Simple, sensitive assay of polyamines by high-performance liquid chromatography with electrochemical detection after post-column reaction with immobilized polyamine oxidase.

Abstract

This simple, rapid liquid-chromatographic assay of urinary polyamines (putrescine, spermidine, spermine, and cadaverine) involves electrochemical detection with a post-column immobilized enzyme, polyamine oxidase (EC 1.4.3.6) from soybean seedlings. Polyamines are separated by isocratic ion-pairing reversed-phase chromatography, then enzymatically converted, with release of hydrogen peroxide, via the post-column reactor with immobilized polyamine oxidase; the hydrogen peroxide is detected by electrochemical oxidation on a platinum electrode. The detection limits for injected putrescine, spermidine, and spermine were 0.3, 0.5, 0.6, and 4 pmol, respectively, with linear ranges of two to three orders of magnitude. Reproducibility was also good, with CV values less than 7%. The efficiency of the immobilized enzyme column was not decreased after analysis of 300 urine samples. Putrescine and spermidine excretion in urine from patients with blood cancers and solid cancers was significantly increased.