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Tissue Distribution and Elimination of Florfenicol in Topmouth Culter (Culter Alburnus) After Oral Administration

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Tissue Distribution and Elimination of Florfenicol in Topmouth Culter (Culter Alburnus) After Oral Administration Vol. 27, 2009, No. 3: 316–221 Czech J. Food Sci. Tissue Distribution and Elimination of Florfenicol in Topmouth Culter (Culter alburnus) after Oral Administration Weifen WANG1, Xianjun DAI1, Zhiqiang LI2 and Qingli MENG1 1College of Life Science, China Jiliang University, Hangzhou, Zhejiang Province, China; 2Xiaoshan School of Science and Technology, Zhejiang Ocean University, Hangzhou, Zhejiang Province, China Abstract Wang W., Dai X., Li Z., Meng Q. (2009): Tissue distribution and elimination of florfenicol in topmouth culter (Culter alburnus) after oral administration. Czech J. Food Sci., 27: 216–221. The serum concentration and muscle residue of florfenicol following oral administration were evaluated in topmouth culter (Culter alburnus) under field conditions. Fishes were cultured in tanks and fed a commercial medicated diet containing 2000 mg/kg florfenicol in a single dose. The sampling was conducted at different intervals (0, 0.5, 1, 1.5, 2, 3, 4, 8, 10, 12, 16, 24, 36, 48, 72, 96, 120, 144, 168 h) after the cessation of medication. Drug analysis was carried out by HPLC. The maximum concentration (Cmax) of 10.8 μg/g in serum and 4.2 μg/g in muscle were reached at the same time (tmax) of 8 hours. If the EU MRL value of 1.0 μg/g is quoted for the topmouth culter tissue, the extrapolation of the data indicated that it will be passed after a 40.6 h withdrawal period for florfenicol in topmouth culter muscle. Keywords: serum; elimination; florfenicol; topmouth culter (Culter alburnus) Florfenicol is a fluorinated analogue of thiam- pounds, could become a major drug in veterinary phenicol and its structure also resembles that medicine. of chloramphenicol. Thiamphenicol and chlo- The topmouth culter, Culter alburnus (Basilew- ramphenicol have been used as broad-spectrum sky), belongs to Cypriniformes, Cyprinidae, Culter, veterinary antibiotics (Zhang et al. 2008). Flor- and is widely distributed in large rivers, reservoirs, fenicol is a synthetic broad-spectrum antibiotic and lake areas of China (Chen 1998). The cultured potentially effective in controlling a number of production of the topmouth culter has greatly bacterial infections in the fish in which resistant increased over the past decades, amounting to for bacteria are becoming increasingly prevalent. Stud- about 1500 tons annually. Topmouth culter has ies have revealed its effectiveness against a wide already become one of the most important com- range of pathogens such as Pasteurella piscicida mercial freshwater fishes in China. However, the (Yasunaga et al. 1988), Aeromonas salmonicida research into this species has been very sporadic (Inglis et al. 1991; Nordmo et al. 1994), Vibrio both at home and abroad. anguillarum, and Edwardsiella tarda (Fukui et In aquaculture, florfenicol is mainly administered al. 1987). Powers et al. (1988) predicted that through medicated feeds. This practice may result florfenicol, because of its high potency and great in the antibiotics entering the environment by safety for humans as compared to related com- leaching from uneaten feeds or aquatic animals 216 Czech J. Food Sci. Vol. 27, 2009, No. 3: 216–221 excrements (Robinson et al. 2007). This may result was stored on ice. All samples were kept frozen in adverse ecological effects, including the devel- at –20°C until analysed. opment of resistant bacterial populations, direct Florfenicol analysis protocol. The extraction toxicity to microflora and microfauna, and/or pos- and analysis of florfenicol in serum and muscle sible risks in the transfer of antibiotics resistance were carried out according to the protocol of (Vue to human pathogenic microbes (Hektoen et al. et al. 2002) with some modifications. 5 g of the 1995; Rigos et al. 2004; Cabello 2006). ground muscle sample (plasma) was weighed into The purpose of the present study was to obtain a 40 ml centrifuge tube. 5 ml PBS and 20 ml ethyl information on florfenicol distribution in muscle and acetate (1 ml PBS and 4 ml ethyl acetate for plasma) serum, and to determine florfenicol withdrawal pe- were added, and the mixture was homogenised in a riod for the topmouth culter (Culter alburnus). homogeniser (Model 985-370, Biospec, USA) and then centrifuged at 1500 g for 20 minutes. The supernatant was transferred to a clean centrifugal MateRIALS AND metHodS tube. The extraction step was repeated. The extracts were combined and evaporated to dryness at 60°C Chemicals and apparatus. Florfenicol (purity under a gentle stream of nitrogen. The residue was 99.7%) was obtained from the Chinese National dissolved in 3 ml of mobile phase consisting of Institute for the Control of Pharmaceutical and acetonitrile/water (27/73, v/v) solution and 2 ml Biological Products. Ethyl acetate and hexane were hexane, and then it was mixed. For the plasma of HPLC grade and were purchased from Fisher samples, 0.6 ml mobile phase solution and 0.4 ml Scientific (Fair Lawn, USA). PBS was prepared hexane were added. After centrifugation at 4000 g and kept at 4°C. for 10min, the hexane layer was discharged. The The HPLC system (AgilentTM, HP1100, USA) water-base phase was filtered through filter paper consisted of a pump (HP1100 quaternary pump), a (0.45 μm × 25 mm). The resulting solution was in- manual injection valve with a 20 μl loop, a column jected (20 μl) into the HPLC system. The flow rate (Agilent ZORBAX SBC18 5 μm 25 mm/4.6 mm), was 1 ml/min at a pressure of approximately 100 bar. and a UV detector (G1314 A VWD). Florfenicol was detected at 223 nm. Under these Fish. One hundred healthy topmouth culters, Cul- conditions, the retention time was 6.0 minutes. ter alburnus, were obtained from a certified farm in Standard calibration curve. Acetonitrile stock Xiaoshan Economic and Technological Development solution of florfenicol was prepared at a concentra- Zone in Hangzhou, Zhejiang province, and were tion of 100.0 μg/ml and stored at 4°C. The working reared in a pond. The average weight and length standard calibration curve was drawn by plotting of fish was 50 g and 8 cm, respectively. A similar the known florfenicol concentrations (0, 0.2, 1.0, pond with the same number of fish was used for 2.0, 2.5, 5.0, 10.0, 20.0 μg/ml) prepared from the the non-medicated feed controls. The temperature stock solution by dilution against the average peak was maintained at 23–25°C. The topmouth culters area. The florfenicol concentrations in unknown were acclimatised for 2 weeks and were starved for samples were read from the standard curve. 2 days before the start of the experiment. Detection limit and recovery percentage. The Dosing and sampling. Florfenicol for veterinary detection limit is defined as the concentration of use was evenly mixed with the normal fish ration florfenicol in topmouth culter tissue samples which at the theoretical concentration of 2000 mg of can produce an HPLC-UV signal that is three times drug/kg. The medicated as well as the non-medi- greater than the signal-to-noise ratio. cated feeds were kept at 4°C. The recoveries were calculated by adding known The topmouth culters were treated orally with quantities of florfenicol (range 0.5–20 μg/g) to the a single dose, followed by non-medicated feed homogenised florfenicol-free fish tissues followed administration for the remaining 7 days. by extraction and detection. Blood (2 ml) from the caudal vein and muscle Pharmacokinetic analysis. The mean florfeni- (5 g) of the fish were immediately taken at 0, 0.5, col concentrations in the topmouth culter muscle 1, 1.5, 2, 3, 4, 8, 10, 12, 16, 24, 36, 48, 72, 96, 120, were plotted against time semi-logarithmically. 144, 168 h after the cessation of medication. At The elimination of the drug was followed by linear each sampling time, six fish were randomly selected regression analysis (Campbell et al. 2001). The from the treated and the control specimens. Blood elimination half-life (t1/2) was then calculated 217 Vol. 27, 2009, No. 3: 316–221 Czech J. Food Sci. 12 Figure 1. Serum concentrations of florfenicol m following oral administration g/ ΐ 10 ( ȱ 8 florfenicol ȱ 6 of ȱ 4 2 Concentration 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 Timeȱafterȱadministrationȱ(h) using the equation: t1/2 = Ln 2/β, where β – slope Absorption of florfenicol (elimination rate coefficient) of the logarithmic function: drug concentration against time. The mean concentrations in serum after the ad- The data were fitted to a simple linear model. ministration are shown in Figure 1. The maximum After ensuring that the data were normally dis- concentrations (Cmax) of 10.8 μg/g in serum and tributed (Anderson Darling Test-A2), regression 4.2 μg/g in muscle were reached at the same time analysis was performed to determine the functional (tmax) of 8 hours. After 144 h, the concentrations relationship between the dependent variables of of florfenicol in serum and in muscle were not each treatment and time. detectable (less than 0.03 μg/g). At most sampling times, especially in the later period (from 8 h to 144 h), the concentrations of florfenicol in serum ReSULTS were higher than those in muscle. Detection limit and recovery percentage Persistence of florfenicol in the muscle of fish The detection limit was 0.05 μg/ml while as much as 72.5–113% of the florfenicol amount added to The concentration of florfenicol in fish muscle the tissue homogenates of topmouth culter could exhibited mono-exponential elimination. The be recovered. The recovery percentage was higher muscle concentration–time curve following the in serum than in muscle. oral dose of florfenicol is illustrated in Figure 2, 2.5 ȱ 2.0 1.5 transformed 1.0 ȱ 0.5 concentration ȱ 0.0 drug 0 10 20 30 40 50 60 70 Ȭ0.5 Logarithmically Figure 2.
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