Yunkai Lv et al.

, IJSID, 2012, 2 (6), 617-622

ISSN:2249-5347

IJSID

International Journal of Science Innovations and Discoveries

Research Article

An International peer Review Journal for Science

Available online through www.ijsidonline.info

DETERMINATION OF TETRACYCLINE IN MILK SIMPLES

MAGNETIC DISPERSION EXTRACTION BASED ON RESTRICTED ACCESS MEDIA-MAGNETIC MICROSPHERE FOR College of Chemistry and Environmental Science, Hebei University, Key Laboratory of Analytical Science and Technology of Yun-Kai Lv*, Chen-Xi Zhao, Yan-Dong He, Hui Zhang, Xue Xiong Hebei Province, Baoding 071002, China

Received: 04-11-2012 Accepted: 24-12-2012

*Corresponding Author

ABSTRACT (RAM-MMs) was developed for a single step extraction and clean-up of trace tetracycline antibiotics residues in food. The RAM-MMs were synthesized by the suspension polymerization method using Fe3O4 magnetite as the magnetically susceptible component, respectively. The hydrophilic layer around polymeric magnetic microspheres was created by opening glycidilmethacrylate (GMA) epoxide with perchloric acid. The proposed A novel synthesis method of restricted access media - magnetic microspheres

styrene (St) and divinylbenzene (DVB) as functional monomer and cross-linking agent, method was successfully applied to extraction of trace tetracycline antibiotics residues in milk. The recoveries in milk simple were in the range of 81.5% to 89.4% with the RSD of 0.01538 mg/kg, respectively. milk Keywords: Restricted access materials; Suspension Polymerization; Magnetic microsphere;

Address: Name: Yun-Kai Lv Place: Baoding, China E-mail: lvyunkai@hbu.edu.cn

1.24-2.6%. The limit of detection (LOD) and quantitation (LOQ) were 0.004621 and

INTRODUCTION

INTRODUCTION

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Yunkai Lv et al., IJSID, 2012, 2 (6), 617-622 mycoplasma. The effect of tetracycline was negative bacterias, and it was highly active against staphylococcus, strep- tococcus, and Chinese Ministry of Agriculture have established a maximum residue limit (MRL) of 0.1 mg/kg for tetracycline antibiotics in milk [3-5]. Therefore, a suitable monitor for tetracycline antibiotic residues in food is of great importance. food samples without any preliminary sample preparations. In the present paper, many methods have been described for Tetracycline (TC) is one of the tetracyclines (TCs), a group of broad-spectrum antibiotics, and tetracycline produced by INTRODUCTION

pneumococcus, gonococcus, cholera, dysentery bacillis, pertussis, rickettsia, chlamydia and mycoplasma [1-2]. So tetracycline is

used to a variety of food-producing animals such as cattle, sheep, goats, pigs, poultry and so on, but in some cases, these drug residues can threat human health seriously. Consequently, the US Food and Drug Administration (FDA), European Union (EU) Because analytes are present in trace amount and the matrix is rather complex, therefore, it is difficult to directly analyze

extraction and clean up of tetracycline antibiotics in various edible products, such as liquid-liquid extraction (LLE) [6], solidphase extraction (SPE) [7-9], ultrasonic-assisted extraction (USE) [8], accelerated solvent extraction (ASE) [9, 10], microwaveAlthough these technique have the remarkable merit of higher precision and smaller amounts of extraction solvent, these Consequently, the new technique has received increasing attention in recent years due to their potential application in technique is that magnetic supports can be isolated from sample solutions by the application of an external magnetic field unique physical and chemical properties, it has been widely applied in many fields [14]. without additional centrifugation or filtration, which makes separation easier and faster. Because of its large surface area, and polymerization method using Fe3O4 magnetite as the magnetically susceptible component, styrene (St) and divinylbenzene polymeric magnetic microspheres was created by opening glycidilmethacrylate (GMA) epoxide with perchloric acid. We have tetracycline antibiotics residues in food. Chemicals and Reagents: demonstrated successful preparation of the hydrophilic magnetic microspheres, and applied to extraction of tetracycline EXPERIMENTAL assisted extraction (MAE) [11], solid-phase microextraction extraction (SPME) [12], matrix solid-phase dispersion (MSPD) [13]. environmental and biological samples, which was Magnetic separation technique. A unique and attractive property of this tedious and costly methods can hardly completely separate the low-molecular weight analytes from large biomolecules.

(DVB) as functional monomer and cross-linking agent, respectively, for forming hydrophobic layer. The hydrophilic layer around antibiotics residues in milk samples. The proposed method was found to provide a single step extraction and clean-up of trace

In the present study, a restricted access media - magnetic microsphere (RAM-MM) was synthesized by the suspension

triglyceride (GMA) and divinylbenzene (DVB) were purchased from Nanjing Lianye Chemical Co., Ltd. (Shanghai, China) which analytical or the HPLC grade and used without further disposal. Doubly deionized water (DDW) was used throughout. Samples for HPLC were filtered through a 0.45 m membrane filter.

was cleaned to remove the inhibitor prior to polymerization. Iron (II) chloride tetrahydrate (FeCl24H2O), iron (III) chloride methanol) were obtained from Beijing Chemical Reagent Company (Beijing, China). All the other chemicals were of the 0.1 M of a Na2EDTA-McIlvaine buffer solution was prepared by mixing 1000 ml of 0.1 M citric acid with 625 ml of 0.2 M International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012

hexahydrate (FeCl36H2O), oleinic acid, hydroxyethyl cellulose (HEC), 2,2-Azobisisobutyronitrile (AIBN, recrystallized from

Tetracycline hydrochloride (TC) was obtained from Fluka (Buchs, Switzerland). Styrene (St), methyl methacrylate

disodium hydrogen phosphate (pH adjusted to 4.0 0.05 with NaOH or HCl as needed), and then 60.5 g of Na 2EDTA2H2O was

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added into the above mixture [32]. Chromatographic Conditions:

Yunkai Lv et al., IJSID, 2012, 2 (6), 617-622

detector and RF-10AXL (Shimadzu, Japan). The analytes were separated in a Venusil XBP C18 column (1504.6 mm, 5 m) from Bonna-Agela Technologies (Tianjin, China). The mobile phase was methanol / acetonitrile / 10 mM oxalic acid solution chromatograms were recorded at 350 nm. Preparation of the RAM-MMs: The restricted access media - magnetic microspheres were prepared as followed: (5:25:70, v/v) and the flow rate was 1.0 ml/min at 25 oC. Aliquots of 10 l were injected into the column and the

HPLC analysis was performed using a liquid chromatography system containing a LC-20AT pump, a SPD-20A UVvis

was stirred vigorously at 80 oC and purged with nitrogen gas. Then 40 ml of NaOH solution (26% Wt) was added dropwise approximately 7-8. (b) Fe3O4 (1 g) magnetite was added into a 50 ml beaker, and then 2.0 ml of oleic acid was also added into it. After

into the mixture. After 4.0 h, the temperature of suspension was cooled to below 10 oC. The final pH value was stirred the mixture with a muddler for several minutes and ultrasound dispersed for 5 min, the oleic acid spreaded on the all magnetites evenly. The magnetic precipitates were isolated from the solvent by a permanent magnet. After the grafting reaction, the magnetic precipitates were washed several times with ethanol to remove the unreac ted oleic acid. 50 ml beaker, and the mixture was stirred vigorously and completely. So the preparation of oil phase accomplished. 90 oC, meanwhile, the stirring rate was raised to 650 rpm. The reaction was allowed to proceed for another 5 h. approximately 7. Finally, the separated product was dried in a vacuum oven at 40 oC for 24 h. Sample Preparation:

(a) Typically, FeCl 24H2O (1.6 g) and FeCl 36H2O (4.6 g) were dissolved in distilled water (100 ml), and the mixture

apparatus for several minutes. St (10 ml), DVB (2 ml), GMA (4.6 ml) and oleic-acid-grafted magnetites were added into the completed dissolved, the oil phase added into it. The mixture was stirred at the rate of 550 rpm; meanwhile, the reaction 25 ml of a perchloric acid water solution (20% v/v). The flask was then stried (200 rpm) for 24 h at room temperature. was allowed to proceed at 60 oC. After 1.0 h, AIBN (0.1 g) was added into the actor, and the temperature was then raised to

(c) AIBN (0.1 g) and toluene (0.5 ml) were added into a 50 ml beaker, and stirring the mixture with magnetic stirring

(d) HEC (0.4 g), NaCl (0.5 g) and H2O (100 ml) were added into three-necked flask and stirred. When the solid (e) After the polymerization, the polymers were separated, and washed with water for several times, and added to

When the reaction was stopped, the magnetic polymer was washed with water for several times, a nd the final pH value was

and 20 ml Mcllvaine-Na2EDTA buffer added into it. The mixture was ultrasound - assisted extraction for 5 min at the room the residue in order to extract twice, and extract liquid prepared in the two steps is merged. The extract liquid was evaporated at 45 oC by rotary evaporators [7]. Application of RAM-MMs for Magnetic Dispersion Extraction of TC from Milk Sample: After the extraction was complete, the residues were dissolved with dichloromethane/methanol (92:8, v/v) to 5 ml, and International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012

temperature, and the tubes were centrifuged at 4000 rpm for 10 min. 15 ml Mcllvaine - Na2EDTA buffer solution was added into

The milk samples were obtained from a local supermarket. 5 g of milk samples were weighed into a 50 ml centrifuge tube,

filtered through a 0.45 m syringe filter. The filtrate were added into the beaker and mixed with the magnetic microspheres, and the mixture was stirred for 20 min. The magnetic particles with adsorbed TC were washed with 22.0 ml dichloromethane.

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Finally, the magnetic microspheres with adsorbed TC were eluted by 1 ml methanol solution containing 25% acetic acid three times. The filtrate was applied for HPLC analysis and recovery test. The milk extracts were spiked with TC at three concentration levels of 0.1, 0.2, 0.5 mg/kg. Experiments were repeated three times. Optimization of Preparation Conditions: RESULTS AND DISCUSSION

Yunkai Lv et al., IJSID, 2012, 2 (6), 617-622

So the preparation method of the magnetic microspheres was optimized by changing some of the preparation factors, consist of temperature control.

water/oil mass ratio, stirring rate, the proportion of surfactant, the initiator (AIBN) molar amount in the total moles of monomer, to 650 rpm after 1 h, the micro-size microspheres were prepared, and its diameter uniformity is great good. When the amount of initial temperature was 60 C and the temperature was raised to 90 C after 1 h, the reaction time was moderate; and were prepared under optimal conditions and shown in Figure 1.

The characters of magnetic microspheres include the size of microsphere, magnetism, degree of homogeneity and so on.

reaction steady, and degree of homogeneity was good. When the initial stirring rate was 550 rpm and the stirring rate was raised

surfactant was 0.4 g, the microspheres sticky hardly together; and the sphere diameter was reduced significantly. When the

As can be seen from Table 1, when the initiator (AIBN) molar amount in the total moles of monomer was 0.2 g, the

microspheres didnt sticky together. When the water/oil mass ratio was 5:1, the reaction system not only had characteristic of other proportion or amounts of preparation conditions were also shown in the Table 1 respectively. The magnetic microspheres

high stability, but also meets the requirement of suspension polymerization. Above all the optimization of preparation conditions, Table 1. Effects of the different amounts of initiator and surfactant, water/oil mass ratios (W/O), stirring rates and temperatures on the polymerization. Affecting Factors Initiator (g) Variable 0.08 0.1 0.15 0.2 0.3 0.1 0.2 0.3 0.4 4:1 5:1 6:1 450 550 650 750 60 90 60, 70 60, 90 Particle Size very small, nonuniform small, nonuniform smaller, uniformity small, uniformity larger, uniformity large, nonuniform larger, nonuniform smaller, uniformity small, uniformity Coating Effect bad bad preferably good bad bad preferably good good grume good good good good preferably bad grume grume good good Yield (%) 95.6 96.0 97.3 97.8 98.0 96.5 97.3 97.5 97.8 97.5 97.1 97.6 98.0 97.5 53 96.3 97.2

Surfactant (g) W/O

Initial Stirring Rate (rpm) Temperature (oC)

larger, uniformity larger, uniformity large, uniformity small, uniformity very small, nonuniform smash, nonuniform large, nonuniform smaller, uniformity

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Yunkai Lv et al., IJSID, 2012, 2 (6), 617-622

Magnetic Dispersion Extraction and Analytical Parameters of Method:

the chromatogram of the milk simple has some miscellaneous peak, which indicate that the milk sample are impurity before the clean-up step. But the Figure 2b (the spiked milk after Magnetic microspheres) shows that not only quantities of interferences magnetic microspheres sorbents.
1500 1000 500 0 -500 -1000 0 2 4 6 TC

were removed in the washing step with dichloromethane. It also indicated high selectivity and better clean-up of the hydrophilic

HPLC chromatogram obtained by analyzing milk samples are shown in Figure 2. The Figure 2a (the spiked milk) shows

Figure 1. Photograph of the magnetic microspheres with optimal conditions by the TEM

(a)

1500 1000 500 0

V (b)

TC

-500 -1000 0 2 4 6

the three concentrations (0.1, 0.2 and 0.5 mg/kg), respectively. Relative standard deviations (RSDs) lower than 2.6%, and the LOD (n=3) and LOQ (n=10) were 0.004621 and 0.01538 mg/kg, which calculated based on the analyte concentration producing signal/noise ratio of 3:1 and 10:1, respectively. In this study, we have demonstrated the successful preparation of the hydrophilic magnetic microspheres, and applied to CONCLUSION

Figure 2. Chromatogram of tetracycline in milk samples (a) milk simple spiked with 0.1 mg/kg of TC; (b) spiked milk after magnetic microspheres.

t (min)

t (min)

The recoveries of the TC by the spiked milk simple were in the range of 72.890.1%, 80.593.4.0% and 86.292.5% for

extraction of tetracycline antibiotics residues in milk samples. These magnetic microspheres prepared were applied to the sample pretreatment step, which was quickly, facility and environmental friendly. The proposed analytical method is simple, accurate and sensitive. It is suitable for the determination of pharmaceutical and contaminant residues in food samples. International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012

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Yunkai Lv et al., IJSID, 2012, 2 (6), 617-622 10967126D) and the Natural Science Foundation of Hebei Province (No. B2011201081). REFERENCES
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The authors gratefully appreciate the financial support by the Hebei Provincial Key Basic Research Program (No. ACKNOWLEDGEMENTS

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