Magnetic Chitosan Nanocomposite Used as Cleanup Material to Detect Chloramphenicol in Milk by GC-MS

Magnetic Chitosan Nanocomposite Used as Cleanup Material to Detect Chloramphenicol in Milk by GC-MS Tianshun Liu, Jia Xie, Jianfeng Zhao, Guoxin Song & Yaoming Hu

Food Analytical Methods ISSN 1936-9751 Volume 7 Number 4 Food Anal. Methods (2014) 7:814-819 DOI 10.1007/s12161-013-9686-5

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Author's personal copy Food Anal. Methods (2014) 7:814–819 DOI 10.1007/s12161-013-9686-5

Magnetic Chitosan Nanocomposite Used as Cleanup Material to Detect Chloramphenicol in Milk by GC-MS Tianshun Liu & Jia Xie & Jianfeng Zhao & Guoxin Song & Yaoming Hu

Received: 15 April 2013 / Accepted: 17 July 2013 / Published online: 4 August 2013 # Springer Science+Business Media New York 2013

Abstract A novel method for determination of chloramphenicol in milk samples was introduced. Magnetic Fe3O4@chitosan nanocomposites were synthesized by adding chitosan to the surface of magnetic Fe3O4 using glutaraldehyde as crosslinker. Extraction with ethyl acetate, the study used the magnetic Fe3O4@CS as cleanup materials, without an additional cleanup step, to detect chloramphenicol in milk samples. To obtain maximal cleanup efficiency, several parameters were investigated including amount of magnetic materials, pH of the solution, purification time, and temperature. Under the optimal conditions (the amount of 100 mg/g of magnetic materials, pH=5, 10 min, and 20 °C), the sensitivity of the proposed method had improved about tenfold than that of without magnetic materials. Moreover, the decision limits and detection capability were 0.05 and 0.11 μg/kg, respectively, which were comparable to those measured by most methods. The rapid, simple, solvent-saving, and efficient method was proved to be robust in monitoring [D(−)-theo-2-dichloroacetamido-1-pnitrophenyl-1,3-propanediol] in milk samples. Keywords Chloramphenicol . Milk . Magnetic Fe3O4@CS . Cleanup efficiency . GC-MS

Introduction Antibiotics are widely used in veterinary medicine to control and prevent infectious diseases, as also for animal growth promotion, to improve production efficiency. The use of antibiotics may result in drug residues present in animal foodstuffs, the side effects of which would pose threat to human health.

T. Liu : J. Xie : J. Zhao : G. Song (*) : Y. Hu (*) Research Center of Analysis and Measurement, Fudan University, Shanghai 200433, People’s Republic of China e-mail: [email protected] e-mail: [email protected]

Among them, chloramphenicol [D(−)-theo-2-dichloroacetamido1-p-nitrophenyl-1,3-propanediol] (CAP) is a broad-spectrum antibiotic used as a cheap and effective drug against many Gram-negative and Gram-positive bacteria (D’Aoust 1994). However, researches have shown that the drug has serious adverse effects to human which can cause aplastic anemia, leukemia, and induce gray baby syndrome (Gikas et al. 2004; Huang et al. 2006). These potential hazards have led to a prohibition of its use in food-producing animals in China, Japan, Canada, USA, Australia, the European Union (EU), and some other countries (Wang et al. 2011). For a strict control of this compound, the European Commission has established a minimum required performance limit (MRPL) of the analytical method for the determination of CAP in different animal products of 0.3 μg/kg (Commission Decision 2003/181/EC of 13 March 2003), China even has enforced the implementation of zero-tolerance level of CAP (Xu et al. 2012). Nevertheless, because of its easy access, low cost, and effectiveness, CAP is still illegally abused in animal and traces of it have been found in various food samples, including milk. Therefore, it should be highly desirable to develop a sensitive method that can monitor the residues of CAP in food samples. In recent years, a variety of analytical techniques have been used to determine CAP residues in foods. In these cases, gas chromatography (GC) with electron capture detector (Ding et al. 2005; Pengov et al. 2005) and mass spectrometric (MS) detection (Xie et al. 2005), high-performance liquid chromatography combined with electrospray ionization ion trap tandem mass spectrometry (Hamscher et al. 2005) and ultraviolet detection (Posyniak et al. 2003; Tyrpenou et al. 2002), liquid chromatography (LC; Chen et al. 2008; Moragues et al. 2012), LC-MS (Fujita et al. 2008; Huang et al. 2006; Turnipseed et al. 2003; Wang et al. 2011), LC-MS/MS (Gikas et al. 2004; Shenridan et al. 2008), and enzyme-linked immunosorbent assay (ELISA) (Gaudin et al. 2003; Tajik et al. 2010; Wesongah et al. 2007; Zhang et al. 2006) have been applied for CAP determination in different types of samples.

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However, conventional chromatographic methods for the analysis of CAP require complicated pretreatment of samples, which need expensive apparatus and reagents and they are time consuming. Although ELISA is often used for routine analysis of CAP, it is not reliable because of cross-reaction interference (Biancardi 1997). Therefore, novel, rapid and accurate methods are required to monitor CAP in samples. Chitosan (CS), α-(1-4)-2-amino-2-deoxy-β-D-glucan, is a deacetylated form of chitin, an abundant natural polysaccharide found in crustacean shells. Due to its unique characteristics, the use of chitosan with or without derivation in the field of medicine (Li et al. 2004; Zhu et al. 2007), water and food treatment (Wang et al. 2010; Zhou et al. 2011), and metal recovery (Liu et al. 2009) has been reported in the last decades. It had been found that chitosan could absorb acid dye in waste water (Zhou et al. 2011), remove lipids from cheese whey (Hwang and Damodaran 1995) and control the molecular self-aggregation in the aqueous system with some modification (Chen et al. 2003). Fe3O4@CS nanoparticles have also attracted an increasing interest for application to remove aqueous humic acid (Wang et al. 2010), bovine serum albumin (Zhu et al. 2007), protein (Dong and Ren 2007), and heavy metal ion (Liu et al. 2009) due to their low toxicity, easy preparation, fast separation, and high adsorption ability. However, as far as we know, there are no reports based on the Fe3O4@CS nanoparticles to detect CAP residue in milk samples. In the study, a simple method comprising a single sample extraction with ethyl acetate followed by Fe 3 O 4 @CS nanoparticles which were used as purificatory materials, but without an additional cleanup step, was developed to detect chloramphenicol in milk by GC-MS. The factors that affected the optimization of the method were also investigated.

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1.35 g FeCl3 ·6H2O and 75 mL glycol were added in a dry beaker, and the mixture was stirring until it turned to be transparent. Then, 3.6 g sodium acetate was added to the solution. After being vigorously stirred for 60 min, the mixture was transferred to an autoclave. The autoclave was heated at 200 °C for 16 h, and then allowed to cool down to room temperature. The obtained products were magnetically collected and washed with deionized water/ethanol for several times and vacuum dried at 50 °C for 24 h. In the following step, Fe3O4@CS nanoparticles were synthesized according to the method by Li et al. (2004). The resultant Fe3O4 microspheres were dispersed in 100 mL deionized water, then 0.3 g chitosan in 20 mL 5 % acetic acid solution was added to the mixture followed by 0.5 mL Tween80 with stirring for 30 min. Next, 3 mL 25 % glutaraldehyde was added to the mixture. When it was stirred at room temperature for 12 h, Fe3O4@CS nanoparticles were isolated with a bar of magnet and washed with deionized water and ethanol successively. The products were finally vacuum-dried at 50 °C for 12 h for further analysis. Sample Preparation About 0.3 g milk sample and 1 mL CAP (100 μg/mL in ethyl acetate) and 10 μL internal standard were injected to a test tube followed by 1 mL ethyl acetate. Then, the mixture was subsequently centrifuged at the rate of 1,000 r/min for 5 min. The layer of ethyl acetate was separated, treated with Fe3O4@CS nanoparticles, and further dried under nitrogen flow. The dry residue was silanized with 100 μL of BSTFA:TMCS (99:1) at the temperature of 100 °C for 20 min. After that, 1 μL derivative was injected in GC-MS to analyze. Instrumentation

Materials and Methods Samples and Reagents Milk samples were purchased from local markets in Shanghai, China, which had proved pure without chloramphenicol. Chloramphenicol (CAP) and chloramphenicol-D5 (CAP-D5; internal standard) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Chitosan (≥90 % deacetylation) was acquired from Shanghai chemical reagent company in China. NO-bis-(trimethylsilyl) trifluoroacetamide (BSTFA) with 1 % trimethylchlorosilane (TMCS) was purchased from Supelo (Stintra, Portugal). All other reagents and solvents used were of analytical grade. Synthesis of Fe3O4@CS Nanoparticles Fe3O4 microspheres were synthesized via a solvothermal process according to the method by Meng et al. (2011). Briefly,

The silanized samples were analyzing by a Thermo FocusDSQ GC-MS instrument. A GsBP-5MS (General Separation Technologies, Delaware, USA) capillary column (30 m× 0.25 mm×0.25 μm) coated with 5 % phenyl–95 % dimethyl polysiloxane was used. The carrier gas was helium at the flow of 1 mL/min with a splitless mode. Temperature conditions were as follows: 100 °C maintained for 2 min, then subsequently programmed from 100 to 300 °C at a rate of 30 °C/ min and maintained for 2 min. The injection temperature and electron ionization were set at 250 °C and 70 eV, respectively. Data were obtained with selected ion monitoring mode. The target compound and internal standard were identified on the basis of the retention times and the corresponding ions: m/z=225, 208, 361, and 451 for CAP and m/z=230, 213, 366, and 456 for CAP-D5. Quantification ions were 225 and 230, respectively. The success of forming of Fe3O4@CS was proved by Nicolet Nexus 470 Fourier-transform infrared spectrophotometer, which was purchased from Nicolet in the USA.

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Results and Discussion

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The infrared spectra of (a) chitosan, (b) Fe3O4@CS, and (c) Fe3O4 were shown in Fig. 1. The most intense signal at 573 and 576 cm−1 were Fe–O stretching of Fe3O4 and Fe3O4@CS, respectively. For the spectra of Fe3O4@CS, the broad peak from 3,400 to 3,500 cm−1 was observed as N–H and O–H stretching. The methyl (–CH3) and methylene (–CH2) characteristic vibration bands were observed at 2,922 and 2,854 cm−1, respectively. And the bond at 1,625 cm−1 was related to C=N stretching vibration of Schiff’s base, which proved the reaction between chitosan and glutaraldehyde. What is more, the bond at 1,051 cm−1 was probably assigned to C–O stretching. Therefore, the results demonstrated the success of forming of Fe3O4@CS nanoparticles.

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576 573 3500 3000 2500 2000 1500 1000 wavenumbers(cm-1)

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Fig. 1 FITR spectra of chitosan (a), Fe3O4@CS (b), and Fe3O4 (c)

Fig. 2 The chromatograms of milk before cleanup (a) and after cleanup (b)

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Recovery Abundance

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Due to its hydroxyl group and amino group, Fe3O4@CS was easy to precipitate triglycerides and amino acids via the process of flocculation and chelation. Figure 2 showed the chromatograms of milk samples before cleanup (Fig. 2a) and after cleanup (Fig. 2b). It was clear to see that peak area of CAP in purified sample was much larger than that of unpurified sample. In this study, we took the CAP peak area ratio of purified milk to standard sample as the indicator to evaluate the cleanup efficiency. The dominant parameters that affected the cleanup efficiency, including amount of magnetic materials, pH of solution, purification time, and temperature, were investigated.

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Amounts of Fe3O4@CS Particles The effect of the amount of purified materials on the cleanup efficiency was investigated. The experiments of milk samples were performed using different amounts (from 7.5 to 45 mg) of the synthesized materials; other parameters were set as follows: pH=5, temperature was kept at 20 °C, and time was 10 min. Obtained results were presented in Fig. 3, the cleanup efficiency increased gradually when more materials were used. In particular, when the amounts increased from 22.5 to 30 mg, the cleanup efficiency enhanced sharply. After that, the cleanup efficiency increased slightly even when the amount of materials reached 45 mg. It may be that the matrixes and magnetic materials had basically reached a balance when 30 mg materials were used. Therefore, we selected 30 mg as the optimal amount for the following analysis. Influence of PH Value of Solution The pH value of solution could change the properties of matrixes and magnetic materials (Liu et al. 2009), which would affect the cleanup efficiency. To investigate the influence

Cleanup efficiency

Application of Fe3O4@CS to Purify the Milk Samples

0.6 0.4 0.2 0.0

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Fig. 4 The effect of solution PH on the cleanup efficiency

of pH, the pH was changed from 3 to 9; the amount of materials was set as 30 mg, temperature was kept at 20 °C, and time was 10 min. The results were shown in Fig. 4; it showed that pH had a significant effect on the cleanup efficiency. With the increase of pH from 5 to 9, the amount of adsorbed matrixes on magnetic particles decreased significantly. We found that, at neutral or alkaline condition, magnetic particles had scarcely any function of cleanup in milk samples. And the maximum cleanup efficiency occurred approximately at pH=5, which was close to isoelectric point of protein in milk samples. When the pH decreased from 5 to 3, the cleanup efficiency had a slightly variation. A possible explanation for the influence of pH on the cleanup efficiency might be related to surface charge of magnetic particles and proteins (Peng et al. 2004). At isoelectric point, proteins in milk samples underwent the minimum conformational change, which was propitious to reach a maximal adsorption on magnetic particles. Meanwhile, at the isoelectric point of protein, magnetic particles had a positive charge; the function of electrostatic adhesion between them could contribute to the adsorption. Therefore, pH=5 was chosen as the best value for cleanup.

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Fig. 3 The effect of different amounts of Fe3O4@CS on the cleanup efficiency

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Fig. 5 The effect of time on the cleanup efficiency

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Fig. 6 The effect of temperature on the cleanup efficiency

Purification Time The study of the purification time was carried out with five different minutes: 2, 5, 10, 15, and 20 min. Previously optimized amount of magnetic particles and pH were also used. Purification temperature was set at 20 °C. The obtained results could be observed in Fig. 5. It could be seen that when the purification time reached 5 min, the system basically achieved adsorption balance. Considering higher cleanup efficiency, we selected 10 min as the optimal purification time.

decision limits (CCα), and detection capability (CCβ). Reproducibility and recovery experiments were conducted at the three specified fortified levels; the spiking levels were 1×MRPL, 1.5×MRPL, and 2×MRPL (which corresponded to 0.3, 0.45, and 0.6 μg/kg). As there is no maximum permit level for chloramphenicol, it is more relevant to report the sensitivity of the method as CCα and CCβ. The CCα was calculated from the within-laboratory reproducibility date by spiking 20 blank samples at the above described level. The corresponding concentration at the CCα plus 1.64 times the standard deviation of the within-laboratory reproducibility. All the parallel experiments were repeated six times and the results were listed in Table 1. Compared with other methods, the CCα of chloramphenicol in milk samples of the proposed method was not only below the maximum residue limit, but also comparable to those of most methods (Gasilova and Eremin 2010; Long et al. 1990; Mamani et al. 2009; Wang et al. 2011). Recently, the electrode modified with molecularly imprinted polymer had been used to detect the CAP in milk samples with the limit of detection of 2 ng/mL (Alizadeh et al. 2012); however, this method could only be applied for the CAP determination. More researches are needed to detect multiplicate antibiotics simultaneously with the proposed method in the further study.

Purification Temperature Conclusion After time optimization, an evaluation of the best purification temperature including 20, 30, 40, and 50 °C was carried out. From the observation in Fig. 6, it indicated that the cleanup efficiency decreased gradually with the temperature increasing. Two possible reasons could explain this phenomenon: (1) at the relatively high temperature, the increasing motion of molecules resulted in difficult adsorption between matrixes and magnetic materials. (2) Ethyl acetate hydrolyzed at high temperature, which caused low cleanup efficiency. So, the results verified that 20 °C gave the best cleanup efficiency. Method Validation The validation procedure was realized through a series of experiments of determination of linearity, reproducibility, Table 1 The validation date of cleanup procedure

In this work, Fe3O4 microspheres were synthesized via a solvothermal process; then, CS was introduced to magnetic Fe3O4 so the magnetic Fe3O4@CS materials were synthesized. For extraction with ethyl acetate, we used the magnetic Fe3O4@CS as cleanup materials, without an additional cleanup step, to detect chloramphenicol in milk samples, which was much solvent-saving and efficient than that of rapid liquid–liquid extraction method. Under optimal conditions, the sensitivity had improved about tenfold than that of without magnetic materials. Moreover, the decision limits and detection capability were 0.05 and 0.11 μg/kg, respectively, which were comparable to those measured by most methods. The rapid, simple, solvent-saving, and efficient method was proved to be robust in monitoring CAP in milk samples.

Analyte

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0.3 0.45 0.6

90.3 91.1 89.7

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0.11

Author's personal copy Food Anal. Methods (2014) 7:814–819 Conflict of Interest Tianshun Liu declares that he has no conflict of interest. Jia Xie declares that she has no conflict of interest. Jianfeng Zhao declares that he has no conflict of interest. Guoxin Song declares that he has no conflict of interest. Yaoming Hu declares that he has no conflict of interest. This article does not contain any studies with human or animal subjects

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