultra trace compounds in complex matrices, such as environmental and biological samples.
1. Introduction Sample pretreatment is considered as the most crucial step in a complete analytical process for identification, confirmation and quantification of analytes, especially those in complicated samples, namely environmental, biological, industrial samples etc. [1–4]. In a traditional analytical process, sample pretreatment is time consuming and often devour about 60% of the total time, which does not meet the standard of high efficiency. Thus, considering the low content of analytes and complex interferents, the development of sample pretreatment techniques with high efficiency is urgent. Depending on the phase of extraction material, sample pretreatments can be defined as solvent-based and sorption-based extractions [5]. In the past few years, sorption-
based extraction plays a dominant role in environmental and biological sample pretreatment. For sorption-based extraction, such as solid-phase extraction (SPE) [6], dispersive solid-phase extraction (dSPE) [7], magnetic solid-phase extraction (MSPE) [8], solid-phase microextraction (SPME) [9] and so on, the key element is the sorbent material which determines the sensitivity and selectivity of the method. Therefore, the development of simple, highly efficient, and environmentfriendly sorbent for sample pretreatment is urgently needed. Graphene, a two-dimensional single-atom thick carbon nanomaterial which hexagonally arrayed with sp2-bonded carbon atoms in a closely packed honeycomb crystal lattice, has attracted considerable attention in varieties of applications since it was firstly discovered in 2004 [10]. Due to its large specific surface area, excellent mechanical
Abbreviations: Reduced graphene oxide/ZnFe2O4, rGO/ZnFe2O4; magnetic solid-phase extraction, MSPE; solid-phase extraction, SPE; dispersive solid-phase extraction, dSPE; solid-phase microextraction, SPME; 17β-estradiol, 17β-E2; 17α-estradiol, 17α-E2; estrone, E1; hexestrol, HEX; ethylene glycol, EG; polyethyleneglycol, PEG; graphene oxide, GO; scanning electron microscopy, SEM; Fourier transform-infrared spectroscopy, FT-IR; X-ray diffraction, XRD; vibrating sample magnetometer, VSM; high-performance liquid chromatography, HPLC ∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (M. Lu), [email protected] (Z. Cai). https://doi.org/10.1016/j.talanta.2019.120440 Received 11 June 2019; Received in revised form 18 September 2019; Accepted 3 October 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Wenqi Li, et al., Talanta, https://doi.org/10.1016/j.talanta.2019.120440
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and thermal stability, π delocalized electrons system, and hydrophobic property, graphene is considered as an ideal candidate for extraction and enrichment of carbon-based ring structures in sample preparation [11,12]. Graphene and graphene-based materials were mainly used as sorbents for SPE [13–15] and coating for SPME [16,17]. Compared with traditional column SPE method, dispersive extraction modes including dSPE and MSPE can provide better extraction efficiency because the enhanced contact area between sorbents and target analytes. On the other hand, dSPE and MSPE avoid the packing SPE column that has difficulty in non-professional laboratory [16]. In dSPE, it is often hard to completely retrieve graphene or graphene-based materials from the suspension owing to ultra-light property and extreme dispersity even with high-speed centrifugation [18]. To solve this problem, graphene-based magnetic sorbents are prepared that can be separated from the sample solutions by an external magnetic field. The Fe3O4 nanoparticle is the most commonly used magnetic source because of its good biocompatibility, superparamagnetic property, low toxicity and easy preparation [19]. However, the Fe3O4 nanoparticles usually showed low stability in acidic solution. Therefore, it is desirable to develop an efficient and stable material as magnetic source with a simple and rapid synthesized method. Recently, magnetic spinel ferrites [M(II)Fe(III)2O4, M = Ni, Co, Zn, Mn, etc.] with a face-centred cubic structure have attracted considerable attention because of their uniquely structural, electrical, and magnetic properties [20]. Noormohamadi et al. [21] prepared polyethyleneimine modified cobalt ferrite as magnetic sorbent for extraction of tartrazine from food and water samples. Pastor-Belda and coworkers [22] synthesized oleic acid coated cobalt ferrite nanoparticles as the sorbent of MSPE that coupled to gas chromatography-mass spectrometry for determination of alkylphenols in baby foods. However, the introduction of Co, Ni may lead to the heavy metal pollution to the environment. Due to unique physicochemical properties, good stability, nontoxicity, high specific surface area and excellent magnetism, zinc ferrite (ZnFe2O4) can be used as environmentally friendly magnetic source. Moreover, comparing with conventional Fe3O4 nanoparticle, ZnFe2O4 provide more stable property because it can't be dissolved in most acidic and alkaline medium [23–25]. Endocrine disrupting chemicals refer as exogenous substances are related to diverse health issues in intact organisms or able to alter the endocrine functions of the offspring consequently [25,26]. As a significant group of endocrine disrupting chemicals, estrogens have caught great attentions due to their existence in environmental samples. Study related to estrogens in surface waters has been reported since the early 1980s [27]. Environmental estrogens including endogenous estrogens (17β-estradiol, 17α-estradiol, estrone, estriol) and artificial estrogens (hexestrol, ethinylestradiol, diethylstilbestrol, dienestrol) are the main sources of natural and synthetic hormones which are the excrement of humans and animals. They can get into the aquatic environment through the sewage, agricultural, pharmaceutical and aquaculture waste system [28–30]. Abundant studies have demonstrated that estrogens have potentially adverse health effects to aquatic wildlife and human causing growth, development, and reproduction problems, such as feminization and hermaphroditism [31–34]. Once excessive estrogens get into the human body through food chain, they can interfere the normal functions of the endocrine systems leading to the risk of getting cancers such as prostate cancer and breast cancer [35,36]. Therefore, the development of analytical techniques to efficiently enrich and analyze estrogens at low concentrations is of great significance of science and health issues. Herein, reduced graphene oxide/ZnFe2O4 (rGO/ZnFe2O4) magnetic nanocomposite was synthesized by one-step hydrothermal method, and was used as the sorbent of MSPE for the extraction and preconcentration of estrogens (17β-estradiol (17β-E2), 17α-estradiol (17α-E2), estrone (E1), hexestrol (HEX), the chemical structure referring to Fig. S1 in supplementary material) from river water soil, and fish samples. Comparing with conventional Fe3O4 as magnetic source, ZnFe2O4
provides more stability in acidic solution. The prepared rGO/ZnFe2O4 exhibits excellent extraction efficiency for estrogens in real environmental samples, such as river water and soil samples. 2. Experimental section 2.1. Reagents and material The standard compounds including 17β-estradiol, estrone and hexestrol were purchased from Aladdin Chemistry Co., Ltd (Shanghai, China). 17α-estradiol was supplied by Marcklin Biochemical Co., Ltd (Shanghai, China). Methanol and acetonitrile with HPLC grade were provided by Tedia Company Inc. (Fairfield, USA). Acetone (HPLC grade) was obtained from Anaqua Chemicals Supply (Eldridge Parkway, Houston, USA). Graphite powder (99.95%, 8000 mesh), H2O2 (30%), FeCl3·6H2O (99%), NaOH (96%), isooctane (99.8%), ethyl acetate (99%), H3BO4 (≥99.5%), H2SO4 (98%), HCl (36%), NaNO3 (99%), ZnCl2 (98%) and KMnO4 (≥98%) were achieved from Aladdin Chemistry Co., Ltd (Shanghai, China). Ethylene glycol (EG), polyethyleneglycol (PEG), H3PO4 (≥99.5%), NaAC (99.0%) were purchased from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). Water used throughout the work was produced by a Milli-Q ultrapure water system (Millipore, Bedford, MA, USA). 2.2. Instruments, HPLC-DAD and UPLC-QTOF-MS conditions The morphology of rGO/ZnFe2O4 was characterized by scanning electron microscopy (SEM, JSM-7610F, Japan). Fourier transform infrared spectroscopy (FT-IR) was obtained by a VERTEX 70 spectrometer (Bruker, Germany). X-ray diffraction (XRD) measurement was carried out on Bruker D8 Advance (Bruker, Germany) using Cu Kα radiation. The magnetic property was analyzed using a vibrating sample magnetometer (VSM, Quantum Design, MPMS3). The chromatographic analysis was performed on an Agilent 1260 high-performance liquid chromatography (HPLC) equipped with a diode array detector (DAD). The optimum separation was obtained on a reversed phase Eclipse Plus C18 column (4.6 × 100 mm, 3.5 μm) with a binary mobile phase composed of 45% ultrapure water (solvent A) and 55% acetonitrile (solvent B). The flow rate, column temperature, injection volume and UV wavelength were set at 0.5 mL/min, 5.0 μL, 25 °C and 200 nm, respectively. The UPLC-QTOF-MS analysis was performed on a Waters ACQUITY UPLC/Synapt G2 MS system (Waters, USA) to further confirm the results of real samples that obtained by the developed HPLC-DAD method. The electrospray ionization (ESI) with full scan (m/z, 50–500) in negative ion mode was adopted. The electrospray capillary voltage was set as 3000 V, the source temperature and desolvation temperature was 150 °C and 450 °C, respectively. The cone gas flow was 50 L/h and desolvation gas flow was 450 L/h. The ion source nebulizing gas (N2) pressure was set at 6.5 bar and collision energy was 30 V. 2.3. Preparation of graphene oxide (GO) GO was synthesized from natural flake graphite by a modified Hummers' method [37]. At first, 1.0 g graphite and 0.5 g NaNO3 were dissolved in 23 mL H2SO4 under an ice bath (0–5 °C) for 30 min. 3.0 g KMnO4 was added to the above solution gradually with vigorous stirring and keep the temperature of the solution < 20 °C. The solution was stirred continuously in a water bath at 35 °C for 2 h until it became pasty brownish, then 186 mL ultrapure water was added and stirred for another 30 min, and then 2.5 mL 30 wt% H2O2 was slowly added to the mixture to reduce the residual KMnO4, after which the color of the mixture changed to brilliant yellow. The obtained mixture was centrifuged and was washed by HCl (10%) and ultrapure water until the pH was approached to 7. Finally, the obtained solid was freeze-dried in vacuum freeze dryer to achieve graphite oxide powder. 2
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Fig. 1. Characterization of as-prepared materials. SEM of GO with magnification of 3000 (a), SEM of rGO/ZnFe2O4 with magnification of 50000 (b) and 20000 (c), XRD patterns of GO and rGO/ZnFe2O4 (d), FT-IR spectra of GO and rGO/ZnFe2O4 (e), VSM magnetization curve of rGO/ZnFe2O4 (f).
2.4. Synthesis of rGO/ZnFe2O4
concentrations of estrogens in the initial solution and residual solution (mg/L), respectively. V (L) is the volume of estrogens. M (g) is the amount of adsorbent. The desorption ratio is calculated the following equation [41].
The magnetic rGO/ZnFe2O4 nanocomposite was prepared by a solvothermal method [38]. Briefly, 100 mg of GO was dispersed in 50 mL of EG with ultrasonication for 2 h, then 0.540 g (2 mmol) of FeCl3·6H2O and 0.197 g (1 mmol) of ZnCl2·4H2O were added into the GO dispersion with vigorous stirring. Subsequently, 3.60 g NaAc and 1.00 g PEG were added, followed by stirring for 1 h. The mixture was then transferred into a 100 mL Teflon-lined stainless steel autoclave and maintained at 200 °C for 8 h. During the solvothermal process, EG was used as not only solvent but also agent, meanwhile NaAc would provide an alkaline atmosphere, which could make GO reduce to rGO. The resultant black product was washed with deionized water and ethanol several times and was dried at 50 °C in a vacuum oven for further use.
Qd =
where Qd is the desorption ratio, M1 (g) is the amount of adsorbed target analytes and M2 (g) is the amount of released target analytes. 2.6. Preparation of standard solutions and real samples Stock solutions were prepared at a concentration of 100 μg/mL in methanol. The standard working solutions were stored at 4 °C and daily prepared by appropriate dilutions of the stock solutions with ultra-pure water to obtain the required concentrations prior to use. The environmental water samples were collected from Housi River (Gongyi, China). The water sample was filtered through 0.45 μm filter membrane and stored in the refrigerator at 4 °C. Prior to analysis, the pH of water samples were adjusted to 4, and 30 mL of prepared water sample was subjected to MSPE process before HPLC-DAD analysis. The soil sample was collected from the east suburb of Kaifeng. The soil sample was air dried at room temperature and sieved to a particle size of 0.45 mm before analysis. 10 g soil sample was homogenized in 10 mL of methanol, and was ultrasonicated for 30 min at room temperature. The homogenate was centrifuged at 8000 rpm for 10 min. The residue was extracted again as described above. The two supernatants were combined and dried under a N2 stream, subsequently, the dry residue was redissolved in 500 μL methanol, and 100 μL of the solution was added into 29.9 mL ultra-pure water which was adjusted pH to 4 for MSPE. The fish was purchased from local market (Kaifeng, Henan), which was freeze-dried and stored in the refrigerator at −18 °C 10 g freezedried fish sample was grounded into powder and homogenized in 10 mL of acetonitrile. The mixture was ultrasonicated for 30 min at room temperature. After ultrasonication, the homogenate was centrifuged at 8000 rpm for 15 min. The residue was extracted again as described above, and the subsequent procedures were the same as the preparation of the water and soil samples.
2.5. MSPE procedures In MSPE process, 10 mg of rGO/ZnFe2O4 magnetic composite was added into 30 mL mixed standard solution with a concentration of 200 ng/mL for each analyte. Then, the mixture was vortexed for 1 min in an oscillator and sonicated vigorously for 15 min to make the sorbents dispersed uniformly and fully adsorbed in the mixed standard solution. Subsequently, a strong magnet was placed at the bottom of the beaker to separate the sorbents from the solution. The suspension became limpid gradually and the supernatant was decanted until the supernatant became clear. Then the analytes were eluted from the rGO/ ZnFe2O4 with 3 mL of acetonitrile under ultrasonication for 5 min. After the magnetic separation, the supernatant was dried under a stream of nitrogen at 60 °C and redissolved in 100 μL methanol, afterwards filtered through disposable syringe filters (0.45 μm) into a sample vial and 5 μL of this solution was injected into the HPLC system for analysis. The performance of MSPE is evaluated by adsorption capacity and desorption ratio. The adsorption capacity is investigated by the following equitation [6,39,40].
Qa = [(Co
M2 × 100% M1
C ) × V ]/ M
where Qa (mg/g) is the amount of estrogens adsorbed onto the unit amount of the adsorbent; C0 (mg/L) and C (mg/L) are the 3
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Fig. 2. Factors affecting the adsorption capacity. (a) Adsorption temperature. Other conditions: adsorbent, 10 mg; adsorption time, 10 min; adsorption pH, 7; (b) Adsorption time. Other conditions: adsorbent, 10 mg; adsorption temperature, 30 °C; adsorption pH, 7; (c) pH. Other conditions: adsorbent, 10 mg; adsorption temperature, 30 °C; adsorption time, 15 min.
3. Results and discussion
ZnFe2O4 can be easily separated from the aqueous solution by an external magnetic field.
3.1. Characterization of sorbent
3.2. Optimization of MSPE
The morphology of GO and rGO/ZnFe2O4 composite were characterized by SEM and the results are presented in Fig. 1. The irregular flake-like GO sheets with wrinks are observed in Fig. 1a. It can be seen from Fig. 1b and c that ZnFe2O4 nanoparticles are homogeneously dispersed on the silk-like rGO surface. The size of the ZnFe2O4 nanoparticles on rGO is around 200 nm. The crystalline phase of the GO and rGO/ZnFe2O4 nanocomposites were measured by XRD and illustrated in Fig. 1d. The diffraction pattern of GO showed a strong peak at around 2θ = 10.2°, which was assigned to its (001) plane. In the diffraction patterns of rGO/ZnFe2O4 composites, it was obviously observed that the peak at 2θ = 10.2° was disappeared, which confirmed that oxygen groups have been removed and GO has been flaked and reduced to rGO nanosheets through the solvothermal method. The rGO has no apparent peaks, which may own to its amorphous state, suggesting that the crystal structures of ZnFe2O4 was not altered with the addition of rGO [42]. The other peaks at 2θ values of 29.9°, 35.3°, 42.8°, 53.1°, 56.6°, 62.2° can be indexed to (220), (311), (400), (422), (511), and (440) crystal planes of spinel ZnFe2O4 (JCPDS NO. 22–1012). Furthermore, the diffraction pattern confirmed the formation of the rGO/ZnFe2O4 composites. The FT-IR is another main tool to verify the functional groups of the synthesized rGO/ZnFe2O4. As shown in Fig. 1e, absorption bands of the GO sheets appeared at 3422 cm−1 (O–H stretching band), 1725 cm−1 (C]O stretching band), 1627 cm−1 (skeletal vibrations of aromatic domains), 1396 cm−1 (bending absorption of carboxyl group O]C–O), 1225 cm−1 (O–H bending vibrations), and 1031 cm−1 (C–O stretching vibrations), suggesting that GO was successfully formed from graphite. For rGO/ZnFe2O4 composites, the bands appeared at around 570 cm−1, 450 cm−1 can be attributed to the octahedral Fe3+ (Fe–O mode) stretching vibration and the tetrahedral Zn2+ (Zn–O mode) stretching vibration [43], the peaks corresponding to the oxygen containing functional groups become a slightly weaken, and the characteristic peak at around 1571 cm−1 could be assigned to the stretching vibrations of the conjugated carbon backbone, it was concluded that GO was successfully reduced to rGO during the process of one-pot solvothermal reaction [44]. The intensity of magnetism is another important property for magnetic materials. The magnetic property of the rGO/ZnFe2O4 was studied using a VSM method. The saturation magnetization value for rGO/ZnFe2O4 is 52.6 emu g−1. Fig. 1f shows the magnetic hysteresis loop measured at room temperature. The S-like magnetic hysteresis loop shows no apparent hysteresis, remanence and coercivity, indicating that the rGO/ZnFe2O4 is superparamagnetic and it is a desirable candidate for practical applications in sample pretreatment with great separability and recyclability. The inset of Fig. 1f shows that rGO/
The parameters affecting the extraction efficiency and recovery of analytes were optimized to achieve the best MSPE conditions for extraction of estrogens. 30 mL of the standard solution with a concentration of 200 ng/mL for each analyte was used for optimization. The adsorbents amount, pH, adsorption time, adsorption solvent as well as the desorption temperature, desorption time and eluant volume were systematically investigated as following. 3.2.1. Effect of the amount of rGO/ZnFe2O4 During the MSPE process, the sorbent amount is a major factor that affecting adsorption efficiency. The effect of rGO/ZnFe2O4 amount on adsorption capacity for estrogens was investigated between the ranges from 2 to 15 mg. The results indicated that the adsorption capacity increased rapidly with increase of the amount of rGO/ZnFe2O4 from 2 to 10 mg, demonstrated that the remarkable enrichment ability of rGO/ ZnFe2O4. However, the adsorption capacity was not significantly improved with further increasing the amount of the sorbent. Therefore, 10 mg was selected as the optimum amount of sorbent to ensure the extraction efficiency and quantitative recovery in the following experiments. 3.2.2. Effect of adsorption temperature Considering the fact that extraction efficiency can be influenced by the adsorption temperature as temperature controls the diffusion rate of the target analytes from the sample solution to adsorbent phase, adsorption temperature from 10 to 60 °C was investigated (Fig. 2a). As previously reported that the diffusion rate increase as the temperature rise. But, when the temperatures was higher than 30 °C, there was a significant decrease in adsorption efficiency, which may be due to the exothermic nature of this adsorption [45]. So, 30 °C was selected as the optimum adsorption temperature. 3.2.3. Effect of the adsorption time Adsorption time is another important parameter that influence the extraction efficiency which is directly related to the contact time between analytes and sorbents. Adsorption time ranging from 2 to 20 min was investigated and the result is shown in Fig. 2b. It can be observed that the adsorption capacity for target estrogens was close to the maximum level at 15 min. The adsorption capacity has a slight decrease with further prolonged adsorption time because the adsorption and release was a dynamic equilibrium during the ultrasonication process. Thus, the optimum adsorption time was chosen as 15 min for future experiment. 4
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3.2.4. Effect of pH Estrogens are ionizable compounds, thus, their existing forms in the aqueous solution can be affected by the pH value and the extraction efficiency. Therefore, the effects of solution pH on extraction efficiency were examined between the range of 2.0–9.0 (Fig. 2c). The highest adsorption capacity was observed at pH = 4.0, and the extraction efficiency under alkaline condition is significantly lower than that in acidic condition. The possible reason can be explained as the hydrophobic interaction between the analytes and sorbents was suppressed by the ionized form of estrogens when the pH higher than 7.0 due to the pKa values of target estrogens ranging from 9.80 to 10.34 [45]. On the other hand, the ionization of –OH in the structure of estrogen could be suppressed in acid condition. Therefore, estrogens could be protonated or ionized to form ions at lower or higher pH values, which are not beneficial for adsorption [46,47]. It can be concluded that the adsorption of estrogens with rGO/ZnFe2O4 sorbent was mainly based on hydrophobic interaction and π-π interaction, as well as the intermolecular hydrogen bond between adsorbents and estrogens. Therefore, in the following experiment, pH = 4.0 was chosen as the optimum solution pH.
3 mL. The desorption ratios for estrogens were found to be 92.91% (17β-E2), 93.24% (17α-E2), 95.18% (E1) and 98.54% (HEX), respectively. It is illustrated that 3 mL of acetonitrile is sufficient to obtain satisfactory desorption ratios. 3.3. Reusability of the sorbents The reusability of rGO/ZnFe2O4 sorbent for extraction of estrogens with MSPE was investigated under the optimal conditions and the results are shown in Fig. S2. The results showed that the rGO/ZnFe2O4 composite is still robust and durable after being reused for 15 times, which indicate that the rGO/ZnFe2O4 adsorbent has good reusability, and the extraction efficiency decreased significantly after 20 cycles, this may be due to the shedding of rGO during ultrasonic process. 3.4. Validation of the MSPE-HPLC method The analytical performance of the method including linear range, correlation coefficients (R2), limits of quantification (LOQs, S/N = 10), limits of detection (LODs, S/N = 3), and method precisions were investigated in the standard solutions under the optimized conditions. The enrichment factors were calculated from 241 to 288 according to equation S1 which is presented in supplementary materials. As listed in Table 1, a good linearity was obtained in the concentration range of 0.05–500 ng/mL for all estrogens with R2 between 0.9978 and 0.9993, The LOQs after treatment with the rGO/ZnFe2O4 adsorbent for the four estrogens were calculated to be 0.05 ng/mL. The LODs obtained were 0.01 ng/mL for 17α-estradiol and 0.02 ng/mL for 17β-estradiol, estrone and hexestrol. The repeatability was obtained by analyzing a sample five times during a working day, while the intermediate precision was determined by analyzing a sample once a day over five contiguous days. The RSDs of repeatability and intermediate precision were in the range of 1.24–2.58% and 2.75–7.22%, respectively. It was demonstrated that the developed method possessed reliable and repeatable properties. To verify the repeatability of as-prepared material as sorbent, five different batches of materials were selected for extraction of target analytes. The results demonstrated that the RSDs were not higher than 7.14%, illustrating good synthetic repeatability for preparation of the rGO/ ZnFe2O4. The spiked river water, soil and fish samples with three different concentrations (2.0, 20.0, and 200 ng/mL) were analyzed under the optimal conditions and the results are listed in Table 2. The recoveries for target estrogens were in the range of 73.50–104.13% with RSDs not higher than 8.33%. The results demonstrated that rGO/ZnFe2O4 sorbent had good recovery and intermediate precision, which could used as sorbent for extraction trace estrogens from environmental and biological samples.
3.2.5. Effect of desorption solvents The desorption solvent is another critical parameter during the desorption process. To completely elute the analytes from sorbent and to improve the desorption efficiency, the affinity of desorption solvent toward the target analytes should be higher than that of the sorbent. Different organic solvents including methanol, acetonitrile, acetone, isooctane, ethyl acetate were investigated and the result was shown in Fig. 3a. It can be seen that acetonitrile exhibited the best desorption ratios under the same extraction and desorption conditions. Under this conditions, the desorption ratios for estrogens were found to be 52.50% (17β-E2), 68.15% (17α-E2), 58.08% (E1) and 74.30% (HEX), respectively. Hence, acetonitrile was selected as the desorption solvent. 3.2.6. Effect of desorption time To complete desorption of the analyte from sorbents, the desorption time as an important parameter effect on desorption efficiency was investigated from 1 to 12 min. As shown in Fig. 3b, the desorption process basically reached desorption equilibrium after 5 min. The desorption ratios for estrogens were found to be 52.67% (17β-E2), 69.86% (17α-E2), 59.72% (E1) and 75.02% (HEX), respectively. Therefore, 5 min was selected as desorption time. 3.2.7. Effect of desorption volume To achieve the best desorption ratios for estrogens, an optimum volume of desorption solvent was optimized ranging from 1 to 7 mL. According to Fig. 3c, the desorption ratios were rapid increased upon increasing the volume of acetonitrile from 1 to 3 mL, and there was no obvious enhancement when the volume of acetonitrile was higher than
Fig. 3. Factors affecting the desorption efficiency. (a) Desorption solvents. Other conditions: desorption time, 10 min; desorption solvent volume, 1 mL; (b) Desorption time. Other conditions: desorption solvent, acetonitrile; desorption solvent volume, 1 mL; (c) Desorption solvent volume. Other conditions: desorption solvent, acetonitrile; desorption time, 5 min. 5
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Table 1 The Linear range, correlation coefficients, EFs, LOQs and LODs of the developed MSPE-HPLC method for the analysis of estrogens. Analytes
17β-estradiol 17α-estradiol estrone hexestrol
Linear range (ng/mL)
0.05–500 0.05–500 0.05–500 0.05–500
R2
0.9981 0.9978 0.9980 0.9993
LOQs (ng/mL)
LODs (ng/mL)
0.05 0.05 0.05 0.05
0.02 0.01 0.02 0.02
EFs
241 268 261 288
RSD (%, n = 5) Repeatability
Intermediate
Batch to batch
1.24 2.58 2.50 1.78
4.20 7.22 2.96 2.75
7.14 5.19 5.98 4.72
3.5. Real sample analysis The river water, soil and fish were selected as testing samples to verify the proposed method and the results were listed in Table 2. Among target analytes, 17β-estradiol and hexestrol were detected in river water sample and the content were calculated as 1.92 and 1.30 ng/mL. In the soil sample, the hexestrol was detected with a concentration of 15.9 ng/g. The target analyte was not detected in the fish sample. The chromatograms of water sample are shown in Fig. 4. It can be seen from graph a in Fig. 4 that the target estrogens were not detected in the river water sample by HPLC-DAD. With rGO/ZnFe2O4 as sorbent for extraction and preconcentration of target analytes, two target analytes (17β-estradiol and hexestrol) were detected. Graph C in Fig. 4 demonstrated the analysis of the spiked river water sample (20 ng/mL) by developed MSPE with rGO/ZnFe2O4 as sorbent. The typical chromatograms obtained by the analysis of soil sample and fish sample are presented as Figs. S3 and S4 in the supplementary material. To validate the results of real samples obtained with HPLC method, the extracted solution of water and soil samples with rGO/ZnFe2O4 nanocomposite as sorbent was further analyzed by UPLC-MS, respectively. As shown in Fig. S5, the molecular ion peaks of [M − H]- with m/z at 271.1688 and 269.1532 were observed in water sample, which could be identified as 17β-E2, and HEX. The molecular ion peak of [M − H]- with m/z at 269.1532 was observed in soil sample, which corresponding to HEX. The results obtained with UPLC-MS were same as that of HPLC-DAD.
Fig. 4. The typical chromatograms obtained by direct analysis of river water sample (a), river water sample pretreated with rGO/ZnFe2O4 as MSPE sorbent (b), and spiked river water sample (20 ng/mL) pretreated with developed MSPE procedures (c). Peaks identification: (1) 17β-E2, (2) 17α-E2, (3) E1, (4) HEX.
and Xu et al. [50] both reported magnetic sorbents for extraction of estrogens with LODs in the range of 4.3–7.5 ng/mL and 3.2–20.1 ng/L, respectively. The sorbent amounts were 500 and 200 mg in above two works [49,50]. Zhao and co-workers [51] prepared a molecularly imprinted polymer used to on-line extract four estrogens in milk sample with the LODs of 1–8 ng/g. Wang et al. [52] analyzed the estrogens in water sample using in-tube SPME coupled with HPLC and the LODs were found in the range of 0.05–0.3 ng/mL. Comparing with other reported method, the method developed in current study for analysis of estrogen in water, soil and fish samples displayed a range of advantages including better selectivity, relatively lower LODs, higher enrichment factors and fewer sorbent amount.
3.6. Comparison with other methods The developed MSPE coupled with HPLC-DAD method was compared with other reported methods (Table 3), and the calibrators and samples are subject to the same preparation. Jiang and co-workers [48] reported a method for the determination of estrogens in water samples with LODs ranging from 1.7 to 3.4 ng/mL using ionic liquid as the adsorbents of dispersive liquid-phase microextraction. Tian et al. [49] Table 2 Results for analysis of real samples and the recoveries of method. Samples
River water
Soil
Fish
a b
Analytes
17β-estradiol 17α-estradiol estrone hexestrol 17β-estradiol 17α-estradiol estrone hexestrol 17β-estradiol 17α-estradiol Estrone hexestrol
Found (ng/mL or ng/g)a
1.92 N.d.b N.d. 1.30 N.d. N.d. N.d. 15.9 N.d N.d. N.d N.d
Added 2 ng/mL
Added 20 ng/mL
Added 200 ng/mL
Recoveries (%)
RSD (%) (n = 3)
Recoveries (%)
RSD (%) (n = 3)
Recoveries (%)
RSD (%) (n = 3)
80.24 92.07 92.95 73.69 83.26 81.54 79.94 78.56 74.70 75.97 74.10 73.50
3.81 7.68 6.20 7.66 5.92 3.85 8.33 7.47 4.21 5.93 1.94 1.87
91.57 96.17 96.06 87.17 87.63 86.73 89.57 80.17 82.53 78.43 82.60 74.57
3.19 5.60 4.41 2.29 5.96 3.08 3.88 2.68 6.25 5.80 3.87 4.37
85.32 88.40 91.17 91.55 104.13 90.32 89.21 89.50 89.29 92.63 100.10 95.47
5.65 5.20 2.15 1.68 2.74 3.59 3.54 4.68 4.35 0.82 1.96 3.11
The units are ng/mL for liquid sample and ng/g for solid sample. Not detected. 6
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Table 3 Comparison with other methods for the determination of estrogens. Sorbents IL Fe3O4@TiO2/GO MC MIP Nano-CaCO3 rGO/ZnFe2O4
Samples Water Milk Water Milk Water Water, Soil
Extraction method DLPME MSPE MSPE On-line SPE IT-SPME MSPE
LODs
Linearity
1.7–3.4 ng/mL 4.3–7.5 ng/mL 3.2–20.1 ng/L 1.0–8.0 ng/g 0.05–0.3 ng/mL 0.01–0.02 ng/mL
3
5.0–1.0 × 10 ng/mL 5.0–5.0 × 103 ng/mL 0.1–100 mg/L 3–2500 ng/g 0.15–20 ng/mL 0.05–500 ng/mL
Sorbent amount
Refs.
60 μL 500 mg 200 mg 50 mg 166 mg 10 mg
[48] [49] [50] [51] [52] This work
IL: ionic liquid. MC: magnetic chitosan. MIP: molecularly imprinted polymer. DLPME: dispersive liquid-phase microextraction. IT-SPME: in-tube solid-phase microextraction.
3.7. Potential interferences and the selectivity of the method
References
The potential interferences of environmental samples include benzene compounds, phenolic compounds, nitrobenzene, chlorinated alkanes, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, as well as a variety of organic pesticides and so on. The adsorption mechanism of rGO/ZnFe2O4 mainly contained π-π interaction and hydrogen bonds interaction. Therefore, the rGO/ZnFe2O4 can absorb compounds that containing benzene rings with π-π interaction between rGO and benzene rings. Meanwhile, hydrogen bond interaction also play an important role for the adsorption because the hydroxyl and carboxyl groups on rGO surface tend to form hydrogen bonds with hydroxyl and carboxyl groups in the target analytes. However, the adsorption of the as-prepared material for the target analytes was not the selective adsorption. Therefore, the selectivity of the material was hoped to be improved with other techniques, such as molecular imprinting.
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4. Conclusion In this work, rGO/ZnFe2O4 magnetic composites were prepared by a simple hydrothermal method. The rGO/ZnFe2O4 was used as sorbents for MSPE with many advantages comparing with previously reported methods including lower cost and better precision. In addition, the preparation procedures are simple and environment friendly. Compared with conventional Fe3O4 nanoparticle, ZnFe2O4 is more stable because it doesn't dissolve in most acidic and alkaline medium. The developed MSPE with rGO/ZnFe2O4 as sorbents, coupled with HPLC-DAD, for the analysis of estrogens provided better linearity, lower detection limits, higher extraction efficiency, higher enrichment factors and better precision. The method was also practically applied to analyze target analytes in river water, soil and fish samples, and has great potential for the high efficiency extraction and preconcentration of trace estrogens in other environmental and biological samples. Acknowledgement This work was supported by the National Nature Science Foundation of China (21477033), Program for Science & Technology Innovation Talents in Universities of Henan Province (17HASTIT003), and Ministry of Education Key Laboratory for Analytical Science of Food Safety and Biology Open Fund (Fuzhou University). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120440. 7
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