Determination of melatonin in human plasma with solid‐phase extraction, high‐performance liquid chromatography and fluorescence detection

Abstract

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 μl mobile phase; acetonitrile‐phosphate buffer, 0.01 M pH 7.2 (25:75, v/v). 125 μL was injected into the HPLC system and separation was carried out on a Waters SymmetryShield^™ RP₁₈ column 5 μm (250×4.6 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°C.

• HPLC
• indoles
• N‐acetyl‐5‐methoxytryptamine
• SPE