ORIGINAL_ARTICLE 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. https://macvetrev.mk/Files/Article/2019/10.2478/macvetrev-2019-0016/macvetrev-2019-0016.pdf 2020-10-15T09:00:00 123 130 10.2478/macvetrev-2019-0016 free doxycycline concentration HPLC analysis sheep plasma and milk rabbit Rositsa Mileva rossi_bu@abv.bg false 1 Department of Pharmacology, Physiology of Animals and Physiological Chemistry, Faculty of Veterinary Medicine, Trakia University, 6015 Stara Zagora, Bulgaria LEAD_AUTHOR 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 1 10.1128/MMBR.65.2.232-260.2001 Valentín, S., Morales, A., Sánchez, J.L., Rivera, (2009). Safety and efficacy of doxycycline in the treatment of rosacea. Clin Cosmet Investig Dermatol. 2, 129-140. 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