Determination of Dissolved Free Putrescine, Spermidine and Spermine in Seawater by High Performance Liquid Chromatography

Abstract

A highly sensitive and accurate reversed phase high performance liquid chromatographic (RP-HPLC) method was developed to determine dissolved free polyamines (putrescine, spermidine and spermine) in seawater. This method is based on pre-column derivatization with dansyl chloride (DNS-Cl). The derivatives were separated on a ODS column (150 mm x 4. 6 mm i. d., 5 m particle size, Agilent). The gradient elution was performed with two mobile phases A (acetonitrile) and B (ammonium acetate) at a flow rate of 1.0 mL/min. The column eluate was monitored by fluorescence detection (lambda(ex) = 340 nm; lambda(em) = 515 nm). The detection limits for putrescine, spermidine and spermine were 9.6 x 10(-11), 2. 8 x 10(-10) and 1.0 x 10(-10) mol/L respectively. The reliability of the method was satisfactory in terms of linearity (R > 0. 99), precision(relative standard deviation below 3. 6%) and recovery (from 84.9% to 110. 9%). This analytical technique requires neither preliminary extraction, nor long multi-step procedures. This method can be applied to the determination of dissolved free polyamines (putrescine, spermidine and spermine) in seawater.