Sensitive assay for melatonin in human serum by liquid chromatography

A rapid, sensitive and specific assay was developed for routine determination of melatonin in human serum. Melatonin was measured by liquid chromatography with fluorescence detection (λex = 285 nm, λem = 345 nm), using an isocratic mobile phase consisting of water-acetonitrile (75:25, vv). A simple “solid phase extraction” procedure employing LC-18 and Carbograph as adsorbents was used to isolate and purify the compound of interest from serum samples. The average recovery was 88.9% (range 86.3–91.7%). The method gave satisfactory reproducibility and accuracy, the detection limit of the system was 2 pg for injection and the practical limit of the assay for serum is ca. 3 pg ml−1 for 2 ml of sample. In order to evaluate the specificity, the potential interference of 33 compounds (chiefly monoamines) arising from the metabolism of tyrosine and tryptophan were studied. The melatonin concentration in 21 serum samples measured in the present study was compared by using an radioimmunoassay method. Some results on the melatonin circadian rhythm are presented to illustrate the applicability of this method.