ESTIMATION OF WITHDRAWAL PERIOD OF LEVOFLOXACIN IN LIVER AND BREAST MUSCLE TISSUE OF DUAL PURPOSE CHICKEN

Electronic Journal of Environmental Sciences Vol. 8: 43-48 (2015) ISSN: 0973-9505 (Available online at www.tcrjournals.com) Original Article Indexed in: ProQuest database Abstract, USA ( ProQuest Science journals, Techonology Research database, Illustrata Technology, Environment Science collection and Health and Medical complete), EBSCO databases (USA), Indian Science abstract. ESTIMATION OF WITHDRAWAL PERIOD OF LEVOFLOXACIN IN LIVER AND BREAST MUSCLE TISSUE OF DUAL PURPOSE CHICKEN RAVIKUMAR. C., SANGANAL, J. S., SHRIDHAR. N. B., NARAYANASWAMY, H. D., ANSAR KAMRAN, C. AND RAMACHANDRA, S. G. Department of Veterinary Pharmacology and Toxicology. Veterinary College, Hebbal, Bengaluru 560024, Karnataka. E. mail: ravivet4u@rediffmail.com Received: August 1, 2015; Accepted: September 18, 2015 Abstract: The present study was carried out to evaluate the withdrawal period of levolfoxacin after repeated oral administration for five days in dual purpose chicken. The study was conducted in Indian Rock -3, a dual purpose chicken strain of White Plymoth Rock developed by Karnataka Veterinary Animal Fisheries Sciences University, Bidar. Birds in the age group of thirty days, were divided in to two groups. Group I (n=10) birds served as control, the tissue samples of which were used for the standardization of the instrument, Liquid chromatography-mass spectrometry. Group- II (n=80) birds received levofloxacin at 10 mg per kg body weight orally for five days. Residue analysis in liver and breast muscle were analysed by LC-MS. The residue concentration of levofloxacin were gradually decreased in chicken liver and breast muscle samples starting from day one to day ten after the last dose. Levofloxacin was estimated to have pre slaughter withdrawal period of four to five days in comparison with maximum residue level of 200, and 100 µg / kg for liver and breast muscle respectively as per European economic community council regulations. It was concluded that slower elimination of levofloxacin from the body could be attributed to its liphophilicity and hish tissue perfusion rate. Key words: Levofloxacins, Chicken INTRODUCTION Levofloxacin, a broad spectrum antibiotic of third generation fluoroquinolone is a levo isomer of ofloxacin. Its spectrum of activity includes most strains of gram positive and gram negative anaerobic bacterial pathogens responsible for respiratory, urinary tract, gastrointestinal, abdominal and skin and soft tissue infections [1]. It has excellent broadspectrum activity against Mycoplasma and Chlamydia organisms in veterinary medicine [2]. Levofloxacin and later generation fluoroquinolones are collectively referred to as respiratory quinolones, which exhibited modest activity toward important respiratory pathogen Streptococcus pneumonia [3]. As compared to other fluoroquinolones, such as ofloxacin and levofloxacin, it also has more pronounced bactericidal activity against organisms such as Pseudomonas, Enterobacteriaceae and Klebsiella [4]. Levofloxacin is more extensively distributed into intrapulmonary compartments than levofloxacin and achieved significantly higher steady-state concentrations in plasma and epithelial lining fluid [5] The administration of veterinary drugs to foodproducing animals without an adequate Withdrawal Time (WDT) may lead to violative concentrations 43

Electronic Journal of Environmental Sciences of residues in foods intended for human consumption. These residues represent a risk to public health, including stimulation of bacterial resistance, alterations on intestinal microflora and hypersensitivity reactions. In general terms, the WDT is the period of time required after completion of treatment needed for tissue concentrations of the drug and/or its metabolites to deplete to less than the established Maximum Residue Limits (MRLs) [6] Various analytical techniques such as atomic absorption, spectrometry, polarography, differential pulse polarography, Capillary zone electrophoresis, spectrofluorometry and high performance liquid chromatography, Biphasic, liquid-liquid extraction, Solid phase extraction by Liquid Chromatography Mass Spectroscopy/ Mass Spectroscopy (LC-MS/ MS) and Nuclear Magnetic Resonance were used to extract metabolites in the animal tissues [7]. LC- MS provides superior specificity and sensitivity and can be used to develop highly accurate and reproducible assays. The primary advantage of LC- MS is the ability of analysing a much wider range of components. There were few studies in the assessment of residual status of levofloxacin in the tissues of broilers., making essential to generate tissue depletion data in order to arrive at conclusion regarding MRLs and withdrawal period for levofloxacin drug. In view of the marked species variation in the pharmacokinetic data of antimicrobial drugs, present study was planned to determine the, residue level and withdrawal period of levofloxacin in liver and breast muscle tissue following single oral administration at the dose of 10 mg/kg body weight for five days in the dual purpose chicken. MATERIALS AND METHODS The study was conducted in dual purpose chicken : Indian Rock -3 (IR-3), a strain of White Plymoth Rock developed by KVAFSU, Bidar, The study was performed in animal house at the Department of Poultry Science, Veterinary College, Hebbal, Bengaluru. Healthy birds of thirty days old age (n=90) were selected for the study. Drugs: Levofloxacin and Indomethacin technical grade powder were obtained from Vetoquinol, India Animal Health Private Limited, Mumbai and Sigma Aldrich, (Poole, UK) respectively. HPLC grade formic acid, acetic acid, methanol and acetonitrile were procured from E-merck (Germany). HPLC grade water prepared in house using a Millipore Direct-QTM 5Water System (Millipore, Watford, UK). Filtration of HPLC mobile phase was performed using Sartorius membrane filters [0.45µm] obtained from Sartorius (Epsom, UK) and Solid Phase Extraction cartridges (Orochem Company). Blank tissue samples for the preparation of calibration standards and quality control samples were collected from group- I (n=10) birds and were stored at 20±5 C until analysis. Experimental animals: For residues analysis and withdrawal time calculation, group-ii birds (n=80) were kept under observation for one week prior to commencement of experiment and subjected to clinical examination in order to exclude the possibility of disease. The birds were provided antibiotic-free standard broiler ration for fourteen days. The animal house was maintained at room temperature (25±2 0 C) and at 45 to 65 per cent relative humidity. Food and water were supplied ad libitum with standard management practices to keep the birds free from stress. The experimental protocol was approved by the Institutional Animal Ethics Committee ( LPM/IAEC/181/2014, DATED: 10/01/ 2014. The antibiotic residue analysis was conducted as per European Commission Council Regulation [8]. Levofloxacin was administered to group-ii birds at the dose of 10 mg per kg body weight orally through individual for five days. The birds were euthanized and immediate exsanguinations on day 1,2,3,4, 5,7,9 and 10 after the administration of the last dose of levofloxacin (n=8/day). Samples of liver and breast muscle were collected and the tissue samples were stored at -45 0 C until assayed for concentrations of levofloxacin. The samples were subjected to residual analysis using Liquid Chromatography tandem Mass Spectroscopy (LC-MS). Based on the the residual concentration, the withdrawal time of levofloxacin in dual purpose chicken were established. In general terms, the WDT is the period of time required after completion of treatment needed for tissue concentrations of the drug and/or its metabolites to deplete to less than the established Maximum Residue Limits (MRLs). MRLs calculated from the levofloxacin area at each sampling time were considered for the determination of withdrawal time (WDTs) in 44

muscle, liver and kidney, adopting the European Agency for the Evaluation of Medicinal Products recommendations [9]. Experimental conditions: The chromatography was carried out with LC-MS/MS (Agilent Technologies, Waldbron, Germany) Agilent 1200 RRLC system with a solvent delivery pump, auto-degasser, auto sampler and column oven. Electrospray mass spectrometry (ESI-MS) was carried out using a 3200 Q TRAP triple-quadrupole LC-MS/MS system (Applied Biosystems/MDS Sciex), coupled with a Turbo Ion Spray (TISP) source with ESI mode. Applied Biosystems Sciex Analyst software version 1.5 was employed for data acquisition and processing. The separation was performed on a Thermo Scientific BDS Hypersil C18 RP, 100x4.6 mm, 5 µm. Separation was achieved using a gradient elution with the flow rate of 0.7 ml/min, while the injection volume was 20 µl. Preparation of calibration standard solutions and quality control stocks: Primary stock solution of levofloxacin (LEV) for calibration standard and quality control (QC) samples were prepared in methanol. From the primary stock solution, appropriate dilutions were made using Methanol: water (50:50 % v/v) as a diluent to produce working standard solutions of 2000.000, 4000.000, 10000.000, 20000.000, 40000.000, 80000.000, 120000.000, 160000.000 and 200000.000ng/mL. These solutions were used to prepare relevant calibration curve (CC) standards. Another set of working solutions of levofloxacin was prepared in the diluent (from primary stock) at concentrations of 2000.000, 6000.000, 100000.000 and 180000.000ng/mL respectively for QC samples (LLOQC, LQC, MQC & HQC). The calibration standards and quality control samples were prepared by spiking 0.010mL of the spiking stock solution (Levofloxacin) into 0.190mL of screened blank Ravikumar et al. chicken plasma. Calibration samples were made at concentrations of 100.000, 200.000, 500.000, 1000.000, 2000.000, 4000.000, 6000.000, 8000.000 and 10000.000ng/mL. Quality control samples were prepared at concentrations of 100.000ng/mL (lower limit of quality control, LLOQC), 600.000ng/mL (lower quality control, LQC) 5000.000ng/mL (medium quality control, MQC) and 9000.000ng/mL (higher quality control, HQC). RESULTS AND DISCUSSION In the present study, residual concentrations of levofloxacin in liver and breast muscle tissues of dual purpose chicken were analysed by LC-MS/ MS after oral administration of levofloxacin at 10 mg/kg body weight for five consecutive days. The LC- MS/MS method was selected because of its high specificity and accuracy, which were considered critical factors for this study. Applied Biosystems Sciex Analyst software version 1.5 was employed for data acquisition and processing. The separation was performed on a Thermo Scientific BDS Hypersil C18 RP, 100x4.6 mm, 5 µm. Separation was achieved using a gradient elution with the flow rate of 0.7 ml/ min, while the injection volume was 20 µl.lc-ms- MS has been used for similar purpose by other workers [11,12,]. Liver : High residue level of levofloxacin was observed on day one and gradually decreased up to tenth day in liver tissue samples after the final dose of administration. Highest levofloxacin residue concentration detectable in liver was 1428.89 ± 0.93 µg per kg and there was decreased in residue concentration up to 66.87 ± 0.23 µg per kg on day ten after the last dose of levofloxacin administered. The concentration of levofloxacin on day four was 190.20 ± 0.97 µg/kg. The value was less or almost equal to Maximum Residual Level (MRL) fixed for fluroquinolones (200 µg/kg) in liver tissue samples. Table 1: Intra- and inter-day assay coefficient of variation (CV%) and nominal analysis in liver and breast muscle tissue of dual purpose chicken concentration of levofloxacin for reside Concentration (ng/ml) Mean ±SD (ng/ml) Intra-day assay (n= 8) Inter-day assay (n= 8) CV Mean concentration Mean ±SD (ng/ml) CV Mean concentration 100 (LLQC) 98.86±0.30 10.65 98.76 97.98.46±0.26 11.46 92.84 300 (LQC) 291.68±0.12 12.08 97.23 261.46±0.13 13.41 87.15 5000 (MQC) 4425.78±0.20 6.87 88.52 4671.08±0.21 7.77 93.42 9000 (HQC) 8206.93±6.16 6.61 91.19 7315.38±7.04 7.65 81.28 45

Electronic Journal of Environmental Sciences Fig. 1: Standard curve for levofloxacin extracted blank tissue homogenate sample Fig 2: Levofloxacin concentration (µg per kg) in liver and breast muscle from Day 1 to 10 after the last dose of 46

Table 2: Concentration ( µg per kg) of levofloxacin in dual purpose chicken tissues. Values are expressed as Mean ± SE; n=8 per day. Days Liver Breast muscle 1 1428.89 ± 0.93 467.66 ± 0.92 2 574.74 ± 1.32 218.03 ± 0.62 3 334.04 ± 0.79 179.23 ± 0.71 4 190.20 ± 0.97 128.87 ± 0.76 5 149.53 ± 1.02 82.33 ± 0.62 6 131.75 ± 0.79 54.83 ± 0.74 7 110.64 ± 0.82 46.53 ± 0.56 8 90.79 ± 0.61 32.41± 0.94 9 82.71 ± 0.51 21.29 ± 0.89 10 66.87 ± 0.23 16.51 ± 0.77 The withdrawal period for levofloxacin in liver samples of dual purpose chicken was estimated to be four days approximately. Ravikumar et al. The present findings are in agreement with findings of Kyuchukova et al. [14] who reported that high residue level of the levofloxacin observed in liver tissue on day one was 1051± 648 µg per kg compared to other tissues and decreasing in the residue concentration until day 8 was 56 ± 5 µg per kg after the treatment. Jelena et al. [15] reported high residue levels of enrofloxacin and ciprofloxacin in liver tissue on day one at 1196.1 µg per kg and 187 µg per kg respectively, compared to other tissues and decreasing in the enrofloxacin residue concentration up to ninth day was 24.8 µg per kg after the treatment. Lim et al. [16] reported that high residue concentration of norfloxacin observed in liver tissue was 990 µg per kg on day one compared to other tissues and there was decreased in levofloxacin residue concentration up to 70 µg per kg on fifth day after the treatment. Anadon et al. [17] reported that high residue concentration of difloxacin and its metabolite sarafloxacin in liver tissue were 368.1±52.5 ìg per kg and 10.4±1.2 ìg per kg respectively on day 1 compared to other tissues and decreased in difloxacin residue concentration up to fifth day after administration of the final dose of difloxacin. However on the contrary, Banna et al. [18] who reported that high residue concentration of levofloxacin was noted 2.47±0.23 µg per g or 2470 ± 0.19 µg per kg in kidney tissue on day one compared to the other tissues and decreased in residue concentration of 70 ± 0.01 µg per kg on 9th day after the treatment. Breast muscle: Highest concentration of levofloxacin in breast muscle tissue was 467.66 ± 0.92 µg per kg on day one and decreased in residue concentration up to 16.51 ± 0.77µg per kg on day ten after the administration of the final dose of levofloxacin in dual purpose chicken. In the present study, maximum concentration of levofloxacin was recorded in thigh muscle tissue at 364.64 ± 1.78 µg per kg on day one and the concentration was reduced up to 4.72 ± 0.42 µg per kg day nine after the administration of the last dose of levofloxacin in dual purpose chicken. These findings are in agreement with Kyuchukova et al. [14] who reported that highest residue level of the levofloxacin in the breast muscle was 428±253 µg per kg on day 1 and decreased in the residue concentration up to 56±15 µg per kg on day 8 after the treatment. Banna et al. [18] reported the highest levofloxacin residue concentration in breast muscle was 0.23±0.04 µg per g or 230±0.04 µg per kg on day 1 and decreased in residue concentration up to 50 ± 0.0029 µg per kg or 0.05±0.0029 µg per g on day five after the treatment. Persistence of levofloxacin in animal tissues is attributed to the liphophilicity and tissue perfusion rate of the drug [19,20]. Withdrawal period was calculated based on the residual concentration of the levofloxacin in tissues of dual purpose chicken.[21]. The MRLs were 200 and 100 µg per kg for liver and breast muscles respectively. The present findings are in agreement with findings of Banna et al. [17] who reported that withdrawal period for levofloxacin in live and muscle tissue was four to five days in broiler chicken. Sayed et al. [22] reported the withdrawal period of levofloxacin as five days in normal catfish Anadon et al. [17], reported that withdrawal period of five days was necessary to ensure that the residues of difloxacin were less than MRL or tolerance established by the European Union. Jelena et al. [15] reported the withdrawal period of four days for enrofloxacin in muscle and liver in broiler chicken. Petrovic et al. [23] reported a withdrawal period of four days for enrofloxacin and its metabolite ciprofloxacin residues to decrease to an acceptable level in the meat and liver of the broiler chicken. It was concluded that levofloxacin was estimated to have four days of withdrawal period in liver and five days of withdrawal period in breast muscle tissue of dual purpose chicken. 47

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