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The present paper presents the evaluation of a mixed mode column (reversed phase and cation exchange) performance for the separation of 12 catecholamines, indolamines and their precursors and metabolites. The influence of different parameters, like organic modifier nature and percentage, salts nature and percentage, on the compounds separation was investigated. Good separation of the 12 selected compounds was obtained in the isocratic mode using a mobile phase composed of 90 % methanol and 10% ammonium acetate 20 mM, however the analysis time was excessively long (80 minutes). Thus an elution gradient was optimized and it reduced the analysis time to less than half (35 minutes).