Determination of polyamines by precolumn derivatization with 9-fluorenylmethyl chloroformate and reverse-phase high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) method for the determination of picomole levels of polyamines (putrescine, spermidine, and spermine) is described. Amino groups in polyamines react with 9-fluorenylmethyl chloroformate (FMOC) to form stable and highly fluorescent derivatives which can be separated and quantitatively estimated by HPLC in about 12 min. The mean relative elution times ( n = 14) for putrescine, spermidine and spermine are 4.21 ± 0.02, 10.09 ± 0.02 and 11.19 ± 0.04 min. respectively. The method has been applied to determine polyamine concentration in rat dorsal root ganglia (DRG) without interference with endogenous amino acids. Polyamine content of individual rat DRG has been calculated and the values are as follows: putrescine, 36.8 ± 2.01, spermidine, 1652 ± 131.0 and spermine 388.5 ± 38.4 pmol/DRG. Information on polyamine concentrations in DRG may be useful in understanding the mechanism of action of toxic chemicals on nervous system.