QUANTITATIVE ANALYSIS OF RAW MATERIAL OF TETRACYCLINE AND ITS RELATED SUBSTANCES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

An isocratic method for the analysis of tetracycline (TC) by high performance liquid chromatography (HPLC) using polystyrene-divinylbenzene copolymer packing material is described. The method allows the complete separation of TC, 4-epitetracycline, anhydrotetracycline (ATC), 4-epianhydrotetracycline (EATC). A fermentation impurity, 2-acetyl-2-decarboxamidetetracycline is also resolved from TC. The mobile phase combines tert.-butanol, water and phosphate buffer, tetrabutylammonium sulphate and sodium ethylenediaminetetraacetate at pH 9.0 for elution at a temperature of 60°C. Among the organic modifiers examined, only tetrahydrofuran and tert.-butanol gave good results. For practical reasons, tert.-butanol was retained as the final organic modifier. The preliminary experiments were carried out on polystyrene-divinylbenzene copolymer packing material (PRP-I and PLRP-S columns). The selectivity of the columns is comparable. PLRP-S column was chosen as it shows better separation of tetracycline – anhydrotetracycline. The method was used to analyse the commercial samples.