DETERMINATION OF CATECHOLAMINES BY AUTOMATED PRECOLUMN DERIVATIZATION AND REVERSED-PHASE COLUMN LIQUID-CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

A highly selective and sensitive method for fluorescence determination of catecholamines (CAs) after automated derivatization with 1,2-diphenylethylenediamine (DPE)/potassium ferricyanide-based reagent is described. The reaction is specific for catechol compounds and was shown to be very reliable for analysis of CAs (noradrenaline, adrenaline, dopamine) in plasma and urine. However, in spite of its high sensitivity the method has not yet achieved wide application, probably because of a rather complicated manual derivatization and reaction times of 40-60 min. The present method describes an optimized automated procedure utilizing the CMA/200 refrigerated microsampler. Usually, 10-mu l samples cooled at 4 degrees C were mixed with 13.5 mu l of acetonitrile-ferricyanide reagent and then with 7.5 mu l DPE-bicine as a second reagent; 29 mu l were aspirated into the sampling loop and heated to 80 degrees C. After 6.5 min reaction time, samples were injected onto a 100 x 4 mm column packed with Nucleosil C-18, 3 mu m particle size. CAs (including internal standards alpha-methyl-noradrenaline and isoproterenol) were separated within 8 min using 0.05 M acetate buffer pH 7.0-40% acetonitrile-8% methanol at a flow-rate of 1 ml/min. The detection limits for CAs were 2-5 fmol, which is about 2-4 times better than electrochemical detection used under similar chromatographic conditions. Furthermore, fluorescence detection is more reliable for routine use in clinical laboratories because the detector is much simpler to maintain. The method could be used for automated analysis of CAs in plasma and urine extracts and for microdialysis perfusates.