DETERMINATION OF CATECHOLAMINES BY AUTOMATED PRECOLUMN DERIVATIZATIONAND 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-diphe nylethylenediamine (DPE)/potassium ferricyanide-based reagent is descr ibed. The reaction is specific for catechol compounds and was shown to be very reliable for analysis of CAs (noradrenaline, adrenaline, dopa mine) in plasma and urine. However, in spite of its high sensitivity t he 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 uti lizing the CMA/200 refrigerated microsampler. Usually, 10-mu l samples cooled at 4 degrees C were mixed with 13.5 mu l of acetonitrile-ferri cyanide 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 degree s 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 (incl uding internal standards alpha-methyl-noradrenaline and isoproterenol) were separated within 8 min using 0.05 M acetate buffer pH 7.0-40% ac etonitrile-8% methanol at a flow-rate of 1 ml/min. The detection limit s for CAs were 2-5 fmol, which is about 2-4 times better than electroc hemical detection used under similar chromatographic conditions. Furth ermore, fluorescence detection is more reliable for routine use in cli nical laboratories because the detector is much simpler to maintain. T he method could be used for automated analysis of CAs in plasma and ur ine extracts and for microdialysis perfusates.