Determination of melatonin in human plasma with solid-phase extraction, high-performance liquid chromatography and fluorescence detection.
A new bioanalytical method for the determination of melatonin in plasma with high-performance liquid chromatography (HPLC) and fluorescence detection preceded by solid-phase extraction has been developed and validated. Melatonin was extracted from 3 mL plasma using a Waters Oasis HLB solid-phase extraction cartridge and the elute was evaporated to dryness and dissolved in 200 microl mobile phase; acetonitrile-phosphate buffer, 0.01 M pH 7.2 (25:75, v/v). 125 microL was injected into the HPLC system and separation was carried out on a Waters SymmetryShield RP18 column 5 microm (250 x 4.6 mm). Excitation and emission wavelengths were set to 285 nm and 345 nm, respectively. The HPLC system was able to separate melatonin and internal standard (5-fluorotryptamine) from other endogenous indole compounds such as serotonin and tryptophan. Determination down to 0.10 nmol/L was possible, with an intra-assay precision of about 13%. Melatonin was stable in plasma for at least 30 days at about 23 degrees C.
Römsing
S
S
Department of Clinical Chemistry, Falun Central Hospital, Falun, Sweden. ybq@du.se
Ulfberg
J
J
Bergqvist
Y
Y
D002851:Chromatography, High Pressure Liquid; D005470:Fluorometry / Q000379:methods; D006801:Humans; D008550:Melatonin / Q000097:blood* / Q000302:isolation & purification / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D008550:Melatonin