Anal. Chem. 2007, 79, 8242-8248
Molecularly Imprinted-Matrix Solid-Phase Dispersion for Selective Extraction of Five Fluoroquinolones in Eggs and Tissue Hongyuan Yan,† Fengxia Qiao,‡ and Kyung Ho Row*,†
Department of Chemical Engineering, Inha University, Incheon, 402751, Korea, .and College of Chemical and Environmental Sciences, Hebei University, Baoding, 071002, China
A novel highly selective sample cleanup procedure combining molecular imprinting and matrix solid-phase dispersion (MI-MSPD) was developed for the simultaneous isolation of ofloxacin, pefloxacin, norflorxacin, ciprofloxacin, and enrofloxacin in chicken eggs and swine tissues followed by high-performance liquid chromatography with fluorescence detection. The novel ofloxacin imprinted polymers synthesized in water-containing systems show high selectivity for the five fluoroquinolones in aqueous environment and the affinity can be easily adjusted by the pH of solution. Compared with conventional MSPD methods, using MIPs as selective MSPD sorbents, the five fluoroquinolones could be selectively extracted from a biological matrix and all matrix interferences were eliminated simultaneously. The average recoveries of the five fluoroquinolones were ranged from 85.7 to 104.6% for eggs and 86.8 to 102.7% for tissues with relative standard deviations of less than 7.0%. Detection limits for the identification of the five fluoroquinolones in eggs and tissues ranged from 0.05 to 0.09 ng/g. The technique of molecular imprinting generates synthetic materials that mimic the action of antibodies and enzymes.1-2 Molecularly imprinted polymers (MIPs) can be readily tailored with selectivity for a guest molecule, and it is accomplished by synthesizing a network polymer in the presence of a template molecule.3-6 Removal of the template from the polymeric matrix leaves cavities of complementary size, shape, and chemical functionality to the template. The advantages that MIPs hold over natural receptors, such as their stability at extremes of pH and temperature, high mechanical strength, low cost, and reusability, have led to the development of various MIP applications, including * Corresponding author. Tel: +82-32-860-7470. Fax: +82-32-872-0959. Email:
[email protected]. † Inha University. ‡ Hebei University. (1) Wulff, G. Chem. Rev. 2002, 102, 1-27. (2) Vlatakis, G.; Andersson, L. I.; Miller, R.; Mosbach, K. Nature 1993, 361, 645-647. (3) Mayes, A. G.; Whitcombe, M. J. Adv. Drug Delivery Rev. 2005, 57, 17421778. (4) Katz, A.; Davis, M. E. Nature 2000, 403, 286-289. (5) Busi, E.; Basosi, R.; Ponticelli, F.; Olivucci, M. J. Mol. Catal., A: Chem. 2004, 217, 31-36. (6) Rimmer, S. Chromatography 1998, 46, 470-474.
8242 Analytical Chemistry, Vol. 79, No. 21, November 1, 2007
chromatography,7-9 artificial antibodies,10-13 chemical sensors,14-16 and solid-phase extraction (SPE).17-20 However, due to a common lack of selectivity in aqueous media, MIPs suffered greatly from polar solvent interferences, especially water, with obvious limitations for their further application in environmental and biological matrixes.21-22 Although some MIPs synthesized by the use of specifically designed monomer-solvent combinations or hydrophilic comonomers exhibit recognition properties under aqueous conditions,23-28 the use of additional sample pretreatment procedures were required to remove harmful matrix components and suppress the nonspecific binding. Moreover, in the majority of MIP-based extractions, optimum molecular recognition occurs in aprotic and low polar organic solvents, often the one used in the polymerization process. Synthesis in aqueous media of chemically and mechanically stable MIPs, which demonstrate specific rec(7) Hosoya, K.; Shirasu, Y.; Kimata, K.; Tanaka, N. Anal. Chem. 1998, 70, 943945. (8) Sellergren, B. J. Chromatogr., A 1994, 673, 133-141. (9) Peter, S.; Schweitz, L.; Nilsson, S. Electrophoresis 2003, 24, 3892-3899. (10) Lavignac, N.; Allender, C. J.; Brain, K. R. Anal. Chim. Acta 2004, 510, 139145. (11) Nilsson, K.; Lindell, J.; Norrlow, O.; Sellergren. B. J. Chromatogr., A 1994, 680, 57-61. (12) Ye, L.; Mosbach, K. React. Funct. Polym. 2001, 48, 149-157. (13) Manesiotis, P.; Hall, A. J.; Courtois, J.; Irgum, K.; Sellergren. B. Angew. Chem., Int. Ed. 2005, 44, 3902-3906. (14) Marx, S.; Zaltsman, A.; Turyan, I.; Mandler, D. Anal. Chem. 2004, 76, 120126. (15) Hirayama, K.; Sakai, Y.; Kameoka, K.; Noda, K.; Naganawa, R. Sens. Actuators, B 2002, 86, 20-25. (16) Kriz, O.; Ramstrom, O.; Mosbach, K. Anal. Chem. 1997, 69, 345A-349A. (17) Andersson, L. I. J. Chromatogr., B 2000, 739, 163-173. (18) Lanza, F.; Sellergren, B. Chromatographia 2001, 53, 599-611. (19) Qiao, F.; Sun, H.; Yan, H.; Row, K. H. Chromatographia 2006, 64, 625634. (20) Ariffin, M. M.; Miller, E. I.; Cormack, P. A. G.; Anderson, R. A. Anal. Chem. 2007, 79, 256-262. (21) Koeber, R.; Fleischer, C.; Lanza, F.; Boos, K. S.; Sellergren, B.; Barcelo. D. Anal. Chem. 2001, 73, 2437-2444. (22) Dirion, B.; Cobb, Z.; Schillinger, E.; Andersson, L. I.; Sellergren, B. J. Am. Chem. Soc. 2003, 125, 15101-15109. (23) Hart, B. R.; Shea, K. J. J. Am. Chem. Soc. 2001, 123, 2072-2073. (24) Urraca, J. L.; Hall, A. J.; Moreno-Bondi, M. C.; Sellergren. B. Angew. Chem., Int. Ed. 2006, 45, 5158-5161. (25) Dzygiel, P.; O’Donnell, E.; Fraier, D.; Chassaing, C.; Cormack. P. A. G. J. Chromatogr., B 2007, 853, 346-353. (26) Sellergren. B. Anal. Chem. 1994, 66, 1678-1582. (27) Caro, E.; Marce, R. M.; Cormack, P. A. G.; Sherrington, D. C.; Borrull, F. J. Sep. Sci. 2006, 29, 1230-1236. (28) Urraca, J. L.; Moreno-Bondi, M. C.; Hall, A. J.; Sellergren. B. Anal. Chem. 2007, 79, 695-701. 10.1021/ac070644q CCC: $37.00
© 2007 American Chemical Society Published on Web 10/06/2007
ognition of target molecule in water environment, was still a significant challenge. Fluoroquinolones are among the most important class of synthetic antibiotics in human and veterinary medicines worldwide.29 They are effective in controlling a wide range of bacteria, both Gram-positive and Gram-negative, as well as other bacteria, and are often used in the treatment of a range of illnesses, including respiratory tract, urinary tract, and tissue-based infections.30,31 The wide application of such antibiotics in food-producing animals has led to concerns regarding the residues present in foodstuffs of animal origin, which may be directly toxic or cause pathogen resistance and possible allergic hypersensitivity reactions in humans.32-37 The analysis of fluoroquinolones in eggs is quite complex as they may bind to the lipoproteins and the extraction solvents form emulsions and foams with the egg matrix. Different sample preparation methods have been described for the analysis of fluoroquinolones in eggs and tissue, including liquid-liquid extraction,38-41 pressurized liquid extraction,42 solid-phase (micro)extraction,43-47 diphasic dialysis,48-49 and supercritical fluid extraction.50 The main limitations of these methods include the relatively low recoveries (>50-60%), the inability to simultaneously extract all the fluoroquinolones, and the tedious and time-consuming extraction procedures followed by one or more cleanup processes and enrichment procedures, as well as the special equipment required in some cases (e.g., microdialysis) and the low specificity toward a particular target analyte in SPE. Moreover, eggs and tissue samples are not directly applicable to SPE; dilution, ultrasonic, (29) Martinez, M.; McDermott, P.; Walker. R. Vet. J. 2006, 172, 10-28. (30) Kotlus, B. S.; Wymbs, R. A.; Vellozzi, E. M.; Udell, I. J. Am. J. Ophthalmol. 2006, 142, 726-729. (31) Cohen, A. E.; Lautenbach, E.; Morales, K. H.; Linkin, D. R. Am. J. Med. 2006, 119, 958-963. (32) Spratt, T. E.; Schultz, S. S.; Levy, D. E.; Chen, D.; Schluter, G.; Williams, G. M. Chem. Res. Toxicol. 1999, 12, 809-815. (33) Zhang, L. R.; Wang, Y. M; Chen, B. Y.; Cheng, N. N. Acta Pharmacol. Sin. 2003, 24, 605-609. (34) Pedersen, K.; Wedderkopp, A. J. Appl. Microbiol. 2003, 94, 111-119. (35) Carlucci, G. J. Chromatogr., A 1998, 812, 343-367. (36) Hernandez-Arteseros, J. A.; Barbosa, J.; Compano, R.; Prat, M. D. J. Chromatogr., A 2002, 945, 1-24. (37) Andreu, V.; Blasco, C.; Pico. Y. Trends Anal. Chem. 2007, 26, 534-556. (38) Chu, P. S.; Wang, R. C.; Chu, H. V. J. Agric. Food Chem. 2002, 50, 44524455. (39) Schneider, M.; Donoghue, D. J. Anal. Chim. Acta 2003, 483, 39-49. (40) Zeng, Z.; Dong, A.; Yang, G.; Chen, Z.; Huang, X. J. Chromatogr., B 2005, 821, 202-209. (41) Garcı´a, I.; Sarabia, L.; Ortiz, M. C.; Aldama, J. M. J. Chromatogr., A 2005, 1085, 190-198. (42) Herranz, S.; Moreno-Bondi, M. C.; Marazuela, M. D. J. Chromatogr., A 2007, 1140, 63-70. (43) Bailac, S.; Ballesteros, O.; Jimenez-Lozano, E.; Barron, D.; Sanz-Nebot, V.; Navalon, A.; Vilchez, J. L.; Barbosa, J. J. Chromatogr., A 2004, 1029, 145151. (44) Gigosos, P. G.; Revesado, P. R.; Cadahia, O.; Fente, C. A.; Va´zquez, B. I.; Franco, C. M.; Cepeda, A. J. Chromatogr., A 2000, 871, 31-36. (45) Samanidou, V. F.; Christodoulou, E. A.; Papadoyannis, I. N. J. Sep. Sci. 2005, 28, 555-565. (46) Huang, J. F.; Lin, B.; Yu, Q. W.; Feng, Y. Q. Anal. Bioanal. Chem. 2006, 384, 1228-1235. (47) Lolo, M.; Pedreira, S.; Fente, C.; Va´zquez, B. I.; Franco, C. M.; Cepeda, A. J. Agric. Food Chem. 2005, 53, 2849-2852. (48) Heller, D. N.; Nochetto, C. B.; Rummel, N. G.; Thomas, M. H. J. Agric. Food Chem. 2006, 54, 5267-5278. (49) Lolo, M.; Pedreira, S.; Fente, C.; Vazquez, B. I.; Franco, C. M.; Cepeda, A. Anal. Chim. Acta 2003, 480, 123-130. (50) Shim, J. H.; Lee, M. H.; Kim, M. R.; Lee, C. J.; Kim, I. S. Biosci. Biotechnol. Biochem. 2003, 67, 1342-1348.
centrifugation, and re-extraction were often used during the sample preparation. Matrix solid-phase dispersion (MSPD) is one of the most promising techniques for the simultaneous disruption, extraction, and cleanup of solid, semisolid or highly viscous biological samples.51 This technology involves mechanically blending a small amount of sample matrix with an appropriate sorbent followed by washing and elution of compounds with a small volume of solvent. In MSPD, extraction and cleanup are carried out in a single step, which eliminates most of the complications of performing classical liquid-liquid or solid-phase extractions of solid and semisolid samples, particularly complex biological samples.52-55 Recently, many applications of MSPD have been developed by using octadecylsiloxane (C18, C8, etc.), underivatized silicates (silica gel, sand, etc.) or other organic (graphitic fibers) or inorganic (Florisil, alumina, etc.) solids as dispersant sorbents;56-59 however, due to the fact that sorbents are nonselective, further purification of the extracts is often still required to remove coextractants before further analysis. To our knowledge, this work represents the first attempt of using MIPs as selective MSPD sorbents for the simultaneous determination of five fluoroquinolones in chicken eggs and swine tissues. Using the novel MIPs synthesized in water-containing systems as special MSPD sorbents, fluoroquinolones were selectively isolated from biological samples and all matrix interferences were eliminated simultaneously. The present methods have high separation ability, special selectivity, and sufficient accuracy to be used for trace fluoroquinolones analysis in environmental and biological samples. EXPERIMENTAL SECTION Materials. Ofloxacin, pefloxacin, norflorxacin, ciprofloxacin, enrofloxacin, trimethylolpropane trimethacrylate (TRIM), and trifluoroacetic acid (TFA) were obtained from Sigma (St. Louis, MO). The structures of these fluoroquinolones were shown in Figure 1. Methacrylic acid (MAA) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan) and was purified by distillation. R,R′-Azobis(isobutyronitrile) (AIBN) was the product of Junsei Chemical Co., Ltd. (Tokyo, Japan) and was recrystallized prior to use. Acetonitrile, chloroform, and methanol are all of HPLC grade and from Duksan Pure Chemical Co., Ltd. (Ansan, Korea). All the other reagents used in the experiment were of the highest grade commercially available. Double deionized water was filtered with a 0.45-µm filter membrane before use. HPLC Analysis. HPLC analysis was performed using a Shimadzu HPLC system containing a LC-10A Multisolvent Deliv(51) Barker, S. A. J. Chromatogr., A 2000, 885, 115-127. (52) Blanco, E.; Casais, M. C.; Mejuto, M. C.; Cela, R. Anal. Chem. 2006, 78, 2772-2778. (53) Criado, M. R.; Ferna´ndez, D. H.; Pereiro, I. R.; Torrijos, R. C. J. Chromatogr., A 2004, 1056, 187-194. (54) Totti, S.; Fernandez, M.; Ghini, S.; Pico, Y.; Fini, F.; Manes, J.; Girotti, S. Talanta 2006, 69, 724-729. (55) Bogialli, S.; Curini, R.; Di, Corcia, A.; Nazzari, M.; Polci, M. L. J. Agric. Food Chem. 2003, 51, 4225-4232. (56) Pensado, L.; Casais, M. C.; Mejuto, M. C.; Cela, R. J. Chromatogr., A 2005, 1077, 103-109. (57) Kristenson, E. M.; Ramos, L.; Brinkman, U. A. T. Trends in Anal. Chem. 2006, 25, 96-111. (58) Arribas, A. S.; Bermejo, E.; Chicharro, M.; Zapardiel, A. Talanta 2007, 71, 430-436. (59) Bogialli, S.; Curini, R.; Di, Corcia, A.; Lagana, A.; Rizzuti, G. J. Agric. Food Chem. 2006, 54, 1564-1570.
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Figure 1. Molecular structures of the five fluoroquinolones.
ery System, a DGU-12A on-line degasser, a SCL-10Avp gradient controller, a CTO-10Avp column thermostat, and an RF-10AXL fluorescence detector (Shimadzu, Kyoto, Japan). Excitation and emission wavelengths were set at 280 and 480 nm, respectively. CLASS-VP workstation (Shimadzu) was used as a data acquisition system. The analytical column was packed with ODS C18 stationary phase (VP-ODS, 150 mm × 4.6-mm-i.d., particle size 5 µm, Shimadzu). The column thermostat set at 30 °C. The mobile phase was water-acetonitrile (92:8, v/v) containing 0.02 mol/L tetrabutylammonium bromide, and its flow rate was set at 1.0 mL/min. Preparation of the Imprinted Polymers. The ofloxacin imprinted polymers were prepared by thermal-initiated polymerization within a 50-mL thick-walled glass tube. The polymerization mixture, composed of 1.0 mmol of ofloxacin, 8.0 mmol of MAA, 20.0 mmol of TRIM, and 0.056 g of AIBN, was dissolved in appropriate porogenic solvents (methanol:water ) 8:1, v/v). The solution was sonicated for 10 min, sparged with helium for 5 min to remove oxygen, and then polymerized under 54 °C in a water bath for 36 h. After the polymerization, the polymers were ground and sieved through a 32-µm sieve and then repeatedly suspended in acetone to remove the small particles. Finally, the particles were dried under vacuum, put it into a column, and washed with tetrahydrofuran for 12 h and methanol-acetic acid (80:20, v/v) for 24 h at a flow rate of 0.2 mL/min to remove the templates. After balancing with methanol and drying in a vacuum drying oven (20 °C) 20 h, the obtained particles were stored at ambient temperature until to use. A nonimprinted blank polymer (NIP, in the absence of template) was prepared and treated in an identical manner. Determination of Binding Capacity of the MIPs. In order to investigate the binding capacity of the MIPs in a water environments, a static absorption method was performed by 8244
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placing 20 mg of imprinted particles, 3.00 mL of water, and different concentrations of ofloxacin into 10-mL flasks. After being placed in the dark at room temperature for 48 h, the solutions were centrifuged, filtered, and then determined by a UV spectrophotometer at 293 nm in replicates of three. The data of the static absorption experiment were further processed with the Scatchard equation to estimate the binding parameters of the MIPs. Q is the amount of ofloxacin bound to MIPs at equilibrium, Qmax is the maximum binding capacity, Cfree is the equilibrium concentration of ofloxacin and KD is the dissociation constant.
Scatchard equation:
Q/Cfree ) (Qmax - Q)/KD
Morphological Characteristics of the MIPs. Morphological characteristics including pore analysis and scanning electron microscopy (SEM) analysis of the polymers were also investigated in this experiment. The specific surface areas and porosity of the imprinted polymers were obtained by nitrogen adsorption on an ASAP 2000 accelerated surface area and porosimetry system (Micromeritics). The NIP was analyzed as a control experiment to demonstrate the differences between imprinted and nonimprinted polymers. Microscopic analysis of the MIPs was carried out in an S-4200 SEM (Hitachi) at 15 kV. Procedure of Matrix Solid-Phase Dispersion. Chicken eggs and swine tissue samples used for this study were collected from local markets. Prior to blending with MIP particles, muscle samples were minced with a meat grinder and egg samples were stirred 10 min to blend egg albumen and egg yolk homogeneously. A representative portion of the sample (0.20 g) was placed into a porcelain mortar and mixed with 0.20 g of MIPs by blending gently with a pestle until a homogeneous mixture was obtained (Figure 2). This homogenized samples were introduced into an empty SPE
Figure 2. Schematic procedure of MI-MSPD. Table 1. Effect of Different Porogenic Solvents on Their Separation Characteristicsa no.
template (mmol)
MAA (mmol)
TRIM: (mmol)
porogenic solvents (mL)
mechanical intensity
k1
k2
P1 P2 P3 P4 P5
1 1 1 1 0
4 4 8 8 8
10 20 20 20 20
9.0 mL (MeOH-H2O) 9.0 mL (MeOH-H2O) 9.0 mL (MeOH-H2O) 9.0 mL (chloroform) 9.0 mL (MeOH-H2O)
low middle good good good
12.5 17.7 >20 17.2 3.4
11.3 16.4 >20 6.9 2.5
a The ratio of MeOH-H O was 8:1, v/v; k and k are retention factors of ofloxacin when acetonitrile and water were used as mobile phase, 2 1 2 respectively; column size, 200 mm × 4.6 mm i.d.; mobile phase, acetonitrile or water; flow rate, 1.0 mL/min; detection, 280 nm).
cartridge (0.05 g of MIP particles was prepacked at the bottom), rinsed with 3.0 mL of water, and then eluted with 4.0 mL of acetonitrile-TFA (99:1, v/v). The eluent was evaporated to dryness under vacuum at 25 °C, and the residue was reconstituted into 0.5 mL of mobile phase for further HPLC analysis. Stock standard solutions of fluoroquinolones were prepared in water. Spiked eggs and tissue samples were prepared by adding appropriate volumes of fluoroquinolones standard solution to blank samples, and the volume added was always less than 2% of the final sample volume to preserve the integrity of the samples. After stirring 10 min, the mixture was equilibrated for 60 min at 37 °C in darkness and then extracted according to the above MSPD procedure. The average recoveries of fluoroquinolones were evaluated by spiking three different levels (1.0, 5.0, 25 ng/g) of standard analytes to eggs and tissue samples in replicates of five. Quantitation of fluoroquinolones in extracts was calculated by comparing the peak areas for each compound with those obtained from the injection of standard solutions. RESULTS AND DISCUSSION Preparation and Characteristic of the MIPs. In order to synthesis MIPs, which demonstrates specific recognition for fluoroquinolones in water environment, MIPs prepared by nonpolar solvent (chloroform) and polar porogenic solvents, especially a water-containing system, were investigated and its recognition ability compared in aqueous systems (Table 1). MIPs prepared in a methanol-water system showed higher recognition ability
in a water environment, which presumably was related to strong contact ion pairs forming between MAA and the piperazine ring of fluoroquinolones.8,25 Besides the temperature and proportion of the prepolymeric mixture, the porogenic solvents play an important role in the morphology of the MIP in terms of specific surface area and pore size. The optimal chemical composition of the MIP was obtained in P3. Static absorption data show that water-compatible MIPs have higher binding capacity to analyte, and the two distinct linear portions in Scatchard analysis (Figure 3) indicate two classes of binding sites existed in the imprinted polymer: one is high selectivity or affinity with high binding energy and the other is low affinity with low binding energy. The respective KD and Qmax values are calculated from the slopes and intercepts of the two linear portions of Scatchard analysis, and the results are listed in Table 2. The specific surface areas and pore volumes from nitrogen adsorption experiments were 288.9 m2/g and 0.513 cm3/g for MIP, 279.4 m2/g and 0.502 cm3/g for NIP, respectively. The similar surface areas and pore volumes of MIP and NIP indicated the selectivity of the MIP was due to special imprinted recognition. The microscopic characteristic of the water-compatible MIP was show in Figure 4. When the obtained MIP particles were used as chromatographic stationary phase, fluoroquinolones could not be washed out of the column (200 mm × 4.6 mm i.d.) within 60 min and unrelated molecules with templates such as caffeine, tryptophan, genistein, and catechin were eluted out in less than 15 min when acetonitrile, methanol, or water was used as mobile phase. At the same time, Analytical Chemistry, Vol. 79, No. 21, November 1, 2007
8245
Figure 4. Scanning electron microscopy of the water-compatible MIP.
Figure 3. Adsorption isotherm and Scatchard analysis of ofloxacin imprinted polymers. (Each point in the isotherm was the average values of three replicates; the RSDs for all points were lower than 2.8%.) Table 2. Result of Scatchard Analysis binding sites
linear equation
higher affinity site Q/Cfree ) 1.009-9.413Q lower affinity site Q/Cfree ) 0194-0.695
KD Qmax (mmol/L) (mmol/g) 0.106 1.438
0.107 0.278
fluoroquinolones was quickly washed out of the blank column ( 0.998 (Table 3). Table 4 and Table 5 show the average recoveries of fluoroquinolones from eggs and tissue at three different levels 8248 Analytical Chemistry, Vol. 79, No. 21, November 1, 2007
Table 4. Average Recoveries of Fluoroquinolones from Spiked Egg Samples (n ) 5) 1.0 ng/g
5.0 ng/g
25 ng/g
sample
recovery (%)
RSD (%)
recovery (%)
RSD (%)
recovery (%)
RSD (%)
ofloxacin pefloxacin norflorxacin ciprofloxacin enrofloxacin
93.3 93.9 85.7 95.6 90.9
4.1 3.2 4.6 3.8 7.0
96.4 101.3 93.8 104.6 97.7
4.7 3.5 4.9 5.3 6.1
94.7 98.5 94.5 98.0 94.6
5.8 6.4 3.9 4.7 5.2
Table 5. Average Recoveries of Fluoroquinolones from Spiked Tissue Samples (n ) 5) 1.0 ng/g
5.0 ng/g
25 ng/g
sample
recovery (%)
RSD (%)
recovery (%)
RSD (%)
recovery (%)
RSD (%)
ofloxacin pefloxacin norflorxacin ciprofloxacin enrofloxacin
97.0 94.5 96.8 98.8 102.7
3.9 5.3 3.0 3.4 6.1
101.8 91.0 87.3 88.5 92.0
1.9 4.9 3.8 5.4 4.1
94.2 89.8 95.3 97.3 93.1
4.6 5.1 3.5 4.7 7.0
were ranged from 85.7 to 104.6% with relative standard deviations (RSDs) lower than 7%. Precision was calculated in terms of intraday repeatability (n ) 5) and interday reproducibility (5 different days) on 1.0 and 5.0 ng/g concentration levels for each analyte in eggs and tissue samples. The intraday repeatability evaluated as RSD were ranged from 3 to 7%, and the interday reproducibility was below 9% for all instances. The variations for the precisions of samples may be attributed to the small quality of sample used and the adhesivity of eggs, which can adhere to vessels such as glass beaker and pestle and cause sample loss. The limits of determinations (LODs) ranged from 0.05 to 0.09 ng/g based on a signal-to-noise ratio of 3 and are well below the tolerance levels set by the European Union. CONCLUSION In this work, a multiresidual analytical method for simultaneous determination of five fluoroquinolones in eggs and tissue samples was developed by MI-MSPD followed by HPLC with fluorescence detection. By using the water-compatible MIPs as specific MSPD sorbents, all fluoroquinolones in eggs and tissues samples were selectively isolated and matrix interferences were eliminated simultaneously, which remarkably enhanced the selectivity of MSPD. Moreover, extraction and cleanup were completed in one step, so the procedure of sample preparation was simplified with significant reduction in both sample size and solvent consumption. The performance characteristics, in terms of sensitivity, selectivity, and cleanliness of the extracts, indicate that this method is sufficiently accurate and precise to be used for trace fluoroquinolones analysis in eggs and tissue samples. ACKNOWLEDGMENT The authors gratefully appreciate the financial support by the Center for Advanced Bioseparation Technology of Inha University, Korea. Received for review April 2, 2007. Accepted July 26, 2007. AC070644Q
