Fluorometric determination of spermidine using o-phthalaldehyde: optimum reaction conditions and tests of identity

Spermidine and histamine react with o-phthalaldehyde at alkaline pH, giving rise to fluorescent conjugation products that are stabilized by acidification to pH 2–4. The reaction has been used in the fluorometric assay of both these compounds. In the present study, the reaction conditions have been analyzed with the purpose of improving the sensitivity as well as the specificity of the spermidine assay. The sensitivity was improved by carrying out the condensation at pH 11.0–11.6 for 2 min at 100°C. Under these conditions, the lowest measurable amount of spermidine was 10 ng/ml, and the fluorescence of histamine (below 1–2 μg/ml) was nonmeasurable. Boiling the samples for 1 hr after the acidification did not affect the spermidine fluorescence but abolished residual histamine fluorescence. The spermidine fluorescence failed to develop in the presence of CdCl₂ or SrCl₂, whereas the histamine fluorescence was unaffected. Crude extracts of rat brain gave fluorescence readings similar to those of butanol extracts, suggesting that extensive purification of tissue sperimidine prior to assay is not always necessary.