Abstract
A simple and sensitive high performance liquid chromatographic determination method for free polyamines and their acetylated derivatives was developed. Polyamines were separated on an octadecyl polymer column with isocratic elution. The separated polyamines were introduced into an enzyme reactor containing immobilized acylpolyamine amidohydrolase, amine oxidase (flavin-containing) and putrescine oxidase, in which they were deacetylated and oxidizld to generate hydrogen peroxide. The amount of hydrogen peroxide generated was then determined with an electrochemical detector. When a mixture of nine pure polyamines, i.e., putrescine, acetylputrescine, cadaverine, acetylcadaverine, spermidine, N1-acetylspermidine, N8-acetylspermidine, spermine and acetylspermine, was analyzed with this method, a linear dose response curve up to 1 nmol was obtained for each polyamine except for spermine, which showed linearity in the range of 50 pmol to 1 nmol. The detection limit (S/N>5) for six of polyamines was 1 pmol, but for N8-acetylspermidine and acetylspermine was 10 μmol and for spermine was 30 pmol, respectively. The coefficients of variation were in the range of 1-3%, except those for spermine and N8-acetylspermidine, which were 6.5% and 8.7%, respectively. This method can be applied to the quantitative determination of individual polyamines in clinical specimens such as urine
