1 Maha and Elham Egyptian Journal of Animal Health 1, 2 (2021), 1-10 Evaluation of analytical method for determination of tetracyclines in their dos- age forms by HPLC using different types of chromatographic stationary phases (RP-C 8 and RP-C 18 ) Maha S. Abd Elhafeez* and Elham A. Mobarez Biochemistry, Toxicology and Feed Deficiency Dept., Animal Health Research Insti- tute, Agricultural Research Center, Dokki, Giza, Egypt. ABSTRACT A ntibiotics are extensively used in animal husbandry practices for the treatment of diseases. Four tetracyclines are commercially available as veterinary medicines, of which oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), and doxycycline (DOX). In the present investi- gation, a novel simple, sensitive, and inexpensive method of analysis was veri- fied for tetracyclines by RP-HPLC system in bulk drug substance and pharma- ceutical forms via two stationary phases (C8 & C18). The separation was at- tained on a 250 mm column length, 4.6 mm internal diameter, 5 µm particle size, C18 and C8 columns with a (55:25:20) 5% glacial acetic acid: acetoni- trile: methanolic acid as elution solution at a 355 nm wavelength. The method was performed at a0.8 ml/m inflow rate. This method was applied without an extraction process. The method was validated following ICH and USP guide- lines. It was proved to be reproducible and more economical due to the short retention time of less than 6 min by C8 column and 8 min by C18 column which enabled analysis of tetracycline pharmaceutical products with a rela- tively lower volume of mobile phase. The result of formulation analysis was statistically validated with a high recovery percentage. Furthermore, the devel- oped method was free from the interference of excipients used in the formula- tion. The validated method was accurate, precise, linear, and reproducible. The data suggest the possibility of using the method in quality control labora- tories with better routine analysis of tetracyclines in their dosage forms. INTRODUCTION Antibiotics are extensively used in ani- mal husbandry practices for the treatment and diseases (Moudgil et al. 2019).Tetracyclines are synthetic antibiotics, highly active against a varied range of Gram-negative and Gram- positive microbes. Four tetracyclines are com- mercially available as veterinary medications, of which oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), and doxycy- cline (DOX); their chemical structures showed in figure 1.This group of antibiotics is most commonly used for therapeutic purposes in veterinary practice due to their low cost, easy over-the-counter availability, and broad- spectrum (Martins et al. 2015 Prado et al. 2015 and Jank et al. 2017). Article History Received in 12/1/2021 Received in revised from 8/2/2021 Accepted in 17/2/2021 Keywords: Tetracyclines HPLC Drugs RP-C 8, RP-C 18 . Corresponding author: Maha S. Abd Elhafeez, Biochemistry, Toxicology and Feed Deficiency Dept., Animal Health Research Institute, Agricultural Research Center, Dokki, Giza, Egypt. E-mail: mahasabry86_doctor@hotmail.com DOI: 10.21608/EJAH.2021.160749 Egyptian Journal of Animal Health P-ISSN: 2735-4938 On Line-ISSN: 2735-4946 Journal homepage: htps://ejah.journals.ekb.eg/