Determination of free and total (free plus protein-bound) melatonin in plasma and cerebrospinal fluid by high-performance liquid chromatography with fluorescence detection.
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 microm, 150 x 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.
Rizzo
Vittoria
V
Dipartimento di Biochimica, Sezione Analisi Chimico-Cliniche, IRRCS Policlinico San Matteo, Università degli Studi di Pavia, 27100 Pavia, Italy. vrizzo@venus.it
Porta
Camillo
C
Moroni
Mauro
M
Scoglio
Enrico
E
Moratti
Remigio
R
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008550:Melatonin / Q000097:blood* / Q000134:cerebrospinal fluid*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D008550:Melatonin