Determination of free and total (free plus protein-bound) melatonin in plasma and cerebrospinal fluid by high-performance liquid chromatography with fluorescence detection

摘要

A simple, sensitive and accurate method for the estimation of free and total (free plus protein-bound) melatonin (MLT) in human plasma and cerebrospinal fluid (CSF) is described. Via Chem-Elut cartridges, free and total MLT (the latter obtained after a deproteinization step) were quantified in dichloromethane-extracted samples and analyzed in one chromatographic run by high-performance liquid chromatography (HPLC) with fluorimetric detection. The column used was an Extrasil ODS-2 (3-μm, 150 * 4.6 mm I.D.), while the mobile phase consisted of 75 mM sodium acetate-acetonitrile (72:28, v/v) (pH 5.0). Repeatability and reproducibility of the method were 3.24 and 9.4%, respectively. The recovery of melatonin from plasma and CSF was 99.9 ± 4.0% for non-deproteinized samples and 93.2 ± 4.8% for deproteinized samples. The detection limit of the assay was 0.5 pg/ml. In human plasma, the mean±SD concentrations in the darkness period were 23.18 ± 7.44 pg/ml for free melatonin and 82.5 ± 36.48 pg/ml for total melatonin, while the lowest concentrations detected during daytime were 2.23 ± 2.22 and 7.40 ± 5.68 pg/ml, respectively. Detection of MLT in CSF was 5.01 ± 2.31 and 28.55 ± 6.95 pg/ml for the free and total fraction, respectively.