An Improved Method of Determining Free and Acetylated Polyamines by HPLC Involving an Enzyme Reactor and an Electrochemical Detector1

Abstract

We developed an improved system for the simultaneous measurement of free and acetylated polyamines, which comprised a HPLC pump, a separation column, an enzyme reactor, and an electrochemical detector, connected in series. Polyamines were separated with an isocratic elution system, and the separated polyamines were introduced into the enzyme reactor, in which they were deacetylated and oxidized to generate hydrogen peroxide. The amount of hydrogen peroxide generated was then determined with the electrochemical detector. Analysis of a mixture of nine standard polyamines including both free forms and acetylated derivatives with this method revealed that the analytical variables were satisfactory. For the analysis of polyamines in urine, pretreatment of samples with a weakly acidic ion exchange resin was necessary to reduce the interfering substances present in the urine. On successive determinations of polyamines in a urine sample, the coefficients of variation obtained were below 5.4%, except that for spermine (27.6%), and the analytical recovery rates were above 90%, except that for acetylputrescine (78.5%). The correlation coefficient between the total polyamine content in urine estimated by our method and that obtained by means of a commercially available enzymatic assay system was calculated to be 0.98, and the regression equation was expressed as y = 0.81 x + 0.89.