Polyamine detection system for high-performance liquid chromatography involving enzymatic and chemiluminescent...

We have developed a new specific detection system for polyamines, separated by high-performance liquid chromatography, to replace the usual o-phthalaldehyde (OPA) postlabeling method. This system utilizes a chemiluminescent reaction to determine the amount of hydrogen peroxide generated through the enzymatic oxidation of polyamines. With this method, as little as 5 pmol of putrescine could be accurately determined (S/N = 4), and linearity between the amount of this polyamine and the relative chemiluminescence intensity was observed up to 500 pmol. In the cases of cadaverine, spermidine, and spermine, the linearity continued to 1 nmol. The within-assay coefficients of variation for these four polyamines ranged between 0.97% and 2.03%. As compared with the OPA method, the new method is highly specific for polyamines due to the high specificity of the enzyme used. Another advantage of the new method is the direct determination of polyamines in urine samples that contain various compounds that interfere with the OPA method.