Yunkai Lv et al., IJSID, 2012, 2 (6), 617-622 ISSN:2249-5347 IJSID International Journal of Science Innovations and Discoveries An International peer Review Journal for Science Research Article Available online through www.ijsidonline.info MAGNETIC DISPERSION EXTRACTION BASED ON RESTRICTED ACCESS MEDIA-MAGNETIC MICROSPHERE FOR DETERMINATION OF TETRACYCLINE IN MILK SIMPLES Yun-Kai Lv*, Chen-Xi Zhao, Yan-Dong He, Hui Zhang, Xue Xiong
College of Chemistry and Environmental Science, Hebei University, Key Laboratory of Analytical Science and Technology of
A novel synthesis method of restricted access media - magnetic microspheres
(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 suspensionpolymerization method using Fe3O4 magnetite as the magnetically susceptible component,styrene (St) and divinylbenzene (DVB) as functional monomer and cross-linking agent,respectively. The hydrophilic layer around polymeric magnetic microspheres was createdby opening glycidilmethacrylate (GMA) epoxide with perchloric acid. The proposedmethod 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
1.24-2.6%. The limit of detection (LOD) and quantitation (LOQ) were 0.004621 and0.01538 mg/kg, respectively. INTRODUCTION Place: Keywords: Restricted access materials; Suspension Polymerization; Magnetic microsphere; E-mail: INTRODUCTION International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012 Yunkai Lv et al., IJSID, 2012, 2 (6), 617-622 INTRODUCTION
Tetracycline (TC) is one of the tetracyclines (TCs), a group of broad-spectrum antibiotics, and tetracycline produced by
mycoplasma. The effect of tetracycline was negative bacterias, and it was highly active against staphylococcus, strep- tococcus,pneumococcus, gonococcus, cholera, dysentery bacillis, pertussis, rickettsia, chlamydia and mycoplasma [1-2]. So tetracycline isused to a variety of food-producing animals such as cattle, sheep, goats, pigs, poultry and so on, but in some cases, these drugresidues can threat human health seriously. Consequently, the US Food and Drug Administration (FDA), European Union (EU)and Chinese Ministry of Agriculture have established a maximum residue limit (MRL) of 0.1 mg/kg for tetracycline antibiotics inmilk [3-5]. Therefore, a suitable monitor for tetracycline antibiotic residues in food is of great importance.
Because analytes are present in trace amount and the matrix is rather complex, therefore, it is difficult to directly analyze
food samples without any preliminary sample preparations. In the present paper, many methods have been described forextraction and clean up of tetracycline antibiotics in various edible products, such as liquid-liquid extraction (LLE) , solid-phase extraction (SPE) [7-9], ultrasonic-assisted extraction (USE) , accelerated solvent extraction (ASE) [9, 10], microwave-assisted extraction (MAE) , solid-phase microextraction extraction (SPME) , matrix solid-phase dispersion (MSPD) . Although these technique have the remarkable merit of higher precision and smaller amounts of extraction solvent, thesetedious and costly methods can hardly completely separate the low-molecular weight analytes from large biomolecules. Consequently, the new technique has received increasing attention in recent years due to their potential application inenvironmental and biological samples, which was Magnetic separation technique. A unique and attractive property of thistechnique is that magnetic supports can be isolated from sample solutions by the application of an external magnetic fieldwithout additional centrifugation or filtration, which makes separation easier and faster. Because of its large surface area, andunique physical and chemical properties, it has been widely applied in many fields .
In the present study, a restricted access media - magnetic microsphere (RAM-MM) was synthesized by the suspension
polymerization method using Fe3O4 magnetite as the magnetically susceptible component, styrene (St) and divinylbenzene(DVB) as functional monomer and cross-linking agent, respectively, for forming hydrophobic layer. The hydrophilic layer aroundpolymeric magnetic microspheres was created by opening glycidilmethacrylate (GMA) epoxide with perchloric acid. We havedemonstrated successful preparation of the hydrophilic magnetic microspheres, and applied to extraction of tetracyclineantibiotics residues in milk samples. The proposed method was found to provide a single step extraction and clean-up of tracetetracycline antibiotics residues in food. EXPERIMENTAL Chemicals and Reagents:
Tetracycline hydrochloride (TC) was obtained from Fluka (Buchs, Switzerland). Styrene (St), methyl methacrylate
triglyceride (GMA) and divinylbenzene (DVB) were purchased from Nanjing Lianye Chemical Co., Ltd. (Shanghai, China) whichwas cleaned to remove the inhibitor prior to polymerization. Iron (II) chloride tetrahydrate (FeCl2·4H2O), iron (III) chloridehexahydrate (FeCl3·6H2O), oleinic acid, hydroxyethyl cellulose (HEC), 2,2-Azobisisobutyronitrile (AIBN, recrystallized frommethanol) were obtained from Beijing Chemical Reagent Company (Beijing, China). All the other chemicals were of theanalytical 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.
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
disodium hydrogen phosphate (pH adjusted to 4.0 ± 0.05 with NaOH or HCl as needed), and then 60.5 g of Na2EDTA·2H2O was
International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012 Yunkai Lv et al., IJSID, 2012, 2 (6), 617-622
added into the above mixture . Chromatographic Conditions:
HPLC analysis was performed using a liquid chromatography system containing a LC-20AT pump, a SPD-20A UV–vis
detector and RF-10AXL (Shimadzu, Japan). The analytes were separated in a Venusil XBP C18 column (150×4.6 mm, 5 m) from Bonna-Agela Technologies (Tianjin, China). The mobile phase was methanol / acetonitrile / 10 mM oxalic acid solution (5:25:70, v/v) and the flow rate was 1.0 ml/min at 25 oC. Aliquots of 10 μl were injected into the column and the chromatograms were recorded at 350 nm. Preparation of the RAM-MMs:
The restricted access media - magnetic microspheres were prepared as followed:(a) Typically, FeCl2·4H2O (1.6 g) and FeCl3·6H2O (4.6 g) were dissolved in distilled water (100 ml), and the mixture
was stirred vigorously at 80 oC and purged with nitrogen gas. Then 40 ml of NaOH solution (26% Wt) was added dropwiseinto the mixture. After 4.0 h, the temperature of suspension was cooled to below 10 oC. The final pH value wasapproximately 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
stirred the mixture with a muddler for several minutes and ultrasound dispersed for 5 min, the oleic acid spreaded on theall magnetites evenly. The magnetic precipitates were isolated from the solvent by a permanent magnet. After the graftingreaction, the magnetic precipitates were washed several times with ethanol to remove the unreacted oleic acid.
(c) AIBN (0.1 g) and toluene (0.5 ml) were added into a 50 ml beaker, and stirring the mixture with magnetic stirring
apparatus for several minutes. St (10 ml), DVB (2 ml), GMA (4.6 ml) and oleic-acid-grafted magnetites were added into the50 ml beaker, and the mixture was stirred vigorously and completely. So the preparation of oil phase accomplished.
(d) HEC (0.4 g), NaCl (0.5 g) and H2O (100 ml) were added into three-necked flask and stirred. When the solid
completed dissolved, the oil phase added into it. The mixture was stirred at the rate of 550 rpm; meanwhile, the reactionwas 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 to90 oC, meanwhile, the stirring rate was raised to 650 rpm. The reaction was allowed to proceed for another 5 h.
(e) After the polymerization, the polymers were separated, and washed with water for several times, and added to
25 ml of a perchloric acid water solution (20% v/v). The flask was then stried (200 rpm) for 24 h at room temperature. When the reaction was stopped, the magnetic polymer was washed with water for several times, and the final pH value was approximately 7. Finally, the separated product was dried in a vacuum oven at 40 oC for 24 h. Sample Preparation:
The milk samples were obtained from a local supermarket. 5 g of milk samples were weighed into a 50 ml centrifuge tube,
and 20 ml Mcl vaine-Na2EDTA buffer added into it. The mixture was ultrasound - assisted extraction for 5 min at the room temperature, and the tubes were centrifuged at 4000 rpm for 10 min. 15 ml Mcl vaine - Na2EDTA buffer solution was added into 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 . 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
filtered through a 0.45 m syringe filter. The filtrate were added into the beaker and mixed with the magnetic microspheres, andthe mixture was stirred for 20 min. The magnetic particles with adsorbed TC were washed with 2×2.0 ml dichloromethane. International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012 Yunkai Lv et al., IJSID, 2012, 2 (6), 617-622
Finally, the magnetic microspheres with adsorbed TC were eluted by 1 ml methanol solution containing 25% acetic acid threetimes. The filtrate was applied for HPLC analysis and recovery test. The milk extracts were spiked with TC at three concentrationlevels of 0.1, 0.2, 0.5 mg/kg. Experiments were repeated three times. RESULTS AND DISCUSSION Optimization of Preparation Conditions:
The characters of magnetic microspheres include the size of microsphere, magnetism, degree of homogeneity and so on.
So the preparation method of the magnetic microspheres was optimized by changing some of the preparation factors, consist ofwater/oil mass ratio, stirring rate, the proportion of surfactant, the initiator (AIBN) molar amount in the total moles of monomer,temperature control.
As can be seen from Table 1, when the initiator (AIBN) molar amount in the total moles of monomer was 0.2 g, the
reaction steady, and degree of homogeneity was good. When the initial stirring rate was 550 rpm and the stirring rate was raisedto 650 rpm after 1 h, the micro-size microspheres were prepared, and its diameter uniformity is great good. When the amount ofsurfactant was 0.4 g, the microspheres sticky hardly together; and the sphere diameter was reduced significantly. When theinitial temperature was 60 °C and the temperature was raised to 90 °C after 1 h, the reaction time was moderate; andmicrospheres didn’t sticky together. When the water/oil mass ratio was 5:1, the reaction system not only had characteristic ofhigh stability, but also meets the requirement of suspension polymerization. Above all the optimization of preparation conditions,other proportion or amounts of preparation conditions were also shown in the Table 1 respectively. The magnetic microsphereswere prepared under optimal conditions and shown in Figure 1. Table 1. Effects of the different amounts of initiator and surfactant, water/oil mass ratios (W/O), stirring rates and Affecting Factors Variable Particle Size Coating Effect Yield (%) International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012 Yunkai Lv et al., IJSID, 2012, 2 (6), 617-622 Figure 1. Photograph of the magnetic microspheres with optimal conditions by the TEM Magnetic Dispersion Extraction and Analytical Parameters of Method:
HPLC chromatogram obtained by analyzing milk samples are shown in Figure 2. The Figure 2a (the spiked milk) shows
the chromatogram of the milk simple has some miscel aneous peak, which indicate that the milk sample are impurity before theclean-up step. But the Figure 2b (the spiked milk after Magnetic microspheres) shows that not only quantities of interferenceswere removed in the washing step with dichloromethane. It also indicated high selectivity and better clean-up of the hydrophilicmagnetic microspheres sorbents. t (min) t (min) Figure 2. Chromatogram of tetracycline in milk samples (a) milk simple spiked with 0.1 mg/kg of TC; (b) spiked milk after
The recoveries of the TC by the spiked milk simple were in the range of 72.8–90.1%, 80.5–93.4.0% and 86.2–92.5% for
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 producingsignal/noise ratio of 3:1 and 10:1, respectively. CONCLUSION
In this study, we have demonstrated the successful preparation of the hydrophilic magnetic microspheres, and applied to
extraction of tetracycline antibiotics residues in milk samples. These magnetic microspheres prepared were applied to thesample 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 Yunkai Lv et al., IJSID, 2012, 2 (6), 617-622 ACKNOWLEDGEMENTS
The authors gratefully appreciate the financial support by the Hebei Provincial Key Basic Research Program (No.
10967126D) and the Natural Science Foundation of Hebei Province (No. B2011201081). REFERENCES
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