Determination of six indolic compounds, including melatonin, in rat pineal using high-performance liquid chromatography with serial fluorimetric-electrochemical detection.

A reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, N-acetylserotonin, tryptophan, 5-hydroxytryptamine (serotonin) and melatonin in rat pineal using a buffered aqueous eluent containing acetonitrile and methanol as organic modifiers and an ion-pairing agent to assist in controlling the retention of compounds containing an amine group. Serial fluorimetric-electrochemical detection provided additional assurance of compound identity. Analyte preparation simply involved the sonication of pineals in dilute perchloric acid containing an antioxidant and a chelating agent, followed by centrifugation to clarify. The method simplifies the determination of this range of indolic compounds, which normally would require at least two separate runs with different eluents. Detection limits for melatonin were 60 and 135 pg for fluorimetric and electrochemical detection, respectively. (This represented the "worst case" considering the levels and detection limits of all compounds present.) Using flow programming and a flow-rate varying between 1 and 1.5 ml/min, the analysis time was 27.5 min, which made the determination of ten samples in a working day possible.