Simultaneous analysis of amino acid and biogenic polyamines by high-performance liquid chromatography after pre-column derivatization with N-(9-fluorenylmethoxycarbonyloxy)succinimide

A high-performance liquid chromatography method for the simultaneous analysis of amino acids and biogenic polyamines, using a new procedure for pre-column derivatization of amino groups with N-(9-fluorenylmethoxycarbonyloxy)succinimide is described. The separation of 20 amino acids and 4 biogenic polyamines was achieved within 32 min on a sequence of three short (50 mm) reversed-phase C18, 5 μm columns by elution buffers based on dibutylamine phosphate. The method linearity, calculated for each amino acid and polyamine, has a correlation coefficient higher than 0.991, in concentrations ranging from 0.2 to 50 μM, except for spermine and methionine, where the correlation coefficients were r=0.984 and r=0.979, respectively. The stability of derivatives in acidified samples at 4 °C and room temperature was demonstrated. The limit of quantitation was estimated to be around 50 pM in 50 μl sample injection. The repeatability of the method, expressed as R.S.D., ranged from 1.1 to 6.7%. The presented method was applied for the quantitation of amino acid and polyamine contents in beer, wine, and cell culture samples, using 2-aminoheptanoic acid or 1,7-diaminoheptane as internal standard.