Yun Kai Lv et al.

, IJSID, 2012, 2 (6), 610-616

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

FOR DETERMINATION OF THE FLORFENICOL RESIDUE IN MILK

PREPARATION OF MOLECULARLY IMPRINTED MICROSPHERES FOR SOLID-PHASE EXTRACTION COUPLED WITH HPLC College of Chemistry and Environmental Science, Hebei University, Key Laboratory of Analytical Science and Technology of Yun-Kai Lv*, Xiao-Hu Wang, Wei Zhang, Lei Yang, Ping Liu Hebei Province, Baoding 071002, China

Received: 03-11-2012 Accepted: 16-12-2012

*Corresponding Author

ABSTRACT developed based on molecular imprinting technique in combination with modified as the template, methacrylic acid as organic functional monomer, A novel preparation method of molecularly imprinted microspheres was

suspension polymerization for selective solid-phase extraction (SPE) of florfenicol residues in milk. The molecularly imprinted microspheres were prepared using florfenicol 5%, 10% and 70% methanol aqueous solution. Under the optimized conditions, the ethylene were optimized, and the optimal loading, washing and eluting solution respectively were

glycoldimethacrylate as the cross-linking agent. The solid-phase extraction conditions Address: Name: Yun-Kai Lv Place: Baoding, China E-mail: lvyunkai@hbu.edu.cn average recoveries of three chloramphenicol antibiotics spiked milk at 0.005, 0.05 and kg-1 and 0.0209-0.082 mg kg-1, respectively.

chloramphenicols residues in milk were determined by the SPE-HPLC method. The 0.20 mg kg-1 were in the range of 73.4-92.0% with the precision of 1.7-4.3%. The limits of Keywords: Molecularly imprinted microspheres; Florfenicol; Solid-phase extraction; Milk detection and quantitation of the proposed method were in a range of 0.0063-0.0246 mg INTRODUCTION

INTRODUCTION

International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012

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Yun Kai Lv et al., IJSID, 2012, 2 (6), 610-616 family of antibiotics with a broad antibacterial spectrum. Compared with similar chloramphenicol (CAP) nand thiamphenicol, clinic. Although FF is a more secure drug than CAP and TAP, its use in animal husbandry has the potential to result in the mg/kg for florfenicol in milk was established by Chinese Ministry of Agriculture [2]. fluid extraction have all been employed for this purpose selectivity and fast kinetics becomes very important. chemical, medical and molecular biology fields solution polymerization
[14] [5].

florfenicol has high antimicrobial activity and no potential to cause aplastic anemia and plays an important role in the veterinary combating microbial infections in humans. To ensure the existence of FF antibiotics, the maximum residue limit (MRL) of 0.1 There are many methods for the determination of FF in animal tissues, such as HPLC, LC-MS
[3],

Florfenicol (FF), a fluorinated analogue of thiamphenicol (TAP), is a new semisynthetic member of the amphenicol INTRODUCTION

presence of immune toxicity, embryo toxicity, and the blood system toxicity that could have potential health risks to humans [1]. GC and GC-MS
[4].

Nowadays, antibiotic resistance has become a global threat because existing antibiotics are becoming increasingly ineffective in However, sample preparation procedures are required prior to chromatographic quantitation which often need laborious, timeconsuming and expensive. Liquid-liquid extraction, solid-phase extraction (SPE), solid-phase micro-extraction and supercritical the most extensive. SPE has the following advantages: simplicity, low cost and easy automation, coupled to both HPLC and GC [6]. Molecular imprinting technique (MIT) is a new kind of molecular recognition technology, refers to artificially prepared
[7, 8]. [11], [9, 10].

But, the sorbents typically used in SPE can present low selectivity. So, to development of new SPE sorbents which have high with the presence of specific molecules selectively binding sites of molecularly imprinted polymers (MIPs), to achieve a specific recognition of the target molecule, separation, Purification and enrichment. MIPs have been extensively studied and applied in important microspheres (MIMs) due to their excellent properties and wide applications methods including suspension polymerization and surface imprinting technique emulsion polymerization
[15]Among [12],

In all of the sample pretreatment method, the application of SPE is

Considerable interest has been aroused in the synthesis of molecularly them suspension polymerization has the following precipitation polymerization Recently, the MIMs synthesis
[13],

advantages compared with the other polymerization processes: Easy heat removal and temperature control; low dispersion viscosity; low levels of impurities in the polymer product; low separation costs; and final product in particle form [16]. Suspension polymerization is a heterogeneous radical polymerization process that uses mechanical agitation to mix a

aqueous

monomer or mixture of monomers in a liquid, such as water, while the monomers polymerize, forming spheres of polymer. The and hinder monomer drops from coming together [17].

reaction mixture consists of two phases, a liquid matrix and monomer droplets. The monomer and initiator are insoluble in the liquid phase, so they form drops within the liquid matrix. A suspension agent is usually added to stabilize the monomer droplets polymerization, used it for the solid phase extraction of adsorbent, coupled to high performance liquid chromatography, establish a kind of a new method to analysis of florfenicol in milk. The purpose of this experiment is to synthesize a new florfenicol imprint polymer microspheres via suspension In present study, we describe a simple approach to preparing molecularly imprinted microspheres by the suspension

polymerization. Florfenicol- methacrylic acid complex formed in acidification chloroform containing trifluoroacetic acid and important microsphere was used as SPE packing for selective extraction of chloramphenicol residues from milk samples.

acetic acid, which solved FF slightly soluble problem. When sodium dodecyl sulfate (SDS) added to the PVA suspension International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012

polymerization dispersion system, microspheres adhesion problem was improved. Under the optimized conditions, the FF-

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Yun Kai Lv et al., IJSID, 2012, 2 (6), 610-616 MATERIALS AND METHODS Instrumentation and Reagents: detector and RF-10AXL (Shimadzu, Japan). The analytes were separated in a Venusil XBP C18 column (2504.6 mm, 5 m) from Bonna-Agela Technologies (Tianjin, China). 12-Ports Vacuum SPE Manifold system was purchased from Beijing peaksharp analytical Instrument Co, Ltd. (Beijing, China).. Methacrylic acid (MAA) was purchased from Tianjin Chemical Reagent Research Institute (Tianjin, China) 2, 2Azobisisobutyronitrile (AIBN) was purchased from Beijing Chemical Reagent Company (Beijing, China). Ethylene glycoldimethacrylate (EGDMA) were obtainedfrom Fluka (Steinheim, USA). SDS and Polyvinyl alcohol (PVA, d. p=1788, trifluoroacetic acid (TFA), acetic acid and HPLC-grade methanol, and other materials were obtained from Tianjing filtered through a 0.45 m membrane filter. Preparation of Microspheres: studies [9]. a. b. c. d. For preparing FF-imprinted microspheres, initially four types of solutions were prepared as described in previous in a 10 mL vial and all the components were dissolved by stirring the solution for 10 min at room temperature. Then, in the next step the solution was stirred at 0for 30 min. stirred for 10 min at room temperature. cooled at room temperature. EGDMA (2.8275 mL, 15 mmol), chloroform (2 mL) and AIBN (0.15 g) were taken in a 50 mL flat bottom flask and SDS (1 g) was dissolved in water (20 mL) by stirring in a 250 mL flask for 2 min at room temperature. saponification value = 88%) was from SINOPEC Shanghai Petrochemical Co. Ltd. (Shanghai, China). Analytical grade methanol, chemicalreagent company (Tianjing, China). Doubly deionized water (DDW) was used throughout. Samples for HPLC were Chloramphenicol (CAP), florfenicol (FF) and thiamphenicol (TAP) were purchased from Fluka (Buchs, Switzerland). HPLC analysis was performed in using a liquid chromatography system containing a LC-20AT pump, a SPD-20A UVvis

FF(0.2687 g0.75 mmol), MAA (0.255 mL, 3 mmol), chloroform (4 mL), acetic acid (0.8 mL),TFA (0.53 mL) were taken

PVA (3.45 g) was dissolved in distilled water (130 mL) by stirring in a 250mL flask for 30min at 80-85 0and then Then, (a), (b), (c) and (d) were combined together. These solutions were stirred separately for 2 h and then mixed in a

three-necked double jacket glass vessel, purged with N 2 for 5 min, and stirred at 650 rpm for 60 min at room temperature. polymerization process resulted in MIMs. Afterwards, the polymer microspheres were filtered; the MIMs were filtered and at 50 under vacuum for 12 h [10]. washed three times sequentially with deionized water. Subsequently, the MIMs were washed with washed with methanol in a soxhlet apparatus successively until no FF could be eluted, with final confirmation by HPLC analysis. The MIMs were then dried Molecularly Imprinted Solid-Phase Extraction Procedure:

Polymerization was carried out at 60 0 for 24 h under N2 atmosphere, and with continuous stirring at 650 rpm. This

was conditioned with the following solvents (in order): 3 mL of methanol and 4 mL of 5% methanol-water (v/v), and then 0.8 of 0.2 mL min-1. Subsequently, the columns were washed with 3 mL of 10% methanol-water (v/v) and eluted with 3 mL of 70% methanol-water (v/v) [7]. The collected solutions were analyzed by HPLC-UV detection at 225 nm. International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012

mg/mL of FF solution which in 3.0 mL of 5% methanol-water (v/v) was passed through the cartridges respectively at a flow rate

Accurate take 50 mg dry MIMs, which packed into empty SPE cartridges between two filters respectively. The cartridge

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Yun Kai Lv et al., IJSID, 2012, 2 (6), 610-616 Sample Treatment: perchloric acid solution, 10 mL ethyl acetate, Vortex 10 min, the mixture were centrifuged at 9000 rpm for 10 min, take the supernatant. Then use 10 mL ethyl acetate extract repeated 1 time, combined the supernatant, the ethyl acetate extract under reduced pressure at 30 distilled to give dry [18]. The residues dissolved with methanol /water solution (40:60, v/v) to 5 mL. HPLC Analysis: 5 mL solution which residue solution filtrated through a 0.45 m syringe filter, the filtrate was passed through the The milk samples were obtained from a local supermarket. Accurate take 5 g sample, respectively add 0.25 mL 5%

MISPE cartridges. The above-mentioned MISPE procedure was used to separate and detect FF in milk. The milk samples were repeated three time. The mobile phase was methanol -water solution (40:60, v/v) and the flow rate was 1.0 mLmin-1 at 25. Aliquots of 10 L were injected into the column and the chromatograms were recorded at 225 nm. RESULTS AND DISCUSSION Optimization of Synthesis Conditions: Regular shape of imprinted particles is advantageous in chromatographic applications, and can facilitate system
[10],

spiked with chloramphenicol antibiotics at three concentration levels of 0.005, 0.05, 0.20 mg kg-1, and experiments were

homogenization and mass transfer. A kind of monodisperse microspheres is what we want in order to improve the efficiency of synthesis scheme is according to the literature serious, and it can be seen in Figure 1a. This result prevents the further use in SPE. Many factors influence the synthesis of sulfate (SDS) added to the PVA suspension polymerization dispersion system, microspheres adhesion problem was improved 1.5 and 3.0 m.

analysis. Suspension polymerization seems to be one of the best suited methods for the synthesis of MIMs. Our preliminary to change the above factors in order to achieve uniform size microspheres, but the effect is not ideal. When sodium dodecyl

monodisperse microspheres in suspension polymerization, such as the reaction temperature and the stirring speed. So we tried and the microspheres particle size also become uniform. AS can be seen in Figure 1b, the particles are between approximately

but the preparation of the microspheres particle size uneven and adhesion

Figure 1. Images of the microspheres were obtained. (a) Microspheres synthesized according to the proposed method; (b) Optimization of Solid-Phase Extraction Conditions: optimized. After pre-conditioning, the loading step was optimized. 3.0 mL of different loading solvents (5%, 10%, 15%, 20% International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012 In order to optimize the selectivity of MISPE conditioning, loading, washing and elution steps were evaluated and images of the microspheres when the PVA suspension polymerization dispersion system after adding SDS.

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methanol-water (v/v)) was passed through cartridges packed with 50 mg of the MIMs. The FF was eluted with 3 mL of methanol and the recovery respectively: 100.1%, 95.6%, 90.3%, and 80.9% (loading concentration is 0.48 mg mL1 of FF). FF was completely retained in the column when using 5% methanol-water (v/v). Therefore, 5% methanol-water (v/v) was selected as loading solvent for further investigations. In order to enhance the selectivity of MIMS for FF and decrease the cross-reactivity, a washing and elution step in the

Yun Kai Lv et al., IJSID, 2012, 2 (6), 610-616

MISPE procedure was investigated using 3mL ten different proportion of methanol-water (methanol content of =10% 100%, v/v) as potential washing solvents. Use washing solvents for abscissa and the recovery rate of FF for ordinate established washing-elution curve. The results were shown in Figure 2. after adding SDS.

1 0 0

R e c o v e r y (% )

8 0 6 0 4 0 2 0 0 0 2 0 4 0 6 0 8 0 1 0 0

methanol content in more than 70% the target is basically all washed out. In order to wash off more nonspecific binding unnecessary component, select 70% methanol-water as eluting solvent. Analytical parameters:

impurities, we choose 10% methanol-water (v/v) as washing solvent and in order to avoid solvent is too strong to wash out In order to evaluate the efficiency of the proposed method, the linearity of the method was tested with the

As can been seen in Figure 2, when methanol content fewer than 10%, FF was basically do not have to wash out; when

Figure 2. Washing-elution curve of MISPE column using different solvents

M e t h a n o (l % )

chloramphenicols concentration in the 0.05-5.0 mg L-1 under the optimal conditions. Linear regression equations, correlation coefficient (r) and instrument detection limits for three chloramphenicols were shown in Table 1. The calibration curve of each chloramphenicols showed good linearity with correlation coefficient (R) more than 0.9965. The instrument detection limits for chloramphenicols were calculated three times of the signal noise ratio according to the lowest concentration point in standard curve and shown in Table 1. Analyte FF Linear equation Y = 87 + 3.29104 C Table 1. Linear regression equations, correlation coefficient (r) and instrument detection limits for three chloramphenicols. TAP Y = 3006 + 2.21104 C CAP Y = 455 + 2.38104 C Sample analysis: r 0.9983 0.9965 0.9986 Linear range (mg L-1) 0.005 - 0.5 0.00244 0.00227 Instrument detection limits (mg L-1) 0.00164

To demonstrate the feasibility of using MISPE to extract FF from the milk samples, 5 g of the milk samples was treated International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012

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using the protocol described in the experimental section; the obtained solution was analyzed by HPLC. The chromatograms of

Yun Kai Lv et al., IJSID, 2012, 2 (6), 610-616

the spiked fish and spiked milk after MISPE were shown in Figure 3a and Figure 3b, respectively. All chloramphenicols were selectively extracted and no interferences from the milk were observed after MISPE process, which demonstrates the high selectivity and better clean-up of the FF-MIMs. The mean recoveries of FF, TAP, and CAP were 90.5-92.0%, 73.4-75.1% and 78.981.1%, respectively, spiked concentrations in milk at 0.005, 0.05 and 0.10 mg kg-1 with relative standard deviations (RSD) lower respectively. Table 2. Average recoveries (R), relative standard deviations (RSD, n = 3), limit of detection (LOD) and limit of quantitation (LOQ) of three CAP obtained after MISPE of the spiked milk samples (n=5) Analyte Spiked level (mg kg-1) Detected (mg kg-1) R (%) RSD (%) LOD a (mg kg-1) LOQ b (mg kg-1) 0.005 0.0475 90.5 1.8 0.0125 0.0416 FF 0.01 0.00921 92.0 2.1 0.20 0.1826 91.3 1.7 0.005 0.00367 73.4 3.5 0.0063 0.0209 TAP 0.01 0.00751 75.1 3.0 0.20 0.1492 74.6 3.6 0.005 0.003945 78.9 3.9 0.0246 0.0820 CAP 0.010 0.00808 80.8 4.3 0.20 0.1622 81.1 4.0 a LOD calculated as 3 times the signal-to-noise ratiob LOQ calculated as 10 times the signal-to-noise ratio. than 4.3% (Table 2). The limits of determination (LOD, S/N = 3) and the limits of quantitation (LOQ, S/N = 10) of the proposed

method were 0.0125 and 0.0416 mg kg-1 for FF, 0.0063 and 0.0209 mg kg-1 for TAP, 0.0246 and 0.0820 mg kg-1 for CAP,

1800 1200 600 0 0 5 10 15

(a)
TAP

1800 1200 TAP 600

(b)

FF

CAP
0

FF

CAP

20

10

15

20

Figure 3. Chromatograms obtained from the extraction of florfenicol (FF), Chloramphenicol (CAP) and thiamphenicol (TAP) from the milk samples. (a) the spiked milk; (b) the spiked milk after MISPE. Mobile phase: methanol-water solution (40: 60, v/v). Flow rate: 1.0 mLmin1. Injection volume: 10 L. CONCLUSION We have demonstrated successful fabrication of new MIMs for selective solid-phase extraction (SPE) of florfenicol

t (min)

t (min)

antibiotics residues in milk. The prepared microspheres have regular spherical shapes with rough and porous surfaces and International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012

very low agglomeration. This kind of microspheres was applied in solid phase extraction, which showed good rigidity,

compressive ability, adsorption and fast adsorption-desorption characteristics. We expect that synthesis and application

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protocol of the MIMs is promising as a general strategy for the fabrication of high selective imprinted Microspheres materials and solid-phase extraction of veterinary drug residues in food. 10967126D) and the Natural Science Foundation of Hebei Province (No. B2011201081). 1. 2. 3. 4. 5. 6. 7. 8. 9. REFERENCES Florfenicol - summary report, EMEA/MRL/589/99-FINAL, 1999. 2002, 12. The authors gratefully appreciate the financial support by the Hebei Provincial Key Basic Research Program (No. ACKNOWLEDGEMENTS

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