Reversed-Phase Ion-Pairing Liquid Chromatographic Separation and Fluorimetric Detection of Polyamines

Abstract

A rapid and specific reversed-phase ion-pairing high performance liquid chromatographic procedure for putrescine, spermidine and spermine is reported. The ion-pairing reagent, heptanesulfonate, was employed and o-phthalaldehyde and 2-mercaptoethanol were used for on-line post-column derivatization and subsequent fluorescence detection. Experiments were carried out to determine the effects of several variables such as pH, concentration of the aqueous buffer, counter-ion concentration, and the percentage of organic modifier in the moving phase. The minimum detection limits for the polyamines ranged from 120 pmol for spermine to 12 pmol for putrescine. The method includes a gradient program which provides complete separation from amino acids and specificity for the 3 polyamines. The procedure was applied successfully to urine and serum samples.