Detection of Norfloxacin in Muscle, Liver and Kidney of Broiler Chicken

Antibiotic residues occur in various types of foods of animal origin, including milk, egg and meat due to large-scale application of antibiotics in veterinary practice and create problems not only in the food industry but also have immense public health significance (Kozarova, 2001). Antibiotic residues are procedurally measured on uncooked meat, but most chicken meat undergoes further processing prior to consumption (thermal or food additive treatments or both) for the purpose of increasing palatability and shelf-life. Since most types of foods are cooked before consumption, more information about the effect of cooking on residues are required to give a more accurate estimate of consumer exposure to these chemicals and any breakdown products (Heshmati et al., 2015). Effective control of food matrices is necessary to ensure that they are not present at levels that may pose health risks to consumers (Pena et al., 2010). Fluoroqionolones constitute the second generation of quinolones and their main difference from the classic quinolones is that they contain a fluorine atom at C-3 position. Norfloxacin have a cyclopropyl group in addition. The addition of fluorine enhances DNA gyrase inhibition and activity against Staphylococci. Cyclopropyl group enhances the potency of antibiotics (Oliphant and Green, 2002).
 
Norfloxacin is a first generation synthetic antibacterial agent used for the treatment of common and complicated urinary tract infections (Anderson et al., 2001). Other applications include prostatitis due to E. coli and in ophthalmic preparations for the treatment of conjunctival infections. It is also administered along with a nitroimidazole for the treatment of amoebiasis associated with diarrhoea. The antibacterial action of norfloxacin results from inhibition of bacterial DNA gyrase and Topoisomerase II and IV which are required for bacterial DNA replication and recombination (Hassouan et al., 2007).
        
The present study was therefore aimed to determine the residues of norfloxacin in meat samples of chicken outlets located in and around Jabalpur.

Location and place of work
 
The proposed work was conducted in the Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University (N.D.V.S.U.) Jabalpur, Madhya Pradesh, India.
 
Collection of meat samples
 
A total of 180 broiler meat samples including muscle, liver and kidney consisting of 60 each were collected from slaughtered poultry of selected targeted area (government and private sector poultry farms) located in and around Jabalpur from January to February 2018. Approximately 10 gram of muscle, liver and kidney samples each of the same bird were aseptically collected and transported to the laboratory in thermo-cooled container jacket with ice and were stored in refrigerator at 4°C till processing.
 
Chromatography condition
 
The unit of High Performance Liquid Chromatography (HPLC) Mass Spectrometer (LCMS-8030, Shimadzu, Japan), consisted of mobile phase reservoir, degasser, HPLC pump, sample injector, gourd column, main column, detector, data collection unit, waste or fraction collector with NEXERA software was used for quantification of ciprofloxacin residues. Chromatographic condition was maintained as described with significant modification. Particle separation was done using hypersil column with C₁₈ selectively (Supelco USA, column dimension: 150×2.1 mm, particle size: 1.9 µm) and the temperature of column was set at 30°C. The mobile phase consisting of 1 ml Ortho phosphoric acid (85 per cent v/v), 100 ml water HPLC grade with acitonitrile HPLC grade, 87:12 (v/v) then pH was adjusted to 2.3 with triethylamine HPLC Grade, The Mobile phase was filtered by 0.22 µ nylon syringe filter before use. Flow rate for the mobile phase was 1ml.min^-1. The temperature of column oven was 25±0.5°C. The effluent was monitored at 278 nm wavelength.
 
Chemicals
 
The chemicals used for extraction, detection and quantification of residual concentration of ciprofloxacin was: Methanol: HPLC grade, Chromadolv® Sigma-Aldrich, Acetonitrile: HPLC grade, Chromadolv® Sigma-Aldrich, Ortho Phosphoric acid 85 per cent: CDH, Water: HPLC grade, Chromadolv® Sigma-Aldrich, Triethylamine: HPLC grade, Rankem, Trichloro acetic acid AR., 99 per cent: HPLC grade, Hi Media.
 
Preparation of standard
 
Stock standard solution of norfloxacin was prepared by dissolving in 10 mg of compound in 10 ml of methanol to obtain a final concentration of 1mg/ml. The solution was stored at -20°C and was stable for at least 4 months. These solutions were diluted to obtain a series of working standard solutions prepared daily.
 
Determination of LOD and LOQ
 
The limit of detection and limit of quantification were analysed with serial dilutions of antibiotic, followed by area obtained at various concentrations
 
Samples preparation
 
The sample preparation or pretreatment procedure involved sample extraction and cleanup processes before analysis by chromatographic technique. In the present study extraction and cleanup procedure, as described below for norfloxacin in chicken muscle, liver and kidney samples were done as per the method described by Ammar et al., (2016) with slight modification:
 
1.  Two g of each muscle, liver and kidney were homogenized in mortar and pastel. Placed in a centrifuge tube and 8 ml (5 per cent) trichloroacetic acid (HPLC grade) was added.
2.  The sample was vortexed well for homogenization upto1 minute and then centrifuged at 14000 rpm for 5 minutes.
3.  The supernatant was filtrated through 0.22 µ nylon filter
4.  The 20 µl of filtrate is put in autosampler glass vial having septa and then put into the auto sampler of HPLC apparatus for analysis. 

Quantitative HPLC analysis of norfloxacin in chicken meat samples
 
The HPLC analysis of norfloxacin in muscle, liver and kidney samples of chicken meat was done by using the mobile phase acetonitrile HPLC grade, (87:12 v/v ratio) exhibited good separation of norfloxacin from matrix with a mean retention time of 6.1 minutes at wavelength of 278 nm. The limit of detection (LOD) of norfloxacin was 0.028 µg/kg and limit of quantification (LOQ) was 0.01 µg/kg. The recovery percentage of norfloxacin standard was ranging from 82-94 per cent (Fig 2).
 

        
The calibration curve of norfloxacin was generated using linear regression of peak areas versus concentration of calibration standard. Chromatogram exhibited sharp peak and purity at different concentration levels of norfloxacin. Norfloxacin standard was used in concentrations of 15, 7.5, 3.75, 1.85, 0.93, 0.46, 0.23, 0.115, 0.057 and 0.028 µg.ml^-1 and their peak area (mAu) was calculated to be 9576352, 4773355, 2156586, 1077560, 521579, 263508, 141272, 67463, 26786 and 13344, respectively (Fig 1).
 

 
Quantification of residual concentrations
 
Mean residual concentration of norfloxacin in chicken meat samples (muscle, liver and kidney) obtained from 10 target areas of Jabalpur district was analysed quantitatively using HPLC. A total of 180 chicken meat samples were analysed consisting 18 samples each of muscle, liver and kidney samples collected from each target stations. Out of 180 samples analysed, 3 liver samples of target stations, viz. T₃, T₆ and T₁₀ were found positive for norfloxacin residues with concentrations of 2.00, 2.60 and 2.31 µg/kg, respectively. However all concentration values of residues were below MRL as indicated by European Union Codex.
        
Samples collected from other target stations did not show residual concentration of norfloxacin in muscle, liver and kidney samples. Mean concentration of norfloxacin in muscle, liver and kidney samples of various target stations has been depicted in Table 1 (Fig 3).
 

 

        
In current study, residual concentration of norfloxacin was detected in 3 liver samples ranging from 2.00 to 2.60 µg/kg. However, muscle and kidney samples did not show norfloxacin residue. The study of Mustafa and Ghamdi (2001) also indicated the maximum residual concentration of norfloxacin in liver samples as compared to muscle. The study of Prescott and Vaggot (1993) stated that norfloxacin is well distributed in body tissues and its higher concentration is found in excretory organs especially in liver, which may be a possible reason for detection of norfloxacin residues more in liver samples.
        
In present study, the residual concentration of norfloxacin detected in liver samples was maximum to 2.60 µg/kg. However, other co-workers reported higher residual concentration of norfloxacin in poultry meat. The report of Pena et al., (2010) detected residual concentration of norfloxacin to the extent of 20 µg/kg in chicken meat samples which is higher as compared to the residual concentration of norfloxacin obtained in the present study. The above findings differ due to time of collection of samples and withdrawal period of antibiotics used in poultry.
        
The recovery of percentage in chicken meat samples was in the range 82-94 per cent for norfloxacin. The chicken meat samples exhibited good separation of both analytes from matrix without any interference which clearly indicates that extraction and cleanup procedure for detection of antibiotic residues were accurate and reliable. The results of chromatographic analysis of norfloxacin with reference to recovery percentage has been reported by Kowalski et al., (2007) who also found norfloxacin recovery ranging from 88-93 per cent using HPLC.
        
The limit of detection for norfloxacin in chicken meat samples was 0.028 µg/kg. On the other hand, limit of quantification was 0.01 µg/kg was obtained for norfloxacin in chicken meat samples. The chromatographic technique used in the study generated good data in terms of limit of detection and limit of quantification, indicating an appropriate and precise limit of detection for norfloxacin by using HPLC. The limit of detection for norfloxacin was also estimated by Hee-Jung et al., (2011) as 3 µg/kg in poultry meat.

Based on the information gathered from the present study, out of 180 analyzed samples, only three liver samples were found positive for the residual concentration of norfloxacin. In all positive samples, the concentrations were below MRL. However, even this concentration, may pose a potential hazard to the consumer. So food safety authorities should control the use of antibiotics in poultry farms and banned their use as growth promoter.

The authors would like to acknowledge the Vice-Chancellor for providing financial assistance, Director Research Services and Teaching faculty of Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science and Animal Husbandry, NDVSU, Jabalpur Madhya Pradesh for providing all invaluable insights and regular encouragement throughout the whole study period.