Determination of melatonin and monoamines in rat pineal using reversed-phase ion-interaction chromatography with fluorescence detection

A method is reported for the ion-interaction, reversed-phase separation of 24 compounds (chiefly monoamines) arising from the metabolism of tyrosine and tryptophan. These compounds were separated as two groups. The first group comprised 3,4-dihydroxyphenylethylene glycol, tyrosine, 3-methoxy-4-hydroxyphenyl glycol, 5-hydroxytryptophan, norepinephrine, 3,4-dihydroxyphenylacetic acid, epinephrine, 5-hydroxyindole-3-acetic acid, homovanillic acid, 5-hydroxytryptophol, dopamine, tryptophan, N-acetylserotonin, N-acetyltryptophan, 5-methoxytryptophan and serotonin. The mobile phase consisted of a 6.8:93.2 (v/v) mixture of acetonitrile and an aqueous solution containing 0.16 M ammonium phosphate, 0.06 M citric acid, 0.15 mM disodium EDTA, 10 mM dibutylamine and 6 mM sodium 1-octanesulphonate at pH 4.50. The second group of compounds comprised 6-hydroxymelatonin, 5-methoxyindole-3-acetic acid, indole-3-acetic acid, 5-methoxytryptamine, tryptamine, 5-methoxytryptophol, melatonin and tryptophol. The mobile phase consisted of a 16:84 (v/v) mixture of acetonitrile and an aqueous solution containing 0.05 M ammonium phosphate, 0.05 M citric acid, 0.15 mM disodium EDTA, 25 mM dibutylamine and 5 mM sodium 1-octanesulphonate at pH 5.30. Detection was by fluorescence measurement (λex = 280 nm, λem = 340 nm). The proposed method exhibited linear calibration over the biochemically significant concentration range, with detection limits in the 10–200 pg range. Excellent precision for peak areas and retention times was observed, even over a period of 24 h. The applicability of amperometric detection (at 0.72V) is also demonstrated. The method is applied to the determination of monoamines in individual rat pineals. Low nanogram levels of tyrosine, norepinephrine, 5-hydroxyindole-3-acetic acid, tryptophan, serotonin and 6-hydroxymelatonin, and picogram levels of 5-hydroxytryptophan, 5-hydroxytryptophol, 5-methoxyindole-3-acetic acid, indole 3-acetic acid, 5-methoxytryptophol and melatonin were indicated in most of the samples