A sensitive fluorometric method for the determination of aliphatic diamines and polyamines in biological materials by high-speed liquid chromatography

A high-speed liquid chromatographic method for the determination of fluores-camine-labeled diamines and polyamines was applied to rat liver, human serum, and urine. The samples, after addition of 1,6-hexanediamine and N-3-aminopropyl-1,6-hexanediamine as internal standards, were appropriately pretreated by perchloric acid precipitation and/or acid hydrolysis. The resultant filtrates were subjected to CM-cellulose column chromatography in pyridine-acetic acid buffer. Stepwise elution with 0.2 and 1 m buffer gave fairly pure diamine and polyamine fractions. The evaporated fractions were dissolved in borate buffer and reacted with fluorescamine by either Procedure I or II, depending on the amine content. Aliquots of the reaction mixture were subjected to high-speed liquid chromatography for measurement of the amines. It was possible by this method to assay biological samples containing less than 100 pmol of the amines with about 5% relative standard deviation.