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Macedonian Veterinary Review


p-ISSN 1409-7621
e-ISSN 1857-7415

Co-publishing with:
De Gruyter

Abstract / References

Original Scientific Article

Determination of free doxycycline concentrations in the plasma and milk of sheep and in the plasma of rabbits by using the HPLC method
Rositsa Mileva
Department of Pharmacology, Physiology of Animals and Physiological Chemistry, Faculty of Veterinary Medicine, Trakia University, 6015 Stara Zagora, Bulgaria


A fast and sensitive HPLC method was validated in order to analyze doxycycline in plasma and milk of sheep and in plasma of rabbits. The samples were processed with trifluoroacetic acid (TFA). After the centrifugation step, a supernatant containing extracted doxycycline and internal standard oxytetracycline was injected into the HPLC system with PDA detection. The method showed linearity in the range of 0.125 - 2.5 μg/mL for ovine plasma, 0.125 – 5.0 μg/mL for ovine milk, and 0.125 – 1 μg/mL for rabbit plasma. The inter-assay precision varied between 5.69 – 13.55 %. Values for intraassay precision were between 0.62 – 8.67 %. Accuracy was higher than 90% in all of the tested concentrations in the three types of biological matrices. The mean extraction recovery was higher than 90 % for all matrices. In order to handle only with free drug concentrations, microfiltration of standard solutions with low (0.25mg/mL), medium (0.5mg/mL) and high (1.0mg/mL) concentration was performed. A percentage for correction of the quantified doxycycline was calculated. The most significant adjustments should be made at the low concentrations. The correction for rabbit plasma is 24.63±5.03%, for ovine plasma is 20.10±8.01% and for milk –16.68±0.04 %. This method can be used for routine determination of doxycycline concentrations for pharmacokinetic studies and further dosage adjustment.
Key words: free doxycycline concentration, HPLC analysis, sheep plasma and milk, rabbit

Mac Vet Rev 2019; 42 (2): i-viii
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Available Online First: 9 June 2019

    1. Chopra, I., Roberts, M. (2001). Tetracycline antibiotics: Mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev. 65 (2): 232-260.
    PMid:11381101 PMCid:PMC99026

    2. Valentín, S., Morales, A., Sánchez, J.L., Rivera, A. (2009). Safety and efficacy of doxycycline in the treatment of rosacea. Clin Cosmet Investig Dermatol. 2, 129-140.
    PMid:21436975 PMCid:PMC3047926

    3. Agwuh, K. N., MacGowan, A. (2006). Pharmacokinetics and pharmacodynamics of the tetracyclines including glycylcyclines. J Antimicrob Chemother. 58, 256-265.

    4. Redelsperger, I. M., Taldone T., Riedel, E. R., Lepherd, M. L., Lipman, N. S., Wolf, F. R. (2016). Stability of doxycycline in feed and water and minimal effective doses in tetracycline - inducible systems. J Am Assoc Lab Anim Sci. 55 (4): 467–474.
    PMid:27423155 PMCid:PMC4943619

    5. Ahmed, M. M., Rahman, M. M., Mahbub, K. R., Wahiduzzaman, M. (2011). Characterization of antibiotic resistant Salmonella spp. isolated from chicken eggs of Dhaka City. J Sci Res. 3 (1): 191-196.

    6. Naz, S., Hanif, A., Maqbool, A., Ahmed, S., Muhammand, K. (2012). Isolation, characterization and monitoring of antibiotic resistance in Pasteurella multocida isolates from buffalo (Bubalus bubalis) herds around Lahore. J Anim Plant Sci. 22 (3): 242-245.

    7. Tejedor-Junco, M. T., González-Martín, M., Bermeo-Garrido, E., Villasana-Loaiza, R., Carretón-Gómez, E. (2018). Doxycycline treatment for Dirofilaria immitis in dogs: impact on Staphylococcus aureus and Enterococcus antimicrobial resistance. Vet Res Commun. 42 (3): 227–232.

    8. Maaland, M.G., Papich, M.G., Turnidge, J., Guardabassi, L. (2013). Pharmacodynamics of doxycycline and tetracycline against Staphylococcus pseudintermedius: proposal of canine-specific breakpoints for doxycycline. J Clin Microbiol. 51 (11): 3547-3554.
    PMid:23966509 PMCid:PMC3889732

    9. Meijer, L. A., Ceyssens, K. G. F., de Greve, B. I. J. A. C., de Bruijn, W.1993). Pharmacokinetics and bioavailability of doxycycline hyclate after oral administration in calves. Vet Q. 15, 1-5.

    10. Laczay, P., Semjen, G., Lehel, J., Nagy, G. (2001). Pharmacokinetics and bioavailability of doxycycline in fasted and nonfasted broiler chickens. Acta Vet Hung. 49, 31-37.

    11. Shabir, G. A. (2003). Validation of high-performance liquid chromatography methods for pharmaceutical analysis. Understanding the differences and similarities between validation requirements of the US Food and Drug Administration, the US Pharmacopeia and the International Conference on Harmonization. J Chromatogr A. 987, 57–66.

    12. European Medicines Agency: [Internet]. Guideline on bioanalytical method validation. [cited 2011; last assessed on 09 November 2018].

    13. Fu, H., Zhang, L., Zhang, W. (2011). Preparation of doxycycline hyclate microcapsule and its pharmacokinetic in rabbit. Acta Vet et Zootechnica Sinica. 42 (10): 1438 – 1444.

    14. Castro, L. J., Sahagun, A. M., Diez, M. J., Fernandez, N., Sierra, M., Garcia, J. J. (2009). Pharmacokinetics of doxycycline in sheep after intravenous and oral administration. Vet J. 180, 389 – 395.

    15. Zellner, M., Winkler, W., Hayden, H., Diestinger, M., Eliasen, M., Gesslbauer, B., et al. (2005). Quantitative validation of different protein precipitation methods in proteome analysis of blood platelets. Electrophoresis 26, 2481–2489.

    16. Riond, J.L., Riviere, J.E. (1989). Doxycycline binding to plasma albumin of several species. J Vet Pharmacol Ther. 12 (3): 253-260.

    17. Zeitlinger, M. A., Derendorf, H., Mouton, J. W., Cars, O., Craig, W. A., Andes, D., Theuretzbacher, U. (2011). Protein binding: do we ever learn? Antimicrob Agents Chemother. 55 (7) : 3067–3074.
    PMid:21537013 PMCid:PMC3122431

    18. Chaturvedi P., Esposito, C., Koroma, J., Cannon, E.P., Tanaka, S.K. (2003). In vitro assessment of plasma protein binding and metabolic stability of PTK 0796 (BAY 73-6944). Book of abstracts of the 43th Interscience conference of antimicrobial agents and chemotherapy. F-760, Chicago, USA.





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