Ion-pair HPLC determination of hydroxycinnamic acid monoconjugates of putrescine, spermidine and spermine

Abstract

The terminal hydroxycinnamic acid monoconjugates of putrescine, spermidine and spermine were resolved and quantified in extracts of plant tissues using sodium hexanesulphonate as an ion-pair reagent and a double methanol gradient. Extracts were prepared from acidic methanol:water (80:20) that had been back-extracted with hexane, and fractionated on C-18 solid phase extraction and carboxymethyl cation exchange cartridges. The chromatographic conditions were capable of separating N1- and N8-coumaroyl and feruloyl spermidine, which differ only by the disposition of the secondary amine in the polyamine side chain. Reliable detection limits for most compounds were less than 5 µg/mL (approximately 10−5 M in the plant extract) and extraction efficiencies were 91% as determined using the novel internal standard, N1-coumaroyl hexanediamine. Copyright © 2000 John Wiley & Sons, Ltd.