134 Dan Liu1 Jiutong Ma1 Yan Jin2 Xiqian Li3 Xiao Zhou4 Qiong Jia1 Weihong Zhou1 ∗ 1 College
of Chemistry, Jilin University, Changchun, China 2 Shenyang Entry & Exit Inspection and Quarantine Bureau of China, Shenyang, China 3 Obstetrics & Gynecology, China-Japan Union Hospital, Jilin University, Changchun, China 4 Jilin Entry & Exit Inspection and Quarantine Bureau of China, Changchun, China Received August 18, 2014 Revised October 16, 2014 Accepted October 16, 2014
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Research Article
Preparation of a monolith functionalized with zinc oxide nanoparticles and its application in the enrichment of fluoroquinolone antibiotics This study describes the enrichment ability of ZnO-modified methacrylic acid-co-ethylene dimethacrylate polymer monoliths as stationary phases for the simultaneous determination of antibiotics (ofloxacin, ciprofloxacin, enoxacin, and pefloxacin) combined with highperformance liquid chromatography. The prepared monolith was characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, Fourier-transformed infrared spectroscopy, and thermogravimetric analysis. The polymer monolith microextraction method has been applied to the enrichment of fluoroquinolone antibiotics and satisfactory results were obtained in the analysis of water samples. Compared with the conventional methacrylic acid based monolith, the developed monolith exhibited a higher enrichment capacity because of the introduction of zinc oxide into the preparation process. Keywords: Fluoroquinolone / High-performance liquid chromatography / Monoliths / Zinc oxide DOI 10.1002/jssc.201400893
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction In recent years, much research attention has been focused on the application of nanoparticles to the modification of chemical materials. The potential of nanoparticles such as carbon nanomaterials [1, 2], silica [3, 4], magnetic and nonmagnetic metal oxides [5–7], silver and gold nanoparticles [8–11] have been widely recognized. Modified nanomaterials have some important applications in separation and enrichment owing to the novel physical and chemical properties of nanoparticles associated with their size and shape, e.g., in the field of polymer monolith microextraction (PMME). Some researchers have devoted themselves to controlling the surface chemistry of monoliths by adding or grafting nanoparticles into the monolith supports by copolymerization with monomers or attachment onto the pore surface of monolithic matrix. In 2005,
Correspondence: Professor Qiong Jia, Renmin Street 5988#, Changchun, China E-mail: [email protected]
Abbreviations: ACN, acetonitrile; AIBN, azobisisobutyronitrile; CIP, ciprofloxacin; EDMA, ethylene dimethacrylate; ENO, enoxacin; FQ, fluoroquinolone antibiotic; ␥-MAPS, ␥-methacryloxypropyltrimethoxysilane; MeOH, methanol; MMA, methacrylic acid; OFL, ofloxacin; PEF, pefloxacin; PMME, polymer monolith microextraction; ZnO NP, zinc oxide nanoparticle C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Li et al. first incorporated single-walled carbon nanotubes into a polymerization mixture to prepare a poly(vinylbenzyl chloride-ethylene dimethacrylate-single-walled carbon nanotubes) monolith, which was successfully used for HPLC and CEC [2]. Porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith modified with gold nanoparticles was prepared from the reaction of epoxide moieties with cysteamine to afford a monolith rich in surface thiol groups by Frechet and coworkers [8]. The new monolithic materials realized the selective capture of cysteine-containing peptides and reduced the complexity of peptide mixtures. Feng’s group prepared SiO2 /TiO2 composite monolithic capillary by sol– gel technology and the materials had excellent selectivity toward phosphopeptides as metal oxide affinity, which offered the promising application of the monolith on phosphoproteomics study [3]. Zinc oxide nanoparticles (ZnO NPs) have extensive applications in different industries because of their UV-protective, catalytic, optical, and antimicrobial actions as well as low cost. Many studies have been done toward the surface modification of ZnO NPs through chemical bonding. Tang et al. synthesized nano-ZnO/polymethacrylic acid composite particles via grafting of the copolymer onto the surface of ZnO NPs. The formed poly(zinc methacrylate) complex possessed excellent dispersion performance [12, 13]. Xu et al. prepared magnetic ∗ Additional corresponding author: Professor Weihong Zhou, E-mail: [email protected]
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ZnO surface-imprinted polymers by grafting molecularly imprinted polymer layers on the surface of ZnO nanorods embedded with ␥-Fe2 O3 nanoparticles, which was applied to selective recognition of antibiotics [14]. Antibiotics have drawn worldwide increasing public concern because of their release into the environment including water, soil, and sediments through ecological cycle. As a group of important synthetic antibiotics, fluoroquinolone antibiotics (FQs) have been used in human medicine, animal husbandry, and aquatic breeding. Many studies indicated that FQs are not fully metabolized and have low biodegradability in the body, which may result in disturbed ecosystems and lead to humans and animals more susceptible to antibioticresistant bacteria. Therefore, enormous efforts have been devoted to developing efficient strategies to determine FQs. Common analytical techniques for the determination of FQs are based on chemiluminescence [15,16], HPLC [17–24], LC– MS/MS [25–28], CE [29], etc. Among these methods, HPLC with UV or fluorescence detection is a commonly used analytical method and has been widely employed for the determination of FQs in various environmental samples. Due to their low concentration level in environment, it is a great challenge to directly determine FQs at trace level. Therefore, designing a simple, effective, and economical pretreatment procedure is necessary for the enrichment of FQs to enhance the detection signals. In this work, we synthesized ZnO@poly(methacrylic acid-co-ethylene dimethacrylate) (ZnO@poly(MAA-coEDMA)) monolithic columns as pretreatment materials coupled to HPLC for the analysis and enrichment of FQs (ofloxacin, ciprofloxacin, enoxacin, and pefloxacin). Extraction conditions affecting the extraction efficiency were investigated and optimized. Results indicated that the developed method is feasible, rapid, simple, and practical to be applied to the determination of the FQs in real samples.
2 Materials and methods 2.1 Chemicals and materials Ofloxacin (OFL), ciprofloxacin (CIP), enoxacin (ENO), pefloxacin (PEF), formic acid (FA), tetrabutylammonium bromide, methacrylic acid (MAA), ethylene dimethacrylate (EDMA), dodecanol, and ␥-methacryloxypropyltrimethoxysilane (␥-MAPS) were purchased from Aladdin Reagent (Shanghai, China). Methanol (MeOH) and acetonitrile (ACN) were supplied by Fisher Scientific (shanghai, China). Azobisisobutyronitrile (AIBN), sodium dihydrogen phosphate (NaH2 PO4 ), sodium hydrogen phosphate (Na2 HPO4 ), and TFA 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. Silica capillaries (530 m i.d. × 690 m o.d.) were purchased from Hebei Yongnian Optical Conductive Fiber Plant (Handan, China). A Milli-Q SP system (Millipore, Milford, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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MA, USA) was used to prepare ultrapure water. An LSP01– 1A programmable syringe pump (Baoding Longer Precision Pump, Hebei, China) equipped with a polyethylene syringe was employed for the delivery of the solution. An LD5–2A centrifuge (Beijing Jingli Centrifuge, China) was used for centrifuging. A PB-10 digital pH meter (Shanghai Rex Instruments Factory, China) was used for pH measurements of sample solutions. 2.2 Instrumentation SEM images were recorded on a JSM-6700F (JEOL Company, Japan) microscope. X-ray photoelectron spectroscopy data were obtained with an Escalab 250Xi electron spectrometer (Thermo Fisher Scientific, UK). FTIR spectra were recorded by using a Thermo Nicolet 670 FTIR instrument (Thermo Nicolet Corporation, USA). Thermogravimetric analysis was carried out on a Q500 (TA, USA) from 25 to 700⬚C with a heating rate of 10⬚C/min under air atmosphere. 2.3 Preparation of poly(MAA-co-EDMA) and ZnO@poly(MAA-co-EDMA) monolithic columns Prior to the polymerization, the inner wall of the capillary was vinylized with ␥-MAPS to enable covalent attachment of the monolith. Briefly, the fused-silica capillary was washed sequentially with water, 1.0 mol/L NaOH, water, 1.0 mol/L HCl, water, and acetone each for 30 min at the flow rate of 1.0 mL/min. Then the inner wall of the fused-silica capillary was modified with ␥-MAPS (30% in acetone, v/v) to enable the covalent attachment of monolith. After sealing the two ends of the capillary with silicon rubber, the reaction was allowed to perform at 50⬚C for 12 h. Subsequently, the capillary was washed with acetone and purged with nitrogen for 1 h. In a typical process, the polymerization mixture for preparing the poly(MAA-co-EDMA) monolith, consisting of monomer (48 L MAA), cross-linker (390 L EDMA), porogen (115 L MeOH + 1030 L dodecanol), and initiator (4.5 mg AIBN), was degassed by ultrasonication for about 30 min. Then the homogeneous solution was filled into the pretreated capillary and sealed at both ends with rubber stoppers. The polymerization was initiated at 60⬚C for 24 h [30]. Finally, the prepared capillary was washed with MeOH to remove the unreacted components. The preparation of ZnO NPs modified monolith was carried out in a flask. At first, 5 mg ZnO NPs was added to 48 L MAA and this mixture was stirred at room temperature for 2 h. The hydroxyl groups on ZnO NPs can interact with carboxylic groups in MAA to form zinc methacrylate complex. Then the prepared zinc methacrylate was added to the polymerization solution (390 L EDMA, 115 L MeOH, 1030 L dodecanol, and 4.5 mg AIBN). Before polymerization, the mixture was treated by ultrasound for 30 min to obtain a stable homogenous dispersion, then the reaction mixture was maintained at 60⬚C for 24 h in vinylized capillary to prepare ZnO@poly(MAA-co-EDMA) monolith. The www.jss-journal.com
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Figure 1. The procedure for the preparation of ZnO@poly(MAAco-EDMA) monolith.
procedure for the preparation of ZnO@poly(MAA-co-EDMA) monolith is illustrated in Fig. 1.
2.4 Standard solutions and PMME procedure The mixed stock solutions of OFL, CIP, ENO, and PEF were prepared in MeOH containing 0.1% v/v 1 mol/L NaOH solution, where NaOH was used to enhance the solubility of the compounds [27]. The stock solution was stored in a refrigerator at 4⬚C. The working solutions were freshly prepared by appropriate dilution with ultrapure water. The pretreatment device consisted of a syringe pump and a plastic syringe (5 mL). The critical part of PMME was the initial pinhead which was replaced by a polymer monolithic column. All the solvents were filtered through a 0.22 m Millipore filter before analysis. The extraction procedure included preconditioning, sampling, evacuation, and desorption. Prior to extraction, 0.2 mL MeOH was passed through the polymer monolithic column at the flow rate of 0.05 mL/min, and then 0.8 mL sample solution was injected to realize the adsorption at the flow rate of 0.02 mL/min. Subsequently, an empty and clean syringe was employed for driving out the residual solution in the polymer monolithic column. For the desorption step, FQs adsorbed on the polymer monolithic column were eluted with 0.05 mL ACN containing 0.1% v/v TFA at the flow rate of 0.05 mL/min, and the eluent was collected into a vial for HPLC analysis.
2.5 HPLC equipment and separation conditions HPLC analysis was performed on an Agilent 1100 (Agilent Technologies, Palo Alto, CA, USA) LC system, equipped with a quaternary gradient pump, a solvent degasser system, a thermostatted autosampler and column compartment, and a multiple wavelength diode array detector. Chem-Station software was utilized to control the system and analyze the chromatographic data. A RP Agilent Zorbax Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 m) was employed for the chromatographic separations. A Phenomenex C18 security guard column (4.0 mm × 3.0 mm) was used to protect the analytical column. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
A total of 10 mmol/L tetrabutylammonium bromide solutions containing 0.1% FA (mobile phase A) and 100% ACN (mobile phase B) were employed for the separation of the target FQs at the flow rate of 0.8 mL/min. A gradient program was conducted through the following steps: 100% A at first; 0−10% B within 1 min; and then 90−100% A by a linear gradient within 18 min until the next injection. The injection volumes for all samples and standards were 5 L. The column was kept at room temperature (25⬚C). The detection wavelengths were set at 280 nm for CIP, ENO, PEF, and 294 nm for OFL. 2.6 Water samples analysis Environmental water samples were filtered through a 0.22 m Millipore filter before analysis. All the sample solutions were spiked with OFL, CIP, ENO, and PEF standard solutions at different concentration levels to assess the matrix effects, Level 1 (0.5 g/mL) and Level 2 (1.0 g/mL).
3 Results and discussion 3.1 Characteristics of the ZnO@poly(MAA-co-EDMA) monolithic column In our preliminary experiments, amounts of ZnO NPs added into the preparation process were optimized. Amounts of 5, 8, and 10 mg ZnO NPs were employed to prepare the monoliths. Results showed that 10 mg ZnO NPs could not be dissolved well in 48 L MAA after stirring at room temperature for 2 h. When 8 mg ZnO NPs was introduced, solutions could not pass through the column fluently, implying poor permeability of the column. Finally, 5 mg ZnO NPs was selected to be added into the monolith. The morphology of the prepared ZnO@poly(MAA-coEDMA) monolith was observed by SEM. It could be seen from Fig. 2A that ZnO NPs possessed excellent dispersion performance in monolith and the monolith exhibited a well-distributed channel network. X-ray photoelectron spectroscopy data are shown in Fig. 2B, indicating that typical Zn 2p peaks appeared at 1045.3 and 1021.8 eV. There is no significant shift in the peak positions of Zn 2p spectrum, indicating that the bonding of ZnO NPs to monolith surface is through the oxygen atoms of the carboxylic groups [31, 32]. The relative contents of C, O, N, and Zn of ZnO@poly(MAAco-EDMA) monolith were calculated as 92.2, 7.1, 0.2, and 0.5%, respectively. Compared with the relative contents of C, O, and N of poly(MAA-co-EDMA) monolith (C 94.8, O 5.0, and N 0.2%), it could be observed that the Zn content increased. The results demonstrated that ZnO NPs were successfully introduced into the monolith. In a typical FTIR spectrum (Fig. 2C), the peaks at 3500 and 447 cm−1 corresponded to –OH and Zn−O bonds on ZnO NPs, respectively [33]. The strong absorption bands at 1570 and 1400 cm−1 should belong to the interaction between COO− and ZnO to form poly(zinc methacrylate) complex [13]. The thermogravimetric www.jss-journal.com
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Figure 2. (A) SEM images of ZnO@poly(MAA-co-EDMA) monolith, (B) XPS spectra, (C) FTIR spectra, and (D) TGA curves.
analysis curve in Fig. 2D revealed that thermal transition occurred in two temperature ranges. The decrease during 120– 190⬚C may be attributed to disintegration of ZnO NPs, and the weight loss in the range of 360–450⬚C may correspond to the monolithic material. All these results could confirm that the poly(MAA-co-EDMA) column was successfully modified with ZnO NPs. 3.2 Optimization of PMME conditions To obtain a high extraction efficiency for FQs, conditions of PMME process, including eluent type, sample pH, sample volume, and eluent flow rate were investigated. The peak area of the target compounds as the HPLC response was used to evaluate the extraction efficiency under various experimental conditions. The effect of eluent type was optimized because it can affect extraction efficiency of the targets from monolith. Various solvents, including MeOH, MeOH/water (60:40, v/v), 0.1% TFA in MeOH, ACN, ACN/water (60:40, v/v), and 0.1% TFA in ACN were adopted to investigate the eluent effects. Results were shown in Fig. 3A. The peak areas using ACN containing 0.1% TFA as the elution solvent were slightly higher than those of the other five. So ACN containing 0.1% TFA was selected as the optimized elution solvent to obtain good extraction efficiency. Solution pH is an important parameter affecting the sorption behaviors because it can affect the ionization forms of C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
target compounds in solution. Results in Fig. 3B showed that the peak area reached maximum at nearby pH 7.0 and decreased when pH was lower or higher. Since FQs are zwitterionic compounds with two pKa values, pKa1 of 5.2–6.3 and pKa2 of 7.4–8.9 [23], 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 ZnO surface sites supported by hydrogen bonding of a neighboring carbonyl group [34]. Because the pHzpc of ZnO is 9.5 [35] and FQs are zwitterionic, the negative charge on FQs and the positive charge on ZnO at pH 7.0 allowed the strong interaction between them [36]. When pH is higher than 7.0, the hydrogen bonding interaction becomes weaker with decreasing carboxylic groups in MAA because of the hydrolytic action. On the other hand, electrostatic repulsion interactions are stronger between FQs anions and the negative surface charge of ZnO [22]. Both the two interactions led to the decrease of adsorption capacity of FQs on the ZnO@poly(MAA-co-EDMA) monolith. Finally, pH 7.0 was chosen for further experiments. The sample volume dependency was conducted within the range of 0.02–2.0 mL at a constant flow rate of 0.02 mL/min. Results were demonstrated in Fig. 3C, illustrating that a significant increasing tendency of the peak areas was observed when the sample volume increased from 0.02 to 2.0 mL and the extraction equilibrium did not reach maximum even after 2.0 mL sample volume was employed. www.jss-journal.com
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Figure 3. Effects of (A) eluent type, (B) sample pH, (C) sample volume, and (D) eluent flow rate. Standards: 2.0 g/mL.
Taking the time of experiment into consideration, 0.8 mL sample volume was selected in the present work. The influence of eluent flow rate was investigated by varying the flow rate from 0.01 to 0.07 mL/min with a total eluent loading volume of 0.05 mL. Figure 3D indicated that the highest elution efficiency was obtained when the eluent flow rate was 0.05 mL/min. Therefore, 0.05 mL/min was employed as the eluent flow rate. 3.3 Effect of salt concentration It has been reported in many papers that the addition of salts may increase or decrease the adsorbed amount of differential adsorbents. Further experiments were conducted to investigate the effect of salt concentration on the adsorption of OFL, CIP, ENO, and PEF (NaCl was selected to investigate the effect of salt concentration in this study). As shown in Fig. 4A, the peak areas rapidly decreased with the increasing of NaCl concentration from 1 to 10 mmol/L. When the concentration increased from 10 to 100 mmol/L, the tendency of decrease was much weaker. This phenomenon might ascribe that NaCl reduced the electrostatic interaction between carboxylate anion in FQs and ZnO. Moreover, the addition of salt can decrease the solubility of target compounds in the aqueous sample. These two reasons led to the decrease of signal intensity [37]. The electrostatic interaction became minor or was completely shielded when NaCl concentration was high enough. Under this condition, surface complexation, hydrogen bonding, and hydrophobic interaction contributed to the adsorption of FQs onto the monolith materials, implying an C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
almost constant value of the adsorbed targets. On the basis of above experimental results, NaCl was not added to the aqueous sample in the experiments.
3.4 Effect of organic acid concentration In the environment samples, some organic acids may have competitive adsorption with the targets onto the monolithic column. Humic acid was selected to investigate effects of organic acid in this study [23]. Sample solutions containing different amount of humic acids were extracted by the developed PMME method. As can be seen from Fig. 4B, no obvious changes of peak areas of the entire target FQs were observed when the concentration of humic acid changed from 0.01 to 10 g/mL. Hence humic acid did not interfere with FQs adsorption, implying the good anti-interference ability of the monolithic materials to organic acids.
3.5 Method performance Under the above optimized conditions, the method was validated. Within the concentration range of 0.05−5 g/mL, the square of correlation coefficient (R2 ) was found to be in the range of 0.9949–0.9982. LOD and LOQ, calculated with S/N = 3 and 10, respectively, were determined in the range of 0.007–0.034 and 0.026–0.115 g/mL, respectively. The intraday and interday precisions of the target FQs were determined to be in the range of 3.8–8.0% and 4.7–9.8%, respectively. Furthermore, to investigate the reproducibility of monolith, the www.jss-journal.com
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Figure 4. Effects of (A) salt concentration and (B) organic acid concentration.
intrabatch and interbatch RSDs were also determined to be lower than 7.8 and 8.2%. The present PMME–HPLC method was compared to some preconcentration methods for the extraction of FQs. The LOD values are comparable to the ones obtained with other methods, such as solid-phase sorbents extraction with LC–MS/MS (1.7−2.5 ng/L) [38], MAE–HPLC (0.02 g/mL) [39], and SPE–HPLC (0.5−8.1 g/L) [40]. Although it could not provide the best LOD level, the developed PMME-HPLC method was very simple to operate and did not need any expensive instruments and complex preparation steps. Under the optimal experimental conditions, the analytical performance after the enrichment using ZnO@poly(MAAco-EDMA) column was compared with poly(MAA-co-EDMA) column enrichment and direct HPLC analysis of OFL, CIP, ENO, and PEF. Results are shown in Fig. 5, of which the experiments were carried out in triplicate and the average results were presented. The peak areas with ZnO@poly(MAAco-EDMA) column were significantly higher than those with the other two methods, which may be due to the following. The adsorbed FQs on the poly(MAA-co-EDMA) column were only based on hydrophobic interaction and hydrogen bonding in the extraction process. When ZnO NPs were introduced into the polymerization mixture, ZnO NPs brought high surface-to-volume ratio and special surface chemistry to monolithic columns. The carboxylic groups in FQs are critical for the adsorption onto metal oxide [41], and additional surface complexation and electrostatic attraction have been well
Figure 6. Chromatograms of water samples obtained by the preconcentration procedures: Blank sample, sample spiked with Level 1 (all 0.5 g/mL), and sample spiked with Level 2 (all 1.0 g/mL).
established for the sorption of fluoroquinolone compounds to metal oxides [42]. These interactions contributed to the higher preconcentration ability for FQs with ZnO@poly(MAA-coEDMA) column than that with poly(MAA-co-EDMA) column. 3.6 Application to real samples To assess the applicability of the ZnO@poly(MAA-co-EDMA) monolithic column in real samples, different six kinds of water samples (seawater, lake, river, and underground water) were selected for PMME–HPLC analysis. Supporting Information Table S1 and Fig. 6 exhibit the average recoveries of the four target compounds (70.7–108.3%) and typical chromatograms obtained from spiked samples. These results indicated the utility of the ZnO@poly(MAA-co-EDMA) monolithic column in the analysis of FQs in environmental water samples.
4 Conclusion Figure 5. Comparison of the analytical performance of OFL, CIP, ENO, and PEF enrichment by direct HPLC analysis, poly(MAAco-EDMA), and ZnO@poly(MAA-co-EDMA) monolithic columns under the optimized conditions (n = 3).
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A novel ZnO@poly(MAA-co-EDMA) monolithic column was prepared and used as a medium for the microextraction of FQs. The parameters affecting the adsorption performance, such as eluent type, sample pH, sample volume, www.jss-journal.com
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eluent flow rate, and the concentration of inorganic salt and organic acid, were investigated. The ZnO@poly(MAAco-EDMA) monolithic column exhibited satisfactory recovery values for the determination of water samples. The high adsorption capacity, excellent stability, and high reproducibility of the developed material render it a promising material for preconcentration. The project was supported by National Natural Science Foundation of China (21205047), Administration of Quality Supervision, Inspection and Quarantine (No.2012IK165), and Jilin Provincial Science & Technology Department (201105102). The authors have declared no conflict of interest.
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of Chemistry, Jilin University, Changchun, China 2 Shenyang Entry & Exit Inspection and Quarantine Bureau of China, Shenyang, China 3 Obstetrics & Gynecology, China-Japan Union Hospital, Jilin University, Changchun, China 4 Jilin Entry & Exit Inspection and Quarantine Bureau of China, Changchun, China Received August 18, 2014 Revised October 16, 2014 Accepted October 16, 2014
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Research Article
Preparation of a monolith functionalized with zinc oxide nanoparticles and its application in the enrichment of fluoroquinolone antibiotics This study describes the enrichment ability of ZnO-modified methacrylic acid-co-ethylene dimethacrylate polymer monoliths as stationary phases for the simultaneous determination of antibiotics (ofloxacin, ciprofloxacin, enoxacin, and pefloxacin) combined with highperformance liquid chromatography. The prepared monolith was characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, Fourier-transformed infrared spectroscopy, and thermogravimetric analysis. The polymer monolith microextraction method has been applied to the enrichment of fluoroquinolone antibiotics and satisfactory results were obtained in the analysis of water samples. Compared with the conventional methacrylic acid based monolith, the developed monolith exhibited a higher enrichment capacity because of the introduction of zinc oxide into the preparation process. Keywords: Fluoroquinolone / High-performance liquid chromatography / Monoliths / Zinc oxide DOI 10.1002/jssc.201400893
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction In recent years, much research attention has been focused on the application of nanoparticles to the modification of chemical materials. The potential of nanoparticles such as carbon nanomaterials [1, 2], silica [3, 4], magnetic and nonmagnetic metal oxides [5–7], silver and gold nanoparticles [8–11] have been widely recognized. Modified nanomaterials have some important applications in separation and enrichment owing to the novel physical and chemical properties of nanoparticles associated with their size and shape, e.g., in the field of polymer monolith microextraction (PMME). Some researchers have devoted themselves to controlling the surface chemistry of monoliths by adding or grafting nanoparticles into the monolith supports by copolymerization with monomers or attachment onto the pore surface of monolithic matrix. In 2005,
Correspondence: Professor Qiong Jia, Renmin Street 5988#, Changchun, China E-mail: [email protected]
Abbreviations: ACN, acetonitrile; AIBN, azobisisobutyronitrile; CIP, ciprofloxacin; EDMA, ethylene dimethacrylate; ENO, enoxacin; FQ, fluoroquinolone antibiotic; ␥-MAPS, ␥-methacryloxypropyltrimethoxysilane; MeOH, methanol; MMA, methacrylic acid; OFL, ofloxacin; PEF, pefloxacin; PMME, polymer monolith microextraction; ZnO NP, zinc oxide nanoparticle C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Li et al. first incorporated single-walled carbon nanotubes into a polymerization mixture to prepare a poly(vinylbenzyl chloride-ethylene dimethacrylate-single-walled carbon nanotubes) monolith, which was successfully used for HPLC and CEC [2]. Porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith modified with gold nanoparticles was prepared from the reaction of epoxide moieties with cysteamine to afford a monolith rich in surface thiol groups by Frechet and coworkers [8]. The new monolithic materials realized the selective capture of cysteine-containing peptides and reduced the complexity of peptide mixtures. Feng’s group prepared SiO2 /TiO2 composite monolithic capillary by sol– gel technology and the materials had excellent selectivity toward phosphopeptides as metal oxide affinity, which offered the promising application of the monolith on phosphoproteomics study [3]. Zinc oxide nanoparticles (ZnO NPs) have extensive applications in different industries because of their UV-protective, catalytic, optical, and antimicrobial actions as well as low cost. Many studies have been done toward the surface modification of ZnO NPs through chemical bonding. Tang et al. synthesized nano-ZnO/polymethacrylic acid composite particles via grafting of the copolymer onto the surface of ZnO NPs. The formed poly(zinc methacrylate) complex possessed excellent dispersion performance [12, 13]. Xu et al. prepared magnetic ∗ Additional corresponding author: Professor Weihong Zhou, E-mail: [email protected]
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ZnO surface-imprinted polymers by grafting molecularly imprinted polymer layers on the surface of ZnO nanorods embedded with ␥-Fe2 O3 nanoparticles, which was applied to selective recognition of antibiotics [14]. Antibiotics have drawn worldwide increasing public concern because of their release into the environment including water, soil, and sediments through ecological cycle. As a group of important synthetic antibiotics, fluoroquinolone antibiotics (FQs) have been used in human medicine, animal husbandry, and aquatic breeding. Many studies indicated that FQs are not fully metabolized and have low biodegradability in the body, which may result in disturbed ecosystems and lead to humans and animals more susceptible to antibioticresistant bacteria. Therefore, enormous efforts have been devoted to developing efficient strategies to determine FQs. Common analytical techniques for the determination of FQs are based on chemiluminescence [15,16], HPLC [17–24], LC– MS/MS [25–28], CE [29], etc. Among these methods, HPLC with UV or fluorescence detection is a commonly used analytical method and has been widely employed for the determination of FQs in various environmental samples. Due to their low concentration level in environment, it is a great challenge to directly determine FQs at trace level. Therefore, designing a simple, effective, and economical pretreatment procedure is necessary for the enrichment of FQs to enhance the detection signals. In this work, we synthesized ZnO@poly(methacrylic acid-co-ethylene dimethacrylate) (ZnO@poly(MAA-coEDMA)) monolithic columns as pretreatment materials coupled to HPLC for the analysis and enrichment of FQs (ofloxacin, ciprofloxacin, enoxacin, and pefloxacin). Extraction conditions affecting the extraction efficiency were investigated and optimized. Results indicated that the developed method is feasible, rapid, simple, and practical to be applied to the determination of the FQs in real samples.
2 Materials and methods 2.1 Chemicals and materials Ofloxacin (OFL), ciprofloxacin (CIP), enoxacin (ENO), pefloxacin (PEF), formic acid (FA), tetrabutylammonium bromide, methacrylic acid (MAA), ethylene dimethacrylate (EDMA), dodecanol, and ␥-methacryloxypropyltrimethoxysilane (␥-MAPS) were purchased from Aladdin Reagent (Shanghai, China). Methanol (MeOH) and acetonitrile (ACN) were supplied by Fisher Scientific (shanghai, China). Azobisisobutyronitrile (AIBN), sodium dihydrogen phosphate (NaH2 PO4 ), sodium hydrogen phosphate (Na2 HPO4 ), and TFA 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. Silica capillaries (530 m i.d. × 690 m o.d.) were purchased from Hebei Yongnian Optical Conductive Fiber Plant (Handan, China). A Milli-Q SP system (Millipore, Milford, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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MA, USA) was used to prepare ultrapure water. An LSP01– 1A programmable syringe pump (Baoding Longer Precision Pump, Hebei, China) equipped with a polyethylene syringe was employed for the delivery of the solution. An LD5–2A centrifuge (Beijing Jingli Centrifuge, China) was used for centrifuging. A PB-10 digital pH meter (Shanghai Rex Instruments Factory, China) was used for pH measurements of sample solutions. 2.2 Instrumentation SEM images were recorded on a JSM-6700F (JEOL Company, Japan) microscope. X-ray photoelectron spectroscopy data were obtained with an Escalab 250Xi electron spectrometer (Thermo Fisher Scientific, UK). FTIR spectra were recorded by using a Thermo Nicolet 670 FTIR instrument (Thermo Nicolet Corporation, USA). Thermogravimetric analysis was carried out on a Q500 (TA, USA) from 25 to 700⬚C with a heating rate of 10⬚C/min under air atmosphere. 2.3 Preparation of poly(MAA-co-EDMA) and ZnO@poly(MAA-co-EDMA) monolithic columns Prior to the polymerization, the inner wall of the capillary was vinylized with ␥-MAPS to enable covalent attachment of the monolith. Briefly, the fused-silica capillary was washed sequentially with water, 1.0 mol/L NaOH, water, 1.0 mol/L HCl, water, and acetone each for 30 min at the flow rate of 1.0 mL/min. Then the inner wall of the fused-silica capillary was modified with ␥-MAPS (30% in acetone, v/v) to enable the covalent attachment of monolith. After sealing the two ends of the capillary with silicon rubber, the reaction was allowed to perform at 50⬚C for 12 h. Subsequently, the capillary was washed with acetone and purged with nitrogen for 1 h. In a typical process, the polymerization mixture for preparing the poly(MAA-co-EDMA) monolith, consisting of monomer (48 L MAA), cross-linker (390 L EDMA), porogen (115 L MeOH + 1030 L dodecanol), and initiator (4.5 mg AIBN), was degassed by ultrasonication for about 30 min. Then the homogeneous solution was filled into the pretreated capillary and sealed at both ends with rubber stoppers. The polymerization was initiated at 60⬚C for 24 h [30]. Finally, the prepared capillary was washed with MeOH to remove the unreacted components. The preparation of ZnO NPs modified monolith was carried out in a flask. At first, 5 mg ZnO NPs was added to 48 L MAA and this mixture was stirred at room temperature for 2 h. The hydroxyl groups on ZnO NPs can interact with carboxylic groups in MAA to form zinc methacrylate complex. Then the prepared zinc methacrylate was added to the polymerization solution (390 L EDMA, 115 L MeOH, 1030 L dodecanol, and 4.5 mg AIBN). Before polymerization, the mixture was treated by ultrasound for 30 min to obtain a stable homogenous dispersion, then the reaction mixture was maintained at 60⬚C for 24 h in vinylized capillary to prepare ZnO@poly(MAA-co-EDMA) monolith. The www.jss-journal.com
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Figure 1. The procedure for the preparation of ZnO@poly(MAAco-EDMA) monolith.
procedure for the preparation of ZnO@poly(MAA-co-EDMA) monolith is illustrated in Fig. 1.
2.4 Standard solutions and PMME procedure The mixed stock solutions of OFL, CIP, ENO, and PEF were prepared in MeOH containing 0.1% v/v 1 mol/L NaOH solution, where NaOH was used to enhance the solubility of the compounds [27]. The stock solution was stored in a refrigerator at 4⬚C. The working solutions were freshly prepared by appropriate dilution with ultrapure water. The pretreatment device consisted of a syringe pump and a plastic syringe (5 mL). The critical part of PMME was the initial pinhead which was replaced by a polymer monolithic column. All the solvents were filtered through a 0.22 m Millipore filter before analysis. The extraction procedure included preconditioning, sampling, evacuation, and desorption. Prior to extraction, 0.2 mL MeOH was passed through the polymer monolithic column at the flow rate of 0.05 mL/min, and then 0.8 mL sample solution was injected to realize the adsorption at the flow rate of 0.02 mL/min. Subsequently, an empty and clean syringe was employed for driving out the residual solution in the polymer monolithic column. For the desorption step, FQs adsorbed on the polymer monolithic column were eluted with 0.05 mL ACN containing 0.1% v/v TFA at the flow rate of 0.05 mL/min, and the eluent was collected into a vial for HPLC analysis.
2.5 HPLC equipment and separation conditions HPLC analysis was performed on an Agilent 1100 (Agilent Technologies, Palo Alto, CA, USA) LC system, equipped with a quaternary gradient pump, a solvent degasser system, a thermostatted autosampler and column compartment, and a multiple wavelength diode array detector. Chem-Station software was utilized to control the system and analyze the chromatographic data. A RP Agilent Zorbax Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 m) was employed for the chromatographic separations. A Phenomenex C18 security guard column (4.0 mm × 3.0 mm) was used to protect the analytical column. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
A total of 10 mmol/L tetrabutylammonium bromide solutions containing 0.1% FA (mobile phase A) and 100% ACN (mobile phase B) were employed for the separation of the target FQs at the flow rate of 0.8 mL/min. A gradient program was conducted through the following steps: 100% A at first; 0−10% B within 1 min; and then 90−100% A by a linear gradient within 18 min until the next injection. The injection volumes for all samples and standards were 5 L. The column was kept at room temperature (25⬚C). The detection wavelengths were set at 280 nm for CIP, ENO, PEF, and 294 nm for OFL. 2.6 Water samples analysis Environmental water samples were filtered through a 0.22 m Millipore filter before analysis. All the sample solutions were spiked with OFL, CIP, ENO, and PEF standard solutions at different concentration levels to assess the matrix effects, Level 1 (0.5 g/mL) and Level 2 (1.0 g/mL).
3 Results and discussion 3.1 Characteristics of the ZnO@poly(MAA-co-EDMA) monolithic column In our preliminary experiments, amounts of ZnO NPs added into the preparation process were optimized. Amounts of 5, 8, and 10 mg ZnO NPs were employed to prepare the monoliths. Results showed that 10 mg ZnO NPs could not be dissolved well in 48 L MAA after stirring at room temperature for 2 h. When 8 mg ZnO NPs was introduced, solutions could not pass through the column fluently, implying poor permeability of the column. Finally, 5 mg ZnO NPs was selected to be added into the monolith. The morphology of the prepared ZnO@poly(MAA-coEDMA) monolith was observed by SEM. It could be seen from Fig. 2A that ZnO NPs possessed excellent dispersion performance in monolith and the monolith exhibited a well-distributed channel network. X-ray photoelectron spectroscopy data are shown in Fig. 2B, indicating that typical Zn 2p peaks appeared at 1045.3 and 1021.8 eV. There is no significant shift in the peak positions of Zn 2p spectrum, indicating that the bonding of ZnO NPs to monolith surface is through the oxygen atoms of the carboxylic groups [31, 32]. The relative contents of C, O, N, and Zn of ZnO@poly(MAAco-EDMA) monolith were calculated as 92.2, 7.1, 0.2, and 0.5%, respectively. Compared with the relative contents of C, O, and N of poly(MAA-co-EDMA) monolith (C 94.8, O 5.0, and N 0.2%), it could be observed that the Zn content increased. The results demonstrated that ZnO NPs were successfully introduced into the monolith. In a typical FTIR spectrum (Fig. 2C), the peaks at 3500 and 447 cm−1 corresponded to –OH and Zn−O bonds on ZnO NPs, respectively [33]. The strong absorption bands at 1570 and 1400 cm−1 should belong to the interaction between COO− and ZnO to form poly(zinc methacrylate) complex [13]. The thermogravimetric www.jss-journal.com
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Figure 2. (A) SEM images of ZnO@poly(MAA-co-EDMA) monolith, (B) XPS spectra, (C) FTIR spectra, and (D) TGA curves.
analysis curve in Fig. 2D revealed that thermal transition occurred in two temperature ranges. The decrease during 120– 190⬚C may be attributed to disintegration of ZnO NPs, and the weight loss in the range of 360–450⬚C may correspond to the monolithic material. All these results could confirm that the poly(MAA-co-EDMA) column was successfully modified with ZnO NPs. 3.2 Optimization of PMME conditions To obtain a high extraction efficiency for FQs, conditions of PMME process, including eluent type, sample pH, sample volume, and eluent flow rate were investigated. The peak area of the target compounds as the HPLC response was used to evaluate the extraction efficiency under various experimental conditions. The effect of eluent type was optimized because it can affect extraction efficiency of the targets from monolith. Various solvents, including MeOH, MeOH/water (60:40, v/v), 0.1% TFA in MeOH, ACN, ACN/water (60:40, v/v), and 0.1% TFA in ACN were adopted to investigate the eluent effects. Results were shown in Fig. 3A. The peak areas using ACN containing 0.1% TFA as the elution solvent were slightly higher than those of the other five. So ACN containing 0.1% TFA was selected as the optimized elution solvent to obtain good extraction efficiency. Solution pH is an important parameter affecting the sorption behaviors because it can affect the ionization forms of C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
target compounds in solution. Results in Fig. 3B showed that the peak area reached maximum at nearby pH 7.0 and decreased when pH was lower or higher. Since FQs are zwitterionic compounds with two pKa values, pKa1 of 5.2–6.3 and pKa2 of 7.4–8.9 [23], 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 ZnO surface sites supported by hydrogen bonding of a neighboring carbonyl group [34]. Because the pHzpc of ZnO is 9.5 [35] and FQs are zwitterionic, the negative charge on FQs and the positive charge on ZnO at pH 7.0 allowed the strong interaction between them [36]. When pH is higher than 7.0, the hydrogen bonding interaction becomes weaker with decreasing carboxylic groups in MAA because of the hydrolytic action. On the other hand, electrostatic repulsion interactions are stronger between FQs anions and the negative surface charge of ZnO [22]. Both the two interactions led to the decrease of adsorption capacity of FQs on the ZnO@poly(MAA-co-EDMA) monolith. Finally, pH 7.0 was chosen for further experiments. The sample volume dependency was conducted within the range of 0.02–2.0 mL at a constant flow rate of 0.02 mL/min. Results were demonstrated in Fig. 3C, illustrating that a significant increasing tendency of the peak areas was observed when the sample volume increased from 0.02 to 2.0 mL and the extraction equilibrium did not reach maximum even after 2.0 mL sample volume was employed. www.jss-journal.com
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Figure 3. Effects of (A) eluent type, (B) sample pH, (C) sample volume, and (D) eluent flow rate. Standards: 2.0 g/mL.
Taking the time of experiment into consideration, 0.8 mL sample volume was selected in the present work. The influence of eluent flow rate was investigated by varying the flow rate from 0.01 to 0.07 mL/min with a total eluent loading volume of 0.05 mL. Figure 3D indicated that the highest elution efficiency was obtained when the eluent flow rate was 0.05 mL/min. Therefore, 0.05 mL/min was employed as the eluent flow rate. 3.3 Effect of salt concentration It has been reported in many papers that the addition of salts may increase or decrease the adsorbed amount of differential adsorbents. Further experiments were conducted to investigate the effect of salt concentration on the adsorption of OFL, CIP, ENO, and PEF (NaCl was selected to investigate the effect of salt concentration in this study). As shown in Fig. 4A, the peak areas rapidly decreased with the increasing of NaCl concentration from 1 to 10 mmol/L. When the concentration increased from 10 to 100 mmol/L, the tendency of decrease was much weaker. This phenomenon might ascribe that NaCl reduced the electrostatic interaction between carboxylate anion in FQs and ZnO. Moreover, the addition of salt can decrease the solubility of target compounds in the aqueous sample. These two reasons led to the decrease of signal intensity [37]. The electrostatic interaction became minor or was completely shielded when NaCl concentration was high enough. Under this condition, surface complexation, hydrogen bonding, and hydrophobic interaction contributed to the adsorption of FQs onto the monolith materials, implying an C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
almost constant value of the adsorbed targets. On the basis of above experimental results, NaCl was not added to the aqueous sample in the experiments.
3.4 Effect of organic acid concentration In the environment samples, some organic acids may have competitive adsorption with the targets onto the monolithic column. Humic acid was selected to investigate effects of organic acid in this study [23]. Sample solutions containing different amount of humic acids were extracted by the developed PMME method. As can be seen from Fig. 4B, no obvious changes of peak areas of the entire target FQs were observed when the concentration of humic acid changed from 0.01 to 10 g/mL. Hence humic acid did not interfere with FQs adsorption, implying the good anti-interference ability of the monolithic materials to organic acids.
3.5 Method performance Under the above optimized conditions, the method was validated. Within the concentration range of 0.05−5 g/mL, the square of correlation coefficient (R2 ) was found to be in the range of 0.9949–0.9982. LOD and LOQ, calculated with S/N = 3 and 10, respectively, were determined in the range of 0.007–0.034 and 0.026–0.115 g/mL, respectively. The intraday and interday precisions of the target FQs were determined to be in the range of 3.8–8.0% and 4.7–9.8%, respectively. Furthermore, to investigate the reproducibility of monolith, the www.jss-journal.com
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Figure 4. Effects of (A) salt concentration and (B) organic acid concentration.
intrabatch and interbatch RSDs were also determined to be lower than 7.8 and 8.2%. The present PMME–HPLC method was compared to some preconcentration methods for the extraction of FQs. The LOD values are comparable to the ones obtained with other methods, such as solid-phase sorbents extraction with LC–MS/MS (1.7−2.5 ng/L) [38], MAE–HPLC (0.02 g/mL) [39], and SPE–HPLC (0.5−8.1 g/L) [40]. Although it could not provide the best LOD level, the developed PMME-HPLC method was very simple to operate and did not need any expensive instruments and complex preparation steps. Under the optimal experimental conditions, the analytical performance after the enrichment using ZnO@poly(MAAco-EDMA) column was compared with poly(MAA-co-EDMA) column enrichment and direct HPLC analysis of OFL, CIP, ENO, and PEF. Results are shown in Fig. 5, of which the experiments were carried out in triplicate and the average results were presented. The peak areas with ZnO@poly(MAAco-EDMA) column were significantly higher than those with the other two methods, which may be due to the following. The adsorbed FQs on the poly(MAA-co-EDMA) column were only based on hydrophobic interaction and hydrogen bonding in the extraction process. When ZnO NPs were introduced into the polymerization mixture, ZnO NPs brought high surface-to-volume ratio and special surface chemistry to monolithic columns. The carboxylic groups in FQs are critical for the adsorption onto metal oxide [41], and additional surface complexation and electrostatic attraction have been well
Figure 6. Chromatograms of water samples obtained by the preconcentration procedures: Blank sample, sample spiked with Level 1 (all 0.5 g/mL), and sample spiked with Level 2 (all 1.0 g/mL).
established for the sorption of fluoroquinolone compounds to metal oxides [42]. These interactions contributed to the higher preconcentration ability for FQs with ZnO@poly(MAA-coEDMA) column than that with poly(MAA-co-EDMA) column. 3.6 Application to real samples To assess the applicability of the ZnO@poly(MAA-co-EDMA) monolithic column in real samples, different six kinds of water samples (seawater, lake, river, and underground water) were selected for PMME–HPLC analysis. Supporting Information Table S1 and Fig. 6 exhibit the average recoveries of the four target compounds (70.7–108.3%) and typical chromatograms obtained from spiked samples. These results indicated the utility of the ZnO@poly(MAA-co-EDMA) monolithic column in the analysis of FQs in environmental water samples.
4 Conclusion Figure 5. Comparison of the analytical performance of OFL, CIP, ENO, and PEF enrichment by direct HPLC analysis, poly(MAAco-EDMA), and ZnO@poly(MAA-co-EDMA) monolithic columns under the optimized conditions (n = 3).
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A novel ZnO@poly(MAA-co-EDMA) monolithic column was prepared and used as a medium for the microextraction of FQs. The parameters affecting the adsorption performance, such as eluent type, sample pH, sample volume, www.jss-journal.com
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eluent flow rate, and the concentration of inorganic salt and organic acid, were investigated. The ZnO@poly(MAAco-EDMA) monolithic column exhibited satisfactory recovery values for the determination of water samples. The high adsorption capacity, excellent stability, and high reproducibility of the developed material render it a promising material for preconcentration. The project was supported by National Natural Science Foundation of China (21205047), Administration of Quality Supervision, Inspection and Quarantine (No.2012IK165), and Jilin Provincial Science & Technology Department (201105102). The authors have declared no conflict of interest.
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