Determination of Tetracycline residues in red meat available in Oman

 

Sumaiya Al- Kindi¹, Iman Ismail Yaqoob ALBalushi¹, Aisha Yazid Abdulalim Elshaar¹,

Ahlam Al Kharusi², Razna Al Maimani³, Alka Ahuja*¹

¹Department of Pharmaceutics, College of Pharmacy,

National University of Science and Technology, Muscat Oman.

²Muscat Municipality, Muscat Oman.

³Central Laboratory for Food Safety, Muscat Oman.

 

ABSTRACT:

Tetracycline is heavily used in livestock production either for prophylaxis, treatment or as growth promoter. The residues of tetracycline in animal products have been investigated around the world and linked to imbalance in intestinal microflora, human allergic reactions, and other diseases. Researcher stated that the long term use of tetracycline as sub therapeutic dose increased level of antibiotic-resistant pathogens which is a global threat to human health and food security and development. Many studies reported that the tetracycline levels were above the maximum residual limit^1,2. In Oman, few studies have been done using different techniques for investigating the antibiotics levels in animal products. Therefore, the aim of this study was to investigate the tetracycline residues in imported goat meat (liver and muscle) and to compare the levels of tetracycline residues between liver and muscles. A total of 48 fresh muscle and liver samples were taken from 24 Somali goats that were slaughtered at a slaughterhouse in Muscat. The extraction was done using the Agilent Enhanced Matrix Removal—Lipid (EMR—L) product. Four compounds were tested; Tetracycline, Oxytetracycline, Chlortetracycline and Doxycycline. The final extracts were analyzed using LC-MS/MS. The results showed no detection of tetracycline and doxycycline whereas, oxytetracycline and chlorotetracycline were found. 45% of muscle samples had OXY and CTC with concentration range of 6.04-6.23µg/kg and 5.48-8.35µg/kg, respectively. Around 42% of investigated liver samplesshowed OXY and CTC with concentration of 6.04-6.17 µg/Kg and 7.92-8.13µg/kg, respectively. In this study higher concentrations of OXY and CTC were detected in one muscle with values of 403.60035±234.8µg/kg and 274.8491±87.1058, respectively and one liver sample got higher concentration of OXY which was 3201.9±325.1µl/kg. These values were exceeding the MRL GSO 2481/2015, CX/MRL 2-2018 and EU 37/2010. These results might be related to withdrawal time as most of the samples had lower MRL. All samples were studied in triplicates to verify the results and using LCMSMS making data more satisfactory and validated.

 

 

 

INTRODUCTION: 

In animal livestock, the purpose of using antibiotics is for mainly three reasons; as therapeutic, prophylaxis or as growth promoters. For therapeutic purpose, antibiotics are used to treat animal diseases such as respiratory diseases, gastrointestinal diseases, mastitis, brucellosis, arthritis and other bacterial infectious diseases³, whereas for the prophylaxis purpose they are used to prevent predictable diseases or the outbreak of a disease in a herd. The third use is as growth promoters where antibiotics are used to increase the weight gain in a shorter period. There are differences between the purposes in terms of specificity, quantity, length of use and route of administration, dose and frequency. Around 90% of antibiotics in livestock area are used for prophylaxis and for growth promoting purposes⁴. However, high doses of antibiotics caused harmful effects such as hear loss and kidney toxicity, carcinogenicity, effects on thyroid and pituitary functions^5,6. Also, Adding to that, the residues of antibiotics might be mutagenic, teratogenic or cause reduction in reproductive performance in humans⁷.

 

Tetracyclines are one of the most common antibiotics used in animal livestock as they have a broad spectrum of antibacterial activity against both gram-positive/negative bacteria, have low-cost and there is absence of major side-effects⁸. In 1949, the property of tetracycline as a growth promoter was discovered when chickens were being fed with chlortetracycline supplemented feed. The main mechanisms of action of tetracyclines were found as limiting the growth by blocking incoming aminoacyl tRNA from binding to the ribosome acceptor site or by reversible binding to the bacterial 30S ribosomal subunit (bacteriostatic antibiotics) or by interrupting macromolecules synthesis pathways and disturbing cellular processes. (Bactericidal antibiotics)⁹.

 

Tetracyclines are classified into three groups. The first group is naturally produced such as chlortetracycline and oxytetracycline by Streptomyces species. Second group is produced by semi-synthesis of tetracycline such as doxycycline, lymecycline, meclocycline, minocycline and rolitetracycline. The third group is obtained from total synthesis such as tigecycline.

 

The most common tetracyclines used in the veterinary medicine are tetracyclines, chlortetracycline, oxytetracycline, and doxycycline¹⁰.

 

Many studies were done to develop the method for determination of tetracycline¹¹. Liquid chromatography mass spectrometry is one of quantitative methods for measurement of drug (concentrations, solubility and number of compounds present in the sample^12,13. In veterinary medicine, tetracyclines are used to treat diseases of locomotive organ, genito-urinary tract, systemic infections, sepsis,rocky mountain spotted fever, gastrointestinal as well as respiratory and skin bacterial infections^14,15. However, tetracyclines have a potential to be nephrotoxic. High dose of oxytetracycline was given to cattle and fatal renal failure has been reported¹⁶.

 

Around 90% of tetracyclines were used for prophylaxis for cattle and swine and 15% for poultry⁸. In North America and other countries, tetracyclines are used as growth promoters in animal husbandry, whereas they are banned in the European Union¹⁷. The use of antibiotics can lead to formation of antibiotic-resistant strains of bacteria¹⁸.Several studies mentioned that the long term use of tetracycline in sub therapeutic dose increased level of antibiotic-resistant pathogens^8,17. There were four main mechanisms which lead to resistance of microorganism to antimicrobials such as altering the porins through gene mutation, which makes the cell wall less permeable and difficult for antibiotic to enter the cell^19,20.  Although tetracycline has a short half-life (7–10 h), residues levels were detected in liver, milk, kidney and muscles^3,5. Ramatla et al.in 2017 found that the level of tetracyclines was around as 168.02μg/kg which was lower than the acceptable MRLs as recommended by FAO/WHO Expert committee (200, 600, and 1200μg/kg for the liver, muscles, and kidney, respectively²¹. However, the long term exposure of antibiotics residues might cause acute or chronic toxicity to the organs and the entire body. Tertracyclines accumulate in organs and tissues of animals that have been treated for long period of time with tetracycline. This is a big concern as the end product will be available to humans.

 

Abasi and his friends in 2009 investigated the level of tetracyclines residues in beef muscles, kidney and liver using high performance liquid chromatography (HPLC) method. Around 5% of kidney and liver samples and 21.7% of all samples had residues exceeding MRLs of the WHO²². A study in Tirana, capital of Albania using the same technique detected only oxytetracycline (OTC) in four beef muscles out of 37 samples which were exceeding the MRLs. Tetracycline (TC) and chlortetracycline (CTC) were not detected²³. These results might depend on withdrawal period as Agmas and his colleagues in 2018 reported that the beef farmers didn’t stick to drug withdrawal period and they had lack of awareness about antibiotics side effects. The prescribed withdrawal time is considered as the major reason for presence of drug residues if it not followed²⁴. Around 97.67% of the farmers used tetracycline (oxytetracycline) in beef farms in Debre Tabor and Bahir Dar, Northwest Ethiopia²⁵. Therefore, it is very important to investigate the residues of tetracycline in the available meat in the market either produced locally or imported.

 

MATERIALS AND METHODS:

Sampling:

A total of 48 samples of fresh Somali goats (24 liver and 24 muscle) were collected between April and November 2021 from Muscat municipality slaughterhouse. The sample of muscle was taken from gluteobiceps muscle area. The samples were collected in sterilized polyethylene bags, labelled and preserved in an ice box and immediately stored in a deep freezer at -20^oC.

 

All samples were trimmed of the external fat and fascia cut in cubes and grinded by using food processor. The balance was checked before weighing the samples using the certified weights as in figure 1.

 

 

After grinding the sample, the width was taken and the sample was preserved in a sterilized zipper bag after removing the air to avoid oxidation.

 

Figure 1: Certified weights used for checking the balance before every working day

 

Extraction of the drug from the sample:

Two grams (2.000g-2.0008g) of homogenized tissue was weighed and placed into 50‑mL polypropylene tubes and 2ml of LCMS grade water was added into each sample. Then 10mL solution of acetonitrile with 5 % formic acid was added to the sample and mixed with an orbital shaker for 5 minutes (using Geno/grinder with speed of 1500rpm). The samples were centrifuged at 4,000 rpm for 5 minutes. After this, 5mL of the supernatant was added to the 1g EMR—L tube, which had been activated previously with 5 mL of 5 mM ammonium acetate solution. The tube was then vortexed for 5 mins and centrifuged at 4,000 rpm for 5 minutes.  The 5mL of supernatant from this solution was transferred to a 15-mL centrifuge tube to which 2g of MgSO4 were added from the EMR—L pouch with immediate vortexing for 5 min. The samples were centrifuged at 4,000 rpm for 5 minutes. Three layers were formed, only the top layer was taken and filtered using 0.22mm filter disc. Finally, a 200μL extract was transferred into amber glass vial with 800μL of LC-MS water, ready for Aglient 6460 LC-MS/MS analysis.

Chromatographic condition:

The separation of the antibiotic residues was performed using a Poroshell EC-120 EC-C18 analytical column (2.1mm inner diameter I.D × 150mm length, 2.7μm particle size; Agilent). The separation of antibiotic was accomplished at 40⁰C. The flow rate and injection volume was 0.5mL/min and 15μL, respectively. Two mobile phases were used; (A) 0.1% formic acid and water and (B) Acetonitrile. The gradient elution program was planned as follows: starting with 98% A and 2% B for 1min, then 85% A and 15% B for 1.5 min. After that, 70% A and 30% B for 2.5 min, followed by 55% A and 45% B for 6 min was used. This was followed by 20% A and 80% B for 8.5 min, and later 100% B for two stages 10min and 11 mins. Final run method was 98%A and 2% B for 11.2 min.

 

Calibration curve:

The linear curve was obtained from individual standards using five concentrations of 5 ppb, 10 ppb, 20 ppb, 50 ppb, and 100 ppb. For the preparation of the working solutions, stock solutions (1mg/ml in LC-MS -grade methanol) of the antibiotics were diluted to several concentrations by using methanol as a diluent. Chlortetracycline Hydrochloride, Tetracycline Hydrochloride, Oxytetracycline Hydrochloride and Doxycycline Hyclate where purchased from Sigma Aldrich.

 

Recovery Experiments:

The recoveries of the compounds were investigated by the addition of standards to the samples in order to evaluate the validity of the proposed method. The recoveries were found to be in a range of 100 to 103.82% as shown in Table 1.

 

Figure 2. (1) Weighing the sample, (2) Grinding the sample by food processor, (3) Grinded samples in a sterilized zipper bag, (4) Removal of all air, (5-6) Weighing samples in triplicates, (7-8) Samples in Geno grinder machine, (9) Homogenized sample, (10-12) Using EMR—Lipid tube, (13) Using  the EMR—polish

 

Table 1. Recovery percentages of TC, OXY, CTC and DOXY.

Recovery for	(ng/ml)	% Recovery
Tetracycline	25.5111	102.04
Oxytetracycline	25.2015	100.81
Chlortetracycline	25.9539	103.82
Doxycycline	25.7855	103.14

 

Statistical analysis:

Excel program was used for statistical analysis for all obtained data from LCMSMS as descriptive statistics.

 

RESULTS AND DISCUSSION:

Antibiotics use in livestock production is a regulated activity by the Code of Federal Regulations, (Title 21, Part 558). However, the handling of antibiotics among farmers is done improperly sometimes by extra-label, illegal drugs applications, overdose, etc. Adding to that, the lack of knowledge of withdrawal period before to slaughter the animals plays a major role in providing the consumer with unsafe product.

 

The present study investigated TCs residues in Somali goats which were daily slaughtered at Muscat Municipality Slaughterhouses. 48 samples were analysed in total (24 muscles and 24 liver studied in triplicates).  Figure 3 shows the concentration of detected compounds in muscle tissue of Somali goat. TC and DOXY compounds were not detected in all samples when they were analysed using by LCMS/MS.  Conflict result was found in 2006 which was done on the Somali goat in Oman. They reported a presence of TC with mean of 49.8 µl/kg using ELISA. In 2021, Al-Amri with his colleagues used ELISA and HPLC techniques and detected TC with low concentration not exceeding MRL²⁶. All these results might indicate the lower usage of tetracycline compound in production of Somali goat in Somalia or following the guidelines in use and withdrawal period.  However, 45% of muscle samples had OXY and CTC with concentration range of 6.04-6.23 µg/kg and 5.48-8.35 µg/kg, respectively (figure 3). Adding to that, around 42% of investigated liver (figure 4), showed OXY and CTC with concentration of 6.04-6.17 µg/Kg and 7.92-8.13µg/kg, respectively. These ranges were lower than which have been reported by Cammilleriet al., 2019 which were 10.4–40.2 µg kg⁻¹²⁷.  While the ranges of both OXY and CTC in muscle and liver were below the MRL, the health of consumer will be affected by long term exposure to these concentrations. Moreover in this study higher concentrations of OXY and CTC were detected in one muscle with values of 403.60035 ± 234.8 µg/kg and 274.8491±87.1058, respectively and one liver sample got higher concentration of OXY which was 3201.9± 325.1 µl/kg. These values were exceeding the MRL GSO 2481/2015, CX/MRL 2-2018 and EU 37/2010.

 

 

These results might be related to withdrawal time as most of the samples had lower MRL. All samples were studied in triplicates to verify the results and using LCMSMS making data more satisfactory and validated.

 

Figure 3. The bar graph showing the detected OXY and CTC in muscle tissue of Somali goat

 

Figure 4. The bar graph showing the detected OXY and CTC in liver tissue of Somali goat

 

CONCLUSION:

Majority of red meat samples collected from slaughter house were found to be safe and reported antibiotic levels within the safe limit with exception of two samples. However, regular intake of red meat can lead to cumulative increase in antibiotic levels which could be unsafe for the consumers. Studies on more samples can help us to reach at a better conclusion as the number of samples studied in the present study were limited.

 

CONFLICT OF INTEREST:

The authors have no conflict of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

The authors would like to thank Muscat Municipality, Central laboratory for food safety and Slaughterhouse for their kind support during the collection, providing chemicals, and analysis. The authors would also like to thank The Research Council and National University of Science and Technology for funding this research.

 

REFERENCES:

1.      Bahmani K. Shahbazi Y. Nikousefat Z. Monitoring and Risk Assessment of Tetracycline Residues in Foods of Animal Origin. Food Science And Biotechnology. 2020;29(3):441-8.doi: 10.1007/s10068-019-00665-x

2.      Verma M. Ahmad A. Pant D. Patwal P. Evaluation of Oxytetracycline residues in Chicken Meat Samples by HPLC. The Pharma Innovation Journal. 2021; SP-10(4): 155-157.

3.      Orwa JD. Matofari JW. Muliro PS. Lamuka P. Assessment of Sulphonamides and Tetracyclines Antibiotic Residue Contaminants in Rural and Peri Urban Dairy Value Chains in Kenya. International Journal of Food Contamination. 2017;4(1):1-11.

4.      Jayalakshmi K. Paramasivam M. Sasikala M. Tamilam T. Sumithra A. Review on Antibiotic Residues in Animal Products and Its Impact on Environments and Human Health. Journal of Entomology and Zoology Studies. 2017;5(3):1446-51.

5.      Falowo A.Akinmoladun O. Veterinary Drug Residues in Meat and Meat Products: Occurrence, Detection and Implications. In Veterinary Medicine and Pharmaceuticals. 2019; IntechOpen.https://doi.org/10.5772/intechopen.83616.

6.      Simon V. Sreerag K. Sasikumar R. Kanthlal S. High Dose Antibiotics Induced Hepatotoxicity and Altered Markers: An In-Vitro Liver Slices Study. Research Journal of Pharmacy and Technology. 2017;10(6):1742-5.DOI: 10.5958/0974-360X.2017.00307.9

7.      Singh S. Shukla S. Tandia N. Kumar N. Paliwal R. Antibiotic Residues: A Global Challenge. Pharma Science Monitor. 2014;5(3): 184-197.

8.      Chopra I.Roberts M. Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiology and Molecular Biology Reviews. 2001;65(2):232-60.doi: 10.1128/MMBR.65.2.232-260.2001

9.      Saleha T. Faheem AS. Ummar A. Tetracycline: Classification, Structure Activity Relationship And Mechanism of Action As A Theranostic Agent For Infectious Lesions-A Mini Review. Biomedical Journal of Scientific and Technical Research. 2018;7:5787-96.doi: 10.26717/BJSTR.2018.07.001475

10.   Granados-Chinchilla F. Rodríguez C. Tetracyclines in Food and Feedingstuffs: From Regulation To Analytical Methods, Bacterial Resistance, and Environmental and Health Implications. Journal of Analytical Methods in Chemistry. 2017;2017.doi: 10.1155/2017/1315497.

11.   Rao TN. Sreenivasulu D. Reddy E. Devi KS. A Novel Method for Determination of Tetracycline And Its Metabolite Residues In Cow Milk. Research Journal of Pharmacy and Technology. 2014;7(5):513-6.

12.   Nikam SR. Jagdale AS. Boraste SS. Patil SB. Bioanalysis-Method Development, Validation, Sample Preparation, its Detection Techniques and its Application. Asian Journal of Pharmaceutical Analysis. 2021;11(4). doi: 10.52711/2231-5675.2021.00051

13.   Phale MD. Korgaonkar D. Current Advance Analytical Techniques: A Review. Asian Journal Of Research In Chemistry. 2009; 2(3):235-8.

14.   Michalova E. Schlegelova J. Tetracyclines in Veterinary Medicine and Bacterial Resistance to Them. Veterinarni Medicina. 2004;49(3):79. DOI: 10.17221/5681-VETMED

15.   Dighe NS. Pattan SR. Bhawar SB. Dighe SB. Bhosale MS. Tambe VB. et al. Rocky Mountain Fever: A Review. Research Journal of Pharmacology and Pharmacodynamics. 2009;1(3):104-10.

16.   Vaala E. Ehnen J.Divers. J.Acute Renal Failure Associated With Administration Of Excessive Amounts of Tetracycline in A Cow. Journal of the American Veterinary Medical Association. 1987; 191(12), 1601–1603.

17.   Economou V. Gousia P. Agriculture and food animals as a source of antimicrobial-resistant bacteria. Infection and Drug Resistance. 2015;8:49.doi: 10.2147/IDR.S55778. eCollection 2015

18.   Bhardwaj R. A comparative analysis of organic food products vs non organic food products in India. Asian Journal of Management. 2017;8(3):587-90. doi: 10.5958/2321-5763.2017.00094.4

19.   Marnoor SA. A Review on Antimicrobial Resistance and Role of Pharmacist in tackling this Global Threat. Research Journal of Pharmaceutical Dosage Forms and Technology. 2017;9(4):143-6.doi: 10.5958/0975-4377.2017.00023.4

20.   Khushboo K. Saloni B. Rathore KS. Antibiotic Resistance. Asian Journal of Research in Pharmaceutical Science. 2020;10(4).

21.   Ramatla T. Ngoma L. Adetunji M. Mwanza M. Evaluation of Antibiotic Residues in Raw Meat Using Different Analytical Methods. Antibiotics. 2017;6(4):34.doi: 10.3390/antibiotics6040034

22.   Abasi MM. Rashidi MR. Javadi A. Amirkhiz MB. Mirmahdavi S. Zabihi M. Levels of Tetracycline Residues in Cattle Meat, Liver, and Kidney From A Slaughterhouse in Tabriz, Iran. Turkish Journal of Veterinary and Animal Sciences. 2009;33(4):345-9. doi: 10.3906/vet-0711-32

23.   Emiri A. Myftari E. Çoçoli S. Treska E. Determination of Oxytetracycline, Tetracycline And Chlortetracycline in Beef Meat by HPLC-DAD Detector in Albania. Albanian Journal of Agricultural Sciences. 2014:489.

24.   Jeena S. Venkateswaramurthy N. Sambathkumar R. Antibiotic Residues in Milk Products: Impacts on Human Health. Research Journal of Pharmacology and Pharmacodynamics. 2020;12(1):15-20. doi: 10.5958/2321-5836.2020.00004.X

25.   Agmas B. Adugna M. Antimicrobial Residue Occurrence and Its Public Health Risk of Beef Meat in Debre Tabor and Bahir Dar, Northwest Ethiopia. Veterinary World. 2018;11(7):902. doi: 10.14202/vetworld.2018.902-908

26.   Al-Amri I. Kadim IT. Alkindi A. Hamaed A. Al-Magbali R. Khalaf S. et al. Determination of Residues Of Pesticides, Anabolic Steroids, Antibiotics, and Antibacterial Compounds In Meat Products In Oman By Liquid Chromatography/Mass Spectrometry And Enzyme-Linked Immunosorbent Assay. 2021;14(3):709-20. doi: 10.14202/vetworld.2021.709-720

27.   Cammilleri G. Pulvirenti A. Vella A. Macaluso A. Lo Dico GM. Giaccone V. et al. Tetracycline Residues In Bovine Muscle And Liver Samples From Sicily (Southern Italy) By LC-MS/MS Method: A Six-Year Study. Molecules. 2019;24(4):695. doi: 10.3390/molecules24040695

 

 

 

Accepted on 16.02.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(5):2182-2186.