Malaysian Journal of Pharmaceutical Sciences, Vol. 4, No. 2, 49—61(2006) QUANTITATIVE ANALYSIS OF RAW MATERIAL OF TETRACYCLINE AND ITS RELATED SUBSTANCES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY NABILA PERVEEN AND NAEEM HASAN KHAN* Department of Pharmaceutical Chemistry, Kulliyyah of Pharmacy, International Islamic University Malaysia Jalan Istana, Bandar Indera Mahkota 25200 Kuantan, Pahang, Malaysia 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. Keywords: Tetracycline, High performance liquid chromatography, Polystyrene–divinylbenzene copolymer stationary phase INTRODUCTION Tetracycline (TC) was first prepared by the catalytic reduction of chlortetracycline (Boothe et al. 1953; Conover et al. 1953). TC has also been reported to be produced from the Strains of Streptomyces aureofaciens, Streptomyces avellanus, Streptomycs feofaciens, Streptomyces alboflavus and many others (Weinstein and Wagman 1978). Among the TC group of antibiotics, TC is the most widely used in therapeutics. TC has proved to be safe and effective in numerous commonly encountered infections and also in some specific cases of cholera, trachoma, atypical pneumonia, acne, conjunctivitis, plague and many other diseases. TC is used in the form of capsules, oral liquid * Corresponding author: Dr. Naeem Hasan Khan, e-mail: naeemhshirazi@hotmail.com