Antibiotic residues are among new emerging pollutants to be monitored. The main reason has been the fact that their release into the environment, including soil [1], ground [2], surface [3], drinking water [4], and wastewater [5], presenting a great risk to human health. Fluoroquinolones (FQs) as a group of pharmaceutical antibiotics have been intensively used in human clinical, veterinary medicine and particularly in food-producing animal husbandry [6]. The great chemical stability of the heterocyclic ring makes FQs highly persistent contaminants and the relatively high solubility increases their environmental diffusion.
The extraction and removal of FQs from various sample matrices have been carried out largely by supported liquid membrane extraction (SLME) [7], microwave-assisted extraction (MAE) [8, 9], polymer monolith microextraction (PMME) [10], and solid phase extraction (SPE) with different sorbents [11, 12], in conjunction with chromatographic and other analytical methods. SPE is the most widely used preconcentration method due to its convenience, good recovery and low cost. Among the numerous sorbents for SPE, molecularly imprinted polymers (MIPs) are distinctive for their higher sample load capacity, physical robustness, as well as low cost and ease of preparation. Tan et al. [13] successfully synthesized a novel double-layer molecularly imprinted film (MIF)-based biosensor for rapid, sensitive and highly selective detection of the small molecule 17 β -estradiol (E2) that is frequently detected in environmental water samples. Du et al. [14] prepared a restricted access material combine to molecularly imprinted polymers (RAM-MIPs) as sorbent material in SPE for clean-up of 2-methoxyestradiol (2-ME) from plasma samples. These selective sorbents have been widely applied to remove target compounds from mixtures of structural analogs [15-17]. However, the preparation of MIPs with traditional methods presents some problems such as complex chemical bonding and multiple co-polymerization strategy [18]. Using a time saving and solvent free preparation is a possible alternative to overcome the above troubles. The generation of magnetic MIPs (MMIPs) has received considerable attention in sample pretreatment owing to its several advantages over traditional MIPs. In MMIPs, the MIPs have been coated on Fe3O4 or Fe2O3 so that the core-shell magnetic materials simplify the overall procedure. This is because the separation process can be performed quickly by an external magnetic field, and the MIPs gain high selectivity for the target molecules. The core-shell structure exhibits specific selective binding for the template molecules so that the MMIPs give easy accessibility and low mass transfer resistance to template molecules and analogs [19]. Studies about the preparation of MMIPs have been reported [20-22]. However, there have been only a few reports on the application of MMIPs in the analysis of FQs in real samples [23, 24].
Herein we have synthesized a new MMIPs, with acrylamide (AA) to be the functional monomer and N, N′-methylene bisacrylamide (MBAAm) acted as the cross linker, involving the use of norfloxacin (NOR) as template molecule. The obtained MMIPs have been applied to SPE to select FQs in environmental water samples coupled with HPLC. The characterization, adsorption capacity and parameters affecting the extraction efficiency have been thoroughly investigated. The target FQs recognition in real samples has been achieved, with high selectivity and good recovery, by the employment of MMIPs as sorbents.
Formic acid (FA), methacrylic acid (MAA), glycidyl methacrylate (GMA), ethylene dimethacrylate (EDMA), NOR, ciprofloxacin (CIP), enoxacin (ENO), and lomefloxacin (LOM) were purchased from Aladdin Reagent (Shanghai, China). 3-Methacryloxypropyl trimethoxysilane (MPS), AA, and MBAAm were obtained from Sigma-Aldrich (USA). Ferric chloride hexahydrate (FeCl3\56H2O), ethylene glycol (EG), polyethylene glycol 2000 (PEG-2000), sodium acetate (NaAc), isopropanol, and tetraethyl-orthosilicate (TEOS) were purchased from Sinopharm Chemical Reagent (Shanghai, China). Azobisisobutyronitrile (AIBN) was supplied by Tianjin Chemical Plant (Tianjin, China). AIBN was purified by recrystallization from n-hexane and ethanol (EtOH) (Beijing Chemical Works, Beijing, China), followed by being dried under vacuum at room temperature.
HPLC-grade methanol (MeOH) and acetonitrile (ACN) were supplied by Fisher Scientific (USA). Sodium dihydrogen phosphate (NaH2PO4) and sodium hydroxide (NaOH) were obtained from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). All other reagents were obtained from various commercial sources and were of analytical or HPLC grade. Stock solutions of FQs (1 000 mg/L) were prepared in MeOH. The standard working solutions were stored at 4 ℃ and daily prepared by appropriate dilution from the stock solutions to obtain the required concentrations prior to use. Phosphate buffer was prepared from NaH2PO4, and the pH was adjusted using NaOH or H3PO4. The NdFeB magnets were purchased from Yingke (Beijing, China) with cuboid shape of 15 mm×10 mm×3 mm with surface magnetic field of 3 000 G.
The HPLC system was equipped with Agilent 1200 HPLC system (Agilent, USA), which consisted of a quaternary pump (Quat pump-G1311A), an automatic sample injector (ALSG1329A), a column oven (TCC-G1316A), a degasser system (Degasser-G1322A), a detector (DAD-G1315D), and a Chem-Station software for the acquisition and analysis of the chromatographic data. A reversed phase Agilent Zorbax Eclipse XDB-C18 column (150 mm×4.6 mm, 5 μ m) was employed for the chromatographic separations. A gradient program was used with the mobile phases, combining double deionized water (DDW) containing 0.1% (v/v) FA (mobile phase A) and 100% (v/v) ACN (mobile phase B) as follows: 28% B at first, 28% -90% B within 10 min, and then 90% -28% B within 2 min until the next injection. The flow rate was maintained at 0.8 mL/min. The preferential detection wavelength was 280 nm and the column temperature was set at 30 ℃.
A pHS-3C digital pH meter (Shanghai Rex Instrument Factory, Shanghai, China) was used for pH measurement. A Millipore Milli-Q water purification system (Millipore, Bedford, MA, USA) was used to purify DDW, and the DDW produced at 18.2 M Ω \5cm was prepared for eluents and sample solutions. Before use, mobile phases were filtered through a 0.22 μ m filter, and then degassed for 15 min with a Model DOA-P504-BN pump (IDEX, USA). An LD5-2A centrifuge (Beijing Jingli centrifuge Co., Ltd., China) was used for centrifuging. A 79-1 magnetic stirrer (Changzhou Guohua Instrument Co., Ltd., China) was applied. A ZK-82BB electric vacuum drying oven (Shanghai Experimental Instrument Co., Ltd., China) was utilized.
Magnetic properties of the materials were characterized by an MPMS3 vibrating sample magnetometer (VSM, Quantom, USA). Scanning electron microscope (SEM, JSM 6700-F) and transmission electron microscope (TEM, JSM 2000-F, JEOL Company, Japan) were used to characterize the surface morphology. The Fourier transform infrared spectrum (FT-IR) was obtained using a Thermo Nicolet 670 FT-IR instrument (Thermo, USA). Phase identification was conducted by an X-ray diffractometer (XRD, R-AXIS RAPID-F, Rigaku Corporation, Japan). A Q500 thermal gravimetric analysis (TGA) system (TA Company, USA) was employed to investigate the thermal behavior of the sorbents.
The procedure to prepare Fe3O4/SiO2/poly (AA-MBAAm)-NOR MMIPs composites is depicted in Fig. 1. The preparation of Fe3O4/SiO2 composites was performed according to our previous work with some modifications [25, 26]. Firstly, Fe3O4 was gained by the well-known solvothermal method [27]. FeCl3\56H2O (1.5 g) was dissolved in 40 mL EG to form an orange solution, followed by the addition of 3.6 g NaAc and 1.0 g PEG-2000. The mixture was stirred vigorously for 30 min, and then sealed in a 100 mL teflon-lined stainless steel autoclave. The autoclave was maintained at 200 ℃ for 8 h before cooled to room temperature. The obtained Fe3O4 powder was washed with EtOH for three times and dried under vacuum at 60 ℃ for 6 h. Then, the Fe3O4/SiO2 microspheres were prepared using a sol-gel method. Fe3O4 powder (0.1 g) was treated with 2 mol/L HCl and re-dispersed in a mixture of EtOH, DDW, and aqueous ammonia (NH3\5H2O). Subsequently, 0.05 mL TEOS was quickly added to the dispersion with vigorous stirring and the reaction was allowed to proceed for 12 h. Finally, EtOH was added to disperse the obtained Fe3O4/SiO2 microspheres.
MPS was introduced to graft double bonds onto the surface of Fe3O4/SiO2 to form polymerizable sites with AA and MBAAm in the next reaction process [20, 26]. Briefly, Fe3O4/SiO2 nanoparticles (1.0 g) were ultrasonically dispersed in 50 mL DDW for 10 min. After that, 5 mL MPS dispersed in 50 mL EtOH was added to the above solution and the mixtures were stirred at 70 ℃ for 24 h. The MPS-modified Fe3O4/SiO2 was dried at 80 ℃ (6 h) for subsequent use after washing by EtOH and DDW.
MMIPs were prepared by surface-imprinted polymerization method with NOR as template, AA as monomer, and MBAAm as cross linker. ACN as the reaction solvent and the porogen agent because of its excellent porogen ability [28]. AIBN was chosen to be the initiator and the MPS-modified Fe3O4/SiO2 nanoparticles were used as the core to support the surface imprinting. NOR (20 mg) was dissolved in 20 mL ACN in a three-necked flask, then 20 mg AA was added into the solution and kept stirring for 5 h to form a pre-polymer [29], and 100 mg MPS modified Fe3O4/SiO2 nanoparticles were dispersed in the above solution subsequently. Then 0.5 g MBAAm and 20 mg AIBN were added for polymerization into the solution and degassed in an ultrasonic bath for 15 min to remove oxygen. The polymerization was performed at 60 ℃ with nitrogen protection for 24 h. The MMIPs were collected by using NdFeB magnets, and washed by a mixture of MeOH/acetic acid (9 : 1, v/v) to remove the templates and then washed by MeOH for many times until no NOR was detected by HPLC. Finally, the particles were dried to constant weight under vacuum at 40 ℃. At the same time, the corresponding magnetic non-imprinted polymers (MNIPs) were prepared by the same method as MMIPs with the absence of template.
Environmental water samples were mixed in ultrasonic bath for 10 min. The samples were centrifuged at 5 000 r/min for 10 min. The supernatant was then extracted by MMIPs particles. Finally, the eluent was filtered through 0.22 μ m filter for further use. All sample solutions were spiked with NOR, CIP, ENO, and LOM standard solutions at different levels to assess the matrix effects.
In the synthesis process, the types of monomer and cross linker were optimized to achieve the best adsorption efficiency of the MMIPs [30]. According to the results of molecular modelling and adsorption, three groups of monomer and cross linker including AA-MBAAm (MMIP 1), MAA-EDMA (MMIP 2), and GMA-EDMA (MMIP 3) were selected for MMIPs preparation, and NOR was used as the template molecules for all the MMIPs. The adsorption amount, Qe (mg/g), was calculated by Eq. (1),
where C0 and C1 represent the initial and equilibrium FQs mass concentrations (mg/L), respectively; V is the volume of the FQs solution (L); and m is the amount of MMIPs and MNIPs (g).
The recognition ability of MMIPs for FQs was also evaluated by imprinting factor (IF) which is defined as follows:
where QMMIPs and QMNIPs are the adsorption capacities of MMIPs and MNIPs analyses for FQs, respectively.
The Qe and IF of MMIP 1, MMIP 2, MMIP 3 for FQs are calculated and shown in Table 1. It was found that the adsorption capacity of MMIP 1 was much higher than those of the other polymers (MMIP 2 and MMIP 3). These results showed that MMIPs based on AA as functional monomer and MBAAm as cross linker have the highest value of IF to adsorb the FQs (CIP, LOM, ENO, NOR). Because of the MAA and GMA belonging to acidic functional monomers, MMIP 2, and MMIP 3 have extraction efficiency through hydrogen bonding interaction between the analytes and MAA/GMA. Because AA is a neutral function monomer, and there are a large number of acylamino groups on the surface [31], MMIP 1 has a large advantage for the extraction and enrichment of FQs due to the condensation reaction between the amino groups of AA and the carboxylic groups of FQs. The stability of this effect is stronger than that of the hydrogen bonding. In addition, N atoms can form coordination bonds with other electron-deficient groups to show the special activity. Therefore, AA-MBAAm MMIPs was selected for further tests.
The MMIPs were characterized by a series of tests. Fig. 2 shows SEM and TEM images of the obtained MMIPs. The results indicated that almost all of these obtained MMIPs were regular spheres. From the TEM image, the core-shell structure of the magnetic core and surface molecularly imprinted shell could be clearly observed. The gray layers of SiO2 and MIPs were coated on the dark core of magnetite Fe3O4 nanoparticles, and the final thin coating layer had a thickness of about 20 nm (Fig. 2b), which demonstrated that the monomer, cross linker, and template molecule had some influence on the spherical particles' growth during the synthesis procedure.
FT-IR was performed for Fe3O4, NOR, and MMIPs to further ensure the preparation of MMIPs. Compared with the Fe3O4 nanoparticles, the absorption band of Fe-O around 580 cm-1 found in MMIPs proved that Fe3O4 was embedded in these materials (Fig. 3a). The observed feature of MMIPs around 1 120 cm-1 (Si-O asymmetric stretching vibration), and 810 cm-1 (Si-O symmetric stretching vibration) displayed that SiO2 was successfully encapsulated onto the surface of Fe3O4 microspheres. The peaks at 1 710 cm-1 and 1 400 cm-1 in the spectra of NOR and MMIPs represented the stretching vibration peaks of C=O and C-O from the carboxyl groups of NOR. The peaks at 1 580 cm-1, 1 400 cm-1 and 680 cm-1 (MMIPs) were assigned to the shear-type vibration of -NH2, stretching vibration of C-H, and rocking vibration of methylene, respectively, proving that the amino groups of AA were induced. The above characteristic peaks in the FT-IR spectra indicated that the MMIPs were successfully modified on the surfaces of Fe3O4.
The TGA curve (Fig. 3b) was obtained to investigate the thermal stability of the MMIPs, and the experiment was performed at 85-900 ℃ at a ramp rate of 10 ℃/min. From 200 ℃ to 300 ℃, the weight loss may be attributed to the volatilization of adsorbed water or related small molecular organic compounds on the material [32]. The maximum weight losses occurred at approximately 650 ℃ to 800 ℃, possibly due to the decomposition of the MIPs. The TGA curve tended to be constant when the temperature was higher than 800 ℃, indicating complete decomposition of the MIPs. These results indicated that these MMIPs polymers possess adequate thermal stability for analytical applications below 200 ℃.
The crystal phases and purity of Fe3O4 nanoparticles and MMIPs were determined by XRD analysis (Fig. 3c). The 2 θ diffraction peaks of MMIPs appeared at 30.2°, 35.5°, 43.2°, 54.0°, 57.1°, and 63.6° with the XRD peak position and intensity of the nanocrystallite matching well with standard Fe3O4 and without other crystal phases detected, according to the database of magnetite in JCPDS-International Center (JCPDS card: 19-629). This result indicated that the MMIPs were composed of Fe3O4, and it further proved that the synthesis process did not change the crystal phase of Fe3O4.
It is important for magnetic materials to possess sufficient magnetic properties for their practical applications in liquid medium. The magnetic properties of Fe3O4 and MMIPs were characterized by a VSM at room temperature in this work. As shown in Fig. 3d, no hysteresis is found, and the remanence and coercivity are negligible, suggesting that these nanoparticles were superparamagnetic. The saturation magnetizations were 44.48 and 14.35 emu/g for Fe3O4 nanoparticles and MMIPs, respectively. The saturation magnetization of MMIPs decreased in comparison with Fe3O4, which could be assigned to the shielding effect of the silica coating and the nonmagnetic MIPs shell layer on the surface of Fe3O4, but remained strong magnetism and allowed for magnetic separation. As shown in illustrations of Fig. 3d, after being exposed to an external magnetic field, all the MMIPs composites could be rapidly separated from the solution.
To measure the specificity of MMIPs, the recognition of NOR was compared to those of CIP, ENO, and LOM. The structures of the test compounds are described in Fig. 4 (inset). The adsorption capacities of MMIPs and MNIPs for the four FQs were compared with those obtained by direct HPLC analysis. It is obvious that the adsorption capacity of MNIPs is very close to direct HPLC analysis for the four compounds since there are not selective recognition sites in MNIPs and the adsorption for those compounds are all non-selective. The amount of NOR adsorbed on the MMIPs was the highest of the four selected FQs. This result indicated that the MMIPs selectively adsorb the template, confirming functional imprinted cavities were formed on the MMIPs. The adsorption capacities of the MMIPs for CIP and LOM is slightly lower than that for NOR. It may be explained by their close structural homology. MMIPs did not show the satisfied adsorption capacity for ENO, the possible reason is that the naphthyridine ring of ENO has electron receptor effect. It is difficult to enter the sites left by NOR, and it can reduce the hydrogen bonding between the polymeric matrix and the analytes. The result indicated that the recognition property of MMIPs was not only based on the hydrogen bonding interaction between template and functional monomer, but also on the complementary match of "cavity" (site) with template in size and shape [33].
To obtain the best extraction efficiency of MMIPs in the detection of the four FQs, several experimental parameters were optimized, including desorption solvent, adsorbent amount, sample pH, extraction and desorption times. The peak areas of the target compounds as the HPLC response were used to evaluate the extraction efficiency under various experimental conditions.
The procedure of desorption was optimized to achieve accurate quantification of the analytes. The comparison study among EtOH, MeOH, ACN, 0.1% FA in MeOH/water (60 : 40, v/v), and 0.1% FA in ACN/water (60 : 40, v/v) was conducted (Fig. 5). Results demonstrated that 0.1% FA in MeOH/water (60 : 40, v/v) gives the highest desorption efficiency.
The amount of MMIPs was investigated from 5 mg to 50 mg to enrich trace level of FQs from 50 mL environmental water samples. As shown in Fig. 6a, the peak areas of these FQs increase when the MMIPs amount increased from 5 mg to 20 mg, then keep nearly constant with further increase in the range of 20-50 mg, indicating the remarkable enrichment ability of adsorbents to FQs. In the following experiment, 20 mg of MMIPs sorbent was employed.
The extraction efficiency was investigated by increasing the extraction times from 5 to 50 min. As shown in Fig. 6b, the adsorption equilibrium was reached after approximately 20 min. Considering that the longer extraction time did not improve extraction efficiency, 20 min was adopted as the extraction time for subsequent analysis.
Desorption time was also optimized at the same time. In brief, different time intervals (5, 10, 15, 20, 25, 30, and 40 min) were evaluated, respectively. Fig. 6c illustrates that the desorption amounts of the four FQs do not increase along with the time prolonged after 15 min. Thus, 15 min was set as the optimal desorption time in this work.
Sample pH not only influenced the properties of the nanoparticle surface, but also affected the ionization forms of analyte in solution. Therefore, it is an important parameter affecting the adsorption efficiency. Fig. 6d displays the peak areas of FQs onto the MMIPs at different pH values. It could be seen that the highest recovery of ENO is obtained at pH 6.0, and other FQs have the highest extraction efficiencies at pH 7.0. It was reported that FQs are zwitterionic compounds with two pKa values, pKa1 of 5.2-6.3 and pKa2 of 7.4-8.9 [10, 12]. They exist as cation, zwitterions, and anion types. The fraction of FQs anion increased with increasing pH values from 2.0 to 7.0, which might enhance the surface complexation by bidentate complexation of a carboxylate anion to imprinting sites for NOR supported by hydrogen bonding of a neighboring carbonyl group [34]. When pH is higher than 7.0, hydrogen bonding interaction becomes weaker with decreasing carboxylic groups in FQs because of the hydrolytic action. On the other hand, since NOR and AA existed in anionic forms, this led to rapid decrease of the adsorbed amount due to the decrease of the molecular recognition and the electrostatic repulsive interaction between NOR and AA. Overall, the recoveries of all analytes were relatively high at pH 7.0. Thus, pH 7.0 was selected in the following studies.
Effect of ionic strength on the extraction efficiency was examined by adjusting the salinity of solution with NaCl in the range of 0-80 mmol/L. There was no obvious change in the peak areas of the adsorbed FQs (data not shown), which meant that the effect of salting out between the analytes and the adsorbent material was negligible in this work. Based on such an observation, NaCl was not added in the following procedure.
As described above, the optimal extraction parameters were as follows: adsorbent amount, 20 mg; extraction time, 20 min; desorption time, 15 min; sample pH, 7.0; eluent, 0.1% FA in MeOH/water (60 : 40, v/v).
Under the above optimized conditions, the MMIPs-SPE-HPLC method was validated with respect to linear range, correlation coefficient (R2), LODs, and LOQs. The reproducibility of the method was evaluated by the intra-day and inter-day precisions. As shown in Table 2, R2 are not less than 0.990 4. The LODs and LOQs, based on the mass concentration giving the signal-to-noise ratios of 3 and 10, were found in the range of 4.1-21.3 μ g/L and 13.7-71.0 μ g/L, respectively, indicating that the precision of the method was high enough to satisfy the requirement of analysis.
To illustrate the advantages of the MMIPs as a novel extraction material, the comparative study of the present method with other reported sample preparation procedures [10, 12, 35-39] was performed (Table 3). The LODs of this method were close to those reported in other literatures. However, the present method was sensitive, using small sample volume and little consumption of organic reagents, and the separation process could be performed rapidly by an external magnetic field.
In order to investigate the reusability of the adsorbent, 20 mg of MMIPs composites were repeatedly used 12 times in SPE of NOR. The MMIP adsorbents were recycled by washing with 10 mL MeOH and 10 mL DDW in turn, and dried under vacuum at 60 ℃ each time before reusing [40]. Results shown in Fig. 7 indicate that the recoveries of the FQs decreased only slightly when the adsorbent was reused more than 8 times, implying that the present method had acceptable reusabilities.
The present study aimed to provide selective and practical MMIPs as adsorption materials, which could avoid the residual template leakage and apply in the determination of analytes from complicated samples. For this purpose, the MMIPs adsorbent was used for the determination of NOR, CIP, ENO, and LOM in environmental water under the above optimized experimental conditions. All the real samples were spiked with the four FQs standard solutions at different concentration levels to evaluate the matrix effects, level 1 (0.5 mg/L) and level 2 (1.0 mg/L), respectively. As representatives, the chromatograms of River water-1 are exhibited in Fig. 8. The recoveries are calculated and listed in Fig. 9.
The recoveries are obtained in the range of 70.6%-103.6%, implying that the present method is effective for the determination of CIP, LOM, ENO, and NOR in real samples. These results demonstrate that the prepared MMIPs are selective sorbents for the extraction of FQs in environmental water, which could supply some guidance for the determination and separation of antibiotic compounds with low concentration in real samples.
A novel Fe3O4/SiO2/poly (AA-MBAAm)-NOR MMIPs composites were prepared and used as a medium for the SPE of the FQs. All experimental results demonstrated that the MMIPs-SPE-HPLC method had good precision, reproducibility, satisfactory recovery, repeatability, and extraction efficiency. These excellent properties made MMIPs become one of the most promising candidates in various applications, especially in the analysis of the FQs in environmental water samples based on SPE.
2017,
Vol. 35 Issue (3): 291-301
PDF
2017,
Vol. 35 Issue (3): 291-301
PDF