Tylosin and Spiramycin are medium-spectrum macrolide antibiotic used exclusively in veterinary medicine for the treatment of a wide range of infections.This research deals with the determination of optimal conditions for simultaneous separation and d
etermination of two macrolides antibiotics (Tylosin and Spyramicine), using C8 and C18 Chromatographic separation columns and doing the comparison between them in order to develop a rapid and sensitive method which can be used to measure these two compounds using High Performance Liquid Chromatography – Diode array detector (HPLC-DAD).
This study has used the gradient elution for mobile phase and revealed that the best conditions for separation and determination are conjugated with the best retention times and best areas for both studied compounds using a mobile phase consisted of an aqueous solution of anhydrated disodium Hydrogen Phosphate at pH=2.4 and an organic solution of acetonitrile with a ratio of 80:20v/v (solution A) and acetonitrile (solution B) [Na2HPO4(0.04M) pH:2.4/CAN (80:20v/v)]/ ACN, temperature 40°C for both columns, flow ratio of 1ml/min. for the mobile phase and maximum absorption wave length 280 nm, 232nm for Tylosin and Spyramicine respectively. The best peak areas are recorded as 5.759, 5.927 for Tylosin and Spyramicine 0.10ppm respectively, using C8 Chromatographic separation column in comparison with the best peak areas 4.432, 4.212 respectively at the same concentration using C18 Chromatographic separation column. It was noticed that the best retention times for Tylosin and Spyramicine were 7.013, 4.214min. respectively at concentration of 0.10ppm using C8 Chromatographic separation column in comparison with the best retention times 7.641, 5.898min. respectively at the same concentration using C18 Chromatographic separation column. The calibration curves for both separated compounds on C8 Chromatographic separation column showed a good linearity within a concentration range of 0.0010-0.10 ppm ≈ 1-100ppb at the two wave lengths λmax. = 280, 232nm respectively.
