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Screening of Enrofloxacin and Its Metabolite Ciprofloxacin Residues by High-Performance Liquid Chromatography in Cow Milk of District

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Residues in animal products play significant role in development of resistance to antibiotics. A plenty of resistance cases has been demonstrated in farm conditions making the veterinary practice vulnerable. To monitor the levels of enrofloxacin and
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    International Journal of Livestock Research    eISSN : 2277-1964  NAAS Score -5.36   Vol 7 (7) July  ’17   Hosted@ www.ijlr.org  DOI   10.5455/ijlr.20170520035753       P   a   g   e     1    4    0  riginal Research Screening of Enrofloxacin and Its Metabolite Ciprofloxacin Residues by High-Performance Liquid Chromatography in Cow Milk of District Udham Singh Nagar, Uttarakhand   Srinivasu M. * , A. H. Ahmad, Nirbhay Kumar, Disha Pant and Wasif Ahmad Department of Veterinary Pharmacology & Toxicology, College of Veterinary & Animal Sciences, G. B. Pant University of Agriculture & Technology, Pantnagar, Uttarakhand, INDIA *Corresponding author:  drsrinivasgirish@gmail.com     Rec. Date: Mar 18, 2017 06:39 Accept Date: May 20, 2017 15:57 Published Online: June 27, 2017 DOI 10.5455/ijlr.20170520035753   Abstract  Residues in animal products play significant role in development of resistance to antibiotics. A plenty of resistance cases has been demonstrated in farm conditions making the veterinary practice vulnerable. To monitor the levels of enrofloxacin and its metabolite ciprofloxacin residues in the raw milk samples of cow, 100 samples collected from different blocks of U.S.Nagar were analysed for enrofloxacin and ciprofloxacin residues and positive samples were detected and quantified using High Performance Liquid Chromatography (HPLC). Out of 100 milk samples analysed 19 samples (19%) had detectable amount of residue levels for enrofloxacin and ciprofloxacin and 2 samples (10.52%) had residue levels above the recommended Maximum Residue Limit MRL.    A linear calibration curve was obtained by plotting area against concentration with a correlation coefficient of 0.9975 while average recoveries were greater than 86.8% with relative standard deviation (RSD) value of 0.32%.The method described in the present study would be helpful for monitoring of enrofloxacin and ciprofloxacin residues in milk. Key words:  Residues, Enrofloxacin, Ciprofloxacin, Milk Samples, HPLC How to cite: Srinivasu, M., Ahmad, A, H., Kumar, N., Pant, D., & Ahmad, W. (2017). Screening of Enrofloxacin and Its Metabolite Ciprofloxacin Residues by High-Performance Liquid Chromatography in Cow Milk of District Udham Singh Nagar, Uttarakhand. International Journal of Livestock Research, 7(7), 140-145.  http://dx.doi.org/10.5455/ijlr.20170520035753   Introduction Enrofloxacin and its active metabolite Ciprofloxacin, fluoroquinolone antibiotic, which have been approved for use in food animals and are effective against organisms resistant to commonly used antibiotics in veterinary medicine such as β -lactams, aminoglycosides, tetracyclines and macrolides (Hao et al ., 2014 ) . Drug residues may pass on to tissues and milk due to extra label use of these antibiotics.    International Journal of Livestock Research    eISSN : 2277-1964  NAAS Score -5.36   Vol 7 (7) July  ’17   Hosted@ www.ijlr.org  DOI   10.5455/ijlr.20170520035753       P   a   g   e     1    4    1  Accordingly, consuming such animal products may pose a potential hazard for the consumers, causing allergic reactions and also lead to the emergence of drug resistant bacteria. Incidence of adverse effects of fluoroquinolone treatment, particularly in human medicine, has been reported (Lewis and Cook 2014, CVMP 2007) .  MRL for enrofloxacin and ciprofloxacin was set at 100µg/kg for all animal species by European Union (EMEA 2002).   High performance liquid chromatography (HPLC) enables to detect the minute concentration of the antibiotics and their metabolites in different types of matrices. Analysis of enrofloxacin using HPLC has been less reported. The objective of the present study was to screen the presence of enrofloxacin and ciprofloxacin residues above maximum residue limits in raw milk in and around Udham Singh Nagar.  Materials and Methods Raw milk samples (n=100) were collected from different milk suppliers and local vendors in the Udham Singh Nagar district. Milk samples were collected from all four quarters of the udder of the cows in sterilized centrifuge tubes. After collection of samples, they were immediately processed on the same day to avoid any contamination. All the reagents used were HPLC grade. Acentonitrile, methanol, orthophosphoric acid, triethylamine obtained from Merck Specialties Private Limited (Darmstadt, Germany). Enrofloxacin and ciprofloxacin were supplied by Sigma - Aldrich (St. Louis, USA) .Water was purified in a Milli-Q system (Millipore, Bedford, MA, USA). For the extraction procedure, a phosphate buffer of pH 7.4 (0.05  M  ) was prepared by diluting 250 ml of KH 2 PO 4  (0.2  M  ) and 197.5 ml of NaOH (0.2  M  ) to 1 litre with water. The standard drug solution (1mg/ml) was prepared by dissolving 5mg of enrofloxacin and ciprofloxacin in 5 ml of 0.1M NaOH. The stock solutions of enrofloxacin and ciprofloxacin were then further diluted with mobile phase (acetonitrile: methanol: water (17:3:80 v/v/v) containing 0.4% orthophosphoric acid (85% v/v) and 0.4% Triethylamine , pH adjusted to 2.5-3.) to obtain working solutions with different concentrations of 1.0 mg/ml to 1ng/ml range . Fresh milk samples were taken in sterile test tubes and centrifuged at 5000-6000rpm for about 15 mins. The fat layer of the milk is removed and to 1 ml of whey, 2ml of acetonitrile was added. The mixture was vortexed for 1-2 min. Again the mixture was centrifuged at 5000rpm for 15min. The supernatant was purified by solid-phase extraction (SPE) using a Supelco solid phase extraction C-18 cartridges which had previously been conditioned with 6 ml methanol, 6 ml water and 6 ml phosphate buffer, pH 7.4. The eluate was evaporated in solvent evaporator under nitrogen gas. The residue was re- suspended in 100 µl of acetonitrile and diluted to 900µl with water. Mixture was filtered through Millipore 0.22µ filters and aliquot of 20µl injected for HPLC.    International Journal of Livestock Research    eISSN : 2277-1964  NAAS Score -5.36   Vol 7 (7) July  ’17   Hosted@ www.ijlr.org  DOI   10.5455/ijlr.20170520035753       P   a   g   e     1    4    2  HPLC system (Shimadzu Corporation, Kyoto, Japan, Model SPD-10A LC10AT) comprised of double plunger pump; Rheodyne injector with 20µl loop; UV-VIS detector. C-18 reverse phase columns (Lichrospher 100 RP 18e (5µm); Merck Kga A, 64271 Dramstadt Germany. The mobile phase consisted of acetonitrile: methanol: water (17:3:80 v/v/v) containing 0.4% orthophosphoric acid (85% v/v) and 0.4% triethylamine, ph adjusted to 2.5-3, and the flow rate was 0.6 ml/min. The injection volume was 20 μl and the column temperature was 20°C. UV detection was at 278nm.   Results and Discussion The results were interpreted based on the result of the standard curve and the standard chromatogram (Fig.1 & Fig.2.). Fig. 1: Chromatogram of standard Enrofloxacin and Ciprofloxacin   Standard curves prepared by spiking standards were linear in the range of 0.05- 5 μg/ml with correlation coefficient (r 2 )0.9975. Based on the peak area versus concentration data, the unknown sample concentration was determined. The retention time value of standard enrofloxacin and ciprofloxacin was 5.55 and 7.11 minutes respectively. The recovery of samples ranged from 85-90%. In this study LOD was 0.05μg/ml(50 PPB) and LOQ is 0.1μg/ml (100 PPB).The results of this study indicated that out of total 100 milk samples only 19 samples (19%) were found to be positive for Ciprofloxacin and out of 19 positive samples, 2 samples (10.52%) were found to be above MRL(maximum residue limit). To detect antibiotic residues, screening methods and chromatographic methods have been developed to detect at very low concentrations. The screening tests are generally performed by microbiological (Babapour et al ., 2012), enzymatic and immunological methods (Strasser et al ., 2003). FSSAI has recommended analytical methods for detection and quantification of fluoroquinolone residues by HPLC-MS/MS method.    International Journal of Livestock Research    eISSN : 2277-1964  NAAS Score -5.36   Vol 7 (7) July  ’17   Hosted@ www.ijlr.org  DOI   10.5455/ijlr.20170520035753       P   a   g   e     1    4    3  Fig.2: Chromatogram of Ciprofloxacin in milk sample   The main problem associated with milk extraction for subsequent determination of antibiotics is the high protein content. In most methods reported in the literature, the preparation of milk samples for residue analysis involves protein precipitation followed by solid phase extraction(SPE) through the use of appropriate SPE cartridges(Junza et al ., 2011). Safe levels of residues in milk and other animal products result from the participation of all activities involved in the food chain from stable to tables (Serratosa et al ., 2006). Fluoroquinolones play important role in combating bacterial and protozoan diseases in veterinary medicine. The committee for veterinary medicinal products considers that the sum of all substances belonging to the fluoroquinolones group in raw milk should not exceed 100µg/kg (EMEA, 2002). Considering the issue of public health hazards, milk and milk products contaminated with antibiotics and other chemical contaminants beyond given residue level, are considered unfit for human consumption (Goffova et al ., 2012). Occurrences of veterinary drug residues pose the broad range of health consequences in the consumers. The residue of antibacterials may present pharmacological, toxicological, microbiological, and immunopathological health risks for humans (Drackova et al ., 2009). Residues of antibacterial are detectable in livestock products if proper withdrawal time is not followed by the farmers. For these, fluoroquinolones withdrawal period should be followed very strictly as these are most commonly used antibiotics in farm animals (Lopez et al ., 2015). The results of the present study show that about 19.0% of samples were positive for ciprofloxacin, which demands a scrutiny assay for regular inspection of livestock products. The European Agency has set the MRL of 100ppb for the evaluation of enrofloxacin and its metabolites in the bovine milk. But this study shows up to 228ppm of enrofloxacin and its metabolites were detected in the milk samples which is a hazard for the consumers. So precise regulation and frequent inspection ought to be taken for the safety of consumers. Compared to steers, enrofloxacin is more rapidly metabolized to ciprofloxacin by lactating dairy cows and even rapid clearance into milk in dairy cows (Idowu et al  .,    International Journal of Livestock Research    eISSN : 2277-1964  NAAS Score -5.36   Vol 7 (7) July  ’17   Hosted@ www.ijlr.org  DOI   10.5455/ijlr.20170520035753       P   a   g   e     1    4    4  2010).Approximately 88% of the residues are accounted for enrofloxacin and its metabolite ciprofloxacin in cow’s milk, 6 to 24 hours after treatment (EMEA 1998). Due to extra label use of veter  inary drugs or negligence in obeying withdrawal periods, much higher residue levels appear in edible tissue products (Bostsoglou and Fletouris., 2001). As resistance is of growing concern and being at the high risk group, fluoroquinolones (WHO1998; CVMP 2007), withdrawal period for these antimicrobials should be given importance in field conditions. Hence this study fulfils the need of the hour to highlight the importance of withdrawal period of drugs. Conclusion The present study provides a simple ad accessible method for detection of enrofloxacin in biological samples like milk. This could be applied for other animal products like meat and egg samples to detect minor quantity of residues using HPLC. Monitoring and surveillance studies for residues must be focused to avoid resistance among farm animals and human beings consuming milk containing antibiotics residues. An efficient control of the residue in milk is very important to ensure the safety of milk and milk products. References 1.   Babapour A, Azami L and Fartashmehr J.2012. Overview of antibiotic residues in beef and mutton in Ardebil,North West of Iran. World Applied Sciences Journal , 19:1417-1422. 2.   Botsoglou NA, Fletouris DJ. 2001. Drug Residues in Foods.Pharmacology,Food safety and analysis. Marcel Dekker, New York. 3.   CVMP .2007.Public statement on the use of fuoroquinolones in food-producing animals in the European Union: development of resistance and impact on human and animal health. 4.   Drackova M, Navartilova P, Hadra L,Vorlova L and Hudcova L. 2009.Determination residues of penicillin G and Cloxacillin in raw Cow milk Using Fourier transform near infrared spectroscopy.  Acta Veterinaria Brno ,78: 685-690. 5.   EMEA/MRL/389/98- Final, 1998.Committee for veterinary medicinal products, Enrofloxacin (extension to sheep,rabbits and lactating cows) summary report(3). 6.   EMEA/MRL/820/02- Final, 2002.Committee for veterinary medicinal products, Enrofloxacin (extension to all food producing species) summary report (5). 7.   Goffova ZS, Kozarova I, Mate D, Marcincak S, Gondova Z and Sopkova D.2012.Comparison of detection sensitivity of five microbial inhibition tests for the screening of aminoglycoside residues in fortified milk  . Czech Journal of Food Sciences , 30: 314-320. 8.   Hao H, Cheng G, Iqbal Z, Ai X, Hussain HI, Huang L, Yuan Z. 2014.Benefits and risks of antimicrobial use in food-producing animals. Frontiers in Microbiology ; 5:288. 9.   Idowu OR. 2010 .Comparative pharmacokinetics of enrofloxacin and ciprofloxacin in lactating dairy cows and beef steers following intravenous administration of enrofloxacin. Res. Vet. Sci.; 89(2):230-5. 10.   Junza A, Amatya R, Barron D and Barbosa J.2011.Comparative study of the LC-323 MS/MS and UPLC-MS/MS for the multi-residue analysis of Quinolones,324 pencillins and cephalosporins in cow milk, and validation according to 325 regulation 2002/657/EC.  Journal   of Chromatography ,B 879:2601-2610. 11.   Lewis T, Cook J. 2014.Fluoroquinolones and Tendinopathy: A Guide for Athletes and Sports Clinicians and a Systematic Review of the Literature.  Journal of Athletic Training ; 49(3):422-427.
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