Food Chemistry 149 (2014) 71–75
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Analytical Methods
Determination of residual enrofloxacin in food samples by a sensitive method of chemiluminescence enzyme immunoassay Fei Yu a, Songcheng Yu a, Lanlan Yu b, Yanqiang Li c, Yongjun Wu a,⇑, Hongquan Zhang a, Lingbo Qu d, Peter de B. Harrington e a
College of Public Health, Zhengzhou University, Zhengzhou 450001, China Department of Chemistry, Zhengzhou University, Zhengzhou 450001, China Zhengzhou Tobacco Research Institute of China Tobacco Corporation, Zhengzhou 450001, China d College of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450001, China e Department of Chemistry and Biochemistry, Center for Intelligent Chemical Instrumentation, Ohio University, Athens, OH 45701, USA b c
a r t i c l e
i n f o
Article history: Received 28 December 2012 Received in revised form 11 September 2013 Accepted 6 October 2013 Available online 26 October 2013 Keywords: Enrofloxacin (ENR) Chemiluminescence enzyme immunoassay (CLEIA) Enzyme-linked immunosorbent assay (ELISA) High-performance liquid chromatography (HPLC)
a b s t r a c t A chemiluminescence enzyme immunoassay (CLEIA) based on the HRP-luminol-H2O2 chemiluminescence system for highly sensitive detection of enrofloxacin (ENR) was proposed in this study. Key factors that affect the precision and accuracy for the determination of ENR residues were optimised. Under the optimal conditions, the proposed method showed an excellent performance. The linearity range for method developed for determination of ENR was 0.35–1.0 ng/mL with a correlation coefficient greater than 0.994. The limit of detection was 0.03 ng/mL and the relative standard deviations (RSDs) were less than 9.4% and 13.0% for intra-day and inter-day assays. The proposed method was satisfactorily applied to determine ENR in milk, eggs, and honey samples at three spiked levels (0.4, 0.7, and 1.0 ng/mL) and the recoveries ranged from 92.4% to 104.2% for milk, 93.8% to 103.2% for eggs and 94.1% to 105.0% for honey, respectively. Compared the results of CLEIA with those of ELISA and HPLC, the advantages of the CLEIA were further confirmed. Moreover, one 96-well microtiter plate coated with anti-ENR can be used to detect multiple samples at the same time, which indicated that the CLEIA using HRP-luminol-H2O2 system was a sensitive, high throughput and real-time method for ENR residues analysis. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction In animal agriculture, infectious disease is an important factor affecting the profitability of livestock industries. To increase the outcome and meet the demand of the society, many antibacterial drugs are often used to prevent and treat the infectious diseases in farming practices. It is reported that the use of antibiotics in animal feeds has increased year by year all over the world (Babaahmady & Khosravi, 2011). Fluoroquinolone antibiotics are widely used in the practices of veterinary clinics because of their rapid effect and broad-spectrum antibacterial activity. Enrofloxacin is an important member of fluoroquinolone family, and has an excellent activity against mycoplasma, Gram-positive and Gram-negative pathogens by inhibiting DNA synthesis. As a consequence of its broader antibiotic spectrum, ENR is more popular than other fluoroquinolones (Babaahmady & Khosravi, 2011). However, the long-term application of fluoroquinolone would lead to an increase of resistant ⇑ Corresponding author. Address: No. 100 of Kexue Avenue, Zhengzhou City, China. Tel.: +86 37167781450. E-mail address: [email protected] (Y. Wu). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.10.024
Campylobacter species in poultry and also drug residues in animal muscle and tissue, which is a significant threat against human health through the food chain. The results of a growing number of worldwide studies have suggested that ENR is toxic to the living things. For example, ENR could inhibit cell proliferation and induce morphological changes in equine tendon and canine tendon cell cultures (Lim, Hossain, Park, Choi, & Kim, 2008; Yoon et al., 2004). Aral et al. proved that certain dosages of ENR could reduce the sperm count, deteriorate the sperm motility, and cause sperm morphological abnormalities in male mice (Aral, Karacal, & Baba, 2008). ENR showed some toxic effects on growth rate of Scenedesmus obliquus. It made the algal cells generate reactive oxygen species that damaged some biological macromolecules, and changed the biomembrane permeability (Qin, Chen, Lu, Zhao, & Yuan, 2012). ENR also showed weak effects on the total number of intestinal microbiota, caused enteric dysbacteriosis which could decrease the disease-resistant ability of resident flora in the human intestine (Chen, Yuan, Feng, Wei, & Hua, 2011). Many countries and organisations have defined the upper protection levels of ENR residues in animal foods. For example, the European Union (EU) stipulates that the maximum residue limits of ENR residues in milk and muscle tissues are 100 lg/L and
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30 lg/kg, respectively. Therefore, it is essential to establish a simple and sensitive method for detecting the ENR residues in foods to implement legislative regulations. Many analytical methods have been developed for the measurement of the ENR residues in animal foods, including high-performance liquid chromatography with diode-array detection (Cinquina et al., 2003), high-performance liquid chromatography coupled to fluorescence detection (Choi et al., 2011), ultra-performance liquid chromatography–tandem mass spectrometry (Herrera-Herrera, Hernandez-Borges, Rodriguez-Delgado, Herrero, & Cifuentes, 2011), chemiluminescence analysis (CL) (Pulgarin, Molina, & Munoz, 2011), capillary electrophoresis (Lombardo-Agui, Garcia-Campana, Gamiz-Gracia, & Blanco, 2010) and enzymelinked immunosorbent assay (ELISA) (Zhang et al., 2011). These methods have been proven sensitive and accurate, but they are not suitable for the high throughput monitoring of ENR residues because they are expensive, time-consuming, and are not environmentally friendly. Chemiluminescence enzyme immunoassay (CLEIA), which combines ELISA with CL technique and possesses the advantages of the two methods, is a simple, rapid, sensitive, low cost and specific screening assay for detection of residues in a large amount of sample. Recently, CLEIA has been widely used in variety of fields for trace analysis, including medicine (Wu et al., 2011), environment (Long, Shi, He, Sheng, & Wang, 2009), food (Fang, Chen, Ying, & Lin, 2011) and so on (Fu et al., 2012; Lin, Chu, & We, 2012). For the CLEIA, it is important to choose an appropriate chemiluminescence (CL) detection system. We have developed a CLEIA method to detect the ENR residues using the alkaline phosphatase (ALP)-adamantane (AMPPD) system as the CL detection system (Yu et al., 2012). But luminol-H2O2-horseradish peroxidase (HRP) is one of the most widely used systems in the field of CL, and it is markedly more sensitive than other CL system (Egorov, Gavrilova, & Sakharov, 2000). In this study, the chemiluminescence system of HRP-luminol-H2O2 has been used to establish the more sensitive CLEIA method to detect ENR residues in milk, egg and honey samples, and the results obtained by the proposed method were compared with those by HPLC and ELISA to illustrate the superiority of CLEIA.
2. Material and methods 2.1. Chemicals and solutions The ENR standard was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The monoclonal anti-ENR (ENR-Ab) was purchased from Santa Cruz Biotechnology (USA). Horseradish peroxidase (HRP) was obtained from ROCHE Inc. (Specific activity > 250 l/mg). Bovine serum albumin (BSA) was purchased from Sigma Chemical Corporation (USA). Reagents A and B of the luminol-H2O2 system were purchased from Sino-American Biotechnology Company (Beijing, China). All other chemical reagents were of analytical grade and Milli-Q water was used throughout the experiments. The solutions used in this experiment were as follows. The incubation solution was 0.01 mol/L of sodium phosphate buffered saline (PBS), pH of 7.2. The coating solution was 0.05 mol/L of carbonate buffer, pH of 9.6. The washing and dilution solution was 0.01 mol/L of PBS buffer containing 0.05% (v/v) Tween-20 (PBST) with pH of 7.2. The blocking solution was 0.01 mol/L of PBS buffer with 1% (w/v) BSA and 4% (w/v) cane sugar, and pH of 7.2. Because of the physicochemical property of ENR (pK1 of 5.88, pK2 of 7.74 in water), 1.0 mg/mL ENR stock solution was made by 50 mg ENR standard dissolved in 0.03 mol/L sodium hydroxide
solution (NaOH). The standard solutions were prepared by serial dilutions of the stored solution with 0.03 mol/L NaOH; and their respective concentrations were 0.35, 0.45, 0.55, 0.75, 0.85, and 1.00 ng/mL. The prepared calibrants were stored at 4 °C for use. 2.2. Apparatus The chemiluminescence signal was detected by an MP280 Chemiluminescence Immunoassay Analyser (Beijing Tai Geke letter Biological Technology Co., Ltd.). A UV-2450 UV–vis Spectrophotometre (Shimadzu) was used to identify the HRP–ENR conjugates. The 96-well polystyrene microtiter plates (Shenzhen Jincanhua Industrial Co., Ltd) were used as the solid-phase support for the antibodies. Others instruments applied in this paper for comparative analysis included the United States Diane HPLC (P680 HPLC Pump, ASI-100 Automated Sample Injector, Thermostatted Column Compartment Tcc-100, UVD170 and Chromeleon Chromatography Work Station) and the Enzyme-labelled metre (Multi-skan MK3, Thermo). 2.3. CLEIA-developed method 2.3.1. Preparation of solid phase antibody The direct immobilising method was used for the preparation of the solid phase anti-ENR. At first, the ENR-Ab solution was diluted to 3.0 lg/mL with carbonate buffer solution (pH of 9.6), and each well of the microtiter plates was coated with 100 lL ENR-Ab solution for physical adsorption. The sealed plates were allowed to stand for 150 min at 37 °C. Then the plates were washed with PBST solution for 5 times and were gently tapped against tissue paper to eliminate the free antibodies. After that, 150 lL of blocking solution was added into each well and the plates were heated at 25 °C for 2 h to block the uncoated active sites. Subsequently, the blocking solution was aspirated and the plates were washed with PBST solution for 5 times repeatly. Finally, the coated plates were made dry and stored at 4 °C for use. 2.3.2. Preparation of HRP–ENR conjugates The HRP–ENR conjugates were synthesised in two steps based on the linkage of 2 imine-carbon (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, EDC), which followed the procedures described by Watanabe (Watanabe, Satake, Kido, & Tsuji, 2002) with a further improvement. The detailed steps are as follows, a total of 20 mg ENR was dissolved in 0.1 mol/L NaOH solutions. Then 1.0 mg Nhydroxysuccinimide (NHS), 12.5 mg EDC and 1 mL dimethylformamide (DMF) were added, and the solution was mixed well and placed at 25 °C for 24 h. After that 4.0 mg of HRP dissolved in 1.0 mL of 0.01 mol/L PBS buffer (pH of 7.2) was added into the above solution slowly and stirred for 4 h in the dark at room temperature. Then the mixture was dialyzed against PBS solution (pH of 7.2) for 3 days to remove the unreacted materials. Finally, the obtained solution of HRP–ENR conjugates was stored at 4 °C not exceeding 1 month. 2.3.3. Procedure for measurement of CLEIA In the procedure of CLEIA, a total of 50 lL of ENR calibrant or food sample and 50 lL of HRP–ENR conjugate were added into each well of the coated test plates. Then the mixture was incubated at 37 °C for 1 h, in which ENR and HRP–ENR conjugate competed to combine with the ENR antibody coated on the plates. After the competition reaction, the fluid was discarded. The microplate was washed five times with PBST washing solution and blotted dry with tissue paper. Finally, 25 lL of CL substrate solution of luminol-H2O2 was added into the bottom of the test plates and incubated for 3 min at room temperature (away from the light), and the chemiluminescent signal was measured and expressed as
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relative light unit (RLU). The principles of CLEIA are demonstrated in Fig. 1. 2.4. Preparation of the food samples The milk, egg, and honey samples were all purchased from the local market. The milk and egg samples were pretreated as described in the literature (Liu et al., 2008)) and the honey sample was prepared according to references (GB/T 21330–2007). The nonfat milk sample was diluted 10-fold with PBS buffer. Approximate 5.0 g of homogeneous egg sample was weighed and added PBS buffer into it. After vibration and centrifugal separation, the supernatant solution was diluted by PBS buffer for further use. 1.00 g of honey sample was dissolved in 2 mL PBS buffer solution, then added 8 mL methylene chloride in it. After ultrasonic homogenisation and centrifugation, the supernatant solution was discarded and the remainder was dried by blowing nitrogen at 50 °C, then redissolved in PBS buffer solution for the following determination. Each sample was spiked with known variable amounts of ENR stock solution at the concentrations of 0.40, 0.70, and 1.00 ng/mL, and then analysed as described above for ENR determined by the proposed method directly. 3. Results and discussion 3.1. Optimisation of the method 3.1.1. Optimisation of the coating steps The preparation of solid phase anti-ENR was a key factor influencing the sensitivity of CLEIA. In this study, the coating buffer of ENR-Ab, the concentration of ENR-Ab, and the incubation conditions were studied and optimised. At first, dilution of ENR-Ab to 2.0 lg/mL was performed by using the following buffers: phosphate salt–citric acid (PSCA, pH of 5.0), 0.05 mol/L carbonate buffer (CB, pH of 9.6), 0.01 mol/L phosphate buffered saline (PBS, pH of 7.2), and 0.1 mol/L Tris–HCl buffer (pH of 8.5). The results for the carbonate buffer of 0.05 mol/L gave the best correlation between log RUL and log CENR and the highest RLU. Thus, 0.05 mol/L of carbonate buffer was selected for this experiment. Secondly, the concentration of ENR-Ab was investigated. The ENR-Ab solution was diluted to 0.5, 1.0, 2.0, 3.0, and 5.0 lg/mL by the carbonate buffer. The RUL and the relationship of the logRLU and log CENR were compared, it is obvious from Fig. 2 that 0.5 and 3.0 lg/mL was the best concentration of ENR-Ab. Considering the sensitivity of the method and linear relationship, the concentration of ENR-Ab was selected as 3.0 lg/mL. Finally, two incubation conditions, including incubation at 37 °C for 60–180 min and incubation at 4 °C for 16–24 h were investigated. It was found that 37 °C incubation for 150 min resulted in a higher RLU and a better
Fig. 2. The effects of concentration of ENR-Ab on chemiluminscence intensity (n = 3). The five curves correspond to different concentrations of ENR-Ab (lg/mL).
correlation coefficient. Therefore, 37 °C incubation for 150 min was finally chosen for this experiment.
3.1.2. Optimisation of the assay of Chemiluminescence In CLEIA, the chemiluminescent reaction time and substrate volume would influence the light intensity and thus the sensitivity of the method. To investigate the effect of reaction time on RLU, the kinetics curves of the chemiluminescence reaction with different concentration of ENR were measured (Fig. 3). It can be seen that the RLUs increased in the first few minutes and were stable for a short time, and then began to decline, which showed that this chemiluminescence reaction was fast and longtime-last. When the reaction time was in the range of 0–3 min, the linear range, correlation and sensitivity were satisfactory, and the reaction time of 3 min was selected as the optimal incubation time. Meanwhile, the volume of the chemiluminescent substrate was evaluated, it was found the RLUs increased by not more than one order of magnitude with the increase of substrate volume from 25 to 50 lL per well, and the smaller well size can provided adequate sensitivity. Therefore, 25 lL per well was selected in the experiment for reducing the costs.
3.2. Performance of the CLEIA system for ENR measurements 3.2.1. Calibration curve and linear range Under the selected conditions, a calibration curve for ENR was developed. The calibration curve equation of Log (y) = 6.3904– 0.3059 Log (x) with R2 = 0.9949 was obtained by plotting the
Fig. 1. Principle diagram of CLEIA for the determination of ENR.
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the mean of the blank sample plus three times the standard deviation of the blank). The minimum detectable concentration was calculated to be 0.03 ng/mL (running 5 replicate measurements), which was lower than those of ELISA and HPLC (Table 1). 3.2.3. Precision Three different ENR solutions with the concentrations of 350, 650, and 1000 pg/mL were measured for five times on the same day to obtain the intra-assay precision and measured for five times in six different days for the inter-assay precision evaluation. The intra-assay relative standard deviations varied from 7.8% to 9.4%, and the inter-assay relative standard deviations varied from 9.2% to 13.1%, which were all less than 15%. 3.3. Sample analysis and accuracy Fig. 3. The kinetics curves of the chemiluminescence reaction. The six curves correspond to different concentrations of ENR (pg/mL).
Table 1 The calibration curve, linear range, detection limit of CLEIA, ELISA and HPLC. Method
Calibration curve equation
R2
Linear range (ng/mL)
Detection limit (ng/mL)
CLEIA ELISA HPLC
y = 0.3059 +6.3904 y = 0.3054 +1.1527 y = 1.7704 +0.0509
0.9949 0.9953 0.9991
0.35–1.0 10–1000 10–1000
0.03 3.6 6.0
Table 2 The accuracies and precisions of CLEIA, ELISA and HPLC (n = 5). Method
Sample
Theoretical concentration (ng/mL)
Detectable concentration (ng/mL)
Average recovery (%)
RSD (%)
CLEIA
Milk
0.4 0.7 1.0 0.4 0.7 1.0 0.4 0.7 1.0
0.417 0.652 0.924 0.413 0.657 0.945 0.420 0.662 0.941
104.2 93.1 92.4 103.2 93.8 94.5 105 94.6 94.1
10.04 7.06 7.72 9.19 6.37 8.37 11.17 6.12 8.10
10 500 1000 10 500 1000 10 500 1000
10.75 456.71 839.24 10.644 456.60 853.32 10.676 431.50 880.99
107.5 91.3 83.9 106.4 91.3 85.3 106.8 86.3 88.1
13.92 10.96 5.96 9.97 11.88 6.35 9.55 12.99 7.66
10 500 1000 10 500 1000 10 500 1000
8.7 458.0 867.8 8.87 459.0 918.4 8.78 447.0 903.2
87.0 91.6 86.8 88.7 91.8 91.8 87.8 89.4 90.3
6.67 3.05 1.41 2.78 4.57 2.04 4.70 4.71 1.42
Egg
Honey
ELISA
Milk
Egg
Honey
HPLC
Milk
Egg
Honey
logarithm of RLU with respect to the logarithm of ENR concentration, and the linear range was 0.35–1.0 ng/mL (Table 1). 3.2.2. Limit of detection (LOD) The limit of detection (LOD) defined as the minimum amount of ENR that can be distinguished from the background signal S0 (as
The proposed CLEIA method was used to detect ENR in milk, egg, and honey samples prepared as described in Section 2.4, and the accuracy was calculated by the recovery experiments. Each sample was repeated for five times and the average recoveries are reported in Table 2. 3.4. Methods comparison To evaluate the CLEIA proposed in this paper, two other popular methods of ELISA and HPLC were performed to detect ENR in milk, egg, and honey samples. In ELISA assay, the ENR calibrators were 0, 50, 300, 500, 700, and 1000 ng/mL. The samples were prepared according to the references (GB/T 21330–2007; Liu et al., 2008), and spiked with ENR stock solution to furnish the following concentrations of ENR 10, 500, and 1000 ng/mL. Then the calibration curve, linear range, detection limit and recoveries were measured and reported in Tables 1 and 2. For HPLC analysis, conditions were as follows: column, Hypersil BDS C18 (250 mm 4.6 mm, 5 lm), mobile phase acetonitrilephosphoric buffer (contain 0.2% triethylamine, 86/14 v/v, pH of 2.2), flow rate 1.0 mL/min, detector wavelength 270 nm and column temperature 30 °C. The calibrants were 10, 50, 100, 300, 500, 750, and 1000 ng/mL. The samples were prepared according to the references (Liu, Xie, & Ding, 2004; Yue et al., 2008; Tang et al., 2005), and known concentration of ENR (10, 500 and 1000 ng/mL) was added to the samples for recovery test. Then the calibration curve, linear range, detection limit and recoveries were organised and given in Tables 1 and 2. Compared the developed CLEIA method with ELISA and HPLC methods, the results indicated that the detection limit obtained by CLEIA was the lowest(0.03 ng/mL) and the recovery of CLEIA(92.4–104.2% for milk, 93.8–103.2% for eggs, 94.1–105.0% for honey)was the best. The precision of CLEIA was not as well as HPLC, but it could meet the needs of quantification determination. 4. Conclusion A highly sensitive, specific and high-throughput screening method CLEIA based on luminol-H2O2-HRP chemiluminescence reaction system has been developed for the measurement of ENR, and has been successfully applied for the determination of ENR in spiked milk, egg, and honey samples. Compared with ELISA and HPLC, CLEIA gave better analytical performance for the detection limit and accuracy. The result suggests that the CLEIA system would be a potential and good screening tool for the rapid analysis of ENR in food samples, and also can be applied for analysis of other drug residues in food samples.
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Conflict of interest None declared. Acknowledgments This work was supported by Henan Province Major Public Research Project (No. 101100911300) and Natural Science Foundation of Henan Educational Committee (No.2011A330005). References Aral, F., Karacal, F., & Baba, F. (2008). The effect of enrofloxacin on sperm quality in male mice. Research in Veterinary Science, 84, 95–99. Babaahmady, E., & Khosravi, A. (2011). Toxicology of baytril (enrofloxacin). African Journal of Pharmacy and Pharmacology, 5, 2042–2045. Chen, T. T., Yuan, J., Feng, X., Wei, H., & Hua, W. (2011). Effects of enrofloxacin on the human intestinal microbiota in vitro. International Journal of Antimicrobial Agents, 37, 567–571. Choi, J. H., Mamun, M. I. R., EI-Aty, Abd, Park, J. H., Shin, E. H., Park, J. Y., et al. (2011). Development of a single-step precipitation cleanup method for the determination of enrofloxacin, ciprofloxacin, and danofloxacin in porcine plasma. Food Chemistry, 127, 1878–1883. Cinquina, A. L., Roberti, P., Giannetti, L., Longo, F., Draisci, R., Fagiolo, A., et al. (2003). Determination of enrofloxacin and its metabolite ciprofloxacin in goat milk by high-performance liquid chromatography with diode-array detection optimization and validation. Journal of Chromatography A, 987, 221–226. Egorov, A. M., Gavrilova, E. M., & Sakharov, I. Y. (2000). Applications of peroxidases in modern biotechnology. In H. Greppin, C. Penel, W. J. Broughton, & R. Strasser (Eds.), Integrated plant systems (pp. 369–385). University of Geneva. Fang, L. Q., Chen, H., Ying, X. T., & Lin, J. M. (2011). Micro-plate chemiluminescence enzyme immunoassay for aflatoxin B1 in agricultural product. Talanta, 84, 216–222. Fu, X. L., Meng, M., Zhang, Y., Yin, Y. M., Zhang, X. S., & Xi, R. M. (2012). Chemiluminescence enzyme immunoassay using magnetic nanoparticles for detection of neuron specific enolase in human serum. Analytica Chimica Acta, 722, 114–118. General Administration of Quality Supervision (2007). Inspection and quarantine of the People’s Republic of China, GB/T 21330–2007 determination of streptomycin residues of animal origin-enzyme-linked immunosorbent assay. Beijing: Standards Press of China. Herrera-Herrera, A. V., Hernandez-Borges, J., Rodriguez-Delgado, M. A., Herrero, M., & Cifuentes, A. (2011). Determination of quinolone residues in infant and young children powdered milk combining solid-phase extraction and ultraperformance liquid chromatography–tandem mass spectrometry. Journal of Chromatography A, 1218, 7608–7614.
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Lim, S., Hossain, M. A., Park, J., Choi, S. H., & Kim, G. (2008). The effects of enrofloxacin on canine tendon cells and chondrocytes proliferation in vitro. Veterinary Research Communications, 32, 243–253. Lin, J. H., Chu, P. F., & We, Z. J. (2012). A new dual immunoassay for tumor markers based on chemiluminescence signal amplification by magnetic mesoporous silica and enzyme modified gold nanoparticles. Analytical Sciences, 28, 21–25. Liu, B. H., Xiong, N., Wang, X. L., Shi, D. S., Li, X. Y., Peng, D. P., et al. (2008). Competitive direct ELISA for rapid detection of sulfadiazim residues in edible animal products. Chinese Journal of Veterinary Drug, 42, 16–19. Liu, Y., Xie, M. X., & Ding, L. (2004). Simultaneous determination of four luoroquinolones in eggs by high performance liquid chromatography. Chinese Journal of Analytical Chemistry, 32, 352–355. Lombardo-Agui, M., Garcia-Campana, A. M., Gamiz-Gracia, L., & Blanco, C. C. (2010). Laser induced fluorescence coupled to capillary electrophoresis for the determination of fluoroquinolones in foods of animal origin using molecularly imprinted polymers. Journal of Chromatography A, 1217, 2237–2242. Long, F., Shi, H. C., He, M., Sheng, J. W., & Wang, J. F. (2009). Sensitive and rapid chemiluminescence enzyme immunoassay for microcystin-LR in water samples. Analytica Chimica Acta, 649, 123–127. Pulgarin, J. A. M., Molina, A. A., & Munoz, S. R. (2011). Rapid chemiluminescent determination of enrofloxacin in eggs and veterinary drugs. Analytical Letters, 44, 2194–2208. Qin, H. W., Chen, L. F., Lu, N., Zhao, Y. H., & Yuan, X. (2012). Toxic effects of enrofloxacin on Scenedesmus obliquus. Frontiers of Environmental Science and Engineering in China, 6, 107–116. Tang, Y., He, H., Wu, B., Chen, H. L., Shen, C. Y., & Jiang, Y. (2005). Multiresidue determination of ten sulfonamides in honey by solid- phase extraction and high performance liquid chromatography. Journal of Instrumental Analysis, 24, 96–99. Watanabe, H., Satake, A., Kido, Y., & Tsuji, A. (2002). Monoclonal-based enzymelinked immunosorbent assay and immunochromatographic assay for enrofloxacin in biological matrices. Analyst, 127, 98–103. Wu, Y. J., Yu, S. C., Yu, F., Yan, N. L., Qu, L. B., & Zhang, H. Q. (2011). Chemiluminescence enzyme immunoassay for the determination of sulfamethoxydiazine. Spectrochimica Acta A, 81, 544–547. Yoon, J. H., Brooks, R. L., Khan, A., Pan, H., Bryan, J., Zhang, J., et al. (2004). The effect of enrofloxacin on cell proliferation and proteoglycans in horse tendon cells. Cell Biology and Toxicology, 20, 41–54. Yu, F., Wu, Y. J., Yu, S. C., Zhang, H. L., Zhang, H. Q., Qu, L. B., et al. (2012). A competitive chemiluminescence enzyme immunoassay for rapid and sensitive determination of enrofloxacin. Spectrochimica Acta Part A, 93, 164–168. Yue, Z. F., Xie, L. Q., Chen, X. X., Ji, C. N., Lin, X. Y., Hou, L. X., et al. (2008). Determination of 16 quinolones residues in milk by high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Journal of Instrumental Analysis, 27, 240–243. Zhang, H. T., Jiang, J. Q., Wang, Z. L., Chang, X. Y., Liu, X. Y., Wang, S. H., et al. (2011). Development of an indirect competitive ELISA for simultaneous detection of enrofloxacin and ciprofloxacin. Journal of Zhejiang University Science B, 12, 884–891.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Determination of residual enrofloxacin in food samples by a sensitive method of chemiluminescence enzyme immunoassay Fei Yu a, Songcheng Yu a, Lanlan Yu b, Yanqiang Li c, Yongjun Wu a,⇑, Hongquan Zhang a, Lingbo Qu d, Peter de B. Harrington e a
College of Public Health, Zhengzhou University, Zhengzhou 450001, China Department of Chemistry, Zhengzhou University, Zhengzhou 450001, China Zhengzhou Tobacco Research Institute of China Tobacco Corporation, Zhengzhou 450001, China d College of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450001, China e Department of Chemistry and Biochemistry, Center for Intelligent Chemical Instrumentation, Ohio University, Athens, OH 45701, USA b c
a r t i c l e
i n f o
Article history: Received 28 December 2012 Received in revised form 11 September 2013 Accepted 6 October 2013 Available online 26 October 2013 Keywords: Enrofloxacin (ENR) Chemiluminescence enzyme immunoassay (CLEIA) Enzyme-linked immunosorbent assay (ELISA) High-performance liquid chromatography (HPLC)
a b s t r a c t A chemiluminescence enzyme immunoassay (CLEIA) based on the HRP-luminol-H2O2 chemiluminescence system for highly sensitive detection of enrofloxacin (ENR) was proposed in this study. Key factors that affect the precision and accuracy for the determination of ENR residues were optimised. Under the optimal conditions, the proposed method showed an excellent performance. The linearity range for method developed for determination of ENR was 0.35–1.0 ng/mL with a correlation coefficient greater than 0.994. The limit of detection was 0.03 ng/mL and the relative standard deviations (RSDs) were less than 9.4% and 13.0% for intra-day and inter-day assays. The proposed method was satisfactorily applied to determine ENR in milk, eggs, and honey samples at three spiked levels (0.4, 0.7, and 1.0 ng/mL) and the recoveries ranged from 92.4% to 104.2% for milk, 93.8% to 103.2% for eggs and 94.1% to 105.0% for honey, respectively. Compared the results of CLEIA with those of ELISA and HPLC, the advantages of the CLEIA were further confirmed. Moreover, one 96-well microtiter plate coated with anti-ENR can be used to detect multiple samples at the same time, which indicated that the CLEIA using HRP-luminol-H2O2 system was a sensitive, high throughput and real-time method for ENR residues analysis. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction In animal agriculture, infectious disease is an important factor affecting the profitability of livestock industries. To increase the outcome and meet the demand of the society, many antibacterial drugs are often used to prevent and treat the infectious diseases in farming practices. It is reported that the use of antibiotics in animal feeds has increased year by year all over the world (Babaahmady & Khosravi, 2011). Fluoroquinolone antibiotics are widely used in the practices of veterinary clinics because of their rapid effect and broad-spectrum antibacterial activity. Enrofloxacin is an important member of fluoroquinolone family, and has an excellent activity against mycoplasma, Gram-positive and Gram-negative pathogens by inhibiting DNA synthesis. As a consequence of its broader antibiotic spectrum, ENR is more popular than other fluoroquinolones (Babaahmady & Khosravi, 2011). However, the long-term application of fluoroquinolone would lead to an increase of resistant ⇑ Corresponding author. Address: No. 100 of Kexue Avenue, Zhengzhou City, China. Tel.: +86 37167781450. E-mail address: [email protected] (Y. Wu). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.10.024
Campylobacter species in poultry and also drug residues in animal muscle and tissue, which is a significant threat against human health through the food chain. The results of a growing number of worldwide studies have suggested that ENR is toxic to the living things. For example, ENR could inhibit cell proliferation and induce morphological changes in equine tendon and canine tendon cell cultures (Lim, Hossain, Park, Choi, & Kim, 2008; Yoon et al., 2004). Aral et al. proved that certain dosages of ENR could reduce the sperm count, deteriorate the sperm motility, and cause sperm morphological abnormalities in male mice (Aral, Karacal, & Baba, 2008). ENR showed some toxic effects on growth rate of Scenedesmus obliquus. It made the algal cells generate reactive oxygen species that damaged some biological macromolecules, and changed the biomembrane permeability (Qin, Chen, Lu, Zhao, & Yuan, 2012). ENR also showed weak effects on the total number of intestinal microbiota, caused enteric dysbacteriosis which could decrease the disease-resistant ability of resident flora in the human intestine (Chen, Yuan, Feng, Wei, & Hua, 2011). Many countries and organisations have defined the upper protection levels of ENR residues in animal foods. For example, the European Union (EU) stipulates that the maximum residue limits of ENR residues in milk and muscle tissues are 100 lg/L and
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30 lg/kg, respectively. Therefore, it is essential to establish a simple and sensitive method for detecting the ENR residues in foods to implement legislative regulations. Many analytical methods have been developed for the measurement of the ENR residues in animal foods, including high-performance liquid chromatography with diode-array detection (Cinquina et al., 2003), high-performance liquid chromatography coupled to fluorescence detection (Choi et al., 2011), ultra-performance liquid chromatography–tandem mass spectrometry (Herrera-Herrera, Hernandez-Borges, Rodriguez-Delgado, Herrero, & Cifuentes, 2011), chemiluminescence analysis (CL) (Pulgarin, Molina, & Munoz, 2011), capillary electrophoresis (Lombardo-Agui, Garcia-Campana, Gamiz-Gracia, & Blanco, 2010) and enzymelinked immunosorbent assay (ELISA) (Zhang et al., 2011). These methods have been proven sensitive and accurate, but they are not suitable for the high throughput monitoring of ENR residues because they are expensive, time-consuming, and are not environmentally friendly. Chemiluminescence enzyme immunoassay (CLEIA), which combines ELISA with CL technique and possesses the advantages of the two methods, is a simple, rapid, sensitive, low cost and specific screening assay for detection of residues in a large amount of sample. Recently, CLEIA has been widely used in variety of fields for trace analysis, including medicine (Wu et al., 2011), environment (Long, Shi, He, Sheng, & Wang, 2009), food (Fang, Chen, Ying, & Lin, 2011) and so on (Fu et al., 2012; Lin, Chu, & We, 2012). For the CLEIA, it is important to choose an appropriate chemiluminescence (CL) detection system. We have developed a CLEIA method to detect the ENR residues using the alkaline phosphatase (ALP)-adamantane (AMPPD) system as the CL detection system (Yu et al., 2012). But luminol-H2O2-horseradish peroxidase (HRP) is one of the most widely used systems in the field of CL, and it is markedly more sensitive than other CL system (Egorov, Gavrilova, & Sakharov, 2000). In this study, the chemiluminescence system of HRP-luminol-H2O2 has been used to establish the more sensitive CLEIA method to detect ENR residues in milk, egg and honey samples, and the results obtained by the proposed method were compared with those by HPLC and ELISA to illustrate the superiority of CLEIA.
2. Material and methods 2.1. Chemicals and solutions The ENR standard was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The monoclonal anti-ENR (ENR-Ab) was purchased from Santa Cruz Biotechnology (USA). Horseradish peroxidase (HRP) was obtained from ROCHE Inc. (Specific activity > 250 l/mg). Bovine serum albumin (BSA) was purchased from Sigma Chemical Corporation (USA). Reagents A and B of the luminol-H2O2 system were purchased from Sino-American Biotechnology Company (Beijing, China). All other chemical reagents were of analytical grade and Milli-Q water was used throughout the experiments. The solutions used in this experiment were as follows. The incubation solution was 0.01 mol/L of sodium phosphate buffered saline (PBS), pH of 7.2. The coating solution was 0.05 mol/L of carbonate buffer, pH of 9.6. The washing and dilution solution was 0.01 mol/L of PBS buffer containing 0.05% (v/v) Tween-20 (PBST) with pH of 7.2. The blocking solution was 0.01 mol/L of PBS buffer with 1% (w/v) BSA and 4% (w/v) cane sugar, and pH of 7.2. Because of the physicochemical property of ENR (pK1 of 5.88, pK2 of 7.74 in water), 1.0 mg/mL ENR stock solution was made by 50 mg ENR standard dissolved in 0.03 mol/L sodium hydroxide
solution (NaOH). The standard solutions were prepared by serial dilutions of the stored solution with 0.03 mol/L NaOH; and their respective concentrations were 0.35, 0.45, 0.55, 0.75, 0.85, and 1.00 ng/mL. The prepared calibrants were stored at 4 °C for use. 2.2. Apparatus The chemiluminescence signal was detected by an MP280 Chemiluminescence Immunoassay Analyser (Beijing Tai Geke letter Biological Technology Co., Ltd.). A UV-2450 UV–vis Spectrophotometre (Shimadzu) was used to identify the HRP–ENR conjugates. The 96-well polystyrene microtiter plates (Shenzhen Jincanhua Industrial Co., Ltd) were used as the solid-phase support for the antibodies. Others instruments applied in this paper for comparative analysis included the United States Diane HPLC (P680 HPLC Pump, ASI-100 Automated Sample Injector, Thermostatted Column Compartment Tcc-100, UVD170 and Chromeleon Chromatography Work Station) and the Enzyme-labelled metre (Multi-skan MK3, Thermo). 2.3. CLEIA-developed method 2.3.1. Preparation of solid phase antibody The direct immobilising method was used for the preparation of the solid phase anti-ENR. At first, the ENR-Ab solution was diluted to 3.0 lg/mL with carbonate buffer solution (pH of 9.6), and each well of the microtiter plates was coated with 100 lL ENR-Ab solution for physical adsorption. The sealed plates were allowed to stand for 150 min at 37 °C. Then the plates were washed with PBST solution for 5 times and were gently tapped against tissue paper to eliminate the free antibodies. After that, 150 lL of blocking solution was added into each well and the plates were heated at 25 °C for 2 h to block the uncoated active sites. Subsequently, the blocking solution was aspirated and the plates were washed with PBST solution for 5 times repeatly. Finally, the coated plates were made dry and stored at 4 °C for use. 2.3.2. Preparation of HRP–ENR conjugates The HRP–ENR conjugates were synthesised in two steps based on the linkage of 2 imine-carbon (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, EDC), which followed the procedures described by Watanabe (Watanabe, Satake, Kido, & Tsuji, 2002) with a further improvement. The detailed steps are as follows, a total of 20 mg ENR was dissolved in 0.1 mol/L NaOH solutions. Then 1.0 mg Nhydroxysuccinimide (NHS), 12.5 mg EDC and 1 mL dimethylformamide (DMF) were added, and the solution was mixed well and placed at 25 °C for 24 h. After that 4.0 mg of HRP dissolved in 1.0 mL of 0.01 mol/L PBS buffer (pH of 7.2) was added into the above solution slowly and stirred for 4 h in the dark at room temperature. Then the mixture was dialyzed against PBS solution (pH of 7.2) for 3 days to remove the unreacted materials. Finally, the obtained solution of HRP–ENR conjugates was stored at 4 °C not exceeding 1 month. 2.3.3. Procedure for measurement of CLEIA In the procedure of CLEIA, a total of 50 lL of ENR calibrant or food sample and 50 lL of HRP–ENR conjugate were added into each well of the coated test plates. Then the mixture was incubated at 37 °C for 1 h, in which ENR and HRP–ENR conjugate competed to combine with the ENR antibody coated on the plates. After the competition reaction, the fluid was discarded. The microplate was washed five times with PBST washing solution and blotted dry with tissue paper. Finally, 25 lL of CL substrate solution of luminol-H2O2 was added into the bottom of the test plates and incubated for 3 min at room temperature (away from the light), and the chemiluminescent signal was measured and expressed as
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relative light unit (RLU). The principles of CLEIA are demonstrated in Fig. 1. 2.4. Preparation of the food samples The milk, egg, and honey samples were all purchased from the local market. The milk and egg samples were pretreated as described in the literature (Liu et al., 2008)) and the honey sample was prepared according to references (GB/T 21330–2007). The nonfat milk sample was diluted 10-fold with PBS buffer. Approximate 5.0 g of homogeneous egg sample was weighed and added PBS buffer into it. After vibration and centrifugal separation, the supernatant solution was diluted by PBS buffer for further use. 1.00 g of honey sample was dissolved in 2 mL PBS buffer solution, then added 8 mL methylene chloride in it. After ultrasonic homogenisation and centrifugation, the supernatant solution was discarded and the remainder was dried by blowing nitrogen at 50 °C, then redissolved in PBS buffer solution for the following determination. Each sample was spiked with known variable amounts of ENR stock solution at the concentrations of 0.40, 0.70, and 1.00 ng/mL, and then analysed as described above for ENR determined by the proposed method directly. 3. Results and discussion 3.1. Optimisation of the method 3.1.1. Optimisation of the coating steps The preparation of solid phase anti-ENR was a key factor influencing the sensitivity of CLEIA. In this study, the coating buffer of ENR-Ab, the concentration of ENR-Ab, and the incubation conditions were studied and optimised. At first, dilution of ENR-Ab to 2.0 lg/mL was performed by using the following buffers: phosphate salt–citric acid (PSCA, pH of 5.0), 0.05 mol/L carbonate buffer (CB, pH of 9.6), 0.01 mol/L phosphate buffered saline (PBS, pH of 7.2), and 0.1 mol/L Tris–HCl buffer (pH of 8.5). The results for the carbonate buffer of 0.05 mol/L gave the best correlation between log RUL and log CENR and the highest RLU. Thus, 0.05 mol/L of carbonate buffer was selected for this experiment. Secondly, the concentration of ENR-Ab was investigated. The ENR-Ab solution was diluted to 0.5, 1.0, 2.0, 3.0, and 5.0 lg/mL by the carbonate buffer. The RUL and the relationship of the logRLU and log CENR were compared, it is obvious from Fig. 2 that 0.5 and 3.0 lg/mL was the best concentration of ENR-Ab. Considering the sensitivity of the method and linear relationship, the concentration of ENR-Ab was selected as 3.0 lg/mL. Finally, two incubation conditions, including incubation at 37 °C for 60–180 min and incubation at 4 °C for 16–24 h were investigated. It was found that 37 °C incubation for 150 min resulted in a higher RLU and a better
Fig. 2. The effects of concentration of ENR-Ab on chemiluminscence intensity (n = 3). The five curves correspond to different concentrations of ENR-Ab (lg/mL).
correlation coefficient. Therefore, 37 °C incubation for 150 min was finally chosen for this experiment.
3.1.2. Optimisation of the assay of Chemiluminescence In CLEIA, the chemiluminescent reaction time and substrate volume would influence the light intensity and thus the sensitivity of the method. To investigate the effect of reaction time on RLU, the kinetics curves of the chemiluminescence reaction with different concentration of ENR were measured (Fig. 3). It can be seen that the RLUs increased in the first few minutes and were stable for a short time, and then began to decline, which showed that this chemiluminescence reaction was fast and longtime-last. When the reaction time was in the range of 0–3 min, the linear range, correlation and sensitivity were satisfactory, and the reaction time of 3 min was selected as the optimal incubation time. Meanwhile, the volume of the chemiluminescent substrate was evaluated, it was found the RLUs increased by not more than one order of magnitude with the increase of substrate volume from 25 to 50 lL per well, and the smaller well size can provided adequate sensitivity. Therefore, 25 lL per well was selected in the experiment for reducing the costs.
3.2. Performance of the CLEIA system for ENR measurements 3.2.1. Calibration curve and linear range Under the selected conditions, a calibration curve for ENR was developed. The calibration curve equation of Log (y) = 6.3904– 0.3059 Log (x) with R2 = 0.9949 was obtained by plotting the
Fig. 1. Principle diagram of CLEIA for the determination of ENR.
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the mean of the blank sample plus three times the standard deviation of the blank). The minimum detectable concentration was calculated to be 0.03 ng/mL (running 5 replicate measurements), which was lower than those of ELISA and HPLC (Table 1). 3.2.3. Precision Three different ENR solutions with the concentrations of 350, 650, and 1000 pg/mL were measured for five times on the same day to obtain the intra-assay precision and measured for five times in six different days for the inter-assay precision evaluation. The intra-assay relative standard deviations varied from 7.8% to 9.4%, and the inter-assay relative standard deviations varied from 9.2% to 13.1%, which were all less than 15%. 3.3. Sample analysis and accuracy Fig. 3. The kinetics curves of the chemiluminescence reaction. The six curves correspond to different concentrations of ENR (pg/mL).
Table 1 The calibration curve, linear range, detection limit of CLEIA, ELISA and HPLC. Method
Calibration curve equation
R2
Linear range (ng/mL)
Detection limit (ng/mL)
CLEIA ELISA HPLC
y = 0.3059 +6.3904 y = 0.3054 +1.1527 y = 1.7704 +0.0509
0.9949 0.9953 0.9991
0.35–1.0 10–1000 10–1000
0.03 3.6 6.0
Table 2 The accuracies and precisions of CLEIA, ELISA and HPLC (n = 5). Method
Sample
Theoretical concentration (ng/mL)
Detectable concentration (ng/mL)
Average recovery (%)
RSD (%)
CLEIA
Milk
0.4 0.7 1.0 0.4 0.7 1.0 0.4 0.7 1.0
0.417 0.652 0.924 0.413 0.657 0.945 0.420 0.662 0.941
104.2 93.1 92.4 103.2 93.8 94.5 105 94.6 94.1
10.04 7.06 7.72 9.19 6.37 8.37 11.17 6.12 8.10
10 500 1000 10 500 1000 10 500 1000
10.75 456.71 839.24 10.644 456.60 853.32 10.676 431.50 880.99
107.5 91.3 83.9 106.4 91.3 85.3 106.8 86.3 88.1
13.92 10.96 5.96 9.97 11.88 6.35 9.55 12.99 7.66
10 500 1000 10 500 1000 10 500 1000
8.7 458.0 867.8 8.87 459.0 918.4 8.78 447.0 903.2
87.0 91.6 86.8 88.7 91.8 91.8 87.8 89.4 90.3
6.67 3.05 1.41 2.78 4.57 2.04 4.70 4.71 1.42
Egg
Honey
ELISA
Milk
Egg
Honey
HPLC
Milk
Egg
Honey
logarithm of RLU with respect to the logarithm of ENR concentration, and the linear range was 0.35–1.0 ng/mL (Table 1). 3.2.2. Limit of detection (LOD) The limit of detection (LOD) defined as the minimum amount of ENR that can be distinguished from the background signal S0 (as
The proposed CLEIA method was used to detect ENR in milk, egg, and honey samples prepared as described in Section 2.4, and the accuracy was calculated by the recovery experiments. Each sample was repeated for five times and the average recoveries are reported in Table 2. 3.4. Methods comparison To evaluate the CLEIA proposed in this paper, two other popular methods of ELISA and HPLC were performed to detect ENR in milk, egg, and honey samples. In ELISA assay, the ENR calibrators were 0, 50, 300, 500, 700, and 1000 ng/mL. The samples were prepared according to the references (GB/T 21330–2007; Liu et al., 2008), and spiked with ENR stock solution to furnish the following concentrations of ENR 10, 500, and 1000 ng/mL. Then the calibration curve, linear range, detection limit and recoveries were measured and reported in Tables 1 and 2. For HPLC analysis, conditions were as follows: column, Hypersil BDS C18 (250 mm 4.6 mm, 5 lm), mobile phase acetonitrilephosphoric buffer (contain 0.2% triethylamine, 86/14 v/v, pH of 2.2), flow rate 1.0 mL/min, detector wavelength 270 nm and column temperature 30 °C. The calibrants were 10, 50, 100, 300, 500, 750, and 1000 ng/mL. The samples were prepared according to the references (Liu, Xie, & Ding, 2004; Yue et al., 2008; Tang et al., 2005), and known concentration of ENR (10, 500 and 1000 ng/mL) was added to the samples for recovery test. Then the calibration curve, linear range, detection limit and recoveries were organised and given in Tables 1 and 2. Compared the developed CLEIA method with ELISA and HPLC methods, the results indicated that the detection limit obtained by CLEIA was the lowest(0.03 ng/mL) and the recovery of CLEIA(92.4–104.2% for milk, 93.8–103.2% for eggs, 94.1–105.0% for honey)was the best. The precision of CLEIA was not as well as HPLC, but it could meet the needs of quantification determination. 4. Conclusion A highly sensitive, specific and high-throughput screening method CLEIA based on luminol-H2O2-HRP chemiluminescence reaction system has been developed for the measurement of ENR, and has been successfully applied for the determination of ENR in spiked milk, egg, and honey samples. Compared with ELISA and HPLC, CLEIA gave better analytical performance for the detection limit and accuracy. The result suggests that the CLEIA system would be a potential and good screening tool for the rapid analysis of ENR in food samples, and also can be applied for analysis of other drug residues in food samples.
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Conflict of interest None declared. Acknowledgments This work was supported by Henan Province Major Public Research Project (No. 101100911300) and Natural Science Foundation of Henan Educational Committee (No.2011A330005). References Aral, F., Karacal, F., & Baba, F. (2008). The effect of enrofloxacin on sperm quality in male mice. Research in Veterinary Science, 84, 95–99. Babaahmady, E., & Khosravi, A. (2011). Toxicology of baytril (enrofloxacin). African Journal of Pharmacy and Pharmacology, 5, 2042–2045. Chen, T. T., Yuan, J., Feng, X., Wei, H., & Hua, W. (2011). Effects of enrofloxacin on the human intestinal microbiota in vitro. International Journal of Antimicrobial Agents, 37, 567–571. Choi, J. H., Mamun, M. I. R., EI-Aty, Abd, Park, J. H., Shin, E. H., Park, J. Y., et al. (2011). Development of a single-step precipitation cleanup method for the determination of enrofloxacin, ciprofloxacin, and danofloxacin in porcine plasma. Food Chemistry, 127, 1878–1883. Cinquina, A. L., Roberti, P., Giannetti, L., Longo, F., Draisci, R., Fagiolo, A., et al. (2003). Determination of enrofloxacin and its metabolite ciprofloxacin in goat milk by high-performance liquid chromatography with diode-array detection optimization and validation. Journal of Chromatography A, 987, 221–226. Egorov, A. M., Gavrilova, E. M., & Sakharov, I. Y. (2000). Applications of peroxidases in modern biotechnology. In H. Greppin, C. Penel, W. J. Broughton, & R. Strasser (Eds.), Integrated plant systems (pp. 369–385). University of Geneva. Fang, L. Q., Chen, H., Ying, X. T., & Lin, J. M. (2011). Micro-plate chemiluminescence enzyme immunoassay for aflatoxin B1 in agricultural product. Talanta, 84, 216–222. Fu, X. L., Meng, M., Zhang, Y., Yin, Y. M., Zhang, X. S., & Xi, R. M. (2012). Chemiluminescence enzyme immunoassay using magnetic nanoparticles for detection of neuron specific enolase in human serum. Analytica Chimica Acta, 722, 114–118. General Administration of Quality Supervision (2007). Inspection and quarantine of the People’s Republic of China, GB/T 21330–2007 determination of streptomycin residues of animal origin-enzyme-linked immunosorbent assay. Beijing: Standards Press of China. Herrera-Herrera, A. V., Hernandez-Borges, J., Rodriguez-Delgado, M. A., Herrero, M., & Cifuentes, A. (2011). Determination of quinolone residues in infant and young children powdered milk combining solid-phase extraction and ultraperformance liquid chromatography–tandem mass spectrometry. Journal of Chromatography A, 1218, 7608–7614.
75
Lim, S., Hossain, M. A., Park, J., Choi, S. H., & Kim, G. (2008). The effects of enrofloxacin on canine tendon cells and chondrocytes proliferation in vitro. Veterinary Research Communications, 32, 243–253. Lin, J. H., Chu, P. F., & We, Z. J. (2012). A new dual immunoassay for tumor markers based on chemiluminescence signal amplification by magnetic mesoporous silica and enzyme modified gold nanoparticles. Analytical Sciences, 28, 21–25. Liu, B. H., Xiong, N., Wang, X. L., Shi, D. S., Li, X. Y., Peng, D. P., et al. (2008). Competitive direct ELISA for rapid detection of sulfadiazim residues in edible animal products. Chinese Journal of Veterinary Drug, 42, 16–19. Liu, Y., Xie, M. X., & Ding, L. (2004). Simultaneous determination of four luoroquinolones in eggs by high performance liquid chromatography. Chinese Journal of Analytical Chemistry, 32, 352–355. Lombardo-Agui, M., Garcia-Campana, A. M., Gamiz-Gracia, L., & Blanco, C. C. (2010). Laser induced fluorescence coupled to capillary electrophoresis for the determination of fluoroquinolones in foods of animal origin using molecularly imprinted polymers. Journal of Chromatography A, 1217, 2237–2242. Long, F., Shi, H. C., He, M., Sheng, J. W., & Wang, J. F. (2009). Sensitive and rapid chemiluminescence enzyme immunoassay for microcystin-LR in water samples. Analytica Chimica Acta, 649, 123–127. Pulgarin, J. A. M., Molina, A. A., & Munoz, S. R. (2011). Rapid chemiluminescent determination of enrofloxacin in eggs and veterinary drugs. Analytical Letters, 44, 2194–2208. Qin, H. W., Chen, L. F., Lu, N., Zhao, Y. H., & Yuan, X. (2012). Toxic effects of enrofloxacin on Scenedesmus obliquus. Frontiers of Environmental Science and Engineering in China, 6, 107–116. Tang, Y., He, H., Wu, B., Chen, H. L., Shen, C. Y., & Jiang, Y. (2005). Multiresidue determination of ten sulfonamides in honey by solid- phase extraction and high performance liquid chromatography. Journal of Instrumental Analysis, 24, 96–99. Watanabe, H., Satake, A., Kido, Y., & Tsuji, A. (2002). Monoclonal-based enzymelinked immunosorbent assay and immunochromatographic assay for enrofloxacin in biological matrices. Analyst, 127, 98–103. Wu, Y. J., Yu, S. C., Yu, F., Yan, N. L., Qu, L. B., & Zhang, H. Q. (2011). Chemiluminescence enzyme immunoassay for the determination of sulfamethoxydiazine. Spectrochimica Acta A, 81, 544–547. Yoon, J. H., Brooks, R. L., Khan, A., Pan, H., Bryan, J., Zhang, J., et al. (2004). The effect of enrofloxacin on cell proliferation and proteoglycans in horse tendon cells. Cell Biology and Toxicology, 20, 41–54. Yu, F., Wu, Y. J., Yu, S. C., Zhang, H. L., Zhang, H. Q., Qu, L. B., et al. (2012). A competitive chemiluminescence enzyme immunoassay for rapid and sensitive determination of enrofloxacin. Spectrochimica Acta Part A, 93, 164–168. Yue, Z. F., Xie, L. Q., Chen, X. X., Ji, C. N., Lin, X. Y., Hou, L. X., et al. (2008). Determination of 16 quinolones residues in milk by high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Journal of Instrumental Analysis, 27, 240–243. Zhang, H. T., Jiang, J. Q., Wang, Z. L., Chang, X. Y., Liu, X. Y., Wang, S. H., et al. (2011). Development of an indirect competitive ELISA for simultaneous detection of enrofloxacin and ciprofloxacin. Journal of Zhejiang University Science B, 12, 884–891.
