STUDIES ON OXYTETRACYCLINE RESIDUES DEPLETION IN RABBIT MEAT

MORSHDY, A.M.^*; HUSSEIN, M.A.^* and EL-GOHARY, A.E.^**

^*Food Control Dept.-Faculty of Vet. Medicine-Zagazig, University-Zagazig, Egypt.

^** Food Control Lab. –Dammietta Seaport-Animal Health Research Institute, Egypt.

Email: elged2010@yahoo.com

INTRODUCTION

Rabbit meat is considered a high quality product due to its high protein and low saturated fatty acids, cholesterol and sodium content. Antibiotics are widely used in veterinary medicine and subsequently drug residues may persist in foods derived from animals, which may pose an adverse health effect for the consumer. Today antimicrobial drugs are used to control, prevent and treat infection, and to enhance animal growth and feed efficiency. Tetracyclines are most widely used antibiotics in veterinary medicine in Egypt due to its broad spectrum of antimicrobial activity, availability and low cost. Violation of the maximum residue limit (MRL) regulation can only be proven with fully validated chromatographic methods such as high performance liquid chromatography (HPLC) (Moats, 1997). The use of antimicrobials in food-producing animals may result in the presence of residues in foodstuffs of animal origin. Protection of public health against possible harmful effects of veterinary drug residues is a relatively recent preoccupation. The initial intention for adequate consumer protection led to the desire to achieve a complete elimination of all traces of drug residues in food commodities. Therefore, animal drugs were initially approved based on a “no residue” tolerance policy, but actually the “zero” tolerance represented the sensitivity of the analytical method used to monitor for drug residues. As analytical methods improved, the “no residue” tolerance was continually being lowered. Ultimately, a policy of negligible tolerance, based on toxicology data, was developed (Boisseau, 1993; Teske, 1993). The legislation for pharmaceutical and veterinary products established a withdrawal period of antibiotics of 28 days for fattening rabbits, limiting the addition of antibiotics to the first days of fattening(Badiola et al., 2007). Since the most of foods-producing animals are always cooked before consumption and the variations in oxytetracycline levels in the meat are dependent on type of cooking. More findings about the effect of cooking on oxytetracycline residue are needed to accurately determine consumer exposure to this drug. The boiling for 30 minutes and roasting at 150°C for 30 minutes caused a complete degradation of drug residues. Freezing at -20°C ensured gradual degradation of the residues remained in different meats of the medicated rabbits(Gehad, 2002). The acceptable MRLs for tetracyclines as recommended by the Joint FAO/WHO Expert Committee on Food Additives is 200, 600, and 1200 μg/kg for muscles, liver, and kidney, respectively, (Mehran et al., 2009). The abundant and improper use of tetracycline antibiotics may result in the presence of their residues in edible animal meats, which can be toxic and dangerous for human health and potentially cause allergic reactions. In addition, low-level doses of antibiotic in foodstuffs consumed for long periods can lead to the spread of drug-resistant microorganisms (Yu et al., 2011). Therefore, this work was designed to determine the residue depletion of oxytetracycline in rabbit meats and the effect of boiling and freezing on oxytetracycline residues.

MATERIALS and METHODS

I. Materials:

1- HPLC grade acetonitrile, methanol and water.

2- AR/GR grade disodium hydrogen phosphate (Na2HPO4), citric acid and oxalic acid.

3- Oasis HLB cartridge 6 cm3 (200 mg).

4- From pure standard of oxytetracyline (assay 96.2% in HPLC), as their hydrochloride, individual stock standard solution at 1mg/mL (free base) was prepared in methanol in an amber color volumetric flask and was stored at -20°C in the dark for maximum period of 2 months. A composite working standard solutions of 300, 250, 200, 160, 80, 40 and 20 μg/mL were prepared by diluting stock solution with methanol. As it is unstable at room temperature, so prepared daily and stored at 4°C. For analysis, 0.01 M oxalic acid (pH 1.6), 0.1 M citric acid and 0.2 M disodium hydrogen phosphate (Na2HPO4) buffer were prepared in Milli-Q water and filtered through 0.22 μm cellulose filter.

5- 0.01 M methanolic oxalic acid (pH 1.86) was prepared in methanol.

6- McIIvaine buffer (pH 3.85) was prepared by mixing 278 mL of 0.1 M citric acid solution in 222 mL of 0.2 M Na2HPO4 solution and the pH was adjusted to 3.85 with extra citric acid solution. All these buffer solutions were stored at 4°C.

II. Methods:

1. Injection of rabbits with oxytetracyclines:

Hundred rabbits weighed about 1000-1500 grams(free from any antibiotics by feeding the rabbits on a balanced ration free from antibiotics for three weeks) were divided into two groups; control group (50) and test group (50) which were injected subcutaneously with oxytetracycline hydrochloride 20 mg/kg body weight for five successive days.

2. Slaughtering of rabbits:

Five rabbits from each group were slaughtered at zero time, 24 hr, 48 hr, 72 hr, 96 hr, 120 hr, 144 hr, 168 hr (1 week), 2 weeks and 3 weeks.

3. Effect of boiling and freezing on oxytetracycline residues in rabbit meats.

The samples were taken from meat, liver and kidney directly after slaughtering and were divided as follow:

1st group: Consists of 100 samples from each type (meat, liver and kidney).The oxytetracycline residues were determined using HPLC without treatment (boiling or freezing).

2nd group: Consists of 100 samples from each type (meat, liver and kidney).The oxytetracycline residues were determined using HPLC after boiling for 30 min.

3rd group: Consists of 100 samples from each type (meat, liver and kidney).The oxytetracycline residues were determined using HPLC after freezing at - 20º C for one, two and three months.

4. Determination of oxytetracycline residues by HPLC method:- 

The method recommended by Biswas et al. (2007)was used.

4.1. Instrumentation:

HPLC and uv detector.The analytical column was a Luna 5 μ C8 (RP- C8) column (4.6 × 250 mm, 5μm particle size). The optimized mobile phase for desorption and separation was a mixture of 0.01 M oxalic acid/acetonitrile/methanol (77:18:5, v/v/v), and the flow rate was 0.6 mL/min. The detection was performed at 355 nm with scanning range 340-360 nm.

4.2. Method of extraction:

1- Frozen meat samples were thawed and finely diced with scissors after trimming off external fat and fascia.

2- The finely cut samples were blended in a high speed   (15,000 rpm) meat blender for 2 min.

3- A representative portion of this sample (10 g) was weighed into a polypropylene tube and homogenized with 10 mL of Milli-Qwater for 1.5 min using Ultra-Turrex T25 meats homogenizer (Janke and Kenkel, IKA, LaborTecnik, USA).

4- Then an aliquot (0.5 g) of homogenized sample was transferred into a glass test tube, fortified with 50 μL of variable concentrations of the working standard solution, leaving the analytes in contact with meat sample for 30min.

5- After 3 mL of McIIvaine buffer was added, the mixture was vortexed at high speed, incubated for 5 minutes at room temperature and centrifuged at 3,500 rpm for10 min in a refrigerated centrifuge.

6- The extraction was repeated by adding 2 mL of McIIvaine buffer and the supernatant was pooled.

7- The supernatant was filtered and loaded on an Oasis HLB6 cm3 (200 mg) polymeric cartridge previously conditioned with 3 mL of methanol and 2 mL of water. The cartridge containing the sample was washed with 5 mLof water, and then tetracyclines were eluted with 4.5 mL of 0.01 M methanolic oxalic acid (pH 1.80).    

8- One milliliter of eluent was filtered through 0.22 μm nylon filter, vortexed and centrifuged, and then 20 μL of the aliquot was injected into the HPLC system.

RESULTS

Table 1: Minimum, maximum and mean values of oxytetracycline hydrochloride residues in injected rabbits meat (µg/kg) before and after boiling (n=5).

Table 2: Minimum, maximum and mean values of oxytetracycline hydrochoride residues in injected rabbits meat (µg/kg) before and after freezing (n=5).

Table 3: Minimum, maximum and mean values of oxytetracycline hydrochloride residues in injected rabbits kidneys (µg/kg) before and after boiling (n=5).

Table 4: Minimum, maximum and mean values of oxytetracycline hydrochloride residues in injected rabbits kidneys (µg/kg) before and after freezing (n=5).

Table 5: Minimum, maximum and mean values of oxytetracycline hydrochloride residues in injected rabbits livers (µg/kg) before and after boiling (n=5).

Table 6: Minimum, maximum and mean values of oxytetracycline hydrochloride residues in injected rabbits livers (µg/kg) before and after freezing (n=5).

Min. = Minimum, Max. = Maximum, N.D = Not detected and S.D= Standard Deviation.

Figure1: HPLC chromatogram of meat sample of the group slaughtered at zero time after the last dose of oxytetracycline (6235.1 µg/kg).

Figure 2: HPLC chromatogram of meat sample of the group slaughtered one weeks after the Last dose of oxytetracycline and freezed for three months (87.5 µg/kg).

Figure 3: HPLC chromatogram of kidney sample of the group slaughtered two weeks after the Last dose of oxytetracycline and freezed for one month (438.2 µg/kg).

DISCUSSION

The obtained results in table (1) showed that the  mean values of oxytetracycline in meat after slaughter time zero, one day, two days, three days, four days, five days, six days, one week, two weeks and three weeks from the last dose (20 mg/kg body weight for 5 successive days) estimated by HPLC was 4565.1±1.640, 3801.4±0.341, 3433.2±0.904, 3238.5±0.906, 2938.2±0.755, 2612.1±0.611, 2024.5±0.559, 1533.6±0.636, 198.2±0.204 and  1.4±0.003 µg/kg, respectively, but after boiling they were 2842.1±0.988, 3126.9±0.885, 3219.4±0.984, 2845.5±1.013, 2474.7±0.665, 1568.3±0.900, 1321.1±0.747, 1071.9±0.733, 45.3±0.042and undetected µg/kg, respectively, while after  freezing results in table (2) showed that for one month they were 3370.6±0.709, 3290.3±0.769, 3125.6±0.946, 2919.3±1.016, 2573.5±0.651, 1889.9±0.667, 1705.9±0.664, 1276.4±0.322, 60.8±0.059 and undetected µg/kg, respectively, after freezing for two months they were 1950.7±0.166, 1771.1±0.198, 1606.1±0.220, 1396.2±0.174, 1192.9±0.114, 1043.8±0.094, 871.3±0.064, 725.3±0.150, 1.7±0.003 and undetected µg/kg, respectively and after freezing for three months they were 828.6±0.322, 645.4±0.223, 539.5±0.244, 346.9±0.147, 195.2±0.089, 107.4±0.012, 87.3±0.019, 55.4±0.052, undetected and undetected µg/kg, respectively. It is clearly appeared that the time of slaughter after the last injected dose of oxytetracycline significantly affects the concentration of oxytetracycline in muscle, as slaughtering of rabbits after prolonged time of last dose of oxytetracycline decreased greatly the residual level until the concentration became very low at slaughter time 3 weeks after the last dose of oxytetracycline. These results are in agreement approximately with Rome (1991) and  Villa et al. (2001) who concln.ded that there were significant differences among the sacrifice times (0, 24, 48 and 72 hours post- treatment) in all matrixes collected in rabbits and by increasing the slaughter time after the last dose, the oxytetracycline concentration decreases. Boiling significantly decreases oxytetracycline level in muscle till complete disappearance of oxytetracycline at slaughter time 3 weeks after the last dose of oxytetracycline. Oxytetracycline residues decreased by boiling (Nashwa, 2012). Cooking significantly affect the oxytetracycline level in turkey meat(Javedi, 2011). These results were nearly similar to previously recorded results Javedi (2011) and Nashwa (2012) and disagreed with those recorded by Mohammed (1997). Freezing caused partial degradation of oxytetracycline in relation to the slaughter time after the last dose injected and the period of freezing, as long storage of rabbit muscles at freezing temperature (-20º c) decreased the oxyteracycline residues until disappeared completely from the rabbit muscles which slaughtered 2 weeks and 3 weeks after the last dose injected and stored at -20º c for 3 months. These results were in a partial agreement with Hagsmaa (1993), Kan (1995), Mansour (2000), Abd El-Monem et al. (2002), Gehad (2002), Hanaa (2002) and Tamer (2012),as they concluded that tetracycline residues were degraded by freezing at -18º c to -20º c. While these results were disagreed with those obtained by Gehan (1991).

Table (3) showed that the mean values of oxytetracycline in kidney samples after slaughter time zero, one day, two days, three days, four days, five days, six days, one week, two weeks and three weeks was 35355.5±8.524,  22811.7±4.275, 15329.5±3.619, 10929.2±4.680, 7409.4±3.579, 5872.9±2.677, 5247±2.011, 2540±0.894, 965.1±0.543 and 17.7±0.014µg/kg, respectively, while after boiling the  mean values of oxytetracycline was 14626.7±7.537, 10245.1±4.137, 7217.3±2.944, 7979.9±3.484, 5294.6±3.059, 3819.6±1.866, 3201±1.712, 1155.5±0.620, 510±0.364and  undetected µg/kg, respectively, and table(4) showed that after freezing for one month the  mean values of oxytetracycline was 26964.3±8.531, 17324.9±3.796, 10865.6±2.095, 9108.7±2.812, 6242.7±3.962, 4428.4±2.147, 4088.4±1.894, 1927.6±0.769, 720±0.516 and undetected µg/kg, respectively, after freezing for two months the mean values was 5297.8±0.614, 4196.7±0.675, 3077.2±0.162, 2806.7±0.414, 1882±0.318, 1696.6±0.377, 1384.5±0.308, 701.7±0.434, 20.3±0.021 and undetected µg/kg, respectively and after freezing for three months they were 1834.3±0.416, 1482.6±0.386, 1271.6±0.294, 1074.5±0.216, 911.7±0.308, 767.8±0.199, 466.3±0.152, 187.1±0.118, 5±0.005 and undetected µg/kg, respectively. High oxytetracycline concentrations were detected in the kidney and this is due to the kidney is the route of excretion of oxytetracycline. The level of oxytetracycline decreased with the increase of slaughter time.  Nearly similar results were reported by Rome (1991) and Villa et al. (2001). Data illustrated in table (5) showed that the  mean values of oxytetracycline after slaughter time at zero, one day, two days, three days, four days, five days, six days, one week, two weeks and three weeks was 7393.1±1.832, 6475.6±1.766,  4838.5±1.216, 4111.2±1.147, 3626.5±0.755, 3321.3±0.671, 2962.2±0.642, 2609.8±0.624, 1037.6±0.422 and 3.8±0.006µg/kg, respectively, while after boiling they were 5196.9±1.781, 4418.1±1.449, 3454.9±0.834, 3223.4±0.598, 2821.9±0.781, 2615.6±0.662, 2164.4±0.525, 1538.4±0.713, 481.7±0.267 and undetected µg/kg, respectively, and as showed in table (6) after freezing for one month they were 6545.5±2.021, 5949.1±1.327, 4196.4±1.052, 3637.3±0.874, 3356.7±0.314, 2918.6±0.715, 2408.3±0.565, 2157.7±0.655, 899.8±0.437 and undetected µg/kg, respectively, after freezing for two months they were 5512.9±1.507, 4978.9±1.161, 4353.6±0.849, 3532.2±0.881, 3096.9±0.822, 2725.9±0.711, 2261.6±0.518, 1942.6±0.648, 433±0.559 and undetected µg/kg, respectively and after freezing for three months they were 3328.5±0.875, 3112.7±0.909, 2959.9±0.799, 2581.5±0.588, 2122.8±0.420, 1908.7±0.451, 1441.4±0.303, 719.1±0.474, 262.9±0.372 and undetected µg/kg, respectively. The liver is the site of metabolism of oxytetracycline so the level of residual oxytetracycline is high in liver and begin gradually to be decreased with increasing the slaughter time after the last dose till reached a very low concentration after slaughter time 3 weeks. These results were nearly similar to those obtained by Rome (1991) and Villa et al. (2001). These results disagreed with those recorded by Mohammed (1997), Hanaa (2002) and Tamer (2012)that clarified that oxytetracycline completely disappeared by boiling regardless the initial concentration before boiling.

The recovery rates for oxytetracycline in rabbit meat, kidneys and livers was 102%, 92% and 86%, respectively, at concentration of 20 µg/kg of spiked samples. 

It can be concluded that neither boiling nor freezing could be considered as a reliable method to get rid completely from oxytetracycline as they caused a partial degradation of oxytetracycline residues. The only solution depending on the obtained results is to delay the slaughter time till the oxytetracycline metabolized in liver and excreted from the kidneys, and this takes a withdrawal period about 3 weeks after a course of treatment 20 mg/kg body weight subcutaneously for five successive days.

REFERENCES

Abd El-Monem, K.M.; Soliman, M.R. and Saad, S.M. (2002): Oxytetracycline residues in broiler carcasses produced by closed and open system. J. Egypt Vet. Med. Ass., 62(6a):   119-124.

Badiola, J.I.; González, J.; Aloy, N. and Pérez de Rozas, A.M. (2007): El uso de antimicrobianos en granjas cunícolas: necesidades de registro, uso prn. dente y medidas alternativas. In Proc. II Congreso Ibérico de Cunicultura, 2007 May, Vila-Real , Portugal, 173-181.

BiswAs, A.K.; RAo, G.s.; KoNdAiAh, N.; ANjANeyulu, A.S.R.; MeNdiRAttA, S.K.; PRASAd, R. and MAliK, J.K. (2007): A Simple Multiresidue Method for Determination of Oxytetracycline, Tetracycline and Chlortetracycline in Export Buffalo Meat by HPLC-Photodiode Array Detector. Journal of Food and Drug Analysis, Vol. 15, No. 3, 2007, Pages 278-284.

Boisseau, J. (1993): Basis for the evaluation of the microbiological risks due to veterinary drug residues in food. Vet. Microbiol. 35, 187-192.

Gehad, F.A. (2002): Stability of some Veterinary Drug Residues in animal meats, During storage, Preparation and Processing. Ph.D.V.Sc. Thesis Fac. Vet. Med.

Gehan, R.M. (1991): Studies on the withdrawal time of some antibiotics from broilers and rabbits. M.V.Sc. Thesis Fac. Vet. Med. Zagazig University, Moshtohor Branch.

Hagsmaa, N. (1993): Stability of veterinary drug residues during storage and processing. Proceeding of the European Residue conference, Veldhoren, Netherland, 1:41–48.

Hanaa, M.M. Sultan (2002): Residual status of some growth promoters and chemotherapeutics in meat and offals of slaughtered cattle and sheep. Ph.D.V.Sc. Dept. of food hygiene and control, CairoUniversity.

Javadi, A. (2011): Effect of roasting, boiling and microwaving cooking method on Doxycline residues in edible meats of poultry by microbial method. African Journal of Pharmacy and Pharmacology Vol. 5(8), pp. 1034-1037.

Kan, C.A. (1995): Residues probability and their occurrence and efficiency of their detection. Misset- World poultry J. 11(10): 67-73.

Mansour, A.H.M. (2000): Stn.dies on antibiotic residues in turkey meat and offal. Ph. D. Thesis, Dept. of food control, Faculty of Vet. Medicine, Moshtohor, Zagazig univ., Benha branch, Egypt.

Mehran Mesgari Abasi; Mohammad Reza Rashidi; Afshin Javadi; Maryam Bannazadeh Amirkhiz; Solmaz Mirmahdavi and Maryam Zabihi. (2009): Levels of tetracycline residues in cattle meat, liver, and kidney from a slaughterhouse in Tabriz, Iran. Turk. J. Vet. Anim. Sci. 2009; 33(4): 345-349.

Moats, W.A. (1997): Advances in determination of antibiotic residues. Journal of AOAC international, 80, 1± 4.

Mohammed, H.I. (1997): Antibiotic residues in rabbit carcasses with special reference to the effect of some processing on its stability. M.V.Sc. Thesis, Fac. Vet. Med.

Nashwa, M.Z. (2012): Seasonal variation of antibiotic residues in some slaughtered animal. Ph.D. of Vet. Med. Sciences, Dept. of food control, ZagazigUniversity.

  Rome, A. (1991): Residues of some veterinary drugs in animals and food. FAO / Food and Nutrition papers, 97–119.

Tamer, M.A.G. (2012): Effect of age and sex of animal on the antibiotic residues in meats of some slaughtered animals. Ph.D.V.Sc., food control dept., ZagazigUniversity.

Teske, R. (1993): Microbiological significance of drug residues in food welcome and introduction. Vet. Hum. Toxicol. 35, 1-2.

Villa, R.; Cagnardi, P.; Bacchetta, S.; Sonzogni, O.; Faustini, M. and Carli, S. (2001): Meat distripution and residue depletion of oxytetracycline in the rabbit. World rabbit Science 2001, vol. 9(4), 159-164.

Yu, H; Tao, Y.; Chen, D.; Wang, Y. and Yuan, Z. (2011): Development of an HPLC-UV method for the simultaneous determination of tetracyclines in muscle and liver of porcine, chicken and bovine with accelerated solvent extraction. Food Chem. 124: 1131–1138.