Pefloxacin Residues in Tissues and Organs of Treated Rabbits

Thirty balady rabbits of both sexes (weighing 1.5-2 Kg wt) were used for studding pefloxacin residues. The effect of heat treatment and freezing on the presence of these residues were also studied. The drug was injected intramuscularly (10 mg/Kg body weight) for 5 successive days. Animals were slaughtered at different intervals, samples from shoulder, thigh, back muscle, liver, and kidneys were examined. Parts of samples from animals slaughtered at 12 hr, 24 hr, 48 hr, 72 hr, and 96 hr after treatment, were examined for presence of drug residues and then boiled for 45 minutes and tested while the rest of these samples were frozen and examined weekly for presence of tested drug using HPLC. The result showed that pefloxacin highest concentration level was detected in kidneys at 12 hr from last dose (28.32±2.261 μg/g) and decreased till not detected at 144hr followed by liver (26.32±2.31 μg/g) then shoulder muscle (18.21±1.011 μg/g) and its level showed significant decrease at (p< 0.05) till not detected at 72 hr. concentration level showed in shoulder muscle, thigh and back muscles was (18.21±1.11, 13.5±1.023, 12.6±1.031 μg/g). Regarding the effect of boiling of the drug in kidney sample it is evident that pefloxacin concentration was decreased from 28.32 ±2.261 to 16.516 ±0.421 μg/g). Such decrease was detected in the samples examined at the following hours until disappeared. Meanwhile frozen kidneys sample at 10°C showed significant decrease after 1 week and decreased till not detected at 5 week (14.240±0.351, 6.425±0.052, 1.253±0.322 μg/g) followed by liver (4.250±0.33, 2.872±0.251, 1.205±0.158, 0.38±0.241 μg/g). INTRODUCTION Antibacterial agents are widely used for prophylactic and treatment of various diseases in animals. The first antimicrobials based on the 4-quinolone ring were nalidixic acid and oxolinic acids, which are active in vitro against a wide range of Gram-negative bacteria. The included problems associated with their application were restricted spectrum of activity and the relatively rapid emergence of resistant mutants, which led to the discovery of fluroquinolones. One of them is pefloxacin (1-ethyl-6-fluoro-1,4-dihydro7-(4-methyl-1-piperazinyl)-4-oxo-3quinoline carboxylic acid. As other fluoroquinolones, Pefloxacin achieves rapid bactericidal 155 Bull High Inst Public Health Vol.38 No.1 [2008] activity by inhibiting the bacterial DNA gyrase Chu and Fernandes, (1991) – Marie et al., (2000). The drug possesses good in vitro activity against a variety of pathogens, including Gram-positive and Gram-negative bacteria. Pefloxacin is relatively similar to other fluroquinolones such as Enrofloxacin, Ciprofloxacin, and Marbofloxacin (Van-Custen et al., (1990) – Spreng et al., (1995) – Brown, (1996) in possessing a wide spectrum of activity, a large volume of distribution, and their activity at low concentration. Residues of veterinary medicinal products which are defined by European Union, are pharmacologically active substances (whether active principals, expepients, or degradation products), and their metabolites remain in food stuffs obtained from animals to which the veterinary medicinal products in question has been administrated Vander Creek, (1984.There has been an increased concern amoung consumer about antibiotic and other drug or chemical residues Katz and Brady, (1993).The potential problems associated with drug residues may be aesthetics, all ergic reactions, direct toxic effects, and change in the resistance patterns of bacteria exposed to antibiotic Weaver. (1992). The drug is metabolized in the animal's body and broken down or excreted, within the directed withdrawal time. Organs such as the kidney and liver remove residual drug and greatly reduce the content present in red meat or milk. These organs are the tissues tested for residues but content in red meat are less by many times Glott et al., (1979).The aim of this study is to detect the light pefloxacin residues in different tissues as well as the effect of heat treatment (boiling) and freezing on tissue residues. MATERIAL AND METHODS Material i. Drug: Pefloxacin (pefloadad 10% solution) Structural formula Mona O Abou El-Nil 156 The chemical name: (1-ethyl-6-fluoro-1,4dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3quinoline carboxylic acid. 1) Chemical Formula: C17H20FN3O3 2) Dose: Pefloxacin is available in an injectable solution to be given intramuscularly at a dose of 10 mg/kg body weight. ii. Experimental Rabbits: Thirty healthy rabbits (Balady bread) of both sexes weighing about 1.5-2 Kg Body weight were used as experiment animals throughout this study. They were kept for 15 days under well hygienic conditions and fed on concentrated mixture of barseem barley in a pellet form free from any antibacterial agents and water ad. Lid. Methods 1) Two rabbits (control) were slaughtered and tested for the absence of any antibacterial residues. 2) The rest of rabbits were given pefloxacin intramuscularly (10 mg/Kg body weight) for 5 successive days. 3) Two rabbits were slaughtered at 12, 24, 48, 72, 96,120, and 144 hours after the last treatment. 4) Shoulder, thigh, back muscle, liver and kidneys were obtained from each rabbit at slaughtering time.  Effect of Heat: Part of samples (12 hours post treatment) were boiled in distilled water for 45 minutes and used to detect the drug residues.  Effect of Freezing: The rest of samples (12 hours post treatment) were kept at -10°C in freezer and examined weekly for detecting residues for one month. Detection of Residues: Pefloxacin residues were determined using High Performance Liquid Chromatography (HPLC) Knoure, Ink Germany, according to the method described by Groeneveld and Brouwers, (1986. Pefloxacin was extracted from 157 Bull High Inst Public Health Vol.38 No.1 [2008] samples with dichloromethane and 0.1 M sodium phosphate buffer pH 7.4. Chromatography was performed on an amino-exchange column with the mobile phase and tested using UV detector, UV absorbance was monitored at 278 nm. Into a 10 ml extraction tube of 1 g of homogenized tissue (shoulder, thigh, back muscle, liver, and kidney) and 1 ml of 0.1 M phosphate buffer pH 7.4 were added. After adding 5 ml dichloromethane, the tube was stoppered and gently shaken at 100 cycle/min for 10 minutes and centrifuged at 4000 rpm for 10 minutes at room temperature. After removing the aqueous layer, the organic layer was transferred into another tube and dried under nitrogen at -50°C. The residues were dissolved in 1 ml mobile phase using Vortex mixer and sonication, before HPLC analysis. Depending on concentration, 5-


1) Chemical Formula: C17H20FN3O3
2) Dose: Pefloxacin is available in an injectable solution to be given intramuscularly at a dose of 10 mg/kg body weight.

ii. Experimental Rabbits:
Thirty healthy rabbits (Balady bread) of both sexes weighing about 1.5-2 Kg Body weight were used as experiment animals throughout this study.
They were kept for 15 days under well hygienic conditions and fed on concentrated mixture of barseem barley in a pellet form free from any antibacterial agents and water ad.Lid.

1)
Two rabbits (control) were slaughtered and tested for the absence of any antibacterial residues.
2) The rest of rabbits were given pefloxacin intramuscularly (10 mg/Kg body weight) for 5 successive days.After adding 5 ml dichloromethane, the tube was stoppered and gently shaken at 100 cycle/min for 10 minutes and centrifuged at 4000 rpm for 10 minutes at room temperature.After removing the aqueous layer, the organic layer was transferred into another tube and dried under nitrogen at -50°C.The residues were dissolved in 1 ml mobile phase using Vortex mixer and sonication, before HPLC analysis.Depending on concentration, 5-20l were injected.

Standard preparation
Pefloxacin standard solution was prepared from drug pure 100% by dissolving a weight amount of drug in distilled water to make stock solution

Statistical analysis
It was carried out according to  Freezing for 3 months also failed to destroy the residues in kidney completely.Gyhan (1997) 36 found that apramycin sulphate residues in chicken tissues after boiling at

Conclusions and Recommendations
It is clear that the use of antibiotic for prophylaxis and treatment of some injectionus diseases of poultry and rabbits shortly before slaughter resulted in the presence of their residues in muscles and organs.To safe consumer against the hazards which result from consumption of such in rabbit meat and organs containing antibiotic residues, the following instructions should be recommended: 1. Prohibit administration of antibiotic before slaughter is necessary for withdrawal of any residues (Garrod, 1964) 42 .
2. An administration of antibiotics should be done under the supervision of veterinarians.
3. Regular examination of slaughtered animal for detection of antibiotic residues.

-
the 4-quinolone ring were nalidixic acid and oxolinic acids, which are active in vitro against a wide range of Gram-negative bacteria.The included problems associated with their application were restricted spectrum of activity and the relatively rapid emergence of resistant mutants, which led to the discovery of fluroquinolones.One of them is pefloxacin (1-ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3quinoline carboxylic acid.As other fluoroquinolones, Pefloxacin achieves rapid bactericidal Bull High Inst Public Health Vol.38 No.1 [2008] activity by inhibiting the bacterial DNA gyrase Chu and Fernandes, (1991) 1 -Marie et al., (2000) 2 .The drug possesses good in vitro activity against a variety of pathogens, including Gram-positive and Gram-negative bacteria.Pefloxacin is relatively similar to other fluroquinolones such as Enrofloxacin, Ciprofloxacin, and Marbofloxacin (Van-Custen et al., (1990) 3 Spreng et al., (1995) 4 -Brown, (1996) 5 in possessing a wide spectrum of activity, a large volume of distribution, and their activity at low concentration.has been an increased concern amoung consumer about antibiotic and other drug or chemical residues Katz and Brady, (1993) 7 .The potential problems associated with drug residues may be aesthetics, all ergic reactions, direct toxic effects, and change in the resistance patterns of bacteria exposed to antibiotic Weaver.(1992) 8 .The drug is metabolized in the animal's body and broken down or excreted, within the directed withdrawal time.Organs such as the kidney and liver remove residual drug and greatly reduce the content present in red meat or milk.These organs are the tissues tested for residues but content in red meat are less by many times Glott et al., (1979) 9 .The aim of this study is to detect the light pefloxacin residues in different tissues as well as the effect of heat treatment (boiling) and freezing on tissue residues.: (1-ethyl-6-fluoro-1,4dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3quinoline carboxylic acid.
in poultry farms and rabbit batteries for prophylaxis and treatment purposes of various bacterial diseases as well as for growth promotion.Some of these antibiotics leave residues in the animal tissues and may couse hazards to human beings consuming such tissues.Residues in meat of animals and poultry form a great problem facing food hygienists and constitute a real hazard to human consumers in the recent years.These residues are responsible for inducing allergic reactions such as urticaria, eczema, and other forms of dermatitis as well as increasing resistance of pathogenic micro-organisms in man, in addition to their harmful effects on the microflora and Mona O Abou El-Nil 158 consequently the produced vitamins (Mol, 1971) 12 .Growth promoters are mostly given during the whole life time, while prophylactic and therapeutic regimens of antibiotics are given only for a short period (Van Schothors et al., 1978) 13 .Organs such as liver and kidney remove residual drug and greatly reduce the content in meat.These organs and tissues are tested for detecting drug residues (Glott et al., 1979) 9 .In the present study, pefloxacin administrated intramuscularly to rabbits at dose of 10 mg/Kg body weight for 5 successive days.

100°C for 45 minutes
failed to detect in liver, kidney, gizzard and fat after 48 hours from drug administration and were failed to detect in breast and thigh muscles after 72 hours from last oral and residues disappeared from breast and thigh muscles, liver, kidney, gizzard and fat after the 3 rd day from freezing and disappeared from skin after 2 days from freezing samples.Pouliques and Morvan, (2002) 37 determined the residues of oxolonic acid (OA) and flumequine (Flu) in freeze-dried salmon muscle with attached skin, using reversed-phase high performance liquid chromatography.They concluded that, the limits of detection were 3.2 and 16 ng/g wet weight tissue respectively.Mean extraction recoveries of OA and Flu freeze-dried tissue were 85.5 and 85.2%, respectively.Abd el-Aty and Goudah, (2000) 38 found that after intravenous and intramuscular administration of pefloxacin at dose of 10 mg/Kg body weight at a single dose to lactating goats, drug distributed rapidly and was detected in serum 5 min after injection and its concentration decreased gradually till reading lowest detectable level.Abou El-Nil, (2003) 39 revealed that Danfloxacin, Ciprofloxacin, and Flumequine administration orally to chicken at dose of 5 mg/Kg body weight for 5 successive days the highest concentration level was detected in liver at 1 st day from drug administration.Abou El-Nil, (2006) 40 reported that the highest concentration level of Ciprofloxacin detected in chicken liver at 1 st day and decreased till not detected at 9 th day and the lowest concentration level was detected in thigh muscle 12.6 ± 1.025 µg/g.concentration of antibiotic decreased by heat treatment frozen storage of liver, kidney, breast, and thigh muscle resulted in gradual decrease in concentration from 1 st week till not detected at 5 th week.Habib et el, (2006) 41showed that the effect of cooking on the decomposition ond concentration of flumequine and oxolonic was observed.

AJW, Brouwer SJ.
Katz SE, Brady MS.Antibiotic residues in food and their significance in antimicribials in food.Edited cy P. Michael Davidson.Alfred Larry Branen.Newyork.1993;pp 353-370.8-Weaver LD.Antibiotic residues in milk and meat precipitations and relatities.