Ion-exchange chromatographic systems for separation of catecholamines were developed using a carboxylic acid type ion-exchange resin (Amberlite IRC-50) as a stationary phase and buffers of pH 6.3 or 4.0-4.3 containing boric acid as eluents. At the higher pH (pH 6-7), catecholamines were eluted faster when an eluent containing higher concentration of boric acid was used, although at the lower pH (e. g. pH 4), increase of boric acid concentration had little effect on the elution volume of catecholamines. This result seems to be due to formation of borate complex of catecholamines having less net charge at the higher pH. We used eluents containing 0.35M or 0.66M boric acid. At pH 6.3, epinephrine, norepinephrine and dopamine were eluted in the order described, whereas at pH 4, norepinephrine was eluted first, followed by epinephrine, dopamine and deoxyepinephrine. Isoproterenol overlapped with deoxyepinephrine. Since isoproterenol and deoxyepinephrine do not occur in human plasma and in urine, these amines could be used as the internal standard. Size of the column was 0.8cm in diameter and 12-28cm in length, and 1.0ml of sample solution could be injected without loss of resolution. Flow rate of buffer through the column was 0.7-1.0ml per min and separation of catecholamines was completed within an hour.
Amines were extracted from a deproteinized and neutralized extract or urine by adsorption on an ion-exchange column (Amberlite CG-50, 0.4×12 or 0.5×12cm, buffered at pH 6.5). The column was washed with water and catecholamines were eluted with 0.66M boric acid solution (pH 6.1). pH of the eluate was adjusted to the pH of the eluent to be used for chromatography and diluted to 3 or 4 ml, and when it is not used promptly, it could be stored in a refrigerator for several days. Catecholamines in the eluate were determined fluorimetrically by a modified ethylenediamine condensation method, trihydroxyindole method or 4-aminobenzoic acid-hexacyanoferrate (III) oxidation method. The former method was used for the determination of catecholamines in human urine, the second method was used for the determination of epinephrine, norepinephrine and the third method for dopamine with higher sensitivity. 20-40 picograms of each catecholamine in a sample solution (1.0ml) could be detected.