Determination of Polyamines by Pre-Column Derivatization and Electrochemical Detection

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Abstract
Precolumn derivatization with o-phtalaldehyde (OPA) and thiol, combined with HPLC was used to measure the polyamines spermine, spermidine, putrescine and cadaverine. The derivatives were quantified by electrochemical detection instead of fluorescence; the optimum potential from the working electrodes was + 0.65 V. Best results were obtained with a derivatization mixture containing excess OPA and thiol, methanol and borate buffer, pH 9.5. Spermine gave two derivative peaks; the broad minor peak seemed to be an intermediate which could be converted to the major derivative by increasing either the methanol content of the derivatization mixture or the reaction time. The best reaction time was 10 min; the detector response was linear from 1 pmole to 10 nmoles and the sensitivity limit was from 200 fmoles for spemidine to 1.5 pmoles for spermine. The column retention times could be reduced by the addition of methanol or dimethylcyclohexylamine (DCA) to the eluent. Substitution of H3PO4 by HC104 improved peak quality. The best elution of standards was obtained with HC104/DCA and 80% methanol. A gradient from 60 to 90% methanol gave improved separation when required for biological samples.