Quantification of biogenic amines in human plasma based on the derivatization with N-hydroxy-succinimidyl fluorescelin-O-acetate by high-performance liquid chromatography

An HPLC method for the determination of biogenic amines based on the precolumn derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA) has been developed. The derivatization was performed at 45 degrees C for 30 min in borate buffer (pH 8.0). The derivatives were separated on a ZORBAX Eclipse XDB-C8 column (150 mm x 4.6 mm id; 5 mu m) and monitored by fluorescence detection (excitation, 469 nm; emission, 512 nm). The LODs (S/N = 3) for spermine, spermidine, putrescine, cadaverine, and phenethylamine were 0.4, 0.2, 0.3, 0.5, and 0.4 nM, respectively. The developed method has been successfully applied to the determination of biogenic amines in human plasma of three healthy volunteers and four cancer patients. Average recoveries for plasma samples ranged from 94 to 106% and coefficients of variation ranged from 1.8 to 4.6%. Deproteinization of plasma was accomplished with ACN to precipitate interfering substances and the centrifuged supernatant was used directly for analysis.